Molecular malaria surveillance using a novel protocol for extraction and analysis of nucleic acids retained on used rapid diagnostic tests

The use of malaria rapid diagnostic tests (RDTs) as a source for nucleic acids that can be analyzed via nucleic acid amplification techniques has several advantages, including minimal amounts of blood, sample collection, simplified storage and shipping conditions at room temperature. We have systematically developed and extensively evaluated a procedure to extract total nucleic acids from used malaria RDTs. The co-extraction of DNA and RNA molecules from small volumes of dried blood retained on the RDTs allows detection and quantification of P. falciparum parasites from asymptomatic patients with parasite densities as low as 1 Pf/µL blood using reverse transcription quantitative PCR. Based on the extraction protocol we have developed the ENAR (Extraction of Nucleic Acids from RDTs) approach; a complete workflow for large-scale molecular malaria surveillance. Using RDTs collected during a malaria indicator survey we demonstrated that ENAR provides a powerful tool to analyze nucleic acids from thousands of RDTs in a standardized and high-throughput manner. We found several, known and new, non-synonymous single nucleotide polymorphisms in the propeller region of the kelch 13 gene among isolates circulating on Bioko Island, Equatorial Guinea

Emergence and spread of SARS-CoV-2 lineage B.1.620 with variant of concern-like mutations and deletions.

Distinct SARS-CoV-2 lineages, discovered through various genomic surveillance initiatives, have emerged during the pandemic following unprecedented reductions in worldwide human mobility. We here describe a SARS-CoV-2 lineage - designated B.1.620 - discovered in Lithuania and carrying many mutations and deletions in the spike protein shared with widespread variants of concern (VOCs), including E484K, S477N and deletions HV69Δ, Y144Δ, and LLA241/243Δ. As well as documenting the suite of mutations this lineage carries, we also describe its potential to be resistant to neutralising antibodies, accompanying travel histories for a subset of European cases, evidence of local B.1.620 transmission in Europe with a focus on Lithuania, and significance of its prevalence in Central Africa owing to recent genome sequencing efforts there. We make a case for its likely Central African origin using advanced phylogeographic inference methodologies incorporating recorded travel histories of infected travellers

The evolving SARS-CoV-2 epidemic in Africa: Insights from rapidly expanding genomic surveillance

INTRODUCTION Investment in Africa over the past year with regard to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) sequencing has led to a massive increase in the number of sequences, which, to date, exceeds 100,000 sequences generated to track the pandemic on the continent. These sequences have profoundly affected how public health officials in Africa have navigated the COVID-19 pandemic. RATIONALE We demonstrate how the first 100,000 SARS-CoV-2 sequences from Africa have helped monitor the epidemic on the continent, how genomic surveillance expanded over the course of the pandemic, and how we adapted our sequencing methods to deal with an evolving virus. Finally, we also examine how viral lineages have spread across the continent in a phylogeographic framework to gain insights into the underlying temporal and spatial transmission dynamics for several variants of concern (VOCs). RESULTS Our results indicate that the number of countries in Africa that can sequence the virus within their own borders is growing and that this is coupled with a shorter turnaround time from the time of sampling to sequence submission. Ongoing evolution necessitated the continual updating of primer sets, and, as a result, eight primer sets were designed in tandem with viral evolution and used to ensure effective sequencing of the virus. The pandemic unfolded through multiple waves of infection that were each driven by distinct genetic lineages, with B.1-like ancestral strains associated with the first pandemic wave of infections in 2020. Successive waves on the continent were fueled by different VOCs, with Alpha and Beta cocirculating in distinct spatial patterns during the second wave and Delta and Omicron affecting the whole continent during the third and fourth waves, respectively. Phylogeographic reconstruction points toward distinct differences in viral importation and exportation patterns associated with the Alpha, Beta, Delta, and Omicron variants and subvariants, when considering both Africa versus the rest of the world and viral dissemination within the continent. Our epidemiological and phylogenetic inferences therefore underscore the heterogeneous nature of the pandemic on the continent and highlight key insights and challenges, for instance, recognizing the limitations of low testing proportions. We also highlight the early warning capacity that genomic surveillance in Africa has had for the rest of the world with the detection of new lineages and variants, the most recent being the characterization of various Omicron subvariants. CONCLUSION Sustained investment for diagnostics and genomic surveillance in Africa is needed as the virus continues to evolve. This is important not only to help combat SARS-CoV-2 on the continent but also because it can be used as a platform to help address the many emerging and reemerging infectious disease threats in Africa. In particular, capacity building for local sequencing within countries or within the continent should be prioritized because this is generally associated with shorter turnaround times, providing the most benefit to local public health authorities tasked with pandemic response and mitigation and allowing for the fastest reaction to localized outbreaks. These investments are crucial for pandemic preparedness and response and will serve the health of the continent well into the 21st century

Rapid Identification of SARS-CoV-2 Variants of Concern Using a Portable peakPCR Platform

The need for tools that facilitate rapid detection and continuous monitoring of SARS-CoV-2 variants of concern (VOCs) is greater than ever, as these variants are more transmissible and therefore increase the pressure of COVID-19 on healthcare systems. To address this demand, we aimed at developing and evaluating a robust and fast diagnostic approach for the identification of SARS-CoV-2 VOC-associated spike gene mutations. Our diagnostic assays detect the E484K and N501Y single-nucleotide polymorphisms (SNPs) as well as a spike gene deletion (HV69/70) and can be run on standard laboratory equipment or on the portable rapid diagnostic technology platform peakPCR. The assays achieved excellent diagnostic performance when tested with RNA extracted from culture-derived SARS-CoV-2 VOC lineages and clinical samples collected in Equatorial Guinea, Central-West Africa. Simplicity of usage and the relatively low cost are advantages that make our approach well suitable for decentralized and rapid testing, especially in resource-limited settings.ISSN:1520-6882ISSN:0003-270

Development and evaluation of PlasmoPod: A cartridge-based nucleic acid amplification test for rapid malaria diagnosis and surveillance.

Malaria surveillance is hampered by the widespread use of diagnostic tests with low sensitivity. Adequate molecular malaria diagnostics are often only available in centralized laboratories. PlasmoPod is a novel cartridge-based nucleic acid amplification test for rapid, sensitive, and quantitative detection of malaria parasites. PlasmoPod is based on reverse-transcription quantitative polymerase chain reaction (RT-qPCR) of the highly abundant Plasmodium spp. 18S ribosomal RNA/DNA biomarker and is run on a portable qPCR instrument which allows diagnosis in less than 30 minutes. Our analytical performance evaluation indicates that a limit-of-detection as low as 0.02 parasites/μL can be achieved and no cross-reactivity with other pathogens common in malaria endemic regions was observed. In a cohort of 102 asymptomatic individuals from Bioko Island with low malaria parasite densities, PlasmoPod accurately detected 83 cases, resulting in an overall detection rate of 81.4%. Notably, there was a strong correlation between the Cq values obtained from the reference RT-qPCR assay and those obtained from PlasmoPod. In an independent cohort, using dried blood spots from malaria symptomatic children living in the Central African Republic, we demonstrated that PlasmoPod outperforms malaria rapid diagnostic tests based on the PfHRP2 and panLDH antigens as well as thick blood smear microscopy. Our data suggest that this 30-minute sample-to-result RT-qPCR procedure is likely to achieve a diagnostic performance comparable to a standard laboratory-based RT-qPCR setup. We believe that the PlasmoPod rapid NAAT could enable widespread accessibility of high-quality and cost-effective molecular malaria surveillance data through decentralization of testing and surveillance activities, especially in elimination settings

DiaxxoPCR and PlasmoPod setup for rapid molecular malaria detection.

A) The DiaxxoPCR instrument is a pyramid-shaped stand-alone device for rapid qPCR cycling and fluorescence acquisition. (B) The PlasmoPod assay is based on a single-use cartridge which is pre-loaded with all qPCR reagents in dried form. (C) The PlasmoPod laboratory setup consisting of the diaxxoPCR device, a heatblock, a pipette and PlasmoPod cartridges. (D) Finger prick blood is sampled and stored as dried blood spots (DBS). (E) The setup for rapid NA extraction from DBS. (F) The NA are amplified and signals analysed using the diaxxoPCR device and its integrated analysis software. Images A and B are republished from diaxxo AG under a CC BY license, with permission from Dr Michele Gregorini (who is an author of this manuscript), original copyright [diaxxo AG, 2020–2022]. The granted permission to the manuscript has been uploaded. Partially created with Biorender.com.</p

Analysis of asymptomatic malaria cohort.

(A) Correlation of of Cq valuesvales obtained from reference RT-qPCR run on the Biorad CFX96 instrumentintrument and PlasmoPod run on diaxxoPCR. Samples negative for PlasmoPod were assigned a Cq value of -1. (B) Detection probability for PlasmoPod modelled based on reference Cq values. The grey area represents the 95% confidence interval. (C) Detection rate of PlasmoPod stratified by age group. (D) Cq values stratified by age group.</p

Parasitological and demographic characteristics of the study population selected for evaluation of PlasmoPod from Central African Republic.

Parasitological and demographic characteristics of the study population selected for evaluation of PlasmoPod from Central African Republic.</p