De Novo Genome Assembly of Auanema Melissensis, a Trioecious Free-Living Nematode.

Nematodes of the genus Auanema are interesting models for studying sex determination mechanisms because their populations consist of three sexual morphs (males, females, and hermaphrodites) and produce skewed sex ratios. Here, we introduce a new undescribed species of this genus, Auanema melissensis n. sp., together with its draft nuclear genome. This species is also trioecious and does not cross with the other described species A. rhodensis or A. freiburgensis. Similar to A. freiburgensis, A. melissensis' maternal environment influences the hermaphrodite versus female sex determination of the offspring. The genome of A. melissensis is ~60 Mb, containing 11,040 protein-coding genes and 8.07% of repeat sequences. Using the estimated ancestral chromosomal gene content (Nigon elements), it was possible to identify putative X chromosome scaffolds.

Assessment of public health laboratory preparedness and response in WHO South-East Asia region during the COVID-19 pandemic: lessons learned and future directions.

This Health Policy reviews the preparedness and response of public health laboratories in the WHO South-East Asia Region (SEAR) during the COVID-19 pandemic. Through a scoping review and in-depth interviews with key stakeholders, the study identifies successes, challenges, and lessons learned from available literature and the perspective of senior laboratory leaders. Key themes include human resources, health information systems, diagnostic capacity, public risk communication, biosafety, biosecurity, funding, and laboratory network coordination. The findings provide a comprehensive overview of the adaptive capacities of laboratories, the contextual factors influencing their response, and the implications for future pandemic preparedness. This study demonstrates the resilience and adaptability of diagnostic networks in the face of a pandemic but also emphasises the need for strategic resource allocation, highlighting the importance of flexible and scalable networks in managing public health crises. The success of these deployments highlights the necessity for continual investment and coordination of national, regional, and global resources in diagnostic infrastructure to improve preparedness for future public health crises.

Diagnostic accuracy of DPP Fever Panel II Asia tests for tropical fever diagnosis.

BACKGROUND: Fever is the most frequent symptom in patients seeking care in South and Southeast Asia. The introduction of rapid diagnostic tests (RDTs) for malaria continues to drive patient management and care. Malaria-negative cases are commonly treated with antibiotics without confirmation of bacteraemia. Conventional laboratory tests for differential diagnosis require skilled staff and appropriate access to healthcare facilities. In addition, introducing single-disease RDTs instead of conventional laboratory tests remains costly. To overcome some of the delivery challenges of multiple separate tests, a multiplexed RDT with the capacity to diagnose a diverse range of tropical fevers would be a cost-effective solution. In this study, a multiplex lateral flow immunoassay (DPP Fever Panel II Assay) that can detect serum immunoglobulin M (IgM) and specific microbial antigens of common fever agents in Asia (Orientia tsutsugamushi, Rickettsia typhi, Leptospira spp., Burkholderia pseudomallei, Dengue virus, Chikungunya virus, and Zika virus), was evaluated. METHODOLOGY/PRINCIPAL FINDINGS: Whole blood (WB) and serum samples from 300 patients with undefined febrile illness (UFI) recruited in Vientiane, Laos PDR were tested using the DPP Fever Panel II, which consists of an Antibody panel and Antigen panel. To compare reader performance, results were recorded using two DPP readers, DPP Micro Reader (Micro Reader 1) and DPP Micro Reader Next Generation (Micro Reader 2). WB and serum samples were run on the same fever panel and read on both micro readers in order to compare results. ROC analysis and equal variance analysis were performed to inform the diagnostic validity of the test compared against the respective reference standards of each fever agent (S1 Table). Overall better AUC values were observed in whole blood results. No significant difference in AUC performance was observed when comparing whole blood and serum sample testing, except for when testing for R. typhi IgM (p = 0.04), Leptospira IgM (p = 0.02), and Dengue IgG (p = 0.03). Linear regression depicted R2 values had ~70% agreement across WB and serum samples, except when testing for leptospirosis and Zika, where the R2 values were 0.37 and 0.47, respectively. No significant difference was observed between the performance of Micro Reader 1 and Micro Reader 2, except when testing for the following pathogens: Zika IgM, Zika IgG, and B pseudomallei CPS Ag. CONCLUSIONS/SIGNIFICANCE: These results demonstrate that the diagnostic accuracy of the DPP Fever Panel II is comparable to that of commonly used RDTs. The optimal cut-off would depend on the use of the test and the desired sensitivity and specificity. Further studies are required to authenticate the use of these cut-offs in other endemic regions. This multiplex RDT offers diagnostic benefits in areas with limited access to healthcare and has the potential to improve field testing capacities. This could improve tropical fever management and reduce the public health burden in endemic low-resource areas.

Laboratory-acquired infections and pathogen escapes worldwide between 2000 and 2021: a scoping review.

Laboratory-acquired infections (LAIs) and accidental pathogen escape from laboratory settings (APELS) are major concerns for the community. A risk-based approach for pathogen research management within a standard biosafety management framework is recommended but is challenging due to reasons such as inconsistency in risk tolerance and perception. Here, we performed a scoping review using publicly available, peer-reviewed journal and media reports of LAIs and instances of APELS between 2000 and 2021. We identified LAIs in 309 individuals in 94 reports for 51 pathogens. Eight fatalities (2·6% of all LAIs) were caused by infection with Neisseria meningitidis (n=3, 37·5%), Yersinia pestis (n=2, 25%), Salmonella enterica serotype Typhimurium (S Typhimurium; n=1, 12·5%), or Ebola virus (n=1, 12·5%) or were due to bovine spongiform encephalopathy (n=1, 12·5%). The top five LAI pathogens were S Typhimurium (n=154, 49·8%), Salmonella enteritidis (n=21, 6·8%), vaccinia virus (n=13, 4·2%), Brucella spp (n=12, 3·9%), and Brucella melitensis (n=11, 3·6%). 16 APELS were reported, including those for Bacillus anthracis, SARS-CoV, and poliovirus (n=3 each, 18·8%); Brucella spp and foot and mouth disease virus (n=2 each, 12·5%); and variola virus, Burkholderia pseudomallei, and influenza virus H5N1 (n=1 each, 6·3%). Continual improvement in LAI and APELS management via their root cause analysis and thorough investigation of such incidents is essential to prevent future occurrences. The results are biased due to the reliance on publicly available information, which emphasises the need for formalised global LAIs and APELS reporting to better understand the frequency of and circumstances surrounding these incidents.

The Biosafety Research Road Map: The Search for Evidence to Support Practices in the Laboratory-SARS-CoV-2.

Introduction: The SARS-CoV-2 virus emerged as a novel virus and is the causative agent of the COVID-19 pandemic. It spreads readily human-to-human through droplets and aerosols. The Biosafety Research Roadmap aims to support the application of laboratory biological risk management by providing an evidence base for biosafety measures. This involves assessing the current biorisk management evidence base, identifying research and capability gaps, and providing recommendations on how an evidence-based approach can support biosafety and biosecurity, including in low-resource settings. Methods: A literature search was conducted to identify potential gaps in biosafety and focused on five main sections, including the route of inoculation/modes of transmission, infectious dose, laboratory-acquired infections, containment releases, and disinfection and decontamination strategies. Results: There are many knowledge gaps related to biosafety and biosecurity due to the SARS-CoV-2 virus's novelty, including infectious dose between variants, personal protective equipment for personnel handling samples while performing rapid diagnostic tests, and laboratory-acquired infections. Detecting vulnerabilities in the biorisk assessment for each agent is essential to contribute to the improvement and development of laboratory biosafety in local and national systems.

The Biosafety Research Road Map: The Search for Evidence to Support Practices in the Laboratory-Foot and Mouth Disease Virus.

Introduction: Foot and mouth disease (FMD) is a highly contagious infection of cloven-hoofed animals. The Biosafety Research Road Map reviewed scientific literature regarding the foot and mouth disease virus (FMDV). This project aims to identify gaps in the data required to conduct evidence-based biorisk assessments, as described by Blacksell et al., and strengthen control measures appropriate for local and national laboratories. Methods: A literature search was conducted to identify potential gaps in biosafety and focused on five main sections: the route of inoculation/modes of transmission, infectious dose, laboratory-acquired infections, containment releases, and disinfection and decontamination strategies. Results: The available data regarding biosafety knowledge gaps and existing evidence have been collated. Some gaps include the need for more scientific data that identify the specific safety contribution of engineering controls, support requirements for showering out after in vitro laboratory work, and whether a 3- to 5-day quarantine period should be applied to individuals conducting in vitro versus in vivo work. Addressing these gaps will contribute to the remediation and improvement of biosafety and biosecurity systems when working with FMDV.

The Biosafety Research Road Map: The Search for Evidence to Support Practices in the Laboratory-Crimean Congo Haemorrhagic Fever Virus and Lassa Virus.

Introduction: Crimean Congo Hemorrhagic Fever (CCHF) virus and Lassa virus (LASV) are zoonotic agents regarded as high-consequence pathogens due to their high case fatality rates. CCHF virus is a vector-borne disease and is transmitted by tick bites. Lassa virus is spread via aerosolization of dried rat urine, ingesting infected rats, and direct contact with or consuming food and water contaminated with rat excreta. Methods: The scientific literature for biosafety practices has been reviewed for both these two agents to assess the evidence base and biosafety-related knowledge gaps. The review focused on five main areas, including the route of inoculation/modes of transmission, infectious dose, laboratory-acquired infections, containment releases, and disinfection and decontamination strategies. Results: There is a lack of data on the safe collection and handling procedures for tick specimens and the infectious dose from an infective tick bite for CCHF investigations. In addition, there are gaps in knowledge about gastrointestinal and contact infectious doses for Lassa virus, sample handling and transport procedures outside of infectious disease areas, and the contribution of asymptomatic carriers in viral circulation. Conclusion: Due to the additional laboratory hazards posed by these two agents, the authors recommend developing protocols that work effectively and safely in highly specialized laboratories in non-endemic regions and a laboratory with limited resources in endemic areas.

The Biosafety Research Road Map: The Search for Evidence to Support Practices in the Laboratory-Zoonotic Avian Influenza and Mycobacterium tuberculosis.

Introduction: The Biosafety Research Road Map reviewed the scientific literature on a viral respiratory pathogen, avian influenza virus, and a bacterial respiratory pathogen, Mycobacterium tuberculosis. This project aims at identifying gaps in the data required to conduct evidence-based biorisk assessments, as described in Blacksell et al. One significant gap is the need for definitive data on M. tuberculosis sample aerosolization to guide the selection of engineering controls for diagnostic procedures. Methods: The literature search focused on five areas: routes of inoculation/modes of transmission, infectious dose, laboratory-acquired infections, containment releases, and disinfection and decontamination methods. Results: The available data regarding biosafety knowledge gaps and existing evidence have been collated and presented in Tables 1 and 2. The guidance sources on the appropriate use of biosafety cabinets for specific procedures with M. tuberculosis require clarification. Detecting vulnerabilities in the biorisk assessment for respiratory pathogens is essential to improve and develop laboratory biosafety in local and national systems.

The Biosafety Research Road Map: The Search for Evidence to Support Practices in the Laboratory-Mpox/Monkeypox Virus.

Introduction: The virus formerly known as monkeypox virus, now called mpoxv, belongs to the Orthopoxvirus genus and can cause mpox disease through both animal-to-human and human-to-human transmission. The unexpected spread of mpoxv among humans has prompted the World Health Organization (WHO) to declare a Public Health Emergency of International Concern (PHEIC). Methods: We conducted a literature search to identify the gaps in biosafety, focusing on five main areas: how the infection enters the body and spreads, how much of the virus is needed to cause infection, infections acquired in the lab, accidental release of the virus, and strategies for disinfecting and decontaminating the area. Discussion: The recent PHEIC has shown that there are gaps in our knowledge of biosafety when it comes to mpoxv. We need to better understand where this virus might be found, how much of it can spread from person-to-person, what are the effective control measures, and how to safely clean up contaminated areas. By gathering more biosafety evidence, we can make better decisions to protect people from this zoonotic agent, which has recently become more common in the human population.

The Biosafety Research Road Map: The Search for Evidence to Support Practices in Human and Veterinary Laboratories.

Introduction: Lack of evidence-based information regarding potential biological risks can result in inappropriate or excessive biosafety and biosecurity risk-reduction strategies. This can cause unnecessary damage and loss to the physical facilities, physical and psychological well-being of laboratory staff, and community trust. A technical working group from the World Organization for Animal Health (WOAH, formerly OIE), World Health Organization (WHO), and Chatham House collaborated on the Biosafety Research Roadmap (BRM) project. The goal of the BRM is the sustainable implementation of evidence-based biorisk management of laboratory activities, particularly in low-resource settings, and the identification of gaps in the current biosafety and biosecurity knowledge base. Methods: A literature search was conducted for the basis of laboratory design and practices for four selected high-priority subgroups of pathogenic agents. Potential gaps in biosafety were focused on five main sections, including the route of inoculation/modes of transmission, infectious dose, laboratory-acquired infections, containment releases, and disinfection and decontamination strategies. Categories representing miscellaneous, respiratory, bioterrorism/zoonotic, and viral hemorrhagic fever pathogens were created within each group were selected for review. Results: Information sheets on the pathogens were developed. Critical gaps in the evidence base for safe sustainable biorisk management were identified. Conclusion: The gap analysis identified areas of applied biosafety research required to support the safety, and the sustainability, of global research programs. Improving the data available for biorisk management decisions for research with high-priority pathogens will contribute significantly to the improvement and development of appropriate and necessary biosafety, biocontainment and biosecurity strategies for each agent.

The Biosafety Research Road Map: The Search for Evidence to Support Practices in the Laboratory-Bacillus anthracis and Brucella melitensis.

Introduction: Brucella melitensis and Bacillus anthracis are zoonoses transmitted from animals and animal products. Scientific information is provided in this article to support biosafety precautions necessary to protect laboratory workers and individuals who are potentially exposed to these pathogens in the workplace or other settings, and gaps in information are also reported. There is a lack of information on the appropriate effective concentration for many chemical disinfectants for this agent. Controversies related to B. anthracis include infectious dose for skin and gastrointestinal infections, proper use of personal protective equipment (PPE) during the slaughter of infected animals, and handling of contaminated materials. B. melitensis is reported to have the highest number of laboratory-acquired infections (LAIs) to date in laboratory workers. Methods: A literature search was conducted to identify potential gaps in biosafety and focused on five main sections including the route of inoculation/modes of transmission, infectious dose, LAIs, containment releases, and disinfection and decontamination strategies. Results: Scientific literature currently lacks information on the effective concentration of many chemical disinfectants for this agent and in the variety of matrices where it may be found. Controversies related to B. anthracis include infectious dose for skin and gastrointestinal infections, proper use of PPE during the slaughter of infected animals, and handling contaminated materials. Discussion: Clarified vulnerabilities based on specific scientific evidence will contribute to the prevention of unwanted and unpredictable infections, improving the biosafety processes and procedures for laboratory staff and other professionals such as veterinarians, individuals associated with the agricultural industry, and those working with susceptible wildlife species.

The Biosafety Research Road Map: The Search for Evidence to Support Practices in the Laboratory-Shigella spp.

Introduction: Shigella bacteria cause shigellosis, a gastrointestinal infection most often acquired from contaminated food or water. Methods: In this review, the general characteristics of Shigella bacteria are described, cases of laboratory-acquired infections (LAIs) are discussed, and evidence gaps in current biosafety practices are identified. Results: LAIs are undoubtedly under-reported. Owing to the low infectious dose, rigorous biosafety level 2 practices are required to prevent LAIs resulting from sample manipulation or contact with infected surfaces. Conclusions: It is recommended that, before laboratory work with Shigella, an evidence-based risk assessment be conducted. Particular emphasis should be placed on personal protective equipment, handwashing, and containment practices for procedures that generate aerosols or droplets.

Evaluation of the Panbio Leptospira IgM ELISA among Outpatients Attending Primary Care in Southeast Asia.

Despite estimates suggesting Leptospira spp. being endemic in Southeast Asia, evidence remains limited. Diagnostic accuracy evaluations based on Leptospira ELISA mainly rely on hospitalized and severe patients; therefore, studies measuring the pathogen burden may be inaccurate in the community. We evaluated the Panbio Leptospira ELISA IgM among 656 febrile outpatients attending primary care in Chiangrai, Thailand, and Hlaing Tha Yar, Yangon, Myanmar. ELISA demonstrated limited diagnostic accuracy for the detection of acute leptospiral infection using the manufacturer recommended cutoff, with a sensitivity of 71.4% and specificity of 36.4%, and an area under the receiver operator characteristic curve value of 0.65 (95% CI: 0.41-0.89), compared with our reference test, the PCR assay. ELISA also performed poorly as a screening tool for detecting recent exposure to Leptospira spp. compared with the "gold-standard" microscopic agglutination test, with a specificity of 42.7%. We conclude that the utility of the Leptospira IgM ELISA for both serodiagnosis and seroprevalence is limited in our setting.

Selection of Diagnostic Cutoffs for Murine Typhus IgM and IgG Immunofluorescence Assay: A Systematic Review.

Murine typhus is a neglected but widespread infectious disease that results in acute fever. The immunofluorescence assay (IFA) is the "gold standard" to identify IgM or IgG antibodies, although there is a lack of standardization in methodologies. The objective of this review is to summarize 1) the differences in published methodologies, 2) the diagnostic cutoff titers, and 3) the justification of diagnostic cutoffs. Searches were performed by combining the following search terms: "murine typhus," "rickettsia typhi," "immunofluorescence," "IFA," and "serologic" with restrictions (i.e., "rickettsia typhi" or "murine typhus," and "IFA" or "immunofluorescence," or "serologic*"). The search identified 78 studies that used IFA or immunoperoxidase assay (IIP) antibody cutoffs to diagnose murine typhus, 39 of which were case series. Overall, 45 studies (57.7%) provided little to no rationale as to how the cutoff was derived. Variation was seen locally in the cutoff titers used, but a 4-fold or greater increase was often applied. The cutoffs varied depending on the antibody target. No consensus was observed in establishing a cutoff, or for a single-value diagnostic cutoff. In conclusion, there is a lack of consensus in the establishment of a single-value cutoff. Further studies will need to be executed at each distinct geographic location to identify region-specific cutoffs, while also considering background antibody levels to distinguish between healthy and infected patients.

Diagnostic accuracy of an in-house Scrub Typhus enzyme linked immunoassay for the detection of IgM and IgG antibodies in Laos.

Scrub typhus is a major cause of morbidity and mortality in Southeast Asia. Diagnosis of scrub typhus is difficult due to a lack of accessible validated diagnostic tools. Despite its objectivity, the diagnostic accuracy of ELISA tests is influenced by methodological and patient factors. This study aims to evaluate the performance of a novel in-house ELISA developed in the Mahidol Oxford Tropical Medicine Research Unit (MORU) for anti-scrub typhus group IgM and IgG compared to the "gold standard" reference IFA and PCR, and to determine whether the in-house ELISA can be used as a seroepidemiological screening tool and/or stand-alone test for scrub typhus. A total of 1,976 admission and 1,438 participant follow-up sera collected in the Lao PDR (Laos) were tested with ELISA for IgM and IgG. Samples with an ELISA OD≥0.50 were tested with IFA for IgM and/or IgG. A strong positive relationship was present between ELISA ODs and IFA titers for admission IgM (r2: 0.70, p <0.005) and IgG (r2: 0.76, p<0.005), and for follow-up IgM and IgG (both r2: 0.76, p<0.005) samples. The best compromise between sensitivity and specificity for the ELISA OD cut-off is likely to be between 0.8-1.0 for IgM antibodies and 1.2-1.8 for IgG antibodies. These results demonstrate that the diagnostic accuracy of the MORU in-house scrub typhus group ELISA is comparable to that of IFA, with similar results as reported for the commonly used InBios Scrub Typhus Detect ELISA, validating the use of the in-house ELISA. The optimal ELISA cut-off would depend on the use of the test, and the desired sensitivity and specificity. Further studies are required to authenticate the use of these cut-offs in other endemic regions. This in-house ELISA has the potential to replace the imperfect IFA, which could ultimately reduce the burden of scrub typhus by improving the rate of scrub typhus diagnoses in endemic low-resource areas.