Usefulness of a multiplex immunodot in case of discordant results between automated COVID-19 serological assays.

BACKGROUND: At present, the only reliable test for COVID-19 diagnosis is RT-qPCR. Serological assays have been widely used to increase the detection sensitivity of infected population. Hereby, we report the performance of a new pan-IgG multiplex Enzyme Immunoassay (immunodot) method for exploration of discrepant SARS-COV-2 serological results. METHODS: A retrospective study on 38 residual serum samples from recovered COVID-19 subjects with discordant serological results on Roche and Snibe platforms, were reanalyzed on a new semi-automated pan-IgG immunodot Enzyme Immunoassay, namely COVIDOT-TEST, in order to find the source of discrepancies and to evaluate the latter method. All samples were analyzed on the BlueDiver® Instrument and all strips were read by the BlueScan® Scanner using Dr DOT® Software. RESULTS: Based on our data, subject samples showed specific IgG reactions on ≥ 2 different antigens on immunodot strips. Of these 38 samples, 97.4 % of samples showed specific IgG reaction against S1 + S2 antigens, 89.5 % showed against RBD antigen, 86.8 % against S2 antigen reaction on the COVIDOT-TEST kit. Specific IgG-S1 antigen and IgG-N antigen reactions were detected in 73.7 % and 65.8 % of the samples, respectively. CONCLUSION: The new semi-automated pan-IgG immunodot Enzyme Immunoassay method appeared to be a reliable assay to confirm suspicious COVID-19 serological screening results.

Short Communication: An Insertion of Seven Amino Acids in the Envelope Cytoplasmic Tail of HIV-2 Selected During Disease Progression Enhances Viral Replication.

The cytoplasmic tail (CT) of the HIV-2 envelope glycoprotein (Env) includes amino acid (aa) sequences that are similar to lentiviral lytic peptides (LLP) described in other lentiviruses. Within the putative LLP-2 region, we previously observed insertions of 3 or 7 aa in sequences deduced from plasma viral RNA of symptomatic HIV-2-infected individuals. Based on these observations, we reproduced the insertions in a molecular clone to assess their impact on replicative fitness and cell death in vitro. Using a molecular clone of the HIV-2ROD reference strain, site-directed mutagenesis experiments allowed the generation of plasmids with the insertion L791TAI or L791QRALTAI in the Env protein. The clone with 7 aa insertion enhanced viral release 8 to 11 times in infected T cells and cell viability was impaired by more than 20%, compared with the wild-type HIV-2ROD virus. The effect of the 3 aa insertion was milder, with a nonsignificant trend to enhance viral replication and cell death compared with the wild-type virus. Interestingly, the insertions in the Env proteins did not induce a significant increase of viral infectivity, as revealed by the infectivity assay using TZM-bl cells. The insertions in the Env CT observed in vivo from disease progressors may, therefore, be involved in the higher viral load observed in these individuals. This study may open the way to the development of a prognostic marker related to the HIV-2 infection progression.

Specific and quantitative detection of human polyomaviruses BKV and JCV by LightCycler real-time PCR.

BACKGROUND: BK virus (BKV) and JC virus (JCV) are the only two known human polyomavirus that typically establish subclinical persistent infections. In immunocompromised hosts reactivation of the JCV infection is the cause of the central nervous system disease progressive multifocal leucoencephalopathy (PML), while BKV may cause renal nephropathy and haemorrhagic cystitis. OBJECTIVES: The goal of this study was to develop a specific quantification assay for each polyomavirus by LightCycler real-time polymerase chain reaction (PCR) based on SYBR Green I detection. STUDY DESIGN: DNA fragments of 138bp and 233bp from the "large T antigen" region of JCV and BKV, respectively, were amplified. The ability of the designed primer sets to separately quantify BKV DNA or JCV DNA and the PCR efficiency were assessed on reference DNA samples. Known amounts of cloned JCV DNA and BKV DNA from TEBU-BIO nv (Boechout, Belgium) were used to generate standard curves for the quantification assays. Species-specificity of the PCR was evaluated with cloned DNA and with DNA from patient samples. RESULTS: The assay allowed a specific quantification over a 7log dynamic range. Seventeen copies each of the viral genes were reproducibly and accurately detected. The primer sets generated specific DNA fragments for each virus confirmed by agarose gel analysis and by cycle sequencing. The similarities of the amplified gene sequences by BLAST analysis were 99% and 100% for BKV and JCV, respectively. There was no cross-reactivity within the dynamic range of the standard dilutions. CONCLUSIONS: We developed LightCycler real-time PCR assay based on SYBR Green I detection that provided rapid and specific quantification of polyomavirus load.