Low cost, low tech SNP genotyping tools for resource-limited areas: Plague in Madagascar as a model

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

Rodent control to fight plague : field assessment of methods based on rat density reduction

Research funding: Directorate General for International Relations and Strategy. Grant Number: 2018‐SB‐024‐18SSEOC049‐PMG7‐SSA5‐IPMMADAGASCAR ACKNOWLEDGMENTS: We are especially grateful to the health authorities and the population in Miantso and Ankazobe for allowing us to do this work and being so helpful. We thank the staff of the Plague Unit, Institut Pasteur de Madagascar, for helping with the field and laboratory work, especially Alain Berthin Rakotoarisoa and Andrianiaina Parfait Rakotonindrainy. This work was supported by a Directorate General for International Relations and Strategy grant (2018‐SB‐024‐18SSEOC049‐PMG7‐SSA5‐IPMMADAGASCAR) covering the project “Développement de contre‐mesures médicales à la peste à Madagascar” with scientific support of IRBA (French Armed Forces Biomedical Research Institute), within the framework of French MoD's involvement in G7 Global partnership. The French Agency for International Technical Expertise (AFETI) ensures the proper financial execution of the project and contributes to the implementation of cooperation actions under the control of the Directorate General for International Relations and Strategy. This research was also funded in part by the Wellcome Trust [095171/Z/10/Z] and the Institut Pasteur de Madagascar. For the purpose of Open Access, the authors have applied a CC BY public copyright license to any Author Accepted Manuscript version arising from this submission. K.S. was supported by the Biotechnology and Biological Sciences Research Council (BBSRC) under the EastBio DTP (grant number BB/M010996/1).Peer reviewedPublisher PD

Temporal phylogeography of Yersinia pestis in Madagascar : Insights into the long-term maintenance of plague

Data Availability: All relevant data are within the paper and its Supporting Information files except for the sequence read archives for 31 newly sequenced strains that are available at NCBI under the accession numbers: SRR4175414-SRR4175444. The direct link to this data is: https://www.ncbi.nlm.nih.gov/sra/?term=SRP086709. Funding: Funding for this study was provided by the US Department of Homeland Security’s Science and Technology Directorate award number HSHQDC-10-C-00139 to PK; the Cowden Endowment at Northern Arizona University; and Wellcome fellowships 081705 and 095171 to ST. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.Peer reviewedPublisher PD

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

Preparation and characterization of antibacterial cobalt-exchanged natural zeolite/poly(vinyl alcohol) hydrogels

In the present study, potential application of the local clinoptilolite-rich natural zeolite in formulation of antibacterial hydrogels was investigated. The zeolite powder exchanged with cobalt(II) ions was used in preparation of the zeolite/poly(vinyl alcohol) hydrogel films in different amounts. The films were physically crosslinked by the freezing-thawing method and characterized for their crystallinity, surface and cross sectional morphology, chemical composition, thermal behaviour, mechanical properties, swelling and dissolution behaviours, and antibacterial activities against a Gram-negative bacteria. The films with 0.48 wt% and higher cobalt-exchanged zeolite contents showed antibacterial activity. Addition of the zeolite powder in the formulations did not cause significant changes in the other properties of the films.Turkish Republic Prime Ministry State Planning Organization (DPT-2006 K120690

The impact of COVID-19 on clinical research for Neglected Tropical Diseases (NTDs): A case study of bubonic plague.

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

Development and Evaluation of Two Simple, Rapid Immunochromatographic Tests for the Detection of Yersinia pestis Antibodies in Humans and Reservoirs

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

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

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

Immune Responses to Plague Infection in Wild Rattus rattus, in Madagascar: A Role in Foci Persistence?

Plague is endemic within the central highlands of Madagascar, where its main reservoir is the black rat, Rattus rattus. Typically this species is considered susceptible to plague, rapidly dying after infection inducing the spread of infected fleas and, therefore, dissemination of the disease to humans. However, persistence of transmission foci in the same area from year to year, supposes mechanisms of maintenance among which rat immune responses could play a major role. Immunity against plague and subsequent rat survival could play an important role in the stabilization of the foci. In this study, we aimed to investigate serological responses to plague in wild black rats from endemic areas of Madagascar. In addition, we evaluate the use of a recently developed rapid serological diagnostic test to investigate the immune response of potential reservoir hosts in plague foci.We experimentally infected wild rats with Yersinia pestis to investigate short and long-term antibody responses. Anti-F1 IgM and IgG were detected to evaluate this antibody response. High levels of anti-F1 IgM and IgG were found in rats one and three weeks respectively after challenge, with responses greatly differing between villages. Plateau in anti-F1 IgM and IgG responses were reached for as few as 500 and 1500 colony forming units (cfu) inoculated respectively. More than 10% of rats were able to maintain anti-F1 responses for more than one year. This anti-F1 response was conveniently followed using dipsticks.Inoculation of very few bacteria is sufficient to induce high immune response in wild rats, allowing their survival after infection. A great heterogeneity of rat immune responses was found within and between villages which could heavily impact on plague epidemiology. In addition, results indicate that, in the field, anti-F1 dipsticks are efficient to investigate plague outbreaks several months after transmission

Human plague: An old scourge that needs new answers

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