Evaluation of two automated real-time PCR-based quantification methods for whole blood Epstein-Barr viral load.

Quantification of EBV DNA is important in transplantation settings for the diagnosis of post-transplantation. We evaluated the performance of the AltoStar® EBV PCR Kit 1.5 on whole blood specimens: limit of detection, linearity, accuracy, and precision were determined using the WHO NIBSC 09/260 international standard. Results of 69 clinical samples were compared between the AltoStar® EBV PCR Kit 1.5 (altona Diagnostics) and the RealTime EBV assay (Abbott). The LoD of the AltoStar® Kit was 148 IU/mL and linearity was between 375 and 500000. A high concordance was found between nominal value of the NIBSC dilutions and the AltoStar EBV result. The total variation ranged from 2.2% to 9.6%. Out of 69 clinical samples tested, there was a high concordance between the 22 paired results within the overlapping linear ranges of both tests. The AltoStar® EBV assay is reliable and accurate for EBV viral load determination on whole blood samples.

Qualitative plasma viral load determination as a tool for screening of viral reservoir size in PWH.

OBJECTIVES: Suppression of viral replication in patients on antiretroviral therapy (ART) is determined by plasma viral load (pVL) measurement. Whenever pVL reaches values below the limit of quantification, the qualitative parameter 'target detected' or 'target not detected' is available but often not reported to the clinician. We investigated whether qualitative pVL measurements can be used to estimate the viral reservoir size. DESIGN: The study recruited 114 people with HIV (PWH) who are stable on ART between 2016 and 2018. The percentage of pVL measurements qualitatively reported as 'target detected' (PTD) within a 2-year period was calculated. METHODS: t-DNA and US-RNA were used to estimate viral reservoir size and were quantified on peripheral blood mononuclear cells (PBMCs) using droplet digital PCR. RESULTS: A median of 6.5 pVL measurements over a 2-year period was evaluated for each participant to calculate PTD. A positive correlation was found between t-DNA and PTD (r = 0.24; P = 0.011) but not between US-RNA and PTD (r = 0.1; P = 0.3). A significantly lower PTD was observed in PWH with a small viral reservoir, as estimated by t-DNA less than 66 copies/106 PBMCs and US-RNA less than 10 copies/106 PBMCs, compared with PWH with a larger viral reservoir (P = 0.001). We also show that t-DNA is detectable whenever PTD is higher than 56% and that ART regimen does not affect PTD. CONCLUSION: Our study shows that PTD provides an efficient parameter to preselect participants with a small viral reservoir based on already available pVL data for future HIV cure trials.

Healthcare-Associated SARS-CoV-2 Reinfection after 3 Months with a Phylogenetically Distinct Omicron Variant: A Case Report.

This case report describes a 60-year-old female patient suffering from systemic sclerosis, for which she received immunomodulatory drugs. Her first SARS-CoV-2-positive nasopharyngeal sample was obtained in the emergency department, on 31 January 2022. Whole genome sequencing confirmed infection with Omicron BA.1.1. Her hospital stay was long and punctuated by many complications, including admission to the intensive care unit. At the beginning of April 2022, she started complaining of increased coughing, for which another SARS-CoV-2 RT-qPCR test was performed. The latter nasopharyngeal swab showed a strongly positive result. To support the theory of healthcare-associated reinfection, whole genome sequencing was performed and confirmed reinfection with Omicron BA.2. Since this patient was one of ten positive cases in this particular ward, a hospital outbreak investigation was performed. Whole genome sequencing data were available for five of these ten patients and showed a cluster of four patients with ≤2 small nucleotide polymorphisms difference.

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.

Evaluation of four laboratory-based SARS-CoV-2 IgG antibody immunoassays.

OBJECTIVES: To evaluate the diagnostic performances of four SARS-CoV-2 IgG antibody immunoassays. METHODS: Following immunoassays were studied: Abbott's SARS-CoV-2 IgG assay, Diasorin's Liaison SARS-CoV-2 S2/S2 IgG assay, Euroimmun's Anti-SARS-CoV-2 IgG ELISA, and Roche's Elecsys Anti-SARS-CoV-2 assay. Specificity was retrospectively evaluated with 38 samples from 2019. Sensitivity samples (n = 147) were taken from SARS-CoV-2 real-time PCR-positive patients who developed COVID-19 symptoms ten days earlier. RESULTS: Mean specificity was 96.6%. Mean sensitivity was 62.7% from ten days after onset of symptoms, 84.4% from 15 days after onset of symptoms, and 87.5% from 20 days after onset of symptoms. CONCLUSIONS: Specificity was high, while Abbott and Roche were 100% specific. Sensitivity increased over time, with Abbott and Roche having the highest sensitivity at all time points with ≥90% from 20 days after symptoms' onset. These findings may assist in selecting SARS-CoV-2 IgG antibody immunoassays for additional diagnostics, epidemiological research, and vaccine development.

High frequency of new recombinant forms in HIV-1 transmission networks demonstrated by full genome sequencing.

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.

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.

Exploring HIV-1 Transmission Dynamics by Combining Phylogenetic Analysis and Infection Timing.

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.

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.

Multicenter evaluation of the cobas® HIV-1 quantitative nucleic acid test for use on the cobas® 4800 system for the quantification of HIV-1 plasma viral load.

BACKGROUND AND OBJECTIVES: Measurement of HIV-1 viral load (VL) is necessary to monitor treatment efficacy in patients receiving antiretroviral therapy. We evaluated the performance of the cobas® HIV-1 quantitative nucleic acid test for use on the cobas® 4800 system ("cobas 4800 HIV-1"). METHODS: Limit of detection, linearity, accuracy, precision, and specificity of cobas 4800 HIV-1, COBAS® AmpliPrep/COBAS® Taqman® HIV-1 version 2.0 (CAP/CTM HIV-1 v2) and Abbott RealTime HIV-1 were determined in one or two out of three sites. RESULTS: The limit of detection of the cobas 4800 HIV-1 for 400 µL and 200 µL input volumes was 14.2 copies/mL (95% CI: 12.5-16.6 copies/mL) and 43.9 copies/mL (37.7-52.7 copies/mL), respectively. Cobas 4800 HIV-1 demonstrated 100% specificity, and results were linear for all analyzed group M HIV-1 subtypes. Precision was high (SD < 0.19 log10) across all measured ranges, reagent lots and input volumes. Correlation between cobas 4800 HIV-1 and CAP/CTM HIV-1 v2 or RealTime HIV-1 was high (R2 ≥ 0.95). Agreement between cobas 4800 HIV-1 and CAP/CTM HIV-1 v2 was 96.5% (95.0%-97.7%) at a threshold of 50 copies/mL, and 97.2% (95.8%-98.3%) at 200 copies/mL. Agreement between cobas 4800 HIV-1 and RealTime HIV-1 was 96.6% (93.4%-98.5%) at 50 copies/mL, and 97.0% (94.0%-98.8%) at 200 copies/mL. The mean difference between cobas 4800 HIV-1 and CAP/CTM HIV-1 v2 or RealTime HIV-1 was -0.10 log10 or 0.01 log10, respectively. CONCLUSIONS: The cobas 4800 HIV-1 test is highly sensitive, accurate and correlated well with other assays, including agreement around clinically relevant thresholds, indicating minimal overall VL quantification differences between tested platforms.

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.

Decision tree for accurate infection timing in individuals newly diagnosed with HIV-1 infection.

BACKGROUND: There is today no gold standard method to accurately define the time passed since infection at HIV diagnosis. Infection timing and incidence measurement is however essential to better monitor the dynamics of local epidemics and the effect of prevention initiatives. METHODS: Three methods for infection timing were evaluated using 237 serial samples from documented seroconversions and 566 cross sectional samples from newly diagnosed patients: identification of antibodies against the HIV p31 protein in INNO-LIA, SediaTM BED CEIA and SediaTM LAg-Avidity EIA. A multi-assay decision tree for infection timing was developed. RESULTS: Clear differences in recency window between BED CEIA, LAg-Avidity EIA and p31 antibody presence were observed with a switch from recent to long term infection a median of 169.5, 108.0 and 64.5 days after collection of the pre-seroconversion sample respectively. BED showed high reliability for identification of long term infections while LAg-Avidity is highly accurate for identification of recent infections. Using BED as initial assay to identify the long term infections and LAg-Avidity as a confirmatory assay for those classified as recent infection by BED, explores the strengths of both while reduces the workload. The short recency window of p31 antibodies allows to discriminate very early from early infections based on this marker. BED recent infection results not confirmed by LAg-Avidity are considered to reflect a period more distant from the infection time. False recency predictions in this group can be minimized by elimination of patients with a CD4 count of less than 100 cells/mm3 or without no p31 antibodies. For 566 cross sectional sample the outcome of the decision tree confirmed the infection timing based on the results of all 3 markers but reduced the overall cost from 13.2 USD to 5.2 USD per sample. CONCLUSIONS: A step-wise multi assay decision tree allows accurate timing of the HIV infection at diagnosis at affordable effort and cost and can be an important new tool in studies analyzing the dynamics of local epidemics or the effects of prevention strategies.

Effect of Genital Sampling Site on the Detection and Quantification of Ureaplasma Species with Quantitative Polymerase Chain Reaction during Pregnancy.

Objective. This study aimed to compare the qualitative and quantitative reproducibility of quantitative PCR (qPCR) for Ureaplasma species (Ureaplasma spp.) throughout pregnancy and according to the genital sampling site. Study Design. Between 5 and 14 weeks of gestation (T1), vaginal, fornix, and two cervical samples were taken. Sampling was repeated during the 2nd (T2) and 3rd (T3) trimester in randomly selected T1 positive and negative women. Qualitative and quantitative reproducibility were evaluated using, respectively, Cohen's kappa (κ) and interclass correlation coefficients (ICC) and repeated measures ANOVA on the log-transformed mean number of DNA copies for each sampling site. Results. During T1, 51/127 women were positive for U. parvum and 8 for U. urealyticum (4 patients for both). Sampling was repeated for 44/55 women at T2 and/or T3; 43 (97.7%) remained positive at the three timepoints. κ ranged between 0.83 and 0.95 and the ICC for cervical samples was 0.86. Conclusions. Colonization by Ureaplasma spp. seems to be very constant during pregnancy and vaginal samples have the highest detection rate.

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.

Species identification of clinical Prevotella isolates by matrix-assisted laser desorption ionization-time of flight mass spectrometry.

The performance of matrix-assisted laser desorption-ionization time of flight mass spectrometry (MALDI-TOF MS) for species identification of Prevotella was evaluated and compared with 16S rRNA gene sequencing. Using a Bruker database, 62.7% of the 102 clinical isolates were identified to the species level and 73.5% to the genus level. Extension of the commercial database improved these figures to, respectively, 83.3% and 89.2%. MALDI-TOF MS identification of Prevotella is reliable but needs a more extensive database.

Modified real-time PCR for detecting, differentiating, and quantifying Ureaplasma urealyticum and Ureaplasma parvum.

We evaluated a previously described quantitative real-time PCR (qPCR) for quantifying and differentiating Ureaplasma parvum and U. urealyticum. Because of nonspecific reactions with Staphylococcus aureus DNA in the U. parvum PCR, we developed a modified qPCR and designed new primers. These oligonucleotides eradicated cross-reactions, indicating higher specificity. The detection limits of the qPCR were determined at 1 and 3 colony-forming units/ml for U. parvum and U. urealyticum, respectively. The quantification limits of the assay for both Ureaplasma species ranged from 2.10(6) to 2.10(1) copy numbers per PCR. A total of 300 patient samples obtained from the lower genital tract were tested with this newly designed qPCR assay and compared with culture results. Of the samples, 132 (44.0%) were culture positive, whereas 151 (50.3%) tested positive using qPCR. The U. parvum and U. urealyticum species were present in 79.5% and 12.6% of the qPCR-positive samples, respectively. Both species were found in 7.9% of those samples. Quantification of U. parvum and U. urealyticum in the samples ranged from less than 2.5 × 10(3) to 7.4 × 10(7) copies per specimen. In conclusion, the modified qPCR is a suitable method for rapid detection, differentiation, and quantification of U. parvum and U. urealyticum.

Association of abnormal vaginal flora and Ureaplasma species as risk factors for preterm birth: a cohort study.

OBJECTIVE: To find out whether the presence of Ureaplasma species (U. spp.) in combination with an alteration of the normal vaginal flora is an independent risk factor for preterm delivery. DESIGN: Prospective observational study. SETTING: Department of Obstetrics, University Hospital in Brussels. POPULATION: A total of 1,988 singleton pregnancies were included. METHODS: From each woman, a cervical culture for U. spp. was obtained and the vaginal flora evaluated at the first prenatal visit. The presence of known risk factors for preterm delivery was recorded. Preterm birth was defined as delivery < 37 weeks. RESULTS: There were 97 (4.9%) preterm births. In patients delivered before 37 weeks, abnormal vaginal flora was detected in 22.7% and U. spp. in 53.6% of these. The conditions were found together in 17.5%. In patients delivered at term, an abnormal vaginal flora was detected in 14.4% and U. spp. in 41.4% of these women, while they co-existed in 8.2%. Using a logistic regression analysis taking into account known risk factors for preterm birth and the microbiological parameters, preterm delivery was correlated with the presence of U. spp. (odds ratio (OR) 1.64; 95% confidence interval (CI) 1.08-2.48; p = 0.02) and abnormal vaginal flora in combination with U. spp. (OR 2.35; 95% CI 1.35-4.10; p = 0.003). No significant correlation between the presence of abnormal vaginal flora and preterm delivery (p = 0.09) was found. CONCLUSIONS: Preterm delivery was significantly correlated with the presence of U. spp. The risk for preterm delivery increased when U. spp. was associated with an abnormal vaginal flora.