An open-label, randomized, non-inferiority trial of the efficacy and safety of ciprofloxacin versus an aminoglycoside + ciprofloxacin in the treatment of bubonic plague (IMASOY): study protocol for a randomized control trial-an update to the published protocol.

This article reports an update to the protocol of the IMASOY trial, which was prospectively registered on clinicaltrials.gov (NCT04110340) in October 2019.

Review of genotyping methods for Yersinia pestis in Madagascar.

BACKGROUND: Plague, a zoonotic disease caused by Yersinia pestis, was responsible for 3 historical human pandemics that killed millions of people. It remains endemic in rodent populations in Africa, Asia, North America, and South America but human plague is rare in most of these locations. However, human plague is still highly prevalent in Madagascar, which typically records a significant part of all annual global cases. This has afforded an opportunity to study contemporary human plague in detail using various typing methods for Y. pestis. AIM: This review aims to summarize the methods that have been used to type Y. pestis in Madagascar along with the major discoveries that have been made using these approaches. METHODS: Pubmed and Google Scholar were used to search for the keywords: "typing Yersinia pestis Madagascar," "evolution Yersinia pestis Madagascar," and "diversity Yersinia pestis Madagascar." Eleven publications were relevant to our topic and further information was retrieved from references cited in those publications. RESULTS: The history of Y. pestis typing in Madagascar can be divided in 2 periods: the pre-genomics and genomics eras. During the pre-genomics era, ribotyping, direct observation of plasmid content and plasmid restriction fragment length polymorphisms (RFLP) were employed but only revealed a limited amount of diversity among Malagasy Y. pestis strains. Extensive diversity only started to be revealed in the genomics era with the use of clustered regularly interspaced palindromic repeats (CRISPR), multiple-locus variable number tandem repeats (VNTR) analysis (MLVA), and single-nucleotide polymorphisms (SNPs) discovered from whole genome sequences. These higher-resolution genotyping methods have made it possible to highlight the distribution and persistence of genotypes in the different plague foci of Madagascar (Mahajanga and the Central and Northern Highlands) by genotyping strains from the same locations across years, to detect transfers between foci, to date the emergence of genotypes, and even to document the transmission of antimicrobial resistant (AMR) strains during a pneumonic plague outbreak. Despite these discoveries, there still remain topics that deserve to be explored, such as the contribution of horizontal gene transfer to the evolution of Malagasy Y. pestis strains and the evolutionary history of Y. pestis in Madagascar. CONCLUSIONS: Genotyping of Y. pestis has yielded important insights on plague in Madagascar, particularly since the advent of whole-genome sequencing (WGS). These include a better understanding of plague persistence in the environment, antimicrobial AMR and multi-drug resistance in Y. pestis, and the person-to-person spread of pneumonic plague. Considering that human plague is still a significant public health threat in Madagascar, these insights can be useful for controlling and preventing human plague in Madagascar and elsewhere, and also are relevant for understanding the historical pandemics and the possible use of Y. pestis as a biological weapon.

Socio-ecological risk factors associated with human flea infestations of rural household in plague-endemic areas of Madagascar.

Plague is a flea-borne fatal disease caused by the bacterium Yersinia pestis, which persists in rural Madagascar. Although fleas parasitizing rats are considered the primary vectors of Y. pestis, the human flea, Pulex irritans, is abundant in human habitations in Madagascar, and has been found naturally infected by the plague bacterium during outbreaks. While P. irritans may therefore play a role in plague transmission if present in plague endemic areas, the factors associated with infestation and human exposure within such regions are little explored. To determine the socio-ecological risk factors associated with P. irritans infestation in rural households in plague-endemic areas of Madagascar, we used a mixed-methods approach, integrating results from P. irritans sampling, a household survey instrument, and an observational checklist. Using previously published vectorial capacity data, the minimal P. irritans index required for interhuman bubonic plague transmission was modeled to determine whether household infestations were enough to pose a plague transmission risk. Socio-ecological risk factors associated with a high P. irritans index were then identified for enrolled households using generalized linear models. Household flea abundance was also modeled using the same set of predictors. A high P. irritans index occurred in approximately one third of households and was primarily associated with having a traditional dirt floor covered with a plant fiber mat. Interventions targeting home improvement and livestock housing management may alleviate flea abundance and plague risk in rural villages experiencing high P. irritans infestation. As plague-control resources are limited in developing countries such as Madagascar, identifying the household parameters and human behaviors favoring flea abundance, such as those identified in this study, are key to developing preventive measures that can be implemented at the community level.

Multiple Introductions of Yersinia pestis during Urban Pneumonic Plague Epidemic, Madagascar, 2017.

Pneumonic plague (PP) is characterized by high infection rate, person-to-person transmission, and rapid progression to severe disease. In 2017, a PP epidemic occurred in 2 Madagascar urban areas, Antananarivo and Toamasina. We used epidemiologic data and Yersinia pestis genomic characterization to determine the sources of this epidemic. Human plague emerged independently from environmental reservoirs in rural endemic foci >20 times during August-November 2017. Confirmed cases from 5 emergences, including 4 PP cases, were documented in urban areas. Epidemiologic and genetic analyses of cases associated with the first emergence event to reach urban areas confirmed that transmission started in August; spread to Antananarivo, Toamasina, and other locations; and persisted in Antananarivo until at least mid-November. Two other Y. pestis lineages may have caused persistent PP transmission chains in Antananarivo. Multiple Y. pestis lineages were independently introduced to urban areas from several rural foci via travel of infected persons during the epidemic.

Reproductive ecology of the black rat (Rattus rattus) in Madagascar: the influence of density-dependent and -independent effects.

The black rat (Rattus rattus) poses a severe threat to food security and public health in Madagascar, where it is a major cause of pre- and post-harvest crop losses and an important reservoir for many zoonotic diseases, including plague. Elsewhere, ecologically based rodent management (EBRM) strategies have been developed using ecological information to inform decisions on where and when to target control. EBRM could deliver improved health and well-being outcomes in Madagascar if adapted to the local ecological context. Using data collected from removal studies, we explored spatio-temporal patterns in the breeding activity of the black rat (R. rattus) in domestic and agricultural habitats across Madagascar and investigated to what extent these trends are influenced by rainfall and rat density. We identified clear spatio-temporal variation in the seasonality of R. rattus reproduction. Reproduction was highly seasonal both inside and outside of houses, but seasonal trends varied between these two habitats. Seasonal trends were explained, in part, by variation in rainfall; however, the effect of rainfall on reproductive rates did itself vary by season and habitat type. A decline in breeding intensity with increasing rat density was recorded outside of houses. This has important implications for control, as populations may compensate for removal through increased reproduction. We recommend that sustained control initiated before the main breeding season, combined with improved hygiene and adequate rodent-proofing in homes and grain stores, could curtail population growth and reduce pre- and post-harvest losses provided that these measures overcome the compensatory response of rodent populations.

Population dynamics of plague vector fleas in an endemic focus: implications for plague surveillance.

Plague is a zoonotic vector-borne disease caused by the bacterium Yersinia pestis. In Madagascar, it persists in identified foci, where it is a threat to public health generally from September to April. A more complete understanding of how the disease persists could guide control strategies. Fleas are the main vector for transmission between small mammal hosts and humans, and fleas likely play a role in the maintenance of plague. This study characterized the dynamics of flea populations in plague foci alongside the occurrence of human cases. From 2018 to 2020, small mammals were trapped at sites in the central Highlands of Madagascar. A total of 2,762 small mammals were captured and 5,295 fleas were collected. The analysis examines 2 plague vector species in Madagascar (Synopsyllus fonquerniei and Xenopsylla cheopis). Generalized linear models were used to relate flea abundance to abiotic factors, with adjustments for trap location and flea species. We observed significant effects of abiotic factors on the abundance, intensity, and infestation rate by the outdoor-associated flea species, S. fonquerniei, but weak seasonality for the indoor-associated flea species, X. cheopis. A difference in the timing of peak abundance was observed between the 2 flea species during and outside the plague season. While the present study did not identify a clear link between flea population dynamics and plague maintenance, as only one collected X. cheopis was infected, the results presented herein can be used by local health authorities to improve monitoring and control strategies of plague vector fleas in Madagascar.

Assessing the effectiveness of intervention to prevent plague through community and animal-based survey.

Bubonic plague, transmitted by infected flea bites, is the most common form of plague and, left untreated, can progress to the pneumonic form, which is highly contagious. Surveillance focusing on reservoir and vector is considered to be the main approach to prevent plague. Common rodent control methods include the use of rodenticide and snap traps but, in a plague context, the dispersal of fleas from killed animals may pose a serious health threat. Therefore, there is a need for strategies which address reservoir and vector control. The aim of this study was to assess the effects of combination of reservoir and vector control through community-based surveillance. Activities were implemented by local previously trained community agents in two active plague foci in Madagascar. Kartman bait stations containing rodenticide and insecticide were placed indoors while live traps were set outdoors. Small mammals were identified and killed with their fleas. Effectiveness of control measures was evaluated by comparison of plague incidence two years before and after intervention using data on reported human cases of plague from the Central Laboratory of Plague. A total of 4,302 small mammals were captured, with the predominance of the black rat Rattus rattus. Our results found a reduction in plague incidence in the treated site for at least two years after treatment. Community-based interventions played an important role in reducing contact between humans-rodents-fleas. Our study confirms the importance of animal surveillance during the low plague transmission season. The combination of reservoir and vector control with community involvement may be effective at reducing the risks of plague spillover to humans. The strategy of using Kartman bait stations indoors with live traps outdoors can be used to refine proactive plague prevention, however, due to the potential development of resistance to pesticides in flea and rat populations, overuse should be considered.

The surveillance of plague among rodents and dogs in Western Iran.

BACKGROUND: The causative agent of plague, Yersinia pestis, is maintained in nature via a flea-rodent cycle. Western Iran is an old focus for plague, and recent data indicate that rodents and dogs in this region have serological evidence of Y. pestis infection. The purpose of this study was to conduct a large-scale investigation of Y. pestis infection in shepherd dogs, rodents, and their fleas in old foci for plague in Western Iran. MATERIALS AND METHODS: This study was conducted in Hamadan province from 2014 to 2020. Rodents and fleas were collected from various locations throughout this region. Y. pestis was investigated in rodent spleen samples and fleas using culture, serology, and real-time PCR methods. Additionally, sera samples were collected from carnivores and hares in this region, and the IgG antibody against the Y. pestis F1 antigen was assessed using an ELISA. RESULTS: In this study, 927 rodents were captured, with Meriones spp. (91.8%) and Microtus qazvinensis (2.6%) being the most prevalent. A total of 6051 fleas were collected from rodents and carnivores, most of which were isolated from Meriones persicus. None of the rodents or fleas examined tested positive for Y. pestis using real-time PCR and culture methods. Meanwhile, IgG antibodies were detected in 0.32% of rodents. All serologically positive rodents belonged to M. persicus. Furthermore, none of the sera from the 138 carnivores (129 sheepdogs, five Vulpes vulpes, four Canis aureus), and nine hares tested positive in the ELISA test. CONCLUSION: This primary survey of rodent reservoirs shows serological evidence of Y. pestis infection. Western Iran is an endemic plague focus, and as such, it requires ongoing surveillance.

A self-amplifying RNA vaccine provides protection in a murine model of bubonic plague.

Mice were immunized with a combination of self-amplifying (sa) RNA constructs for the F1 and V antigens of Yersinia pestis at a dose level of 1 µg or 5 µg or with the respective protein sub-units as a reference vaccine. The immunization of outbred OF1 mice on day 0 and day 28 with the lowest dose used (1 µg) of each of the saRNA constructs in lipid nanoparticles protected 5/7 mice against subsequent sub-cutaneous challenge on day 56 with 180 cfu (2.8 MLD) of a 2021 clinical isolate of Y. pestis termed 10-21/S whilst 5/7 mice were protected against 1800cfu (28MLD) of the same bacteria on day 56. By comparison, only 1/8 or 1/7 negative control mice immunized with 10 µg of irrelevant haemagglutin RNA in lipid nanoparticles (LNP) survived the challenge with 2.8 MLD or 28 MLD Y. pestis 10-21/S, respectively. BALB/c mice were also immunized with the same saRNA constructs and responded with the secretion of specific IgG to F1 and V, neutralizing antibodies for the V antigen and developed a recall response to both F1 and V. These data represent the first report of an RNA vaccine approach using self-amplifying technology and encoding both of the essential virulence antigens, providing efficacy against Y. pestis. This saRNA vaccine for plague has the potential for further development, particularly since its amplifying nature can induce immunity with less boosting. It is also amenable to rapid manufacture with simpler downstream processing than protein sub-units, enabling rapid deployment and surge manufacture during disease outbreaks.

Socioenvironmental determinants as indicators of plague risk in the central highlands of Madagascar: Experience of Ambositra and Tsiroanomandidy districts.

BACKGROUND: Human plague cases are reported annually in the central highland regions of Madagascar, where the disease is endemic. The socioenvironmental characteristics and lifestyles of the populations of the central highland localities could be linked to this endemicity. The aim of this study was to determine socioenvironmental determinants that may be associated with plague risk and explain this variation in epidemiological contexts. METHODS: The current study was based on the distribution of plague cases between 2006 and 2015 that occurred in localities of districts positioned in the central highlands. Household surveys were performed from June to August 2017 using a questionnaire and direct observations on the socioenvironmental aspects of households in selected localities. Bivariate and multivariate analyses were performed to highlight the socioenvironmental parameters associated with plague risk in both districts. RESULTS: A total of 503 households were surveyed, of which 54.9% (276/503) were in Ambositra and 45.1% (227/503) were in Tsiroanomandidy. Multivariate analyses showed that thatched roofs [adjusted odds ratio (AOR): 2.63; 95% confidence interval (95% CI): 1.78-3.88] and ground floor houses [AOR: 2.11; 95% CI: 1.3-3.45-] were significantly associated with the vulnerability of a household to plague risk (p value<0.05). CONCLUSIONS: Plague risk in two districts of the Malagasy central highlands is associated with human socioenvironmental characteristics. Socioenvironmental characteristics are parameters expressing spatial heterogeneity through the difference in epidemiological expression of the plague in Ambositra and Tsiroanomandidy. These characteristics could be used as indicators of vulnerability to plague risk in plague-endemic areas.

Knockdown resistance mutations are common and widely distributed in Xenopsylla cheopis fleas that transmit plague in Madagascar.

BACKGROUND: Plague, caused by the bacterium Yersinia pestis, remains an important disease in Madagascar, where the oriental rat flea, Xenopsylla cheopis, is a primary vector. To control fleas, synthetic pyrethroids (SPs) have been used for >20 years, resulting in resistance in many X. cheopis populations. The most common mechanisms of SP resistance are target site mutations in the voltage-gated sodium channel (VGSC) gene. METHODOLOGY/PRINCIPAL FINDINGS: We obtained 25 collections of X. cheopis from 22 locations across Madagascar and performed phenotypic tests to determine resistance to deltamethrin, permethrin, and/or dichlorodiphenyltrichloroethane (DDT). Most populations were resistant to all these insecticides. We sequenced a 535 bp segment of the VGSC gene and identified two different mutations encoding distinct substitutions at amino acid position 1014, which is associated with knockdown resistance (kdr) to SPs in insects. Kdr mutation L1014F occurred in all 25 collections; a rarer mutation, L1014H, was found in 12 collections. There was a significant positive relationship between the frequency of kdr alleles and the proportion of individuals surviving exposure to deltamethrin. Phylogenetic comparisons of 12 VGSC alleles in Madagascar suggested resistant alleles arose from susceptible lineages at least three times. Because genotype can reasonably predict resistance phenotype, we developed a TaqMan PCR assay for the rapid detection of kdr resistance alleles. CONCLUSIONS/SIGNIFICANCE: Our study provides new insights into VGSC mutations in Malagasy populations of X. cheopis and is the first to report a positive correlation between VGSC genotypes and SP resistance phenotypes in fleas. Widespread occurrence of these two SP resistance mutations in X. cheopis populations in Madagascar reduces the viability of these insecticides for flea control. However, the TaqMan assay described here facilitates rapid detection of kdr mutations to inform when use of these insecticides is still warranted to reduce transmission of plague.

Phylogenetic analysis of the origin and spread of plague in Madagascar.

BACKGROUND: Plague is a zoonotic disease caused by the bacterium Yersinia pestis, highly prevalent in the Central Highlands, a mountainous region in the center of Madagascar. After a plague-free period of over 60 years in the northwestern coast city of Mahajanga, the disease reappeared in 1991 and caused several outbreaks until 1999. Previous research indicates that the disease was reintroduced to the city of Mahajanga from the Central Highlands instead of reemerging from a local reservoir. However, it is not clear how many reintroductions occurred and when they took place. METHODOLOGY/PRINCIPAL FINDINGS: In this study we applied a Bayesian phylogeographic model to detect and date migrations of Y. pestis between the two locations that could be linked to the re-emergence of plague in Mahajanga. Genome sequences of 300 Y. pestis strains sampled between 1964 and 2012 were analyzed. Four migrations from the Central Highlands to Mahajanga were detected. Two resulted in persistent transmission in humans, one was responsible for most of the human cases recorded between 1995 and 1999, while the other produced plague cases in 1991 and 1992. We dated the emergence of the Y. pestis sub-branch 1.ORI3, which is only present in Madagascar and Turkey, to the beginning of the 20th century, using a Bayesian molecular dating analysis. The split between 1.ORI3 and its ancestor lineage 1.ORI2 was dated to the second half of the 19th century. CONCLUSIONS/SIGNIFICANCE: Our results indicate that two independent migrations from the Central Highlands caused the plague outbreaks in Mahajanga during the 1990s, with both introductions occurring during the early 1980s. They happened over a decade before the detection of human cases, thus the pathogen likely survived in wild reservoirs until the spillover to humans was possible. This study demonstrates the value of Bayesian phylogenetics in elucidating the re-emergence of infectious diseases.

Analytical framework to evaluate and optimize the use of imperfect diagnostics to inform outbreak response: Application to the 2017 plague epidemic in Madagascar.

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.

Plague in Tanzania: first report of sylvatic plague in Morogoro region, persistence in Mbulu focus, and ongoing quiescence in Lushoto and Iringa foci.

Objectives: Plague has been a threat to human health in Tanzania since 1886. This zoonotic disease has established several endemic foci in the country, posing a risk of outbreaks. This study was conducted to investigate the presence of Yersinia pestis in small mammals in five districts. These districts were selected because of recent (Mbulu), past (40-18 years ago: Lushoto) and historic (>100 years ago: Iringa and Kilolo) human cases of plague. In addition, one region that has not had any reported human cases of plague was included (Morogoro-Mvomero). Methods: Blood from 645 captured small mammals was screened for antibodies against the fraction 1 (F1) antigen of Y. pestis using indirect enzyme-linked immunosorbent assay (ELISA) and competitive-blocking ELISA. Results: Specific antibodies against Y. pestis F1 antigens were detected in six (0.93%) animals belonging to Mastomys natalensis. Of these, four animals were captured in the active focus in Mbulu, and two animals were captured from an area with no history of human plague (Morogoro-Mvomero). Conclusion: These results provide evidence of the circulation of Y. pestis in small mammals in Tanzania. Furthermore, evidence of the circulation of Y. pestis in Morogoro-Mvomero highlights the importance of carrying out plague surveillance in areas with no history of human plague, which can help to predict areas where future outbreaks may occur.

Characterization of Klebsiella pneumoniae isolated from patients suspected of pulmonary or bubonic plague during the Madagascar epidemic in 2017.

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 ß-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.

Tracking of Mammals and Their Fleas for Plague Surveillance in Madagascar, 2018-2019.

Plague, a zoonotic disease caused by the bacterium Yersinia pestis, remains a major public health threat in Madagascar. To better understand the risk of transmission to humans and to guide targeted plague prevention and control measures, a survey of Y. pestis infection and exposure in mammals and their fleas was implemented. Small mammals were captured in five districts of Madagascar ranging in levels of plague endemicity, as measured by notified cases, from none to active foci. Blood and spleen samples and fleas were collected from small mammals for the detection of anti-Y. pestis F1 antibodies by ELISA, F1 antigens by rapid diagnostic tests, and pla, caf1, and inv genes by polymerase chain reaction. Some rodent fleas were kept alive and reared in the insectary to assess susceptibility to insecticides. Blood was also collected from 15 dogs and tested for anti-F1 antibodies. A total of 557 spleens, 484 sera, and 1,539 fleas were collected from 557 rodents and shrews. Nineteen (3.4%) spleens were positive for F1 antigen, most from Toamasina (N = 13), a historical plague focus. One dog was also found seropositive in Toamasina. Twenty-two (4.5%) serologic specimens from small mammals were positive for anti-F1 antibodies. The flea index was highest in the city of Antananarivo (8.8). No flea was positive for Y. pestis DNA. Flea populations exhibited resistance to various insecticides weakening the efficacy of vector control. This study highlights the potential use of animal-based surveillance to identify the risk of plague transmission in endemic and nonendemic foci for targeted prevention and control.

Proteomic Signatures of Antimicrobial Resistance in Yersinia pestis and Francisella tularensis.

Antimicrobial resistance (AMR) is a well-recognized, widespread, and growing issue of concern. With increasing incidence of AMR, the ability to respond quickly to infection with or exposure to an AMR pathogen is critical. Approaches that could accurately and more quickly identify whether a pathogen is AMR also are needed to more rapidly respond to existing and emerging biological threats. We examined proteins associated with paired AMR and antimicrobial susceptible (AMS) strains of Yersinia pestis and Francisella tularensis, causative agents of the diseases plague and tularemia, respectively, to identify whether potential existed to use proteins as signatures of AMR. We found that protein expression was significantly impacted by AMR status. Antimicrobial resistance-conferring proteins were expressed even in the absence of antibiotics in growth media, and the abundance of 10-20% of cellular proteins beyond those that directly confer AMR also were significantly changed in both Y. pestis and F. tularensis. Most strikingly, the abundance of proteins involved in specific metabolic pathways and biological functions was altered in all AMR strains examined, independent of species, resistance mechanism, and affected cellular antimicrobial target. We have identified features that distinguish between AMR and AMS strains, including a subset of features shared across species with different resistance mechanisms, which suggest shared biological signatures of resistance. These features could form the basis of novel approaches to identify AMR phenotypes in unknown strains.

High Rickettsial Diversity in Rodents and Their Ectoparasites From the Central Highlands of Madagascar.

Rickettsioses are among emerging infectious diseases around the world. In Madagascar, little information is available regarding Rickettsia (Rickettsiales: Rickettsiaceae) diversity and their potential impacts on public health. In fact, molecular screening of ectoparasites of mammals reported the presence of three species, Rickettsia africae, Rickettsia typhi, and Rickettsia felis. The present study aims to investigate the diversity of Rickettsia in small mammals and associated ectoparasites (fleas and ticks) using a molecular approach. In September and December 2016, fieldworks were undertaken in two districts of Madagascar to capture small mammals using standard traps (Tomahawk and Sherman traps) and collect associated ectoparasites. In total, 12 taxa of ectoparasites (5 flea and 7 tick species) were collected from 89 individuals of four species of terrestrial small mammals. Rickettsia spp. were molecularly identified in one specimen of Rattus rattus (Rodentia: Muridae), one specimen of Pulex irritans (Siphonaptera: Pulicidae) as well as four specimens of Ixodes cf. colasbelcouri (Ixodida: Ixodidae). This study showed the presence of three phylogenetically distinct taxa of Rickettsia in small mammals and their ectoparasites. The current study broadens our knowledge on the diversity of Rickettsia in the Central Highlands of Madagascar and highlights for the first time the presence of Ri. felis in R. rattus and in tick, I. cf. colasbelcouri in Madagascar. Additional studies are needed to have exhaustive information on Rickettsia in small mammals and their ectoparasites, to determine their pathogenicity as well as their potential effects on public health in order to update the national policy for the control of emerging infectious diseases in Madagascar.

Local-scale diversity of Yersinia pestis: A case study from Ambohitromby, Ankazobe District, Madagascar.

Plague is a re-emerging zoonotic disease and a major public health concern in several portions of the world, especially in Madagascar. We report on the presence of different subtypes of Yersinia pestis co-occurring in the same locality. After confirmation of a human plague case in Ambohitromby Commune (Ankazobe District) via isolation of Y. pestis, we undertook small mammal trapping to identify the circulation of Y. pestis amongst rodents in this locality; blood samples were collected from rodents for seroprevalence analysis. Of the 60 individuals of Rattus rattus captured, one yielded an isolate of Y. pestis, 13 others were positive for F1 antigen of Y. pestis using a rapid diagnostic test, and 4 were PCR positive targeting the caf1 and pla genes; 28/60 (46.7%) of the captured R. rattus were seropositive for Y. pestis. Whole-genome SNP analyses revealed that the two isolates obtained from the human case, and the R. rattus belonged to two different subtypes of Y. pestis (s05 and s13, respectively) that were circulating concurrently in Ambohitromby in 2016. Three Y. pestis subtypes (s03, s05 and s13) have now been isolated from Ambohitromby. Subtype s05 had been persisting there for >10 years but one or both of the other subtypes may have been introduced from the Central Highlands region as they were not observed in previous years (s13) or only observed once previously (s03). High seroprevalence against Y. pestis in R. rattus suggests that a portion of the local murine population may have acquired resistance to Y. pestis. Future research should focus on genomically characterizing Y. pestis strains circulating in Ankazobe District and other plague-endemic regions of Madagascar to better understand the overall phylogeography of Y. pestis.

Transmission of Antimicrobial Resistant Yersinia pestis During a Pneumonic Plague Outbreak.

BACKGROUND: Pneumonic plague (PP), caused by Yersinia pestis, is the most feared clinical form of plague due to its rapid lethality and potential to cause outbreaks. PP outbreaks are now rare due to antimicrobial therapy. METHODS: A PP outbreak in Madagascar involving transmission of a Y. pestis strain resistant to streptomycin, the current recommended first-line treatment in Madagascar, was retrospectively characterized using epidemiology, clinical diagnostics, molecular characterization, and animal studies. RESULTS: The outbreak occurred in February 2013 in the Faratsiho district of Madagascar and involved 22 cases, including 3 untreated fatalities. The 19 other cases participated in funeral practices for the fatal cases and fully recovered after combination antimicrobial therapy: intramuscular streptomycin followed by oral co-trimoxazole. The Y. pestis strain that circulated during this outbreak is resistant to streptomycin resulting from a spontaneous point mutation in the 30S ribosomal protein S12 (rpsL) gene. This same mutation causes streptomycin resistance in 2 unrelated Y. pestis strains, one isolated from a fatal PP case in a different region of Madagascar in 1987 and another isolated from a fatal PP case in China in 1996, documenting this mutation has occurred independently at least 3 times in Y. pestis. Laboratory experiments revealed this mutation has no detectable impact on fitness or virulence, and revertants to wild-type are rare in other species containing it, suggesting Y. pestis strains containing it could persist in the environment. CONCLUSIONS: Unique antimicrobial resistant (AMR) strains of Y. pestis continue to arise in Madagascar and can be transmitted during PP outbreaks.