Abstract The rapid emergence and global dissemination of SARS-CoV-2 highlighted a need for robust, adaptable surveillance systems. However, financial and infrastructure requirements for whole genome sequencing (WGS) mean most surveillance data have come from higher-resource geographies, despite unprecedented investment in sequencing in low-middle income countries (LMICs) throughout the SARS-CoV-2 pandemic. Consequently, the molecular epidemiology of SARS-CoV-2 in some LMICs is limited, and there is a need for more cost-accessible technologies to help close data gaps for surveillance of SARS-CoV-2 variants. To address this, we have developed two high-resolution melt curve (HRM) assays that target key variant-defining mutations in the SARS-CoV-2 genome, which give unique signature profiles that define different SARS-CoV-2 variants of concern (VOCs). Extracted RNA from SARS-CoV-2 positive samples collected from 205 participants (112 in Burkina Faso, 93 in Kenya) on the day of enrolment in the MALCOV study (Malaria as a Risk Factor for COVID-19) between February 2021 and February 2022 were analysed using our optimised HRM assays and compared to Next Generation Sequencing (NGS) on Oxford Nanopore MinION . With NGS as a reference, two HRM assays, HRM-VOC-1 and HRM-VOC-2, demonstrated sensitivity/specificity of 100%/99.29% and 92.86/99.39%, respectively, for detecting Alpha, 90.08%/100% and 92.31%/100% for Delta and 93.75%/100% and 100%/99.38% for Omicron. The assays described here provide a lower-cost approach (<$1 per sample) to conducting molecular epidemiology, capable of high-throughput testing. We successfully scaled up the HRM-VOC-2 assay to screen a total of 506 samples from which we were able to show the replacement of Alpha with the introduction of Delta and the replacement of Delta by the Omicron variant in this community in Kisumu, Kenya. These assays are readily adaptable and can focus on local epidemiological surveillance questions or be updated quickly to accommodate the emergence of a novel variant or adapt to novel and emerging pathogens.
The COVID-19 pandemic has given rise to numerous commercially available antigen rapid diagnostic tests (Ag-RDTs). To generate and to share accurate and independent data with the global community requires multisite prospective diagnostic evaluations of Ag-RDTs. This report describes the clinical evaluation of the OnSite COVID-19 rapid test (CTK Biotech, CA, USA) in Brazil and the United Kingdom. A total of 496 paired nasopharyngeal (NP) swabs were collected from symptomatic health care workers at Hospital das Clínicas in São Paulo, Brazil, and 211 NP swabs were collected from symptomatic participants at a COVID-19 drive-through testing site in Liverpool, United Kingdom. Swabs were analyzed by Ag-RDT, and results were compared to quantitative reverse transcriptase PCR (RT-qPCR). The clinical sensitivity of the OnSite COVID-19 rapid test in Brazil was 90.3% (95% confidence interval [CI], 75.1 to 96.7%) and in the United Kingdom was 75.3% (95% CI, 64.6 to 83.6%). The clinical specificity in Brazil was 99.4% (95% CI, 98.1 to 99.8%) and in the United Kingdom was 95.5% (95% CI, 90.6 to 97.9%). Concurrently, analytical evaluation of the Ag-RDT was assessed using direct culture supernatant of SARS-CoV-2 strains from wild-type (WT), Alpha, Delta, Gamma, and Omicron lineages. This study provides comparative performance of an Ag-RDT across two different settings, geographical areas, and populations. Overall, the OnSite Ag-RDT demonstrated a lower clinical sensitivity than claimed by the manufacturer. The sensitivity and specificity from the Brazil study fulfilled the performance criteria determined by the World Health Organization, but the performance obtained from the UK study failed to do. Further evaluation of Ag-RDTs should include harmonized protocols between laboratories to facilitate comparison between settings. IMPORTANCE Evaluating rapid diagnostic tests in diverse populations is essential to improving diagnostic responses as it gives an indication of the accuracy in real-world scenarios. In the case of rapid diagnostic testing within this pandemic, lateral flow tests that meet the minimum requirements for sensitivity and specificity can play a key role in increasing testing capacity, allowing timely clinical management of those infected, and protecting health care systems. This is particularly valuable in settings where access to the test gold standard is often restricted.
The U.S. Department of Defense (DoD) must evaluate whether military officers being considered for appointments, promotions, or retirements meet an exemplary conduct standard. The authors of this report analyze DoD and Service policies and processes for tracking and reporting adverse and other unfavorable information about military officers and identify opportunities to better align Senate, DoD, and Service expectations, policies, and processes.
ABSTRACT A loop-mediated isothermal amplification (LAMP) assay for open reading frame 1 (ORF1) of the glutamine synthetase gene of Neisseria gonorrhoeae was able to tolerate urea concentrations of ≤1.8 M, compared with a PCR assay that was functional at concentrations of <100 mM. The LAMP assay was as sensitive as the PCR assay while being faster and simpler to perform.
Abstract The worldwide dissemination of Mycobacterium tuberculosis strains has led to the study of their genetic diversity. One of the most used genotyping methods is spoligotyping, based on the detection of spacers in the clustered regularly interspaced short palindromic repeats (CRISPR) locus. This study assessed the performance of a microbead-based spoligotyping assay using samples extracted from Ziehl-Neelsen-stained smear-microscopy preparations and described the genetic diversity of Mycobacterium tuberculosis among new TB patients in Southern Nations, Nationalities and Peoples’ Region (SNNPR) in Ethiopia. Among the 91 samples analysed, 59 (64.8%) generated spoligotyping patterns. Fifty (84.7%) samples were classified into 12 clusters (mostly Lineage 4 or 3) comprising 2–11 samples and nine had unique spoligotyping patterns. Among the 59 spoligotyping patterns, 25 belonged to the T1 sublineage, 11 to the T3-ETH, 5 to the URAL, 4 to the H3 and 14 to other L4 sublineages. There was a remarkable variation in genetic distribution in SNNPR compared to other regions of the country. Microbead-based spoligotyping is an easy-to-perform, high-throughput assay that can generate genotyping information using material obtained from smear microscopy preparations. The method provides an opportunity to obtain data of the M. tuberculosis genetic epidemiology in settings with limited laboratory resources.
Travel to resource-limited settings is a known risk for acquisition of extended-spectrum β-lactamase-producing Enterobacterales (ESBL-PE) and carbapenem-resistant Enterobacterales (CRE), which are both associated with increased morbidity and mortality. We investigated the ESBL-PE and CRE baseline prevalence in British service personnel (SP).
Abstract Objectives Neonatal sepsis, a major cause of death amongst infants in sub-Saharan Africa, is often gut derived. Gut colonisation by Enterobacteriaceae producing extended spectrum beta-lactamase (ESBL) or carbapenemase enzymes can lead to antimicrobial-resistant (AMR) or untreatable infections. We sought to explore the rates of colonisation by ESBL or carbapenemase producers in two neonatal units (NNUs) in West and East Africa. Methods Stool and rectal swab samples were taken at multiple timepoints from newborns admitted to the NNUs at the University College Hospital, Ibadan, Nigeria and the Jaramogi Oginga Odinga Teaching and Referral Hospital, Kisumu, western Kenya. Samples were tested for ESBL and carbapenemase genes using a previously validated qPCR assay. Kaplan–Meier survival analysis was used to examine colonisation rates at both sites. Results In total 119 stool and rectal swab samples were taken from 42 infants admitted to the two NNUs. Colonisation with ESBL (37 infants, 89%) was more common than with carbapenemase producers (26, 62.4%; P = 0.093). Median survival time before colonisation with ESBL organisms was 7 days and with carbapenemase producers 16 days ( P = 0.035). The majority of ESBL genes detected belonged to the CTX-M-1 (36/38; 95%), and CTX-M-9 (2/36; 5%) groups, and the most prevalent carbapenemase was bla NDM (27/29, 93%). Conclusions Gut colonisation of neonates by AMR organisms was common and occurred rapidly in NNUs in Kenya and Nigeria. Active surveillance of colonisation will improve the understanding of AMR in these settings and guide infection control and antibiotic prescribing practice to improve clinical outcomes.
This study evaluated detection methods for Salmonella Typhi (S. Typhi) in the environment, to establish a novel pathway from field sampling to isolation of viable organisms and molecular confirmation from complex environmental samples, thus enabling environmental surveillance of typhoid.Multiple media were assessed using clinical isolates from the Public Health England's (PHE) Culture collection. The culture pathway selected consisted of a primary 2% bile broth and secondary Selenite F broth, followed by modified Chromogenic Agar for Salmonella Esterase (mCASE). A qPCR assay was adapted from a validated S. Typhi PCR panel for confirmation of isolates, with comparison to biochemical and serological tests showing good specificity. Sampling locations in Blantyre, Malawi were used to compare sampling methods. Viable S. Typhi were isolated from a mixture of trap and grab river water samples on six occasions.Culture of viable S. Typhi from environmental samples was possible using effective capture and culture techniques.Whilst several studies have attempted to detect S. Typhi from the environment, this is the first successful attempt to isolate the organism from river water since the 1980s. Supplementing clinical data with environmental screening offers the potential for enhanced surveillance, which might inform interventions and assess vaccination programmes.
Summary Background The ongoing outbreaks of mpox, the disease caused by monkeypox virus (MPXV) highlight the urgent need for a rapid and low-cost MPXV diagnostic test to accurately detect and control mpox disease. In this study we assessed the diagnostic accuracy of three brands of rapid diagnostic tests for antigen detection (Ag–RDT) of MPXV. Methods We estimated the clinical diagnostic performance of FlowFlex Monkeypox Virus Antigen Rapid Test (ACON Biotech Co., Ltd., Hangzhou, China), Ecotest Monkeypox Antigen Rapid Test (Assure Tech. Co., Ltd, Hangzhou, China), and STANDARD Q Monkeypox Ag Test (SD Biosensor, Inc. Republic of Korea) using skin lesion swabs (SS) and upper–respiratory tract swabs (URS) from 68 participants in the Democratic Republic of the Congo (DRC) and from 16 in the United Kingdom (UK). The limit of detection (LOD) of the three Ag–RDT brands was evaluated using viral culture of the MPXV of the IIb lineage (EVAg, France). Findings Although the specificity of the three Ag–RDT was high (100%), sensitivity was estimated as 15·79% (95% CI, 5·52–37·57%) for Flowflex and Ecotest and as 10·53% (95% CI, 2·94–31·39%) for Standard–Q using SS in the DRC. The sensitivity was estimated as 0.00% (95% CI, 0·0–20·6%) among URS in the DRC. In the UK, the sensitivity of the three Ag-RDT was 0.00% among SS (95% CI, 0·0–12·7%) and among URS (95% CI, 0·0–21·5%). The LOD of all Ag–RDT was determined to be 1·0× 10 4 pfu/ml (1·3×10 5 copies/mL) using viral culture. Interpretation None of the three Ag–RDT reached the target clinical sensitivity and thus we do not recommend these as diagnostic or screening tool for suspected mpox cases. Marked improvement in sensitivity are needed for these Ag–RDT prior adoption. The recommended analytical LOD in the WHO target product profile (TPP) is at 10 6 pfu/mL, being fulfilled by the three brands of Ag–RDTs suggesting that the analytical LOD stated in the TPP does not align with the desired clinical sensitivity. There remains an urgent need for accurate rapid Ag-RDTs for mpox diagnosis.
Infections with Enterobacterales (E) are increasingly difficult to treat due to antimicrobial resistance. After ceftriaxone replaced chloramphenicol (CHL) as empiric therapy for suspected sepsis in Malawi in 2004, extended-spectrum beta-lactamase (ESBL)-E rapidly emerged. Concurrently, resistance to CHL in Escherichia coli and Klebsiella spp. decreased, raising the possibility of CHL re-introduction. However, many phenotypically susceptible isolates still carry CHL acetyltransferase (cat) genes. To understand the molecular mechanisms and stability of this re-emerging CHL susceptibility we use a combination of genomics, phenotypic susceptibility assays, experimental evolution, and functional assays for CAT activity. Here, we show that of 840 Malawian E. coli and Klebsiella spp. isolates, 31% have discordant CHL susceptibility genotype-phenotype, and we select a subset of 42 isolates for in-depth analysis. Stable degradation of cat genes by insertion sequences leads to re-emergence of CHL susceptibility. Our study suggests that CHL could be reintroduced as a reserve agent for critically ill patients with ESBL-E infections in Malawi and similar settings and highlights the ongoing challenges in inferring antimicrobial resistance from sequence data.
'%3e%3cpath fill='%23012169' d='M0 0v30h60V0z'/%3e%3cpath stroke='%23fff' stroke-width='6' d='m0 0 60 30m0-30L0 30'/%3e%3cpath stroke='%23C8102E' stroke-width='4' d='m0 0 60 30m0-30L0 30' clip-path='url(%23b)'/%3e%3cpath stroke='%23fff' stroke-width='10' d='M30 0v30M0 15h60'/%3e%3cpath stroke='%23C8102E' stroke-width='6' d='M30 0v30M0 15h60'/%3e%3c/g%3e%3c/svg%3e)
