Isolation of camel single domain antibodies against Yersinia pestis V270 antigen based on a semi-synthetic single domain antibody library and development of a VHH-based lateral flow assay.

BACKGROUND: Antibodies have been proven effective as diagnostic agents for detecting zoonotic diseases. The variable domain of camel heavy chain antibody (VHH), as an antibody derivative, may be used as an alternative for traditional antibodies in existing immunodiagnostic reagents for detecting rapidly spreading infectious diseases. OBJECTIVES: To expedite the isolation of specific antibodies for diagnostic purposes, we constructed a semi-synthetic camel single domain antibody library based on the phage display technique platform (PDT) and verified the validity of this study. METHODS: The semi-synthetic single domain antibody sequences consist of two parts: one is the FR1-FR3 region amplified by RT-PCR from healthy camel peripheral blood lymphocytes (PBLs), and the other part is the CDR3-FR4 region synthesised as an oligonucleotide containing CDR3 randomised region. The two parts were fused by overlapping PCR, resulting in the rearranged variable domain of heavy-chain antibodies (VHHs). Y. pestis low-calcium response V protein (LcrV) is an optional biomarker to detect the Y. pestis infection. The semi-synthetic library herein was screened using recombinant (LcrV) as a target antigen. RESULTS: After four cycles of panning the library, four VHH binders targeting 1-270 aa residues of LcrV were isolated. The four VHH genes with unique sequences were recloned into an expression vector and expressed as VHH-hFc chimeric antibodies. The purified antibodies were identified and used to develop a lateral flow immunoassay (LFA) test strip using latex microspheres (LM) for the rapid and visual detection of Y. pestis infection. CONCLUSIONS: These data demonstrate the great potential of the semi-synthetic library for use in isolation of antigen-specific nanobodies and the isolated specific VHHs can be used in antigen-capture immunoassays.

Screening and identification of DNA nucleic acid aptamers against F1 protein of Yersinia pestis using SELEX method.

BACKGROUND: Yersinia pestis is a bacterium that causes the disease plague. It has caused the deaths of many people throughout history. The bacterium possesses several virulence factors (pPla, pFra, and PYV). PFra plasmid encodes fraction 1 (F1) capsular antigen. F1 protein protects the bacterium against host immune cells through phagocytosis process. This protein is specific for Y. pestis. Many diagnostic techniques are based on molecular and serological detection and quantification of F1 protein in different food and clinical samples. Aptamers are small nucleic acid sequences that can act as specific ligands for many targets.This study, aimed to isolate the high-affinity ssDNA aptamers against F1 protein. METHODS AND RESULTS: In this study, SELEX was used as the main strategy in screening aptamers. Moreover, enzyme-linked aptamer sorbent assay (ELASA) and surface plasmon resonance (SPR) were used to determine the affinity and specificity of obtained aptamers to F1 protein. The analysis showed that among the obtained aptamers, the three aptamers of Yer 21, Yer 24, and Yer 25 were selected with a KD value of 1.344E - 7, 2.004E - 8, and 1.68E - 8 M, respectively. The limit of detection (LoD) was found to be 0.05, 0.076, and 0.033 µg/ml for Yer 21, Yer 24, and Yer 25, respectively. CONCLUSION: This study demonstrated that the synthesized aptamers could serve as effective tools for detecting and analyzing the F1 protein, indicating their potential value in future diagnostic applications.

Development and evaluation of a multi-target droplet digital PCR assay for highly sensitive and specific detection of Yersinia pestis.

BACKGROUND: Plague, caused by the bacterium Yersinia pestis, is a zoonotic disease that poses considerable threats to human health. Nucleic acid tests are crucial for plague surveillance and the rapid detection of Y. pestis. However, inhibitors in complex samples such as soil and animal tissues often hamper nucleic acid detection, leading to a reduced rate of identifying low concentrations of Y. pestis. To address this challenge, we developed a sensitive and specific droplet digital polymerase chain reaction (ddPCR) assay for detecting Y. pestis DNA from soil and animal tissue samples. METHODS: Three genes (ypo2088, caf1, and pla) from Y. pestis were used to develop a multi-target ddPCR assay. The limits of detection (LoD), reproducibility, and specificity were assessed for bacterial genomic DNA samples. The ability of the assay to detect low concentrations of Y. pestis DNA from simulated soil and mouse liver tissue samples was respectively evaluated and compared with that of quantitative real-time PCR (qPCR). RESULTS: The results showed that the ddPCR LoDs ranged from 6.2 to 15.4 copies/reaction for the target genes, with good reproducibility and high specificity for Y. pestis. By testing 130 soil and mouse liver tissue samples spiked with Y. pestis, the ddPCR assay exhibited a better sensitivity than that of the qPCR assay used in the study, with LoDs of 102 colony forming units (CFU)/100 mg soil and 103 CFU/20 mg liver. Moreover, the assay presented good quantitative linearity (R2 = 0.99) for Y. pestis at 103-106 CFU/sample for soil and liver samples. CONCLUSION: The ddPCR assay presented good performance for detecting Y. pestis DNA from soil and mouse tissue samples, showing great potential for improving the detection rate of low concentrations of Y. pestis in plague surveillance and facilitating the early diagnosis of plague cases.

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

BACKGROUND: Plague is a zoonotic disease that, despite affecting humans for more than 5000 years, has historically been the subject of limited drug development activity. Drugs that are currently recommended in treatment guidelines have been approved based on animal studies alone-no pivotal clinical trials in humans have yet been completed. As a result of the sparse clinical research attention received, there are a number of methodological challenges that need to be addressed in order to facilitate the collection of clinical trial data that can meaningfully inform clinicians and policy-makers. One such challenge is the identification of clinically-relevant endpoints, which are informed by understanding the clinical characterisation of the disease-how it presents and evolves over time, and important patient outcomes, and how these can be modified by treatment. METHODOLOGY/PRINCIPAL FINDINGS: This systematic review aims to summarise the clinical profile of 1343 patients with bubonic plague described in 87 publications, identified by searching bibliographic databases for studies that meet pre-defined eligibility criteria. The majority of studies were individual case reports. A diverse group of signs and symptoms were reported at baseline and post-baseline timepoints-the most common of which was presence of a bubo, for which limited descriptive and longitudinal information was available. Death occurred in 15% of patients; although this varied from an average 10% in high-income countries to an average 17% in low- and middle-income countries. The median time to death was 1 day, ranging from 0 to 16 days. CONCLUSIONS/SIGNIFICANCE: This systematic review elucidates the restrictions that limited disease characterisation places on clinical trials for infectious diseases such as plague, which not only impacts the definition of trial endpoints but has the knock-on effect of challenging the interpretation of a trial's results. For this reason and despite interventional trials for plague having taken place, questions around optimal treatment for plague persist.

Engagement with Traditional Healers for Early Detection of Plague in Uganda.

In rural Uganda, many people who are ill consult traditional healers prior to visiting the formal healthcare system. Traditional healers provide supportive care for common illnesses, but their care may delay diagnosis and management of illnesses that can increase morbidity and mortality, hinder early detection of epidemic-prone diseases, and increase occupational risk to traditional healers. We conducted open-ended, semi-structured interviews with a convenience sample of 11 traditional healers in the plague-endemic West Nile region of northwestern Uganda to assess their knowledge, practices, and attitudes regarding plague and the local healthcare system. Most were generally knowledgeable about plague transmission and its clinical presentation and expressed willingness to refer patients to the formal healthcare system. We initiated a public health outreach program to further improve engagement between traditional healers and local health centers to foster trust in the formal healthcare system and improve early identification and referral of patients with plaguelike symptoms, which can reflect numerous other infectious and noninfectious conditions. During 2010-2019, 65 traditional healers were involved in the outreach program; 52 traditional healers referred 788 patients to area health centers. The diagnosis was available for 775 patients; malaria (37%) and respiratory tract infections (23%) were the most common diagnoses. One patient had confirmed bubonic plague. Outreach to improve communication and trust between traditional healers and local healthcare settings may result in improved early case detection and intervention not only for plague but also for other serious conditions.

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.

Preliminary investigation and intervention of the suspected plague outbreak in Madunga, Babati District-Tanzania.

BACKGROUND: Rodents are known to be reservoirs of plague bacteria, Yesinia pestis in the sylvatic cycle. A preliminary investigation of the suspected plague outbreak was conducted in Madunga Ward, Babati District Council in Manyara Region December-2019-January 2020 Following reported two cases which were clinically suspected as showing plague disease symptoms. METHOD: The commensal and field rodents were live trapped using Sherman traps in Madunga Ward, where plague suspect cases were reported and, in the Nou-forest reserve areas at Madunga Ward, Babati District Council, to assess plague risk in the area. Fleas were collected inside the houses using light traps and on the rodents 'body after anaesthetizing the captured rodent to determine flea indices which are used to estimate the risk of plague transmission. Lung impression smears were made from sacrificed rodents to examine for possible bipolar stained Yersinia spp bacilli. RESULTS: A total of 86 rodents consisting of ten rodent species were captured and identified from the study sites. Nine forest rodent species were collected. Field/fallow rodent species were dominated by Mastomys natalensis. whereas domestic rodent species captured was Rattus rattus. Overall lung impression smear showed bipolar stain were 14 (16.28%) while House Flea Index (HFI) was 3.1 and Rodent Flea Index (RFI) was 1.8. CONCLUSION: The findings of this study have shown that, the presence of bipolar stained bacilli in lung impression smears of captured species of rodents indicates (not confirmed) possible circulation of Yesrsinia pests in rodents and the high flea indices in the area which included the most common flea species known to be plague vectors in Tanzania could have played transmission role in this suspected outbreak. The study recommends surveillance follow-up in the area and subject collected samples to the standard plague confirmatory diagnosis.

Evaluation of a multi-species Protein A-ELISA assay for plague serologic diagnosis in humans and other mammal hosts.

BACKGROUND: The Hemagglutination assay (HA) is widely used in plague diagnosis, however, it has a subjective interpretation and demands high amounts of antigen and other immunobiological supplies. On the other hand, the conventional Anti-IgG ELISA is limited by the need of specific conjugates for multiple plague hosts, which leaves a gap for new diagnostic methods able to cover both the diagnosis of human cases and the epidemiological surveillance of multiple sentinel species. METHODS: We developed an ELISA Protein A-peroxidase method to detect anti-F1 antibodies across several species, including humans. To determine the cut-off and performance rates, HA results from 288 samples (81 rabbits, 64 humans, 66 rodents and 77 dogs) were used as reference. Next, we evaluated the agreement between Protein A-ELISA and Anti-IgG ELISA in an expanded sample set (n = 487). RESULTS: Optimal conditions were found with 250ng/well of F1 and 1:500 serum dilution. Protein A-ELISA showed high repeatability and reproducibility. We observed good correlation rates between the Protein A and IgG ELISAs optical densities and a higher positive/negative OD ratio for the Protein A-ELISA method. The overall sensitivity, specificity and area under the curve for Protein A-ELISA were 94%, 99% and 0.99, respectively. Similar results were observed for each species separately. In the analysis of the expanded sample set, there was a strong agreement between Protein A and IgG assays (kappa = 0.97). Furthermore, there was no cross-reaction with other common infectious diseases, such as dengue, Zika, Chagas disease, tuberculosis (humans) and ehrlichiosis, anaplasmosis and leishmaniasis (dogs). CONCLUSIONS: Altogether, the Protein A-ELISA showed high performance when compared both to HA and Anti-IgG ELISA, with a polyvalent single protocol that requires reduced amounts of antigen and can be employed to any plague hosts.

Plague in Disguise: The Discovery of Occult Buboes on Surgical Procedure or Autopsy.

Introduction: Bubonic plague classically manifests as a painful, swollen superficial lymph node (bubo) that is readily apparent on physical examination. However, patients occasionally present with buboes formed in deep lymph nodes, which are difficult to detect and can lead to delays in diagnosis and treatment. To better characterize this phenomenon, we conducted a review of the published literature to identify reports of occult buboes among patients with plague. Methods: Articles were identified from two sources: a systematic review on plague treatment, and a search of the PubMed Central database. Articles were eligible if they described a patient with plague who had (1) no evidence of lymphadenopathy on examination; and (2) at least one bubo discovered during surgery or autopsy. Results: Six patients with occult buboes were identified among 5120 articles screened. The majority were male (n = 4/6) and three were <15 years of age. Fever (n = 6/6), leukocytosis (n = 5/6), and abdominal pain or distention (n = 4/6) were the most common signs and symptoms. Initial diagnoses included other bacterial infections, appendicitis, or acute abdomen. Four patients received at least one antimicrobial effective against Yersinia pestis; however, some experienced delayed treatment due to late diagnosis of plague. Occult buboes were discovered in retroperitoneal (n = 2), inguinal/femoral (n = 2), mesenteric (n = 2), axillary (n = 1), and mediastinal (n = 1) regions. Four of the six patients died. Conclusions: Patients with occult buboes experienced delays in the diagnosis of plague and a high fatality rate. Clinicians in plague-endemic areas should consider the presence of occult buboes among patients with compatible symptoms and exposure history.

Performance assessment of a new indirect rapid diagnostic test for plague detection in humans and other mammalian hosts.

Plague is a flea-borne zoonosis that affects a wide range of mammals and still causes outbreaks in human populations yearly across several countries. While crucial for proper treatment, early diagnosis is still a major challenge in low- and middle-income countries due to poor access to laboratory infrastructure in rural areas. To tackle this issue, we developed and evaluated a new Fraction 1 capsular antigen (F1)-based rapid diagnostic test (RDT) as an alternative method for plague serological diagnosis and surveillance in humans and other mammals. In this study, 187 serum samples from humans, dogs, rodents and rabbits were retrospectively assessed using the plague RDT method. To calculate its performance, results were compared to those obtained by traditional hemagglutination (HA) and ELISA, which are well-established methods in the plague routine serodiagnosis. Remarkably, the results from RDT were in full agreement with those from the ELISA and HA assays, resulting in 100% (CI 95% = 95.5-100%) of sensitivity and 100% (CI 95% = 96.6-100%) of specificity. Accordingly, the Cohen's Kappa test coefficient was 1.0 (almost perfect agreement). Moreover, the RDT showed no cross-reaction when tested with sera from individuals positive to other pathogens, such as Y. pseudotuberculosis, Yersinia enterocolitica, Anaplasma platys, Ehrlichia canis and Leishmania infantum. Although preliminary, this study brings consistent proof-of-concept results with high performance of the Plague RDT when compared to HA and ELISA. Although further human and animal population-based studies will be necessary to validate these findings, the data presented here show that the plague RDT is highly sensitive and specific, polyvalent to several mammal species and simple to use in field surveillance or point-of-care situations with instant results.

Improved Selective BIN Agar for a Better Rate of Yersinia pestis Isolation from Primary Clinical Specimens in Suspected Madagascar Plague Cases.

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.

Critical Comparison between Large and Mini Vertical Flow Immunoassay Platforms for Yersinia Pestis Detection.

Yersinia pestis is a Gram-negative bacterium that is the causative agent of plague and is widely recognized as a potential biological weapon. Due to the high fatality rate of plague when diagnosis is delayed, the development of rapid, sensitive, specific, and cost-effective methods is needed for its diagnosis. The Y. pestis low calcium response V (LcrV) protein has been identified as a potential microbial biomarker for the diagnosis of plague. In this paper, we present a highly sensitive, paper-based, vertical flow immunoassay (VFI) prototype for the detection of LcrV and the diagnosis of plague. An antigen-capture assay using monoclonal antibodies is employed to capture and detect the LcrV protein, using a colorimetric approach. In addition, the effect of miniaturizing the VFI device is explored based on two different sizes of VFI platforms, denoted as "large VFI" and "mini VFI." Also, a comparative analysis is performed between the VFI platform and a lateral flow immunoassay (LFI) platform to exhibit the improved assay sensitivity suitable for point-of-care (POC) diagnostics. The analytical sensitivity or limit of detection (LOD) in the mini VFI is approximately 0.025 ng/mL, that is, 10 times better than that of the large VFI platform or 80 times over a standard lateral flow configuration. The low LOD of the LcrV VFI appears to be highly suitable for testing clinical samples and potentially diagnosing plague at earlier time points. In addition, optimization of the gold nanoparticle (AuNP) concentration, nanomaterial plasmonic properties, and flow velocity analysis could improve the performance of the VFI. Furthermore, we developed automated image analysis software that shows potential for integrating the diagnostic system into a smartphone. These methods and findings demonstrate that the VFI platform is a highly sensitive device for detecting the LcrV and potentially many other biomarkers.

Potential for community based surveillance of febrile diseases: Feasibility of self-administered rapid diagnostic tests in Iquitos, Peru and Phnom Penh, Cambodia.

Rapid diagnostic tests (RDTs) have the potential to identify infectious diseases quickly, minimize disease transmission, and could complement and improve surveillance and control of infectious and vector-borne diseases during outbreaks. The U.S. Defense Threat Reduction Agency's Joint Science and Technology Office (DTRA-JSTO) program set out to develop novel point-of-need RDTs for infectious diseases and deploy them for home use with no training. The aim of this formative study was to address two questions: 1) could community members in Iquitos, Peru and Phnom Penh, Cambodia competently use RDTs of different levels of complexity at home with visually based instructions provided, and 2) if an RDT were provided at no cost, would it be used at home if family members displayed febrile symptoms? Test kits with written and video (Peru only) instructions were provided to community members (Peru [n = 202]; Cambodia [n = 50]) or community health workers (Cambodia [n = 45]), and trained observers evaluated the competency level for each of the several steps required to successfully operate one of two multiplex RDTs on themselves or other consenting participant (i.e., family member). In Iquitos, >80% of residents were able to perform 11/12 steps and 7/15 steps for the two- and five-pathogen test, respectively. Competency in Phnom Penh never reached 80% for any of the 12 or 15 steps for either test; the percentage of participants able to perform a step ranged from 26-76% and 23-72%, for the two- and five-pathogen tests, respectively. Commercially available NS1 dengue rapid tests were distributed, at no cost, to households with confirmed exposure to dengue or Zika virus; of 14 febrile cases reported, six used the provided RDT. Our findings support the need for further implementation research on the appropriate level of instructions or training needed for diverse devices in different settings, as well as how to best integrate RDTs into existing local public health and disease surveillance programs at a large scale.

Pulse-Controlled Amplification-A new powerful tool for on-site diagnostics under resource limited conditions.

BACKGROUND: Molecular diagnostics has become essential in the identification of many infectious and neglected diseases, and the detection of nucleic acids often serves as the gold standard technique for most infectious agents. However, established techniques like polymerase chain reaction (PCR) are time-consuming laboratory-bound techniques while rapid tests such as Lateral Flow Immunochromatographic tests often lack the required sensitivity and/or specificity. METHODS/PRINCIPLE FINDINGS: Here we present an affordable, highly mobile alternative method for the rapid identification of infectious agents using pulse-controlled amplification (PCA). PCA is a next generation nucleic acid amplification technology that uses rapid energy pulses to heat microcyclers (micro-scale metal heating elements embedded directly in the amplification reaction) for a few microseconds, thus only heating a small fraction of the reaction volume. The heated microcyclers cool off nearly instantaneously, resulting in ultra-fast heating and cooling cycles during which classic amplification of a target sequence takes place. This reduces the overall amplification time by a factor of up to 10, enabling a sample-to-result workflow in just 15 minutes, while running on a small and portable prototype device. In this proof of principle study, we designed a PCA-assay for the detection of Yersinia pestis to demonstrate the efficacy of this technology. The observed detection limits were 434 copies per reaction (purified DNA) and 35 cells per reaction (crude sample) respectively of Yersinia pestis. CONCLUSIONS/SIGNIFICANCE: PCA offers fast and decentralized molecular diagnostics and is applicable whenever rapid, on-site detection of infectious agents is needed, even under resource limited conditions. It combines the sensitivity and specificity of PCR with the rapidness and simplicity of hitherto existing rapid tests.

WHO guidelines for plague management: revised recommendations for the use of rapid diagnostic tests, fluoroquinolones for case management and personal protective equipment for prevention of post-mortem transmission

Plague has killed millions of people during the past 25 centuries, and the disease reappeared in several countries during the 1990s. Consequently, plague was categorized as a re-emerging disease. Human plague outbreaks continue to be reported, including an outbreak of pneumonic plague in Madagascar in 2017. Plague is an acute bacterial infection caused by Yersinia pestis. Although effective antimicrobials are available, plague still has high mortality because most outbreaks take place in remote places, where proper diagnosis and treatment remain challenging. Early identification of the disease is crucial to ensure prompt treatment and better outcomes. Pneumonic plague is highly contagious and of particular concern because of the high risk of triggering epidemics. Thus, plague is both a medical and a public health emergency. These guidelines were developed in accordance with the WHO handbook for guideline development. A WHO Steering Group, led by the responsible technical officer, developed the draft scope of the guidelines and the key questions to be addressed. The Steering Group selected the members of the Guideline Development Group (GDG) to ensure diverse areas of expertise were represented, including clinicians, microbiologists, public health professionals, researchers and an anthropologist. The Steering Group also commissioned technical advisers to lead the Evidence Review Team and provide methodological support. The GDG assisted with developing the final scope of the guideline and defining the key areas to be addressed, and also formulated the recommendations. Three key areas were selected to be addressed: (i) the use of rapid diagnostic tests (RDTs) for diagnosing plague in different contexts; (ii) the choice of antimicrobials for treating the different forms of plague, including whether fluoroquinolones should be introduced as a first-line medicine of choice; and (iii) the use of personal protective equipment in case of exposure to the dead body of a person who was infected with plague. The Evidence Review Team conducted systematic reviews to address each of the three key areas. At a meeting in Antananarivo, Madagascar, on 20­21 September 2019, the GDG interpreted the main findings of the systematic reviews as they applied to each key question and formulated evidence-based recommendations following the GRADE (Grading of Recommendations Assessment, Development and Evaluation) approach. For each key question, there was discussion about the certainty of the evidence, desirable and undesirable effects, values and preferences, cost, acceptability, equity, feasibility and barriers to implementation. The GRADE evidence-to-decision tables were used to facilitate consensus and record the decision of the GDG. The GDG developed final recommendations where possible and graded each of them as strong or conditional. The final guidelines were written by the Evidence Review Team.

Development and evaluation of loop-mediated isothermal amplification for detection of Yersinia pestis in plague biological samples.

BACKGROUND: Several tests are available for plague confirmation but bacteriological culture with Yersinia pestis strain isolation remains the gold standard according to the World Health Organization. However, this is a time consuming procedure; requiring specific devices and well-qualified staff. In addition, strain isolation is challenging if antibiotics have been administered prior to sampling. Here, we developed a loop-mediated isothermal amplification (LAMP) technique, a rapid, simple, sensitive and specific technique that would be able to detect Y. pestis in human biological samples. METHODS: LAMP primers were designed to target the caf1 gene which is specific to Y. pestis. The detection limit was determined by testing 10-fold serial dilution of Y. pestis DNA. Cross-reactivity was tested using DNA extracts from 14 pathogens and 47 residual samples from patients suffering from non-plague diseases. Specificity and sensitivity of the LAMP caf1 were assessed on DNA extracts of 160 human biological samples. Then, the performance of the LAMP caf1 assay was compared to conventional PCR and bacteriological culture. RESULTS: The detection limit of the developed Y. pestis LAMP assay was 3.79 pg/µl, similar to conventional PCR. The result could be read out within 45 min and as early as 35 minutes in presence of loop primer, using a simple water bath at 63°C. This is superior to culture with respect to time (requires up to 10 days) and simplicity of equipment compared to PCR. Furthermore, no cross-reactivity was found when tested on DNA extracts from other pathogens and human biological samples from patients with non-plague diseases. Compared to the gold standard, LAMP sensitivity and specificity were 97.9% (95% CI: 89.1%-99.9%) and 94.6% (95% CI: 88.6%-97.9%), respectively. CONCLUSION: LAMP detected Y. pestis effectively with high sensitivity and specificity in human plague biological samples. It can potentially be used in the field during outbreaks in resource limited countries such as Madagascar.

An invasive disease, sylvatic plague, increases fragmentation of black-tailed prairie dog (Cynomys ludovicianus) colonies.

CONTEXT: A disease can be a source of disturbance, causing population declines or extirpations, altering species interactions, and affecting habitat structure. This is particularly relevant for diseases that affect keystone species or ecosystem engineers, leading to potentially cascading effects on ecosystems. OBJECTIVE: We investigated the invasion of a non-native disease, plague, to a keystone species, prairie dogs, and documented the resulting extent of fragmentation and habitat loss in western grasslands. Specifically, we assessed how the arrival of plague in the Conata Basin, South Dakota, United States, affected the size, shape, and aggregation of prairie dog colonies, an animal species known to be highly susceptible to plague. METHODS: Colonies in the prairie dog complex were mapped every 1 to 3 years from 1993 to 2015. Plague was first confirmed in 2008 and we compared prairie dog complex and colony characteristics before and after the arrival of plague. RESULTS: As expected the colony complex and the patches in colonies became smaller and more fragmented after the arrival of plague; the total area of each colony and the mean area per patch within a colony decreased, the number of patches per colony increased, and mean contiguity of each patch decreased, leading to habitat fragmentation. CONCLUSION: We demonstrate how an emerging infectious disease can act as a source of disturbance to natural systems and lead to potentially permanent alteration of habitat characteristics. While perhaps not traditionally thought of as a source of ecosystem disturbances, in recent years emerging infectious diseases have shown to be able to have large effects on ecosystems if they affect keystone species.