Jeremy A. Garson

The Omicron variant of SARS-CoV-2 (B.1.1.529), first identified during November 2021, is rapidly spreading throughout the world, replacing the previously dominant Delta variant. Omicron has a high number of mutations in the spike gene, some of which are associated with greatly increased transmissibility and immune evasion. The BA.1 sublineage has been most prevalent but there is recent evidence that the BA.2 sublineage is increasing in proportion in many countries. Genome sequencing is the gold standard for Omicron identification but is relatively slow, resource intensive, of limited capacity and often unavailable. We therefore developed a simple, rapid reverse transcription PCR (RT-PCR) method for sensitive and specific detection of the Omicron variant, including both the BA.1 and BA.2 sublineages. The assay targets a total of 5 nucleotide mutations in the receptor binding domain of the spike gene that give rise to 4 amino acid substitutions at G339D, S371L, S373P and S375F. The forward primer was designed as a double-mismatch allele specific primer (DMAS) with an additional artificial mismatch located four nucleotides from the 3’ end to enhance binding specificity. Assay specificity was confirmed by testing a wide range of previously-sequenced culture-derived viral isolates and clinical samples including the Alpha, Beta and Delta variants and ‘wild type’ SARS-CoV-2. Respiratory syncytial virus and influenza A were also tested. The assay can be run in singleplex format, or alternatively as a multiplex RT-PCR to enable Omicron and Delta variants to be detected and distinguished within the same reaction by means of probes labelled with different fluorescent dyes. Sublineages BA.1 and BA.2 can be differentiated if required. The methods presented here can readily be established in any PCR laboratory and should provide valuable support for epidemiologic surveillance of Omicron infections, particularly in those regions that lack extensive sequencing facilities.

The highly transmissible Delta variant of SARS-CoV-2 (B.1.617.2), first identified in India, is currently replacing pre-existing variants in Europe, the USA, and many other parts of the world. It is essential to monitor efficiently its spread to help guide public health policies. Genome sequencing is the gold standard for identification of Delta, but is time-consuming, expensive, and unavailable in many regions. We describe here a rapid and relatively inexpensive alternative to sequencing for specific identification of the Delta variant, by application of double-mismatch allele-specific RT-PCR (DMAS-RT-PCR). The technique exploits forward and reverse allele-specific primers, targeting two spike gene mutations, L452R and T478K, within the same amplicon. The discriminatory power of each primer is enhanced by the presence of an additional mismatch located at the fourth nucleotide from the 3′ end. Amplicons are detected in real-time by means of a conventional fluorescently-labelled hydrolysis probe. Specificity was assessed by testing a range of well characterised cell culture-derived viral isolates and clinical samples, most of which had previously been fully sequenced. In all cases the results of viral genotyping by DMAS-RT-PCR were entirely concordant with the results of sequencing, and the assay was shown to discriminate reliably between the Delta variant and other variants of concern (Alpha, B.1.1.7 and Beta, B.1.351), and ‘wild-type’ SARS-CoV-2. Other respiratory viruses, including influenza A and respiratory syncytial virus, were non-reactive in the assay. The sensitivity of DMAS-RT-PCR matched that of the diagnostic SARS-CoV-2 RT-qPCR screening assay, which targets the E gene. Several samples that could not be sequenced due to insufficient virus could successfully be genotyped by DMAS-RT-PCR. The method we describe would be simple to establish in any laboratory that has the ability to conduct PCR assays and should greatly facilitate monitoring of the spread of the Delta variant throughout the world, and its proportional representation in any SARS-CoV-2-infected population.

Over the past two decades a number of studies have demonstrated activity of the retroviral enzyme reverse transcriptase in the serum of patients with sporadic amyotrophic lateral sclerosis (ALS). Known human exogenous retroviruses such as HIV-1 have been eliminated as possible sources of this activity and investigators have therefore considered the possibility that human endogenous retroviruses (HERVs) might be involved. HERV-K (HML-2) is the most recent retroviral candidate to be proposed following the observation of elevated HERV-K expression in cortical and spinal neurons of ALS patients and the demonstration of HERV-K envelope protein neurotoxicity in vitro and in transgenic mice. This retroviral hypothesis is an attractive one, not least because it raises the possibility that ALS might become treatable using antiretroviral drugs. In the present study we have attempted independent confirmation of the observation that HERV-K RNA levels are elevated in ALS brain. Total RNA was extracted from the postmortem premotor cortex of 34 patients with ALS and 23 controls. Quantitative real-time reverse transcription PCR (RT-qPCR) was performed according to the MIQE guidelines using HERV-K gag, pol and env primer sets. Data was analysed by the 2-∆∆Ct method with normalisation against two reference genes, GAPDH and XPNPEP1. Geometric mean HERV-K RNA expression levels in the premotor cortex of ALS patients were not found to be different from the expression levels in non-ALS controls. Our findings do not confirm the recently reported association between elevated cortical HERV-K RNA levels and ALS, and thus raise doubts about the role of this endogenous retrovirus in ALS pathogenesis. The results of this study may have implications for ongoing clinical trials aiming to suppress HERV-K activity with antiretroviral drugs.

Background: Japanese encephalitis virus (JEV) is a leading cause of central nervous system (CNS) infections in Asia and results in significant morbidity and mortality. JEV RNA is rarely detected in serum or cerebrospinal fluid (CSF), and diagnosis of JEV infection is usually based on serological tests that are frequently difficult to interpret. Unlike serum or CSF, urine is relatively easy to obtain, but, to date, there has been minimal work on the feasibility of testing urine for JEV RNA. Methods: We investigated the use of lysis buffer and a Microsep device to optimize urine storage for detection of JEV RNA by reverse transcription real-time polymerase chain reaction (RT-qPCR). The best of the studied methods was then evaluated in consecutive patients admitted to the hospital with suspected CNS infections in Laos. Results: We demonstrated degradation of JEV RNA in urine after even short storage periods at 4°C or –80°C. Although there was no advantage in using a Microsep concentration device alone, immediate addition of lysis buffer to fresh urine improved the detection of JEV RNA at the limit of detection. Conclusions: In 2 studies of 41 patients with acute encephalitis syndrome, 11 (27%) were positive for JEV IgM in CSF and/or serum, and 2 (4.9%) were JEV RT-qPCR positive from throat swabs. JEV RNA was not detected in any of these patients’ urine samples. However, lysis buffer was only used during a prospective study, that is, for only 17/41 (41%) patient urine samples. Our findings suggest a need for larger studies testing urine for JEV RNA, with urine collected at different times from symptom onset, and using lysis buffer, which stabilizes RNA, for storage.

Summary: Objective: The aim of our work was to establish a semi‐automated high‐throughput DNA amplification method for the universal screening of bacteria in platelet concentrates (PCs).Background: Among cases of transfusion transmission of infectious agents, bacterial contamination ranks first in the number of events, morbidity and mortality. Transmission occurs mainly by transfused PCs. Automated culture is adopted by some blood banks for screening of bacterial contamination, but this procedure is expensive and has a relatively long turnaround time.Methods: PCs were spiked with suspensions of five different bacterial species in a final concentration of 1 and 10 colony‐forming units (CFU) per millilitre. After incubation, the presence of bacteria was investigated by real‐time polymerase chain reaction (PCR) and by the Enhanced Bacterial Detection System (eBDS, Pall) assay as a reference method. Real‐time PCR amplification was performed with a set of universal primers and probes targeting the 16S rRNA gene. Co‐amplification of human mitochondrial DNA served as an internal control.Results: Using the real‐time PCR method, it was possible to detect the presence of all bacterial species tested with an initial concentration of 10 CFU mL⁻¹ 24 h after contamination, except for Staphylococcus hominis. The PCR assay also detected, at 24 h, the presence of Serratia marcescens and Enterobacter cloacae with an initial concentration of 1 CFU mL⁻¹.Conclusions: The real‐time PCR assay may be a reliable alternative to conventional culture methods in the screening of bacterial contamination of PCs, enabling bacterial detection even with a low initial concentration of microorganisms.

Japanese encephalitis virus (JEV) is a major cause of encephalitis in Asia, and the commonest cause of mosquito-borne encephalitis worldwide. Detection of JEV RNA remains challenging due to the characteristic brief and low viraemia, with 0–25% of patients positive, and the mainstay of diagnosis remains detection of anti-JEV IgM antibody. We performed a systematic review of published RT-PCR protocols, and evaluated them in silico and in vitro alongside new primers and probes designed using a multiple genome alignment of all JEV strains >9,000nt from GenBank, downloaded from the NCBI website (November 2016). The new assays included pan-genotype and genotype specific assays targeting genotypes 1 and 3. Ten RT-qPCR assays were compared, a pre-existing in-house assay, three published assays and six newly designed assays, using serial RNA dilutions. We selected three assays, one published and two novel assays, with the lowest limit of detection (LOD) for further optimisation and validation. One of the novel assays, detecting NS2A, showed the best results, with LOD approximately 4 copies/ reaction, and no cross-reaction on testing closely related viruses in the JEV serocomplex, West Nile Virus and St. Louis Virus. The optimised assays were validated in consecutive patients with central nervous system infections admitted to hospitals in Laos, testing paired CSF and serum samples. We succeeded in developing a JEV specific RT-qPCR assay with at least 1 log₁₀ improved sensitivity as compared to existing assays. Further evaluation is required, field-testing the assay in a larger group of patients.