Evaluation of the Genmark ePlex® and QIAstat-Dx® respiratory pathogen panels in detecting bacterial targets in lower respiratory tract specimens.

BACKGROUND: The ePlex® and QIAstat-Dx® respiratory pathogen panels detect multiple respiratory pathogens, mainly viruses but also Legionella pneumophila, Mycoplasma pneumoniae and Bordetella pertussis. The assays have been marketed for use in nasopharyngeal swab specimens. For diagnosing bacterial pneumonia, lower respiratory tract (LRT) specimens are indicated. Aim of this study was to evaluate the performance of these syndromic panels for these three bacterial targets in samples from the LRT. Fifty-six specimens were collected from our repositories, five negative samples and fifty-one samples which had been previously tested positive with the routine diagnostic real-time PCR assays for Legionella spp. (N = 20), Bordetella spp. (N = 16) or M. pneumoniae (N = 15). RESULTS: The QIAstat-Dx Respiratory Panel V2 (RP) assay detected all of the L. pneumophila and B. pertussis positive samples but only 11/15 (73.3 %) of the M. pneumoniae targets. The ePlex Respiratory Pathogen Panel (RPP) assay detected 10/14 (71.4 %) of the L. pneumophila targets, 8/12 (66.7 %) of the B. pertussis positive samples and 13/15 (86.7 %) of the M. pneumoniae targets. CONCLUSIONS: No false-positive results were reported for all three bacterial pathogens by both assays. The clinical performance of both assays depended highly on the bacterial load in the sample and the type of specimen under investigation.

Hypertrophic cardiomyopathy in a 20-year-old woman.

A case of a young woman with complaints of shortness of breath and recurrent collapses is presented, including echocardiographic and cardiac MRI images showing extremely hypertrophied myocardium due to hypertrophic cardiomyopathy. The patient was referred for therapy and genetic counselling.

Increased proportion of perforin-expressing CD8+T-cells indicates control of herpesvirus reactivation in children after stem cell transplantation.

Hematopoietic stem cell transplantation (HSCT) is frequently complicated by viral reactivations. Early diagnosis of viral reactivations and preemptive therapy relies on frequent viralload monitoring. An easy marker of effective cytotoxicity in lymphopenia is lacking and therefore we studied perforin-expression in CD8+T-cells in children following HSCT. Prospectively, we weekly monitored viral loads and perforin-expression of CD8+T-cells in whole blood by FACS, until 4months after HSCT in children. 27 patients were included (median age 4,3, range 0.3-20,1years) of whom 19 developed viral reactivations. These patients showed higher percentages of perforin-expressing CD8+T-cells (17,2%, range 0-63%) than those without (6,8%; range 0-16%) (p=0.001). The increased percentage of perforin-expressing CD8+T-cells coincided with a decrease in viral load with a median interval between maximum viral load and maximum level of perforin-expression of 0,4weeks (range 0.1-7.1). We conclude that perforin-expression in CD8+T-cells may be a marker for effective antiviral T-cell reconstitution early after HSCT in children.

Laboratory-based surveillance in the molecular era: the TYPENED model, a joint data-sharing platform for clinical and public health laboratories.

Laboratory-based surveillance, one of the pillars of monitoring infectious disease trends, relies on data produced in clinical and/or public health laboratories. Currently, diagnostic laboratories worldwide submit strains or samples to a relatively small number of reference laboratories for characterisation and typing. However, with the introduction of molecular diagnostic methods and sequencing in most of the larger diagnostic and university hospital centres in high-income countries, the distinction between diagnostic and reference/public health laboratory functions has become less clear-cut. Given these developments, new ways of networking and data sharing are needed. Assuming that clinical and public health laboratories may be able to use the same data for their own purposes when sequence-based testing and typing are used, we explored ways to develop a collaborative approach and a jointly owned database (TYPENED) in the Netherlands. The rationale was that sequence data - whether produced to support clinical care or for surveillance -can be aggregated to meet both needs. Here we describe the development of the TYPENED approach and supporting infrastructure, and the implementation of a pilot laboratory network sharing enterovirus sequences and metadata.

Integrated surveillance of human respiratory viruses in addition to SARS-CoV-2 in a public testing facility in the Netherlands.

BACKGROUND: SARS-CoV-2 prevention measures impact the circulation of other respiratory viruses. Surveillance in the network of general practitioners is hampered by widespread testing for SARS-CoV-2 in public testing facilities. OBJECTIVES: To evaluate integrated community surveillance of SARS-CoV-2 and other respiratory viruses and describe epidemiological trends. STUDY DESIGN: Respiratory surveillance was set up within an existing SARS-CoV-2 public testing facility. Community-dwelling (a)symptomatic persons provided consent for completion of a questionnaire and additional testing on residual material from swabs taken for SARS-CoV-2 RT-PCR (Allplex Seegene). Daily, a random subset was tested for sixteen respiratory viruses by multiplex realtime PCRs (Seegene). RESULTS: Between October 6th (week 40) 2021 and April 22nd (week 16) 2022, 3,969 subjects were tested. The weekly median age ranged from 23 to 39 years. The prevalence of respiratory symptoms ranged from 98.5% (week 40) to 27.4% (week 1). The prevalence of detection of any respiratory virus (including SARS-CoV-2), ranged from 19.6% in week 49 to 75.3% in week 14. SARS-CoV-2 prevalence ranged from 2.2% (week 40) to 63.3% (week 14). Overall, SARS-CoV-2 was detected most frequently (27.3%), followed by rhinoviruses (14.6%, range 3.5-47.8%) and seasonal coronaviruses (3.7%, range 0-10.4%, mostly 229E and OC43). Influenzavirus was detected in 3.0% of participants from week 6 onwards. CONCLUSIONS: Integrated respiratory viral surveillance within public testing facilities is feasible and informative. Prevalences may be affected by changes in SARS-CoV-2 prevention and testing policies. Population characteristics help to interpret trends over time. Integrated surveillance may inform policymakers and hospitals for adequate response measures during respiratory seasons.

First analysis of human herpesvirus 6T-cell responses: specific boosting after HHV6 reactivation in stem cell transplantation recipients.

Early human herpesvirus 6 (HHV6) reactivation after hematopoietic stem cell transplantation (HSCT) is associated with poor survival. We characterized HHV6 immuneresponses in HSCT patients during lymphopenia. Prospectively, HHV6 DNA-load was measured weekly by realtime-PCR. Numbers of IFNγ-producing HHV6-T-cells were retrospectively determined by enzyme-linked immunospot assay 2 months after HSCT. HHV6-specific T-cell proliferative capacity was analyzed with a newly developed assay using antigen-presenting autologous HHV6-infected PBMC. Fifty-six patients were included (median age 4.6 years; range 0.2-21.2 years). HHV6-reactivation occurred in 29/56 (52%) patients with a median time of 14 (range 1-41) days after HSCT. The median number of IFN-γ producing HHV6-specific T-cells at 2 months and the HHV6-specific CD8+ T-cell proliferative capacity at 6 months after HSCT was increased after HHV6-reactivation compared to non-reactivating patients (P=0.006 and p=0.019). In conclusion, HHV6-specific immuneresponses can be initiated during lymphopenia early after HSCT, which implicates a potential window for development of HHV6-specific (immuno)therapy.

High level of perforin expression in T cells: An early prognostic marker of the severity of herpesvirus reactivation after allogeneic stem cell transplantation in adults.

BACKGROUND: Epstein-Barr virus (EBV) and cytomegalovirus reactivations are frequent complications of hematopoeitic allogeneic stem cell transplantation (SCT) because of a lack of T cell control after immunosuppression. Early diagnosis of reactivation and subsequent preemptive therapy relies on frequent viral load measurement. Additional virus-specific T cell reconstitution data could improve the predictive value of viral load detection for viral complications after transplantation. Here, we studied perforin expression in CD8(+) T cells as a measure of cytotoxic T cell capacity in relation to the occurrence of viral reactivation. METHODS: In a prospective study, we monitored 40 patients during the first 3 months after transplantation and measured viral loads in combination with intracellular perforin expression in CD8(+) T cells. RESULTS: Median perforin expression in CD8(+) T cells throughout follow-up was higher in patients with viral reactivations than in patients without viral reactivations (4.9% vs 2.3%; P = .001). The median percentage of perforin-expressing CD8(+) T cells in patients with high viral reactivations exceeding 1000 copies/mL (10.7%) was statistically significantly higher than that in patients with minor reactivations of 50-1000 copies (4.0%), that in patients with detectable EBV loads that did not exceed the detection limit of 50 copies/mL (2.9%), and that in patients without reactivations (0.8%). Patients with high viral reactivations reached a high percentage of perforin-expressing CD8(+) T cells (>10.2%) more often and faster than did patients with low viral loads (1000 copies/mL) or without viral reactivations. High perforin expression preceded high viral loads. CONCLUSION: Perforin-expressing CD8(+) T cells may be useful as an easy-to-measure prognostic marker for identifying patients at risk for severe viral reactivation very soon after SCT.

Multicenter external quality assessment of molecular methods for detection of human herpesvirus 6.

The purpose of this study was to evaluate the performance of laboratories for the detection and quantification of human herpesvirus 6 (HHV-6) by an external quality assessment (EQA) evaluation. The HHV-6 EQA panel consisted of eight samples containing various concentrations of HHV-6 type A (strain GS) or type B (strain Z29), two samples containing other herpesviruses (i.e., human cytomegalovirus [HCMV] and Epstein-Barr virus [EBV]), and two HHV-6-negative samples. Panel samples were prepared in human plasma, heat inactivated, and lyophilized. Panel distribution, data management, and analysis were coordinated by Quality Control for Molecular Diagnostics (QCMD), Glasgow, United Kingdom. Fifty-one laboratories participated and submitted 57 data sets. Eleven (19.3%) data sets were generated using conventional in-house assays, 11 (19.3%) data sets using commercial real-time PCR assays, and 35 (61.4%) data sets using in-house real-time PCR assays. The presence of HHV-6 DNA at viral loads exceeding 6,000 copies/ml was detected by all participants, and over 80% of the participants still reported correct qualitative results for the sample containing just over 200 copies/ml. The false-positivity rate was 1.8% for both the negative samples and the samples containing HCMV or EBV DNA. The majority (23/33; 69.7%) of quantitative data sets were generated using in-house real-time PCR assays. The standard deviations of the geometric means of the samples ranged from 0.5 to 0.7 log(10). The results of this first international EQA demonstrate encouraging analytical sensitivity for the detection of HHV-6-DNA in human plasma, although we observed extensive interlaboratory variation of quantitative HHV-6 DNA results. Standardization needs to be improved to allow further elucidation of the clinical significance of HHV-6 loads.

T-cell response to viral antigens in adults and children with common variable immunodeficiency and specific antibody deficiency.

Several T cell abnormalities have been described in common variable immunodeficiency (CVID), a B cell disorder of mainly unknown origin. A subset of CVID patients suffers from frequent reactivations of herpes viruses. We studied T cell function in CVID [and in a subset of paediatric patients with specific antibody deficiency (SAD)] by measuring T cell proliferation and cytokine production in response to herpes virus-antigens in paediatric CVID patients (n=9) and paediatric SAD patients (n=5), in adult CVID patients (n=14) and in healthy controls. Paediatric CVID patients, but not SAD patients, displayed moderately increased CD8+ T cell proliferation in response to cytomegalovirus, human herpes virus type 6B (HHV6-B) and herpes simplex virus compared to controls. CD8+ T cell responses in adult CVID patients tended to be increased in response to cytomegalovirus and herpes simplex virus. In response to stimulation with herpes virus antigens, the proinflammatory cytokines interleukin (IL)-1beta, IL-6, tumour necrosis factor (TNF)-alpha and interferon inducible protein (IP)-10 were produced. Overall, no major differences were detected in cytokine production upon stimulation between patients and controls, although higher IL-10 and IL-12 production was detected in paediatric patients. In conclusion, cellular immunity against herpes virus antigens appears undisturbed in CVID patients, although defects in subpopulations of CVID patients cannot be excluded.

Hepatitis C virus transmission in a Dutch haemodialysis unit: detailed outbreak investigation using NS5A gene sequencing.

BACKGROUND: Haemodialysis is a risk factor for hepatitis C virus (HCV) transmission. Two patients receiving haemodialysis in a Dutch dialysis unit in The Hague were found to seroconvert to HCV in December 2016 after the yearly routine control for blood-borne viruses. Following the presumed time of infection, three chronically infected HCV patients were identified as possible index cases. AIM: To confirm inter-patient transmission and to identify the source. METHODS: Molecular investigation and review of medical records were performed. FINDINGS: Both of the incident cases and one of the three possible index cases were demonstrated to be infected with HCV genotype 2b based on 5'UTR sequencing. Epidemiological relatedness between these viruses was further investigated by sequencing of the NS5A region. Phylogenetic analysis clearly identified the incident cases and the index case to represent a cluster distinct from unrelated controls with HCV genotype 2b. Detailed review of the medical records identified two possible incidents that might have resulted in the HCV transmission cases: contamination of the venous pressure-sensing port due to high venous pressures or incomplete compliance with infection control precautions of the unit staff during handling of two incidents, that occurred at the same time in a single haemodialysis session with the index patient as well as both incident cases present. CONCLUSION: This study demonstrates that detailed incident recording in combination with state-of-the-art molecular investigations such as sequencing of the NS5A region resulted in unravelling a set of two HCV transmissions that occurred at a haemodialysis unit.

HIV-1 drug resistance genotyping quality assessment: results of the ENVA7 Genotyping Proficiency Programme.

BACKGROUND: Drug-resistance testing plays a critical role in selection of optimal treatment regimens for HIV infected individuals. Laboratories performing testing must implement quality control measures including external quality assessment. OBJECTIVES: The ENVA7 Programme (2007) was organised by QCMD to assess the performance of laboratories testing for drug-resistance mutations in the HIV-1 Protease and Reverse Transcriptase genes. STUDY DESIGN: The ENVA7 panel consisted of 5 lyophilised plasma samples (HIV-1 subtypes B, C and F). The viruses harboured wild type or resistant genotypes at various positions of the PR and RT genes. All IAS-defined resistance-associated codons were scored in comparison to the consensus sequence for each sample using a scoring system developed to allow simple and standardised comparisons between laboratories and/or technologies. RESULTS: 111 laboratories from 44 countries participated of which 95 submitted 98 datasets. 36 datasets were generated using ViroSeq (Abbott), 27 using TruGene (Siemens) and 35 using in-house assays. CONCLUSIONS: All technologies successfully genotyped each of the panel samples, irrespective of the virus subtype. While the assays for genotypic HIV drug-resistance determination have evolved into reliable and technically capable procedures of generating high quality results, variation in the quality of results is still observed between laboratories.

Syndromic sample-to-result PCR testing for respiratory infections in adult patients.

BACKGROUND: Syndromic sample-to-result (SS2R) poly merase chain reaction (PCR) can rapidly identify causative pathogens of respiratory tract infections (RTI). We evaluated diagnostic accuracy and applicability of one of the current SS2R diagnostics, the FilmArray® Respiratory Viral Panel. METHODS: We performed a prospective study among adults presenting with symptoms of RTI at the Emergency Department of the University Medical Centre Utrecht (the Netherlands) during the 2016-2017 viral respiratory season. Clinical data were collected. We compared SS2R results on nasopharyngeal swabs to conventional real time PCR, calculated turnaround times (TAT) and explored implementation barriers using questionnaires. RESULTS: 62 Patients were included (64.5 yr [interquartile range (IQR) 44.3-75.0]). SS2R sensitivity was 82.9% [95% confidence interval (CI) 67.9-92.9] and specificity was 95.2% [95% CI 76.2-99.9] for detection of all present viruses (n = 60). Kappa agreement (0.73 [95% CI 0.56-0.90]) was good (p = 0.000). Median SS2R TAT was 2:06 hours [IQR 1:45-3:17] compared to 32:00 hours [IQR 26:50-40:42] of conventional PCR (n = 49, p = 0.000). Ease-of-use and fast TAT were unanimously reported as benefits, and low test capacity with a single SS2R system as drawback. CONCLUSION: SS2R testing for respiratory viruses offers a rapid and reliable diagnostic method which has great potential for more efficient and targeted management in adult patients with RTI.

[AIDS treatment in Africa: the risk of antiretroviral resistance]. / Aidsbehandeling in Afrika: het risico van antiretrovirale resistentie.

--In recent years, implementation of antiretroviral therapy in developing countries with a high prevalence of HIV-1 has been recognised as a public health priority. Consequently, the availability ofantiretroviral combination therapy for people with HIV is increasing rapidly in sub-Saharan Africa. --HIV treatment programmes are implemented according to the standardised, simplified public health guidelines developed by the World Health Organization (WHO). --However, the implementation of treatment programmes in Africa is hindered by several factors, including the lack of adequate immunological and virological laboratory monitoring, insufficient support for adherence to therapy, vulnerable health care systems and the use of suboptimal drug combinations. --These suboptimal treatment conditions increase the risk that resistant virus strains will emerge that are less susceptible to standard first-line combination therapy, thus threatening the long-term success of the treatment programmes. --The WHO has initiated HIVResNet, an international expert advisory board that has developed a global strategy for surveillance and prevention of antiretroviral drug resistance. --The Dutch initiative known as 'PharmAccess African studies to evaluate resistance' (PASER) is contributing to this strategy by creating a surveillance network in sub-Saharan Africa.

Absence of human metapneumovirus co-infection in cases of severe respiratory syncytial virus infection.

It has been suggested that co-infection of human metapneumovirus (hMPV) in severe respiratory syncytial (RSV) virus bronchiolitis is very common. To evaluate the epidemiology of hMPV co-infection in children with severe lower respiratory tract infection caused by RSV virus. This was an observational cohort study in which hMPV and RSV viral load was measured by RT-PCR in tracheal specimens from the target population. hMPV could not be detected in any of the 30 mechanically ventilated children with RSV lower respiratory tract infection. Our study suggests that hMPV co-infection is not very common in severe RSV lower respiratory tract infection.

Contribution of APOBEC3G/F activity to the development of low-abundance drug-resistant human immunodeficiency virus type 1 variants.

Plasma drug-resistant minority human immunodeficiency virus type 1 variants (DRMVs) increase the risk of virological failure to first-line non-nucleoside reverse transcriptase inhibitor antiretroviral therapy (ART). The origin of DRMVs in ART-naive patients, however, remains unclear. In a large pan-European case-control study investigating the clinical relevance of pre-existing DRMVs using 454 pyrosequencing, the six most prevalent plasma DRMVs detected corresponded to G-to-A nucleotide mutations (V90I, V106I, V108I, E138K, M184I and M230I). Here, we evaluated if such DRMVs could have emerged from apolipoprotein B mRNA editing enzyme, catalytic polypeptide 3G/F (APOBEC3G/F) activity. Out of 236 ART-naive subjects evaluated, APOBEC3G/F hypermutation signatures were detected in plasma viruses of 14 (5.9%) individuals. Samples with minority E138K, M184I, and M230I mutations, but not those with V90I, V106I or V108I, were significantly associated with APOBEC3G/F activity (Fisher's P < 0.005), defined as the presence of > 0.5% of sample sequences with an APOBEC3G/F signature. Mutations E138K, M184I and M230I co-occurred in the same sequence as APOBEC3G/F signatures in 3/9 (33%), 5/11 (45%) and 4/8 (50%) of samples, respectively; such linkage was not found for V90I, V106I or V108I. In-frame STOP codons were observed in 1.5% of all clonal sequences; 14.8% of them co-occurred with APOBEC3G/F signatures. APOBEC3G/F-associated E138K, M184I and M230I appeared within clonal sequences containing in-frame STOP codons in 2/3 (66%), 5/5 (100%) and 4/4 (100%) of the samples. In a re-analysis of the parent case control study, the presence of APOBEC3G/F signatures was not associated with virological failure. In conclusion, the contribution of APOBEC3G/F editing to the development of DRMVs is very limited and does not affect the efficacy of non-nucleoside reverse transcriptase inhibitor ART.

Increased polymerase fidelity of lamivudine-resistant HIV-1 variants does not limit their evolutionary potential.

OBJECTIVE: Anti HIV-1 therapy with nucleoside reverse transcriptase inhibitors can select for drug-resistant reverse transcriptase variants with altered enzyme properties. Some of the mutations, e.g. Met184Val and Met184Ile, result in an increase in polymerase fidelity of the enzyme as measured in biochemical assays; however, the effect of such changes on the fidelity during viral replication is largely unknown. In this study, the codon 184 variants were used to investigate whether the mutation at codon 184 affects the mutation spectrum and mutation rate of the mutant viruses. DESIGN AND METHOD: In vitro selection experiments with either wild-type or lamivudine-resistant viruses (Met184Val and Met184Ile) were performed using a protease inhibitor as the selective drug. In addition, a novel selection approach was developed using a mixture of viruses, instead of individual viruses, during the selection process. RESULTS: Comparison of a total of 108 protease-resistant variants revealed no significant difference in the mutational spectrum of the wild-type and the lamivudine-resistant variants. In addition, the selection experiments with the viral mixtures demonstrated no delay in the kinetics of mutation generation in response to an antiviral drug. CONCLUSION: This study demonstrates that the Met184Val and Met184Ile mutations in the HIV-1 reverse transcriptase enzyme do not significantly affect the evolutionary potential of the corresponding viruses.

Increased fitness of drug resistant HIV-1 protease as a result of acquisition of compensatory mutations during suboptimal therapy.

OBJECTIVE: It is thought as a consequence of continuous replication, HIV-1 has acquired an optimal fitness state and that suboptimal antiretroviral therapy selects for drug resistant variants which show impaired fitness in the absence of the drug. In this paper we studied the evolution and fitness of viral populations appearing in a patient who received protease monotherapy. METHODS: Two factors contributing to fitness, drug resistance and protease catalytic activity, were studied at the enzymatic and virological level. RESULTS: The first drug resistant viral variants that were selected in vivo harboured one to three protease substitutions. These mutants showed reduced protease activity and consequently a reduction in viral replication capacity. During continued in vivo replication of these viruses in the presence of the drug, novel variants harbouring additional substitutions in the viral protease appeared. These variants did not display any further increase in drug resistance but demonstrated clearly increased protease activity. Consequently the replication capacity of these viruses was raised to a level at which they replicated better than the original wild-type virus. CONCLUSION: This study indicates that the viral population in the patient does not have to represent the fittest possible variants, and thus antiretroviral therapy may drive the viral population first through a lower fitness level and then to a higher fitness level.

Efficient inhibition of both syncytium-inducing and non-syncytium-inducing wild-type HIV-1 by lamivudine in vivo.

OBJECTIVE: To determine the effect of lamivudine (3TC) on syncytium-inducing (SI) and non-SI (NSI) HIV-1 populations in vivo. DESIGN: Responses in virus load and 3TC resistance in 40 3TC-treated subjects were analysed in relation to the presence or absence of SI HIV-1 variants. METHODS: Peripheral blood samples were collected at regular intervals from 40 HIV-1-infected subjects during 3TC treatment. Virus isolates obtained at the start of treatment were typed for SI-capacity by coculture with MT-2 cells. Changes in levels of viral RNA in plasma were determined by quantitative reverse transcriptase PCR. The relative amount of wild-type and mutant virus at codon 184, associated with HIV resistance to 3TC, was determined using a primer-guided nucleotide incorporation assay after amplification of part of the reverse transcriptase gene. In five subjects the frequency of productively infected CD4+ cells with SI or NSI variants was determined in relation to codon 184 genotype. RESULTS: Twenty-six subjects harboured only NSI variants at baseline, whereas 14 subjects also harboured SI variants. Although baseline plasma viral RNA load and CD4 cell counts were different between the two groups, no differences in the response to 3TC therapy were observed for these parameters. In-depth analysis of five subjects showed that the kinetics of virus load changes and emergence of 3TC resistance mutations were similar in plasma and cells, and comparable for the SI and NSI populations present in one individual. CONCLUSIONS: These data show that, in contrast to didanosine and zidovudine, the pressure exerted by 3TC is similar for SI and NSI M184 populations.