FLASH-TB: an Application of Next-Generation CRISPR to Detect Drug Resistant Tuberculosis from Direct Sputum.

Offering patients with tuberculosis (TB) an optimal and timely treatment regimen depends on the rapid detection of Mycobacterium tuberculosis (Mtb) drug resistance from clinical samples. Finding Low Abundance Sequences by Hybridization (FLASH) is a technique that harnesses the efficiency, specificity, and flexibility of the Cas9 enzyme to enrich targeted sequences. Here, we used FLASH to amplify 52 candidate genes probably associated with resistance to first- and second-line drugs in the Mtb reference strain (H37Rv), then detect drug resistance mutations in cultured Mtb isolates, and in sputum samples. 92% of H37Rv reads mapped to Mtb targets, with 97.8% of target regions covered at a depth ≥ 10X. Among cultured isolates, FLASH-TB detected the same 17 drug resistance mutations as whole genome sequencing (WGS) did, but with much greater depth. Among the 16 sputum samples, FLASH-TB increased recovery of Mtb DNA compared with WGS (from 1.4% [IQR 0.5-7.5] to 33% [IQR 4.6-66.3]) and average depth reads of targets (from 6.3 [IQR 3.8-10.5] to 1991 [IQR 254.4-3623.7]). FLASH-TB identified Mtb complex in all 16 samples based on IS1081 and IS6110 copies. Drug resistance predictions for 15/16 (93.7%) clinical samples were highly concordant with phenotypic DST for isoniazid, rifampicin, amikacin, and kanamycin [15/15 (100%)], ethambutol [12/15 (80%)] and moxifloxacin [14/15 (93.3%)]. These results highlighted the potential of FLASH-TB for detecting Mtb drug resistance from sputum samples.

A comparison of four epidemic waves of COVID-19 in Malawi; an observational cohort study.

BACKGROUND: Compared to the abundance of clinical and genomic information available on patients hospitalised with COVID-19 disease from high-income countries, there is a paucity of data from low-income countries. Our aim was to explore the relationship between viral lineage and patient outcome. METHODS: We enrolled a prospective observational cohort of adult patients hospitalised with PCR-confirmed COVID-19 disease between July 2020 and March 2022 from Blantyre, Malawi, covering four waves of SARS-CoV-2 infections. Clinical and diagnostic data were collected using an adapted ISARIC clinical characterization protocol for COVID-19. SARS-CoV-2 isolates were sequenced using the MinION™ in Blantyre. RESULTS: We enrolled 314 patients, good quality sequencing data was available for 55 patients. The sequencing data showed that 8 of 11 participants recruited in wave one had B.1 infections, 6/6 in wave two had Beta, 25/26 in wave three had Delta and 11/12 in wave four had Omicron. Patients infected during the Delta and Omicron waves reported fewer underlying chronic conditions and a shorter time to presentation. Significantly fewer patients required oxygen (22.7% [17/75] vs. 58.6% [140/239], p < 0.001) and steroids (38.7% [29/75] vs. 70.3% [167/239], p < 0.001) in the Omicron wave compared with the other waves. Multivariable logistic-regression demonstrated a trend toward increased mortality in the Delta wave (OR 4.99 [95% CI 1.0-25.0 p = 0.05) compared to the first wave of infection. CONCLUSIONS: Our data show that each wave of patients hospitalised with SARS-CoV-2 was infected with a distinct viral variant. The clinical data suggests that patients with severe COVID-19 disease were more likely to die during the Delta wave.

Isolation of Vaccine-Like Poliovirus Strains in Sewage Samples From the United Kingdom.

Background: Environmental surveillance (ES) is a sensitive method for detecting human enterovirus (HEV) circulation, and it is used worldwide to support global polio eradication. We describe a novel ES approach using next-generation sequencing (NGS) to identify HEVs in sewage samples collected in London, United Kingdom, from June 2016 to May 2017. Methods: Two different methods were used to process raw sewage specimens: a 2-phase aqueous separation system and size exclusion by filtration and centrifugation. HEVs were isolated using cell cultures and analyzed using NGS. Results: Type 1 and 3 vaccine-like poliovirus (PV) strains were detected in samples collected from September 2016 through January 2017. NGS analysis allowed us to rapidly obtain whole-genome sequences of PV and non-PV HEV strains. As many as 6 virus strains from different HEV serotypes were identified in a single cell culture flask. PV isolates contained only a small number of mutations from vaccine strains commonly seen in early isolates from vaccinees. Conclusions: Our ES setup has high sensitivity for polio and non-PV HEV detection, generating nearly whole-genome sequence information. Such ES systems provide critical information to assist the polio eradication endgame and contribute to the improvement of our understanding of HEV circulation patterns in humans.

Detection and Characterization of Human Pegivirus 2, Vietnam.

We report human pegivirus 2 (HPgV-2) infection in Vietnam. We detected HPgV-2 in some patients with hepatitis C virus/HIV co-infection but not in patients with HIV or hepatitis A, B, or C virus infection, nor in healthy controls. HPgV-2 strains in Vietnam are phylogenetically related to global strains.

Prevalence of Endemic Respiratory Viruses During the COVID-19 Pandemic in Urban and Rural Malawi.

Background: We investigated endemic respiratory virus circulation patterns in Malawi, where no lockdown was imposed, during the COVID-19 pandemic. Methods: Within a prospective household cohort in urban and rural Malawi, adult participants provided upper respiratory tract (URT) samples at 4 time points between February 2021 and April 2022. Polymerase chain reaction (PCR) was performed for SARS-CoV-2, influenza, and other endemic respiratory viruses. Results: 1626 URT samples from 945 participants in 542 households were included. Overall, 7.6% (n = 123) samples were PCR- positive for >1 respiratory virus; SARS-CoV-2 (4.4%) and rhinovirus (2.0%) were most common. No influenza A virus was detected. Influenza B and respiratory syncytial virus (RSV) were rare. Higher virus positivity were detected in the rural setting and at earlier time points. Coinfections were infrequent. Conclusions: Endemic respiratory viruses circulated in the community in Malawi during the pandemic, though influenza and RSV were rarely detected. Distinct differences in virus positivity and demographics were observed between urban and rural cohorts.

Distribution and origins of Mycobacterium tuberculosis L4 in Southeast Asia.

Molecular and genomic studies have revealed that Mycobacterium tuberculosis Lineage 4 (L4, Euro-American lineage) emerged in Europe before becoming distributed around the globe by trade routes, colonial migration and other historical connections. Although L4 accounts for tens or hundreds of thousands of tuberculosis (TB) cases in multiple Southeast Asian countries, phylogeographical studies have either focused on a single country or just included Southeast Asia as part of a global analysis. Therefore, we interrogated public genomic data to investigate the historical patterns underlying the distribution of L4 in Southeast Asia and surrounding countries. We downloaded 6037 genomes associated with 29 published studies, focusing on global analyses of L4 and Asian studies of M. tuberculosis. We identified 2256 L4 genomes including 968 from Asia. We show that 81 % of L4 in Thailand, 51 % of L4 in Vietnam and 9 % of L4 in Indonesia belong to sub-lineages of L4 that are rarely seen outside East and Southeast Asia (L4.2.2, L4.4.2 and L4.5). These sub-lineages have spread between East and Southeast Asian countries, with no recent European ancestor. Although there is considerable uncertainty about the exact direction and order of intra-Asian M. tuberculosis dispersal, due to differing sampling frames between countries, our analysis suggests that China may be the intermediate location between Europe and Southeast Asia for two of the three predominantly East and Southeast Asian L4 sub-lineages (L4.2.2 and L4.5). This new perspective on L4 in Southeast Asia raises the possibility of investigating host population-specific evolution and highlights the need for more structured sampling from Southeast Asian countries to provide more certainty of the historical and current routes of dispersal.

Utilizing river and wastewater as a SARS-CoV-2 surveillance tool in settings with limited formal sewage systems.

The COVID-19 pandemic has profoundly impacted health systems globally and robust surveillance has been critical for pandemic control, however not all countries can currently sustain community pathogen surveillance programs. Wastewater surveillance has proven valuable in high-income settings, but less is known about the utility of water surveillance of pathogens in low-income countries. Here we show how wastewater surveillance of SAR-CoV-2 can be used to identify temporal changes and help determine circulating variants quickly. In Malawi, a country with limited community-based COVID-19 testing capacity, we explore the utility of rivers and wastewater for SARS-CoV-2 surveillance. From May 2020-May 2022, we collect water from up to 112 river or defunct wastewater treatment plant sites, detecting SARS-CoV-2 in 8.3% of samples. Peak SARS-CoV-2 detection in water samples predate peaks in clinical cases. Sequencing of water samples identified the Beta, Delta, and Omicron variants, with Delta and Omicron detected well in advance of detection in patients. Our work highlights how wastewater can be used to detect emerging waves, identify variants of concern, and provide an early warning system in settings with no formal sewage systems.

Utilizing river and wastewater as a SARS-CoV-2 surveillance tool to predict trends and identify variants of concern in settings with limited formal sewage systems.

The COVID-19 pandemic continues to impact health systems globally and robust surveillance is critical for pandemic control, however not all countries can sustain community surveillance programs. Wastewater surveillance has proven valuable in high-income settings, but little is known about how river and informal sewage in low-income countries can be used for environmental surveillance of SARS-CoV-2. In Malawi, a country with limited community-based COVID-19 testing capacity, we explored the utility of rivers and wastewater for SARS-CoV-2 surveillance. From May 2020 - January 2022, we collected water from up to 112 river or informal sewage sites/month, detecting SARS-CoV-2 in 8.3% of samples. Peak SARS-CoV-2 detection in water samples predated peaks in clinical cases. Sequencing of water samples identified the Beta, Delta, and Omicron variants, with Delta and Omicron detected well in advance of detection in patients. Our work highlights wastewater can be used for detecting emerging waves, identifying variants of concern and function as an early warning system in settings with no formal sewage systems.

Cost of wastewater-based environmental surveillance for SARS-CoV-2: Evidence from pilot sites in Blantyre, Malawi and Kathmandu, Nepal.

Environmental surveillance of rivers and wastewater for SARS-CoV-2 detection has been explored as an innovative way to surveil the pandemic. This study estimated the economic costs of conducting wastewater-based environmental surveillance for SARS-CoV-2 to inform decision making if countries consider continuing these efforts. We estimated the cost of two SARS-CoV-2 environmental surveillance pilot studies conducted in Blantyre, Malawi, and Kathmandu, Nepal. The cost estimation accounted for the consumables, equipment, and human resource time costs used for environmental surveillance from sample selection until pathogen detection and overhead costs for the projects. Costs are reported in 2021 US$ and reported as costs per month, per sample and person per year. The estimated costs for environmental surveillance range from $6,175 to $8,272 per month (Blantyre site) and $16,756 to $30,050 (Kathmandu site). The number of samples processed per month ranged from 84 to 336 at the Blantyre site and 96 to 250 at the Kathmandu site. Consumables costs are variable costs influenced by the number of samples processed and are a large share of the monthly costs for ES (ranging from 39% to 72%). The relatively higher costs per month for the Kathmandu site were attributable to the higher allocation of dedicated human resources and equipment to environmental surveillance for SARS-CoV-2 compared to the Blantyre site where these resources were shared with other activities. The average cost per sample ranged from $25 to $74 (Blantyre) and $120 to $175 (Kathmandu). There were associated economies of scale for human resources and equipment costs with increased sample processing and sharing of resources with other activities. The cost per person in the catchment area per year ranged from $0.07 to $0.10 in Blantyre and $0.07 to $0.13 in Kathmandu. Environmental surveillance may be a low-cost early warning signal for SARS-CoV-2 that can complement other SARS-CoV2 monitoring efforts.

A comparison of four epidemic waves of COVID-19 in Malawi; an observational cohort study.

Background: Compared to the abundance of clinical and genomic information available on patients hospitalised with COVID-19 disease from high-income countries, there is a paucity of data from low-income countries. Our aim was to explore the relationship between viral lineage and patient outcome. Methods: We enrolled a prospective observational cohort of adult patients hospitalised with PCR-confirmed COVID-19 disease between July 2020 and March 2022 from Blantyre, Malawi, covering four waves of SARS-CoV-2 infections. Clinical and diagnostic data were collected using an adapted ISARIC clinical characterization protocol for COVID-19. SARS-CoV-2 isolates were sequenced using the MinIONâ"¢ in Blantyre. Results: We enrolled 314 patients, good quality sequencing data was available for 55 patients. The sequencing data showed that 8 of 11 participants recruited in wave one had B.1 infections, 6/6 in wave two had Beta, 25/26 in wave three had Delta and 11/12 in wave four had Omicron. Patients infected during the Delta and Omicron waves reported fewer underlying chronic conditions and a shorter time to presentation. Significantly fewer patients required oxygen (22.7% [17/75] vs. 58.6% [140/239], p<0.001) and steroids (38.7% [29/75] vs. 70.3% [167/239], p<0.001) in the Omicron wave compared with the other waves. Multivariable logistic-regression demonstrated a trend toward increased mortality in the Delta wave (OR 4.99 [95% CI 1.0-25.0 p=0.05) compared to the first wave of infection. Conclusions: Our data show that each wave of patients hospitalised with SARS-CoV-2 was infected with a distinct viral variant. The clinical data suggests that patients with severe COVID-19 disease were more likely to die during the Delta wave. Summary: We used genome sequencing to identify the variants of SARS-CoV-2 causing disease in Malawi, and found that each of the four waves was caused by a distinct variant. Clinical investigation suggested that the Delta wave had the highest mortality.

Distinct clinical and immunological profiles of patients with evidence of SARS-CoV-2 infection in sub-Saharan Africa.

Although the COVID-19 pandemic has left no country untouched there has been limited research to understand clinical and immunological responses in African populations. Here we characterise patients hospitalised with suspected (PCR-negative/IgG-positive) or confirmed (PCR-positive) COVID-19, and healthy community controls (PCR-negative/IgG-negative). PCR-positive COVID-19 participants were more likely to receive dexamethasone and a beta-lactam antibiotic, and survive to hospital discharge than PCR-negative/IgG-positive and PCR-negative/IgG-negative participants. PCR-negative/IgG-positive participants exhibited a nasal and systemic cytokine signature analogous to PCR-positive COVID-19 participants, predominated by chemokines and neutrophils and distinct from PCR-negative/IgG-negative participants. PCR-negative/IgG-positive participants had increased propensity for Staphylococcus aureus and Streptococcus pneumoniae colonisation. PCR-negative/IgG-positive individuals with high COVID-19 clinical suspicion had inflammatory profiles analogous to PCR-confirmed disease and potentially represent a target population for COVID-19 treatment strategies.

Enterovirus D68 in Viet Nam (2009-2015).

BACKGROUND: Since 1962, enterovirus D68 (EV-D68) has been implicated in multiple outbreaks and sporadic cases of respiratory infection worldwide, but especially in the USA and Europe with an increasing frequency between 2010 and 2014. We describe the detection, associated clinical features and molecular characterization of EV-D68 in central and southern Viet Nam between 2009 and 2015. METHODS: Enterovirus/rhinovirus PCR positive respiratory or CSF samples taken from children and adults with respiratory/central nervous system infections in Viet Nam were tested by an EV-D68 specific PCR. The included samples were derived from 3 different observational studies conducted at referral hospitals across central and southern Viet Nam between 2009 and 2015. Whole-genome sequencing was carried out using a MiSeq based approach. Phylogenetic reconstruction and estimation of evolutionary rate and recombination were carried out in BEAST and Recombination Detection Program, respectively. RESULTS: EV-D68 was detected in 21/625 (3.4%) enterovirus/rhinovirus PCR positive respiratory samples but in none of the 15 CSF. All the EV-D68 patients were young children (age range: 11.8 - 24.5 months) and had moderate respiratory infections. Phylogenetic analysis suggested that the Vietnamese sequences clustered with those from Asian countries, of which 9 fell in the B1 clade, and the remaining sequence was identified within the A2 clade. One intra sub-clade recombination event was detected, representing the second reported recombination within EV-D68. The evolutionary rate of EV-D68 was estimated to be 5.12E -3 substitutions/site/year. Phylogenetic analysis indicated that the virus was imported into Viet Nam in 2008. CONCLUSIONS: We have demonstrated for the first time EV-D68 has been circulating at low levels in Viet Nam since 2008, associated with moderate acute respiratory infection in children. EV-D68 in Viet Nam is most closely related to Asian viruses, and clusters separately from recent US and European viruses that were suggested to be associated with acute flaccid paralysis.

Challenges of the Unknown: Clinical Application of Microbial Metagenomics.

Availability of fast, high throughput and low cost whole genome sequencing holds great promise within public health microbiology, with applications ranging from outbreak detection and tracking transmission events to understanding the role played by microbial communities in health and disease. Within clinical metagenomics, identifying microorganisms from a complex and host enriched background remains a central computational challenge. As proof of principle, we sequenced two metagenomic samples, a known viral mixture of 25 human pathogens and an unknown complex biological model using benchtop technology. The datasets were then analysed using a bioinformatic pipeline developed around recent fast classification methods. A targeted approach was able to detect 20 of the viruses against a background of host contamination from multiple sources and bacterial contamination. An alternative untargeted identification method was highly correlated with these classifications, and over 1,600 species were identified when applied to the complex biological model, including several species captured at over 50% genome coverage. In summary, this study demonstrates the great potential of applying metagenomics within the clinical laboratory setting and that this can be achieved using infrastructure available to nondedicated sequencing centres.

A Rapid and Sensitive Diagnostic Screening Assay for Detection of Mycobacteria Including Mycobacterium tuberculosis Directly from Sputum without Extraction.

We report a novel approach utilising a real-time PCR screening assay targeting a 53 bp tandemly repeated element present at various loci within the Mycobacterium tuberculosis (Mtb) genome. Positive samples were identified within a discriminatory melting curve range of 90-94°C, with results obtained in under one hour directly from decontaminated sputum samples without extraction. A panel of 89 smear-positive sputa were used for analytical validation of the assay with 100% concordance, with sensitivity matching that of culture. Cross reactivity was detected within a narrow range of mycobacteria other than tuberculosis (MOTT) (five sputa, three in silico), with the highest sensitivity within M. avium complex (MAC). A year-long head to head evaluation of the test with the GeneXpert platform was carried out with 104 consecutive samples at the Royal Free Hospital, UK. Receiver operating characteristics (ROC) analysis of the data revealed that the two tests are approximately equivalent in sensitivity, with the area under the curve being 0.85 and 0.80 for the GeneXpert and our assay, respectively, indicating that the test would be a cost effective screen prior to GeneXpert testing.