Evaluation of two commercial diagnostic methods for HHV-8 viral load assessment.

Objectives: Human herpesvirus-8 (HHV-8) can cause Kaposi's sarcoma or B lymphoproliferative disorders such as multicentric Castleman disease. Patient follow-up is based on assessing the HHV-8 viral load, which is usually achieved using real-time polymerase chain reaction (PCR). The HHV-8 Premix r-gene kit (BioMérieux) was used by some laboratories in the past, but BioMérieux ceased the production and distribution of this kit in 2021-2022. Other kits need to be tested so that they can be used for diagnostic purposes. Here we evaluated two commercial kits: HHV8 ELITe MGB Kit (ELITech) and Quanty HHV-8 (Clonit) and compared them with the HHV-8 Premix r-gene kit. Methods: We used whole blood samples that had previously been tested with the HHV-8 Premix r-gene kit for diagnostic purposes. Overall, 46 samples (37 HHV-8-positive and 9 HHV-8-negative) were tested with the ELITe MGB Kit and 37 (29 HHV-8-positive and 8 HHV-8-negative) with the Quanty HHV-8 kit. The different methods were compared using Bland-Altman and Passing-Bablok tests with Analyse-it software. Results: Qualitative results were concordant except for one HHV-8 low-positive sample that was found to be negative by the ELITe MGB Kit. The quantitative results were also concordant; both kits showed mean differences of 0.58 log10 copies/ml and 0.73 log10 copies/ml, respectively, compared to the Premix r-gene kit. Conclusions: Both the methods tested produced acceptable results and could be used for diagnostic purposes. It should be remembered that there is no international standard for HHV-8 quantification and that patients should always be followed using the same method.

Accuracy assessment of total or IgG Immunoglobulin to hepatitis A virus tests around immunity threshold.

BACKGROUND: Anti-hepatitis A virus (HAV) antibody titers at 20 IU/L are assumed to correlate with protection against HAV challenge. METHODS: We examined the accuracy and precision of currently in use immunoassays for total or anti-HAV IgG determination, by repeated testing of dilutions of the international anti-HAV standard, within a 10-50 IU/mL concentration range. RESULTS AND CONCLUSION: Eight immunoassays were evaluated. All could confidently identify people who need to be vaccinated, or who might benefit from a booster vaccine: no positive interpretation for the 10 and 15 IU/mL concentrations. However, qualitative interpretation may differ from test to test in the 15-30 IU/mL range. This variation has to be taken into account when comparing seroprevalence data.

Quantitative Assessment of SARS-CoV-2 Virus in Nasopharyngeal Swabs Stored in Transport Medium by a Straightforward LC-MS/MS Assay Targeting Nucleocapsid, Membrane, and Spike Proteins.

Alternative methods to RT-PCR for SARS-CoV-2 detection are investigated to provide complementary data on viral proteins, increase the number of tests performed, or identify false positive/negative results. Here, we have developed a simple mass spectrometry assay for SARS-CoV-2 in nasopharyngeal swab samples using common laboratory reagents. The method employs high sensitivity and selectivity targeted mass spectrometry detection, monitoring nine constitutive peptides representative of the three main viral proteins and a straightforward pellet digestion protocol for convenient routine applications. Absolute quantification of N, M, and S proteins was achieved by addition of isotope-labeled versions of best peptides. Limit of detection, recovery, precision, and linearity were thoroughly evaluated in four representative viral transport media (VTM) containing distinct total protein content. The protocol was sensitive in all swab media with limit of detection determined at 2 × 103 pfu/mL, corresponding to as low as 30 pfu injected into the LC-MS/MS system. When tested on VTM-stored nasopharyngeal swab samples from positive and control patients, sensitivity was similar to or better than rapid immunoassay dipsticks, revealing a corresponding RT-PCR detection threshold at Ct ∼ 24. The study represents the first thorough evaluation of sensitivity and robustness of targeted mass spectrometry in nasal swabs, constituting a promising SARS-CoV-2 antigen assay for the first-line diagnosis of COVID-19 and compatible with the constraints of clinical settings. The raw files generated in this study can be found on PASSEL (Peptide Atlas) under data set identifier PASS01646.

Improving preparedness to respond to cross-border hepatitis A outbreaks in the European Union/European Economic Area: towards comparable sequencing of hepatitis A virus.

IntroductionSequence-based typing of hepatitis A virus (HAV) is important for outbreak detection, investigation and surveillance. In 2013, sequencing was central to resolving a large European Union (EU)-wide outbreak related to frozen berries. However, as the sequenced HAV genome regions were only partly comparable between countries, results were not always conclusive.AimThe objective was to gather information on HAV surveillance and sequencing in EU/European Economic Area (EEA) countries to find ways to harmonise their procedures, for improvement of cross-border outbreak responses.MethodsIn 2014, the European Centre for Disease Prevention and Control (ECDC) conducted a survey on HAV surveillance practices in EU/EEA countries. The survey enquired whether a referral system for confirming primary diagnostics of hepatitis A existed as well as a central collection/storage of hepatitis A cases' samples for typing. Questions on HAV sequencing procedures were also asked. Based on the results, an expert consultation proposed harmonised procedures for cross-border outbreak response, in particular regarding sequencing. In 2016, a follow-up survey assessed uptake of suggested methods.ResultsOf 31 EU/EEA countries, 23 (2014) and 27 (2016) participated. Numbers of countries with central collection and storage of HAV positive samples and of those performing sequencing increased from 12 to 15 and 12 to 14 respectively in 2016, with all countries typing an overlapping fragment of 218 nt. However, variation existed in the sequenced genomic regions and their lengths.ConclusionsWhile HAV sequences in EU/EEA countries are comparable for surveillance, collaboration in sharing and comparing these can be further strengthened.

Hepatitis A outbreak disproportionately affecting men who have sex with men (MSM) in the European Union and European Economic Area, June 2016 to May 2017.

Between 1 June 2016 and 31 May 2017, 17 European Union (EU) and European Economic Area countries reported 4,096 cases associated with a multi-country hepatitis A (HA) outbreak. Molecular analysis identified three co-circulating hepatitis A virus (HAV) strains of genotype IA: VRD_521_2016, V16-25801 and RIVM-HAV16-090. We categorised cases as confirmed, probable or possible, according to the EU outbreak case definitions. Confirmed cases were infected with one of the three outbreak strains. We investigated case characteristics and strain-specific risk factors for transmission. A total of 1,400 (34%) cases were confirmed; VRD_521_2016 and RIVM-HAV16-090 accounted for 92% of these. Among confirmed cases with available epidemiological data, 92% (361/393) were unvaccinated, 43% (83/195) travelled to Spain during the incubation period and 84% (565/676) identified as men who have sex with men (MSM). Results depict an HA outbreak of multiple HAV strains, within a cross-European population, that was particularly driven by transmission between non-immune MSM engaging in high-risk sexual behaviour. The most effective preventive measure to curb this outbreak is HAV vaccination of MSM, supplemented by primary prevention campaigns that target the MSM population and promote protective sexual behaviour.