Testing of four-sample pools offers resource optimization without compromising diagnostic performance of real time reverse transcriptase-PCR assay for COVID-19

Quick identification and isolation of SARS-CoV-2 infected individuals is central to managing the COVID-19 pandemic. Real time reverse transcriptase PCR (rRT-PCR) is the gold standard for COVID-19 diagnosis. However, this resource-intensive and relatively lengthy technique is not ideally suited for mass testing. While pooled testing offers substantial savings in cost and time, the size of the optimum pool that offers complete concordance with results of individualized testing remains elusive. To determine the optimum pool size, we first evaluated the utility of pool testing using simulated 5-sample pools with varying proportions of positive and negative samples. We observed that 5-sample pool testing resulted in false negativity rate of 5% when the pools contained one positive sample. We then examined the diagnostic performance of 4-sample pools in the operational setting of a diagnostic laboratory using 500 consecutive samples in 125 pools. With background prevalence of 2.4%, this 4-sample pool testing showed 100% concordance with individualized testing and resulted in 66% and 59% reduction in resource and turnaround time, respectively. Since the negative predictive value of a diagnostic test varies inversely with prevalence, we re-tested the 4-sample pooling strategy using a fresh batch of 500 samples in 125 pools when the prevalence rose to 12.7% and recorded 100% concordance and reduction in cost and turnaround time by 36% and 30%, respectively. These observations led us to conclude that 4-sample pool testing offers the optimal blend of resource optimization and diagnostic performance across difference disease prevalence settings.

Norovirus Extraction from Frozen Raspberries Using Magnetic Silica Beads

Human noroviruses (HuNoV) are among the main causes of acute gastroenteritis worldwide. Frozen raspberries have been linked to several HuNoV food-related outbreaks. However, the extraction of HuNoV RNA from frozen raspberries remains challenging. Recovery yields are low, and real-time quantitative reverse transcriptase PCR (RT-qPCR) inhibitors limit the sensitivity of the detection methodologies. A new approach using fine magnetic silica beads was developed for the extraction of HuNoV spiked on frozen raspberries. Relatively low recovery yields were observed with both the magnetic silica bead and the reference ISO 15216-1:2017 methods. High RT-qPCR inhibition was observed with the ISO 15216-1:2017 recommended amplification kit but could be reduced by using an alternative kit. Reducing RT-qPCR inhibition is important to limit the number of inconclusive HuNoV assays thus increasing the capacity to assess the HuNoV prevalence in frozen raspberries.

Possible COVID-19 reinfection in a patient with X-linked agammaglobulinaemia

This report highlights the case of a patient with X-linked agammaglobulinaemia (XLA) and resultant bronchiectasis who was discharged from hospital after recovering from real-time reverse transcriptase-PCR positive COVID-19 infection having had a subsequent negative swab and resolution of symptoms, but was readmitted 3 weeks later with recrudescent symptoms and a further positive swab. Although there are reports of COVID-19 infection in XLA, for the first time we report a case of possible reinfection. Lessons learnt from this case include the potential for reinfection of COVID-19 in a patient with a weakened immune system and the importance of repeating COVID-19 swabs in inpatients. Extra caution needs to be taken when providing care in groups of patients who have a weakened or absent immune system.

Normal breathing releases SARS-CoV-2 into the air

This study tests the release of SARS-CoV-2 RNA into the air during normal breathing, without any sign of possible risk of contagion such as coughing, sneezing or talking. Five patients underwent oropharyngeal, nasopharyngeal and salivary swabs for real-time reverse transcriptase PCR (RT-PCR) detection of SARS-CoV-2 RNA. Direct SARS-CoV-2 release during normal breathing was also investigated by RT-PCR in air samples collected using a microbiological sampler. Viral RNA was detected in air at 1 cm from the mouth of patients whose oropharyngeal, nasopharyngeal and salivary swabs tested positive for SARS-CoV-2 RNA. In contrast, the viral RNA was not identified in the exhaled air from patients with oropharyngeal, nasopharyngeal and salivary swabs that tested negative. Contagion of SARS-CoV-2 is possible by being very close to the mouth of someone who is infected, asymptomatic and simply breathing.