How to choose the right real-time RT-PCR primer sets for the SARS-CoV-2 genome detection?

OBJECTIVES: The SARS-CoV-2 pandemic has created an unprecedented need for rapid large-scale diagnostic testing to prompt clinical and public health interventions. Currently, several quantitative reverse-transcription polymerase chain reaction (RT-qPCR) assays recommended by the World Health Organization are being used by clinical and public health laboratories and typically target regions of the RNA-dependent RNA polymerase (RdRp), envelope (E) and nucleocapsid (N) coding region. However, it is currently unclear if results from different tests are comparable. This study aimed to clarify the clinical performances of the primer/probe sets designed by US CDC and Charité/Berlin to help clinical laboratories in assay selection for SARS-CoV-2 routine detection. METHODS: We compared the clinical performances of the recommended primer/probe sets using one hundred nasopharyngeal swab specimens from patients who were clinically diagnosed with COVID-19. An additional 30 "pre-intervention screening" samples from patients who were not suspected of COVID-19 were also included in this study. We also performed sequence alignment between 31064 European SARS-CoV-2 and variants of concern genomes and the recommended primer/probe sets. RESULTS: The present study demonstrates substantial differences in SARS-CoV-2 RNA detection sensitivity among the primer/probe sets recommended by the World Health Organization especially for low-level viral loads. The alignment of thousands of SARS-CoV-2 sequences reveals that the genetic diversity remains relatively low at the primer/probe binding sites. However, multiple nucleotide mismatches might contribute to false negatives. CONCLUSION: An understanding of the limitations depending on the targeted genes and primer/probe sets may influence the selection of molecular detection assays by clinical laboratories.

Low performance of rapid antigen detection test as frontline testing for COVID-19 diagnosis.

BACKGROUND: Ensuring accurate diagnosis is essential to limit the spread of SARS-CoV-2 and for the clinical management of COVID-19. Although real-time reverse transcription polymerase chain reaction (RT- qPCR) is the current recommended laboratory method to diagnose SARS-CoV-2 acute infection, several factors such as requirement of special equipment and skilled staff limit the use of these time-consuming molecular techniques. Recently, several easy to perform rapid antigen detection tests were developed and recommended in some countries as the first line of diagnostic. OBJECTIVES: The aim of this study was to evaluate the performances of the Coris COVID-19 Ag Respi-Strip test, a rapid immunochromatographic test for the detection of SARS-CoV-2 antigen, in comparison to RT-qPCR. RESULTS: 148 nasopharyngeal swabs were tested. Amongst the 106 positive RT-qPCR samples, 32 were detected by the rapid antigen test, given an overall sensitivity of 30.2%. All the samples detected positive with the antigen rapid test were also positive with RT-qPCR. CONCLUSIONS: Higher viral loads are associated with better antigen detection rates. Unfortunately, the overall poor sensitivity of the COVID-19 Ag Respi-Strip does not allow using it alone as the frontline testing for COVID-19 diagnosis.

Comparison of Sanger sequencing for hepatitis C virus genotyping with a commercial line probe assay in a tertiary hospital.

BACKGROUND: The technique most frequently used to genotype HCV is quantitative RT-PCR. This technique is unable to provide an accurate genotype/subtype for many samples; we decided to develop an in-house method with the goal of accurately identifying the genotype of all samples. As a Belgium National Centre of reference for hepatitis, we developed in-house sequencing not only for 5'UTR and core regions starting from VERSANT LiPA amplicons but also for NS5B regions. The sequencing of VERSANT LiPA amplicons might be useful for many laboratories worldwide using the VERSANT LiPA assay to overcome undetermined results. METHODS: 100 samples from Hepatitis C virus infected patients analysed by the VERSANT HCV Genotype 2.0 LiPA Assay covering frequent HCV types and subtypes were included in this study. NS5B, 5'UTR and Core home-made sequencing were then performed on these samples. The sequences obtained were compared with the HCV genomic BLAST bank. RESULTS: All the samples were characterised by the VERSANT LiPA assay (8 G1a, 17 G1b, 6 G2, 11 G3, 13 G4, and 10 G6). It was not possible to discriminate between G6 and G1 by the VERSANT LiPA assay for 8 samples and 27 had an undetermined genotype. Forty-one samples were sequenced for the three regions: NS5B, 5'UTR and Core. Twenty-three samples were sequenced for two regions: 5' UTR and Core and 36 samples were sequenced only for NS5B. Of the 100 samples included, 64 samples were analysed for 5'UTR and Core sequencing and 79 samples were analysed for NS5B sequencing. The global agreement between VERSANT LiPA assay and sequencing was greater than 95%. CONCLUSIONS: In this study, we describe a new, original method to confirm HCV genotypes of samples not discriminated by a commercial assay, using amplicons already obtained by the screening method, here the VERSANT LiPA assay. This method thus saves one step if a confirmation assay is needed and might be of usefulness for many laboratories worldwide performing VERSANT LiPA assay in particular.

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.

Use of anti-HBc positive allografts in adult liver transplantation: toward a safer way to expand the donor pool.

The use of livers from anti-hepatitis B core (HBc) positive donors can alleviate donor shortage. Nineteen of 367 (6%) adults receiving anti-HBc positive allografts [three were hepatitis B antigen (HBsAg) negative, hepatitis B antibody (HBsAb) positive; four were HBsAg positive and 12 were not exposed to hepatitis B viral (HBV) infection] were retrospectively reviewed. In HBsAg negative recipients, immunoprophylaxis (IP) was guided by viral serology and immunohistochemistry (IH) of day 0 and day 7 liver biopsies. If IH was negative, IP was stopped. None of three HBsAg negative, HBsAb positive recipients infected; one (replicating) of four HBsAg positive recipients reinfected and seven of eight (87.5%) HBsAg, HBsAb negative recipients, who did not receive long-term IP, infected after a median time of 2 years (range 1-5); one patient died of liver failure. Four HBsAg, HBsAb negative recipients, receiving life-long IP, remained infection free. Anti-HBc positive donor livers must be directed selectively first to HBsAg positive recipients, next to recipients having HBV antibodies and finally to HBV-naive recipients. Identification of both donor and recipient risk factors for HBV infection before transplantation allows indiscriminate use of antiviral prophylaxis. The necessity for IP therapy should be guided by HBV-DNA testing of donor liver tissue and serum. IH of early liver biopsies is an unreliable marker for predicting antiviral treatment requirements.

Human immunodeficiency virus type 1 (HIV-1) proviral DNA load in purified CD4+ cells by LightCycler real-time PCR.

BACKGROUND: The human immunodeficiency virus type 1 (HIV-1) proviral DNA persists in infected cells, even after prolonged successful HAART. In the present study, a relative quantification assay of HIV-1 proviral DNA by LightCycler real-time PCR based on SYBR Green I detection was developed in comparison to the number of purified CD4+ cells as estimated by the quantification of the beta-globin gene. METHODS: The ability of the designed gag primers to quantify HIV-1 Group M and the PCR efficiency were assessed on HIV-1 reference isolate subtypes A, B, C and D. The 8E5 cell line containing a single defective copy of HIV-1 proviral DNA was used as a standard for both the HIV-1 target gene and the beta-globin reference gene. The assay was applied on thirty consecutive patient samples received for RNA viral load determinations and on retrospective samples from fifteen patients undergoing 2 years of structured treatment interruption (STI). RESULTS: The lower limit of quantification was 50 HIV-1 DNA proviral copies per CD4+ cell sample. The dynamic range was from 50 to 106 HIV-1 DNA copies per CD4+ cell sample with intra- and inter-assay coefficients of variability ranging from 3.1% to 37.1%. The beta-globin reference gene was quantified down to a limit of 1.5 pg of DNA/microl (approximately 5 cells) with intra- and interassay coefficients of variability ranging from 1.8% to 21%. DNA proviral load varies widely among HIV-1 infected patients. Proviral load and plasma viral load rebound were high in STI patients who took longer to achieve an undetectable plasma viral load under therapy. A statistically significant correlation was observed between DNA proviral load and RNA steady state viral load in STI patients (p-value = 0.012). CONCLUSION: We have developed a fast, sensitive and specific relative quantification assay of HIV-1 proviral DNA in purified CD4+ cells. The assay enables the monitoring of HIV-1 proviral load, which may be useful to monitor therapy efficacy especially in patients with undetectable plasma RNA viral load, and allows the exploration of viral reservoirs.

The seroepidemiology of human T-lymphotropic viruses: types I and II in Europe: a prospective study of pregnant women.

BACKGROUND: Up to 20 million persons are infected with the human retroviruses human T-lymphotropic virus (HTLV)-I and HTLV-II globally. Most data on the seroprevalence of HTLV-I and HTLV-II in Europe are from studies of low-risk blood donors or high-risk injection drug users (IDUs). Little is known about the general population. METHODS: A prospective anonymous study of HTLV-I and HTLV-II seroprevalence among 234,078 pregnant women in Belgium, France, Germany, Italy, Portugal, Spain, and the United Kingdom was conducted. Maternal antibody status was determined by standard methods using sera obtained for routine antenatal infection screens or eluted from infant heel prick dried blood spots obtained for routine neonatal metabolic screens. RESULTS: Anti-HTLV-I/II antibodies were detected and confirmed in 96 pregnant women (4.4 per 10,000, 95% confidence interval [CI]: 3.5-5.2). Of these, 73 were anti-HTLV-I, 17 were anti-HTLV-II, and 6 were specifically anti-HTLV but untyped. The seroprevalence ranged from 0.7 per 10,000 in Germany to 11.5 per 10,000 in France. CONCLUSIONS: Pregnant women better reflect the general population than blood donors or IDUs. The seroprevalence of HTLV-I and HTLV-II in Western Europe is 6-fold higher among pregnant women (4.4 per 10,000) than among blood donors (0.07 per 10,000). These data provide a robust baseline against which changes in HTLV-I and HTLV-II seroprevalence in Europe can be measured.

Stable hepatitis C virus RNA detection by RT-PCR during four days storage.

BACKGROUND: Suboptimal specimen processing and storage conditions of samples which contain hepatitis C virus (HCV) RNA may result in a decline of HCV RNA concentration or false-negative results in the detection of HCV RNA in serum. We evaluated the stability of HCV RNA in serum and clotted blood samples stored at room temperature or at 4 degrees C for 4 days with the aim of optimizing the standard procedures of processing and storage of samples. METHODS: Blood from five HCV RNA positive patients was collected in tubes with and without separator gel, centrifuged 1 or 6 hours after collection. Samples were then left 6, 24, 48, 72 or 96 h at room temperature (21.5 - 25.4 degrees C) or at 4 degrees C before determining their HCV RNA level using the COBAS AMPLICOR HCV MONITOR Test, vs 2.0 (Roche Diagnostic Systems). RESULTS: The logarithm of the HCV RNA level measurements remained within a 0.3 value of the means for 4 days at both temperatures (room temperature or 4 degrees C). CONCLUSIONS: We conclude that blood samples may be collected and aliquoted within 6 h of collection and can be stored at 4 degrees C for 72 hours as proposed by the manufacturer without significant differences in measured HCV RNA level. Our results indicate that lapses in this scheme may still yield reliable results.