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Background: The cobas Plasma Separation Card (PSC; Roche Diagnostics) was developed for HIV viral load testing. This study evaluates the performance of HIV and Treponema pallidum (Tp) antibody (Ab) detection on PSCs as an alternative to dried blood spots (DBSs)., Methods: EDTA whole blood samples were collected from HIV-positive (n = 100), HIV-negative (n = 50), Tp-positive (n = 100), and Tp-negative patients (n = 50) and spotted on DBS and PSC. Antibody detection performance was evaluated for HIV Ab using the Genscreen ULTRA HIV Ag-Ab test (Bio-Rad) and for Tp Ab using the Syphilis Total Ab test (Bio-Rad). Plasma was used as a reference specimen., Results: Receiver operating characteristic curve analysis for DBS and PSC generated areas under the curve (AUC + 95% confidence interval) of 0.985 (0.960-1.000) and 0.987 (0.973-1.000) for HIV Ab and 1.000 (1.000-1.000) and 0.996 (0.983-1.000) for Tp Ab, respectively. Receiver operating characteristic areas under the curve were not significantly different between DBS and PSC for HIV or TP Ab. At selected cutoff values rendering at least 99% sensitivity for HIV Ab detection, the specificity was 96% on DBS and 68% on PSC. For Tp Ab detection at 90% sensitivity, 100% specificity is reached on both DBS and PSC (exceeding the required 95%). However, the median quantitative HIV and Tp Ab signal of positive samples significantly decreased in PSC compared with DBS and plasma., Conclusions: Although receiver operating characteristic analysis does not seem to indicate significant differences in performance between DBS and PSC, the significant reduction in quantitative Ab detection signal dictates card composition optimization before its use for HIV and Tp Ab detection can be advised., Competing Interests: Conflict of Interest and Sources of Funding: None to declare., (Copyright © 2023 American Sexually Transmitted Diseases Association. All rights reserved.)

Vaccine-induced seroreactivity/positivity (VISR/P) poses a significant and common challenge to HIV vaccine implementation, as up to 95% of vaccine recipients may be misclassified as having HIV infection by current HIV screening and confirmatory serological assays. We investigated whether internal HIV proteins could be used to overcome VISR and discovered a set of 4 antigens (gp41 endodomain, p31 integrase, p17 matrix protein, and Nef) that are recognized by antibodies produced in individuals with HIV infection but not in vaccinated individuals. When evaluated in a multiplex double-antigen bridging ELISA, this antigen combination had specificities of 98.1% prevaccination and 97.1% postvaccination, demonstrating the assay is minimally impacted by vaccine-induced antibodies. The sensitivity was 98.5%, further increasing to 99.7% when p24 antigen testing was included. Results were similar across HIV-1 clades. Although more technical advancements will be desired, this research provides the groundwork for the development of new fourth-generation HIV tests unaffected by VISR. IMPORTANCE While the detection of HIV infection is accomplished by several methods, the most common are serological tests that detect host antibodies produced in response to viral infection. However, the use of current serological tests may present a significant challenge to the adoption of an HIV vaccine in the future because the antibodies to HIV antigens detected in currently available tests also tend to be included as antigens in the HIV vaccines in development. The use of these serological tests may thus result in the misclassification of vaccinated HIV-negative individuals, which can have potential for significant harms for individuals and could prevent the widespread adoption and implementation of HIV vaccines. Our study aimed to identify and evaluate target antigens for inclusion in new serological tests that can be used to identify HIV infections without interference from vaccine-induced antibodies but also fit within existing platforms for HIV diagnostics., Competing Interests: The authors declare a conflict of interest. O.L., J.V., and W.C. are current employees of Janssen Pharmaceutica NV, and D.J.S., V.O.M., and M.G.P. are current employees of Janssen Vaccines & Prevention B.V., both being Johnson & Johnson Companies and they may own stock or stock options in that company. L.L. is a consultant for Janssen Vaccines and Prevention B.V. The remaining co-authors have no conflicts of interest with the content of this article.

The risk of infection after exposure to clade IIb mpox virus (MPXV) is unknown, and potential presymptomatic shedding of MPXV remains to be demonstrated. High-risk contacts of mpox patients were followed-up in a prospective longitudinal cohort study. Individuals reporting sexual contact, >15 min skin-to-skin contact, or living in the same household with an mpox patient were recruited in a sexual health clinic in Antwerp, Belgium. Participants kept a symptom diary, performed daily self-sampling (anorectal, genital, and saliva), and presented for weekly clinic visits for physical examination and sampling (blood and oropharyngeal). Samples were tested for MPXV by PCR. Between June 24 and July 31, 2022, 25 contacts were included, of which 12/18 (66.0%) sexual and 1/7 (14.0%) nonsexual contacts showed evidence of infection by MPXV-PCR. Six cases had typical mpox symptoms. Viral DNA was detected as early as 4 days before symptom onset in 5 of them. In 3 of these cases, replication-competent virus was demonstrated in the presymptomatic phase. These findings confirm the existence of presymptomatic shedding of replication-competent MPXV and emphasize the high risk of transmission during sexual contact. Sexual contacts of mpox cases should abstain from sex during the incubation period, irrespective of symptoms., (© 2023 The Authors. Journal of Medical Virology published by Wiley Periodicals LLC.)

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.)

The magnitude of the 2022 multi-country monkeypox virus (MPXV) outbreak has surpassed any preceding outbreak. It is unclear whether asymptomatic or otherwise undiagnosed infections are fuelling this epidemic. In this study, we aimed to assess whether undiagnosed infections occurred among men attending a Belgian sexual health clinic in May 2022. We retrospectively screened 224 samples collected for gonorrhea and chlamydia testing using an MPXV PCR assay and identified MPXV-DNA-positive samples from four men. At the time of sampling, one man had a painful rash, and three men had reported no symptoms. Upon clinical examination 21-37 days later, these three men were free of clinical signs, and they reported not having experienced any symptoms. Serology confirmed MPXV exposure in all three men, and MPXV was cultured from two cases. These findings show that certain cases of monkeypox remain undiagnosed and suggest that testing and quarantining of individuals reporting symptoms may not suffice to contain the outbreak., (© 2022. The Author(s).)

Background: Hepatitis C virus (HCV) screening is critical to HCV elimination efforts. Simplified diagnostics are required for low-resource settings and difficult-to-reach populations. This retrospective study assessed performance of rapid diagnostic tests (RDTs) for detection of HCV antibodies., Methods: Two lots of 13 RDTs were evaluated at 3 laboratories using archived plasma samples from 4 countries (Nigeria, Georgia, Cambodia, and Belgium). HCV status was determined using 3 reference tests according to a composite algorithm. Sensitivity and specificity were evaluated in HIV-infected and HIV-uninfected populations. Operational characteristics were also assessed., Results: In total, 1710 samples met inclusion criteria. In HIV-uninfected samples (n = 384), the majority of RDTs had sensitivity ≥98% in 1 or both lots and most RDTs had specificity ≥99%. In HIV-infected samples (n = 264), specificity remained high but sensitivity was markedly lower than in HIV-uninfected samples; only 1 RDT reached >95%. The majority of HIV-infected samples for which sensitivity was low did not have detectable HCV viral load/core antigen. Interreader variability, lot-to-lot variability, and rate of invalid runs were low for all RDTs (<2%)., Conclusions: HCV RDTs should be evaluated in the intended target population, as sensitivity can be impacted by population factors such as HIV status., Clinical Trials Registration: NCT04033887., (© The Author(s) 2020. Published by Oxford University Press for the Infectious Diseases Society of America.)

Background: To assess the prevalence and evolution of transmitted drug resistance (TDR) in Belgium, a total of 3708 baseline human immunodeficiency virus (HIV)-1 polymerase sequences from patients diagnosed between 2013 and 2019 were analyzed., Methods: Protease and reverse-transcriptase HIV-1 sequences were collected from the 7 national Aids Reference Laboratories. Subtype determination and drug resistance scoring were performed using the Stanford HIV Drug Resistance Database. Trends over time were assessed using linear regression, and the maximum likelihood approach was used for phylogenetic analysis., Results: A total of 17.9% of the patients showed evidence of TDR resulting in at least low-level resistance to 1 drug (Stanford score  ≥15). If only the high-level mutations (Stanford score ≥60) were considered, TDR prevalence dropped to 6.3%. The majority of observed resistance mutations impacted the sensitivity for nonnucleoside reverse-transcriptase inhibitors (NNRTIs) (11.4%), followed by nucleoside reverse-transcriptase inhibitors (6.2%) and protease inhibitors (2.4%). Multiclass resistance was observed in 2.4%. Clustered onward transmission was evidenced for 257 of 635 patients (40.5%), spread over 25 phylogenetic clusters., Conclusions: The TDR prevalence remained stable between 2013 and 2019 and is comparable to the prevalence in other Western European countries. The high frequency of NNRTI mutations requires special attention and follow-up. Phylogenetic analysis provided evidence for local clustered onward transmission of some frequently detected mutations., (© The Author(s) 2022. Published by Oxford University Press on behalf of Infectious Diseases Society of America.)

COVID-19 Antibody Detecting Rapid Diagnostic Tests (COVID-19 Ab RDTs) are the preferred tool for SARS-CoV-2 seroprevalence studies, particularly in low- and middle-income countries. The present study challenged COVID-19 Ab RDTs with pre-pandemic samples of patients exposed to tropical pathogens. A retrospective study was performed on archived serum ( n = 94) and EDTA whole blood ( n = 126) samples obtained during 2010-2018 from 196 travelers with malaria ( n = 170), schistosomiasis ( n = 25) and dengue ( n = 25). COVID-19 Ab RDTs were selected based on regulatory approval status, independent evaluation results and detecting antigens. Among 13 COVID-19 Ab RDT products, overall cross-reactivity was 18.5%; cross-reactivity for malaria, schistosomiasis and dengue was 20.3%, 18.1% and 7.5%, respectively. Cross-reactivity for current and recent malaria, malaria antibodies, Plasmodium species and parasite densities was similar. Cross-reactivity among the different RDT products ranged from 2.7% to 48.9% (median value 14.5%). IgM represented 67.9% of cross-reactive test lines. Cross-reactivity was not associated with detecting antigens, patient categories or disease (sub)groups, except for schistosomiasis (two products with ≥60% cross-reactivity). The high cross-reactivity for malaria, schistosomiasis and-to a lesser extent-dengue calls for risk mitigation when using COVID-19 Ab RDTs in co-endemic regions.

Myotonic dystrophy type 1 (DM1) is caused by expansion of a CTG repeat in the DMPK gene, where expansion size and somatic mosaicism correlates with disease severity and age of onset. While it is known that the mismatch repair protein MSH2 contributes to the unstable nature of the repeat, its role on other disease-related features, such as CpG methylation upstream of the repeat, is unknown. In this study, we investigated the effect of an MSH2 knock-down (MSH2KD) on both CTG repeat dynamics and CpG methylation pattern in human embryonic stem cells (hESC) carrying the DM1 mutation. Repeat size in MSH2 wild-type (MSH2WT) and MSH2KD DM1 hESC was determined by PacBio sequencing and CpG methylation by bisulfite massive parallel sequencing. We found stabilization of the CTG repeat concurrent with a gradual loss of methylation upstream of the repeat in MSH2KD cells, while the repeat continued to expand and upstream methylation remained unchanged in MSH2WT control lines. Repeat instability was re-established and biased towards expansions upon MSH2 transgenic re-expression in MSH2KD lines while upstream methylation was not consistently re-established. We hypothesize that the hypermethylation at the mutant DM1 locus is promoted by the MMR machinery and sustained by a constant DNA repair response, establishing a potential mechanistic link between CTG repeat instability and upstream CpG methylation. Our work represents a first step towards understanding how epigenetic alterations and repair pathways connect and contribute to the DM1 pathology., (© The Author(s) 2020. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Background: This study evaluated performance of two hepatitis C virus (HCV) rapid diagnostic tests (RDTs) performed by intended users in resource-limited settings., Methods: Testing was conducted at three facilities in two countries (Georgia, Cambodia) using matched fingerstick whole blood, plasma and serum samples. Investigational RDTs were compared with a composite reference standard (CRS) comprised of three laboratory tests, and a reference RDT., Results: In matched samples from 489 HCV positive and 967 HCV negative participants, specificity with both investigational RDTs was high using either reference method (≥98.4% in all sample types). Sensitivity was lower in whole blood versus plasma and serum for both RDTs compared with the CRS (86.5-91.4% vs 97.5-98.0% and 97.3-97.1%) and reference RDT (93.6-97.8% vs 100% and 99.4%). Sensitivity improved when considering only samples with detectable HCV viral load., Conclusion: Sensitivity was highest in serum and plasma versus whole blood. The World Health Organization prequalification criterion (≥98%) was narrowly missed by both RDTs in serum, and one in plasma, possibly due to the intended user factor. Performance in whole blood was considered adequate, given potential roles of HCV infection history, improved sensitivity with detectable viral load and performance similarities to the reference RDT., Competing Interests: The authors have declared that no competing interests exist.

This retrospective study evaluated the reactivity of 3 human immunodeficiency virus (HIV) confirmatory assays (INNO-LIA, Geenius, and MP) and 7 HIV rapid tests on samples from 2 different study populations in Belgium. For the early-treated cohort (83 HIV-1 adult patients treated within 3 months after infection), HIV-1 diagnosis was not obtained in at least 1 confirmatory assay in 12.0% (10/83) and in an HIV rapid test in 31.3% (26/83). Confirmation assay sensitivities ranged from 87.5% to 95.2%, whereas rapid test assay sensitivities ranged from 75.9% to 100%. The time to treatment initiation or the length of time on treatment did not have a statistical influence on the probability to obtain a false-negative test result. The fastest reversion was demonstrated after 4 months of treatment. Among the long-term treated cohort (390 HIV-1 patients with ≥ 9 years of undetectable viral load), false-negative test results were found in at least 1 HIV confirmatory assay for 2.1% (8/390) of the patients and in a HIV rapid test for 4.9% (19/390). Confirmation assay sensitivities ranged from 98.1% to 99.5%, whereas rapid test sensitivities ranged from 96.2% to 100%. Longer treatment increased nonreactivity of the HIV rapid tests (P = .033). Undetectable viral load decreases the sensitivities of HIV diagnostic tests, and further monitoring of the performance of serological assays is advised., (© The Author(s) 2020. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, e-mail: journals.permissions@oup.com.)

The HIV-1 epidemic in Belgium is primarily driven by MSM. In this patient population subtype B predominates but an increasing presence of non-B subtypes has been reported. We aimed to define to what extent the increasing subtype heterogeneity in a high at risk population induces the formation and spread of new recombinant forms. The study focused on transmission networks that reflect the local transmission to an important extent. One hundred and five HIV-1 transmission clusters were identified after phylogenetic analysis of 2849 HIV-1 pol sequences generated for the purpose of baseline drug resistance testing between 2013 and 2017. Of these 105 clusters, 62 extended in size during the last two years and were therefore considered as representing ongoing transmission. These 62 clusters included 774 patients in total. From each cluster between 1 and 3 representative patients were selected for near full-length viral genome sequencing. In total, the full genome sequence of 101 patients was generated. Indications for the presence of a new recombinant form were found for 10 clusters. These 10 clusters represented 105 patients or 13.6% of the patients covered by the study. The findings clearly show that new recombinant strains highly contribute to local transmission, even in an epidemic that is largely MSM and subtype B driven. This is an evolution that needs to be monitored as reshuffling of genome fragments through recombination may influence the transmissibility of the virus and the pathology of the infection. In addition, important changes in the sequence of the viral genome may challenge the performance of tests used for diagnosis, patient monitoring and drug resistance analysis., Competing Interests: Declaration of Competing Interest None., (Copyright © 2020 Elsevier B.V. All rights reserved.)

HIV-1 pol sequences obtained through baseline drug resistance testing of patients newly diagnosed between 2013 and 2017 were analyzed for genetic similarity. For 927 patients the information on genetic similarity was combined with demographic data and with information on the recency of infection. Overall, 48.3% of the patients were genetically linked with 11.4% belonging to a pair and 36.9% involved in a cluster of ≥3 members. The percentage of early diagnosed (≤4 months after infection) was 28.6%. Patients of Belgian origin were more frequently involved in transmission clusters (49.7% compared to 15.3%) and diagnosed earlier (37.4% compared to 12.2%) than patients of Sub-Saharan African origin. Of the infections reported to be locally acquired, 69.5% were linked (14.1% paired and 55.4% in a cluster). Equal parts of early and late diagnosed individuals (59.9% and 52.4%, respectively) were involved in clusters. The identification of a genetically linked individual for the majority of locally infected patients suggests a high rate of diagnosis in this population. Diagnosis however is often delayed for >4 months after infection increasing the opportunities for onward transmission. Prevention of local infection should focus on earlier diagnosis and protection of the still uninfected members of sexual networks with human immunodeficiency virus (HIV)-infected members.