A colorimetric RT-LAMP assay and LAMP-sequencing for detecting SARS-CoV-2 RNA in clinical samples.

The coronavirus disease 2019 (COVID-19) pandemic caused by the SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2) coronavirus is a major public health challenge. Rapid tests for detecting existing SARS-CoV-2 infections and assessing virus spread are critical. Approaches to detect viral RNA based on reverse transcription loop-mediated isothermal amplification (RT-LAMP) have potential as simple, scalable, and broadly applicable testing methods. Compared to RT quantitative polymerase chain reaction (RT-qPCR)-based methods, RT-LAMP assays require incubation at a constant temperature, thus eliminating the need for sophisticated instrumentation. Here, we tested a two-color RT-LAMP assay protocol for detecting SARS-CoV-2 viral RNA using a primer set specific for the N gene. We tested our RT-LAMP assay on surplus RNA samples isolated from 768 pharyngeal swab specimens collected from individuals being tested for COVID-19. We determined the sensitivity and specificity of the RT-LAMP assay for detecting SARS-CoV-2 viral RNA. Compared to an RT-qPCR assay using a sensitive primer set, we found that the RT-LAMP assay reliably detected SARS-CoV-2 RNA with an RT-qPCR cycle threshold (CT) number of up to 30, with a sensitivity of 97.5% and a specificity of 99.7%. We also developed a swab-to-RT-LAMP assay that did not require a prior RNA isolation step, which retained excellent specificity (99.5%) but showed lower sensitivity (86% for CT < 30) than the RT-LAMP assay. In addition, we developed a multiplexed sequencing protocol (LAMP-sequencing) as a diagnostic validation procedure to detect and record the outcome of RT-LAMP reactions.

SARS-CoV-2 RNA Extraction Using Magnetic Beads for Rapid Large-Scale Testing by RT-qPCR and RT-LAMP.

Rapid large-scale testing is essential for controlling the ongoing pandemic of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The standard diagnostic pipeline for testing SARS-CoV-2 presence in patients with an ongoing infection is predominantly based on pharyngeal swabs, from which the viral RNA is extracted using commercial kits, followed by reverse transcription and quantitative PCR detection. As a result of the large demand for testing, commercial RNA extraction kits may be limited and, alternatively, non-commercial protocols are needed. Here, we provide a magnetic bead RNA extraction protocol that is predominantly based on in-house made reagents and is performed in 96-well plates supporting large-scale testing. Magnetic bead RNA extraction was benchmarked against the commercial QIAcube extraction platform. Comparable viral RNA detection sensitivity and specificity were obtained by fluorescent and colorimetric reverse transcription loop-mediated isothermal amplification (RT-LAMP) using a primer set targeting the N gene, as well as RT-qPCR using a primer set targeting the E gene, showing that the RNA extraction protocol presented here can be combined with a variety of detection methods at high throughput. Importantly, the presented diagnostic workflow can be quickly set up in a laboratory without access to an automated pipetting robot.

Molecular surveillance of drug-resistance associated mutations of Plasmodium falciparum in south-west Tanzania.

BACKGROUND: In Tanzania, drug-resistant malaria parasites are an increasing public health concern. Because of widespread chloroquine (CQ) resistance Tanzania changed its first line treatment recommendations for uncomplicated malaria from CQ to sulfadoxine-pyrimethamine (SP) in 2001. Loss of SP sensitivity is progressing rapidly. SP resistance is associated with mutations in the dihydrofolate reductase (pfdhfr) and dihydropteroate synthase (pfdhps) genes. METHODS: In samples from 86 patients with uncomplicated Plasmodium falciparum malaria from Mbeya and Matema, Mbeya region, south-western Tanzania, the occurrence of mutations was investigated in the pfcrt and pfmdr1 genes which are associated with CQ resistance and in pfdhfr and pfdhps, conferring SP resistance, as well in cytb which is linked to resistance to atovaquone. RESULTS: Pfcrt T76 occurs in 50% and pfmdr1 Y86 in 51.7%. Pfdhfr triple mutations coexisting with pfdhps double mutations were detected in 64.3% of the P. falciparum isolates. This quintuple mutation is seen as a possible predictive molecular marker for SP treatment failure. Mutations of the cytb gene were not detected. CONCLUSION: These findings of a high prevalence of mutations conferring SP resistance correspond to data of in vivo SP efficacy studies in other regions of Tanzania and underline the recommendation of changing first-line treatment to artemisinin-based combination therapy.

High carriage rate of ESBL-producing Enterobacteriaceae at presentation and follow-up among travellers with gastrointestinal complaints returning from India and Southeast Asia.

BACKGROUND: International travel contributes to the spread of multidrug-resistant microorganisms including extended spectrum beta-lactamase-producing Enterobacteriaceae (ESBL-PE). We assessed the proportion of faecal carriers of ESBL-PE among 211 patients with gastrointestinal symptoms who returned to Berlin, Germany, after international travel. METHODS: ESBL-PE were screened for on chromogenic agar, antimicrobial susceptibility testing was performed, and ESBL-genes were genotyped. Travel-related data were assessed by questionnaire. RESULTS: Diarrhoea, abdominal pain and nausea were the main symptoms. Half of the travellers carried ESBL-PE (97% Escherichia coli); the proportion was highest for returnees from India (72%) and mainland Southeast Asia (59%), and comparatively lower for Africa (33%) and Central America (20%). Co-resistance to fluoroquinolones (particularly in isolates from India), gentamicin and cotrimoxazole was frequent but all isolates were carbapenem-susceptible. ESBL-PE carriage decreased with increasing timespan from return to presentation, and with age. At revisit of initially ESBL-PE positive patients half a year later, 28% (17/61) of the individuals were still carriers, CTX-M groups being congruent with the initial isolates. CTX-M groups 9 and 1/9, vegetarian diet and cat ownership tended to be associated with ESBL-PE carriage upon revisit. CONCLUSIONS: Travellers, particularly those returning from India and Southeast Asia, constitute a relevant source of potential spread of ESBL-PE. Carriage declines over time but ESBL-PE persist for at least 6 months in a substantial proportion of individuals. Both genetic characteristics of the bacteria and lifestyle factors seem to contribute to persistent carriage of ESBL-PE. A recent, extra-European travel history argues for ESBL-PE screening and contact precautions for patients admitted to hospital.