bubonic plague research paper

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• Published: 15 June 2022

The source of the Black Death in fourteenth-century central Eurasia

• Maria A. Spyrou   ORCID: orcid.org/0000-0002-3615-3936 1 , 2 , 3 ,
• Lyazzat Musralina 2 , 3 , 4 , 5 ,
• Guido A. Gnecchi Ruscone   ORCID: orcid.org/0000-0002-6490-8101 2 , 3 ,
• Arthur Kocher   ORCID: orcid.org/0000-0002-9499-6472 2 , 3 , 6 ,
• Pier-Giorgio Borbone 7 ,
• Valeri I. Khartanovich   ORCID: orcid.org/0000-0002-5533-0686 8 ,
• Alexandra Buzhilova   ORCID: orcid.org/0000-0001-6398-2177 9 ,
• Leyla Djansugurova   ORCID: orcid.org/0000-0002-6745-9903 4 ,
• Kirsten I. Bos   ORCID: orcid.org/0000-0003-2937-3006 2 , 3 ,
• Denise Kühnert   ORCID: orcid.org/0000-0002-5657-018X 2 , 3 , 6 , 10 ,
• Wolfgang Haak   ORCID: orcid.org/0000-0003-2475-2007 2 , 3 ,
• Philip Slavin   ORCID: orcid.org/0000-0002-6460-145X 11 &
• Johannes Krause   ORCID: orcid.org/0000-0001-9144-3920 2 , 3  

Nature volume  606 ,  pages 718–724 ( 2022 ) Cite this article

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• Archaeology
• Evolutionary genetics

The origin of the medieval Black Death pandemic ( ad  1346–1353) has been a topic of continuous investigation because of the pandemic’s extensive demographic impact and long-lasting consequences 1 , 2 . Until now, the most debated archaeological evidence potentially associated with the pandemic’s initiation derives from cemeteries located near Lake Issyk-Kul of modern-day Kyrgyzstan 1 , 3 , 4 , 5 , 6 , 7 , 8 , 9 . These sites are thought to have housed victims of a fourteenth-century epidemic as tombstone inscriptions directly dated to 1338–1339 state ‘pestilence’ as the cause of death for the buried individuals 9 . Here we report ancient DNA data from seven individuals exhumed from two of these cemeteries, Kara-Djigach and Burana. Our synthesis of archaeological, historical and ancient genomic data shows a clear involvement of the plague bacterium Yersinia pestis in this epidemic event. Two reconstructed ancient Y. pestis genomes represent a single strain and are identified as the most recent common ancestor of a major diversification commonly associated with the pandemic’s emergence, here dated to the first half of the fourteenth century. Comparisons with present-day diversity from Y. pestis reservoirs in the extended Tian Shan region support a local emergence of the recovered ancient strain. Through multiple lines of evidence, our data support an early fourteenth-century source of the second plague pandemic in central Eurasia.

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The Black Death, caused by the bacterium Y. pestis 10 , was the initial wave of a nearly 500-year-long pandemic termed the second plague pandemic and is one of the largest infectious disease catastrophes in human history 1 , 11 , 12 . Estimated to have claimed the lives of up to 60% of the western Eurasian population over its eight-year course 1 , 12 , the Black Death had a profound demographic and socioeconomic impact in all affected areas, with the European historical record being the most extensively studied resource until now 2 , 13 , 14 , 15 .

Despite intense multidisciplinary research on this topic, the geographical source of the second plague pandemic remains unclear. Hypotheses based on historical records and modern genomic data have put forward a number of putative source locations ranging from western Eurasia to eastern Asia (Supplementary Information  1 ). In recent years, comparisons between ancient and modern Y. pestis genomes have shown the Black Death to be associated with a star-like emergence of four major lineages (branches 1, 2, 3 and 4) 16 , 17 , the descendants of which are dispersed among rodent foci in Eurasia, Africa and the Americas. Although extant lineages that diverged before this event have been identified in central and eastern Eurasia 16 , 18 , 19 , complementary ancient DNA (aDNA) data from such regions are lacking. Until now, analyses of the historical record and ancient Y. pestis data have largely focused on the pandemic’s progression in western Eurasia 12 , 17 , 20 , 21 . Although efforts to expand historical investigations and provide a wider spatiotemporal perspective are under way 9 , 11 , 22 , 23 , 24 , 25 , 26 , the prevailing Eurocentric focus has hampered an identification of the origins of the Second Pandemic.

A fourteenth-century epidemic in central Eurasia

To explore possible evidence associated with the early history of the second plague pandemic, we investigated the cemeteries of Kara-Djigach and Burana, located in the Chüy Valley near Lake Issyk-Kul of modern-day Kyrgyzstan. Excavations of these cemeteries between 1885 and 1892 revealed a unique archaeological assemblage potentially associated with an epidemic that affected the region during the fourteenth century (Fig. 1 and Supplementary Information  2 ). On the basis of tombstone inscriptions, these cemeteries showed a disproportionally high number of burials dating between 1338 and 1339, with some inscriptions stating that the cause of death was due to an unspecified pestilence 9 , 27 (Fig. 1 , Extended Data Fig. 1 , Supplementary Fig. 1 , Supplementary Table 1 and Supplementary Information  2 ). Given the location, timing and associated demographic pattern, early interpretations considered these characteristics as indicative of a plague epidemic 3 , 27 and have since triggered a long-lasting debate about the epidemic’s association with the onset of the second plague pandemic 1 , 3 , 4 , 5 , 6 , 7 , 8 , 9 , 26 (Supplementary Information 2 ).

a , Locations of the Kara-Djigach and Burana archaeological sites in modern-day Kyrgyzstan. Regions encompassing Y. pestis foci at present are highlighted in blue (as in refs. 18 , 19 ). The map was created using QGIS v.3.22.1 (ref. 51 ) and uses Natural Earth vector map data from  https://www.naturalearthdata.com/ . b , Area within the Kara-Djigach cemetery, referred to as ‘Chapel 1’ with the highest concentration of excavated burials dating between 1338 and 1339. Burial dates were determined on the basis of their associated tombstones (Supplementary Information  2 ). The site map has been redrawn based on the original created by N. Pantusov in 1885. Individuals from graves 6, 9, 20, 22 and 28 (the numbers in bold) were investigated using aDNA in this study. Burials shown with stripe patterns were associated with individuals BSK001, BSK003 and BSK007, which showed evidence of Y. pestis infections. c , Annual numbers of tombstones from Kara-Djigach ( n  = 456) and Burana ( n  = 11) (Supplementary Table 1 ). Dataset updated from ref. 9 (see Supplementary Information  2 for details). d , Tombstone from the Kara-Djigach cemetery with legible pestilence-associated inscription. The inscription is translated as ‘In the Year 1649 [= ad  1338], and it was the Year of the Tiger, in Turkic Bars. This is the tomb of the believer Sanmaq. [He] died of pestilence [=mawtānā]’. For a tracing of the inscription, see Extended Data Fig. 1 .

To better understand the contexts of Kara-Djigach and Burana, we translated and analysed surviving archival information from their excavations (Supplementary Information  2 and Supplementary Figs. 1 – 4 ). Furthermore, we generated human genomic data from 7 individuals (5 from Kara-Djigach and 2 from Burana) through a hybridization capture of approximately 1.24 million ancestry-informative single-nucleotide polymorphisms (SNPs) 28 , which resulted in 4 individuals with sufficient genomic coverage for population genetic analyses (>30,000 SNPs). Using principal component analysis and ancestry modelling, we found these individuals to be falling broadly within the variability of ancient and present-day populations from central Eurasia. However, precise connections could not be determined given the scarcity of contemporaneous human genomic data from this region (Supplementary Information  3 , Supplementary Fig. 5 and Supplementary Tables 2 – 5 ). On the basis of the available tombstone inscriptions, burial artefacts, coin hoards and historical records, we found that the Chüy Valley housed ethnically diverse communities that relied on trade and maintained connections with several regions across Eurasia (Supplementary Information  2 ). Such links may have contributed to the spread of infectious diseases to and from this region during the fourteenth century.

Ancient pathogen DNA screening

To investigate traces of ancient pathogen DNA that could explain the cause of the suspected epidemic, shotgun metagenomic data generated from all seven individuals were taxonomically classified using the HOPS pipeline 29 (Supplementary Table 6 ). Of those, three individuals exhumed from the Kara-Djigach cemetery (BSK001, BSK003 and BSK007) displayed potential evidence of ancient Y. pestis DNA (Supplementary Table 7 ) as well as low edit distances in reads mapping against the CO92 reference genome, and the presence of chemical alterations characteristic of aDNA (Supplementary Fig. 6 and Supplementary Table 8 ). As such, the respective DNA libraries were subjected to whole-genome Y. pestis capture ( Methods ).

The ancestor of a fourteenth-century polytomy

Whole-genome Y. pestis capture yielded 6.7-fold and 2.8-fold average coverage for BSK001 and BSK003, respectively. Coverage across all three Y. pestis plasmids ranged from 24.7-fold to 4.7-fold (Supplementary Tables 9 and 10 ). For BSK007, genomic coverage was lower, approximately 0.13-fold, resulting from poorer aDNA preservation that was also reflected in the shotgun screening and human DNA enrichment data (Supplementary Tables 2, 3 and 8 ). Nevertheless, this sample was considered a true Y. pestis -positive because of the even distribution of mapping reads against the CO92 reference chromosome and the presence of aDNA-associated damage (Extended Data Figs. 2 and 3 and Supplementary Tables 9 – 11 ). Furthermore, a metagenomic classification of BSK007 reads aligning to the pCD1, pMT1 and pPCP1 plasmids identified >99% as Y. pestis -specific (Extended Data Fig. 3 ).

To evaluate whether the higher-coverage Y. pestis genomes BSK001 and BSK003 represented distinct bacterial strains, we compared their SNP profiles. To limit variant calls deriving from environmental contamination, particularly given the high amounts of multi-allelic sites identified in both genomes (Supplementary Fig. 7 ), we performed a taxonomy-informed metagenomic filtering using MALT ( Methods and Supplementary Table 11 ). We identified 20 sites differing between BSK001 and BSK003, all of which are unique variants in the lower-coverage BSK003 (Supplementary Table 12 ). On the basis of previously defined authenticity criteria 30 , 31 ( Methods ), all such variants were consistent with residual exogenous contamination, suggesting that the two genomes were probably identical. Recovery of identical strains from both individuals is consistent with published evidence showing low diversity in Y. pestis genomes isolated from single epidemic contexts 10 , 17 , 20 , 21 , 32 . On the basis of their associated tombstones, BSK001, BSK003 and BSK007 were buried during the epidemic year 1338–1339 (Fig. 1 and Supplementary Information  2 ) and our data further support a Y. pestis involvement in this event.

We performed a comparative SNP analysis between the Kara-Djigach genomes and previously published historical and currently circulating Y. pestis diversity (Fig. 2a , Supplementary Tables 13 – 15 ). For this, BSK001 and BSK003 were combined (BSK001/003) to achieve an increased genomic resolution (combined coverage of 9.5-fold; Supplementary Table 9 ). Our analysis revealed one SNP unique to BSK001/003 when compared against 203 modern and 46 historical Y. pestis chromosomal genomes (Extended Data Fig. 4 and Supplementary Tables 16 and 17 ). This SNP was found in a region with persistent multi-allelic sites; therefore, it is considered artefactual 31 (Supplementary Fig. 8 ). Consistent with previous research on the evolutionary history of Y. pestis 16 , our inferred maximum likelihood phylogeny exhibited five major branches, designated 0, 1, 2, 3 and 4, with published Second Pandemic genomes being associated with branch 1 (Fig. 2b ). The placement of BSK001/003 is ancestral to all published fourteenth-century genomes from western Eurasia (Fig. 2b and Extended Data Fig. 5 ), separated by one SNP from LAI009, an isolate from the Volga region in eastern Europe 17 , and by two SNPs from five genetically identical Black-Death-associated genomes from southern, central and northern Europe 17 , 21 . Specifically, BSK001/003 is positioned on a node previously designated N07 (ref. 16 ), which preceded the multifurcation of branches 1–4. To evaluate whether missing data affected the accuracy of our phylogenetic placements, we investigated all BSK001 and BSK003 variant calls for shared positions with lineages deriving from the N07 node and those directly preceding it. BSK001/003 carries the ancestral state in all covered diagnostic SNPs defining branches 1–4 and 0.ANT3, which is the closest related branch 0 lineage to BSK001/003, as well as the derived state in all positions leading from 0.ANT3 to N07 (Fig. 2c , Extended Data Fig. 6 and Supplementary Table 18 ). At our current resolution, we conclude that BSK001/003 represents the direct progenitor of the branch 1–4 polytomy.

a , Map of all historical Y. pestis genomes used in the present study ( n  = 48). The colours represent different genome ages on a scale between 1300 and 1800, as depicted in b . The colour scale is maintained across all panels of this figure. To aid visibility in overlapping symbols, a jitter option was implemented for plotting genomes on the map. The map was created with QGIS v.3.22.1 (ref. 51 ) and uses Natural Earth vector map data from  https://www.naturalearthdata.com/ . b , Y. pestis maximum likelihood phylogeny based on 2,960 SNPs, visualized using GrapeTree 50 . The depicted portion of the phylogeny contains the closest related lineages to BSK001/003. (For a fully labelled tree, see Extended Data Fig. 5 ). The colours of published historical strains are consistent with a . The scale denotes the number of substitutions per genomic site. c , Abundance of diagnostic SNP sharing in fourteenth-century Y. pestis genomes. The number of diagnostic SNPs ( n ) shared between all modern genomes on branch 1, and therefore defining this branch, were retrieved from a comparative SNP table of 203 modern Y. pestis genomes. SNP sharing was assessed by determining the allele status of each diagnostic position according to a threefold SNP calling threshold. The error bars denote the degree of missing data ( n ) in the respective ancient genome. Refer to Extended Data Fig. 6 and Supplementary Table 18 for an overview of diagnostic SNP sharing on different phylogenetic branches.

Divergence time for the branch 1–4 polytomy

The polytomy of branches 1–4 is a major event in the evolution of Y. pestis given its association with the Black Death 9 , 26 , 33 and the rich genetic diversity that emerged from it 16 (Fig. 2b ). Estimates on the timing of this diversification have so far yielded wide ranges spanning from the tenth to the fourteenth centuries 16 , 34 . Recently, a narrower time frame was proposed that placed this emergence in the early thirteenth century, more than 100 years before the Black Death 22 , 26 . As BSK001/003 represents the common ancestor of branches 1–4, we used this genome from 1338 to 1339 to construct a time-calibrated phylogeny and re-estimate an age range for this diversification with BEAST2 (Supplementary Figs. 9 and 10 and Supplementary Table 19 ). After evaluating a number of demographic models (Supplementary Table 20 ), our resulting estimates based on the coalescent Bayesian skyline model revealed overlapping ages for the divergence of BSK001/003 (95% highest posterior density (HPD): 1308–1339), as well as for that of branch 1 from branches 2–4 (95% HPD: 1317–1345) (Fig. 3 ). As BEAST2 only infers bifurcating trees, we also used TreeTime 35 to infer a time-calibrated phylogeny that can retain polytomies. Consistent with our estimates above, we inferred a 1316–1340 date for the split time of branches 1–4 (Supplementary Fig. 11 ), although we caution that this method does not account for age uncertainties in ancient genomes. Taken together, the present results support an age range spanning the first half of the fourteenth century for the timing of the branch 1–4 polytomy.

a , Maximum clade credibility time-calibrated phylogenetic tree. The tree is based on 167 genomes (historical and modern) and was estimated using the coalescent skyline tree prior and a log-normal relaxed clock. Collapsed branches contain modern and ancient isolates dating after AD 1400 (post-Black Death). The coloured arrows mark the nodes, for which equivalent posterior age distributions are shown in b . The estimated divergence dates (95% HPD intervals) of modern branches are shown on each corresponding node. b , Estimated posterior distributions based on the coalescent Bayesian skyline tree prior for the divergence of Y. pestis branches 1–4 (blue), for the estimated divergence of BSK001/003 (purple) and for the entire dataset used for this analysis (time to the most recent common ancestor of branches 1–4 and 0.ANT3, shown in grey). The dotted lines indicate mean posterior estimates and are annotated with the corresponding 95% HPD intervals.

Furthermore, to quantify the proportion of present-day Y. pestis genetic diversity that emerged from this polytomy, we computed mean pairwise distances (MPDs) and Faith’s phylogenetic diversity (FPD) indices in 203 genomes comprising our entire modern dataset, as well as 130 genomes comprising branches 1–4 ( Methods ). In our dataset, 64% (130 out of 203) of modern Y. pestis strains belonged to branches 1–4, reflecting the high worldwide frequency known for these lineages 16 , 36 , 37 . We estimate that branches 1–4 represent approximately 40% of the overall phylogenetic diversity within present-day Y. pestis based on our full dataset (MPD ratio: 41%; 95%  percentile interval (PI): 35.3–46.4; FPD ratio: 35.9%; 95% PI: 31.6–39.5). This value is marginally reduced after equalizing the number of genomes in branches 1–4 and branch 0 (MPD ratio: 36.8%; 95% PI: 32.0–41.9; FPD ratio: 33.9%; 95% PI: 29.4–37.7) (Extended Data Fig. 7 ). Given that the known history of Y. pestis  reaches back at least 5,000 years 38 , it is notable that a substantial fraction of its surviving genetic diversity accumulated since the fourteenth century.

Plague reservoirs in the Tian Shan area

To address existing hypotheses on the Black Death’s geographical origins (Supplementary Information  1 ), we investigated the possibility of a local emergence versus an introduction of the BSK001/003 strain into the Chüy Valley  from a different area. For this, we assessed the geographical distribution of the most closely related ancestral branching lineages to BSK001/003 and identified 164 present-day 0.ANT strains with record of their isolation locations (Supplementary Table 21 ). Consistent with previous interpretations 9 , 18 , 26 , we found that all such strains were retrieved from foci in eastern Kazakhstan, eastern Kyrgyzstan and the Xinjiang Uygur Autonomous Region of northwestern China (Fig. 4 and Extended Data Fig. 8 ). Although we cannot exclude a different geographical range for these lineages in the past, the current data are consistent with a local emergence of BSK001/003 within the extended Tian Shan region. Intriguingly, the oldest recovered genome associated with 0.ANT was also identified in the Tian Shan region (third century  AD ) 39 and forms part of an extinct clade that caused the first plague pandemic (sixth to eighth centuries  AD ) 30 . As noted previously 18 , 26 , 33 , 40 , most extant 0.ANT strains have been isolated from marmots and their ectoparasites known to be the primary Y. pestis reservoirs in these areas (Supplementary Table 21 ). Therefore, such species could represent possible candidates for the spillover that led to the second plague pandemic.

a , Maximum likelihood phylogenetic tree, based on 2,441 genome-wide variant positions. The tree was constructed to indicate the genetic relationships between available 0.ANT genomes depicted on the map and BSK001/003. Modern branches were collapsed to enhance tree clarity (see Extended Data Fig. 8 for a full tree). b , Map depicting the geographical isolation locations of 0.ANT strains (Supplementary Table 21 ), which belong to the closest ancestral branching lineages to the Kara-Djigach strain. The map includes both whole-genome data (further specified as 0.ANT lineages 1, 2, 3 and 5) and PCR-genotyped isolates that are broadly defined as 0.ANT, belonging to any of the 4 lineages. For strains in which exact geographical coordinates were unavailable, locations were approximated according to their associated plague reservoirs. To aid visibility in overlapping symbols, a jitter option was implemented for plotting objects on the map. The map was created with QGIS v.3.22.1 (ref. 51 ) and uses Natural Earth vector map data from https://www.naturalearthdata.com/ .

The power of ancient metagenomics lies in its potential to provide direct evidence for testing long-standing historical hypotheses and reveal phylogeographical patterns of microbial diversity through time 41 . One such debate concerns the events that triggered the second plague pandemic, as well as the time and place of its emergence. Recently, an analysis of historical, genetic and ecological data led to the suggestion that the emergence of Y. pestis branches 1–4 occurred more than a century before the beginning of the Black Death. According to the proposed model, this initial diversification was mediated by people and was linked with territorial expansions of the Mongol Empire across Eurasia during the early thirteenth century 22 , 26 , 42 . By contrast, we present ancient Y. pestis data from central Eurasia that support a fourteenth-century emergence; therefore, earlier outbreak attributions remain to be explored. At present, the narrow-focused sampling chosen for this study does not allow for an assessment of the spread of the BSK001/003 strain. Previous studies have shown that Y. pestis can disseminate rapidly without accumulation of genetic diversity 17 , 21 , thus potentiating a contemporaneous presence of the same strain across a large geographical range. Nevertheless, the known range of extant plague foci associated with lineages ancestral to BSK001/003 provide support for its emergence in central Eurasia and possibly in the extended Tian Shan region. Although the dynamics that triggered the bacterium’s emergence in this region are unknown, previous studies showed that environmental factors, such as natural disasters and sudden changes in temperature and precipitation can have an impact on Y. pestis host ecologies and, as a result, can trigger outbreaks in human populations 43 , 44 , 45 , 46 . Although we have no evidence to infer such connections with the Kara-Djigach epidemic, we envision that our precise 1338–1339 date will serve as a reference point for future environmental, archaeological and historical research focusing on the events that caused a Y. pestis introduction into human populations and precipitated the second plague pandemic.

The onset of the Black Death has been conventionally associated with outbreaks that occurred around the Black Sea region in 1346 (refs. 1 , 47 ), eight years after the Kara-Djigach epidemic. At present, the exact means through which Y. pestis reached western Eurasia are unknown, primarily due to large pre-existing uncertainties around the historical and ecological contexts of this process. Previous research suggested that both warfare and/or trade networks were some of the main contributors in the spread of  Y. pestis 21 , 22 , 26 , 47 , 48 . Yet, related studies have so far either focused on military expeditions that were arguably unrelated to initial outbreaks 47 or others that occurred long before the mid-fourteenth century 22 , 26 . Moreover, even though preliminary analyses exist to support an involvement of Eurasian-wide trade routes in the spread of the disease 48 , their systematic exploration has so far been conducted only for restricted areas of western Eurasia 21 , 47 . The placement of the Kara-Djigach settlement in proximity to trans-Asian networks 9 , 49 , as well as the diverse toponymic evidence and artefacts identified at the site (Supplementary Information  2 ) lend support to scenarios implicating trade in Y. pestis dissemination. Therefore, an investigation of early-to-mid-fourteenth-century connections across Asia, interpreted alongside genomic evidence, will be important for disentangling the bacterium’s westward dispersals.

Past and present experiences have demonstrated that reconciling the source of a pandemic is a complex task that cannot be accomplished by a single research discipline. Although the ancient Y. pestis genomes reported in this Article offer biological evidence to settle an old debate, it is the unique historical and archaeological contexts that define our study’s scope and importance. As such, we envision that future synergies will continue to reveal important insights for a detailed reconstruction of the processes that triggered the second plague pandemic.

Sampling, DNA extraction, partial uracil DNA glycosylase library preparation and sequencing

We obtained permission from the Kunstkamera, Peter the Great Museum of Anthropology and Ethnography in St Petersburg for the sampling and ancient DNA analysis of 7 tooth specimens, excavated between 1885 and 1892 from the medieval cemeteries of Kara-Djigach and Burana (Supplementary Information  2 ). No statistical methods were used to predetermine the number of samples used in this study. All laboratory procedures were carried out in the dedicated aDNA facilities of the Max Planck Institute for the Science of Human History and the Max Planck Institute for Evolutionary Anthropology. The detailed procedures used for tooth sampling can be found in ref. 52 . In brief, teeth were sectioned in the dentin–enamel junction using an electric saw with a diamond blade. After tooth sectioning, approximately 50 mg of powder was removed from the surface of the pulp chamber of each tooth using rounded dental drill bits.

The recovered tooth powder was used for DNA extractions using a previously established protocol optimized for the recovery of short fragments of DNA 53 . The exact steps and modifications of the procedure used have been made available in ref. 54 . In brief, the tooth powder was incubated overnight (12–16 h) at 37 °C in 1 ml of DNA lysis buffer containing EDTA (0.45 M, pH 8.0) and proteinase K (0.25 mg ml −1 ). After incubation, DNA binding and isolation was performed using a custom GuHCl-based binding buffer and purification using High Pure Viral Nucleic Acid Large Volume Kit (Roche). Finally, DNA was eluted in 100 μl of Tris-EDTA-Tween containing Tris-HCl (10 mM), EDTA (1 mM, pH 8.0) and Tween-20 (0.05%). For procedure monitoring, extraction blanks and positive extraction controls were included throughout the laboratory processing steps.

All DNA extracts were converted into one-to-two double-stranded DNA libraries for Illumina sequencing, using 25 μl of input extract per library with an initial partial uracil DNA glycosylase (UDG) and endonuclease VIII treatment (USER enzyme; New England Biolabs) according to established protocols 55 , 56 . The detailed library preparation procedure, including the blunt-end repair, adaptor ligation and adaptor fill-in reaction steps can be found in ref. 57 . After library preparation, each library was quantified using a quantitative PCR system (LightCycler 96 Instrument) using the IS7 and IS8 primers 55 . For multiplex sequencing, we performed double indexing of all libraries using previously published procedures 58 , outlined in detail in ref. 59 . A combination of unique index primers containing 8 base pair (bp) identifiers were assigned to each library. To aid amplification efficiency, libraries were then split into multiple PCR reactions for the indexing step based on their initial IS7/IS8 quantification. The number of indexing PCR reactions performed for each library was determined so that every reaction was assigned an input of no more than 1.5 × 10 10 DNA copies. Each reaction was set up using the Pfu Turbo Cx Hotstart DNA Polymerase (Agilent Technologies) and was run for 10 cycles using the following conditions: initial denaturation at 95 °C for 2 min followed by a cycling of 95 °C for 30 s, 58 °C for 30 s and 72 °C for 1 min, as well as a final elongation step at 72 °C for 10 min. All PCR products were purified using the MinElute DNA Purification Kit (QIAGEN), with some modifications to the manufacturer’s protocol 59 . Finally, all indexing PCR products were qPCR-quantified (LightCycler 96 Instrument) using the IS5 and IS6 primer combination 58 , 59 . To avoid heteroduplex formation, indexed libraries were amplified to  10 13 DNA copies per reaction with the Herculase II Fusion DNA Polymerase (Agilent Technologies) and quantified using a 4200 Agilent TapeStation Instrument using a D1000 ScreenTape system (Agilent Technologies). Libraries were diluted to 10 nM and pooled equimolarly for sequencing. We performed shotgun DNA sequencing on an Illumina HiSeq 4000 platform using a 76-cycle kit (1 × 76 + 8 + 8 cycles).

Shotgun next-generation sequencing read processing and metagenomic screening

After demultiplexing, raw shotgun sequenced reads were preprocessed in the EAGER pipeline v.1.92.58 using AdapterRemoval v.2.2.0 (ref. 60 ), which was used to remove Illumina adaptors (minimum overlap of 1 bp), as well as for read filtering according to sequencing quality (minimum base quality of 20) and length (retaining reads ≥30 bp). Subsequently, all datasets were screened for the presence of pathogen DNA traces using the metagenomic pipeline HOPS 29 . First, preprocessed reads were aligned against a custom RefSeq database 61 (November 2017) containing all complete bacterial and viral genome assemblies, a subset of eukaryotic pathogen assemblies and the GRCh38 human reference genome. Genome assemblies that contained the word ‘unknown’ were removed from the database, retaining a total of 15,361 entries. The database retained a number of Yersinia species entries: Yersinia aldovae ( n  = 1), Yersinia aleksiciae ( n  = 1), Yersinia enterocolitica ( n  = 16), Yersinia entomophaga ( n  = 1), Yersinia frederiksenii ( n  = 3), Yersinia intermedia ( n  = 1), Yersinia kristensenii ( n  = 2), Y. pestis ( n  = 39), Yersinia phage ( n  = 17), Yersinia pseudotuberculosis ( n  = 13), Yersinia rohdei ( n  = 1), Yersinia ruckeri ( n  = 4), Yersinia similis ( n  = 1) and Yersinia sp. FDA-ARGOS ( n  = 1). MALT v0.4 62 was run using the following parameters: -id 90 -lcaID 90 -m BlastN -at SemiGlobal -topMalt 1 -sup 1 -mq 100 -verboseMalt 1 -memoryMode load -additionalMaltParameters. The resulting alignment files were post-processed with MALTExtract for a qualitative assessment against a predefined list of 356 target taxonomic entries ( https://github.com/rhuebler/HOPS/blob/external/Resources/default_list.txt ). Specifically, reads were assessed according to their edit distance against a specific pathogen sequence in the database and the potential occurrence of mismatches that could signify the presence of aDNA damage 29 . In cases in which both parameters were met, the corresponding pathogen alignment was considered a strong candidate. Preprocessed reads were mapped against the Y. pestis CO92 (NC_003143.1) and human ( hg19 ) reference genomes with the Burrows–Wheeler Aligner (BWA). Mapping parameters were set to 0.01 for the edit distance (-n) and seed length was disabled (-l 9999). Subsequently, we used SAMtools v.1.3 (ref. 63 ) to remove reads with mapping quality lower than 37 (for CO92) or 30 (for hg19 ); PCR duplicates were removed with MarkDuplicates v1.140 ( http://broadinstitute.github.io/picard/ ). Finally, patterns of aDNA damage were assessed with mapDamage v.2.0 (ref. 64 ).

Single-stranded DNA library preparation and hybridization capture

For specimens BSK001 and BSK003, extra single-stranded DNA libraries were constructed from an input DNA extract of 30 μl. We performed library preparation at the Max Planck Institute for Evolutionary Anthropology using an automated protocol that is publicly available 65 . Single-stranded and double-stranded libraries from individuals BSK001, BSK003 and BSK007 were enriched using DNA probes covering the whole Y. pestis genome, as well as 1.24 million genome-wide SNP sites of the human genome 66 , 67 . For capture preparation, all libraries were amplified for the necessary number of PCR cycles to achieve 1–2 μg of input DNA. PCR reactions were carried out using the Herculase II Fusion DNA Polymerase. They were then purified using the MinElute DNA Purification Kit and eluted in EB elution buffer containing 0.05% Tween 20. Finally, library concentrations (ng μl −1 ) were quantified using a NanoDrop spectrophotometer (Thermo Fisher Scientific). For the in-solution Y. pestis captures, the probe set design was based on a set of publicly available modern genomes, specifically the Y. pestis CO92 chromosome (NC_003143.1), CO92 plasmid pMT1 (NC_003134.1), CO92 plasmid pCD1 (NC_003131.1), KIM10 chromosome (NC_004088.1), Pestoides F chromosome (NC_009381.1) and the Y. pseudotuberculosis IP32953 chromosome (NC_006155.1). For the in-solution human DNA captures, the probe set design was created to target 1,237,207 variants across the genome that are informative for studying the genetic history of worldwide human populations 28 , 67 . Both human DNA and Y. pestis hybridization captures were carried out for two rounds as described previously 28 , 69 , 68 , 67 , 66 , in which partially UDG-treated libraries from the same individual were pooled in equimolar ratios for capture and single-stranded libraries were captured separately.

Post-capture Y. pestis data processing

After Y. pestis whole-genome capture, libraries were sequenced on a HiSeq 4000 platform (1 × 76 + 8 + 8 cycles or 2 × 76 + 8 + 8 cycles) at a depth of approximately 11–27 million raw reads. The preprocessing of raw demultiplexed reads was carried out as described in the ‘Shotgun next-generation sequencing read processing and metagenomic screening’ section. At this stage, the datasets produced from partially UDG-treated libraries from the same individual were pooled and terminal bases were trimmed using fastx_trimmer (FASTX Toolkit 0.0.14,  http://hannonlab.cshl.edu/fastx_toolkit/ ) to avoid damaged site interference with SNP calling during further processing. The following steps for read mapping, PCR duplicate removal and aDNA damage calculation were carried out in the EAGER pipeline 70 . We performed read mapping with BWA v.0.7.12 against the Y. pestis CO92 reference genome (NC_003143.1). For the pooled and trimmed partial UDG-treated libraries, BWA parameters were set to 0.1 for the edit distance (-n) and seed length was disabled (-l 9999). Given that the single-stranded libraries constructed for this study retained aDNA-associated damage, the BWA parameters were set to 0.01 for the edit distance (-n) to allow for an increased number of mismatches that could derive from deamination; seed length was disabled (-l 9999). We performed read mapping against the plasmids using the same parameters against a concatenated reference of all three Y. pestis plasmids (pMT1: NC_003134.1; pPCP1: NC_003132.1; and pCD1: NC_003131.1), masking the problematic pPCP1 region between nucleotides 3000 and 4200 that was shown to have high similarity to expression vectors used in laboratory reagents 71 . SAMtools v.1.3 (ref. 63 ) was used to remove all reads with mapping quality lower than 37 (-q), whereas MarkDuplicates was used to remove PCR duplicates. Deamination patterns associated with aDNA damage were retrieved with mapDamage v.2.0 (ref. 64 ). We used MALT 62 for a taxonomic classification of mapped reads, to attempt a retention of reads that are more likely to be endogenous Y. pestis . MALT was run against the same database as described in the section ‘Shotgun next-generation sequencing read processing and metagenomic screening’, using the following parameters: -m BlastN -at SemiGlobal -top 1 -sup 1 -mq 100 -memoryMode load -ssc -sps. The minimum percentage identity parameter was set to default (-id 0.0), as opposed to a 90% identity filter used for running HOPS 29 , to avoid any reference bias that might arise from the removal of endogenous reads with a higher number of mismatches. After run completion, to retain the maximum number of reads accounting for the naive lowest common ancestor algorithm, we extracted reads that were assigned to the Yersinia genus node or summarized under the Y. pseudotuberculosis complex node. Reads were extracted in FASTA format from MEGAN v.6.4.12 (ref. 72 ). Subsequently, FASTA files were converted into FASTQ format with the reformat.sh script in BBMap from the BBtools suite (version 38.86,  https://sourceforge.net/projects/bbmap/ ). FASTQ files were then remapped against the CO92 reference genome using the same parameters as described previously in this section. For single-stranded libraries, mapDamage v.2.0 (ref. 64 ) was used to rescale quality scores in read positions at which potential deamination-associated mismatches to the reference were identified. Subsequently, BAM files corresponding to the same individual were concatenated after mapping quality filtering and PCR duplicate removal. We performed concatenation using the SAMtools ‘merge’ command and with the AddOrReplaceReadGroups tool in Picard ( http://broadinstitute.github.io/picard/ ) for assigning a single read group to all reads in each new file.

SNP calling, heterozygosity estimates and SNP filtering

Variant calling was carried out for BSK001 and BSK003, both before and after MALT 62 filtering using the UnifiedGenotyper in the Genome Analysis Toolkit (GATK) v.3.5 (ref. 73 ). GATK was run using the EMIT_ALL_SITES option, which produced a call for every position on the chromosomal CO92 reference genome. The resulting genomic profiles of BSK001 and BSK003 were compared against a set of 233 modern and 46 historical Y. pestis genomes, as well as against the Y. pseudotuberculosis reference genome IP32953 (NC_006155.1), using the Java tool MultiVCFAnalyzer v.0.85 ( https://github.com/alexherbig/MultiVCFAnalyzer ). MultiVCFAnalyzer v.0.85 was run with the following parameters. SNPs were called at a minimum coverage of threefold and in cases of heterozygous positions, calls were made at a 90% minimum support threshold. In addition, SNPs were called at a minimum genotyping quality of 30. Furthermore, previously defined non-core and repetitive regions, as well as regions containing homoplasies, ribosomal RNAs, transfer-messenger RNAs and transfer RNAs were excluded from comparative SNP calling 16 , 32 . A set of 6,567 total variant sites were identified in the present dataset.

To investigate the extent of possible exogenous contamination within the BSK001 and BSK003 datasets, we estimated the number of ambiguous heterozygous variants beyond the SNP calling threshold. For this, MultiVCFAnalyzer v.0.85 (ref. 74 ) was used to generate an SNP table of alternative allele frequencies ranging between 10 and 90%. The results were then used to create ‘heterozygosity’ histogram plots of the estimated frequencies in R v.3.6.1 (ref. 75 ). Heterozygosity plots were created both before and after MALT filtering (see ‘Post-capture Y. pestis data processing’) to investigate whether taxonomy-informed filtering could aid the elimination of contaminant sequences in the investigated datasets (Supplementary Fig. 7 ).

An SNP table created with MultiVCFAnalyzer v.0.85, containing all variant positions across the present dataset, was filtered to identify SNP differences between the BSK001 and BSK003 genomes. The identified differences ( n  = 20) were then evaluated with the Java tool SNP_Evaluation 30 (build date 13 August 2018;  https://github.com/andreasKroepelin/SNP_Evaluation ). The variant table and the VCF files of each genome were used as input for SNP_Evaluation. Furthermore, each identified private variant was evaluated within a 50 bp window and was considered ‘true’ when fulfilling the following criteria established in studies published previously 17 , 21 , 30 , 76 : (1) no multi-allelic sites were permitted within the evaluated window unless they were consistent with aDNA deamination (signified as spurious C-to-T or G-to-A substitutions); (2) the evaluated SNP position itself was not consistent with aDNA damage (no bases overlapping the SNP were downscaled by mapDamage v.2.0 (ref. 64 )); (3) no gaps in genomic coverage were identified in the evaluated window; (4) reads overlapping the SNP sites showed specificity to the Y. pseudotuberculosis complex when screened with BLASTn ( https://blast.ncbi.nlm.nih.gov/Blast.cgi ).

Finally, to gain phylogenetic resolution, the BSK001 and BSK003 Y. pestis datasets were concatenated. We performed concatenation of BAM files, MALT 62 filtering and aDNA damage rescaling (with mapDamage v.2.0 (ref. 64 )) as described in the section ‘Post-capture Y. pestis data processing’. Moreover, the dataset was included in the comparative SNP analysis using MultiVCFAnalyzer v.0.85 (ref. 74 ) as described above. Finally, unique SNPs were evaluated with SNP_Evaluation 30 according to the four criteria listed above.

Phylogenetic reconstruction and diversity estimations

Phylogenetic analysis was used to explore 233 Y. pestis genomes as part of the modern comparative dataset. An SNP alignment produced by MultiVCFAnalyzer v.0.85 (ref. 74 ) was used to construct a phylogenetic tree in MEGA7, using the maximum parsimony approach with 95% partial deletion (6,032 SNPs). Of the 233 modern Y. pestis genomes in the current dataset, 30 displayed extensive private branch lengths (Supplementary Fig. 12 ). Such an effect in bacterial phylogenies could result either from true biological diversity or from technical artefacts associated with false SNP incorporation during computational genome reconstruction. Although we cannot exclude the presence of several strains with exceedingly higher mutation rates in the current dataset, previous studies showed that modern Y. pestis strains with ‘mutator’ profiles are uncommon 16 , 36 . In this study, 27 out of 30 genomes that showed disparities in their private SNP counts compared to the rest of the dataset, were derived from assemblies for which the quality of SNP calls could not be evaluated (raw data unavailable). Because potential mis-assemblies or false-positive SNP calls can affect evolutionary inferences and diversity estimations, these genomes were excluded from further analyses. Therefore, we performed phylogenetic analysis using a subset of 203 modern Y. pestis genomes (Supplementary Table 13 ). The list of excluded genomes is as follows: 2.MED1_139 (ref. 19 ), 2.MED1_A-1809 (ref. 18 ), 2.MED1_A-1825 (ref. 19 ), 2.MED1_A-1920 (ref. 19 ), 2.MED0_C-627 (ref. 19 ), 2.MED1_M-1484 (ref. 19 ), 2.MED1_M-519 (ref. 19 ), 0.ANT5_A-1691 (ref. 18 ), 0.ANT5_A-1836 (ref. 18 ), 0.PE2_C-678 (ref. 77 ), 0.PE2_C-370 (ref. 77 ), 0.PE2_C-700 (ref. 77 ), 0.PE2_C-746 (ref. 77 ), 0.PE2_C-535 (ref. 77 ), 0.PE2_C-824 (ref. 77 ), 0.PE2_C-712 (ref. 77 ), 0.PE2b_G8786 (ref. 16 ), 0.PE4_I-3446 (ref. 78 ), 0.PE4_I-3517 (ref. 78 ), 0.PE4t_A-1815 (ref. 18 ), 0.PE4_I-3447 (ref. 78 ), 0.PE4_I-3518 (ref. 78 ), 0.PE4_I-3443 (ref. 78 ), 0.PE4_I-3442 (ref. 78 ), 0.PE4_I-3519 (ref. 78 ), 0.PE4_I-3516 (ref. 78 ), 0.PE4_I-3515 (ref. 78 ), 0.PE4_Microtus91001 (ref. 79 ), 0.PE5_I-2238 (ref. 80 ) and 0.PE7b_620024 (ref. 16 ).

A genome-wide SNP alignment consisting of 203 modern-day and 48 historical Y. pestis genomes (Supplementary Table 14 ), as well as the Y. pseudotuberculosis IP32953 genome, was used as input to construct a maximum likelihood phylogeny including 2,960 SNPs and up to 4% missing data. We performed phylogenetic analysis with RAxML 81 v.8.2.9 using the generalized time-reversible (GTR) substitution model with 4 gamma rate categories. Finally, 1,000 bootstrap replicates were used to estimate node support for the resulting tree topology. After run completion, the maximum likelihood phylogenies were visualized with FigTree v.1.4.4 ( http://tree.bio.ed.ac.uk/software/figtree/ ) and GrapeTree (v1.5.0) 50 .

To estimate the proportion of modern Y. pestis diversity descending from BSK001/003, we used the R package picante v1.8.2 82 to compute the MPD and FPD 83 from the reconstructed maximum likelihood substitution tree. Measures made on a subset of the tree corresponding to the subclade descending from BSK001/003 (branches 1–4) were compared to that of the complete Y. pestis phylogeny. In both cases, only modern strains were included in the calculation. We used a bootstrapping approach to assess the sensitivity of our results with regard to sampling and phylogenetic uncertainty 84 . For each of the 1,000 RAxML bootstrap trees, we randomly resampled modern strains with replacement and only kept branches of the tree corresponding to the sampled strains. Diversity measures were performed for each of the obtained resampled bootstrap trees, from which median estimates and 95% percentile intervals were derived.

To assess the potential impact of uneven sampling among branches (branches 1–4 contained 130 modern strains whereas branch 0 contained only 73), we repeated the same analysis but adding an initial step intended to equalize the number of genomes in both parts of the tree. We subsampled branches 1–4 to the same number of strains as in branch 0 using sequence clustering in branches 1–4 to obtain representative subsamples. We performed hierarchical clustering based on pairwise phylogenetic distances (derived from the maximum likelihood phylogenetic tree) and the resulting tree was cut to define 73 clusters (functions hclust 85 and cutree in R v.4.0.3). For each bootstrap tree, clusters were randomly downsampled to one strain, resulting in an equal number of strains between branch 1–4 and branch 0. Resampling with replacement was then applied as previously to each of the downsampled trees before computing diversity measures.

Plasmid SNP analysis

To investigate possible genetic variation among the plasmids of historical genomes, we performed read mapping of BSK001, BSK003 and BSK001/003 with BWA as well as SNP calling with GATK v.3.5 as described in the above section 'SNP calling, heterozygosity estimates and SNP filtering' against each of the three Y. pestis plasmids (pMT1: NC_003134.1; pPCP1: NC_003132.1; and pCD1: NC_003131.1). We then performed comparative SNP calling using MultiVCFAnalyzer v0.85 (ref. 74 ) against a set of 46 historical Y. pestis genomes as well as the modern reference strains CO92, KIM5 and 0.PE4-Microtus91001. Variants were filtered in individual genomes using SNP_Evaluation according to previously defined criteria (see the ‘SNP calling, heterozygosity estimates and SNP filtering’ section). In the present dataset, we identified ten variants in pCD1, eight in pMT1 and two in pPCP1 (Supplementary Table 15 ).

Time-calibrated phylogenetic analysis

To estimate the timing for the divergence of Y. pestis branches 1–4 using the BSK001/003 genomes as a new calibration point, we used a dataset comprising all modern genomes from branches 1–4 used for phylogenetic analysis ( n  = 130), genomes of the ancestral branching lineage 0.ANT3 ( n  = 8) and all 29 historical (fourteenth–eighteenth century) genomes in our dataset representing unique genotypes. In cases of identical genomes, the highest coverage genome was chosen for this analysis. We applied a molecular clock test using a maximum likelihood method in MEGA7 (ref. 86 ), using a GTR substitution model in which differences in evolutionary rates among sites were estimated using a discrete gamma distribution with four rate categories. On the basis of this molecular clock test, the null hypothesis of equal evolutionary rates across tested phylogenetic branches was rejected, which is consistent with previous studies showing substitution rate variation across Y. pestis lineages 16 , 17 . Therefore, a log-normal relaxed clock model was used for all subsequent molecular dating analyses.

For the molecular dating analysis, we used the Bayesian statistical framework BEAST2 v.6.6 (ref. 87 ). The ages of all ancient isolates were used as calibration points to construct a time-calibrated phylogeny with their radiocarbon or archaeological context age ranges set as uniform priors (see Supplementary Table 19 for all used age ranges). The ages of all modern isolates were set to 0 years before the present. We tested a number of coalescent tree priors such as the coalescent constant size, Bayesian skyline 88 and exponential population models, all of which have been used or tested in previous ancient pathogen genomic studies 17 , 89 , 90 , 91 . We also tested the birth–death skyline tree prior, which has gained traction in recent years 91 , 92 , 93 because it can account for epidemiological variables and can model sampling disparities through time 94 . Moreover, we used jModelTest v.2.1.10 (ref. 95 ) to identify the substitution model of best fit for our dataset. The indicated transversion model was implemented in BEAUti by using a GTR model (4 gamma rate categories) and the AG substitution rate parameter fixed to 1.0 (as indicated previously 93 ). All tree priors were used in combination with a log-normal relaxed clock rate with a uniform prior distribution ranging between 1 × 10 −3 and 1 × 10 −6 substituions per site per year for the SNP alignment (1,405 sites after a 95% partial deletion), corresponding to a range of 3 × 10 −7 to 3 × 10 −10 across the entire genome, which is within the range of previous estimates 17 . As part of the phylogenetic topology set-up, all branch 1–4 genomes (ancient plus modern) as well as the 0.ANT3 lineage were constrained to be independent monophyletic clades. For the constant population size and exponential population tree priors, all other parameters were set to default. For the coalescent skyline tree prior, a Jeffreys prior distribution (1/ x ) was used for the population sizes and a dimension of 5 was used to permit variations in the group and population sizes through time, with an upper bound of 380,000 for the effective population size (default). Moreover, for the birth–death skyline tree prior, we used a uniform prior for the rate to become non-infectious that ranged between 0.03 and 70, to account for possible infectious periods ranging from 30 years (lifelong infections in rodent reservoirs 96 , 97 ) to 5 days (average infectious period for bubonic plague 98 ). We used a prior beta distribution with mean = 0.1 (alpha = 10.0, beta = 90.0) for the sampling probability ρ at time 0 and a uniform distribution ranging between 0 and 0.1 for the sampling proportion s . For the latter, two shifts were allowed through time. Finally, the reproductive number R was allowed to vary between 0 and 4.0 using a long normal prior distribution of median = 1.0 and s.d. = 0.7, which is within the range of previous estimates for bubonic and pneumonic plague during medieval epidemics 98 .

The suitability of all tree priors was evaluated using path sampling as implemented in the model selection package of BEAST2 v.6.6. Path sampling was run in 50 steps, with 20 million states as the chain length for each step. The resulting log-marginal likelihoods favoured with ‘strong support’ 99 the coalescent skyline model for the present analysis (log Bayes factor= 8.35 when compared against the second best model) (Supplementary Table 20 ). Therefore, the coalescent skyline model was chosen for further analysis. To evaluate the temporal signal in the present dataset, we used TempEst v.1.5.3 to estimate the root-to-tip distance against specimen ages in a linear regression analysis 100 . For TempEst, we used a maximum parsimony tree computed in MEGA7 (ref. 86 ) in NEXUS format. Moreover, we used the midpoint of the archaeological or radiocarbon date ranges for all ancient genomes as tip dates. All modern genome ages were set to 0 years before the present. The resulting correlation coefficient r (0.39) and R 2 (0.16) values supported the existence of a temporal signal in the present dataset. Furthermore, we used the BETS approach 101 for a temporal signal assessment that takes into account all analysis parameters. BETS compares the (log)-marginal likelihood estimations produced from an isochronous model (all sampling dates set to 0 years before the present) against a heterochronous model (including real sampling times). As previously, path sampling was run in 50 steps with 20 million states as the chain length for each step. The estimated (log)-Bayes factor of 129.33 was in strong support of the heterochronous model; therefore, it indicated the presence of a temporal signal in the present dataset.

For the molecular dating analysis using a coalescent skyline model set-up, we performed Markov chain Monte Carlo sampling using 2 independent chains of 300–400 million states each. After completion, runs were combined using LogCombiner v.2.6.7 and convergence was evaluated using Tracer v.1.6 ( http://tree.bio.ed.ac.uk/software/tracer/ ) ensuring that the effective sample sizes were greater than 200 for each estimated posterior distribution after a 10% burn-in. Maximum clade credibility trees were constructed using TreeAnnotator in the BEAST2 v.6.6 package 87 with a 10% burn-in and were then visualized in FigTree v.1.4.4. In parallel with the molecular dating analysis, we performed a sampling from the prior analysis to test for possible overfitting of the prior to the data. We performed Markov chain Monte Carlo sampling for 2 independent chains of 600 million states each. After run completion, runs were combined and convergence was evaluated after a 30% burn-in. The results indicate that the posterior distributions of the uncorrelated log-normal relaxed clock and the time to the most recent common ancestor estimates are not concordant with those obtained when using a data-informed analysis (Supplementary Fig. 13 ).

Because most Bayesian phylogenetic frameworks (such as BEAST2) are based on bifurcating trees and hence are poor at resolving multifurcating nodes, we complemented our approach by using TreeTime v.0.8.4 (ref. 35 ) to infer a time-calibrated phylogeny using a maximum likelihood approach. TreeTime has been shown to resolve polytomies in a way that is consistent with specimen tip dates. We generated a rooted maximum likelihood phylogeny using RAxML (Supplementary Fig. 10 ) from the same SNP alignment as the one used for BEAST2 (95% partial deletion). The maximum likelihood tree was then used as input for TreeTime, which was run using all known sampling dates for modern genomes and the midpoint of the age range for the ancient genomes (Supplementary Table 22 ). TreeTime was run using the Kingman coalescent tree prior with the skyline setting. An appropriate substitution model was chosen for the data using the -gtr infer option. The time-scaled phylogeny was inferred using an uncorrelated relaxed clock and with the branch length optimization, keep-root and keep-polytomies options. Moreover, the divergence time intervals were estimated from the highest likelihood tree using the -confidence option. Analyses were run using a maximum number of 500 and 1,000 iterations (maximum number of iterations option) and produced consistent outputs. The resulting time tree can be found in Supplementary Fig. 11 .

Reporting summary

Further information on research design is available in the  Nature Research Reporting Summary linked to this paper.

Data availability

The raw sequence data produced in this study, the Y. pestis aligned reads after metagenomic filtering and the human aligned reads are available through the European Nucleotide Archive under accession no. PRJEB46734 . More data are available in the Supplementary Information .

Code availability

No specialized custom code was used for this study. All software used for the data analyses in this study is publicly available.

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Acknowledgements

We thank A. Herbig, G. Neumann and A. Andrades Valtueña and all other members of the Molecular Palaeopathology and Computational Pathogenomics working groups of the Max Planck Institute for Evolutionary Anthropology for helpful discussions throughout the course of this study; C. Posth and R. Barquera for comments on early versions of the manuscript; T. Hermes for helpful discussions during the review of this study; M. O’Reilly for graphical support; R. I. Tukhbatova, N. Martins, F. Aron, A. Wissgott, R. Radzeviciute and G. Brandt at the Max Planck Institute for the Science of Human History in Jena as well as S. Nagel at the Max Planck Institute for Evolutionary Anthropology in Leipzig for laboratory support; K. Prüfer and S. Clayton for computational assistance. Radiocarbon dating took place at the Curt-Engelhorn-Zentrum Archäometrie in Mannheim, Germany; we thank R. Freidrich for assisting with the interpretation of the radiocarbon dating results. Moreover, we thank V. I. Selezneva from the Peter the Great Museum of Anthropology and Ethnography for initial aid with specimen sampling and N. Smelova, currently at the University of Oslo, for providing essential contextual information during the initial phase of this project. This project received funding from the European Research Council under the European Union’s Horizon 2020 research and innovation programme under grant no. 771234 (PALEoRIDER to W.H.). L.M. and L.D. were supported by grant no. AP08856654 from the Ministry of Education and Science of the Republic of Kazakhstan. M.A.S., G.A.G.R., A.K., K.I.B., D.K. and J.K. were also supported by the Max Planck Society.

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Institute for Archaeological Sciences, Eberhard Karls University of Tübingen, Tübingen, Germany

Maria A. Spyrou

Department of Archaeogenetics, Max Planck Institute for Evolutionary Anthropology, Leipzig, Germany

Maria A. Spyrou, Lyazzat Musralina, Guido A. Gnecchi Ruscone, Arthur Kocher, Kirsten I. Bos, Denise Kühnert, Wolfgang Haak & Johannes Krause

Department of Archaeogenetics, Max Planck Institute for the Science of Human History, Jena, Germany

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Lyazzat Musralina & Leyla Djansugurova

Kazakh National University by al-Farabi, Almaty, Kazakhstan

Lyazzat Musralina

Transmission, Infection, Diversification & Evolution Group, Max Planck Institute for the Science of Human History, Jena, Germany

Arthur Kocher & Denise Kühnert

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Valeri I. Khartanovich

Research Institute and Museum of Anthropology, Lomonosov Moscow State University, Moscow, Russian Federation

Alexandra Buzhilova

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Contributions

M.A.S., P.S. and J.K. conceived and led the investigation. M.A.S., K.I.B., P.S. and J.K. designed the study. M.A.S. and L.M. performed the laboratory work. M.A.S. and A.K. performed the bacterial genomic data analysis. M.A.S., A.K. and D.K. performed the molecular dating analysis. G.A.G.R. performed the human population genetic analysis. P.-G.B. and P.S. assembled, analysed and translated the historical, archaeological and epigraphic context information. A.B. and V.I.K. provided access to the archaeological material and contextual information. M.A.S., A.K., L.D., K.I.B., D.K., W.H., P.S. and J.K. aided in interpreting the results. M.A.S. and P.S. wrote the paper with contributions from all co-authors.

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Correspondence to Maria A. Spyrou , Philip Slavin or Johannes Krause .

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Extended data figures and tables

Extended data fig. 1 available tombstone pictures from kara-djigach..

a–c , Available tombstone pictures from individuals investigated as part of this study. For a translation of the tombstone inscriptions, see individual descriptions within Supplementary Information  2 . Tombstone dates are as follows: Grave 9 (1338-9 CE), Grave 19/20 (1338-9 CE), Grave 6 (year not inscribed). Picture credits to P.-G. Borbone. d+e , Tombstones identified in Kara-Djigach containing pestilence-stating inscriptions, dating to the years 1338 and 1339 CE. These tombstones do not correspond to individuals analysed within our aDNA dataset. The original tombstone on panel d (without traced inscription) is shown in Fig. 1 . Complete  translations are available within Supplementary Information  2 .

Extended Data Fig. 2 Ancient DNA damage substitution frequencies for all Y. pestis captured libraries.

C-to-T substitution frequencies characteristic of post-mortem deamination of ancient DNA are shown for the 5′ ends of sequenced reads aligned against the CO92 Y. pestis reference genome (NC_003143.1).

Extended Data Fig. 3 Evaluation of BSK007 after whole-genome Y. pestis capture.

a , Post-capture coverage distribution of BSK007 across the Y. pestis CO92 chromosome. Mean coverage was estimated across the genome in 4,000 bp windows. The dotted gray line indicates the mean coverage across the entire genome (0.125-fold). b , Krona plots showing the taxonomic classification of BSK007 reads mapping against all Y. pestis CO92 elements (chromosome NC_003143.1, pMT1 NC_003134.1, pPCP1 NC_003132.1 and pCD1 NC_003131.1). Numbers in brackets next to element designations correspond to the number of assigned reads in MALT. The colours of Krona sectors represent different taxonomic levels and their completeness is proportional to the relative abundance of summarised reads at each corresponding taxonomic node. The shown percentages indicate the species-level (outermost circle) proportion of reads aligned to taxa other than Y. pestis and Y. pseudotuberculosis .

Extended Data Fig. 4 Read length and ancient DNA damage distribution of contaminant SNP regions in the combined BSK001/003 dataset prior to metagenomic filtering.

Overlayed length distributions of reads mapping against the CO92 Y. pestis reference genome, calculated for the entire dataset (gray) as well as for 117 regions surrounding putatively contaminant SNPs (blue). Regions were extracted within a 150 bp window surrounding each putatively contaminant SNP. Dotted lines represent average fragment lengths for the entire dataset and for the 117 putatively contaminant SNP regions in gray and blue, respectively. Reads comprising contaminant SNP regions show a distinct length distribution compared to the one observed across the entire BSK001/003 genome, with a marked shift towards longer read lengths. The 76 bp fragment length peak represents the uppermost possible read length of single-end sequenced reads, which comprised the majority of data within the present dataset. Ancient DNA damage patterns were compared between the entire dataset (upper panel) and the putatively contaminant SNP regions (lower panel), showing a near 3-fold reduction in the latter as estimated for the terminal 5′ base.

Extended Data Fig. 5 Phylogenetic comparisons between BSK001/003 against ancient and modern Y. pestis diversity.

Full length maximum likelihood phylogenetic tree using 1,000 bootstrap iterations for estimating node support and visualised using FigTree v1.4.4. The tree is constructed with 203 modern and 48 historical Y. pestis genomes, and is based on 2,960 SNPs (96% partial deletion). Scale denotes the number of substitutions per genomic site.

Extended Data Fig. 6 Evaluation of phylogenetically diagnostic SNPs across 14 th century Y. pestis genomes.

The estimated variant calls were retrieved from a SNP table comprising 203 modern and 48 historical Y. pestis genomes (full dataset contains 3,533 SNPs). Error bars indicate uncertainty due to the presence of missing data (Ns) within the variant calls of the respective genomes.

Extended Data Fig. 7 Comparison of phylogenetic diversity measures computed for the complete modern Y. pestis phylogeny against the Branch 1-4 subclade.

The left panels show maximum likelihood substitution trees considering only extant Y. pestis genomes, annotated based on the compared clades and their corresponding Faith’s phylogenetic diversity index (FPD). Phylogenetic branches considered for the Branch 1-4 FPD computation are shown in green (full dataset) and red (subsampled dataset). The right panels show violin plots indicating the distribution and mean of pairwise phylogenetic distances based on maximum likelihood trees (MPD). Median estimates and 95% percentile intervals were derived from the resampled bootstrap trees (1,000 bootstrap iterations). Points within violin plots indicate the mean estimated phylogenetic distance for all datasets.

Extended Data Fig. 8 Phylogenetic relationships of 0.ANT lineages.

Maximum likelihood phylogenetic tree based on 2,441 genome-wide variant positions (all SNPs). The tree was constructed to indicate the genetic relationships between all previously published extant 0.ANT genomes and BSK001/003. Scale denotes the number of substitutions per genomic site. Node support was determined by 1,000 bootstrap iterations.

Supplementary information

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Supplementary Sections 1–4 and References.

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Spyrou, M.A., Musralina, L., Gnecchi Ruscone, G.A. et al. The source of the Black Death in fourteenth-century central Eurasia. Nature 606 , 718–724 (2022). https://doi.org/10.1038/s41586-022-04800-3

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Article Contents

Public health measures, yersinia pestis and bubonic plague, the biology of bubonic plague, spread of bubonic plague to humans, manifestations of bubonic plague in humans, manifestations of haemorrhagic plague, why yersinia pestis was not responsible for the plagues of europe, profile of an epidemic of haemorrhagic plague, reed and frost modelling, determination of the characteristics of the disease, nature of the pathogen, origins of haemorrhagic plague, origin of the ccr5-δ32 mutation.

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What caused the Black Death?

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C J Duncan, S Scott, What caused the Black Death?, Postgraduate Medical Journal , Volume 81, Issue 955, May 2005, Pages 315–320, https://doi.org/10.1136/pgmj.2004.024075

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For the whole of the 20th century it was believed that the Black Death and all the plagues of Europe (1347–1670) were epidemics of bubonic plague. This review presents evidence that this view is incorrect and that the disease was a viral haemorrhagic fever, characterised by a long incubation period of 32 days, which allowed it to be spread widely even with the limited transport of the Middle Ages. It is suggested that haemorrhagic plague emerged from its animal host in Ethiopia and struck repeatedly at European/Asian civilisations, before appearing as the Black Death. The CCR5-Δ32 mutation confers protection against HIV-1 in an average of 10% of the people of European origin today. It is suggested that all the Δccr5 alleles originated from a single mutation event that occurred before 1000 bc and the subsequent epidemics of haemorrhagic plague gently forced up its frequency to 5×10 −5 at the time of the Black Death. Epidemics of haemorrhagic plague over the next three centuries then steadily raised the frequency in Europe (but not elsewhere) to present day values.

Immediately on its arrival in 1347 in the port of Messina in Sicily the Great Pestilence (or Black Death as it was named in 1823 because of the black blotches caused by subcutaneous haemorrhages that appeared on the skin of victims) was recognised as a directly infectious disease. Michael of Piazza, a Franciscan friar who wrote 10 years after the Black Death had arrived, said “The infection spread to everyone who had any intercourse with the disease”. 1 Indeed, they believed (incorrectly) that priests who heard the confessions of the dying “were immediately overcome by death, so that some even remained in the rooms of the dying.” 1 Case mortality was 100%. They realised that safety lay in fleeing but this, very effectively, served only to spread the infection.

The Black Death moved as a wave northwards through Europe at an average speed of about 4 km per day and reached the Arctic Circle by 1350, remarkable progress in the days of very limited means of transport. 2–4 Even more impressively: it had earlier appeared in Asia Minor and the Crimea and moved south through Antioch; it was present in the Levant and spread along the north African coastlands and to Mecca in Saudi Arabia, covering, in all, some seven million square km. When it had burnt itself out, 40% of the population of Europe had been killed. This outbreak was a pandemic on a scale never before experienced (or since).

But this unknown disease had not disappeared completely and there were epidemics scattered through Europe during the 1350s. 5 Thereafter, the plague was permanently established in France with epidemics every year that cycled round the main trading routes. From there, infected travellers carried the disease by road and river across the continental landmass and by sea to Britain and Ireland. But all these peripheral epidemics died out completely and were restarted by fresh infectives coming from the focus in France. 4

The epidemics progressively increased in spread, frequency, and ferocity ( fig 1 ) with a pronounced rise after 1550 because transport improved and the population of the towns steadily grew (that is, there was a greater number of susceptibles). Contemporary accounts, pattern of spread, and mortality all confirm that the same pathogen was responsible for all the plagues, including the first strike of the Black Death.

 Number of places in Europe reporting a plague epidemic, 1350–1670. Note the increased frequency after 1550. Data from Biraben. 5

Even in the 14th century the health authorities in northern Italy had established the importance of a 40 day quarantine period, which became the gold standard for continental Europe for the next 300 years. The 40 day quarantine was not adopted in England until the 16th century and even then it was changed to 30 days only to find that this was completely ineffective, whereupon this regulation was speedily rescinded.

The complete success of the quarantine period confirms that the plague was a directly infectious disease and it also shows that it had a long incubation period. Towns in France gradually realised that the danger lay in the arrival of an infected traveller who may well have come from a considerable distance. Entry was denied if they had come from a town that had suffered an epidemic. Later, in addition to inspecting travellers on arrival, the authorities also required proof that all the towns through which they had journeyed were completely free of plague.

Once an epidemic had erupted, those displaying symptoms were removed to emergency primitive isolation hospitals called pest (an abbreviation of pestilence) houses, which were hurriedly erected outside the town. Once a plague case had been identified, the family was locked up in the house, the well known cross was daubed on the door, and a watchman was appointed to stand guard. These measures were less successful in containing an epidemic because, as shown below, victims were more infectious before the appearance of the symptoms.

Despite only sketchy medical knowledge at the time, the epidemiology of the plague was fully understood at least by the middle of the 17th century. Daniel Defoe 6 had perspicaciously noted that, in the Great Plague of London in 1665, “because of its infectious nature, the disease may be spread by apparently healthy people who harbour the disease but have not yet exhibited the symptoms. Such a person was in fact a poisoner, a walking destroyer perhaps for a week or a fortnight before his death, who might have ruined those that he would have hazarded his life to save… breathing death upon them, even perhaps his tender kissing and embracings of his own children.”

Clearly, they recognised that victims were infectious before the symptoms appeared, the lengthy duration of the incubation period, the necessity of a 40 day quarantine, and the dangers of droplet infection. But there were many features of the epidemics that were mystifying and they also clung to their beliefs in divine intervention, transmission via contaminated clothing and bedding, movements of the planets, and poisonous miasmas.

Even before 1347, bubonic plague had been grumbling along for centuries in Asia with occasional severe epidemics. But from the mid-19th century the disease gathered momentum and erupted in Canton and Hong Kong in 1894, Calcutta in 1895, and Bombay in 1896 and the pandemic of the 20th century had begun. Steamships carried infected rats and fleas from the infested warehouses of the Chinese ports to many of the warmer parts of the world, wherever suitable rodent hosts could be found. But endemic bubonic plague never became established in Europe, despite numerous introductions in the 20th century.

The complex aetiology and biology of bubonic plague was elucidated by Yersin 7 and the Plague Commission of India 8 : it is a disease of wild rodents in which the bacterial pathogen, Yersinia pestis , is spread by infected fleas. Occasionally today it is transmitted to humans from peridomestic rats and there are some 1600 cases a year. 9 The characteristic (but not specific) symptom of bubonic plague in humans is the appearance of the bubo.

However, once Yersin had announced his seminal results, it was realised that victims of haemorrhagic plague also sometimes presented with swollen lymph glands. It was, apparently, immediately assumed that the Black Death was caused by bubonic plague. Nobody compared the two diseases objectively and for the whole of the 20th century this view, based solely on the appearance of one symptom, was universally accepted without question.

To be able to refute unequivocally the belief that Yersinia pestis was the pathogen of the Black Death it is necessary to understand fully the complicated biology of bubonic plague. The key lies in the difference between susceptible and resistant rodent species. Susceptible species, like rats, die from the infection and an outbreak of human bubonic plague was often presaged by the appearance of hundreds of dead rats. Obviously, no outbreak can be maintained for any length of time and bubonic plague cannot become endemic where all the local species are susceptible. In central Colorado an isolated colony of prairie dogs ( Cynomys gunnisoni ) was wiped out when Yersinia pestis was introduced. 10

In Siberia and Mongolia, for example, susliks and tarabagans are susceptible and subject to recurrent, short term outbreaks that might eventually eliminate the plague focus through lack of hosts; but the local gerbils and voles are more resistant to Y pestis and so they can serve to maintain the endemic state in the area because they do not die from an infection. 10

In warm climates, rodents breed throughout the year and may produce up to nine litters. In this way, the density of a population of rodents living under favourable conditions increases rapidly and outbreaks of bubonic plague can occur at any time of year. The turnover of a rodent population in a subtropical area can be very high: as fast as they breed, infections (including Yersinia ) and predators act to reduce their numbers. This process generates regular cycles in the population of rodents and prevents the establishment of a stable, plague resistant population. Therefore, any focus of bubonic plague in rodents in subtropical climates is in a continual state of population flux.

The spread of bubonic plague through a rodent population is critically dependent on active fleas. At least 30 species of flea have been proved to be vectors and, as more than 200 species of rodent can carry plague, the host-vector permutations in the Asian subcontinent are formidable and the population dynamics complex. 3, 10

Flea reproduction is strongly dependent on environmental and other factors: temperature and humidity greatly affect both egg laying and the development of the larvae. Temperatures between 18°C and 27°C and a relative humidity of 70% are ideal, whereas temperatures below 7°C are deleterious to all developmental stages except the adult. The fleas, the rats, the resistant rodents, and the susceptible rodents each need specific conditions for the successful completion of their life cycle and the overall maintenance of their populations. All are potentially capable of prodigious reproduction. These life cycles and the environmental requirements for reproduction have to intermesh successfully if an infection of Yersinia pestis is to be established in rodents. The dynamics of bubonic plague are complicated but rodents usually keep within their home range and the disease spreads only slowly through the countryside. 3, 10

If an infected wild rodent strays near human habitations and then shares its fleas with rats living around the settlement, Yersinia can spread from rodent to rat, and from rat to man. The rat is just an intermediary and is not a reservoir of bubonic plague: its role is to die and then pass on the infection. A number of dead rats will usually be found during an outbreak of bubonic plague in humans: in a small village perhaps just a few; in a large South African township perhaps many barrow loads.

There are several other ways in which Yersinia pestis can spread from a focus among local rodents to humans: when humans go out and invade an area where the rodents are infected—for example when hunting or picnicking—they may catch bubonic plague directly from the fleas living on the wild rodents. 10

Patients with bubonic plague, who have been bitten by an infected flea, are not normally infectious to other people and can be nursed in open wards. Notably, the incubation period is typically two to six days after exposure and the characteristic symptom is the bubo. Typically the onset is sudden with chills and rigors and a rise of temperature to 38.8°C–39.4°C. The patient has a severe, splitting headache and often pains in the limbs, the back, and abdomen. They become confused, restless, irritable or apathetic, their speech slurred, and they may vomit. Within a day or two the person is prostrate with all the symptoms of shock. Most patients die between the third and sixth day: if they are alive on the seventh day they may struggle through to recovery. 10

However, in about 5% of the cases of bubonic plague the Yersinia reaches the lungs and the patient coughs out the bacteria in the sputum, which may be inhaled by anyone in close contact who then gets pneumonic plague. The victim dies between the third and sixth day and, without medical treatment, pneumonic plague is invariably fatal. 10

Pneumonic plague cannot occur in the absence of the bubonic form, not can it persist independently. While pneumonic plague increased the mortality locally in an epidemic focus, it was rarely responsible for spreading Yersinia pestis over any distance—mortally sick people were unable to move very far in the few days before death. Furthermore, transmissibility of pneumonic plague is low: the average number of secondary cases per primary case ( R   o ) based on past outbreaks was only 1.3. 11

We believe that the Black Death was caused by a disease that was completely different from bubonic plague and, to avoid confusion, have named it haemorrhagic plague. Case mortality was 100% and the disease was directly infectious. In the Plague of Athens, victims were stricken suddenly with severe headaches, inflamed eyes, and bleeding in their mouths and throats. The next symptoms were coughing, sneezing, and chest pains followed by stomach cramps, intensive vomiting and diarrhoea, and unquenchable thirst. The skin was flushed, livid, and broken with small blisters and open sores. The patients burned with fever so extreme that they could not tolerate being covered, choosing rather to go naked. Their desire was to cast themselves into cold water, and many of those who were unsupervised did throw themselves into public cisterns, consumed as they were by unceasing thirst. Many became delirious. 3 In the Black Death, prolonged bleeding from the nose and vomiting blood was regarded as a fatal prognostic. 1 Some of the earliest signs were blisters (the “blains”) or carbuncles on the skin and these were followed by the buboes. Gui de Chauliac, physician to the Papal Court at Avignon during the Black Death, saw clearly that the buboes were by no means an invariable symptom and that the mortality was of two types. One died from the first in three days. The second presented with apostumes and carbuncles on the external parts, principally on the armpits and groin and, from this, the victim died in five days. 2 But the most feared signs were the haemorrhagic spots (God’s tokens), which varied in size and colour and could occur anywhere, although the neck, breast, back, and thighs were the most common site. Necropsies showed general necrosis of the internal organs.

The following is a brief summary of the evidence:

There were two authentic plague epidemics in Iceland in the 15th century that persisted through the freezing conditions of winter. 12 No rats were present on the island and the conditions were inimical for flea activity. 3

The brown rat ( Rattus norwegicus ) did not arrive in Europe until 60 years after the plagues had disappeared. 3 The black rat ( Rattus rattus ) was absent in rural England 13 ; no rat species were available to spread the disease throughout the country.

There are no resistant rodents present in Europe (and never were), which are essential for the establishment of focus of bubonic plague. 3, 10

The plagues were confined to Europe where the CCR5-Δ32 mutation is now found, whereas bubonic plague, which was not a serious disease until the late 19th century, was confined to Asia where CCR5-Δ32 is absent 4, 14 (see below).

The case mortality in haemorrhagic plague was 100% and the total mortality recorded in an epidemic was very much greater (usually at least 10-fold) than that in an outbreak of bubonic plague: humans can be infected with Yersinia pestis without suffering from the disease and there are clinical forms (pestis minor) where there is no danger of dying. In victims who present with fever and the bubo, between 30% and 50% die if not treated. 10 This level of mortality is insufficient to force up the CCR5-Δ32 mutation to present day levels. 15

Flea reproduction was impossible in the climatic conditions of northern Europe. 3

The Black Death spread remarkably rapidly—from Sicily to the Arctic Circle in less than three years and covered vast areas of Europe. Many of the subsequent epidemics jumped over 300 km. This is in complete contrast with an epidemic of bubonic plague that moves very slowly 4, 14 ; the black rat has a home range of 100 metres and rarely strays outside it. 16

The bacterium Yersinia does not use the CCR5 receptor. 17

The 40 day quarantine for the plague was rigorously established and completely successful for 300 years. It corresponds with the long incubation period that has been established for the plagues of Europe. 4 Quarantine measures are not applicable to bubonic plague. 4, 14

The plagues were recognised as a directly infectious disease and it was established that it was not safe to come within four metres of an infected person. 4

Both normal and CCR5 deficient mice have been infected with Yersinia pestis but there were no differences between the two groups in either bacterial growth or survival time. 17

The report that DNA specific for Y pestis was amplified from 16th and 18th century human teeth believed to be from French plague victims 18 and 14th century French Black Death victims 19 has not been confirmed and the results have been ascribed to contamination. 20

A full scale plague epidemic developed only in a town above a certain minimum size. Mortality was low in villages. Figure 2 illustrates the profile of a typical epidemic that began in the spring. It follows the pattern of a person to person infection, but is characterised by its slow generation and its long duration of eight or nine months. The epidemic falls into three stages:

 Weekly plague burials at Newcastle upon Tyne after the start of the epidemic on 14 May 1636.

Rising. Mortality rises exponentially, dependent on the transmission rate. Once the epidemic has killed a proportion of the population, the transmission rate starts to fall and, concomitantly, the mortality rate decreases because there are fewer susceptibles around to infect.

Plateau. When the transmission rate  = 1, the mortality rate remains static.

Decaying. Once the pool of remaining susceptibles is depleted below a critical level, the transmission rate falls to <1 and, inevitably, the epidemic fizzles out.

The unpublished mathematical model of the epidemics of directly infectious diseases developed by Reed and Frost 21 may be summarised as follows. In a closed population of size N within which people intermingle fairly uniformly, it is assumed that, in a certain period of time t , every person will have about the same number of contacts with other individuals, K . If t is made equal to the serial generation time, the individuals infected during one period will then be infectious during the next.

The probability of an adequate contact between any two given individuals during time t will be

will be the probability of any given person avoiding adequate contact with any other given person during time t .

Thus, the population is at any time, t , composed of cases, C t , and susceptibles, S t , and the probability of any given person avoiding contact with any of the cases will be q and, with all the C t cases, will be

And the probability of any given person having at least one adequate contact with any of the cases will be

In the next time period ( t +1), the number of contacts between cases and susceptibles is given by

Computer modelling of Reed and Frost dynamics shows that the duration of an epidemic is strongly dependent on the serial generation time of the disease. 4 The epidemic at Newcastle ( fig 2 ) suggests a long serial generation time of 22 days and a low R o of 3.

These conclusions are illustrated in figure 3 , which shows the results of modelling epidemics of influenza (incubation period  = 2–3 days) and a hypothetical plague (incubation period  = 32 days), with R o standardised at 3. N  = 1200.

 Computer modelling of epidemics of influenza (incubation period  = 2–3 days) and a plague (incubation period  = 32 days) in the same community, with R o standardised at 3. N  = 1200.

From the Elizabethan period vicars and parish clerks were required to mark the registers of plague burials with a “P” or “Pest”. Detailed analysis of some 100 plague epidemics recorded in parish registers, coupled with family reconstitution enabled the tracing of the lines of infection both within and between households and the determination of the vital characteristics of the pathogen 4 :

An epidemic was often specifically recorded as being started by a visiting stranger or by a resident returning from a visit to a place where the plague was raging.

There was a considerable delay, often more than 15 days, between the death of the primary case and the first secondary case.

Transmission was much easier within, rather than between, households.

Transmission was much more difficult in the colder months and probably impossible out of doors in the depth of winter.

Four metres was established as a safe distance of separation out of doors.

From April to October, R o  = 3 to 4, but with a range of 1 to more than 20, depending on circumstances.

The characteristic symptom was the appearance of haemorrhagic spots.

By the following of the lines of infection, particularly in the early and late stages of an epidemic, it is possible to determine the following:

Latent period  = 12 days (occasionally 10 days)

Infectious period before symptoms  = 20–22 days

Incubation period  = 32 days

Period of symptoms  = 5 to 6 days (range  = 2–15 days). Victim probably less infectious during this time.

Total infectious period  = 25–27 days

Total time from point of infection to death  = 37–38 days, in agreement with the 40 day quarantine instituted in the 14th century.

It was the very long incubation period that, even in the days of very limited transport, allowed travellers and traders to spread the plague widely throughout Europe and across the sea to England, Ireland, and Iceland.

During the Black Death in 1348 there was evidence of a few people who were resistant to the disease. For example, a monk who was the sole survivor in a monastic community, having nursed and buried his fellow inmates. By the 17th century, inspection of the burials registers of London suggests that the percentage of the resident population showing resistance had risen considerably, with the greatest mortality among naive immigrant apprentices and maidservants from the provinces. 4

Current studies in molecular biology throw light on this phenomenon. The transmembrane CCR5 chemokine receptor is used by HIV strains to enter cells of the immune system. 22–24 The CCR5-Δ32 deletion prevents the expression of the receptor and provides almost complete resistance to HIV-1 infection in homozygous people and partial resistance in the heterozygous state. 25–29 The average frequency of the CCR5-Δ32 deletion allele is estimated at 10% in European populations, but is virtually absent among native sub-Sahara African, Asian, and American Indian populations 25, 29–31 —that is, the CCR5-Δ32 mutation is found today only in the area that was once ravaged by plague. The age of the CCR5-Δ32 bearing haplotype has been computed to be about 700 years old (but with a wide range of 275–1875 years) and it has been suggested that it was driven upwards to the present day frequencies of 5% to 15% by a historic, strongly selective event, probably an enormous mortality mediated by a widespread epidemic of a pathogen that, like HIV-1, utilised CCR5 for entry into lymphoid cells. 30 The Black Death is an excellent candidate for such a catastrophic event 30 but this single pandemic would have raised the frequency of the mutation from the estimated value of 5×10 −5 to only, at most, 10 −4 . Rather, we suggest that the virus of haemorrhagic plague used the CCR5 receptor as a means of entry and that the continuous epidemics for the following 300 years acted as a strong selection pressure that drove up the frequency of the mutation to present day values in Europe of 10 −1 . Recent research has shown that Yersinia pestis , the bacterium of bubonic plague, cannot enter via the CCR5 receptor. 17

There is no known disease today that presents with the symptoms of haemorrhagic plague. The studies with the CCR-5 receptor suggest that the pathogen was viral and the symptoms and necropsy reports of haemorrhagic plague are closest to those of Ebola and Marburg, particularly the necrosis of the internal organs and the haemorrhagic manifestations, 32 suggesting that the pathogen may have been a filovirus. “Filoviruses are the prototypical emerging pathogens: they cause a haemorrhagic disease of high case-fatality associated with explosive outbreaks due to person-to-person transmission, have no known treatment, occur unpredictably, and have an unknown reservoir”. 32 We suggest, therefore, that the plagues were an emergent haemorrhagic fever, probably caused by a filovirus.

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We suggest that haemorrhagic plague first emerged in Ethiopia, the cradle of human evolution; it is in this area that humans have been in longest association with animals. A number of Arabic sources have traced plague back to there and the disease was carried down the Nile Valley by caravan traffic 33 to Sudan and Egypt and North Africa. We can trace sporadic epidemics of haemorrhagic plague that occurred widely over the eastern Mediterranean area during a very long time span, from the earliest writings. Presumably, the plague was active in the Nile valley, albeit unrecorded, well before these times. Box 1 summarises the written evidence for the historical sequence of epidemics.

Haemorrhagic fevers in the Nile valley in Pharaonic Egypt, 1500–1350 bc .

Viral haemorrhagic fevers were reported in ancient Mesopotamia “If … his epigastrium [has] a piercing pain, blood flows incessantly [from his mouth], his arms are continually weak, depression continually falls upon him [and] his eyes are suffused with blood [it is] ‘Hand of Marduk’; he will be worried and die.’ (Mesopotamian diagnostic handbook circa 721–453 bc ). 36

Plague of Athens 430–427 bc . Originated in Ethiopia. The description of the symptoms given by Thucydides correspond closely with those of haemorrhagic plague. 37, 38

Plague of Justinian ad 541–2; continued sporadically until ad 700. Originated in Ethiopia. The description of the symptoms given by Procopius correspond closely with those of haemorrhagic plague. 39

Plagues of Islam ad 627 to 744. 33

Haemorrhagic plague in Asia minor and the Levant (the plague focus), 1345–48. 2, 40

Haemorrhagic plagues of Europe, 1347–1670.

Epidemics of haemorrhagic plague in Denmark and Sweden, 1710–11. 41

Sporadic epidemics in Poland through the 18th century. 41

It is generally agreed that most, if not all, Δccr5 alleles originate from a single mutation event that is estimated to have taken place either 3500 (400–13 000) or 1400 (375–3675) years ago. 31 We see from box 1 that this critical mutation could readily have occurred within this predicted time scale, and grumbling and sporadic epidemics during a period of over 2000 years could have gently forced up the mutation to the estimated frequency of 5×10 −5 by the time of the Black Death. Traces of the CCR5-Δ32 mutation have been found in Bronze Age skeletons taken from a cave at Liechenstein dating from 900 bc . 34

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Drancourt   M , Aboudharam   G , Signoli   M , et al.    Detection of 400-year-old Yersinia pestis DNA in human dental pulp: an approach to the diagnosis of ancient septicaemia . Proc Natl Acad Sci USA 1998 ; 95 : 12637 – 40 .

Raoult   D , Aboudharam   G , Crubezy   E , et al.    Molecular identification by “suicide PCR” of Yersinia pestis as the agent of medieval black death . Proc Natl Acad Sci USA 2000 ; 97 : 12800 – 3 .

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Maia J de   OC . Some mathematical developments on the epidemic theory formulated by Reed and Frost . Hum Biol 1952 ; 24 : 167 – 200 .

Alkhatib   G , Combadiere   C , Broder   CC , et al.    CCCKR5: a RANTES, MIP-1α, MIP-1β receptor as a fusion cofactor for macrophage-tropic HIV-1 . Science 1996 ; 272 : 1955 – 8 .

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A systematic review of the clinical profile of patients with bubonic plague and the outcome measures used in research settings

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Background Plague is a zoonotic disease that, despite affecting humans for more than 5000 years, has historically been the subject of limited drug development activity. Drugs that are currently recommended in treatment guidelines have been approved based on animal studies alone – no pivotal clinical trials in humans have yet been completed. As a result of the sparse clinical research attention received, there are a number of methodological challenges that need to be addressed in order to facilitate the collection of clinical trial data that can meaningfully inform clinicians and policy-makers. One such challenge is the identification of clinically-relevant endpoints, which are informed by understanding the clinical characterisation of the disease – how it presents and evolves over time, and important patient outcomes, and how these can be modified by treatment.

Methodology/ Principal findings This systematic review aims to summarise the clinical profile of 1343 patients with bubonic plague described in 87 publications, identified by searching bibliographic databases for studies that meet pre-defined eligibility criteria. The majority of studies were individual case reports. A diverse group of signs and symptoms were reported at baseline and post-baseline timepoints – the most common of which was presence of a bubo, for which limited descriptive and longitudinal information was available. Death occurred in 15% of patients; although this varied from an average 10% in high-income countries to an average 17% in low- and middle-income countries. The median time to death was 1 day, ranging from 0 to 16 days.

Conclusions/ Significance This systematic review elucidates the restrictions that limited disease characterisation places on clinical trials for infectious diseases such as plague, which not only impacts the definition of trial endpoints but has the knock-on effect of challenging the interpretation of a trial’s results. For this reason and despite interventional trials for plague having taken place, questions around optimal treatment for plague persist.

Author summary Plague is an infectious disease that, despite affecting humans for more than 5000 years, has historically been the subject of limited drug development activity. In fact, the drugs currently used to treat plague have been approved based on experimental data alone – no major clinical trials have yet been completed that demonstrate the efficacy and safety of one treatment over another in humans. A major barrier to accomplishing this is that few research studies have taken place to date that can meaningfully inform the design of a clinical trial. We conducted this systematic review to gather and summarise all the existing information on the clinical profile of plague patients to understand whether sufficient information exists on which to design informative clinical trials that would provide clinicians and policy-makers with the information needed to make treatment decisions for patients.

This study however found that, based on the existing literature, there is insufficient data that can be used to develop methodologies for future trials. Either time must be invested in to collecting robust clinical data or innovative trial designs need to be identified that can simultaneously collect much-needed observational data while also evaluating much-needed interventions.

Competing Interest Statement

The authors have declared no competing interest.

Funding Statement

Author declarations.

I confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained.

I confirm that all necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived, and that any patient/participant/sample identifiers included were not known to anyone (e.g., hospital staff, patients or participants themselves) outside the research group so cannot be used to identify individuals.

I understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance).

I have followed all appropriate research reporting guidelines, such as any relevant EQUATOR Network research reporting checklist(s) and other pertinent material, if applicable.

Data Availability

The dataset used in this systematic review can be obtained upon reasonable request to the corresponding author.

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Black Death

By: History.com Editors

Updated: March 28, 2023 | Original: September 17, 2010

The Black Death was a devastating global epidemic of bubonic plague that struck Europe and Asia in the mid-1300s. The plague arrived in Europe in October 1347, when 12 ships from the Black Sea docked at the Sicilian port of Messina. People gathered on the docks were met with a horrifying surprise: Most sailors aboard the ships were dead, and those still alive were gravely ill and covered in black boils that oozed blood and pus. Sicilian authorities hastily ordered the fleet of “death ships” out of the harbor, but it was too late: Over the next five years, the Black Death would kill more than 20 million people in Europe—almost one-third of the continent’s population.

How Did the Black Plague Start?

Even before the “death ships” pulled into port at Messina, many Europeans had heard rumors about a “Great Pestilence” that was carving a deadly path across the trade routes of the Near and Far East. Indeed, in the early 1340s, the disease had struck China, India, Persia, Syria and Egypt.

The plague is thought to have originated in Asia over 2,000 years ago and was likely spread by trading ships , though recent research has indicated the pathogen responsible for the Black Death may have existed in Europe as early as 3000 B.C.

Symptoms of the Black Plague

Europeans were scarcely equipped for the horrible reality of the Black Death. “In men and women alike,” the Italian poet Giovanni Boccaccio wrote, “at the beginning of the malady, certain swellings, either on the groin or under the armpits…waxed to the bigness of a common apple, others to the size of an egg, some more and some less, and these the vulgar named plague-boils.”

Blood and pus seeped out of these strange swellings, which were followed by a host of other unpleasant symptoms—fever, chills, vomiting, diarrhea, terrible aches and pains—and then, in short order, death.

The Bubonic Plague attacks the lymphatic system, causing swelling in the lymph nodes. If untreated, the infection can spread to the blood or lungs.

How Did the Black Death Spread?

The Black Death was terrifyingly, indiscriminately contagious: “the mere touching of the clothes,” wrote Boccaccio, “appeared to itself to communicate the malady to the toucher.” The disease was also terrifyingly efficient. People who were perfectly healthy when they went to bed at night could be dead by morning.

Did you know? Many scholars think that the nursery rhyme “Ring around the Rosy” was written about the symptoms of the Black Death.

Understanding the Black Death

Today, scientists understand that the Black Death, now known as the plague, is spread by a bacillus called Yersinia  pestis . (The French biologist Alexandre Yersin discovered this germ at the end of the 19th century.)

They know that the bacillus travels from person to person through the air , as well as through the bite of infected fleas and rats. Both of these pests could be found almost everywhere in medieval Europe, but they were particularly at home aboard ships of all kinds—which is how the deadly plague made its way through one European port city after another.

Not long after it struck Messina, the Black Death spread to the port of Marseilles in France and the port of Tunis in North Africa. Then it reached Rome and Florence, two cities at the center of an elaborate web of trade routes. By the middle of 1348, the Black Death had struck Paris, Bordeaux, Lyon and London.

Today, this grim sequence of events is terrifying but comprehensible. In the middle of the 14th century, however, there seemed to be no rational explanation for it.

No one knew exactly how the Black Death was transmitted from one patient to another, and no one knew how to prevent or treat it. According to one doctor, for example, “instantaneous death occurs when the aerial spirit escaping from the eyes of the sick man strikes the healthy person standing near and looking at the sick.”

How Do You Treat the Black Death?

Physicians relied on crude and unsophisticated techniques such as bloodletting and boil-lancing (practices that were dangerous as well as unsanitary) and superstitious practices such as burning aromatic herbs and bathing in rosewater or vinegar.

Meanwhile, in a panic, healthy people did all they could to avoid the sick. Doctors refused to see patients; priests refused to administer last rites; and shopkeepers closed their stores. Many people fled the cities for the countryside, but even there they could not escape the disease: It affected cows, sheep, goats, pigs and chickens as well as people.

In fact, so many sheep died that one of the consequences of the Black Death was a European wool shortage. And many people, desperate to save themselves, even abandoned their sick and dying loved ones. “Thus doing,” Boccaccio wrote, “each thought to secure immunity for himself.”

Black Plague: God’s Punishment?

Because they did not understand the biology of the disease, many people believed that the Black Death was a kind of divine punishment—retribution for sins against God such as greed, blasphemy, heresy, fornication and worldliness.

By this logic, the only way to overcome the plague was to win God’s forgiveness. Some people believed that the way to do this was to purge their communities of heretics and other troublemakers—so, for example, many thousands of Jews were massacred in 1348 and 1349. (Thousands more fled to the sparsely populated regions of Eastern Europe, where they could be relatively safe from the rampaging mobs in the cities.)

Some people coped with the terror and uncertainty of the Black Death epidemic by lashing out at their neighbors; others coped by turning inward and fretting about the condition of their own souls.

Flagellants

Some upper-class men joined processions of flagellants that traveled from town to town and engaged in public displays of penance and punishment: They would beat themselves and one another with heavy leather straps studded with sharp pieces of metal while the townspeople looked on. For 33 1/2 days, the flagellants repeated this ritual three times a day. Then they would move on to the next town and begin the process over again.

Though the flagellant movement did provide some comfort to people who felt powerless in the face of inexplicable tragedy, it soon began to worry the Pope, whose authority the flagellants had begun to usurp. In the face of this papal resistance, the movement disintegrated.

How Did the Black Death End?

The plague never really ended and it returned with a vengeance years later. But officials in the port city of Ragusa were able to slow its spread by keeping arriving sailors in isolation until it was clear they were not carrying the disease—creating social distancing that relied on isolation to slow the spread of the disease.

The sailors were initially held on their ships for 30 days (a trentino ), a period that was later increased to 40 days, or a quarantine — the origin of the term “quarantine” and a practice still used today. 

Does the Black Plague Still Exist?

The Black Death epidemic had run its course by the early 1350s, but the plague reappeared every few generations for centuries. Modern sanitation and public-health practices have greatly mitigated the impact of the disease but have not eliminated it. While antibiotics are available to treat the Black Death, according to The World Health Organization, there are still 1,000 to 3,000 cases of plague every year.

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Home — Essay Samples — History — Black Death — A Research Paper On Bubonic Plague

A Research Paper on Bubonic Plague

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Published: Apr 11, 2022

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Introduction, infection and transmission, treatment, detection, and prevention.

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Open Access

Peer-reviewed

Research Article

A systematic review of the clinical profile of patients with bubonic plague and the outcome measures used in research settings

Roles Conceptualization, Data curation, Formal analysis, Investigation, Methodology, Project administration, Visualization, Writing – original draft, Writing – review & editing

* E-mail: [email protected]

Affiliation ISARIC, Pandemic Sciences Institute, University of Oxford, Oxford, United Kingdom

Roles Data curation, Investigation, Validation

Affiliation Institut Pasteur de Madagascar, Antananarivo, Madagascar

Roles Conceptualization, Methodology, Supervision, Writing – review & editing

Roles Data curation, Investigation, Methodology

Roles Conceptualization, Resources, Supervision, Writing – review & editing

Roles Conceptualization, Methodology, Resources, Supervision, Writing – review & editing

• Josephine Bourner, 
• Lovarivelo Andriamarohasina, 
• Alex Salam, 
• Nzelle Delphine Kayem, 
• Rindra Randremanana, 
• Piero Olliaro

• Published: November 9, 2023
• https://doi.org/10.1371/journal.pntd.0011509
• Peer Review
• Reader Comments

Plague is a zoonotic disease that, despite affecting humans for more than 5000 years, has historically been the subject of limited drug development activity. Drugs that are currently recommended in treatment guidelines have been approved based on animal studies alone–no pivotal clinical trials in humans have yet been completed. As a result of the sparse clinical research attention received, there are a number of methodological challenges that need to be addressed in order to facilitate the collection of clinical trial data that can meaningfully inform clinicians and policy-makers. One such challenge is the identification of clinically-relevant endpoints, which are informed by understanding the clinical characterisation of the disease–how it presents and evolves over time, and important patient outcomes, and how these can be modified by treatment.

Methodology/Principal findings

This systematic review aims to summarise the clinical profile of 1343 patients with bubonic plague described in 87 publications, identified by searching bibliographic databases for studies that meet pre-defined eligibility criteria. The majority of studies were individual case reports. A diverse group of signs and symptoms were reported at baseline and post-baseline timepoints–the most common of which was presence of a bubo, for which limited descriptive and longitudinal information was available. Death occurred in 15% of patients; although this varied from an average 10% in high-income countries to an average 17% in low- and middle-income countries. The median time to death was 1 day, ranging from 0 to 16 days.

Conclusions/Significance

This systematic review elucidates the restrictions that limited disease characterisation places on clinical trials for infectious diseases such as plague, which not only impacts the definition of trial endpoints but has the knock-on effect of challenging the interpretation of a trial’s results. For this reason and despite interventional trials for plague having taken place, questions around optimal treatment for plague persist.

Author summary

Plague is an infectious disease that, despite affecting humans for more than 5000 years, has historically been the subject of limited drug development activity. In fact, the drugs currently used to treat plague have been approved based on experimental data alone–no major clinical trials have yet been completed that demonstrate the efficacy and safety of one treatment over another in humans. A major barrier to accomplishing this is that few research studies have taken place to date that can meaningfully inform the design of a clinical trial. We conducted this systematic review to gather and summarise all the existing information on the clinical profile of plague patients to understand whether sufficient information exists on which to design informative clinical trials that would provide clinicians and policy-makers with the information needed to make treatment decisions for patients.

This study however found that, based on the existing literature, there is insufficient data that can be used to develop methodologies for future trials. Either time must be invested in to collecting robust clinical data or innovative trial designs need to be identified that can simultaneously collect much-needed observational data while also evaluating much-needed interventions.

Citation: Bourner J, Andriamarohasina L, Salam A, Kayem ND, Randremanana R, Olliaro P (2023) A systematic review of the clinical profile of patients with bubonic plague and the outcome measures used in research settings. PLoS Negl Trop Dis 17(11): e0011509. https://doi.org/10.1371/journal.pntd.0011509

Editor: Vladimir L. Motin, University of Texas Medical Branch at Galveston, UNITED STATES

Received: July 8, 2023; Accepted: October 14, 2023; Published: November 9, 2023

Copyright: © 2023 Bourner et al. This is an open access article distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Data Availability: All relevant data are within the paper and its Supporting Information files.

Funding: This work was supported by the UK Foreign, Commonwealth and Development Office ( https://www.gov.uk/government/organisations/foreign-commonwealth-development-office ) and Wellcome ( https://wellcome.org/ ) (Grant ref: 215091/Z/18/Z). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Competing interests: The authors have declared that no competing interests exist.

Introduction

Plague is a zoonotic infectious disease caused by Yersinia pestis , Gram-negative bacteria most commonly found in small mammals and transmitted by fleas [ 1 ].

Transmission to humans typically occurs following a bite from an infected flea, contact with contaminated bodily fluids or tissues, or inhalation of respiratory droplets [ 1 ]. Once infected, human plague manifests in three main clinical forms: bubonic, pneumonic and septicaemic–the most common of which is bubonic plague, but the disease can also progress to include secondary clinical forms [ 1 , 2 ].

Other than characteristic swollen, painful lymph nodes–or “buboes”–symptoms of bubonic plague are often non-specific and can typically include fever, headache, chills and weakness [ 1 , 2 ]. Estimates of the case fatality rate (CFR) of bubonic plague are wide-ranging, extending from 24% to 60% overall [ 1 , 3 ], but a lower CFR of ~5% has been reported in treated cases and in research settings [ 4 ].

In 2018, the last year for which global data exist, 248 cases of plague were reported– 98% of which derive from just two countries, Madagascar and the Democratic Republic of Congo [ 5 ]. However, since 2013, cases have been consistently reported within a number of countries across Asia, North America, South America, and Africa and the global distribution of natural plague foci is thought to extend far beyond the countries that have recently reported cases [ 5 , 6 ]. Due to the disease’s ability to lie dormant for years and even decades, [ 7 ] there is potential for cases to arise in locations in which plague has not been reported for a significant amount of time.

While several treatment regimens for plague are recommended in both national and international treatment guidelines, there are no robust clinical data to support the drugs that are commonly used in clinical practice against plague. No pivotal randomised controlled trials have successfully taken place to generate sufficient evidence for plague treatment regimens. A number of drugs, such as streptomycin, ciprofloxacin and doxycycline [ 8 ], have been approved for the treatment of plague based on the U.S. Food and Drug Administration’s (FDA) so-called ‘Animal rule’–which permits the approval of drugs on the basis of well-controlled animal studies when human studies are neither ethical nor feasible [ 9 ]. Other drugs, which have not received FDA approval, such as gentamicin, are also commonly used to treat plague based on experimental data and clinical experience [ 10 ].

Some of these drugs however have significant drawbacks. Until recently, guidelines relied heavily on the use of aminoglycosides, with streptomycin in particular being the drug of choice for the treatment of plague in many settings [ 11 , 12 – 14 ]. While effective against Gram-negative bacterial infections, patients treated with aminoglycosides are at higher risk of experiencing ototoxic or nephrotoxic side effects than when treated with other drugs [ 15 ], requiring onerous clinical monitoring–which many low-resource health facilities in areas with the highest burden of plague are not equipped to carry out. Streptomycin also comes with a high cost and is currently in short supply on the global market. Other treatment regimens, such as those using doxycycline, are bacteriostatic, have only oral formulations available and therefore are not adapted for more severe forms of plague. Both aminoglycosides and tetracyclines are contraindicated in pregnancy [ 16 ].

Robust clinical studies are therefore required to identify safe and effective treatment regimens for plague. To date, only a small number of interventional trials have attempted to evaluate the efficacy of plague treatment regimens, two of which have published data [ 4 , 17 ]. Neither of these studies were able to generate sufficient evidence to demonstrate clinical benefit of their investigational drugs. This is in part due to the low numbers of patients enrolled in the trials–five and 65 patients were enrolled respectively–influenced by logistical challenges and the low numbers of cases reported in the areas in which the studies were conducted. The other challenge is methodological. One trial was designed as a non-randomised trial without a control group and the other was designed to recruit a small number of cases–for which the sample size estimation was based on data generated in a substantively different patient population–to assess a broadly-defined composite endpoint of cure or improvement in condition.

It is therefore clear that more work needs to be done to refine clinical trial methodologies for plague, keeping in mind the logistical challenges and the current low case numbers that might prevent the success of large-scale trials. Understanding the clinical characterisation of plague would be beneficial to overcoming some of the methodological challenges evident in the two studies referenced above, such as identifying viable, clinically relevant endpoints.

This review therefore aims to summarise the clinical profile of patients with bubonic plague–including clinical characteristics at baseline and post-treatment, and clinical outcomes–as described in published, peer-reviewed scientific literature.

We conducted a systematic review to describe the clinical characteristics and outcomes of patients with suspected or confirmed bubonic plague from presentation to last recorded observation.

A search was conducted on bibliographic databases and clinical trial registries, including PubMED, Cochrane CENTRAL, clinicaltrials.gov, ISRCTN and the International Clinical Trials Registry Platform (ICTRP) for peer-reviewed publications describing the clinical characteristics of patients with bubonic plague. A supplementary search was conducted on JSTOR to identify older reports (pre-1970) of plague, although no formal search was conducted to obtain these publications. A full list of search terms and filters that were used for each database and registry can be found in S1 Text .

To obtain data relating to the clinical characteristics of bubonic plague, all study designs were eligible for inclusion providing the publication contained individual patient data for adults or children of any age with suspected or confirmed bubonic plague, and described signs, symptoms and outcomes. There were no restrictions placed on language or publication year. Non-human, non-clinical, post-mortem and vaccine studies were excluded.

Two reviewers independently conducted screening in Rayyan [ 18 ] and was completed in two stages by reviewing titles and abstracts then reviewing full-text articles of remaining publications. Data were extracted by the first reviewer on to a standardised data capture form in Excel ( S1 Data ). A second reviewer verified the search and performed a quality control review on 30% of the data records. Any disagreement was discussed between the two reviewers and a third independent reviewer was involved if a disagreement remained unresolved.

Risk of bias was evaluated within the included studies by one reviewer using the Joanna Briggs Institute Critical Appraisal tools [ 19 ]. Case reports were assessed using the case reports tools, randomised controlled trials were assessed using the randomised controlled trials tool and non-randomised interventional studies were assessed using the quasi-experimental studies tool.

The protocol and data dictionary associated with this review can be accessed upon reasonable request to the corresponding author.

The analysis was completed using R v.4.2.2.

The screening and inclusion processes are summarised in the PRISMA flow diagram ( S1 Table ). A summary of all included studies is provided in a table detailing the study title, study type, country in which the study was conducted, number of bubonic plague cases in each study, the ratio of males to females, and the median and range of ages of the study population in years.

Two bar charts summarise the number of included studies per year per country, and the size of the patient population in the studies per year per country.

Demographic data were extracted from each study to summarise the ratio of males to females, and age (median and range) across the entire patient population of all included studies. The total number and percent of pregnant women and patients with comorbidities among the patient population is also provided.

The signs and symptoms are summarised for patients who received a confirmed clinical or laboratory diagnosis of bubonic plague and are reported at baseline and post-baseline timepoints. Baseline is defined at the first interaction the patient has with a health professional and post-baseline includes all timepoints that occur after the day of the initial interaction. Signs and symptoms are summarised as the number and percentage of patients in whom each sign or symptom was reported on at least one occasion. The denominator is based on the sum of the sample sizes of studies in which at least one patient reported the sign or symptom; in order to eliminate studies in which the sign or symptom may not have been assessed or captured during data collection, studies which do not report the sign or symptom for at least one patient have been excluded from the denominator calculation.

The number and percentage of patients receiving treatment or no treatment is provided. Treatment information is summarised according to antimicrobial class, as defined in a recent systematic review by the U.S. Centres for Disease Control and Prevention [ 20 ]. Aminoglycosides, tetracyclines, fluoroquinolones, sulphonamides, and amphenicols were considered to be “high-efficacy”. All other antimicrobials were classified as “other antibiotics”. The number and percentage of deaths among those who received high-efficacy, other antibiotics or no treatment has been provided.

Patient outcomes are summarised as the number and percentage of deaths among the entire patient population, and according to the patient population in studies taking place in high-income countries and low- and middle-income countries. The outcome of the bubo–as present or not present–at the last recorded observation has been reported as the number and percentage of patients reporting the outcome among the patient population with a known bubo.

In total, 2023 publications were identified in the search ( Fig 1 ). After removing duplicates, 1984 publications were screened for inclusion. Following title, abstract and full-text screening, 1897 records were excluded, resulting in the inclusion of 87 publications for data synthesis.

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https://doi.org/10.1371/journal.pntd.0011509.g001

Of the included studies, 72 (83%) were case reports, 11 (13%) were cohort studies, four (4%) were interventional studies, of which one (1%) was randomised and three (3%) were non-randomised ( Table 1 ).

https://doi.org/10.1371/journal.pntd.0011509.t001

There was a moderate risk of bias across all study types ( S2 Table and S1 Fig ). Case reports contained limited demographic, treatment and follow-up information, although baseline signs and symptoms, and diagnostic information was well-described. In interventional and cohort studies, outcomes were generally reliably described and assessed, although the completeness of follow-up information was often unclear.

All studies were published between 1902 and 2021 ( Fig 2A ).

https://doi.org/10.1371/journal.pntd.0011509.g002

Of the 87 included studies, 73 (84%) were conducted in the United States, three (3%) in the United Kingdom and Vietnam, two (2%) in Zambia, and 1 (1%) study each in Israel, Libya, Madagascar, Mongolia, Tanzania, and Uganda ( Fig 2A ).

These studies contain data on 1343 probable and confirmed cases of bubonic plague (including patients with secondary pneumonic or septicaemic plague). Of the included population, 870 (65%) derive from one study conducted in Madagascar, 307 (23%) from studies conducted in the United States, 76 (6%) from Vietnam, 65 (5%) from Tanzania, eight (1%) from Zambia, six (<1%) from each the United Kingdom, four (<1%) from Uganda, three (<1%) from Israel, and two (<1%) from Mongolia and Libya ( Fig 2B ).

However, over time the international distribution of included individuals has changed. From the 1960s to the 1980s reports of plague mainly derived from the United States; during this period, 209 (73%) individuals reported in this review were assessed in the United States (mostly in case reports of single patients or case series of small numbers of patients), and the remainder in Vietnam. Since the 1990s, the focus has shifted more towards the African region; during this time, 91% of cases included in this review were assessed in Madagascar, Tanzania, Zambia, Uganda and Libya, with the remainder assessed in the United States and Mongolia.

The study population consists of 769 (57%) males and 588 (43%) females with ages ranging from 0 to 82 years and a median age of 17 years. Among the population are five (<1%) patients who were pregnant at the time of their plague diagnosis. Comorbidities were reported for 17 (<1%) patients, including 13 (<1%) who tested positive for malaria. Other comorbidities include chronic cough, chronic renal insufficiency, idiopathic thrombocytopenia, and heart murmur.

Reported signs and symptoms

Baseline signs and symptoms..

The median time from symptom onset to admission was 2 days, with a range of 0 to 18 days.

A diverse group of signs and symptoms were reported at baseline and post-baseline timepoints for patients with confirmed bubonic plague within the articles included in this review ( S3 Table ).

The most commonly reported symptom at baseline was the presence of a bubo–an enlarged lymph node–which was recorded in 1216/1265 (96%) cases ( Table 1 ). Of these cases, the median number of buboes reported per patient was one. Nineteen patients reported having multiple buboes, for whom the median number of buboes per patient was two. One patient was recorded as having 10 enlarged inguinal or suprapubic nodes, which were detected through the use of ultrasound. The location of the bubo was reported in 1090 (90%) of these patients. Buboes were most commonly reported in the inguinal area (590/1090, 54%), followed by the axillary (276/1090, 25%) and cervical areas (159/1090, 15%), and less frequently in other locations, such as in femoral and epitrochlear areas. Pain at the site of the bubo was recorded in 230/320 (72%) patients and bubo size was infrequently recorded– 19/88 (22%) publications noted the size of at least one patient’s bubo. The median recorded bubo size was 30mm, with a range of 1mm to 150mm. The method of bubo measurement was reported only in one case report describing one patient, in which ultrasound was used.

Following the presence of a bubo, fever was the most frequently reported systemic feature at baseline, present in 1004/1279 (78%) patients, and for which the median recorded temperature was 39.5°C (range: 36°C to 41.5°C) ( Table 2 ). Headache was present in 127/244 (52%) patients, followed by altered mental status (33/84, 39%), chills (75/195, 38%) and malaise (22/68, 32%).

https://doi.org/10.1371/journal.pntd.0011509.t002

Fewer patients presented with clinical signs and symptoms indicative of severe illness at baseline, such as seizure (4/34, 12%), septic shock (5/59, 8%), and respiratory distress, arrest or failure (3/5, 60%).

All other signs and symptoms are reported in S3 Table .

Post-baseline signs and symptoms.

Reporting of signs and symptoms decreased following the baseline assessment. Patients were followed up for a median of 7 days from admission (IQR 2 days to 20 days).

Reports of fever and headache persisted each in 43% of patients, and there were infrequent reports of other common symptoms at baseline, such as malaise, fatigue, abdominal pain, vomiting, cough, nausea, hypotension, and diarrhoea. While altered mental status and chills were reported in relatively high proportions of patients at baseline, no reports of these symptoms were made post-baseline.

Only 19/88 (22%) articles provided information about the presence of the bubo at any timepoint post-baseline for at least one patient in the study’s cohort. Within this population, a persistent bubo was recorded for 61/95 (64%) patient. A repeated bubo measurement is described in two case reports, in which one repeated the measurement at 3 days post-baseline and demonstrated increasing inguinal lymphadenopathy, which subsequently reduced to 10mm 26 days post-baseline–a reduction of 25mm in total; and the other repeated the measurement at 29 days post-baseline and demonstrated a reduction in size to 5mm–a decrease of 15mm from the baseline measurement–from baseline in an axillary bubo which fully resolved at 39 days post-baseline [ 78 ].

However, the frequency of some signs and symptoms indicative of severe illness increased. After the baseline assessment, respiratory distress, arrest or failure, for example, was recorded in 8/65 (12%) patients and disseminated intravascular coagulation was recorded in 7/41 (17%) patients.

An outcome was recorded for 1300/1343 (97%) patients. At the time of the last recorded observation (median: 7 days; range: 0 to 137 days), 1090/1343 (81%) had fully recovered and death was reported in 208/1343 (15%) patients ( Table 3 ); however, the case fatality ratio but varied from 10% for cases reported in high-income countries (HICs) to 17% in low- and middle-income countries (LMICs). The median time to death was 1 day in HICs and 2 days in LMICs, ranging from 0 to 16 days.

https://doi.org/10.1371/journal.pntd.0011509.t003

A bubo outcome was reported in 93/1279 (7%) cases in whom a bubo was reported at baseline. In 32/93 (34%) patients the bubo had completely disappeared at the last reported observation and in 61/93 (66%) cases the bubo was still present at the time of the last reported observation, which ranged from 0 to 137 days post-admission.

Information on therapeutic intervention–or no intervention where specifically indicated–was available for 306/1343 (23%) patients, of whom 271/306 (89%) received a high-efficacy antimicrobial (with or without other antibiotics), 24/306 (8%) received only other antibiotics and 11/306 (4%) received no antibiotics ( Table 4 ).

https://doi.org/10.1371/journal.pntd.0011509.t004

Streptomycin was the most frequently administered high-efficacy plague therapeutic that was administered–received by 141/306 (46%) patients either alone or in combination with other drugs–followed by gentamicin, tetracycline and doxycycline received by 91/306 (30%), 85/306 (28%), and 42/306 (14%) patients, respectively. All other drugs were administered in small numbers of patients.

Of those who received a high-efficacy antimicrobial at any time following initial presentation, 15/271 (6%) died. Of the 24 patients who received only other antibiotics, 6 (25%) died and of the 11 patients who received no antibiotic treatment, 5 (45%) died.

This systematic review was designed to summarise the clinical profile of patients with bubonic plague, as described in academic literature, with the objective of clarifying the endpoint(s) that could be used as the primary efficacy endpoint in therapeutic trials.

While this study has captured data on the signs, symptoms and outcomes of substantial number of confirmed cases of plague, it is limited to published studies written in English. There are several other studies, written in other languages, from which data have not been extracted. It must be acknowledged that studies describing cases in China, where plague cases have been well-documented [ 106 ], are not included in this review and only one study describing two cases from Mongolia has been included.

Overall the CFR that occurred across the patient population included in this review (15%) indicates that mortality might not be a suitable primary endpoint for a clinical trial–a future trial using a single mortality endpoint would likely require sample sizes that may not be attainable to detect significant treatment effects between arms. A rough sample size estimation designed to detect a 50% reduction in mortality with a significance level of 5%, 80% power and 10% lost to follow-up would yield a target sample size of 203 patients per arm, which might require several years to complete, or not be attainable at all.

The data on CFR collected in the scope of this review–which varies according to context and type of treatment received by the patient–may however not be representative of bubonic plague in the 21 st century. The majority of included studies are case reports which derive from the United States between the 1960s and 1980s and represent a large proportion of the patient population included in this review. In 2018 however–the most recent year for which global data are available– 80% of the global cases of plague derive from Madagascar, and a further 18% from the Democratic Republic of Congo. These are two countries in which healthcare availability and accessibility may be substantially different to that of the United States, which will likely influence patterns in disease presentation and patient outcomes. In Madagascar, the CFR for patients with bubonic plague between 1998 and 2016 was reported to be 15% [ 107 ]. The study reporting this figure however cited several limitations in that diagnostic testing evolved during the course of the study–meaning some earlier cases were potentially missed due to the sub-optimal sensitivity of older testing methods–and that samples from regions in Madagascar that had historically infrequently reported cases of plague were not consistently available. The CFR was higher at 24% during a large, atypical urban plague outbreak that occurred in 2017 [ 3 ]. Uncertainty therefore still remains around the true incidence and CFR for plague in the country with the greatest disease burden.

As survival or mortality may not be feasibly used as the single primary efficacy endpoint in a trial for bubonic plague, alternative or additional clinically-relevant outcomes must be identified. The only randomised controlled trial included in this review used “cure or improvement in condition” as its primary endpoint–a composite outcome measure consisting of 1) fever resolution; 2) resolution of bubo pain and swelling; 3) and recovery from pneumonia or any other symptom of plague present at baseline [ 4 ]. An ongoing trial in Madagascar also uses a composite endpoint of therapeutic response defined as 1) the patient being alive; 2) resolution of fever; 3) a 25% decrease in bubo size; 4) alternative plague treatment not received; 5) no decision to continue treatment beyond day 10 [ 108 ]. Composite primary endpoints such as these provide a useful solution for diseases where low numbers of individual events would prevent the detection of significant differences between arms in a trial with a single endpoint. For example, by identifying several outcomes of interest, the number of outcome events increases, therefore making it more likely that an effect can be detected with a reasonable level of confidence.

Composite endpoints however also have the potential to introduce uncertainty around any discernible differences that are detected and make the interpretation of the trial results challenging. For example, the clinical relevance of the individual criteria that make up the above-referenced composite endpoints is difficult to discern. Fever resolution, for example, was reported only for a small proportion of patients (14%) included in this review, and although the event rate– 161/186 (87%)–was high, its relationship with a patient’s overall clinical status is unclear in the literature as limited longitudinal data are available. The same can be said of the persistence or size of the bubo.

The breadth of the events included in the composite endpoint also create challenges for the accurate interpretation of the trial’s results. The events range from resolution of fever to resolution of pneumonia or any other symptom of plague. It is clear that the severity and clinical importance of these events are vastly different. However, they are weighted equally in this composite endpoint making it difficult to understand the true efficacy of the drug and how it works in this patient population. In the event that Patient A presents with only a fever at baseline and Patient B presents with pneumonia, fever and a bubo, can the recovery of both patients represent the true efficacy of the drug?

While the urgency to provide results for plague, a disease with little evidence to support current treatment recommendations, is understandable, critically, the relationship between individual signs, symptoms and clinical status–as determined by both physical indicators of a patient’s condition and laboratory testing for presence of Y . pestis in blood and/or bubo site–must be established before implementing a primary outcome measure of this nature in a clinical trial.

Given the existing data, however, it is not currently possible to identify a single or composite endpoint for plague, for which clinical relevance has been established and which would generate an achievable sample size. It is clear that robust longitudinal data from an observational study would provide critical information about the overall clinical course of plague illness and illuminate the important outcomes that could be used in either a single or composite endpoint.

However, this will take a substantial amount of time–which could be better spent by taking a more pragmatic approach to a trial. An approach recently proposed for Lassa fever, which exhibits similar challenges in endpoint definition, is to embed an observational cohort within the trial framework to collect data on patient outcomes [ 109 ]. The proposed design involves powering the trial for a mortality endpoint, and in parallel identifying and collecting data on several important outcomes of interest during the first year of the trial. An interim feasibility analysis would be undertaken to determine whether the frequencies of the events are sufficient to generate an achievable sample size and, if so, the trial would amend its primary endpoint. While this design has not been implemented in a trial to date, it may provide a useful path forward for diseases such as plague where little comparable data have been generated and uncertainty surrounds outcome frequencies. This framework may provide a cost-saving and efficient approach to trials for diseases like plague that could minimise delays to testing new drugs and reduce costs associated with implementing both observational studies and trials sequentially.

Overall, this systematic review demonstrates the restrictions that limited disease characterisation places on clinical trials for rare infectious diseases such as plague. The limited data that exist on the clinical course of plague and patient outcomes, not only impact the definition of trial endpoints but has the knock-on effect of challenging the interpretation of a trial’s results. For this reason and despite interventional trials for plague having taken place, questions around optimal treatment for plague persist. Options however exist–including those for pragmatic and innovative trial designs. A bigger challenge may however be to catalyse R&D interest in a disease for which there is limited money to be made in the pharmaceutical industry.

Supporting information

S1 text. search strategy..

https://doi.org/10.1371/journal.pntd.0011509.s001

S1 Table. PRISMA checklist.

https://doi.org/10.1371/journal.pntd.0011509.s002

S2 Table. Risk of bias assessments.

https://doi.org/10.1371/journal.pntd.0011509.s003

S3 Table. Other reported signs and symptoms at baseline and post-baseline.

https://doi.org/10.1371/journal.pntd.0011509.s004

S1 Fig. Summary of risk of bias assessments.

https://doi.org/10.1371/journal.pntd.0011509.s005

S1 Data. Full dataset.

https://doi.org/10.1371/journal.pntd.0011509.s006

Acknowledgments

The authors would like to thank Eli Harris for guidance and support to develop the search strategy in this review.

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• 5. Betherat E. Weekly Epidemiological Record: Plague around the world in 2019. World Health Organisation, 2019.
• 13. Dennis DT, Gage KL, Gratz NG, Poland JD, Tikhomirov E, World Health Organization. Epidemic Disease C. Plague manual: epidemiology, distribution, surveillance and control / principal authors: David T. Dennis– [et al.]. Geneva: World Health Organization; 1999.

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A systematic review of the clinical profile of patients with bubonic plague and the outcome measures used in research settings

Josephine bourner.

1 ISARIC, Pandemic Sciences Institute, University of Oxford, Oxford, United Kingdom

Lovarivelo Andriamarohasina

2 Institut Pasteur de Madagascar, Antananarivo, Madagascar

Nzelle Delphine Kayem

Rindra randremanana, piero olliaro, associated data.

All relevant data are within the paper and its Supporting Information files.

Plague is a zoonotic disease that, despite affecting humans for more than 5000 years, has historically been the subject of limited drug development activity. Drugs that are currently recommended in treatment guidelines have been approved based on animal studies alone–no pivotal clinical trials in humans have yet been completed. As a result of the sparse clinical research attention received, there are a number of methodological challenges that need to be addressed in order to facilitate the collection of clinical trial data that can meaningfully inform clinicians and policy-makers. One such challenge is the identification of clinically-relevant endpoints, which are informed by understanding the clinical characterisation of the disease–how it presents and evolves over time, and important patient outcomes, and how these can be modified by treatment.

Methodology/Principal findings

This systematic review aims to summarise the clinical profile of 1343 patients with bubonic plague described in 87 publications, identified by searching bibliographic databases for studies that meet pre-defined eligibility criteria. The majority of studies were individual case reports. A diverse group of signs and symptoms were reported at baseline and post-baseline timepoints–the most common of which was presence of a bubo, for which limited descriptive and longitudinal information was available. Death occurred in 15% of patients; although this varied from an average 10% in high-income countries to an average 17% in low- and middle-income countries. The median time to death was 1 day, ranging from 0 to 16 days.

Conclusions/Significance

This systematic review elucidates the restrictions that limited disease characterisation places on clinical trials for infectious diseases such as plague, which not only impacts the definition of trial endpoints but has the knock-on effect of challenging the interpretation of a trial’s results. For this reason and despite interventional trials for plague having taken place, questions around optimal treatment for plague persist.

Author summary

Plague is an infectious disease that, despite affecting humans for more than 5000 years, has historically been the subject of limited drug development activity. In fact, the drugs currently used to treat plague have been approved based on experimental data alone–no major clinical trials have yet been completed that demonstrate the efficacy and safety of one treatment over another in humans. A major barrier to accomplishing this is that few research studies have taken place to date that can meaningfully inform the design of a clinical trial. We conducted this systematic review to gather and summarise all the existing information on the clinical profile of plague patients to understand whether sufficient information exists on which to design informative clinical trials that would provide clinicians and policy-makers with the information needed to make treatment decisions for patients.

This study however found that, based on the existing literature, there is insufficient data that can be used to develop methodologies for future trials. Either time must be invested in to collecting robust clinical data or innovative trial designs need to be identified that can simultaneously collect much-needed observational data while also evaluating much-needed interventions.

Introduction

Plague is a zoonotic infectious disease caused by Yersinia pestis , Gram-negative bacteria most commonly found in small mammals and transmitted by fleas [ 1 ].

Transmission to humans typically occurs following a bite from an infected flea, contact with contaminated bodily fluids or tissues, or inhalation of respiratory droplets [ 1 ]. Once infected, human plague manifests in three main clinical forms: bubonic, pneumonic and septicaemic–the most common of which is bubonic plague, but the disease can also progress to include secondary clinical forms [ 1 , 2 ].

Other than characteristic swollen, painful lymph nodes–or “buboes”–symptoms of bubonic plague are often non-specific and can typically include fever, headache, chills and weakness [ 1 , 2 ]. Estimates of the case fatality rate (CFR) of bubonic plague are wide-ranging, extending from 24% to 60% overall [ 1 , 3 ], but a lower CFR of ~5% has been reported in treated cases and in research settings [ 4 ].

In 2018, the last year for which global data exist, 248 cases of plague were reported– 98% of which derive from just two countries, Madagascar and the Democratic Republic of Congo [ 5 ]. However, since 2013, cases have been consistently reported within a number of countries across Asia, North America, South America, and Africa and the global distribution of natural plague foci is thought to extend far beyond the countries that have recently reported cases [ 5 , 6 ]. Due to the disease’s ability to lie dormant for years and even decades, [ 7 ] there is potential for cases to arise in locations in which plague has not been reported for a significant amount of time.

While several treatment regimens for plague are recommended in both national and international treatment guidelines, there are no robust clinical data to support the drugs that are commonly used in clinical practice against plague. No pivotal randomised controlled trials have successfully taken place to generate sufficient evidence for plague treatment regimens. A number of drugs, such as streptomycin, ciprofloxacin and doxycycline [ 8 ], have been approved for the treatment of plague based on the U.S. Food and Drug Administration’s (FDA) so-called ‘Animal rule’–which permits the approval of drugs on the basis of well-controlled animal studies when human studies are neither ethical nor feasible [ 9 ]. Other drugs, which have not received FDA approval, such as gentamicin, are also commonly used to treat plague based on experimental data and clinical experience [ 10 ].

Some of these drugs however have significant drawbacks. Until recently, guidelines relied heavily on the use of aminoglycosides, with streptomycin in particular being the drug of choice for the treatment of plague in many settings [ 11 , 12 – 14 ]. While effective against Gram-negative bacterial infections, patients treated with aminoglycosides are at higher risk of experiencing ototoxic or nephrotoxic side effects than when treated with other drugs [ 15 ], requiring onerous clinical monitoring–which many low-resource health facilities in areas with the highest burden of plague are not equipped to carry out. Streptomycin also comes with a high cost and is currently in short supply on the global market. Other treatment regimens, such as those using doxycycline, are bacteriostatic, have only oral formulations available and therefore are not adapted for more severe forms of plague. Both aminoglycosides and tetracyclines are contraindicated in pregnancy [ 16 ].

Robust clinical studies are therefore required to identify safe and effective treatment regimens for plague. To date, only a small number of interventional trials have attempted to evaluate the efficacy of plague treatment regimens, two of which have published data [ 4 , 17 ]. Neither of these studies were able to generate sufficient evidence to demonstrate clinical benefit of their investigational drugs. This is in part due to the low numbers of patients enrolled in the trials–five and 65 patients were enrolled respectively–influenced by logistical challenges and the low numbers of cases reported in the areas in which the studies were conducted. The other challenge is methodological. One trial was designed as a non-randomised trial without a control group and the other was designed to recruit a small number of cases–for which the sample size estimation was based on data generated in a substantively different patient population–to assess a broadly-defined composite endpoint of cure or improvement in condition.

It is therefore clear that more work needs to be done to refine clinical trial methodologies for plague, keeping in mind the logistical challenges and the current low case numbers that might prevent the success of large-scale trials. Understanding the clinical characterisation of plague would be beneficial to overcoming some of the methodological challenges evident in the two studies referenced above, such as identifying viable, clinically relevant endpoints.

This review therefore aims to summarise the clinical profile of patients with bubonic plague–including clinical characteristics at baseline and post-treatment, and clinical outcomes–as described in published, peer-reviewed scientific literature.

We conducted a systematic review to describe the clinical characteristics and outcomes of patients with suspected or confirmed bubonic plague from presentation to last recorded observation.

A search was conducted on bibliographic databases and clinical trial registries, including PubMED, Cochrane CENTRAL, clinicaltrials.gov, ISRCTN and the International Clinical Trials Registry Platform (ICTRP) for peer-reviewed publications describing the clinical characteristics of patients with bubonic plague. A supplementary search was conducted on JSTOR to identify older reports (pre-1970) of plague, although no formal search was conducted to obtain these publications. A full list of search terms and filters that were used for each database and registry can be found in S1 Text .

To obtain data relating to the clinical characteristics of bubonic plague, all study designs were eligible for inclusion providing the publication contained individual patient data for adults or children of any age with suspected or confirmed bubonic plague, and described signs, symptoms and outcomes. There were no restrictions placed on language or publication year. Non-human, non-clinical, post-mortem and vaccine studies were excluded.

Two reviewers independently conducted screening in Rayyan [ 18 ] and was completed in two stages by reviewing titles and abstracts then reviewing full-text articles of remaining publications. Data were extracted by the first reviewer on to a standardised data capture form in Excel ( S1 Data ). A second reviewer verified the search and performed a quality control review on 30% of the data records. Any disagreement was discussed between the two reviewers and a third independent reviewer was involved if a disagreement remained unresolved.

Risk of bias was evaluated within the included studies by one reviewer using the Joanna Briggs Institute Critical Appraisal tools [ 19 ]. Case reports were assessed using the case reports tools, randomised controlled trials were assessed using the randomised controlled trials tool and non-randomised interventional studies were assessed using the quasi-experimental studies tool.

The protocol and data dictionary associated with this review can be accessed upon reasonable request to the corresponding author.

The analysis was completed using R v.4.2.2.

The screening and inclusion processes are summarised in the PRISMA flow diagram ( S1 Table ). A summary of all included studies is provided in a table detailing the study title, study type, country in which the study was conducted, number of bubonic plague cases in each study, the ratio of males to females, and the median and range of ages of the study population in years.

Two bar charts summarise the number of included studies per year per country, and the size of the patient population in the studies per year per country.

Demographic data were extracted from each study to summarise the ratio of males to females, and age (median and range) across the entire patient population of all included studies. The total number and percent of pregnant women and patients with comorbidities among the patient population is also provided.

The signs and symptoms are summarised for patients who received a confirmed clinical or laboratory diagnosis of bubonic plague and are reported at baseline and post-baseline timepoints. Baseline is defined at the first interaction the patient has with a health professional and post-baseline includes all timepoints that occur after the day of the initial interaction. Signs and symptoms are summarised as the number and percentage of patients in whom each sign or symptom was reported on at least one occasion. The denominator is based on the sum of the sample sizes of studies in which at least one patient reported the sign or symptom; in order to eliminate studies in which the sign or symptom may not have been assessed or captured during data collection, studies which do not report the sign or symptom for at least one patient have been excluded from the denominator calculation.

The number and percentage of patients receiving treatment or no treatment is provided. Treatment information is summarised according to antimicrobial class, as defined in a recent systematic review by the U.S. Centres for Disease Control and Prevention [ 20 ]. Aminoglycosides, tetracyclines, fluoroquinolones, sulphonamides, and amphenicols were considered to be “high-efficacy”. All other antimicrobials were classified as “other antibiotics”. The number and percentage of deaths among those who received high-efficacy, other antibiotics or no treatment has been provided.

Patient outcomes are summarised as the number and percentage of deaths among the entire patient population, and according to the patient population in studies taking place in high-income countries and low- and middle-income countries. The outcome of the bubo–as present or not present–at the last recorded observation has been reported as the number and percentage of patients reporting the outcome among the patient population with a known bubo.

In total, 2023 publications were identified in the search ( Fig 1 ). After removing duplicates, 1984 publications were screened for inclusion. Following title, abstract and full-text screening, 1897 records were excluded, resulting in the inclusion of 87 publications for data synthesis.

Of the included studies, 72 (83%) were case reports, 11 (13%) were cohort studies, four (4%) were interventional studies, of which one (1%) was randomised and three (3%) were non-randomised ( Table 1 ).

a Median and range only given where article includes >1 case of bubonic plague

b Only median age available

There was a moderate risk of bias across all study types ( S2 Table and S1 Fig ). Case reports contained limited demographic, treatment and follow-up information, although baseline signs and symptoms, and diagnostic information was well-described. In interventional and cohort studies, outcomes were generally reliably described and assessed, although the completeness of follow-up information was often unclear.

All studies were published between 1902 and 2021 ( Fig 2A ).

Of the 87 included studies, 73 (84%) were conducted in the United States, three (3%) in the United Kingdom and Vietnam, two (2%) in Zambia, and 1 (1%) study each in Israel, Libya, Madagascar, Mongolia, Tanzania, and Uganda ( Fig 2A ).

These studies contain data on 1343 probable and confirmed cases of bubonic plague (including patients with secondary pneumonic or septicaemic plague). Of the included population, 870 (65%) derive from one study conducted in Madagascar, 307 (23%) from studies conducted in the United States, 76 (6%) from Vietnam, 65 (5%) from Tanzania, eight (1%) from Zambia, six (<1%) from each the United Kingdom, four (<1%) from Uganda, three (<1%) from Israel, and two (<1%) from Mongolia and Libya ( Fig 2B ).

However, over time the international distribution of included individuals has changed. From the 1960s to the 1980s reports of plague mainly derived from the United States; during this period, 209 (73%) individuals reported in this review were assessed in the United States (mostly in case reports of single patients or case series of small numbers of patients), and the remainder in Vietnam. Since the 1990s, the focus has shifted more towards the African region; during this time, 91% of cases included in this review were assessed in Madagascar, Tanzania, Zambia, Uganda and Libya, with the remainder assessed in the United States and Mongolia.

The study population consists of 769 (57%) males and 588 (43%) females with ages ranging from 0 to 82 years and a median age of 17 years. Among the population are five (<1%) patients who were pregnant at the time of their plague diagnosis. Comorbidities were reported for 17 (<1%) patients, including 13 (<1%) who tested positive for malaria. Other comorbidities include chronic cough, chronic renal insufficiency, idiopathic thrombocytopenia, and heart murmur.

Reported signs and symptoms

Baseline signs and symptoms.

The median time from symptom onset to admission was 2 days, with a range of 0 to 18 days.

A diverse group of signs and symptoms were reported at baseline and post-baseline timepoints for patients with confirmed bubonic plague within the articles included in this review ( S3 Table ).

The most commonly reported symptom at baseline was the presence of a bubo–an enlarged lymph node–which was recorded in 1216/1265 (96%) cases ( Table 1 ). Of these cases, the median number of buboes reported per patient was one. Nineteen patients reported having multiple buboes, for whom the median number of buboes per patient was two. One patient was recorded as having 10 enlarged inguinal or suprapubic nodes, which were detected through the use of ultrasound. The location of the bubo was reported in 1090 (90%) of these patients. Buboes were most commonly reported in the inguinal area (590/1090, 54%), followed by the axillary (276/1090, 25%) and cervical areas (159/1090, 15%), and less frequently in other locations, such as in femoral and epitrochlear areas. Pain at the site of the bubo was recorded in 230/320 (72%) patients and bubo size was infrequently recorded– 19/88 (22%) publications noted the size of at least one patient’s bubo. The median recorded bubo size was 30mm, with a range of 1mm to 150mm. The method of bubo measurement was reported only in one case report describing one patient, in which ultrasound was used.

Following the presence of a bubo, fever was the most frequently reported systemic feature at baseline, present in 1004/1279 (78%) patients, and for which the median recorded temperature was 39.5°C (range: 36°C to 41.5°C) ( Table 2 ). Headache was present in 127/244 (52%) patients, followed by altered mental status (33/84, 39%), chills (75/195, 38%) and malaise (22/68, 32%).

Fewer patients presented with clinical signs and symptoms indicative of severe illness at baseline, such as seizure (4/34, 12%), septic shock (5/59, 8%), and respiratory distress, arrest or failure (3/5, 60%).

All other signs and symptoms are reported in S3 Table .

Post-baseline signs and symptoms

Reporting of signs and symptoms decreased following the baseline assessment. Patients were followed up for a median of 7 days from admission (IQR 2 days to 20 days).

Reports of fever and headache persisted each in 43% of patients, and there were infrequent reports of other common symptoms at baseline, such as malaise, fatigue, abdominal pain, vomiting, cough, nausea, hypotension, and diarrhoea. While altered mental status and chills were reported in relatively high proportions of patients at baseline, no reports of these symptoms were made post-baseline.

Only 19/88 (22%) articles provided information about the presence of the bubo at any timepoint post-baseline for at least one patient in the study’s cohort. Within this population, a persistent bubo was recorded for 61/95 (64%) patient. A repeated bubo measurement is described in two case reports, in which one repeated the measurement at 3 days post-baseline and demonstrated increasing inguinal lymphadenopathy, which subsequently reduced to 10mm 26 days post-baseline–a reduction of 25mm in total; and the other repeated the measurement at 29 days post-baseline and demonstrated a reduction in size to 5mm–a decrease of 15mm from the baseline measurement–from baseline in an axillary bubo which fully resolved at 39 days post-baseline [ 78 ].

However, the frequency of some signs and symptoms indicative of severe illness increased. After the baseline assessment, respiratory distress, arrest or failure, for example, was recorded in 8/65 (12%) patients and disseminated intravascular coagulation was recorded in 7/41 (17%) patients.

An outcome was recorded for 1300/1343 (97%) patients. At the time of the last recorded observation (median: 7 days; range: 0 to 137 days), 1090/1343 (81%) had fully recovered and death was reported in 208/1343 (15%) patients ( Table 3 ); however, the case fatality ratio but varied from 10% for cases reported in high-income countries (HICs) to 17% in low- and middle-income countries (LMICs). The median time to death was 1 day in HICs and 2 days in LMICs, ranging from 0 to 16 days.

A bubo outcome was reported in 93/1279 (7%) cases in whom a bubo was reported at baseline. In 32/93 (34%) patients the bubo had completely disappeared at the last reported observation and in 61/93 (66%) cases the bubo was still present at the time of the last reported observation, which ranged from 0 to 137 days post-admission.

Information on therapeutic intervention–or no intervention where specifically indicated–was available for 306/1343 (23%) patients, of whom 271/306 (89%) received a high-efficacy antimicrobial (with or without other antibiotics), 24/306 (8%) received only other antibiotics and 11/306 (4%) received no antibiotics ( Table 4 ).

Streptomycin was the most frequently administered high-efficacy plague therapeutic that was administered–received by 141/306 (46%) patients either alone or in combination with other drugs–followed by gentamicin, tetracycline and doxycycline received by 91/306 (30%), 85/306 (28%), and 42/306 (14%) patients, respectively. All other drugs were administered in small numbers of patients.

Of those who received a high-efficacy antimicrobial at any time following initial presentation, 15/271 (6%) died. Of the 24 patients who received only other antibiotics, 6 (25%) died and of the 11 patients who received no antibiotic treatment, 5 (45%) died.

This systematic review was designed to summarise the clinical profile of patients with bubonic plague, as described in academic literature, with the objective of clarifying the endpoint(s) that could be used as the primary efficacy endpoint in therapeutic trials.

While this study has captured data on the signs, symptoms and outcomes of substantial number of confirmed cases of plague, it is limited to published studies written in English. There are several other studies, written in other languages, from which data have not been extracted. It must be acknowledged that studies describing cases in China, where plague cases have been well-documented [ 106 ], are not included in this review and only one study describing two cases from Mongolia has been included.

Overall the CFR that occurred across the patient population included in this review (15%) indicates that mortality might not be a suitable primary endpoint for a clinical trial–a future trial using a single mortality endpoint would likely require sample sizes that may not be attainable to detect significant treatment effects between arms. A rough sample size estimation designed to detect a 50% reduction in mortality with a significance level of 5%, 80% power and 10% lost to follow-up would yield a target sample size of 203 patients per arm, which might require several years to complete, or not be attainable at all.

The data on CFR collected in the scope of this review–which varies according to context and type of treatment received by the patient–may however not be representative of bubonic plague in the 21 st century. The majority of included studies are case reports which derive from the United States between the 1960s and 1980s and represent a large proportion of the patient population included in this review. In 2018 however–the most recent year for which global data are available– 80% of the global cases of plague derive from Madagascar, and a further 18% from the Democratic Republic of Congo. These are two countries in which healthcare availability and accessibility may be substantially different to that of the United States, which will likely influence patterns in disease presentation and patient outcomes. In Madagascar, the CFR for patients with bubonic plague between 1998 and 2016 was reported to be 15% [ 107 ]. The study reporting this figure however cited several limitations in that diagnostic testing evolved during the course of the study–meaning some earlier cases were potentially missed due to the sub-optimal sensitivity of older testing methods–and that samples from regions in Madagascar that had historically infrequently reported cases of plague were not consistently available. The CFR was higher at 24% during a large, atypical urban plague outbreak that occurred in 2017 [ 3 ]. Uncertainty therefore still remains around the true incidence and CFR for plague in the country with the greatest disease burden.

As survival or mortality may not be feasibly used as the single primary efficacy endpoint in a trial for bubonic plague, alternative or additional clinically-relevant outcomes must be identified. The only randomised controlled trial included in this review used “cure or improvement in condition” as its primary endpoint–a composite outcome measure consisting of 1) fever resolution; 2) resolution of bubo pain and swelling; 3) and recovery from pneumonia or any other symptom of plague present at baseline [ 4 ]. An ongoing trial in Madagascar also uses a composite endpoint of therapeutic response defined as 1) the patient being alive; 2) resolution of fever; 3) a 25% decrease in bubo size; 4) alternative plague treatment not received; 5) no decision to continue treatment beyond day 10 [ 108 ]. Composite primary endpoints such as these provide a useful solution for diseases where low numbers of individual events would prevent the detection of significant differences between arms in a trial with a single endpoint. For example, by identifying several outcomes of interest, the number of outcome events increases, therefore making it more likely that an effect can be detected with a reasonable level of confidence.

Composite endpoints however also have the potential to introduce uncertainty around any discernible differences that are detected and make the interpretation of the trial results challenging. For example, the clinical relevance of the individual criteria that make up the above-referenced composite endpoints is difficult to discern. Fever resolution, for example, was reported only for a small proportion of patients (14%) included in this review, and although the event rate– 161/186 (87%)–was high, its relationship with a patient’s overall clinical status is unclear in the literature as limited longitudinal data are available. The same can be said of the persistence or size of the bubo.

The breadth of the events included in the composite endpoint also create challenges for the accurate interpretation of the trial’s results. The events range from resolution of fever to resolution of pneumonia or any other symptom of plague. It is clear that the severity and clinical importance of these events are vastly different. However, they are weighted equally in this composite endpoint making it difficult to understand the true efficacy of the drug and how it works in this patient population. In the event that Patient A presents with only a fever at baseline and Patient B presents with pneumonia, fever and a bubo, can the recovery of both patients represent the true efficacy of the drug?

While the urgency to provide results for plague, a disease with little evidence to support current treatment recommendations, is understandable, critically, the relationship between individual signs, symptoms and clinical status–as determined by both physical indicators of a patient’s condition and laboratory testing for presence of Y . pestis in blood and/or bubo site–must be established before implementing a primary outcome measure of this nature in a clinical trial.

Given the existing data, however, it is not currently possible to identify a single or composite endpoint for plague, for which clinical relevance has been established and which would generate an achievable sample size. It is clear that robust longitudinal data from an observational study would provide critical information about the overall clinical course of plague illness and illuminate the important outcomes that could be used in either a single or composite endpoint.

However, this will take a substantial amount of time–which could be better spent by taking a more pragmatic approach to a trial. An approach recently proposed for Lassa fever, which exhibits similar challenges in endpoint definition, is to embed an observational cohort within the trial framework to collect data on patient outcomes [ 109 ]. The proposed design involves powering the trial for a mortality endpoint, and in parallel identifying and collecting data on several important outcomes of interest during the first year of the trial. An interim feasibility analysis would be undertaken to determine whether the frequencies of the events are sufficient to generate an achievable sample size and, if so, the trial would amend its primary endpoint. While this design has not been implemented in a trial to date, it may provide a useful path forward for diseases such as plague where little comparable data have been generated and uncertainty surrounds outcome frequencies. This framework may provide a cost-saving and efficient approach to trials for diseases like plague that could minimise delays to testing new drugs and reduce costs associated with implementing both observational studies and trials sequentially.

Overall, this systematic review demonstrates the restrictions that limited disease characterisation places on clinical trials for rare infectious diseases such as plague. The limited data that exist on the clinical course of plague and patient outcomes, not only impact the definition of trial endpoints but has the knock-on effect of challenging the interpretation of a trial’s results. For this reason and despite interventional trials for plague having taken place, questions around optimal treatment for plague persist. Options however exist–including those for pragmatic and innovative trial designs. A bigger challenge may however be to catalyse R&D interest in a disease for which there is limited money to be made in the pharmaceutical industry.

Supporting information

Acknowledgments.

The authors would like to thank Eli Harris for guidance and support to develop the search strategy in this review.

Funding Statement

This work was supported by the UK Foreign, Commonwealth and Development Office ( https://www.gov.uk/government/organisations/foreign-commonwealth-development-office ) and Wellcome ( https://wellcome.org/ ) (Grant ref: 215091/Z/18/Z). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Data Availability

• PLoS Negl Trop Dis. 2023 Nov; 17(11): e0011509.

Decision Letter 0

27 Aug 2023

Dear Ms Bourner,

Thank you very much for submitting your manuscript "A systematic review of the clinical profile of patients with bubonic plague and the outcome measures used in research settings" for consideration at PLOS Neglected Tropical Diseases. As with all papers reviewed by the journal, your manuscript was reviewed by members of the editorial board and by several independent reviewers. In light of the reviews (below this email), we would like to invite the resubmission of a significantly-revised version that takes into account the reviewers' comments.

Please, pay attention on comments from Reviewer 3 and try to change the presentation of the data from large Tables to graphical format when it is possible.

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PLOS Neglected Tropical Diseases

Stuart Blacksell

Section Editor

***********************

Reviewer's Responses to Questions

Key Review Criteria Required for Acceptance?

As you describe the new analyses required for acceptance, please consider the following:

-Are the objectives of the study clearly articulated with a clear testable hypothesis stated?

-Is the study design appropriate to address the stated objectives?

-Is the population clearly described and appropriate for the hypothesis being tested?

-Is the sample size sufficient to ensure adequate power to address the hypothesis being tested?

-Were correct statistical analysis used to support conclusions?

-Are there concerns about ethical or regulatory requirements being met?

Reviewer #1: (No Response)

Reviewer #2: The objectives of the study are clear: the authors aim to identify endpoints that could be used as the primary efficacy endpoint in therapeutic trials on bubonic plague.

The study design seems appropriate. The criteria used to include studies are clear. The large number of included studies provides informative data on signs and symptoms.

Reviewer #3: This study was presented as a hypothesis-generating study, with a clearly stated goal to catalog the available data on plague patients, their disease course, and response to treatment. The study design and inclusion of data considered the relatively sparse amount of plague cases, which also justified the analysis. The authors were thoughtful in the inclusion criteria for the analysis. The population was significantly weighted to a single recent large outbreak in Madagascar; in addition the majority of the remaining cases were from the US. These factors may have reduced the study power, but also are somewhat out of the control of the authors.

--------------------

-Does the analysis presented match the analysis plan?

-Are the results clearly and completely presented?

-Are the figures (Tables, Images) of sufficient quality for clarity?

Reviewer #2: Presented analysis match the analysis plan.

Results are clearly and completely presented.

Figures and tables are clear. However, I noted some inconsistencies:

- Table 3 should be revised:

Presentation of the results is not concordant with the number of reported outcomes. The authors should choose between to present the number of death and full recovery among the 1300 reported outcomes and to present the distribution of the outcomes among the 1343 patients and in this case, precise the number of unknown outcomes.

It will be worthy to indicate in table 3, the % of deaths observed in high-income countries and in low-and middle-income countries and the sex ratio of deaths in these 2 categories.

Persistent bubos already are mentioned in table 2 and should be removed from table 3

- There is a discordance in the number of patients with information on intervention. Lines 266-268:information was available for 305 patients, but 271 +24 +11 =306. The same discordance is present also in table 4.

Reviewer #3: The results were presented in tables, with one data figure that depicted where and when the included cases were drawn. It would be easier for the reader if the authors provided figures that supported the conclusions that could be drawn from the very large tables. For example, the authors could display graphically the most common outcomes reported; the authors could display in a graph, a comparison between the outcomes in the US compared to Madagascar, since these were the two largest groups; the authors could compare outcomes over time in all groups using a graph. I think any or all of these would provide clarity for the data that is presented, and might even lead to additional provocative conclusions.

Conclusions

-Are the conclusions supported by the data presented?

-Are the limitations of analysis clearly described?

-Do the authors discuss how these data can be helpful to advance our understanding of the topic under study?

-Is public health relevance addressed?

Reviewer #2: The conclusions are well supported by the data

the bias are presented in the results section

The discussion is interesting and may be helpful to advance in therapeutic trial for bubonic plague

Reviewer #3: The conclusions appear to be supported, but clarity could be improved by the presentation of the data and prevailing hypothesis on endpoints (see above comment). The limitations of the analysis are clearly described, and the authors provide a somewhat limited discussion of how these data can be be helpful to advance the impact of clinical trials for plague.

Editorial and Data Presentation Modifications?

Use this section for editorial suggestions as well as relatively minor modifications of existing data that would enhance clarity. If the only modifications needed are minor and/or editorial, you may wish to recommend “Minor Revision” or “Accept”.

Reviewer #2: Line 49: pestis should be written without upper-case letter

Line 225: range of temperature: 36°C to 103.2°C. Is it possible? or is it a problem of unit?

Table 1: References are missing for 5 studies:

Three cases of bubonic plague arising in England, 1916

Plague : Shasta County, California, 1965

Suspected Case of Imported bubonic plague, 1966

Bubonic Plague – California, 1970

Case report, Lazet, 2018

Reviewer #3: I have no additional editorial suggestions.

Summary and General Comments

Use this section to provide overall comments, discuss strengths/weaknesses of the study, novelty, significance, general execution and scholarship. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. If requesting major revision, please articulate the new experiments that are needed.

Reviewer #1: Bourner J. et al. collected the published papers in English on bubonic plague and systematically review the clinical profile of this disease for understanding its clinical characterization. 87 reports published between 1902 and 2021 were finally included in this study. 1343 probable and confirmed cases of bubonic plague mainly reported in the United States, the United Kingdom, Vietnam, Zambia, and Israel, Libya, Madagascar, Mongolia, Tanzania, and Uganda. With these available data the authors summarized the baseline and post-baseline signs and symptoms, the patients’ outcome, and treatment.

While providing valuable data for understanding the clinical feature of bubonic plague, this review also presents a limitation, which should be discussed in this manuscript. The data collection was only limited in papers in English. There are many reports on bubonic plague around the world in other languages, such as reports in Russian and Chinese. Much more patients have been missed from this statistical analysis. When discuss the limitation, Xu’s paper needs to be cited (Xu, et al. Nonlinear effect of climate on plague during the third pandemic in China. PNAS 2011 June 6.).

The flowing review is also recommended to cite in the section of introduction. Yang et al. Zoonoses (2023) 3:5; DOI 10.15212/ZOONOSES-2022-0040

It is recommended that the big Table 1 should be moved to supplements.

Reviewer #2: This systematic review is very interesting as it is based on bubonic plague cases reported during a long period, in different countries and continents. As few cases were described during the last years, this review gathers many informative descriptions of cases providing data on number, size, and location of bubos, on progression of symptoms and on treatment.

Identification of clinically relevant endpoints for plague at baseline is difficult as they may depend on the clinical status of the patient, the progression of the disease and the treatment. So, this review may help to determine some commonly identified endpoints which could be used in therapeutic trials.

Reviewer #3: This manuscript describes a meta analysis of available clinical information on plague patients, with the goal to compile a robust description of the clinical manifestation of disease and response to antibiotic treatment, in order to help inform endpoint selection for clinical trials on experimental drugs that are intended for use in combination with antibiotics. Overall this is an important goal and the results were interesting. This assessment adds to a number of recent similar papers that have surveyed patient data from plague. The impact of the work would be strengthened by graphical presentation of the data that are used to form the most pronounced conclusions that were discussed in the final section.

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Reviewer #1: Yes: Ruifu Yang

Reviewer #2: No

Reviewer #3: No

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Author response to Decision Letter 0

18 Sep 2023

Submitted filename: Response to reviewers_14Sep2023.docx

Decision Letter 1

14 Oct 2023

We are pleased to inform you that your manuscript 'A systematic review of the clinical profile of patients with bubonic plague and the outcome measures used in research settings' has been provisionally accepted for publication in PLOS Neglected Tropical Diseases.

Before your manuscript can be formally accepted you will need to complete some formatting changes, which you will receive in a follow up email. A member of our team will be in touch with a set of requests.

Please note that your manuscript will not be scheduled for publication until you have made the required changes, so a swift response is appreciated.

IMPORTANT: The editorial review process is now complete. PLOS will only permit corrections to spelling, formatting or significant scientific errors from this point onwards. Requests for major changes, or any which affect the scientific understanding of your work, will cause delays to the publication date of your manuscript.

Should you, your institution's press office or the journal office choose to press release your paper, you will automatically be opted out of early publication. We ask that you notify us now if you or your institution is planning to press release the article. All press must be co-ordinated with PLOS.

Thank you again for supporting Open Access publishing; we are looking forward to publishing your work in PLOS Neglected Tropical Diseases.

Best regards,

***********************************************************

The response to reviewers is satisfactory.

Reviewer #2: The objectives of the study are clear: the authors aim to identify endpoints that could be used as the primary efficacy endpoint in therapeutic trial on bubonic plague.

Reviewer #3: (No Response)

Reviewer #2: Presented analysis match the analysis plan.

Figures and tables are clear.

Reviewer #2: The conclusions are well supported by the data

Reviewer #2: The corrections have been done

Reviewer #2: This systematic review is very interesting as it is based on bubonic plague cases reported during a long period, in different countries and continents. As few cases were described during the last years, this review gathers many informative descriptions of cases providing data on number, size, and location of bubos, on progression of symptoms and on treatment.

Reviewer #3: The authors present a revised manuscript that incorporates suggestions of the previous review. In addition, they have provided a rebuttal to some of the criticisms that did not result in revisions to the manuscript. In general, I don't agree with the points made in the rebuttal, however the criticisms were not aimed at the data directly, but rather the presentation of data to improve clarity. Therefore, since no substantive changes are needed, the revised manuscript is satisfactory in its attempt to address the criticisms of the original manuscript.

Reviewer #2: No

Reviewer #3: No

Acceptance letter

26 Oct 2023

We are delighted to inform you that your manuscript, "A systematic review of the clinical profile of patients with bubonic plague and the outcome measures used in research settings," has been formally accepted for publication in PLOS Neglected Tropical Diseases.

We have now passed your article onto the PLOS Production Department who will complete the rest of the publication process. All authors will receive a confirmation email upon publication.

The corresponding author will soon be receiving a typeset proof for review, to ensure errors have not been introduced during production. Please review the PDF proof of your manuscript carefully, as this is the last chance to correct any scientific or type-setting errors. Please note that major changes, or those which affect the scientific understanding of the work, will likely cause delays to the publication date of your manuscript. Note: Proofs for Front Matter articles (Editorial, Viewpoint, Symposium, Review, etc...) are generated on a different schedule and may not be made available as quickly.

Soon after your final files are uploaded, the early version of your manuscript will be published online unless you opted out of this process. The date of the early version will be your article's publication date. The final article will be published to the same URL, and all versions of the paper will be accessible to readers.

Thank you again for supporting open-access publishing; we are looking forward to publishing your work in PLOS Neglected Tropical Diseases.

Shaden Kamhawi

co-Editor-in-Chief

Paul Brindley

Bubonic Plague - Free Essay Examples And Topic Ideas

The Bubonic Plague, also known as the Black Death, ravaged Europe in the 14th century, leaving a profound imprint on the continent’s demographic, social, and cultural fabric. Essays could delve into the historical unfolding of the plague, examining its origins, transmission pathways, and the devastating death toll it exacted. They might also discuss the societal and economic repercussions, such as the labor shortages, social upheaval, and the questioning of traditional religious and social orders. Discussions could extend to the examination of how the Black Death impacted art, literature, and philosophical thought of the era, and its lasting legacy on European and world history. The discourse may also touch on the scientific understanding of the disease, the historical attempts at containment, and the comparisons between the Bubonic Plague and modern pandemics, exploring the lessons learned and the enduring human struggle against infectious diseases. We’ve gathered an extensive assortment of free essay samples on the topic of Bubonic Plague you can find in Papersowl database. You can use our samples for inspiration to write your own essay, research paper, or just to explore a new topic for yourself.

The Descriptions and Effects of the Bubonic Plague, Septicemia and Pneumonia

The Plague is a word that has horrified many populations over the centuries across the globe. It remains a feared word today. This term describes a number of diseases; the three most common types are referred to as the Bubonic Plague, Septicemia, and Pneumonic. Plague pneumonia, or pneumonic plague, is caused by the same bacterium as bubonic plague but is acquired by inhaling infected droplets from the lungs of a person whose plague infection has spread to the respiratory system. […]

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The famine set the stage in the Black Death, by infecting a lot of Europe's people into hunger and starvation. The famine made people more aware of what is happening around them and in European in the 1300's. Furthermore, in the 1347's, there was a horrible turning point that occurred in Europe called the Black Death. The plague began in a hot, dry summer, which caused a multitude of fleas and rats to come out from other places. The rats […]

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The Black Death was a catastrophic event that caused many people to die, because of 3 different strains of plague. The plague was so strong it killed almost 60 percent of Europe's population, around 25 million people. The most common plague people would get was the Bubonic plague. The Bubonic plague is a bacterial infection that is transmitted by fleas or rodents, causing inflammation in the victim's lymph node. It presented swollen lymph nodes that grew as large as a […]

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Plague: the Black Death in Europe

The Black Death began in Europe in 1347 and had an estimated death toll if 75 to 200 million people. The Black Death, also known as the Bubonic Plague was carried by fleas living on the back of rats, which were normally found on the merchant ships. The plague reached Sicily in October 1347. People gathered on the docks were met with sailors aboard the ships were dead, and those still alive were gravely ill, and covered in black boils […]

About the Black Death in History

Plague is one of the three epidemic diseases that is still a problem to the International Health Regulations and is reported by the World Health Organization. The bacteria Yersinia Pestis is said to be the agent that causes this disease. This type of bacteria is a zoonotic bacteria that is embedded in small animals and fleas (Plague, 2017).Yersenia Pestis bacteria is recognized by humans as being able of causing a pathogenic disease (Stenseth, et al., 2008). The plague has led […]

Black Death in the Late Roman Empire

IN OCTOBER 1348, GENOESE TRADING SHIPS dropped anchor at the port of Messina, Sicily. The ships had come from the Black Sea port of Kaffa, now called Feodosiya. On board were goods from Central Asia, which was then controlled by the Mongol Empire. The sailors were afflicted with strange black swellings (buboes) the size of eggs that oozed blood and pus. These swellings followed by fevers, boils, and black blotches on the skin caused by internal bleeding, After four or […]

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In the annals of history, few events have left an indelible mark as profound as the Bubonic Plague, a merciless scourge that swept through medieval Europe, leaving devastation in its wake. "Catastrophe's Echo: The Lingering Impact of the Bubonic Plague" delves into the enduring consequences of this harrowing epidemic, exploring its far-reaching effects on society, culture, and the collective psyche of an era. The Black Death, as it is often called, descended upon Europe in the mid-14th century, striking fear […]

Bubonic Plague: a Historical Perspective on the Black Death

In the intricate tapestry of history, few threads are as dark and haunting as the Black Death, the Bubonic Plague that descended upon medieval Europe in the mid-14th century. This devastating pandemic, caused by the bacterium Yersinia pestis, marked an epoch of unparalleled suffering, reshaping the contours of society, culture, and the very essence of human existence. The Black Death was no ordinary calamity; it was a cataclysm that struck fear into the hearts of those unfortunate enough to witness […]

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The Bubonic Ballet, an ominous performance on the global stage, unfolded its dark narrative in the 14th century, leaving an unforgettable impression on the canvas of human history. Dubbed the Black Death, this malevolent choreography of disease orchestrated a grim symphony that reverberated across continents, reshaping societies and rewriting the rules of existence. The tale, though often told, bears the distinctive marks of a haunting masterpiece. Originating in the heartlands of Central Asia, the Bubonic Ballet was directed by the […]

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The Bubonic Drama, an ominous tale woven into the fabric of human history, unraveled its dark narrative in the 14th century, leaving an indelible mark across continents. Termed the Shadowed Plague, this relentless saga redefined societal norms, leaving an imprint that echoes through the corridors of time. Emerging from the heartlands of Central Asia, the Bubonic Drama starred Yersinia pestis as the malevolent orchestrator, conducting a symphony of suffering with rodents and fleas as unwitting performers. The bacterium, a clandestine […]

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