Cost-effectiveness analysis of interventions to improve diagnosis and preventive therapy for paediatric tuberculosis in 9 sub-Saharan African countries: A modelling study.

BACKGROUND: Over 1 million children aged 0 to 14 years were estimated to develop tuberculosis in 2021, resulting in over 200,000 deaths. Practical interventions are urgently needed to improve diagnosis and antituberculosis treatment (ATT) initiation in children aged 0 to 14 years and to increase coverage of tuberculosis preventive therapy (TPT) in children at high risk of developing tuberculosis disease. The multicountry CaP-TB intervention scaled up facility-based intensified case finding and strengthened household contact management and TPT provision at HIV clinics. To add to the limited health-economic evidence on interventions to improve ATT and TPT in children, we evaluated the cost-effectiveness of the CaP-TB intervention. METHODS AND FINDINGS: We analysed clinic-level pre/post data to quantify the impact of the CaP-TB intervention on ATT and TPT initiation across 9 sub-Saharan African countries. Data on tuberculosis diagnosis and ATT/TPT initiation counts with corresponding follow-up time were available for 146 sites across the 9 countries prior to and post project implementation, stratified by 0 to 4 and 5 to 14 year age-groups. Preintervention data were retrospectively collected from facility registers for a 12-month period, and intervention data were prospectively collected from December 2018 to June 2021 using project-specific forms. Bayesian generalised linear mixed-effects models were used to estimate country-level rate ratios for tuberculosis diagnosis and ATT/TPT initiation. We analysed project expenditure and cascade data to determine unit costs of intervention components and used mathematical modelling to project health impact, health system costs, and cost-effectiveness. Overall, ATT and TPT initiation increased, with country-level incidence rate ratios varying between 0.8 (95% uncertainty interval [UI], 0.7 to 1.0) and 2.9 (95% UI, 2.3 to 3.6) for ATT and between 1.6 (95% UI, 1.5 to 1.8) and 9.8 (95% UI, 8.1 to 11.8) for TPT. We projected that for every 100 children starting either ATT or TPT at baseline, the intervention package translated to between 1 (95% UI, -1 to 3) and 38 (95% UI, 24 to 58) deaths averted, with a median incremental cost-effectiveness ratio (ICER) of US$634 per disability-adjusted life year (DALY) averted. ICERs ranged between US$135/DALY averted in Democratic of the Congo and US$6,804/DALY averted in Cameroon. The main limitation of our study is that the impact is based on pre/post comparisons, which could be confounded. CONCLUSIONS: In most countries, the CaP-TB intervention package improved tuberculosis treatment and prevention services for children aged under 15 years, but large variation in estimated impact and ICERs highlights the importance of local context. TRIAL REGISTRATION: This evaluation is part of the TIPPI study, registered with ClinicalTrials.gov (NCT03948698).

Immunity certification for COVID-19: ethical considerations.

Restrictive measures imposed because of the coronavirus disease 2019 (COVID-19) pandemic have resulted in severe social, economic and health effects. Some countries have considered the use of immunity certification as a strategy to relax these measures for people who have recovered from the infection by issuing these individuals a document, commonly called an immunity passport. This document certifies them as having protective immunity against severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), the virus that causes COVID-19. The World Health Organization has advised against the implementation of immunity certification at present because of uncertainty about whether long-term immunity truly exists for those who have recovered from COVID-19 and concerns over the reliability of the proposed serological test method for determining immunity. Immunity certification can only be considered if scientific thresholds for assuring immunity are met, whether based on antibodies or other criteria. However, even if immunity certification became well supported by science, it has many ethical issues in terms of different restrictions on individual liberties and its implementation process. We examine the main considerations for the ethical acceptability of immunity certification to exempt individuals from restrictive measures during the COVID-19 pandemic. As well as needing to meet robust scientific criteria, the ethical acceptability of immunity certification depends on its uses and policy objectives and the measures in place to reduce potential harms, and prevent disproportionate burdens on non-certified individuals and violation of individual liberties and rights.

Les restrictions imposées dans le cadre de la lutte contre la pandémie de maladie à coronavirus 2019 (COVID-19) ont eu de lourdes conséquences économiques, sociales et sanitaires. Certains pays ont envisagé la mise en place d'une stratégie visant à alléger ces restrictions pour les individus guéris en leur octroyant un document communément appelé «passeport d'immunité¼. Ce document atteste qu'ils ont développé une immunité protectrice contre le coronavirus 2 du syndrome respiratoire aigu sévère (SARS-CoV-2), le virus à l'origine de la COVID-19. L'Organisation mondiale de la Santé a déconseillé l'usage du certificat d'immunité pour l'instant, car l'incertitude demeure quant à l'existence réelle d'une immunité à long terme pour ceux qui se sont remis de la COVID-19. En outre, la fiabilité des tests sérologiques censés déterminer si l'individu est immunisé n'est pas avérée. Un tel certificat ne peut être instauré que si les seuils scientifiques en matière d'immunité sont respectés, qu'ils soient fondés sur les anticorps ou sur d'autres critères. Néanmoins, même si le certificat d'immunité est désormais bien accepté par la science, il s'accompagne de nombreuses questions d'ordre éthique en ce qui concerne la limitation des libertés individuelles et la mise en œuvre. Dans le présent document, nous examinons les principales considérations à prendre en compte pour garantir l'acceptabilité éthique du certificat d'immunité visant à lever les mesures de restriction pour certaines personnes durant la pandémie de COVID-19. Cette acceptabilité éthique dépend non seulement de son degré de conformité à des critères scientifiques stricts, mais aussi de son usage, des objectifs politiques ainsi que des mesures mises en place pour atténuer les préjudices potentiels et éviter d'imposer une charge disproportionnée sur les individus dépourvus de certificat, ou de bafouer les droits et libertés de tout un chacun.

Las medidas restrictivas impuestas a causa de la pandemia de la enfermedad coronavirus de 2019 (COVID-19) han tenido graves efectos sociales, económicos y sanitarios. Algunos países han considerado la posibilidad de utilizar la certificación de inmunidad como estrategia para flexibilizar dichas medidas para las personas que se han recuperado de la infección mediante la expedición a dichas personas de un documento, comúnmente denominado pasaporte de inmunidad. Este documento certifica que han desarrollado inmunidad protectora contra el coronavirus-2 del síndrome respiratorio agudo severo (SARS-CoV-2), el virus que causa la COVID-19. La Organización Mundial de la Salud ha desaconsejado la aplicación de la certificación de la inmunidad en la actualidad debido a la incertidumbre sobre si existe realmente una inmunidad a largo plazo para quienes se han recuperado de la COVID-19 y a las preocupaciones sobre la fiabilidad del método de prueba serológica propuesto para determinar la inmunidad. La certificación de la inmunidad solo puede considerarse si se cumplen los umbrales científicos para asegurar la inmunidad, ya sea que se basen en anticuerpos o en otros criterios. Sin embargo, incluso si la certificación de la inmunidad llegara a estar bien respaldada por la ciencia, tiene muchas cuestiones éticas en cuanto a las diferentes restricciones de las libertades individuales y su proceso de aplicación. Examinamos las principales consideraciones sobre la aceptabilidad ética de la certificación de la inmunidad para eximir a los individuos de las medidas restrictivas durante la pandemia de la COVID-19. Además de necesitar cumplir criterios científicos sólidos, la aceptabilidad ética de la certificación de inmunidad depende de sus usos y objetivos de política y de las medidas que se apliquen para reducir los posibles daños y evitar que se impongan cargas desproporcionadas a las personas que no cuenten con dicha certificación y se violen las libertades y derechos individuales.

Barriers to Access to New Gonorrhea Point-of-Care Diagnostic Tests in Low- and Middle-Income Countries and Potential Solutions: A Qualitative Interview-Based Study.

BACKGROUND: To assess the potential market for 2 hypothetical diagnostic tests, one for Neisseria gonorrhoeae/Chlamydia trachomatis (NG/CT) detection and one for NG antimicrobial resistance (AMR) marker identification. METHODS: This is a qualitative interview-based study. Semistructured interviews with global- and country-level experts were performed. Interviewees were provided with simplified versions of Foundation for Innovative New Diagnostics/World Health Organization-developed target product profiles for each test. Interviewees were asked to comment on use cases, test characteristics, and factors that may influence test adoption. RESULTS: Twenty-one experts were interviewed, including 15 country-level experts (from South Africa, India, Zimbabwe, Ghana, China, Peru, Kenya, and Cambodia). Interviewees welcomed an NG/CT point-of-care test, with near-universal preference for a test that could detect symptomatic and asymptomatic infections. Interviewees also saw value in a test that could be used to screen high-risk populations. Factors that may drive adoption of the NG/CT test identified by interviewees included price, cost-effectiveness, evidence of public health benefit, and World Health Organization guidance. Interviewees felt that AMR test use would likely be limited to patients failing first-line treatment. CONCLUSIONS: Although the potential target population for an NG/CT diagnostic test in low- and middle-income countries is sizeable, there are areas of uncertainty relating to the price of the test and its intended use, warranting further research to determine the most effective positioning. An NG AMR test would likely be used very selectively.

Comparative performance study of three Ebola rapid diagnostic tests in Guinea.

BACKGROUND: The 2014/15 Ebola outbreak in West Africa resulted in 11,000 deaths and massive strain on local health systems, and the ongoing outbreak in Democratic Republic of Congo has afflicted more than 3000 people. Accurate, rapid Ebola diagnostics suitable for field deployment would enable prompt identification and effective response to future outbreaks, yet remain largely unavailable. The purpose of this study was to assess the accuracy of three novel rapid diagnostic tests (RDTs): an Ebola, an Ebola-Malaria, and a Fever Panel test that includes Ebola, all from a single manufacturer. METHODS: We evaluated the three RDTs in 109 Ebola-positive and 96 Ebola-negative stored serum samples collected during the outbreak in Guinea in 2014/15, and tested by real-time polymerase chain reaction (RT-PCR). Sensitivity, specificity, and overall percent agreement were calculated for each RDT using RT-PCR as a reference standard, stratified by Ct value ranges. RESULTS: All tests performed with high accuracy on samples with low Ct value (high viral load). The Fever Panel test performed with the highest accuracy, with a sensitivity of 89.9% and specificity of 90.6%. The Ebola and Ebola-Malaria tests performed comparably to each other: sensitivity was 77.1 and 78% respectively, and specificity was 91.7% for the Ebola test and 95.8% for the Ebola-Malaria test. CONCLUSIONS: This study evaluated the accuracy of three novel rapid diagnostic tests for Ebola. The tests may have significant public health relevance, particularly the Fever Panel test, which detects seven pathogens including Ebola. Given limitations to the study resulting from uncertain sample quality, further evaluation is warranted. All tests performed with highest accuracy on samples with low Ct value (high viral load), and the data presented here suggests that these RDTs may be useful for point-of-care diagnosis of cases in the context of an outbreak. Restrictions to their use in non-severe Ebola cases or for longitudinal monitoring, when viral loads are lower, may be appropriate. Highlighting the challenge in developing and evaluating Ebola RDTs, there were concerns regarding sample integrity and reference testing, and there is a need for additional research to validate these assays.

A novel chemical lysis method for maximum release of DNA from difficult-to-lyse bacteria.

Molecular detection of microorganisms requires releasing DNA from cells. However, since certain microbial organisms are refractory to lysis by chemical or enzymatic methods, mechanical lysis by bead-beating is typically employed to disrupt difficult-to-lyse microbes. A newly developed chemical lysis method called sporeLYSE enables release of DNA from difficult-to-lyse microbes without bead-beating. The sporeLYSE method was compared to bead-beating and an alkaline/detergent lysis solution for releasing DNA from microbes grown in vitro, including surrogates of Category A bioterrorism agents. sporeLYSE released 83% to 100% of DNA from Mycobacterium smegmatis, Francisella philomiragia, Yersinia enterocolitica, Bacillus thuringiensis, Pseudomonas aeruginosa, Moraxella catarrhalis and Klebsiella pneumoniae. qPCR results indicated that sporeLYSE extracted an equal or greater amount of DNA than either bead-beating or alkaline/detergent lysis from Gram-positive and Gram-negative bacteria. When sporeLYSE was used to extract DNA from saliva and sputum spiked with M. smegmatis and M. tuberculosis, respectively, the qPCR Ct values were 4-8 cycles lower than those for extractions via alkaline/detergent lysis and heat. Mean Ct values for sporesLYSE extractions from spores of Clostridium difficile and C. botulinum were approximately two cycles lower than those of MagNA Pure DNA extractions. Our results suggest that sporeLYSE is an easy-to-use liquid reagent that can efficiently release large amounts of DNA from a variety of bacteria, including spores.

Detection of Staphylococcus aureus enterotoxin production genes from patient samples using an automated extraction platform and multiplex real-time PCR.

To minimize specimen volume, handling and testing time, we have developed two TaqMan(®) multiplex real-time PCR (rtPCR) assays to detect staphylococcal enterotoxins A-E and Toxic Shock Syndrome Toxin production genes directly from clinical patient stool specimens utilizing a novel lysis extraction process in parallel with the Roche MagNA Pure Compact. These assays are specific, sensitive and reliable for the detection of the staphylococcal enterotoxin encoding genes and the tst1 gene from known toxin producing strains of Staphylococcus aureus. Specificity was determined by testing a total of 47 microorganism strains, including 8 previously characterized staphylococcal enterotoxin producing strains against each rtPCR target. Sensitivity for these assays range from 1 to 25 cfu per rtPCR reaction for cultured isolates and 8-20 cfu per rtPCR for the clinical stool matrix.

Use of Measles and Rubella Rapid Diagnostic Tests to Improve Case Detection and Targeting of Vaccinations.

Efforts to control and eliminate measles and rubella are aided by high-quality surveillance data-supported by laboratory confirmation-to guide decision-making on routine immunization strategies and locations for conducting preventive supplementary immunization activities (SIAs) and outbreak response. Important developments in rapid diagnostic tests (RDTs) for measles and rubella present new opportunities for the global measles and rubella surveillance program to greatly improve the ability to rapidly detect and respond to outbreaks. Here, we review the status of RDTs for measles and rubella Immunoglobulin M (IgM) testing, as well as ongoing questions and challenges regarding the operational use and deployment of RDTs as part of global measles and rubella surveillance. Efforts to develop IgM RDTs that can be produced at scale are underway. Once validated RDTs are available, clear information on the benefits, challenges, and costs of their implementation will be critical for shaping deployment guidance and informing country plans for sustainably deploying such tests. The wide availability of RDTs could provide new programmatic options for measles and rubella elimination efforts, potentially enabling improvements and flexibility for testing, surveillance, and vaccination.

Comparative Evaluation of Select Serological Assays for Zika Virus Using Blinded Reference Panels.

In response to the 2015 Zika virus (ZIKV) epidemic that occurred in Brazil, numerous commercial serological assays have been developed for clinical and research applications. Diagnosis of recent infection in pregnant women remains challenging. Having standardized, comparative studies of ZIKV tests is important for implementing optimal diagnostic testing and disease surveillance. This is especially important for serology tests used to detect ZIKV infection given that antibodies against ZIKV can cross-react with other arboviruses in the same virus family, such as dengue virus (DENV), yellow fever virus (YFV) and West Nile virus (WNV). We looked at the sensitivity and specificity of tests detecting ZIKV antibodies (IgM, IgG) from multiple manufacturers using panels of samples previously collected with known exposure to ZIKV and other arboviruses. We found that performance of the IgM tests was highly variable, with only one test (Inbios 2.0 IgM capture ELISA) having both high sensitivity and specificity. All IgG tests showed good sensitivity; however, specificity was highly variable, with some assays giving false-positive results on samples infected by another flavivirus. Overall, the results confirmed that accurate ZIKV antibody testing is challenging, especially in specimens from regions endemic for multiple other flaviviruses, and highlight the importance of available and suitable reference samples to evaluate ZIKV diagnostics.

Standardized evaluation of Zika nucleic acid tests used in clinical settings and blood screening.

Early detection of Zika virus (ZIKV) transmission within geographic regions informs implementation of community mitigation measures such as vector reduction strategies, travel advisories, enhanced surveillance among pregnant women, and possible implementation of blood and organ donor screening or deferral. Standardized, comparative assessments of ZIKV assay and testing lab performance are important to develop optimal approaches to ZIKV diagnostic testing and surveillance. We conducted an expanded blinded panel study to characterize and compare the analytical performance of fifteen diagnostic and blood screening ZIKV NAT assays, including detection among single- and multiplex assays detecting ZIKV, dengue virus (DENV) and chikungunya virus (CHIKV). A 300 member blinded panel was constructed, consisting of 11 serial half-log dilutions ranging from ~104 to 10-1 genome equivalents/mL in 25 replicates each of the Tahitian Asian ZIKV isolate in ZIKV-negative human serum. Additionally, clinical samples from individuals with DENV-like syndrome or suspected ZIKV infection in Brazil were evaluated. The majority of assays demonstrated good specificity. Analytical sensitivities varied 1-2 logs, with a substantially higher limit of detection (LOD) in one outlier. Similar analytical sensitivity for ZIKV RNA detection in singleplex and multiplex assays of the Grifols and ThermoFisher tests were observed. Coefficient of Assay Efficiency (CE), calculated to characterize assays' RNA extraction and amplification efficiency, ranged from 0.13 for the Certest VIASURE multiplex and 0.75 for the Grifols multiplex assays. In general, assays using transcription mediated amplification (TMA) technology had greater CE compared to assays using conventional PCR technology. Donor screening NAT assays were significantly more sensitive than diagnostic RT-qPCR assays, primarily attributable to higher sample input volumes. However, ideal assays to maximize sensitivity and throughput may not be a viable option in all contexts, with other factors such as cost, instrumentation, and regulatory approval status influencing assay availability and selection, particularly in resource constrained settings.

Secondary and tertiary transmission of vaccinia virus from US military service member.

During February and March 2010, the New York State Department of Health investigated secondary and tertiary vaccinia contact transmission from a military vaccinee to 4 close contacts. Identification of these cases underscores the need for strict adherence to postvaccination infection control guidance to avoid transmission of the live virus.

Analysis of Clostridium botulinum serotype E strains by using multilocus sequence typing, amplified fragment length polymorphism, variable-number tandem-repeat analysis, and botulinum neurotoxin gene sequencing.

A total of 41 Clostridium botulinum serotype E strains from different geographic regions, including Canada, Denmark, Finland, France, Greenland, Japan, and the United States, were compared by multilocus sequence typing (MLST), amplified fragment length polymorphism (AFLP) analysis, variable-number tandem-repeat (VNTR) analysis, and botulinum neurotoxin (bont) E gene sequencing. The strains, representing environmental, food-borne, and infant botulism samples collected from 1932 to 2007, were analyzed to compare serotype E strains from different geographic regions and types of botulism and to determine whether each of the strains contained the transposon-associated recombinase rarA, involved with bont/E insertion. MLST examination using 15 genes clustered the strains into several clades, with most members within a cluster sharing the same BoNT/E subtype (BoNT/E1, E2, E3, or E6). Sequencing of the bont/E gene identified two new variants (E7, E8) that showed regions of recombination with other E subtypes. The AFLP dendrogram clustered the 41 strains similarly to the MLST dendrogram. Strains that could not be differentiated by AFLP, MLST, or bont gene sequencing were further examined using three VNTR regions. Both intact and split rarA genes were amplified by PCR in each of the strains, and their identities were confirmed in 11 strains by amplicon sequencing. The findings suggest that (i) the C. botulinum serotype E strains result from the targeted insertion of the bont/E gene into genetically conserved bacteria and (ii) recombination events (not random mutations) within bont/E result in toxin variants or subtypes within strains.

An open-source molecular diagnostic platform approach for outbreak and epidemic preparedness.

BACKGROUND: Diagnostic development for outbreak pathogens has typically followed a disease-specific reactive rather than proactive response. Given the diversity of outbreak pathogens, particularly those prioritised by the World Health Organization Research and Development Blueprint, a more flexible and proactive approach to epidemic preparedness is needed to expand access to critical molecular diagnostic tests in peripheral and resource-constrained deployment settings. OBJECTIVE: New and more sustainable directives are needed to spur the development of high-quality products, particularly for epidemics more often found in low- and middle-income countries. To leverage and de-risk the development process, we present the benefits and challenges of an open-source business model for co-development of molecular diagnostic tests for decentralised settings. METHODS: We identify key outbreak pathogens that are available only for testing in high infrastructure laboratories and compare in-country installed base platforms that could be leveraged for menu expansion. Key strengths and challenges for development are highlighted for both platform and assay developers, with discussion of how to leverage and de-risk the process through an open-source development model. RESULTS: Depending on the specific partner strengths, options for partnership roles are presented. The proposed open-source business model addresses the particular challenges in the detection of outbreak- and epidemic-prone pathogens in low- and middle-income countries, reduces development and deployment risks to support outbreak response, strengthens diagnostic capacity and creates a viable market for product developers. CONCLUSION: We hope this model for a collaborative and open-source approach for molecular diagnostics serves to encourage stakeholders to consider co-development partnerships to improve outbreak preparedness and epidemic/pandemic response.

Target Product Profile for a mobile app to read rapid diagnostic tests to strengthen infectious disease surveillance.

The essential role of rapid diagnostic tests (RDTs) in disease control is compromised every time a test is not performed correctly or its result is not reported accurately and promptly. A mobile app that utilizes the camera and connectivity of a common smartphone can fill this role of supporting the test's proper execution and the automatic transmission of results. In a consensus process with 51 expert participants representing the needs of clinical users, healthcare programs, health information systems, surveillance systems, and global public health stakeholders, we developed a Target Product Profile describing the minimal and optimal characteristics of such an app. We collected feedback over two rounds and refined the characteristics to arrive at a preferred agreement level of greater than 75%, with an average of 92% agreement (range: 79-100%). As per this feedback, such an app should be compatible with many RDTs and mobile devices without needing accessories. The app should assist the user with RDT-specific instructions, include checks to facilitate quality control of the testing process and suggest results with ≥ 95% accuracy across common lighting conditions while allowing the user to determine the final result. Data from the app must be under the control of the health program that operates it, and the app should support at least one of the common data exchange formats HL7, FHIR, ASTM or JSON. The Target Product Profile also lays out the minimum data security and privacy requirements for the app.

Impact of a package of diagnostic tools, clinical algorithm, and training and communication on outpatient acute fever case management in low- and middle-income countries: protocol for a randomized controlled trial.

BACKGROUND: The management of acute febrile illnesses places a heavy burden on clinical services in many low- and middle-income countries (LMICs). Bacterial and viral aetiologies of acute fevers are often clinically indistinguishable and, in the absence of diagnostic tests, the 'just-in-case' use of antibiotics by many health workers has become common practice, which has an impact on drug-resistant infections. Our study aims to answer the following question: in patients with undifferentiated febrile illness presenting to outpatient clinics/peripheral health centres in LMICs, can we demonstrate an improvement in clinical outcomes and reduce unnecessary antibiotic prescription over current practice by using a combination of simple, accurate diagnostic tests, clinical algorithms, and training and communication (intervention package)? METHODS: We designed a randomized, controlled clinical trial to evaluate the impact of our intervention package on clinical outcomes and antibiotic prescription rates in acute febrile illnesses. Available, point-of-care, pathogen-specific and non-pathogen specific (host markers), rapid diagnostic tests (RDTs) included in the intervention package were selected based on pre-defined criteria. Nine clinical study sites in six countries (Burkina Faso, Ghana, India, Myanmar, Nepal and Uganda), which represent heterogeneous outpatient care settings, were selected. We considered the expected seasonal variations in the incidence of acute febrile illnesses across all the sites by ensuring a recruitment period of 12 months. A master protocol was developed and adapted for country-specific ethical submissions. Diagnostic algorithms and choice of RDTs acknowledged current data on aetiologies of acute febrile illnesses in each country. We included a qualitative evaluation of drivers and/or deterrents of uptake of new diagnostics and antibiotic use for acute febrile illnesses. Sample size estimations were based on historical site data of antibiotic prescription practices for malarial and non-malarial acute fevers. Overall, 9 semi-independent studies will enrol a minimum of 21,876 patients and an aggregate data meta-analysis will be conducted on completion. DISCUSSION: This study is expected to generate vital evidence needed to inform policy decisions on the role of rapid diagnostic tests in the clinical management of acute febrile illnesses, with a view to controlling the rise of antimicrobial resistance in LMICs. TRIAL REGISTRATION: Clinicaltrials.gov NCT04081051 . Registered on 6 September 2019. Protocol version 1.4 dated 20 December 2019.

Developing target product profiles for Neisseria gonorrhoeae diagnostics in the context of antimicrobial resistance: An expert consensus.

BACKGROUND: There is a need for a rapid diagnostic point of care test to detect Neisseria gonorrhoeae (NG) infection to prevent incorrect, lack or excess of treatment resulting from current syndromic management in low-resource settings. An assay to identify NG antimicrobial resistance (AMR) is also highly desirable to facilitate antibiotic stewardship. Here we describe the development of two target product profiles (TPPs): one for a test for etiological diagnosis of NG and Chlamydia trachomatis (CT) (TPP1) and one for the detection of NG AMR/susceptibility (TPP2). METHODS: Draft TPPs were initially developed based on a landscape analysis of existing diagnostics and expert input. TPPs were refined via an online Delphi survey with two rounds of input from 68 respondents. TPP characteristics on which <75% of non-industry respondents agreed were further discussed and revised by an expert working group. RESULTS: The need for a test to identify NG in patients with urethral or vaginal discharge was identified as a minimal requirement of TPP1, with a test that can diagnose NG in asymptomatic patients as the optimal requirement. A sensitivity of 80% was considered acceptable, either in context of syndromic management or screening high-risk populations. For TPP2, the agreed minimal requirement was for a test to be used at level 2 healthcare facilities and above, with an optimal requirement of level 1 or above. A lateral flow format was preferred for TPP1, while it was considered likely that TPP2 would require a molecular format. A total of 31 test characteristics were included in TPP1 and 27 in TPP2. CONCLUSIONS: Following the working group revisions, TPPs were posted online for public feedback for two months, and are now finalized. The final TPPs are currently guiding the development of new diagnostics that meet the defined characteristics to reach the market within two years.

Neutralizing monoclonal antibodies directed against defined linear epitopes on domain 4 of anthrax protective antigen.

The anthrax protective antigen (PA) is the receptor-binding subunit common to lethal toxin (LT) and edema toxin (ET), which are responsible for the high mortality rates associated with inhalational Bacillus anthracis infection. Although recombinant PA (rPA) is likely to be an important constituent of any future anthrax vaccine, evaluation of the efficacies of the various candidate rPA vaccines is currently difficult, because the specific B-cell epitopes involved in toxin neutralization have not been completely defined. In this study, we describe the identification and characterization of two murine monoclonal immunoglobulin G1 antibodies (MAbs), 1-F1 and 2-B12, which recognize distinct linear neutralizing epitopes on domain 4 of PA. 1-F1 recognized a 12-mer peptide corresponding to residues 692 to 703; this epitope maps to a region of domain 4 known to interact with the anthrax toxin receptor CMG-2 and within a conformation-dependent epitope recognized by the well-characterized neutralizing MAb 14B7. As expected, 1-F1 blocked PA's ability to associate with CMG-2 in an in vitro solid-phase binding assay, and it protected murine macrophage cells from intoxication with LT. 2-B12 recognized a 12-mer peptide corresponding to residues 716 to 727, an epitope located immediately adjacent to the core 14B7 binding site and a stretch of amino acids not previously identified as a target of neutralizing antibodies. 2-B12 was as effective as 1-F1 in neutralizing LT in vitro, although it only partially inhibited PA binding to its receptor. Mice passively administered 1-F1 or 2-B12 were partially protected against a lethal challenge with LT. These results advance our fundamental understanding of the mechanisms by which antibodies neutralize anthrax toxin and may have future application in the evaluation of candidate rPA vaccines.

Diagnostics for Nipah virus: a zoonotic pathogen endemic to Southeast Asia.

Nipah virus (NiV) is an emerging pathogen that, unlike other priority pathogens identified by WHO, is endemic to Southeast Asia. It is most commonly transmitted through exposure to saliva or excrement from the Pteropus fruit bat, or direct contact with intermediate animal hosts, such as pigs. NiV infection causes severe febrile encephalitic disease and/or respiratory disease; treatment options are limited to supportive care. A number of in-house diagnostic assays for NiV using serological and nucleic acid amplification techniques have been developed for NiV and are used in laboratory settings, including some early multiplex panels for differentiation of NiV infection from other febrile diseases. However, given the often rural and remote nature of NiV outbreak settings, there remains a need for rapid diagnostic tests that can be implemented at the point of care. Additionally, more reliable assays for surveillance of communities and livestock will be vital to achieving a better understanding of the ecology of the fruit bat host and transmission risk to other intermediate hosts, enabling implementation of a 'One Health' approach to outbreak prevention and the management of this zoonotic disease. An improved understanding of NiV viral diversity and infection kinetics or dynamics will be central to the development of new diagnostics, and access to clinical specimens must be improved to enable effective validation and external quality assessments. Target product profiles for NiV diagnostics should be refined to take into account these outstanding needs.

Diagnostic tests for Crimean-Congo haemorrhagic fever: a widespread tickborne disease.

Crimean-Congo haemorrhagic fever (CCHF) is a widespread tickborne disease that circulates in wild and domestic animal hosts, and causes severe and often fatal haemorrhagic fever in infected humans. Due to the lack of treatment options or vaccines, and a high fatality rate, CCHF virus (CCHFV) is considered a high-priority pathogen according to the WHO R&D Blueprint. Several commercial reverse transcriptase PCR (RT-PCR) and serological diagnostic assays for CCHFV are already available, including febrile agent panels to distinguish CCHFV from other viral haemorrhagic fever agents; however, the majority of international laboratories use inhouse assays. As CCHFV has numerous amplifying animal hosts, a cross-sectoral 'One Health' approach to outbreak prevention is recommended to enhance notifications and enable early warning for genetic and epidemiological shifts in the human, animal and tick populations. However, a lack of guidance for surveillance in animals, harmonisation of case identification and validated serodiagnostic kits for animal testing hinders efforts to strengthen surveillance systems. Additionally, as RT-PCR tests tend to be lineage-specific for regional circulating strains, there is a need for pan-lineage sensitive diagnostics. Adaptation of existing tests to point-of-care molecular diagnostic platforms that can be implemented in clinic or field-based settings would be of value given the potential for CCHFV outbreaks in remote or low-resource areas. Finally, improved access to clinical specimens for validation of diagnostics would help to accelerate development of new tests. These gaps should be addressed by updated target product profiles for CCHFV diagnostics.

Diagnostics for Lassa fever virus: a genetically diverse pathogen found in low-resource settings.

Lassa fever virus (LASV) causes acute viral haemorrhagic fever with symptoms similar to those seen with Ebola virus infections. LASV is endemic to West Africa and is transmitted through contact with excretions of infected Mastomys natalensis rodents and other rodent species. Due to a high fatality rate, lack of treatment options and difficulties with prevention and control, LASV is one of the high-priority pathogens included in the WHO R&D Blueprint. The WHO LASV vaccine strategy relies on availability of effective diagnostic tests. Current diagnostics for LASV include in-house and commercial (primarily research-only) laboratory-based serological and nucleic acid amplification tests. There are two commercially available (for research use only) rapid diagnostic tests (RDTs), and a number of multiplex panels for differential detection of LASV infection from other endemic diseases with similar symptoms have been evaluated. However, a number of diagnostic gaps remain. Lineage detection is a challenge due to the genomic diversity of LASV, as pan-lineage sensitivity for both molecular and immunological detection is necessary for surveillance and outbreak response. While pan-lineage ELISA and RDTs are commercially available (for research use only), validation and external quality assessment (EQA) is needed to confirm detection sensitivity for all known or relevant strains. Variable sensitivity of LASV PCR tests also highlights the need for improved validation and EQA. Given that LASV outbreaks typically occur in low-resource settings, more options for point-of-care testing would be valuable. These requirements should be taken into account in target product profiles for improved LASV diagnostics.

Diagnostics for filovirus detection: impact of recent outbreaks on the diagnostic landscape.

Ebolaviruses and Marburg virus (MARV) both belong to the family Filoviridae and cause severe haemorrhagic fever in humans. Due to high mortality rates and potential for spread from rural to urban regions, they are listed on the WHO R&D blueprint of high-priority pathogens. Recent ebolavirus outbreaks in Western and Central Africa have highlighted the importance of diagnostic testing in epidemic preparedness for these pathogens and led to the rapid development of a number of commercially available benchtop and point-of-care nucleic acid amplification tests as well as serological assays and rapid diagnostic tests. Despite these advancements, challenges still remain. While products approved under emergency use licenses during outbreak periods may continue to be used post-outbreak, a lack of clarity and incentive surrounding the regulatory approval pathway during non-outbreak periods has deterred many manufacturers from seeking full approvals. Waning of funding and poor access to samples after the 2014-2016 outbreak also contributed to cessation of development once the outbreak was declared over. There is a need for tests with improved sensitivity and specificity, and assays that can use alternative sample types could reduce the need for invasive procedures and expensive equipment, making testing in field conditions more feasible. For MARV, availability of diagnostic tests is still limited, restricted to a single ELISA test and assay panels designed to differentiate between multiple pathogens. It may be helpful to extend the target product profile for ebolavirus diagnostics to include MARV, as the viruses have many overlapping characteristics.