Prevalence and Evolution of Transmitted Human Immunodeficiency Virus Drug Resistance in Belgium Between 2013 and 2019.

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.

Moderate forms of COVID-19 among healthcare workers in a Belgian hospital and their immune response.

INTRODUCTION: Healthcare professionals have been treating patients with COVID-19 since the pandemic started in early 2020 while also trying to limit disease spread among their coworkers and communities. This study aimed to identify and follow potentially infected healthcare workers in one hospital in order to develop an epidemiological baseline for COVID-19 infection and spread rates in this population. MATERIALS AND METHODS: This prospective study was conducted between 1 April and 30 June 2020 at a single Belgian hospital. Healthcare workers with symptoms consistent with COVID-19 were included. Participants underwent testing for SARS-CoV-2 infection by nasopharyngeal (NP) swab and analysis of blood samples for antibody response at different timepoints (day 0, 7, 14 and day 30 or 60). Patient exposures, symptoms, and disease progression were collected. RESULTS: Of a total of 150 healthcare workers with symptoms compatible with SARS-CoV-2 infection, 31% (47) tested positive for the virus by NP swab. Of the 47 participants with positive NP swabs, 66% also had positive IgG serology. Of the 99 participants with negative NP swabs who underwent blood sample collection, 5% had positive IgG serology results. Of the 29 participants who presented with at least 3 major symptoms, 15 (52%) had positive NP swabs and 14 (48%) had positive serology. CONCLUSIONS: This study demonstrates that evidence of symptoms, even when major, is a poor predictor for SARS-CoV-2 positivity in health care workers and raises the question of the best way to efficiently screen this population especially during the upcoming flu period.

Chronic and Early Antiretroviral Therapy Impact Human Immunodeficiency Virus (HIV) Serological Assay Sensitivity, Leading to More False-Negative Test Results in HIV Diagnosis.

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.

Earlier Initiation of Antiretroviral Treatment Coincides With an Initial Control of the HIV-1 Sub-Subtype F1 Outbreak Among Men-Having-Sex-With-Men in Flanders, Belgium.

Human immunodeficiency virus type 1 (HIV-1) non-B subtype infections occurred in Belgium since the 1980s, mainly amongst migrants and heterosexuals, whereas subtype B predominated in men-having-sex-with-men (MSM). In the last decade, the diagnosis of F1 sub-subtype in particular has increased substantially, which prompted us to perform a detailed reconstruction of its epidemiological history. To this purpose, the Belgian AIDS Reference Laboratories collected HIV-1 pol sequences from all sub-subtype F1-infected patients for whom genotypic drug resistance testing was requested as part of routine clinical follow-up. This data was complemented with HIV-1 pol sequences from countries with a high burden of F1 infections or a potential role in the global origin of sub-subtype F1. The molecular epidemiology of the Belgian subtype F1 epidemic was investigated using Bayesian phylogenetic inference and transmission dynamics were characterized based on birth-death models. F1 sequences were retained from 297 patients diagnosed and linked to care in Belgium between 1988 and 2015. Phylogenetic inference indicated that among the 297 Belgian F1 sequences, 191 belonged to a monophyletic group that mainly contained sequences from people likely infected in Belgium (OR 26.67, 95% CI 9.59-74.15), diagnosed in Flanders (OR 7.28, 95% CI 4.23-12.53), diagnosed at a recent stage of infection (OR 7.19, 95% CI 2.88-17.95) or declared to be MSM (OR 34.8, 95% CI 16.0-75.6). Together with a Spanish clade, this Belgian clade was embedded in the genetic diversity of Brazilian subtype F1 strains and most probably emerged after one or only a few migration events from Brazil to the European continent before 2002. The origin of the Belgian outbreak was dated back to 2002 (95% higher posterior density 2000-2004) and birth-death models suggested that its extensive growth had been controlled (Re < 1) by 2012, coinciding with a time period where delay in antiretroviral treatment initiation substantially declined. In conclusion, phylogenetic reconstruction of the Belgian HIV-1 sub-subtype F1 epidemic illustrates the introduction and substantial dissemination of viral strains in a geographically restricted risk group that was most likely controlled by effective treatment as prevention.

Distribution of HCV genotypes in Belgium from 2008 to 2015.

BACKGROUND: The knowledge of circulating HCV genotypes and subtypes in a country is crucial to guide antiviral therapy and to understand local epidemiology. Studies investigating circulating HCV genotypes and their trends have been conducted in Belgium. However they are outdated, lack nationwide representativeness or were not conducted in the general population. METHODS: In order to determine the distribution of different circulating HCV genotypes in Belgium, we conducted a multicentre study with all the 19 Belgian laboratories performing reimbursed HCV genotyping assays. Available genotype and subtype data were collected for the period from 2008 till 2015. Furthermore, a limited number of other variables were collected: some demographic characteristics from the patients and the laboratory technique used for the determination of the HCV genotype. RESULTS: For the study period, 11,033 unique records collected by the participating laboratories were used for further investigation. HCV genotype 1 was the most prevalent (53.6%) genotype in Belgium, with G1a and G1b representing 19.7% and 31.6%, respectively. Genotype 3 was the next most prevalent (22.0%). Further, genotype 4, 2, and 5 were responsible for respectively 16.1%, 6.2%, and 1.9% of HCV infections. Genotype 6 and 7 comprise the remaining <1%. Throughout the years, a stable distribution was observed for most genotypes. Only for genotype 5, a decrease as a function of the year of analysis was observed, with respectively 3.6% for 2008, 2.3% for 2009 and 1.6% for the remaining years. The overall M:F ratio was 1.59 and was mainly driven by the high M:F ratio of 3.03 for patients infected with genotype 3. Patients infected with genotype 3 are also younger (mean age 41.7 years) than patients infected with other genotypes (mean age above 50 years for all genotypes). The patients for whom a genotyping assay was performed in 2008 were younger than those from 2015. Geographical distribution demonstrates that an important number of genotyped HCV patients live outside the Belgian metropolitan cities. CONCLUSION: This national monitoring study allowed a clear and objective view of the circulating HCV genotypes in Belgium and will help health authorities in the establishment of cost effectiveness determinations before implementation of new treatment strategies. This baseline characterization of the circulating genotypes is indispensable for a continuous surveillance, especially for the investigation of the possible impact of migration from endemic regions and prior to the increasing use of highly potent direct-acting antiviral (DAA) agents.

Phylogenetic analysis of the Belgian HIV-1 epidemic reveals that local transmission is almost exclusively driven by men having sex with men despite presence of large African migrant communities.

To improve insight in the drivers of local HIV-1 transmission in Belgium, phylogenetic, demographic, epidemiological and laboratory data from patients newly diagnosed between 2013 and 2015 were combined and analyzed. Characteristics of clustered patients, paired patients and patients on isolated branches in the phylogenetic tree were compared. The results revealed an overall high level of clustering despite the short time frame of sampling, with 47.6% of all patients having at least one close genetic counterpart and 36.6% belonging to a cluster of 3 or more individuals. Compared to patients on isolated branches, patients in clusters more frequently reported being infected in Belgium (95.1% vs. 47.6%; p < 0.001), were more frequently men having sex with men (MSM) (77.9% vs. 42.8%; p < 0.001), of Belgian origin (68.2% vs. 32.9%; p < 0.001), male gender (92.6% vs. 65.8%; p < 0.001), infected with subtype B or F (87.8% vs. 43.4%; p < 0.001) and diagnosed early after infection (55.4% vs. 29.0%; p < 0.001). Strikingly, Sub-Saharan Africans (SSA), overall representing 27.1% of the population were significantly less frequently found in clusters than on individual branches (6.0% vs. 41.8%; p < 0.001). Of the SSA that participated in clustered transmission, 66.7% were MSM and this contrasts sharply with the overall 12.0% of SSA reporting MSM. Transmission clusters with SSA were more frequently non-B clusters than transmission clusters without SSA (44.4% versus 18.2%). MSM-driven clusters with patients of mixed origin may account, at least in part, for the increasing spread of non-B subtypes to the native MSM population, a cross-over that has been particularly successful for subtype F and CRF02_AG. The main conclusions from this study are that clustered transmission in Belgium remains almost exclusively MSM-driven with very limited contribution of SSA. There were no indications for local ongoing clustered transmission of HIV-1 among SSA.

Screening for Chlamydia trachomatis and Neisseria gonorrhoeae Infections in Men Who Have Sex With Men: Diagnostic Accuracy of Nucleic Acid Amplification Test on Pooled Urine, Anorectal, and Pharyngeal Specimens.

Neisseria gonorrhoeae and Chlamydia trachomatis screening was performed in a cohort of 100 men who have sex with men. A nucleic acid amplification test on a pooled sample of first-pass urine, pharyngeal, and anorectal specimens was compared with results on nonpooled samples. Despite an excellent agreement (Cohen κ, 0.932), pooling specimens reduced test sensitivity to 89.5%.

HIV-1 low-level viraemia assessed with 3 commercial real-time PCR assays show high variability.

BACKGROUND: Current real-time PCR-based HIV-1 viral load (VL) assays allow the detection of residual viraemia in antiretroviral-treated patients. The clinical outcome of HIV1 patients experiencing low-level replication (<50 cop/mL) in comparison with fully suppressed patients is currently debated. We analysed variability of 3 VL assays <50 cop/mL, and evaluated the reproducibility of viral blips <100 cop/mL. METHODS: Three commercial VL assays were tested: Versant HIV-1 RNA 1.0 kPCR (Siemens), Abbott Realtime HIV-1, and Cobas Ampliprep/Cobas Taqman HIV-1 v2.0 (Roche). Ten replicates of a reference sample at 4 low target dilutions were tested to evaluate assay variability. Prospective collection of 181 clinical samples with detectable VL <50 cop/mL was used to evaluate intra-and inter-assay variability by triplicate testing. Samples from 26 patients experiencing a viral blip were retested. RESULTS: All assays showed substantial variability at low VL level: the coefficient of variation at 100, 50, 25 and 12 cop/mL ranged respectively from 32 to 44%, 35 to 68%, 41 to 83% and 33 to 77%. In the intra-assay evaluation of repeatability, 52.5 to 57.5% of detectable VL <50 cop/mL tested in triplicate showed at least one fully undetected result. Variability was similar in the inter-assay arm. The VL blips could only be reproduced in 19% of cases. CONCLUSIONS: The most recent versions of widespread commercial VL assays showed substantial variability at low levels and residual viraemia could not be consistently reproduced. Patient outcome studies comparing residual VL to full suppression are therefore biased when using commercial assays.

Development and evaluation of single sperm washing for risk reduction in artificial reproductive technology (ART) for extreme oligospermic HIV positive patients.

The serodiscordant couples, where the male is HIV-positive, are treated in fertility clinics, using the sperm washing technique by gradient centrifugation. This protocol cannot be carried out in oligo-azoospermic patients, where spermatozoa retrieval from the epididymis and testis must be performed. We developed a single sperm washing technique, where the spermatozoa, after the retrieval, are washed with the aid of a micromanipulator, to obtain virus decontamination and then used for the intracytoplasmic sperm injection (ICSI). The experiment was performed by using sperm samples containing three different viral loads. After one hour of incubation, spermatozoa were taken one by one from the HIV loaded drop and washed in four different microdrops. Before each passage into the next washing drop, the pipette was emptied in a first waste drop and then loaded with new washing medium from a second separate loading drop. After transferring of 10 spermatozoa in these four successive drops, the washing medium and the virus-loaded drops were tested for the HIV RNA presence by the nested RT-PCR technique. The presence of the virus was detected in the waste drop of all three viral loads. The four washing microdrops were each time negative for the presence of HIV-1 RNA, tested by the nested RT-PCR technique. The results show that by rinsing the spermatozoa four times, we are able to diminish the viral load to an undetectable level. Our data demonstrate that single sperm washing can be performed in the cases of extreme male sterility in HIV-positive men. From now on the couples, where the male is oligoazoospermic and HIV positive, could be included in our ICSI program, respecting the usual viral safety level of the ART techniques for the embryo.

Development and evaluation of a qualitative reverse-transcriptase nested polymerase chain reaction protocol for same-day viral validation of human immunodeficiency virus type 1 ribonucleic acid in processed semen.

OBJECTIVE: To develop a method for same-day validation of processed semen in the setting of assisted reproductive techniques (ART) with patients who are seropositive for human immunodeficiency virus, type 1 (HIV-1). DESIGN: Laboratory experiments. SETTING: University hospital. PATIENT(S): Volunteers who are HIV-1 seronegative and seropositive. INTERVENTION(S): Evaluation of the sensitivity of a reverse-transcriptase (RT)-nested polymerase chain reaction (PCR) in HIV-1 RNA-positive blood plasma, in artificially infected blood plasma and semen, and in 85 semen samples of 29 HIV-1-seropositive volunteers. Semen was submitted to gradient separation, followed by swim-up. MAIN OUTCOME MEASURE(S): Qualitative detection of HIV-1 RNA in blood plasma and in different parts of semen preparation by using RT-nested PCR, PCR inhibition control by dilution of samples, and an internal control. RESULT(S): The detection limit of our PCR was 20 HIV-1 RNA copies per milliliter. Among seropositive patients, RNA was detected in 25% of fresh semen, 36.5% of seminal plasma, 27.5% of gradient supernatants, and 7.1% of final preparations before the migration-sedimentation stage. Positive final preparations were observed in patients who had blood viral loads of >/=20,000 HIV-1 RNA copies per milliliter. Inhibition was present in 17.6% of seminal plasma and in 20% gradient supernatants and in 2 final preparations among 69 tested. Among 25 preparations tested after the migration-sedimentation stage, 2 were positive (1 patient; 70,000 HIV-1 RNA copies per milliliter). CONCLUSION(S): The RT-nested PCR detects low viral load and allows the validation of semen preparations of HIV-1-seropositive patients for ART on the day of sampling. For this purpose, the validation is performed on spermatozoa that are obtained after gradient separation before swim-up. Inhibition of the PCR must be controlled by using an internal control that is well-designed to explore the detection limit of the method.