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Background: To improve tuberculosis case-finding, rapid, non-sputum triage tests need to be developed according to the World Health Organization target product profile (TPP) (>90% sensitivity, >70% specificity). We prospectively evaluated and compared artificial intelligence-based, computer-aided detection software, CAD4TBv7, and C-reactive protein assay (CRP) as triage tests at health facilities in Lesotho and South Africa., Methods: Adults (≥18 years) presenting with ≥1 of the 4 cardinal tuberculosis symptoms were consecutively recruited between February 2021 and April 2022. After informed consent, each participant underwent a digital chest X-ray for CAD4TBv7 and a CRP test. Participants provided 1 sputum sample for Xpert MTB/RIF Ultra and Xpert MTB/RIF and 1 for liquid culture. Additionally, an expert radiologist read the chest X-rays via teleradiology. For primary analysis, a composite microbiological reference standard (ie, positive culture or Xpert Ultra) was used., Results: We enrolled 1392 participants, 48% were people with HIV and 24% had previously tuberculosis. The receiver operating characteristic curve for CAD4TBv7 and CRP showed an area under the curve of .87 (95% CI: .84-.91) and .80 (95% CI: .76-.84), respectively. At thresholds corresponding to 90% sensitivity, specificity was 68.2% (95% CI: 65.4-71.0%) and 38.2% (95% CI: 35.3-41.1%) for CAD4TBv7 and CRP, respectively. CAD4TBv7 detected tuberculosis as well as an expert radiologist. CAD4TBv7 almost met the TPP criteria for tuberculosis triage., Conclusions: CAD4TBv7 is accurate as a triage test for patients with tuberculosis symptoms from areas with a high tuberculosis and HIV burden. The role of CRP in tuberculosis triage requires further research., Clinical Trials Registration: Clinicaltrials.gov identifier: NCT04666311., Competing Interests: Potential conflicts of interest. B. v. G. reports that his group received royalties from Delft Imaging Systems and that he holds shares in Thirona. K. R., B. v. G., A. v. H., A. S., K. M., L. L., T. Z., B. K. M. J., S. B., T. M., and M. K. report support related to this work from the European and Developing Countries Clinical Trials Partnership 2 (EDCTP2). K. R. reports grants unrelated to this work from EDCTP2 and Horizon 2020, as well as unpaid participation on a data safety monitoring board or scientific advisory board for evaluation of new TB diagnostics. S. B. reports a grant unrelated to this work from the National Institutes of Health (NIH) and travel support from MSD. T. M. reports travel support from MSD. N. D. L. reports support for travel from ViiV Healthcare and Gilead Sciences Sarl as well as for attending a board meeting from ViiV Healthcare and Pharming. S. B. and T. M. report travel support from MSD. The other authors report no potential conflict of interest. All authors have submitted the ICMJE Form for Disclosure of Potential Conflicts of Interest. Conflicts that the editors consider relevant to the content of the manuscript have been disclosed., (© The Author(s) 2024. Published by Oxford University Press on behalf of Infectious Diseases Society of America.)

The 2018-2020 Ebola virus disease (EVD) outbreak in the Democratic Republic of the Congo (DRC) was the largest since the disease's discovery in 1976. Rapid identification and isolation of EVD patients are crucial during triage. This study aimed to develop a clinical prediction score for EVD using clinical and epidemiological predictors. We conducted a retrospective cross-sectional study using surveillance data from EVD outbreak, collected during routine clinical care at the Ebola Transit Center (ETC) in Beni, DRC, from 2018 to 2020. The Spiegelhalter and Knill-Jones method was used for score development, including potential predictors with an adjusted likelihood ratio above 2 or below 0.50. Validation was performed using a dataset previously published in PLOSOne by Tshomba et al. Among 3725 patients screened, 3698 fulfilled the inclusion criteria, with 571 (15.4%) testing positive for EVD via RT-PCR Test. Seven predictive factors were identified: asthenia, sore throat, conjunctivitis, bleeding gums, hematemesis, contact with a sick person, and contact with a traditional healer. The prediction score achieved an Area under the receiver operating characteristic (AUROC) of 0.764, with 81.4% sensitivity and 53.6% specificity at a -1 cutoff. External validation demonstrated an AUROC of 0.766, with 80.8% sensitivity and 41.4% specificity at the -1 cutoff. Our study developed a screening tool to assess the risk of suspected patients developing EVD and being admitted to ETUs for RT-PCR testing and treatment. External validation results affirmed the model's reliability and generalizability in similar settings, suggesting its potential integration into clinical practice. Given the severity and urgency of EVD as well as the risk nosocomial EVD transmission, it is essential to continuously update these models with real-time data on symptoms, disease progression, patient outcomes and validated RDT during EVD outbreaks. This approach will enhance model accuracy, enabling more precise risk assessments and more effective outbreak management., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Yango et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Background: CD4 measurement is pivotal in the management of advanced HIV disease. VISITECT® CD4 Advanced Disease (AccuBio Limited, Alva, UK; VISITECT) is an instrument-free, point-of-care, semi-quantitative test allowing visual identification of a CD4 ≤200 cells/µl, or >200 cells/µl from finger-prick or venous blood., Methods: As part of a diagnostic accuracy study of FUJIFILM SILVAMP TB LAM (clinicaltrials.gov: NCT04089423), people living with HIV of ≥18 years old were prospectively recruited in seven countries from outpatient departments if a tuberculosis symptom was present, and from inpatient departments. Participants provided venous blood for CD4 measurement using flow cytometry (reference standard) and finger-prick blood for VISITECT (index text), performed at point-of-care. Sensitivity, specificity, and positive and negative predictive values of VISITECT to determine a CD4 ≤200 cells/µl were evaluated., Results: Among 1604 participants, the median flow cytometry CD4 was 367 (IQR 128-626) cells/µl and 521 (32.5%) had a CD4 ≤200 cells/µl. VISITECT sensitivity was 92.7% (483/521, 95% CI 90.1-94.7%) and specificity was 61.4% (665/1083, 95% CI 58.4-64.3%). For participants with a CD4 between 0-100, 101-200, 201-300, 301-500, and >500 cells/µl, VISITECT misclassified 4.5% (95% CI 2.5-7.2%), 12.5 (95% CI 8.0-18.2%), 74.1% (95% CI 67.0-80.5%), 48.0% (95% CI 42.5-53.6%), and 22.6% (95% CI 19.3-26.3%), respectively., Conclusions: VISITECT's sensitivity, but not specificity, met the World Health Organization's minimal sensitivity and specificity threshold of 80% for point-of-care CD4 tests. VISITECT's quality needs to be assessed and its accuracy optimized. VISITECT´s utility as CD4 triage test should be investigated., (© The Author(s) 2024. Published by Oxford University Press on behalf of Infectious Diseases Society of America.)

Background: Estimation of prevalence and diagnostic test accuracy in tuberculosis (TB) prevalence surveys suffer from reference standard and verification biases. The former is attributed to the imperfect reference test used to bacteriologically confirm TB disease. The latter occurs when only the participants screening positive for any TB-compatible symptom or chest X-ray abnormality are selected for bacteriological testing (verification). Bayesian latent class analysis (LCA) alleviates the reference standard bias but suffers verification bias in TB prevalence surveys. This work aims to identify best-practice approaches to simultaneously alleviate the reference standard and verification biases in the estimates of pulmonary TB prevalence and diagnostic test performance in TB prevalence surveys., Methods: We performed a secondary analysis of 9869 participants aged ≥15 years from a community-based multimorbidity screening study in a rural district of KwaZulu-Natal, South Africa (Vukuzazi study). Participants were eligible for bacteriological testing using Xpert Ultra and culture if they reported any cardinal TB symptom or had an abnormal chest X-ray finding. We conducted Bayesian LCA in five ways to handle the unverified individuals: (i) complete-case analysis, (ii) analysis assuming the unverified individuals would be negative if bacteriologically tested, (iii) analysis of multiply-imputed datasets with imputation of the missing bacteriological test results for the unverified individuals using multivariate imputation via chained equations (MICE), and simultaneous imputation of the missing bacteriological test results in the analysis model assuming the missing bacteriological test results were (iv) missing at random (MAR), and (v) missing not at random (MNAR). We compared the results of (i)-(iii) to the analysis based on a composite reference standard (CRS) of Xpert Ultra and culture. Through simulation with an overall true prevalence of 2.0%, we evaluated the ability of the models to alleviate both biases simultaneously., Results: Based on simulation, Bayesian LCA with simultaneous imputation of the missing bacteriological test results under the assumption that the missing data are MAR and MNAR alleviate the reference standard and verification biases. CRS-based analysis and Bayesian LCA assuming the unverified are negative for TB alleviate the biases only when the true overall prevalence is <3.0%. Complete-case analysis produced biased estimates. In the Vukuzazi study, Bayesian LCA with simultaneous imputation of the missing bacteriological test results under the MAR and MNAR assumptions produced overall PTB prevalence of 0.9% (95% Credible Interval (CrI): 0.6-1.9) and 0.7% (95% CrI: 0.5-1.1) respectively alongside realistic estimates of overall diagnostic test sensitivity and specificity with substantially overlapping 95% CrI. The CRS-based analysis and Bayesian LCA assuming the unverified were negative for TB produced 0.7% (95% CrI: 0.5-0.9) and 0.7% (95% CrI: 0.5-1.2) overall PTB prevalence respectively with realistic estimates of overall diagnostic test sensitivity and specificity. Unlike CRS-based analysis, Bayesian LCA of multiply-imputed data using MICE mitigates both biases., Conclusion: The findings demonstrate the efficacy of these advanced techniques in alleviating the reference standard and verification biases, enhancing the robustness of community-based screening programs. Imputing missing values as negative for bacteriological tests is plausible under realistic assumptions., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Keter et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Objectives: In clinical decision-making, physicians take actions such as prescribing treatment only when the probability of disease is sufficiently high. The lowest probability at which the action will be considered, is the action threshold. Such thresholds play an important role whenever decisions have to be taken under uncertainty. However, while several methods to estimate action thresholds exist, few methods give satisfactory results or have been adopted in clinical practice. We piloted the adapted nominal group technique (aNGT), a new prescriptive method based on a formal consensus technique adapted for use in clinical decision-making., Design, Setting and Participants: We applied this method in groups of postgraduate students using three scenarios: treat for rifampicin-resistant tuberculosis (RR-TB), switch to second-line HIV treatment and isolate for SARS-CoV-2 infection., Interventions: The participants first summarise all harms of wrongly taking action when none is required and wrongly not taking action when it would have been useful. Then they rate the statements on these harms, discuss their importance in the decision-making process, and finally weigh the statements against each other., Main Outcome Measures: The resulting consensus threshold is estimated as the relative weights of the harms of the false positives divided by the total harm, and averaged out over participants. In some applications, the thresholds are compared with an existing method based on clinical vignettes., Results: The resulting action thresholds were just over 50% for RR-TB treatment, between 20% and 50% for switching HIV treatment and 43% for COVID-19 isolation. These results were considered acceptable to all participants. Between sessions variation was low for RR-TB and moderate for HIV. Threshold estimates were moderately lower with the method based on clinical vignettes., Conclusions: The aNGT gives sensible results in our pilot and has the potential to estimate action thresholds, in an efficient manner, while involving all relevant stakeholders. Further research is needed to study the value of the method in clinical decision-making and its ability to generate acceptable thresholds that stakeholders can agree on., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2023. Re-use permitted under CC BY. Published by BMJ.)

Because TB control is still hampered by the limitations of diagnostic tools, diagnostic uncertainty is common. The decision to offer treatment is based on clinical decision-making. The therapeutic threshold, test threshold and test-treatment threshold can guide in making these decisions. This review summarizes the literature on methods to estimate the therapeutic threshold that have been applied for TB. Only five studies estimated the threshold for the diagnosis of TB. The therapeutic threshold can be estimated by prescriptive methods, based on calculations, and by descriptive methods, deriving the threshold from observing clinical practice. Test and test-treatment thresholds can be calculated using the therapeutic threshold and the characteristics of an available diagnostic test. Estimates of the therapeutic threshold for pulmonary TB from intuitive descriptive approaches (20%-50%) are higher than theoretical prescriptive calculations (2%-3%). In conclusion, estimates of the therapeutic threshold for pulmonary TB depend on the method used. Other methods exist within the field of decision-making that have yet to be implemented or adapted as tools to estimate the TB therapeutic threshold. Because clinical decision-making is a core element of TB management, it is necessary to find a new, clinician-friendly way to unbiasedly estimate context-specific, agreed upon therapeutic thresholds., (© The Author(s) 2023. Published by Oxford University Press on behalf of Royal Society of Tropical Medicine and Hygiene.)

Introduction: Evidence on the real-world effects of "Treat All" on attrition has not been systematically reviewed. We aimed to review existing literature to compare attrition 12 months after antiretroviral therapy (ART) initiation, before and after "Treat All" was implemented in Sub-Saharan Africa and describe predictors of attrition., Methods: We searched Embase, Google Scholar, PubMed, and Web of Science in July 2020 and created alerts up to the end of June 2023. We also searched for preprints and conference abstracts. Two co-authors screened and selected the articles. Risk of bias was assessed using the modified Newcastle-Ottawa Scale. We extracted and tabulated data on study characteristics, attrition 12 months after ART initiation, and predictors of attrition. We calculated a pooled risk ratio for attrition using random-effects meta-analysis., Results: Eight articles and one conference abstract (nine studies) out of 8179 screened records were included in the meta-analysis. The random-effects adjusted pooled risk ratio (RR) comparing attrition before and after "Treat All" 12 months after ART initiation was not significant [RR = 1.07 (95% Confidence interval (CI): 0.91-1.24)], with 92% heterogeneity (I 2 ). Being a pregnant or breastfeeding woman, starting ART with advanced HIV, and starting ART within the same week were reported as risk factors for attrition both before and after "Treat All"., Conclusions: We found no significant difference in attrition before and after "Treat All" one year after ART initiation. While "Treat All" is being implemented widely, differentiated approaches to enhance retention should be prioritised for those subgroups at risk of attrition., Prospero Number: CRD42020191582 ., (© 2023. BioMed Central Ltd., part of Springer Nature.)

Background: Tumour apparent diffusion coefficient (ADC) from diffusion-weighted magnetic resonance imaging (MRI) is a putative pharmacodynamic/response biomarker but the relationship between drug-induced effects on the ADC and on the underlying pathology has not been adequately defined. Hypothesis: Changes in ADC during early chemotherapy reflect underlying histological markers of tumour response as measured by tumour regression grade (TRG). Methods: Twenty-six patients were enrolled in the study. Baseline, 14 days, and pre-surgery MRI were performed per study protocol. Surgical resection was performed in 23 of the enrolled patients; imaging-pathological correlation was obtained from 39 lesions from 21 patients. Results: There was no evidence of correlation between TRG and ADC changes at day 14 (study primary endpoint), and no significant correlation with other ADC metrics. In scans acquired one week prior to surgery, there was no significant correlation between ADC metrics and percentage of viable tumour, percentage necrosis, percentage fibrosis, or Ki67 index. Conclusions: Our hypothesis was not supported by the data. The lack of meaningful correlation between change in ADC and TRG is a robust finding which is not explained by variability or small sample size. Change in ADC is not a proxy for TRG in metastatic colorectal cancer.

Background: Ebola virus disease (EVD) outbreaks have emerged in Central and West Africa. EVD diagnosis relies principally on RT-PCR testing with GeneXpert®, which has logistical and cost restrictions at the peripheral level of the health system. Rapid diagnostic tests (RDTs) would offer a valuable alternative at the point-of-care to reduce the turn-around time, if they show good performance characteristics. We evaluated the performance of four EVD RDTs against the reference standard GeneXpert® on stored EVD positive and negative blood samples collected between 2018 and 2021 from outbreaks in eastern Democratic Republic of the Congo (DRC)., Methods: We conducted a prospective and observational study in the laboratory on QuickNavi-Ebola™, OraQuick® Ebola Rapid Antigen, Coris® EBOLA Ag K-SeT, and Standard® Q Ebola Zaïre Ag RDTs using left-over archived frozen EDTA whole blood samples. We randomly selected 450 positive and 450 negative samples from the EVD biorepositories in DRC, across a range of GeneXpert® cycle threshold values (Ct-values). RDT results were read by three persons and we considered an RDT result as "positive", when it was flagged as positive by at least two out of the three readers. We estimated the sensitivity and specificity through two independent generalized (logistic) linear mixed models (GLMM)., Findings: 476 (53%) of 900 samples had a positive GeneXpert Ebola result when retested. The QuickNavi-Ebola™ showed a sensitivity of 56.8% (95% CI 53.6-60.0) and a specificity of 97.5% (95% CI 96.2-98.4), the OraQuick® Ebola Rapid Antigen test displayed 61.6% (95% CI 57.0-65.9) sensitivity and 98.1% (95% CI 96.2-99.1) specificity, the Coris® EBOLA Ag K-SeT showed 25.0% (95% CI 22.3-27.9) sensitivity and 95.9% (95% CI 94.2-97.1) specificity, and the Standard® Q Ebola Zaïre Ag displayed 21.6% (95% CI 18.1-25.7) sensitivity and 99.1% (95% CI 97.4-99.7) specificity., Interpretation: None of the RDTs evaluated approached the "desired or acceptable levels" for sensitivity set out in the WHO target product profile, while all of the tests met the "desired level" for specificity. Nevertheless, the QuickNavi-Ebola™ and OraQuick® Ebola Rapid Antigen Test demonstrated the most favorable profiles, and may be used as frontline tests for triage of suspected-cases while waiting for RT-qPCR confirmatory testing., Funding: Institute of Tropical Medicine Antwerp/EDCTP PEAU-EBOV-RDC project., Competing Interests: Declaration of interests US CDC provided the Xpert® Ebola cartridges. FIND purchased OraQuick Ebola Rapid Antigen tests and donated to INRB through US CDC partnership. Institute of Tropical Medicine-Antwerp purchased Coris® Ag K-SeT and Standard® Q line Zaïre Ebola Rapid diagnostic tests with the financial support of the EDCTP PEAU- EBOV-RDC project under grant agreement RIA2018EF-2087, and through the FA5 DRC Program funded by the Directorate General for Development Cooperation and Humanitarian Aid (DGD) of the Belgian government. Hokkaido University provided QuickNavi Ebola rapid tests to INRB via Japanese International Cooperation Agency (JICA). Rodolphe Mérieux INRB-Goma Laboratory supported the study with laboratory supplies. DMB is a PhD fellow supported by the Belgian Directorate-general Development Cooperation and Humanitarian Aid. JB-P, ADW, FE-A, BKJ, FM-M, JDK, HK-M, EI-N, PM-K, PM-K, SM-M, MK, AT, PF, NMM, EMM, MAK-M, ET-T, PNF, SR, AL, AN-N, MEM, JMM, JJM-T, KKA, JvG, SA-M declare no competing interests., (Copyright © 2023. Published by Elsevier B.V.)

Diagnostic accuracy studies in pulmonary tuberculosis (PTB) are complicated by the lack of a perfect reference standard. This limitation can be handled using latent class analysis (LCA), assuming independence between diagnostic test results conditional on the true unobserved PTB status. Test results could remain dependent, however, e.g. with diagnostic tests based on a similar biological basis. If ignored, this gives misleading inferences. Our secondary analysis of data collected during the first year (May 2018 -May 2019) of a community-based multi-morbidity screening program conducted in the rural uMkhanyakude district of KwaZulu Natal, South Africa, used Bayesian LCA. Residents of the catchment area, aged ≥15 years and eligible for microbiological testing, were analyzed. Probit regression methods for dependent binary data sequentially regressed each binary test outcome on other observed test results, measured covariates and the true unobserved PTB status. Unknown model parameters were assigned Gaussian priors to evaluate overall PTB prevalence and diagnostic accuracy of 6 tests used to screen for PTB: any TB symptom, radiologist conclusion, Computer Aided Detection for TB version 5 (CAD4TBv5≥53), CAD4TBv6≥53, Xpert Ultra (excluding trace) and culture. Before the application of our proposed model, we evaluated its performance using a previously published childhood pulmonary TB (CPTB) dataset. Standard LCA assuming conditional independence yielded an unrealistic prevalence estimate of 18.6% which was not resolved by accounting for conditional dependence among the true PTB cases only. Allowing, also, for conditional dependence among the true non-PTB cases produced a 1.1% plausible prevalence. After incorporating age, sex, and HIV status in the analysis, we obtained 0.9% (95% CrI: 0.6, 1.3) overall prevalence. Males had higher PTB prevalence compared to females (1.2% vs. 0.8%). Similarly, HIV+ had a higher PTB prevalence compared to HIV- (1.3% vs. 0.8%). The overall sensitivity for Xpert Ultra (excluding trace) and culture were 62.2% (95% CrI: 48.7, 74.4) and 75.9% (95% CrI: 61.9, 89.2), respectively. Any chest X-ray abnormality, CAD4TBv5≥53 and CAD4TBv6≥53 had similar overall sensitivity. Up to 73.3% (95% CrI: 61.4, 83.4) of all true PTB cases did not report TB symptoms. Our flexible modelling approach yields plausible, easy-to-interpret estimates of sensitivity, specificity and PTB prevalence under more realistic assumptions. Failure to fully account for diagnostic test dependence can yield misleading inferences., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2023 Keter et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Background: As untreated visceral leishmaniasis (VL) is fatal, reliable diagnostics are pivotal for accurate treatment allocation. The current diagnostic algorithm for VL in Ethiopia, which is based on the rK39 rapid diagnostic test and microscopy of tissue smears, lacks sensitivity. This probably leads to missed cases and patients not receiving treatment., Methodology: We conducted a retrospective study on stored microscopically negative spleen and bone marrow smears from suspected VL patients collected at the Leishmaniasis Research and Treatment Center (LRTC) in Gondar, northern Ethiopia between June 2019 and November 2020. Sociodemographic, clinical and treatment data were collected and samples were tested by real-time PCR targeting kinetoplast DNA., Principle Findings: Among the 191 eligible samples (135 spleen and 56 bone marrow) with a microscopically negative and valid PCR result, 119 (62.3%) were positive by PCR, although Ct values for some were high (median 33.0). Approximately three quarters of these undiagnosed primary VL (77.3%) and relapse (69.6%) patients did not receive antileishmanial treatment. Of the 56 microscopically negative bone marrow samples, 46 (82.1%) were PCR positive, which is considerably higher compared to the microscopically negative spleen samples, for which 73 out of 135 (54.1%) were PCR positive. The odds of being PCR positive were significantly higher for bone marrow aspirates and higher when white blood cell values were lower and splenomegaly (in cm) was more pronounced., Conclusions: This study demonstrates that a lot of suspected VL patients remain undiagnosed and untreated. This indicates the urgent need for better diagnostics for VL in the East-African region. The outcomes of PCR positive should be closely monitored and treatment should be provided if the patient deteriorates. In resource limited settings, implementation of PCR on bone marrow aspirate smears of patients with low WBC values and splenomegaly could lead to considerable improvements in patient management., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2023 Melkamu et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Background: Mpox (formerly monkeypox) is a viral disease caused by the mpox virus (MPXV), endemic in Central and West Africa and currently causing a global outbreak of international concern. Much remains unknown about sample types most suited for mpox laboratory diagnosis. While it is established that high viral loads can be found in active skin lesions (currently the recommended mpox laboratory confirmation specimen type), WHO mpox testing guidelines encourage the use of oropharyngeal swabs as an additional sample type for mpox diagnosis and suggest investigating the value of other specimens like blood samples., Objective: In this study, we verified the value of select alternative specimen types for mpox laboratory confirmation., Methods: We included 25 patients with MPXV-confirmed skin lesions to compare diagnostic sensitivity of MPXV PCR testing on EDTA plasma and two upper respiratory specimens: oropharyngeal swabs and saliva., Results: In our patient cohort with MPXV-confirmed skin lesions, diagnostic sensitivity of MPXV PCR was 80% in EDTA plasma, 64% in oropharyngeal swabs, and 88% in saliva. MPXV viral loads were significantly higher in saliva compared to oropharyngeal swabs and EDTA plasma., Discussion: The WHO recommendation to collect oropharyngeal swabs as an additional specimen for mpox diagnosis might need to be revised to include saliva wherever feasible. We suggest investigating saliva as a diagnostic specimen in the absence of active skin lesions or during the phase preceding skin manifestations. Moreover, the relatively high MPXV DNA content of saliva warrants elucidating its potential role in disease transmission., Competing Interests: Declaration of Competing Interest None, (Copyright © 2022 The Author(s). Published by Elsevier B.V. All rights reserved.)

Background: The control of Ebola virus disease (EVD) outbreaks relies on rapid diagnosis and prompt action, a daunting task in limited-resource contexts. This study develops prediction scores that can help healthcare workers improve their decision-making at the triage-point of EVD suspect-cases during EVD outbreaks., Methods: We computed accuracy measurements of EVD predictors to assess their diagnosing ability compared with the reference standard GeneXpert® results, during the eastern DRC EVD outbreak. We developed predictive scores using the Spiegelhalter-Knill-Jones approach and constructed a clinical prediction score (CPS) and an extended clinical prediction score (ECPS). We plotted the receiver operating characteristic curves (ROCs), estimated the area under the ROC (AUROC) to assess the performance of scores, and computed net benefits (NB) to assess the clinical utility (decision-making ability) of the scores at a given cut-off. We performed decision curve analysis (DCA) to compare, at a range of threshold probabilities, prediction scores' decision-making ability and to quantify the number of unnecessary isolation., Results: The analysis was done on data from 10432 subjects, including 651 EVD cases. The EVD prevalence was 6.2% in the whole dataset, 14.8% in the subgroup of suspects who fitted the WHO Ebola case definition, and 3.2% for the set of suspects who did not fit this case definition. The WHO clinical definition yielded 61.6% sensitivity and 76.4% specificity. Fatigue, difficulty in swallowing, red eyes, gingival bleeding, hematemesis, confusion, hemoptysis, and a history of contact with an EVD case were predictors of EVD. The AUROC for ECPS was 0.88 (95%CI: 0.86-0.89), significantly greater than this for CPS, 0.71 (95%CI: 0.69-0.73) (p < 0.0001). At -1 point of score, the CPS yielded a sensitivity of 85.4% and specificity of 42.3%, and the ECPS yielded sensitivity of 78.8% and specificity of 81.4%. The diagnostic performance of the scores varied in the three disease contexts (the whole, fitting or not fitting the WHO case definition data sets). At 10% of threshold probability, e.g. in disease-adverse context, ECPS gave an NB of 0.033 and a net reduction of unnecessary isolation of 67.1%. Using ECPS as a joint approach to isolate EVD suspects reduces the number of unnecessary isolations by 65.7%., Conclusion: The scores developed in our study showed a good performance as EVD case predictors since their use improved the net benefit, i.e., their clinical utility. These rapid and low-cost tools can help in decision-making to isolate EVD-suspicious cases at the triage point during an outbreak. However, these tools still require external validation and cost-effectiveness evaluation before being used on a large scale., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2022 Tshomba et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Background: Long-lasting insecticidal nets (LLINs) are one of the key interventions in the global fight against malaria. Since 2014, mass distribution campaigns of LLINs aim for universal access by all citizens of Burundi. In this context, we assess the impact of LLINs mass distribution campaigns on malaria incidence, focusing on the endemic highland health districts. We also explored the possible correlation between observed trends in malaria incidence with any variations in climate conditions., Methods: Malaria cases for 2011-2019 were obtained from the National Health Information System. We developed a generalised additive model based on a time series of routinely collected data with malaria incidence as the response variable and timing of LLIN distribution as an explanatory variable to investigate the duration and magnitude of the LLIN effect on malaria incidence. We added a seasonal and continuous-time component as further explanatory variables, and health district as a random effect to account for random natural variation in malaria cases between districts., Results: Malaria transmission in Burundian highlands was clearly seasonal and increased non-linearly over the study period. Further, a fast and steep decline of malaria incidence was noted during the first year after mass LLIN distribution (p<0.0001). In years 2 and 3 after distribution, malaria cases started to rise again to levels higher than before the control intervention., Conclusion: This study highlights that LLINs did reduce the incidence in the first year after a mass distribution campaign, but in the context of Burundi, LLINs lost their impact after only 1 year., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2022. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

Background: In application studies of latent class analysis (LCA) evaluating imperfect diagnostic tests, residual dependence among the diagnostic tests still remain even after conditioning on the true disease status due to measured variables known to affect prevalence and/or alter diagnostic test accuracy. Presence of severe comorbidities such as HIV in pulmonary tuberculosis (PTB) diagnosis alter the prevalence of PTB and affect the diagnostic performance of the available imperfect tests in use. This violates two key assumptions of LCA: (1) that the diagnostic tests are independent conditional on the true disease status (2) that the sensitivity and specificity remain constant across subpopulations. This leads to incorrect inferences., Methods: Through simulation we examined implications of likely model violations on estimation of prevalence, sensitivity and specificity among passive case-finding presumptive PTB patients with or without HIV. Jointly conditioning on PTB and HIV, we generated independent results for five diagnostic tests and analyzed using Bayesian LCA with Probit regression, separately for sets of five and three diagnostic tests using four working models allowing: (1) constant PTB prevalence and diagnostic accuracy (2) varying PTB prevalence but constant diagnostic accuracy (3) constant PTB prevalence but varying diagnostic accuracy (4) varying PTB prevalence and diagnostic accuracy across HIV subpopulations. Vague Gaussian priors with mean 1 and unknown variance were assigned to the model parameters with unknown variance assigned Inverse Gamma prior., Results: Models accounting for heterogeneity in diagnostic accuracy produced consistent estimates while the model ignoring it produces biased estimates. The model ignoring heterogeneity in PTB prevalence only is less problematic. With five diagnostic tests, the model assuming homogenous population is robust to violation of the assumptions., Conclusion: Well-chosen covariate-specific adaptations of the model can avoid bias implied by recognized heterogeneity in PTB patient populations generating otherwise dependent test results in LCA., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2022 The Authors.)

Introduction: Despite HIV viral load (VL) monitoring being serial, most studies use a cross-sectional design to evaluate the virological status of a cohort. The objective of our study was to use a simplified approach to calculate viraemic-time: the proportion of follow-up time with unsuppressed VL above the limit of detection. We estimated risk factors for higher viraemic-time and whether viraemic-time predicted mortality in a second-line antiretroviral treatment (ART) cohort in Myanmar., Methods: We conducted a retrospective cohort analysis of people living with HIV (PLHIV) who received second-line ART for a period >6 months and who had at least two HIV VL test results between 01 January 2014 and 30 April 2018. Fractional logistic regression assessed risk factors for having higher viraemic-time and Cox proportional hazards regression assessed the association between viraemic-time and mortality. Kaplan-Meier curves were plotted to illustrate survival probability for different viraemic-time categories., Results: Among 1,352 participants, 815 (60.3%) never experienced viraemia, and 172 (12.7%), 214 (15.8%), and 80 (5.9%) participants were viraemic <20%, 20-49%, and 50-79% of their total follow-up time, respectively. Few (71; 5.3%) participants were ≥80% of their total follow-up time viraemic. The odds for having higher viraemic-time were higher among people with a history of injecting drug use (aOR 2.01, 95% CI 1.30-3.10, p = 0.002), sex workers (aOR 2.10, 95% CI 1.11-4.00, p = 0.02) and patients treated with lopinavir/ritonavir (vs. atazanavir; aOR 1.53, 95% CI 1.12-2.10, p = 0.008). Viraemic-time was strongly associated with mortality hazard among those with 50-79% and ≥80% viraemic-time (aHR 2.92, 95% CI 1.21-7.10, p = 0.02 and aHR 2.71, 95% CI 1.22-6.01, p = 0.01). This association was not observed in those with viraemic-time <50%., Conclusions: Key populations were at risk for having a higher viraemic-time on second-line ART. Viraemic-time predicts clinical outcomes. Differentiated services should target subgroups at risk for a higher viraemic-time to control both HIV transmission and mortality., Competing Interests: The authors have declared that no competing interests exist.

Background: Since the scale-up of routine viral load (VL) testing started in 2016, there is limited evidence on VL suppression rates under programmatic settings and groups at risk of non-suppression. We conducted a study to estimate VL non-suppression (> 1000 copies/ml) and its risk factors using "routine" and "repeat after enhanced adherence counselling (EAC)" VL results., Methods: We conducted an analytic cross-sectional study using secondary VL testing data collected between 2014 and 2018 from a centrally located laboratory. We analysed data from routine tests and repeat tests after an individual received EAC. Our outcome was viral load non-suppression. Bivariable and multivariable logistic regression was performed to identify factors associated with having VL non-suppression for routine and repeat VL., Results: We analysed 103,609 VL test results (101,725 routine and 1884 repeat test results) collected from the country's ten provinces. Of the 101,725 routine and 1884 repeat VL tests, 13.8% and 52.9% were non-suppressed, respectively. Only one in seven (1:7) of the non-suppressed routine VL tests had a repeat test after EAC. For routine VL tests; males (vs females, adjusted odds ratio (aOR) = 1.19, [95% CI 1.14-1.24]) and adolescents (10-19 years) (vs adults (25-49 years), aOR = 3.11, [95% CI 2.9-3.31]) were more at risk of VL non-suppression. The patients who received care at the secondary level (vs primary, aOR = 1.21, [95% CI 1.17-1.26]) and tertiary level (vs primary, aOR = 1.63, [95% CI 1.44-1.85]) had a higher risk of VL non-suppression compared to the primary level. Those that started ART in 2014-2015 (vs < 2010, aOR = 0.83, [95% CI 0.79-0.88]) and from 2016 onwards (vs < 2010, aOR = 0.84, [95% CI 0.79-0.89]) had a lower risk of VL non-suppression. For repeat VL tests; young adults (20-24 years) (vs adults (25-49 years), (aOR) = 3.48, [95% CI 2.16 -5.83]), adolescents (10-19 years) (vs adults (25-49 years), aOR = 2.76, [95% CI 2.11-3.72]) and children (0-9 years) (vs adults (25-49 years), aOR = 1.51, [95% CI 1.03-2.22]) were at risk of VL non-suppression., Conclusion: Close to 90% suppression in routine VL shows that Zimbabwe is on track to reach the third UNAIDS target. Strategies to improve the identification of clients with high routine VL results for repeating testing after EAC and ART adherence in subpopulations (men, adolescents and young adolescents) at risk of viral non-suppression should be prioritised., (© 2022. The Author(s).)

Introduction: The number of individuals initiating antiretroviral pre-exposure prophylaxis (PrEP) is increasing, but we do not fully understand who is coming forward for PrEP, how they use it and how they are followed-up. The objective of this study was to examine PrEP user profiles, dynamics in PrEP use and follow-up over time., Methods: We conducted a cohort analysis of longitudinally collected clinical record and questionnaire data among PrEP users at an HIV centre in Antwerp, Belgium, between June 2017 and March 2020. PrEP follow-up and user profiles were examined using descriptive analyses and bivariate logistic regression. We compared early adopting PrEP users (started before June 2018) with late users. We also calculated the probabilities of switching between daily and on-demand PrEP, and interruption, using a naïve estimator., Results and Discussion: We included 1347 PrEP users in the analysis. After 12 months, retention in care was 72.3%. Median time between PrEP visits was 98 days (IQR 85-119 days). At screening visit, early adopting PrEP users (starting June 2017-May 2018) were significantly more likely to report one or more sexually transmitted infection in the prior 12 months, having used drugs during sex, a higher number of sexual partners and a history of paid sex and PrEP use prior to initiation, compared with PrEP users who initiated later (starting June 2018-February 2020). When taking PrEP daily, the probability of staying on daily PrEP at the next visit was 76%, while this was 73% when taking PrEP on-demand. Those using on-demand PrEP had a higher probability (13%) of interrupting PrEP care than daily PrEP users (7%), whereas those returning to PrEP care would mostly re-start with on-demand (35% vs. 13% for daily)., Conclusions: The majority of PrEP users in this sample remained in care after 12 months. The probability of remaining on the same PrEP regimen at the subsequent visit was high. Though, we observed a diversity of transitions between regimens and interruptions in between visits. Our findings reaffirm the need to provide tailored PrEP services, counselling PrEP users across their life course., (© 2022 The Authors. Journal of the International AIDS Society published by John Wiley & Sons Ltd on behalf of the International AIDS Society.)

Background: The Democratic Republic of the Congo has confronted 13 outbreaks of Ebola virus disease since 1976. Rapid diagnostic tests (RDTs) detecting viral antigens have been developed to circumvent difficulties encountered with RT-PCR for diagnosis in remote low-resource settings, but there is still uncertainty about their performance characteristics and usability during outbreaks. We aimed to assess the field performance of three antigen detection RDTs compared with the gold-standard Cepheid GeneXpert Ebola assay results., Methods: We conducted a retrospective, multicentre observational study using complete and de-identified databases of five mobile laboratories (managed by the Institut National de Recherche Biomédicale) to assess the performance of three Ebola virus disease RDTs (QuickNavi-Ebola, OraQuick Ebola Rapid Antigen Test, and Coris EBOLA Ag K-SeT rapid test) run on blood samples of patients with suspected Ebola virus disease in direct comparison with the Cepheid GeneXpert Ebola assay reference test during the 2018-20 outbreak in the eastern Democratic Republic of the Congo. We estimated the sensitivity and specificity of each test through generalised linear mixed models against the GeneXpert Ebola assay reference test and corrected for cycle threshold value and random site effects., Findings: 719 (7·9%) of 9157 samples had a positive GeneXpert Ebola assay result. The QuickNavi-Ebola RDT had a sensitivity of 87·4% (95% CI 63·6-96·8) around the mean cycle threshold value and a specificity of 99·6% (99·3-99·8). The OraQuick Ebola Rapid Antigen Test had a sensitivity of 57·4% (95% CI 38·8-75·8) and specificity of 98·3% (97·5-99·0), and the Coris EBOLA Ag K-SeT rapid test had a sensitivity of 38·9% (23·0-63·6) against the GeneXpert Ebola assay reference and specificity of 97·4% (85·3-99·6). The QuickNavi-Ebola RDT showed a robust performance with good sensitivity, particularly with increasing viral loads (ie, low cycle threshold values), and specificity., Interpretation: The three RDTs evaluated did not achieve the desired sensitivity and specificity of the WHO target product profile. Although the RDTs cannot triage and rule out Ebola virus infection among clinical suspects, they can still help to sort people with suspected Ebola virus disease into high-risk and low-risk groups while waiting for GeneXpert Ebola assay reference testing., Funding: None., Translation: For the French translation of the abstract see Supplementary Materials section., Competing Interests: Declaration of interests WHO donated GeneXpert cartridges and laboratory supplies used during the tenth outbreak of Ebola virus disease in the Democratic Republic of the Congo. WHO, Africa Centers for Disease Control and Prevention, Institute of Tropical Medicine Antwerp, and Japan International Cooperation Agency donated GeneXpert instruments for response purposes. US National Institute of Allergy and Infectious Diseases/National Institutes of Health provided GeneXpert instruments and laboratory supplies in the context of the PALM randomised controlled trial during the tenth outbreak of Ebola virus disease. Centers for Disease Control and Prevention–Atlanta donated OraQuick Ebola Rapid Antigen tests for the response. Denka and Hokkaido University, Sapporo, Japan, provided QuickNavi-Ebola rapid tests via the Japan International Cooperation Agency. Coris Bioconcept supplied Coris K SeT Ag tests via Institut Pasteur de Dakar. DM-B is a Clinical Research During Outbreaks fellow supported by the Belgian Directorate-general for Development Cooperation and Humanitarian Aid. All other authors declare no competing interests., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

It remains unclear why patients discontinue HIV pre-exposure prophylaxis (PrEP) care and to what extent they remain at risk for HIV when they do. We reviewed routinely collected medical records and patient questionnaires and performed an e-mail/telephone survey to assess reasons for discontinuing PrEP care, ongoing risks for HIV infection, and associated factors. Patients with more than two registered PrEP visits from a PrEP clinic in Antwerp, Belgium between June 2017 and February 2020 were included in this study. Patients who did not return for a visit after October 30, 2019 and who were not transferred out were considered as having discontinued PrEP care. A total of 143/1073 patients were considered as having discontinued PrEP care. Patients who discontinued PrEP care were more likely to be younger than those who remained in care (35 vs. 38 years old, p  < 0.01). The most common reasons for discontinuation were having stopped using PrEP (62/101, 61.4%) and "COVID-19" ( n  = 35, 34.7%). The most common reasons for stopping PrEP use was a decreased sexual activity due to coronavirus disease 2019 (COVID-19; 21/62, 33.9%) or not COVID-19 related (10/62, 16.1%), a monogamous relationship (20/62, 32.3%) and consistent condom use (7/62, 11.3%). Among respondents who reported about current HIV risk the majority reported being at low risk either by still taking PrEP (32/91, 35.2%), consistently using condoms, or limiting number of sex acts or partners (58/91, 52.7%). No HIV seroconversion was reported.

In most low-resource settings, microscopy still is the standard method for diagnosis of cutaneous leishmaniasis, despite its limited sensitivity. In Ethiopia, the more sensitive molecular methods are not yet routinely used. This study compared five PCR methods with microscopy on two sample types collected from patients with a suspected lesion to advise on optimal diagnosis of Leishmania aethiopica. Between May and July 2018, skin scrapings (SS) and blood exudate from the lesion spotted on filter paper (dry blood spot, DBS) were collected for PCR from 111 patients of four zones in Southern Ethiopia. DNA and RNA were simultaneously extracted from both sample types. DNA was evaluated by a conventional PCR targeting ITS-1 and three probe-based real-time PCRs: one targeting the SSU 18S rRNA and two targeting the kDNA minicircle sequence (the 'Mary kDNA PCR' and a newly designed 'LC kDNA PCR' for improved L. aethiopica detection). RNAs were tested with a SYBR Green-based RT-PCR targeting spliced leader (SL) RNA. Giemsa-stained SS smears were examined by microscopy. Of the 111 SS, 100 were positive with at least two methods. Sensitivity of microscopy, ITS PCR, SSU PCR, Mary kDNA PCR, LC kDNA PCR and SL RNA PCR were respectively 52%, 22%, 64%, 99%, 100% and 94%. Microscopy-based parasite load correlated well with real-time PCR Ct-values. Despite suboptimal sample storage for RNA detection, the SL RNA PCR resulted in congruent results with low Ct-values. DBS collected from the same lesion showed lower PCR positivity rates compared to SS. The kDNA PCRs showed excellent performance for diagnosis of L. aethiopica on SS. Lower-cost SL RNA detection can be a complementary high-throughput tool. DBS can be used for PCR in case microscopy is negative, the SS sample can be sent to the referral health facility where kDNA PCR method is available., Competing Interests: The authors have declared that no competing interests exist.

Standard data analysis pipelines for digital PCR estimate the concentration of a target nucleic acid by digitizing the end-point fluorescence of the parallel micro-PCR reactions, using an automated hard threshold. While it is known that misclassification has a major impact on the concentration estimate and substantially reduces accuracy, the uncertainty of this classification is typically ignored. We introduce a model-based clustering method to estimate the probability that the target is present (absent) in a partition conditional on its observed fluorescence and the distributional shape in no-template control samples. This methodology acknowledges the inherent uncertainty of the classification and provides a natural measure of precision, both at individual partition level and at the level of the global concentration. We illustrate our method on genetically modified organism, inhibition, dynamic range, and mutation detection experiments. We show that our method provides concentration estimates of similar accuracy or better than the current standard, along with a more realistic measure of precision. The individual partition probabilities and diagnostic density plots further allow for some quality control. An R implementation of our method, called Umbrella, is available, providing a more objective and automated data analysis procedure for absolute dPCR quantification.

Background: Digital polymerase chain reaction (dPCR) is an increasingly popular technology for detecting and quantifying target nucleic acids. Its advertised strength is high precision absolute quantification without needing reference curves. The standard data analytic approach follows a seemingly straightforward theoretical framework but ignores sources of variation in the data generating process. These stem from both technical and biological factors, where we distinguish features that are 1) hard-wired in the equipment, 2) user-dependent and 3) provided by manufacturers but may be adapted by the user. The impact of the corresponding variance components on the accuracy and precision of target concentration estimators presented in the literature is studied through simulation., Results: We reveal how system-specific technical factors influence accuracy as well as precision of concentration estimates. We find that a well-chosen sample dilution level and modifiable settings such as the fluorescence cut-off for target copy detection have a substantial impact on reliability and can be adapted to the sample analysed in ways that matter. User-dependent technical variation, including pipette inaccuracy and specific sources of sample heterogeneity, leads to a steep increase in uncertainty of estimated concentrations. Users can discover this through replicate experiments and derived variance estimation. Finally, the detection performance can be improved by optimizing the fluorescence intensity cut point as suboptimal thresholds reduce the accuracy of concentration estimates considerably., Conclusions: Like any other technology, dPCR is subject to variation induced by natural perturbations, systematic settings as well as user-dependent protocols. Corresponding uncertainty may be controlled with an adapted experimental design. Our findings point to modifiable key sources of uncertainty that form an important starting point for the development of guidelines on dPCR design and data analysis with correct precision bounds. Besides clever choices of sample dilution levels, experiment-specific tuning of machine settings can greatly improve results. Well-chosen data-driven fluorescence intensity thresholds in particular result in major improvements in target presence detection. We call on manufacturers to provide sufficiently detailed output data that allows users to maximize the potential of the method in their setting and obtain high precision and accuracy for their experiments.