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The epidemiology of Yersinia pestis, the causative agent of plague, involves vectors and reservoirs in its transmission cycle. The passive plague surveillance in Madagascar targets mainly rodent and fleas. However, carnivores are routinely surveyed as sentinels of local plague activity in some countries. The aim of this study is to assess the use of domestic dog (Canis familiaris) as sentinel animal for field surveillance of plague in a highly endemic area in Madagascar. Cross-sectional surveys of plague antibody prevalence in C. familiaris were conducted in endemic areas with contrasting histories of plague cases in humans, as well as a plague free area. Rodent capture was done in parallel to evaluate evidence for Y. pestis circulation in the primary reservoirs. In 2 sites, dogs were later re-sampled to examine evidence of seroconversion and antibody persistence. Biological samplings were performed between March 2008 and February 2009. Plague antibody detection was assessed using anti-F1 ELISA. Our study showed a significant difference in dog prevalence rates between plague-endemic and plague-free areas, with no seropositive dogs detected in the plague free area. No correlation was found between rodents and dog prevalence rates, with an absence of seropositive rodents in some area where plague circulation was indicated by seropositive dogs. This is consistent with high mortality rates in rodents following infection. Re-sampling dogs identified individuals seropositive on both occasions, indicating high rates of re-exposure and/or persistence of plague antibodies for at least 9 months. Seroconversion or seropositive juvenile dogs indicated recent local plague circulation. In Madagascar, dog surveillance for plague antibody could be useful to identify plague circulation in new areas or quiescent areas within endemic zones. Within active endemic areas, monitoring of dog populations for seroconversion (negative to positive) or seropositive juvenile dogs could be useful for identifying areas at greatest risk of human outbreaks.

Background Bubonic plague is the primary manifestation of infection with Yersinia pestis, accounting for 90% of all plague cases and with 75% of global cases reported in Madagascar. All drugs in use for treating plague are registered based on experimental data and anecdotal evidence, and no regimen currently recommended is supported by a randomized clinical trial. The IMASOY trial intends to fill this knowledge gap by comparing two 10-day regimens included in the national guidelines in Madagascar. The primary objective of the trial is to test the hypothesis that ciprofloxacin monotherapy is non-inferior to streptomycin followed by ciprofloxacin for the treatment of bubonic plague, thus avoiding the need for injectable, potentially toxic, aminoglycosides. Methods A two-arm parallel-group randomized control trial will be conducted across peripheral health centres in Madagascar in five districts. Males and non-pregnant females of all ages with suspected bubonic or pneumonic plague will be recruited over the course of three plague ‘seasons’. The primary endpoint of the trial is to assess the proportion of patients with bubonic plague who have a therapeutic response to treatment (defined as alive, resolution of fever, 25% reduction in the size of measurable buboes, has not received an alternative treatment and no clinical decision to continue antibiotics) as assessed on day 11. Discussion If successful, the trial has the potential to inform the standard of care guidelines not just in Madagascar but in other countries afflicted by plague. The trial is currently ongoing and expected to complete recruitment in 2022. Trial registration ClinicalTrials.gov NCT04110340. Registered on 1 October 2019

International audience; In late 2017, Madagascar experienced a large urban outbreak of pneumonic plague, the largest outbreak to date this century. During the outbreak, there were widespread reports of plague patients presenting with atypical symptoms, such as prolonged duration of illness and upper respiratory tract symptoms. Reported mortality among plague cases was also substantially lower than that reported in the literature (25% versus 50% in treated patients). A prospective multicenter observational study was carried out to investigate potential reasons for these atypical presentations. Few subjects among our cohort had confirmed or probable plague, suggesting that, in part, there was overdiagnosis of plague cases by clinicians. However, 35% subjects reported using an antibiotic with anti-plague activity before hospital admission, whereas 55% had antibiotics with anti-plague activity detected in their serum at admission. Although there may have been overdiagnosis of plague by clinicians during the outbreak, the high frequency of community antibiotic may partly explain the relatively few culture-positive sputum samples during the outbreak. Community antibiotic use may have also altered the clinical presentation of plague patients. These issues make accurate detection of patients and the development of clinical case definitions and triage algorithms in urban pneumonic plague outbreaks difficult.

International audience; We conducted a national human serologic study of a hantavirus detected in Madagascar rodents using a commercial kit and a new ELISA targeting the virus. Our results suggest a conservative estimate of 2.7% (46/1,680) IgG seroprevalence. A second single-district study using the new ELISA revealed a higher prevalence (7.2%; 10/139).

During outbreaks, the lack of diagnostic “gold standard” can mask the true burden of infection in the population and hamper the allocation of resources required for control. Here, we present an analytical framework to evaluate and optimize the use of diagnostics when multiple yet imperfect diagnostic tests are available. We apply it to laboratory results of 2,136 samples, analyzed with 3 diagnostic tests (based on up to 7 diagnostic outcomes), collected during the 2017 pneumonic (PP) and bubonic plague (BP) outbreak in Madagascar, which was unprecedented both in the number of notified cases, clinical presentation, and spatial distribution. The extent of these outbreaks has however remained unclear due to nonoptimal assays. Using latent class methods, we estimate that 7% to 15% of notified cases were Yersinia pestis-infected. Overreporting was highest during the peak of the outbreak and lowest in the rural settings endemic to Y. pestis. Molecular biology methods offered the best compromise between sensitivity and specificity. The specificity of the rapid diagnostic test was relatively low (PP: 82%, BP: 85%), particularly for use in contexts with large quantities of misclassified cases. Comparison with data from a subsequent seasonal Y. pestis outbreak in 2018 reveal better test performance (BP: specificity 99%, sensitivity: 91%), indicating that factors related to the response to a large, explosive outbreak may well have affected test performance. We used our framework to optimize the case classification and derive consolidated epidemic trends. Our approach may help reduce uncertainties in other outbreaks where diagnostics are imperfect.

Background Among the many collaterals of the COVID-19 pandemic is the disruption of health services and vital clinical research. COVID-19 has magnified the challenges faced in research and threatens to slow research for urgently needed therapeutics for Neglected Tropical Diseases (NTDs) and diseases affecting the most vulnerable populations. Here we explore the impact of the pandemic on a clinical trial for plague therapeutics and strategies that have been considered to ensure research efforts continue. Methods To understand the impact of the COVID-19 pandemic on the trial accrual rate, we documented changes in patterns of all-cause consultations that took place before and during the pandemic at health centres in two districts of the Amoron’I Mania region of Madagascar where the trial is underway. We also considered trends in plague reporting and other external factors that may have contributed to slow recruitment. Results During the pandemic, we found a 27% decrease in consultations at the referral hospital, compared to an 11% increase at peripheral health centres, as well as an overall drop during the months of lockdown. We also found a nation-wide trend towards reduced number of reported plague cases. Discussion COVID-19 outbreaks are unlikely to dissipate in the near future. Declining NTD case numbers recorded during the pandemic period should not be viewed in isolation or taken as a marker of things to come. It is vitally important that researchers are prepared for a rebound in cases and, most importantly, that research continues to avoid NTDs becoming even more neglected., Author summary The COVID-19 pandemic has forced health systems to re-evaluate service provision, leading to the diversion of resources and widespread disruption of research activities. In particular, the pandemic has jeopardised the progress of research for Neglected Tropical Diseases (NTDs), which has historically faced considerable challenges. Many NTDs suffer a lack of robust clinical evidence to support current treatment recommendations and previous attempts to conduct clinical trials have been hampered by field conditions. This article presents the case of a randomised controlled trial for an NTD that was initiated at the start of the pandemic and explores the factors that may have influenced enrolment. Importantly, this article highlights mitigation strategies that have been considered by the trial team to ensure that trial activities can be maintained. The reduction in reporting of NTDs during the pandemic does not necessarily mean that there are fewer cases at the community level or that this trend will continue. As outbreaks of COVID-19 persist, strategies to mitigate the effect of continued disruption are critical to ensure that, even with a downward trend in NTD case notification, studies addressing an urgent research need can continue and NTDs don’t become even more neglected than they already are.

According to the WHO, 75% of the world’s plague cases are found in Madagascar, with an average of 200 to 700 cases suspected annually (mainly bubonic plague). In 2017, a pneumonic plague epidemic of unusual proportions occurred, which raised several challenges for laboratory confirmation of cases, pointing to the need for the development of Yersinia pestis isolation procedures, especially those that can be performed in remote areas. As the WHO gold standard for plague diagnosis is bacterial culture, we sought to develop a simple method to prepare a highly selective medium, fit for use in remote areas where plague is endemic. The performance of the new medium, named improved BIN, was examined in terms of growth support and selectivity with spiked samples as well in isolating Y. pestis from clinical specimens, and it was compared to the results obtained with commercially available selective media. The preparation of the new medium is less complex and its performance was found to be superior to that of first-generation BIN medium. The growth support of the medium is higher, there is no batch diversity, and it maintains high selectivity properties. In 55 clinical specimens obtained from patients suspected to be infected with Y. pestis, approximately 20% more Y. pestis-positive isolates were identified by the improved BIN medium than were identified by commercially available selective media. The improved BIN medium is notably advantageous for the isolation of Y. pestis from clinical specimens obtained from plague patients, thus offering better surveillance tools and proper promotion of medical treatment to more patients suspected of being infected with Y. pestis.

Madagascar is more seriously affected by plague, a zoonosis caused by Yersinia pestis, than any other country. The Plague National Control Program was established in 1993 and includes human surveillance. During 1998-2016, a total of 13,234 suspected cases were recorded, mainly from the central highlands; 27% were confirmed cases, and 17% were presumptive cases. Patients with bubonic plague (median age 13 years) represented 93% of confirmed and presumptive cases, and patients with pneumonic plague (median age 29 years) represented 7%. Deaths were associated with delay of consultation, pneumonic form, contact with other cases, occurrence after 2009, and not reporting dead rats. A seasonal pattern was observed with recrudescence during September-March. Annual cases peaked in 2004 and decreased to the lowest incidence in 2016. This overall reduction occurred primarily for suspected cases and might be caused by improved adherence to case criteria during widespread implementation of the F1 rapid diagnostic test in 2002.

Klebsiella pneumoniae can lead to a wide range of diseases including pneumonia, bloodstream and urinary tract infections. During a short period of a pulmonary plague epidemic in October 2017 in Madagascar, 12 K. pneumoniae isolates were identified in ten sputum and two buboes aspirate samples. These isolates were from 12 patients suspected of plague, without epidemiological relationships, but were negative for Yersinia pestis in culture. Data were collected from the plague national surveillance system. The isolates were characterized by antimicrobial susceptibility testing and whole-genome sequencing. Real-time PCR was performed to confirm the presence of K. pneumoniae DNA in buboes. All isolates were identified as K. pneumoniae sensu stricto. Five isolates were extended-spectrum β-lactamases producers; eleven different sequence types were identified. Five isolates belonged to known hypervirulent sequence types. Our results demonstrate community-acquired pneumonia caused by K. pneumoniae isolates in patients suspected of plague stressing the importance of bed-side differential diagnosis.

Background Plague is endemic to the central highlands of Madagascar. Sporadic human cases or outbreaks can occur annually in these areas. In Madagascar, the associations between endemicity and the knowledge, attitudes and practices (KAP) of the population with regard to this disease remain poorly documented. The aim of this study was to assess KAP related to plague among the population living in the central highlands. Methods A cross-sectional survey was conducted in the general population from June to August 2017. Based on the reported cases of plague between 2006 and 2015 in two central highland districts, a KAP questionnaire was administered in the population. Based on the proportion of correct answers provided by respondents, KAP scores were classified into three KAP categories: low (< Mean - SD), medium (Mean ± SD) and good (> Mean + SD). Multivariate analyses were performed to determine the associations between population KAP scores related to plague and sociodemographic and epidemiological factors. In addition, individual interviews and focus groups with health professionals were conducted to assess plague perception. Results A total of 597 individuals participated in the survey; 20% (n = 119) had a good KAP score, 62% (n = 370) a medium KAP score and 18% (n = 108) a low KAP score. Among the 119 respondents with good KAP scores, 80% (n = 95) resided in Ambositra district, and 20% (n = 24) resided in Tsiroanomandidy district. According to the health professionals in the two districts, populations in endemic areas are well aware of the plague. There were significant associations (p Conclusion The results of the study showed the need to adapt plague control interventions to the local context to allow a better allocation of human and financial resources. Doing so would minimize delays in patient management care and increase community resilience to plague epidemics.

Background Human plague cases, mainly in the bubonic form, occur annually in endemic regions of the central highlands of Madagascar. The aim of this study was to compare the dynamics of the epidemiological features of the human plague in two districts of the central highlands region. Methods In Madagascar, all clinically suspected plague cases that meet clinical and epidemiological criteria specified in the World Health Organization (WHO) standard case definition are reported to the national surveillance system. Data on plague cases reported between 2006 and 2015 in the districts of Ambositra and Tsiroanomandidy were analysed. Statistical comparisons between the epidemiological characteristics of the two districts were conducted. Results A total of 840 cases of plague were reported over the studied period, including 563 (67%) probable and confirmed cases (P + C). Out of these P + C cases, nearly 86% (488/563) were cases of bubonic plague. Reported clinical forms of plague were significantly different between the districts from 2006 to 2015 (p = 0.001). Plague cases occurred annually in a period of 10 years in the Tsiroanomandidy district. During the same period, the Ambositra district was characterized by a one-year absence of cases. Conclusion The differences in the epidemiological situation with respect to the plague from 2006 to 2015 in the two central highlands districts may suggest that several factors other than biogeographical factors determine the representation of the plague and its dynamics in this region. Considering the epidemiological situations according to the specific contexts of the districts could improve the results in the fight against the plague in Madagascar.

Background Plague, a fatal disease caused by the bacillus, Yersinia pestis, still affects resources-limited countries. Information on antibody response to plague infection in human is scarce. Anti-F1 Ig G are among the known protective antibodies against Y. pestis infection. As a vaccine preventable disease, knowledge on antibody response is valuable for the development of an effective vaccine to reduce infection rate among exposed population in plague-endemic regions. In this study, we aim to describe short and long-term humoral immune responses against Y. pestis in plague-confirmed patients from Madagascar, the most affected country in the world. Methods Bubonic (BP) and pneumonic plague (PP) patients were recruited from plague- endemic foci in the central highlands of Madagascar between 2005 and 2017. For short-term follow-up, 6 suspected patients were enrolled and prospectively investigated for kinetics of the anti-F1 IgG response, whereas the persistence of antibodies was retrospectively studied in 71 confirmed convalescent patients, using an ELISA which was validated for the detection of plague in human blood samples in Madagascar. Results Similarly to previous findings, anti-F1 IgG rose quickly during the first week after disease onset and increased up to day 30. In the long-term study, 56% of confirmed cases remained seropositive, amongst which 60 and 40% could be considered as high- and low-antibody responders, respectively. Antibodies persisted for several years and up to 14.8 years for one individual. Antibody titers decreased over time but there was no correlation between titer and time elapsed between the disease onset and serum sampling. In addition, the seroprevalence rate was not significantly different between gender (P = 0.65) nor age (P = 0.096). Conclusion Our study highlighted that the circulating antibody response to F1 antigen, which is specific to Y. pestis, may be attributable to individual immune responsiveness. The finding that a circulating anti-F1 antibody titer could persist for more than a decade in both BP and PP recovered patients, suggests its probable involvement in patients’ protection. However, complementary studies including analyses of the cellular immune response to Y. pestis are required for the better understanding of long-lasting protection and development of a potential vaccine against plague.

Yersinia pestis, the bacterial causative agent of plague, remains an important threat to human health. Plague is a rodent-borne disease that has historically shown an outstanding ability to colonize and persist across different species, habitats, and environments while provoking sporadic cases, outbreaks, and deadly global epidemics among humans. Between September and November 2017, an outbreak of urban pneumonic plague was declared in Madagascar, which refocused the attention of the scientific community on this ancient human scourge. Given recent trends and plague’s resilience to control in the wild, its high fatality rate in humans without early treatment, and its capacity to disrupt social and healthcare systems, human plague should be considered as a neglected threat. A workshop was held in Paris in July 2018 to review current knowledge about plague and to identify the scientific research priorities to eradicate plague as a human threat. It was concluded that an urgent commitment is needed to develop and fund a strong research agenda aiming to fill the current knowledge gaps structured around 4 main axes: (i) an improved understanding of the ecological interactions among the reservoir, vector, pathogen, and environment; (ii) human and societal responses; (iii) improved diagnostic tools and case management; and (iv) vaccine development. These axes should be cross-cutting, translational, and focused on delivering context-specific strategies. Results of this research should feed a global control and prevention strategy within a “One Health” approach., Author summary The historical aspect of plague makes for fascinating reading, due to its capacity to disrupt human society and its socioeconomic and cultural impacts throughout human history. We argue that the Madagascar outbreak in 2017 is a tipping point in human plague epidemiology and a call to elevate research priorities on plague as a matter of some urgency. In contrast with what occurred with the Ebola virus disease crisis in West Africa between 2013 and 2015 and the new coronaviruses (the emergence of severe acute respiratory syndrome coronavirus [SARS-CoV] and Middle East respiratory syndrome coronavirus [MERS-CoV] as early warnings of the current severe acute respiratory syndrome coronavirus 2 [SARS-CoV-2] pandemic), we have an opportunity to act preventively and enable evidence-based measures to avoid major health crises due to plague outbreaks in the near future.

Background Plague is a highly fatal disease caused by Yersinia pestis. Late diagnosis hampers disease outcome and effectiveness of control measures, induces death and disease spread. Advance on its diagnosis was the use of lateral flow rapid diagnostic test (RDT). Methods We assessed the performance of the plague RDT based on Y. pestis F1 antigen detection more than 15 years after its deployment in Madagascar. We compared the RDT with bacteriological culture results, using data from plague notified cases collected during the periods for which both tests were performed independently and systematically. Results Used with bubonic plague (BP) patient samples, RDTs had a sensitivity of 100% (95% CI: 99.7–100%), a specificity of 67% (95% CI: 64–70%) with a good agreement between bacteriology and RDT results (86%; κ = 0.70, 95% CI 0.67–0.73). For pneumonic plague (PP), RDT had a sensitivity of 100% (95% CI: 91–100%) and a specificity of 59% (95% CI: 49–68%) and concordance between the bacteriological and plague RDT results was moderate (70%; κ = 0.43, 95% CI 0.32–0.55). Analysis focusing on the 2017–2018 plague season including the unprecedented epidemic of PP showed that RDT used on BP samples still had a sensitivity of 100% (95% CI: 85–100%) and a specificity of 82% (95% CI: 48–98%) with a very good agreement with bacteriology 94% (κ = 0.86, 95% CI 0.67–1); for PP samples, concordance between the bacteriological and plague RDT results was poor (61%; κ = − 0.03, 95% CI -0.17 – 0.10). Conclusions RDT performance appeared to be similar for the diagnosis of BP and PP except during the 2017 PP epidemic where RDT performance was low. This RDT, with its good sensitivity on both plague clinical forms during a normal plague season, remained a potential test for alert. Particularly for BP, it may be of great value in the decision process for the initiation of therapy. However, for PP, RDT may deliver false negative results due to inconsistent sample quality. Plague diagnosis could be improved through the development of next generation of RDTs.

International audience; Background: Plague is a life-threatening disease caused by the bacterium, Yersinia pestis. Madagascar is the leading country for human plague cases worldwide. Human plague is a serious disease, particularly in its septicaemic and pneumonic forms. We report a case of pneumonic plague co-infected by a MDR-Stenotrophomonas maltophilia. Case presentation: A 24 year-old man originated from Soavinandriana, a plague focus, felt uneasy and developed high fever with chills. He started treatment by himself, by private medical care and by a traditional healer for nine days moving several times from place to place. His condition had deteriorated when he presented to a district hospital with a syndrome of dyspnea, bronchial rale and altered state of consciousness. Two days later, plague diagnosis, performed as a last resort, revealed a positive F1 antigen on rapid diagnostic test. Additional tests (pla PCR and plague serology) evidenced a Y. pestis infection. However, streptomycin treatment did not achieve a complete recovery as the course of disease was complicated by the presence of MDR-S. maltophilia in his lung. This opportunistic infection could have been favored by an immunosuppression due to Y. pestis pulmonary infection and probably been acquired during his stay at a District Hospital. He was treated with a combination of ciprofloxacin and gentamycin and recovered fully. Conclusions: Pneumonic plague infection may promote another virulent or avirulent bacterial infection particularly when it is not initially suspected. However, coinfection is rarely described and its occurrence frequency is unknown. In middle or low resources areas, which is the case of most plague endemic countries, control and prevention of infections in health facilities is not optimal. Co-infection with an opportunistic pathogen agent, such as S. maltophilia, is a risk which must not be disregarded as demonstrated by this case report. When deciding of a national control strategy, it should be taken into account in the choice of the first line treatment.

International audience; During a pneumonic plague outbreak in Moramanga, Madagascar, we identified 4 confirmed, 1 presumptive, and 9 suspected plague case-patients. Human-to-human transmission among close contacts was high (reproductive number 1.44) and the case fatality rate was 71%. Phylogenetic analysis showed that the Yersinia pestis isolates belonged to group q3, different from the previous outbreak.

We identified mixed infections of pathogenic Leptospira in small mammals across a landscape-scale study area in Madagascar by using primers targeting different Leptospira spp. Using targeted primers increased prevalence estimates and evidence for transmission between endemic and invasive hosts. Future studies should assess rodentborne transmission of Leptospira to humans.

Bubonic is the most prevalent plague form in Madagascar. Indoor ground application of insecticide dust is the conventional method used to control potentially infected rodent fleas that transmit the plague bacterium from rodents to humans. The use of bait stations is an alternative approach for vector control during plague epidemics, as well as a preventive control method during non-epidemic seasons. Bait stations have many advantages, principally by reducing the amount of insecticide used, lowering the cost of the treatment and minimizing insecticide exposure in the environment. A previous study reported promising results on controlling simultaneously the reservoir and vectors, when slow-acting rodenticide was incorporated in bait stations called “Boîtes de Kartman”. However, little evidence of an effective control of the fleas prior to the elimination of rodents was found. In this study, we evaluated bait stations containing insecticide powder and non-toxic attractive rodent bait for their potential to control rat fleas. Its efficacy was compared to the standard method. The impact of both methods on indoor and outdoor rodent fleas, as well as the human household flea Pulex irritans were analyzed at different time points after treatments. Bait stations did not cause any significant immediate or delayed reduction of rat fleas and increasing the number of operational bait stations per household did not significantly improve their efficacy. Insecticide ground dusting appeared to be the most efficient method to control indoor rat fleas. Both methods appeared to have little impact on the density of outdoor rat fleas and human fleas. These results demonstrate limited effectiveness for bait stations and encourage the maintenance of insecticide dusting as a first-line control strategy in case of epidemic emergence of plague, when immediate effect on rodent fleas is needed. Recommendations are given to improve the efficacy of the bait station method., Author summary Insecticide ground dusting inside houses is the recommended measure to control rat fleas responsible for bubonic plague transmission. The main inconvenience of this method is the direct contact of houseowners to the toxic insecticide dust and spillage in environment. A bait station approach, where the insecticide is confined in a box or tunnel containing rodent attractant, seems to be a valuable complementary or alternative vector control tool. However currently, little is known about its real efficacy on reducing or eliminating fleas harbored by rats. Guidelines regarding its implementation (density and duration of use) as vector control tool are lacking. Those questions were addressed during a field trial study, where bait stations were deployed at different densities per household and followed up at different time points. The efficacy of bait station was compared to the standard method. The present study allowed to demonstrate that bait station approach requires more improvements to be efficient. Meanwhile, insecticide ground dusting is still recommended for to control rat fleas during epidemics.

Background: Madagascar accounts for 75% of global plague cases reported to WHO, with an annual incidence of 200–700 suspected cases (mainly bubonic plague). In 2017, a pneumonic plague epidemic of unusual size occurred. The extent of this epidemic provides a unique opportunity to better understand the epidemiology of pneumonic plagues, particularly in urban settings. Methods: Clinically suspected plague cases were notified to the Central Laboratory for Plague at Institut Pasteur de Madagascar (Antananarivo, Madagascar), where biological samples were tested. Based on cases recorded between Aug 1, and Nov 26, 2017, we assessed the epidemiological characteristics of this epidemic. Cases were classified as suspected, probable, or confirmed based on the results of three types of diagnostic tests (rapid diagnostic test, molecular methods, and culture) according to 2006 WHO recommendations. Findings: 2414 clinically suspected plague cases were reported, including 1878 (78%) pneumonic plague cases, 395 (16%) bubonic plague cases, one (

After centuries of epidemics and more than a hundred years since the identification of the causative bacterium, very little is known about the plague dynamics in animal reservoirs, vectors and the changing vulnerabilities for humans. The recent plague epidemic in Madagascar in 2017 highlights these gaps existing within the knowledge of the disease dynamics, the factors influencing it, the performance of diagnostic tests and the best recommended treatment. As the eradication of plague will not be possible due to the widespread existence of the bacterium in wildlife, a One Health approach, drawing on animal, human and environmental health disciplines is needed to better control this poverty-related disease. This article focused on the various aspects of the disease for which more tools and better understanding are required to better control the disease in endemic countries. RésuméAprès des siècles d’épidémies et plus de 100 ans après la découverte de l’agent pathogène, très peu de données sont disponibles sur la dynamiquede la peste dans les réservoirs animaux, sur les vecteurs et sur l’évolution de la vulnérabilité des hommes. L’épidémie de peste récemment observéeà Madagascar (2017) met en évidence les écarts observés en termes de connaissances de la dynamique de la maladie, des facteurs qui l’influencent,de la performance des tests diagnostiques et du traitement optimal recommandé. Puisque l’éradication de la peste est impossible en raison del’omniprésence de la bactérie chez les animaux, l’approche multisectorielle de l’OMS « Un monde, une santé » doit être adoptée en faisant appelaux disciplines relatives à la santé animale, humaine et environnementale afin de mieux contrôler cette maladie liée à la pauvreté. Cet article s’estintéressé aux différents aspects de la maladie pour lesquels davantage d’outils et une meilleure compréhension sont nécessaires afin de mieuxcontrôler la maladie dans les pays endémiques.

International audience; Plague is a rodent-borne disease caused by Yersinia pestis. Most human infections are bubonic plague, as a result of being bitten by infected rodent fleas. Madagascar, Democratic Republic of Congo and Peru are the three most affected countries. Plague was introduced into eastern Madagascar in 1898 by boat from India. It is estimated that the risk of introduction of plague from Madagascar to neighboring islands is very high due to the maritime links. We conducted a study of plague reservoirs and vectors in Longoni Port in Mayotte and Mahajanga Port in Madagascar during two seasons to highlight a non-negligible risk of introduction of Y. pestis to Mayotte. The results showed that two main reservoirs of plague in Madagascar Suncus murinus and Rattus rattus and the main flea vector Xenopsylla cheopis exists in and surrounding the port of Longoni. Y. pestis was isolated from Rattus norvegicus captured close to the port of Mahajanga during this study. Plague bacteria circulate within populations of rodent without causing rodent die-off in Mahajanga. The risk of introduction of plague from Madagascar to Mayotte exists due to the regular exchanges. Continuous surveillance of rat, shrew and flea populations is therefore necessary in all the surrounding countries that have regular exchanges with Madagascar to prevent the spread of the plague.

Identifying key reservoirs for zoonoses is crucial for understanding variation in incidence. Plague re-emerged in Mahajanga, Madagascar in the 1990s but there has been no confirmed case since 1999. Here we combine ecological and genetic data, from during and after the epidemics, with experimental infections to examine the role of the shrew Suncus murinus in the plague epidemiological cycle. The predominance of S. murinus captures during the epidemics, their carriage of the flea vector and their infection with Yersinia pestis suggest they played an important role in the maintenance and transmission of plague. S. murinus exhibit a high but variable resistance to experimental Y. pestis infections, providing evidence of its ability to act as a maintenance host. Genetic analyses of the strains isolated from various hosts were consistent with two partially-linked transmission cycles, with plague persisting within the S. murinus population, occasionally spilling over into the rat and human populations. The recent isolation from a rat in Mahajanga of a Y. pestis strain genetically close to shrew strains obtained during the epidemics reinforces this hypothesis and suggests circulation of plague continues. The observed decline in S. murinus and Xenopsylla cheopis since the epidemics appears to have decreased the frequency of spillover events to the more susceptible rats, which act as a source of infection for humans. Although this may explain the lack of confirmed human cases in recent years, the current circulation of plague within the city highlights the continuing health threat., Author summary The reemergence of plague is related to the persistence and dynamics of its reservoirs and vectors. During the human plague outbreaks that re-occurred in Mahajanga harbor, Madagascar in the 1990s, the shrew Suncus murinus was found infected with Yersinia pestis. Combining field surveys, experimental infections and genetic analysis, we examined the role of Asian shrew Suncus murinus in plague transmission and maintenance comparatively with others potential hosts in Mahajanga. The genomes of nineteen Y. pestis isolates recovered from humans, shrews, rats and fleas in this focus were sequenced and compared. We observed the predominance of S. murinus captured during the epidemics and their carriage of the flea vector. Shrews exhibited high resistance to Y. pestis experimental infections. Genetic analyses of the strains isolated from various hosts were consistent with two partially-linked transmission cycles, with plague persisting within the S. murinus population, occasionally spilling over into the rat and human populations. The isolation of a Y. pestis strain from a rat in Mahajanga in 2014 genetically close to shrew strains reinforces this hypothesis. The current circulation of plague within the city highlights the continuing health threat.

Background Yersinia pestis appears to be maintained in multiple, geographically separate, and phylogenetically distinct subpopulations within the highlands of Madagascar. However, the dynamics of these locally differentiated subpopulations through time are mostly unknown. To address that gap and further inform our understanding of plague epidemiology, we investigated the phylogeography of Y. pestis in Madagascar over an 18 year period. Methodology/Principal findings We generated whole genome sequences for 31 strains and discovered new SNPs that we used in conjunction with previously identified SNPs and variable-number tandem repeats (VNTRs) to genotype 773 Malagasy Y. pestis samples from 1995 to 2012. We mapped the locations where samples were obtained on a fine geographic scale to examine phylogeographic patterns through time. We identified 18 geographically separate and phylogenetically distinct subpopulations that display spatial and temporal stability, persisting in the same locations over a period of almost two decades. We found that geographic areas with higher levels of topographical relief are associated with greater levels of phylogenetic diversity and that sampling frequency can vary considerably among subpopulations and from year to year. We also found evidence of various Y. pestis dispersal events, including over long distances, but no evidence that any dispersal events resulted in successful establishment of a transferred genotype in a new location during the examined time period. Conclusions/Significance Our analysis suggests that persistent endemic cycles of Y. pestis transmission within local areas are responsible for the long term maintenance of plague in Madagascar, rather than repeated episodes of wide scale epidemic spread. Landscape likely plays a role in maintaining Y. pestis subpopulations in Madagascar, with increased topographical relief associated with increased levels of localized differentiation. Local ecological factors likely affect the dynamics of individual subpopulations and the associated likelihood of observing human plague cases in a given year in a particular location., Author summary Plague exists in several geographically separate phylogenetic groups within Madagascar, but little is known about the dynamics of these groups through time. We subtyped 773 Malagasy plague samples and identified 18 phylogenetic groups that showed persistence in the same locations over a period of almost two decades. Locations with more topographical relief contained more phylogenetic groups than flatter areas and different phylogenetic groups varied considerably in the number of samples collected each year. Transfers of plague from one location to another definitely occur, but appear to seldom result in the transplanted phylogenetic group becoming successfully established in a new location. Persistent, local transmission cycles are likely responsible for the long term maintenance of plague in Madagascar, rather than repeated wide scale disease transmission events. Landscape likely plays a role in maintaining different phylogenetic groups, with increased topographical relief associated with increased numbers of phylogenetic groups. Local ecological factors likely affect the dynamics of individual subpopulations and the associated likelihood of observing human plague cases in a given year in a particular location.

International audience; Background: Rickettsiae are obligate intracellular bacteria responsible for many febrile syndromes around the world,including in sub-Saharan Africa. Vectors of these pathogens include ticks, lice, mites and fleas. In order to assessexposure to flea-associated Rickettsia species in Madagascar, human and small mammal samples from an urbanand a rural area, and their associated fleas were tested.Results: Anti-typhus group (TGR)- and anti-spotted fever group rickettsiae (SFGR)-specific IgG were detected in24 (39%) and 21 (34%) of 62 human serum samples, respectively, using indirect ELISAs, with six individuals seropositivefor both. Only two (2%) Rattus rattus out of 86 small mammals presented antibodies against TGR. Out of 117fleas collected from small mammals, Rickettsia typhi, a TGR, was detected in 26 Xenopsylla cheopis (24%) collected fromrodents of an urban area (n = 107), while two of these urban X. cheopis (2%) were positive for Rickettsia felis, a SFGR. R.felis DNA was also detected in eight (31%) out of 26 Pulex irritans fleas.Conclusions: The general population in Madagascar are exposed to rickettsiae, and two flea-associated Rickettsiapathogens, R. typhi and R. felis, are present near or in homes. Although our results are from a single district, theydemonstrate that rickettsiae should be considered as potential agents of undifferentiated fever in Madagascar.

Plague, one of the most devastating infectious diseases in human history, is caused by the bacterial species Yersinia pestis. A live attenuated Y. pestis strain (EV76) has been widely used as a plague vaccine in various countries around the world. Here we compared the whole genome sequence of an EV76 strain used in China (EV76-CN) with the genomes of Y. pestis wild isolates to identify genetic variations specific to the EV76 lineage. We identified 6 SNPs and 6 Indels (insertions and deletions) differentiating EV76-CN from its counterparts. Then, we screened these polymorphic sites in 28 other strains of EV76 lineage that were stored in different countries. Based on the profiles of SNPs and Indels, we reconstructed the parsimonious dissemination history of EV76 lineage. This analysis revealed that there have been at least three independent imports of EV76 strains into China. Additionally, we observed that the pyrE gene is a mutation hotspot in EV76 lineages. The fine comparison results based on whole genome sequence in this study provide better understanding of the effects of laboratory passages on the accumulation of genetic polymorphisms in plague vaccine strains. These variations identified here will also be helpful in discriminating different EV76 derivatives.

Plague is an infectious disease caused by the bacterium Yersinia pestis, which, during the fourteenth century, caused the deaths of an estimated 75–200 million people in Europe. Plague epidemics still occur in Africa, Asia and South America. Madagascar is today one of the most endemic countries, reporting nearly one third of the human cases worldwide from 2004 to 2009. The persistence of plague in Madagascar is associated with environmental and climatic conditions. In this paper we present a case study of the spatio-temporal analysis of plague incidence in Madagascar from 1980 to 2007. We study the relationship of plague with temperature and precipitation anomalies, and with elevation. A joint spatio-temporal analysis of the data proves to be computationally intractable. We therefore develop a spatio-temporal log-Gaussian Cox process model, but then carry out marginal temporal and spatial analyses. We also introduce a spatially discrete approximation for Gaussian processes, whose parameters retain a spatially continuous interpretation. We find evidence of a cumulative effect, over time, of temperature anomalies on plague incidence, and of a very high relative risk of plague occurrence for locations above 800 m in elevation. Our approach provides a useful modeling framework to assess the relationship between exposures and plague risk, irrespective of the spatial resolution at which the latter has been recorded.

Background Genetic analysis of pathogenic organisms is a useful tool for linking human cases together and/or to potential environmental sources. The resulting data can also provide information on evolutionary patterns within a targeted species and phenotypic traits. However, the instruments often used to generate genotyping data, such as single nucleotide polymorphisms (SNPs), can be expensive and sometimes require advanced technologies to implement. This places many genotyping tools out of reach for laboratories that do not specialize in genetic studies and/or lack the requisite financial and technological resources. To address this issue, we developed a low cost and low tech genotyping system, termed agarose-MAMA, which combines traditional PCR and agarose gel electrophoresis to target phylogenetically informative SNPs. Methodology/Principal findings To demonstrate the utility of this approach for generating genotype data in a resource-constrained area (Madagascar), we designed an agarose-MAMA system targeting previously characterized SNPs within Yersinia pestis, the causative agent of plague. We then used this system to genetically type pathogenic strains of Y. pestis in a Malagasy laboratory not specialized in genetic studies, the Institut Pasteur de Madagascar (IPM). We conducted rigorous assay performance validations to assess potential variation introduced by differing research facilities, reagents, and personnel and found no difference in SNP genotyping results. These agarose-MAMA PCR assays are currently employed as an investigative tool at IPM, providing Malagasy researchers a means to improve the value of their plague epidemiological investigations by linking outbreaks to potential sources through genetic characterization of isolates and to improve understanding of disease ecology that may contribute to a long-term control effort. Conclusions The success of our study demonstrates that the SNP-based genotyping capacity of laboratories in developing countries can be expanded with manageable financial cost for resource constraint laboratories. This is a practical formula that reduces resource-driven limitations to genetic research and promises to advance global collective knowledge of infectious diseases emanating from resource limited regions of the world., Author summary Although genetic characterization of pathogenic organisms is a powerful tool for investigating outbreak origins and transmission, associated high upfront costs and demanding technological maintenance exclude this tool for many under-resourced laboratories. Paradoxically, resource constrained regions commonly suffer from high rates of infectious diseases and could benefit most from genetic tracking tools. One such country is Madagascar, which lacks resources to acquire high tech genetic typing equipment, yet suffers from seasonal human plague outbreaks. A serious disease, plague is caused by the clonal bacterium, Yersinia pestis, and is capable of causing human outbreaks. Using plague as a model organism, we developed a genetic typing method that requires only basic, widely used molecular machinery. Our tools target unique single mutations in the Y. pestis genome to assign isolates to distinct phylogenetic groups with known geographical distributions. Transfer of this technology to Madagascar permits genetic characterization of strains from current outbreaks. This eliminates the need for external genetic analysis and expands the research capacity of this resource-constrained laboratory by allowing rapid, in-house strain typing. Ultimately, our goal is to help improve the ability of local institutes to genetically characterize circulating strains, link outbreaks to originating sources, and improve our understanding of the ecology of tropical diseases in resource-limited regions of the world.

International audience; BackgroundLeptospirosis is a widespread but underreported cause of morbidity and mortality. It has rarely been reported in either humans or animals in Madagascar.MethodsWe conducted a cross-sectional survey of the inhabitants in Moramanga, Madagascar, in June 2011, to estimate the prevalence of human infection using the microscopic agglutination test (MAT). This activity was carried out as part of a workshop implemented by the Pasteur Institute of Madagascar, focusing on surveillance with a one week field study and targeting the health staff of the district level.ResultsIn total, we sampled 678 inhabitants from 263 households. The sex ratio (M/F) was 0.65 and the mean age 26.7 years. We obtained a value of 2.9% for the first recorded seroprevalence of this disease in the human community of Moramanga. Questionnaire responses revealed frequent contacts between humans and rodents in Moramanga. However, activities involving cattle were identified as a risk factor significantly associated with seropositivity (OR=3).ConclusionLeptospirosis remains a neglected disease in Madagascar. This study highlights the need to quantify the public health impact of this neglected disease in a more large scale, in all the country and to establish point-of-care laboratories in remote areas.

Multidrug-resistant Yersinia pestis is a serious threat that requires outbreak response strategies., Yersinia pestis, the causative agent of plague, is endemic to Madagascar, particularly to the central highlands. Although plague has not been previously reported in northern Madagascar, an outbreak of pneumonic plague occurred in this remote area in 2011. Over a 27-day period, 17 suspected, 2 presumptive, and 3 confirmed human cases were identified, and all 15 untreated 20 patients died. Molecular typing of Y. pestis isolated from 2 survivors and 5 Rattus rattus rat samples identified the Madagascar-specific 1.ORI3-k single-nucleotide polymorphism genotype and 4 clustered regularly interspaced short palindromic repeat patterns. This outbreak had a case-fatality rate of 100% for nontreated patients. The Y. pestis 1.ORI3-k single-nucleotide polymorphism genotype might cause larger epidemics. Multidrug-resistant strains and persistence of the pathogen in natural foci near human settlements pose severe risks to populations in plague-endemic regions and require outbreak response strategies.

International audience; Fipronil was evaluated as a systemic control agent for the rat flea Xenopsylla cheopis (Rothschild), the main vector of Yersinia pestis (Yersin), the causative agent of plague, in Madagascar. The effectiveness of fipronil as a systemic control agent against X. cheopis was assessed by determining the toxicity values of the " Lethal Dose 50 " (LD 50). Two techniques were used to evaluate the systemic action of the insecticide on the vector: 1) an artificial feeding device filled with blood–fipronil mixture from which X. cheopis was fed and 2) rodent hosts, Rattus norvegicus (Berkenhout) and Rattus rattus (L.), which fed on fipronil-treated bait. As a standardized control method, the susceptibility of X. cheopis to fipronil was evaluated by exposure to impregnated paper within World Health Organization (WHO) insecticide test protocol to compare its effect to the systemic activity of the studied insecticide. Results showed that when administered in a systemic way, fipronil appears to be more effective: the toxicity level was evaluated to be ninefold higher compared with the WHO test. Compared with other methods, which require indiscriminate dusting of rodent burrows and human dwellings, fipronil applied in a systemic way enables the direct targeting of the plague vector. Thus, this method appears to be a superior alternative to fipronil-dusting for the control of the main plague vector in Madagascar. However, subsequent tests in the field are necessary to confirm the suitability of fipronil administration in a systemic way on large scales.

International audience; After 25 years of no cases of plague, this disease recurred near Tobruk, Libya, in 2009. An epidemiologic investigation identified 5 confirmed cases. We determined ribotypes, Not1 restriction profiles, and IS100 and IS1541 hybridization patterns of strains isolated during this outbreak. We also analyzed strains isolated during the 2003 plague epidemic in Algeria to determine whether there were epidemiologic links between the 2 events. Our results demonstrate unambiguously that neighboring but independent plague foci coexist in Algeria and Libya. They also indicate that these outbreaks were most likely caused by reactivation of organisms in local or regional foci believed to be dormant (Libya) or extinct (Algeria) for decades, rather than by recent importation of Yersinia pestis from distant foci. Environmental factors favorable for plague reemergence might exist in this area and lead to reactivation of organisms in other ancient foci.

We describe the development and evaluation of a rapid diagnostic test for Vibrio cholerae O1 and O139 based on lipopolysaccharide detection using gold particles. The specificity ranged between 84 and 100%. The sensitivity of the dipsticks ranged from 94.2 to 100% when evaluated with stool samples obtained in Madagascar and Bangladesh. The dipstick can provide a simple tool for epidemiological surveys.

To the Editor: Yersinia pestis (family Enterobacteriaceae) is a bacterium that can cause high rates of death in susceptible mammals and can provoke septicemic, pneumonic, and bubonic plague in humans (1). This zoonotic pathogen can be transmitted directly by infectious droplets or by contact with contaminated fluid or tissue or indirectly through flea bites (1). Plague was introduced into Madagascar in 1898 from rat-infested steamships that had sailed from affected areas (2). Now, Madagascar is 1 of 2 countries in Africa that have reported cases of human plague every year since 1991 (3). During January 2008–January 2013, the number of human plague cases reported in Madagascar ranged from 312 to 648 per year. Of these, 61.8%–75.5% were laboratory confirmed (National Plague Laboratory of the Ministry of Health, pers. comm.). Most (>83%) confirmed cases were bubonic plague, which most commonly results from flea bites, suggesting that these bites were the most common mode of Y. pestis transmission. In Madagascar, Xenopsylla cheopis fleas have been known as the primary plague vector in urban areas, whereas Synopsyllus fonquerniei fleas have been usually involved in plague transmission in rural areas (2). In January 2013, a total of 9 suspected bubonic plague cases, 3 confirmed, were reported in Soavina, a rural area in the district of Ambatofinandrana, Madagascar. Domestic fleas were collected with candle traps inside 5 houses during 3 nights (Table). Fleas were also caught on small mammals trapped inside houses and outside in the sisal fences and rice fields (Table). A total of 319 fleas belonging to 5 species in 5 genera were collected inside and outside the houses, an average of 44 per house (maximum 71): Pulex irritans, Echidnophaga gallinacea, and Ctenocephalides canis fleas were collected inside the houses (244, 76.5%), and S. fonquerniei and X. cheopis fleas were collected outside (75, 23.5%). The human flea, P. irritans, was the most collected flea species (233, 73.3%), followed by S. fonquerniei (62, 19.4%), X. cheopis (13, 4.1%), E. gallinacea (10, 3.1%), and C. canis (1, 0.3%). Table Fleas collected inside and outside houses in Soavina, Madagascar, January 2013 Bacterial DNA was extracted from 277 fleas of 5 species: 233 P. irritans, 24 S. fonquerniei, 9 X. cheopis, 10 E. gallinacea, and 1 C. canis. PCR to detect Y. pestis was performed by using primers YP1 (5′-ATC TTA CTT TCC GTG AGA AG-3′) and YP2 (5′-CTT GGA TGT TGA GCT TCC TA-3′) to amplify a 478-bp fragment (4). Y. pestis DNA was then amplified and genotyped by Beckman Coulter Genomics Inc. (Takeley, United Kingdom). The positive control was Y. pestis reference strain (strain 6/69, 3 × 108 bacteria/mL; Institut Pasteur de Madagascar). Detection of Y. pestis was carried out on 274 fleas belonging to 5 flea species: 230 P. irritans (181 unfed and 49 engorged), 24 S. fonquerniei (15 unfed and 9 engorged), 9 X. cheopis (8 unfed and 1 engorged), 10 E. gallinacea (blood-feeding status not identified), and 1 unfed C. canis. Y. pestis was detected in 9 P. irritans fleas (7 male [6 unfed and 1 engorged] and 2 [engorged] female) from 3 houses, including the house where a confirmed human case of plague had occurred (Technical Appendix). Eight sequences (GenBank accession nos. {"type":"entrez-nucleotide-range","attrs":{"text":"KJ361938-KJ361945","start_term":"KJ361938","end_term":"KJ361945","start_term_id":"608789158","end_term_id":"608789172"}}KJ361938-KJ361945) were obtained and share 99% nucleotide homology with plasminogen activator genes of Y. pestis published in GenBank (accession nos. {"type":"entrez-nucleotide","attrs":{"text":"AF528537","term_id":"22212811","term_text":"AF528537"}}AF528537, {"type":"entrez-nucleotide","attrs":{"text":"AY305870","term_id":"32250732","term_text":"AY305870"}}AY305870). No Y. pestis was detected in the 24 S. fonquerniei, 9 X. cheopis, 10 E. gallinacea, or 1 C. canis fleas collected. Although only X. cheopis and S. fonqueniei fleas had previously been described as plague vectors in Madagascar, P. irritans fleas were most commonly collected during this field study; engorged and unfed male and female P. irritans fleas carried Y. pestis. Other studies have found P. irritans fleas in the plague risk area in other countries in Africa (5,6); one study found that P. irritans fleas may play a role in plague epidemiology in Tanzania (5). Data on P. irritans fleas in rats make it unlikely that these fleas are involved in rat-to-human transmission of Y. pestis in Madagascar. During 1922–1995, a total of 118,608 rats were caught and examined in Madagascar, but only 148 P. irritans fleas were identified, and none have been found on rats since 1996 (http://www.pasteur.mg/spip.php?rubrique124). The high density of P. irritans fleas we observed in villages where plague outbreaks occurred in late 2012 and early 2013 (http://www.pasteur.mg/spip.php?rubrique124) supports the possibility that P. irritans fleas played a role in domestic human-to-human transmission of Y. pestis during these outbreaks. Technical Appendix: Agarose gel electrophoresis showing positive PCR results for Yersinia pestis in DNA of 9 Pulex irritans fleas collected from 3 houses in an area of Madagascar where plague outbreaks occurred in late 2012 and early 2013. Click here to view.(47K, pdf)

From AD 1347 to AD 1353, the Black Death killed tens of millions of people in Europe, leaving misery and devastation in its wake, with successive epidemics ravaging the continent until the 18th century. The etiology of this disease has remained highly controversial, ranging from claims based on genetics and the historical descriptions of symptoms that it was caused by Yersinia pestis to conclusions that it must have been caused by other pathogens. It has also been disputed whether plague had the same etiology in northern and southern Europe. Here we identified DNA and protein signatures specific for Y. pestis in human skeletons from mass graves in northern, central and southern Europe that were associated archaeologically with the Black Death and subsequent resurgences. We confirm that Y. pestis caused the Black Death and later epidemics on the entire European continent over the course of four centuries. Furthermore, on the basis of 17 single nucleotide polymorphisms plus the absence of a deletion in glpD gene, our aDNA results identified two previously unknown but related clades of Y. pestis associated with distinct medieval mass graves. These findings suggest that plague was imported to Europe on two or more occasions, each following a distinct route. These two clades are ancestral to modern isolates of Y. pestis biovars Orientalis and Medievalis. Our results clarify the etiology of the Black Death and provide a paradigm for a detailed historical reconstruction of the infection routes followed by this disease., Author Summary Several historical epidemic waves of plague have been attributed to Yersinia pestis, the etiologic agent of modern plague. The most famous of these was the second pandemic which was active in Europe from AD 1347 until 1750, and began with the ‘Black Death’. The most informative method to establish the etiological nature of these ancient infections should be the analysis of ancient DNA, but the results of this method have been controversial. Here, by combining ancient DNA analyses and protein-specific detection, we demonstrate unambiguously that Y. pestis caused the Black Death. Furthermore, we show that at least two variants of Y. pestis spread over Europe during the second pandemic. The analysis of up to 20 diagnostic markers reveals that the two variants evolved near the time that phylogenetic branches 1 and 2 separated and may no longer exist. Our results thus resolve a long-standing debate about the etiology of the Black Death and provide key information about the evolution of the plague bacillus and the spread of the disease during the Middle Ages.

Background Tools for plague diagnosis and surveillance are not always available and affordable in most of the countries affected by the disease. Yersinia pestis isolation for confirmation is time-consuming and difficult to perform under field conditions. Serologic tests like ELISA require specific equipments not always available in developing countries. In addition to the existing rapid test for antigen detection, a rapid serodiagnostic assay may be useful for plague control. Methods/Principal Findings We developed two rapid immunochromatography-based tests for the detection of antibodies directed against F1 antigen of Y. pestis. The first test, SIgT, which detects total Ig (IgT) anti-F1 in several species (S) (human and reservoirs), was developed in order to have for the field use an alternative method to ELISA. The performance of the SIgT test was evaluated with samples from humans and animals for which ELISA was used to determine the presumptive diagnosis of plague. SIgT test detected anti-F1 Ig antibodies in humans with a sensitivity of 84.6% (95% CI: 0.76–0.94) and a specificity of 98% (95% CI: 0.96–1). In evaluation of samples from rodents and other small mammals, the SlgT test had a sensitivity of 87.8% (95% CI: 0.80–0.94) and a specificity of 90.3% (95% CI: 0.86–0.93). Improved performance was obtained with samples from dogs, a sentinel animal, with a sensitivity of 93% (95% CI: 0.82–1) and a specificity of 98% (95% CI: 0.95–1.01). The second test, HIgM, which detects human (H) IgM anti-F1, was developed in order to have another method for plague diagnosis. Its sensitivity was 83% (95% CI: 0.75–0.90) and its specificity about 100%. Conclusion/Significance The SIgT test is of importance for surveillance because it can detect Ig antibodies in a range of reservoir species. The HIgM test could facilitate the diagnosis of plague during outbreaks, particularly when only a single serum sample is available., Author Summary Plague is due to the bacterium Yersinia pestis. It is accidentally transmitted to humans by the bite of infected fleas. Currently, approximately 20 developing countries with very limited infrastructure are still affected. A plague case was defined according to clinical, epidemiological and biological features. Rapid diagnosis and surveillance of the disease are essential for its control. Indeed, the delay of treatment is often rapidly fatal for patients and outbreaks may occur. Bubo aspirate is the most appropriate specimen in case of bubonic plague, but its collection is not always feasible. The main current biological approaches for the diagnosis of human plague are F1 antigen detection, serology for antibody detection by ELISA and Y. pestis isolation. The biological diagnosis of plague remains a challenge because the clinical signs are not specific. In this study, we developed some simple, rapid and affordable tests able to detect specific plague antibodies. These tests can be used as alternative methods for plague diagnosis in the field and for plague surveillance.

Background Early detection of cholera outbreaks is crucial for the implementation of the most appropriate control strategies. Methods The performance of an immunochromatographic dipstick test (Institute Pasteur, Paris, France) specific for Vibrio cholerae O1 was evaluated in a prospective study in Beira, Mozambique, during the 2004 cholera season (January-May). Fecal specimens were collected from 391 patients with acute watery nonbloody diarrhea and tested by dipstick and conventional culture. Results The overall sensitivity and specificity of the rapid test compared to culture were 95% (95% confidence interval [CI]: 91%–99%) and 89% (95% CI: 86%–93%), respectively. After stratification by type of sample (rectal swab/bulk stool) and severity of diarrhea, the sensitivity ranged between 85% and 98% and specificity between 77% and 97%. Conclusion This one-step dipstick test performed well in the diagnosis of V. cholerae O1 in a setting with seasonal outbreaks where rapid tests are most urgently needed.