The Outbreak of Unexplained Acute Hepatitis in Children: The Role of Viral Infections in View of the COVID-19 Pandemic.

BACKGROUND AND AIMS: An increase in the number of cases of acute hepatitis of unknown origin (HUO) in children was observed in 2021. Adenovirus and adeno-associated virus 2 (AAV2) infections have been suggested as possible triggers. However, the potential etiology is still unclear. We aimed to characterize a cohort of children with HUO in Israel in view of the COVID-19 pandemic. METHOD: Demographics, clinical data, and laboratory results on the children compatible with the CDC criteria for HUO were collected by the established registry of the Ministry of Health. Available specimens were sent to the Central Virology Laboratory. RESULTS: A total of 39 children were included in the registry. A total of 20 were enrolled prospectively, in which human herpes virus 6 (HHV6) infection or reactivation was identified in 11/19, adenovirus was found in 4/19 of the cases, and AAV2 was detected in 2/16. Past COVID-19 exposure was recorded for 24/39 of the children. A total of 10 children underwent liver biopsy, and 8 were successfully treated with steroids and 2 underwent liver transplantation. CONCLUSIONS: The COVID-19 pandemic and the related containment measures combined with reactivation or active infection with other viruses could have been a trigger for the HUO outbreak. In our cohort, HHV6 was the most abundant finding.

Checklist for studies of HIV drug resistance prevalence or incidence: rationale and recommended use.

HIV drug resistance (HIVDR) is a major challenge to the effectiveness of antiretroviral therapy. Global efforts in addressing HIVDR require clear, transparent, and replicable reporting in HIVDR studies. We describe the rationale and recommended use of a checklist that should be included in reports of HIVDR incidence and prevalence. After preliminary consultations with experts on HIVDR and establishing the need for guidance on HIVDR reporting, we used a sequential, explanatory, mixed methods approach to create the checklist; together with the accompanying articles, the checklist was reviewed by the authors and validated externally. The checklist for studies on HIVDR prevalence or incidence (CEDRIC-HIV) includes 15 recommended items that would enhance transparency and facilitate interpretation, comparability, and replicability of HIVDR studies. CEDRIC-HIV will help authors of HIVDR studies prepare research reports and assist reviewers and editors in assessments of completeness of reporting. The checklist will also facilitate statistical pooling and interpretation of HIVDR data.

Barriers and Facilitators of Hepatitis C Care in Persons Coinfected with Human Immunodeficiency Virus.

Hepatitis C virus (HCV) and human immunodeficiency virus (HIV) are often co-transmitted. Viral coinfection results in worse outcomes. Persons who inject drugs (PWIDs) face barriers to medical treatment, but HCV treatment is indicated and effective even with ongoing active drug use. We aimed to assess access to HCV care and treatment results in patients coinfected with HIV-HCV. This is a real-world retrospective single-center study of patients followed in the HIV clinic between 2002 and 2018. Linkage to care was defined as achieving care cascade steps: (1) hepatology clinic visit, (2) receiving prescription of anti-HCV treatment, and (3) documentation of sustained virologic response (SVR). Of 1660 patients with HIV, 254 with HIV-HCV coinfection were included. Only 39% of them achieved SVR. The rate limiting step was the engagement into hepatology care. Being a PWID was associated with ~50% reduced odds of achieving study outcomes, active drug use was associated with ~90% reduced odds. Older age was found to facilitate treatment success. Once treated, the rate of SVR was high in all populations. HCV is undertreated in coinfected young PWIDs. Further efforts should be directed to improve access to care in this marginalized population.

Evaluation of Biotrin, Serion and Euroimmun commercial assays for the detection of parvovirus B19-specific IgM and IgG antibodies.

Diagnosis of parvovirus B19 (B19) infection in small-medium size clinical laboratories is most often done by nonautomated enzyme immunoassays (EIAs). Using 195 specimens we compared the analytical performance of Biotrin (Dublin, Ireland), Euroimmun (Lubeck, Germany), and Serion (Würzburg, Germany) EIAs. Sensitivity, specificity, and concordance to Biotrin assay were calculated. Overall complete agreement in the IgG and IgM results was 88.7% (173/195) and 75.9% (148/195) samples, respectively. When equivocal results were considered positive, Serion and Euroimmun highly agreed (>93.8%) with Biotrin in the IgG serology. Serion had better IgM sensitivity and specificity than Euroimmun when compared to Biotrin, although more Serion IgM equivocal results needed reflex testing. Clinical interpretation by all three assays was identical in 83% of the samples. We concluded that overall the performance of these assays was similar and both Serion and Euroimmun could be a suitable replacement for the Biotrin.

Ombitasvir/paritaprevir/ritonavir & dasabuvir ± ribavirin following protease inhibitors failure - a prospective multi-centre trial.

BACKGROUND: Hepatitis C virus (HCV) infection is a leading cause of chronic liver disease and hepatocellular carcinoma. Treatment with first generation protease inhibitors (PI) + peg-interferon (pegIFN) and ribavirin (RBV) achieved sustained virologic response (SVR) rates of 65-75% but was associated with multiple side effects. The aim of this study was to evaluate safety and efficacy of Ombitasvir/Paritaprevir/Ritonavir and Dasabuvir (3D) ± RBV in HCV genotype 1 patients that failed previous treatment with first generation PIs. METHODS: An investigator-initiated, open-label, multi-centre clinical trial. HCV Genotype 1 patients who were previously null/partial responders or relapsers to telaprevir, boceprevir or simepravir+pegIFN/RBV and met eligibility criteria were included. 3D ± RBV were administrated for 12 or 24 weeks according to label. The primary outcome was antiviral response (SVR12); Secondary outcomes were patient reported outcomes, adverse events and resistance associated variants. RESULTS: Thirty-nine patients initiated treatment according to study protocol (59% men, age 54.0 ± 8.7 years, BMI 28.7 ± 4.5 kg/m2). Thirty-seven (94.9%) completed the study. Thirty-five patients had genotype 1b (9 cirrhotics) and 4 had genotype 1a (2 cirrhotics). Intention-to-treat SVR12 was 92.3% and per-protocol SVR12 was 97.3%. The rate of advanced fibrosis (FibroScan® score F3-4) declined from 46.2 to 25.7% (P = 0.045). Abnormal ALT levels declined from 84.6 to 8.6% (P < 0.001). Seven patients (17.9%) experienced serious adverse events (3 Psychiatric admissions, 1 pneumonia, 1 ankle fracture, 2 palpitations), and 12 patients (30.8%) experienced self-reported adverse events, mostly weakness. CONCLUSION: 3D ± RBV is safe and effective in achieving SVR among patients with HCV genotype 1 who failed previous first-generation PI treatment. TRIAL REGISTRATION: NCT02646111 (submitted to ClinicalTrials.gov, December 28, 2015).

HIV-1/2 screening in blood centers: implementing a two-step serological screening assay approach to reduce donor deferral.

BACKGROUND: Screening blood donations for human immunodeficiency virus 1/2 (HIV-1/2) infection in blood centers is often done with a highly sensitive 3rd-generation immunoassay which may cause false-positive results. Donations found repeatedly reactive (RR) are discarded regardless of negative HIV-1/2 nucleic acid testing (NAT) and confirmatory assays results. These donors are notified and deferred if RR in a subsequent donation. We evaluated the introduction of a secondary 4th-generation serological assay to the overall algorithm performance. METHODS: All donations collected between January 2016 and May 2018 (574,338) were screened using 3rd-generation immunoassay (PRISM HIV O Plus) and NAT (Procleix Ultrio/Ultrio Elite). Serology RR donations were tested with 4th-generation Architect HIV Ag/Ab Combo and Vidas HIV Duo Ultra and a confirmatory assay (Geenius HIV-1/2). RESULTS: The two 4th-generation assays found that 86% (179/209 on Architect) and 94% (182/193 on Vidas) of the 3rd-generation immunoassay RR were negative for HIV-1/2, which were also negative by confirmatory assay. Only 14% (30/209 on Architect) and 6% (11/193 on Vidas) that were 3rd-generation HIV-1/2 RR required confirmation, of which eight donors were confirmed as HIV positive. The probability of missing an HIV-1 infected donor by this algorithm is one in a million RR cases. CONCLUSION: The introduction of a two-step serological screening algorithm in blood centers whereby 4th-generation assay will be performed for all 3rd-generation RR blood donors will reduce the number of donations requiring confirmation, save time and money, and most importantly, reduce the number of discarded blood donations and allow re-entry processes.

Comparison of the new fully automated extraction platform eMAG to the MagNA PURE 96 and the well-established easyMAG for detection of common human respiratory viruses.

Respiratory viral infections constitute the majority of samples tested in the clinical virology laboratory during the winter season, and are mainly diagnosed using molecular assays, namely real-time PCR (qPCR). Therefore, a high-quality extraction process is critical for successful, reliable and sensitive qPCR results. Here we aimed to evaluate the performance of the newly launched eMAG compared to the fully automated MagNA PURE 96 (Roche, Germany) and to the semi-automated easyMAG (bioMerieux, France) extraction platforms. For this analysis, we assessed and compared the analytic and clinical performance of the three platforms, using 262 archived respiratory samples positive or negative to common viruses regularly examined in our laboratory (influenza A, B, H1N1pdm, Respiratory Syncytial Virus (RSV), human Metapneumovirus (hMPV), parainfluenza-3, adenovirus and negative samples). In addition, quantitated virus controls were used to determine the limit of detection of each extraction method. In all categories tested, eMAG results were comparable to those of the easyMAG and MagNa PURE 96, highly sensitive for all viruses and over 98% clinical specificity and sensitivity for all viruses tested. Together with its high level of automation, the bioMerieux eMAG is a high-quality extraction platform enabling effective molecular analysis and is mostly suitable for medium-sized laboratories.

Immune-escape mutations and stop-codons in HBsAg develop in a large proportion of patients with chronic HBV infection exposed to anti-HBV drugs in Europe.

BACKGROUND: HBsAg immune-escape mutations can favor HBV-transmission also in vaccinated individuals, promote immunosuppression-driven HBV-reactivation, and increase fitness of drug-resistant strains. Stop-codons can enhance HBV oncogenic-properties. Furthermore, as a consequence of the overlapping structure of HBV genome, some immune-escape mutations or stop-codons in HBsAg can derive from drug-resistance mutations in RT. This study is aimed at gaining insight in prevalence and characteristics of immune-associated escape mutations, and stop-codons in HBsAg in chronically HBV-infected patients experiencing nucleos(t)ide analogues (NA) in Europe. METHODS: This study analyzed 828 chronically HBV-infected European patients exposed to ≥ 1 NA, with detectable HBV-DNA and with an available HBsAg-sequence. The immune-associated escape mutations and the NA-induced immune-escape mutations sI195M, sI196S, and sE164D (resulting from drug-resistance mutation rtM204 V, rtM204I, and rtV173L) were retrieved from literature and examined. Mutations were defined as an aminoacid substitution with respect to a genotype A or D reference sequence. RESULTS: At least one immune-associated escape mutation was detected in 22.1% of patients with rising temporal-trend. By multivariable-analysis, genotype-D correlated with higher selection of ≥ 1 immune-associated escape mutation (OR[95%CI]:2.20[1.32-3.67], P = 0.002). In genotype-D, the presence of ≥ 1 immune-associated escape mutations was significantly higher in drug-exposed patients with drug-resistant strains than with wild-type virus (29.5% vs 20.3% P = 0.012). Result confirmed by analysing drug-naïve patients (29.5% vs 21.2%, P = 0.032). Strong correlation was observed between sP120T and rtM204I/V (P < 0.001), and their co-presence determined an increased HBV-DNA. At least one NA-induced immune-escape mutation occurred in 28.6% of patients, and their selection correlated with genotype-A (OR[95%CI]:2.03[1.32-3.10],P = 0.001). Finally, stop-codons are present in 8.4% of patients also at HBsAg-positions 172 and 182, described to enhance viral oncogenic-properties. CONCLUSIONS: Immune-escape mutations and stop-codons develop in a large fraction of NA-exposed patients from Europe. This may represent a potential threat for horizontal and vertical HBV transmission also to vaccinated persons, and fuel drug-resistance emergence.

Diagnosis of HIV-1 infection: Performance of Xpert Qual and Geenius supplemental assays in fourth generation ELISA-reactive samples.

BACKGROUND: Architect (AR) and Vidas (VD) fourth generation HIV screening immunoassays, which identify early stages of HIV infections, could have false positive results especially at low signal/cutoff (S/C) AR values. Geenius HIV1/2 (GS) is a specific confirmation line immunoassay that is not highly sensitive to early HIV infections. An HIV-1 RNA assay may better detect such infections. OBJECTIVES: To evaluate all AR-VD reactive samples with GS results, and to assess Xpert Qual HIV-1 RNA assay (XQ) as an alternative to GS, in the first low S/C AR-VD-reactive samples from a tested individual. STUDY DESIGN: First AR-VD-reactive-GS-tested results from all individuals with resolved HIV status, collected between March 2015 and March 2017 (n = 749), were retrospectively assessed. Samples with AR-VD-reactive-GS-discordant results and those with low S/C AR-VD-reactive results, were tested by XQ. Receiver operating characteristic (ROC) analysis of GS and XQ sensitivity/specificity was performed. RESULTS: Overall, 94.1% (705/749) of AR-VD-reactive results were true HIV-1 positive. All samples with <3 S/C AR values were false positive. XQ resolved all first samples with AR-VD-reactive-GS-discordant results. The diagnostic accuracy of XQ in low (≤33 S/C) AR-VD-reactive samples was better than that of GS (97.6%, 81/83 versus 73.5%, 61/83, p < 0.01). ROC analysis for low S/C AR samples was optimal for pooled XQ and GS results. CONCLUSIONS: Incorporating XQ in the current screening algorithm for the first AR-VD-reactive-GS-discordant samples may significantly reduce overall turn-around time of HIV-1 diagnosis.

Evaluation of Xpert HIV-1 Qual assay for resolution of HIV-1 infection in samples with negative or indeterminate Geenius HIV-1/2 results.

BACKGROUND: Diagnosis of HIV infection is a multistage algorithm. Following screening with 4(th) generation combination immunoassay, confirmation of HIV infection is performed with an antibody assay that differentiates HIV-1 from HIV-2 infection. In the newly updated algorithm, samples that are nonreactive or indeterminate in the differentiation assay are to be tested with an HIV-1 nucleic acid amplification (NAAT) test for resolution. Xpert HIV-1 Qual is a new NAAT assay approved for the identification of HIV infection in whole and dried blood. OBJECTIVES: To assess the performance of Xpert HIV-1 Qual supplementary assay in resolving the clinical status of serum samples reactive by 4(th) generation immunoassays and indeterminate or negative by Geenius HIV-1/2 confirmatory assay. STUDY DESIGN: In a retrospective study, samples from 97 individuals for whom the true HIV-1 status was already known (by follow-up samples) and which were negative or indeterminate by HIV-1/2 Geenius assay were tested with Xpert Qual HIV-1 assay. RESULTS: Xpert Qual assay correctly classified all 97 samples from HIV-1 positive (n=49) and negative (n=48) individuals. The sensitivity and specificity of Xpert Qual when using the true HIV status as a reference were 100% (92.7-100% at 95% confidence interval [CI] and 92.6-100% at 95% CI, respectively). CONCLUSIONS: Applying Xpert Qual HIV-1 assay in the new HIV multi-stage diagnostic algorithm correctly classified 100% of HIV-1 infections including 49 from HIV-1 carriers who have not yet seroconverted. With this assay the total time required for acute HIV diagnosis could be significantly reduced.

Evaluation of GS Junior and MiSeq next-generation sequencing technologies as an alternative to Trugene population sequencing in the clinical HIV laboratory.

Population HIV-1 sequencing is currently the method of choice for the identification and follow-up of HIV-1 antiretroviral drug resistance. It has limited sensitivity and results in a consensus sequence showing the most prevalent nucleotide per position. Moreover concomitant sequencing and interpretation of the results for several samples together is laborious and time consuming. In this study, the practical use of GS Junior and MiSeq bench-top next generation sequencing (NGS) platforms as an alternative to Trugene Sanger-based population sequencing in the clinical HIV laboratory was assessed. DeepChek(®)-HIV TherapyEdge software was used for processing all the protease and reverse transcriptase sequences and for resistance interpretation. Plasma samples from nine HIV-1 carriers, representing the major HIV-1 subtypes in Israel, were compared. The total number of amino acid substitutions identified in the nine samples by GS Junior (232 substitutions) and MiSeq (243 substitutions) was similar and higher than Trugene (181 substitutions), emphasizing the advantage of deep sequencing on population sequencing. More than 80% of the identified substitutions were identical between the GS Junior and MiSeq platforms, most of which (184 of 199) at similar frequency. Low abundance substitutions accounted for 20.9% of the MiSeq and 21.9% of the GS Junior output, the majority of which were not detected by Trugene. More drug resistance mutations were identified by both the NGS platforms, primarily, but not only, at low abundance. In conclusion, in combination with DeepChek, both GS Junior and MiSeq were found to be more sensitive than Trugene and adequate for HIV-1 resistance analysis in the clinical HIV laboratory.

Men who have sex with men, risk behavior, and HIV infection: integrative analysis of clinical, epidemiological, and laboratory databases.

BACKGROUND: Centralized data collection and analytic tools facilitate tracing HIV transmission trends at the patient-population level with increasing resolution, complementing behavioral studies while avoiding sampling biases. By several measures, the rate of HIV infection among men who have sex with men (MSM) in Israel increased in the past several years more rapidly than was expected. We describe features of the data that connect this increase to behavioral changes. METHODS: We retrospectively analyzed data from the national HIV reference laboratory and the national HIV and sexually transmitted infections registries. We examined changes in selected epidemiologic and clinical parameters and in the pattern of drug-resistant virus transmission among MSM in Israel. In particular, virus isolates from 296 MSM (23.8% of all MSM who received a diagnosis) were genotyped, drug-resistance conferring mutations were characterized, and phylogenetic trees were constructed. RESULTS: Compared with earlier years, during 2007-2009 MSM were more often infected with drug-resistant virus before treatment initiation, were coinfected with syphilis, and received a diagnosis during acute retroviral syndrome. Phylogenetic analysis suggested frequent transmission of drug-resistant HIV by drug-treated individuals to >1 partner. Secondary transmission of resistant virus by drug-naive patients is also consistent with the phylogenetic patterns. In addition, non-B HIV subtypes began to appear among MSM. CONCLUSIONS: Together, our findings suggest that the sexual behavior of MSM, both HIV-infected and uninfected, has become riskier, contributing to the number of those seeking early clarification of status, to syphilis comorbidity, and to the spread of drug resistance. These findings call for action by public health planners and community-based organizations aimed at increasing awareness of the risks, bringing a change in attitude and establishing safe sex norms.

Molecular analysis of transitional cell carcinoma using cDNA microarray.

The incidence of transitional cell carcinoma (TCC), the fourth most common neoplasm diagnosed in men, is rising. Despite the development of several noninvasive diagnostic tests, none have gained full recognition by the clinicians. Gene expression profiling of tumors can identify new molecular markers for early diagnosis and disease follow-up. It also allows the classification of tumors into subclasses assisting in disease diagnosis and prognosis, as well as in treatment selection. In this paper, we employed expression profiling for molecular analysis of TCC. A TCC-derived cDNA microarray was constructed and hybridized with 19 probes from normal urothelium and TCC tissues. Hierarchical clustering analysis identified all normal urothelium samples to be tightly clustered and separated from the TCC samples, with 29 of the genes significantly induced (t-test, P<10(-5)) in noninvasive TCC compared to normal urothelium. The identified genes are involved in epithelial cells' functions, tumorigenesis or apoptosis, and could become molecular tools for noninvasive TCC diagnosis. Principal components analysis of the noninvasive and invasive TCC expression profiles further revealed sets of genes that are specifically induced in different tumor subsets, thus providing molecular fingerprints that expand the information gained from classical staging and grading.

Identification of a novel hypoxia-inducible factor 1-responsive gene, RTP801, involved in apoptosis.

Hypoxia is an important factor that elicits numerous physiological and pathological responses. One of the major gene expression programs triggered by hypoxia is mediated through hypoxia-responsive transcription factor hypoxia-inducible factor 1 (HIF-1). Here, we report the identification and cloning of a novel HIF-1-responsive gene, designated RTP801. Its strong up-regulation by hypoxia was detected both in vitro and in vivo in an animal model of ischemic stroke. When induced from a tetracycline-repressible promoter, RTP801 protected MCF7 and PC12 cells from hypoxia in glucose-free medium and from H(2)O(2)-triggered apoptosis via a dramatic reduction in the generation of reactive oxygen species. However, expression of RTP801 appeared toxic for nondividing neuron-like PC12 cells and increased their sensitivity to ischemic injury and oxidative stress. Liposomal delivery of RTP801 cDNA to mouse lungs also resulted in massive cell death. Thus, the biological effect of RTP801 overexpression depends on the cell context and may be either protecting or detrimental for cells under conditions of oxidative or ischemic stresses. Altogether, the data suggest a complex type of involvement of RTP801 in the pathogenesis of ischemic diseases.