Molecular diagnosis of COVID-19 in Burkina Faso: successful challenge

COVID-19 has worsened the health situation in Burkina Faso. In fact, the country has known a peak of the second wave, which began in November, and ended around January 2021. Biological diagnosis has played a key role in the management of COVID-19. The aim of this review paper is to address the practical aspects that laboratories have faced in order to meet the challenge of SARS-CoV-2 diagnosis in Burkina Faso. According to international requirements, Burkina Faso has used real-time Reverse Transcription Polymerase Chain Reaction (rRT-PCR) as the “gold standard” for the diagnosis of COVID-19. From March 9, 2020 to July 31, 2021, in Burkina Faso, laboratories involved in COVID-19 diagnosis analyzed 226,189 samples by molecular tests and 2, 352 samples by rapid antigenic tests, whose peak was in January 2021 with 35,984 samples analyzed. The daily average rate of samples analysis was 456.02 tests. The majority of the individuals requesting COVID-19 tests were travelers (62.00%), followed by contact cases (18.42%), suspected cases (7.95%), voluntary screening (7.57%), and 4.06% of other applicants consisting of health care personnel and at-risk patients. In terms of prevention, vaccines are being administered to the general population. However, some efforts must be made to provide automated sample analysis equipment and complete sequencing of SARS-CoV-2 remains among the challenges

Study protocol: a cluster randomized trial to evaluate the effectiveness and implementation of onsite GeneXpert testing at community health centers in Uganda (XPEL-TB).

BACKGROUND: Delays in diagnosis and treatment of tuberculosis (TB) remain common in high-burden countries. To improve case detection, substantial investments have been made to scale-up Xpert MTB/RIF (Xpert), a cartridge-based nucleic acid amplification test that can detect TB within 2 hours, as a replacement for sputum smear microscopy. However, the optimal strategy for implementation of Xpert testing remains unclear. METHODS: The Xpert Performance Evaluation for Linkage to Tuberculosis Care (XPEL-TB) trial uses an ultra-pragmatic, hybrid type II effectiveness-implementation design to assess the effectiveness and implementation of a streamlined strategy for delivery of Xpert testing in real-world settings. Twenty health centers with TB microscopy units were selected to participate in the trial, with ten health centers randomized to the intervention strategy (onsite molecular testing using GeneXpert Edge, process redesign to facilitate same-day TB diagnosis and treatment, and performance feedback) or routine care (onsite sputum smear microscopy plus referral of sputum samples to Xpert testing sites). The primary outcome is the number of patients with microbiologically confirmed TB who were initiated on treatment within 14 days of presentation to the health center, which reflects successful completion of the TB diagnostic evaluation process. Secondary outcomes include health outcomes (6-month vital status), as well as measures of the reach, adoption, and implementation of the intervention strategy. DISCUSSION: The design elements and implementation approach for the XPEL-TB trial were intentionally selected to minimize disruptions to routine care procedures, with the goal of limiting their influence on key primary and secondary outcomes. Trial findings may result in increased support and funding for rapid, onsite molecular testing as the standard-of-care for all patients being evaluated for TB. TRIAL REGISTRATION: US National Institutes of Health's ClinicalTrials.gov, NCT03044158. Registered 06 February 2017. Pan African Clinical Trials Registry, PACTR201610001763265. Registered 03 September 2016

The identification of aptamers against serum biomarkers of human tuberculosis

>Magister Scientiae - MScTuberculosis (TB) is a global health problem and rated as the second leading cause of death after HIV/AIDS. Transmission of TB from one person to the next is very rapid in crowded communities. Therefore, it is crucial to identify people who are infected as quickly as possible not only to provide treatment but also to prevent the spread of the disease. Current TB diagnostic tests such as the culture and sputum smear tests are time-consuming, while rapid tests make use of antibodies that are costly and have low sensitivity and stability. Great improvement has been observed when aptamers are used in place of antibodies in rapid diagnostic tests such as lateral flow devices (LFDs). Therefore, the current study aims to synthesize and identify aptamers against serum biomarkers for development of rapid TB diagnostic tests such as a lateral flow assay. Several TB serum biomarkers have been identified and can be used for the diagnosis of TB. TB biomarkers expressed in serum samples were identified through in silico approach. The biomarkers were expressed in bacterial systems using recombinant DNA technology. The recombinant proteins were purified by affinity chromatography and further used as targets for the selection of aptamers using Systemic Evolution of Ligands by EXponential enrichment (SELEX). Aptamers for the selected biomarkers were synthesized based on magnetic-bead based SELEX and characterized by electrophoretic mobility shift assay (EMSA), Surface Plasmon resonance (SPR) and MicroScale Thermophoresis (MST). Six putative TB serum biomarker proteins were selected from literature, namely, Insulin-like Growth Factor Binding Protein 6 (IGFBP6), Interferon-stimulated Gene 15 (ISG15), Calcium Binding Protein (S100A9), Retinol Binding Protein 4 (RBP4), Granzyme A (GrA), and Transgelin-2 (TAGLN2). The biomarkers were recombinantly expressed and purified after which they were used as targets in SELEX for aptamers synthesis. Aptamers were analysed by in silico method and the ones with highly conserved motifs were selected. The selected aptamers were synthesized and later characterized. The aptamers that show high affinity and specificity for the biomarkers will be used for the fabrication of a rapid lateral flow device for TB screening. Such a test would allow for a short diagnostic turnaround time, and hence expedite treatment

Tuberculosis in Children: Diagnosis and Epidemiology

Globally, an estimated one-million new tuberculosis (TB) cases occurred in children in 2014. For a long time, research of TB in children has been neglected. Most research and surveillance of TB is performed in adults, and the resulting lack of evidence in children is a major barrier for implementation of rational control strategies for children, including diagnosis. More research on TB in children is of importance as children are more susceptible to developing severe extrapulmonary TB, children require different approaches to both diagnosis and treatment and paediatric TB reflects the ongoing transmission of TB in the population. This research gap on the epidemiology of tuberculosis in children, especially in high burden countries, should be addressed in order to better understand the dynamics of TB transmission in both adults and children. Accurate data are the basis for establishment of effective control strategies. This thesis aims to assess the diagnostic role of microscopic observation drug susceptibility (MODS) and mycobacterial blood culture for diagnosis of TB in children as well as to present the epidemiological characteristics of paediatric TB in northern Vietnam with regard to drug resistance and genotypes of Mycobacterium tuberculosis (MTB), the causative agent of TB, isolated from them. MODS is a low cost non-commercial liquid culture assay to detect MTB by microscopy. MODS was compared with conventional assays including Ziehl-Neelsen smear (ZN) and Löwenstein-Jensen culture (LJ) in a study conducted from 2009 to 2010 at the National Hospital for Paediatrics, a general paediatric hospital, in Hanoi, Vietnam. In suspected paediatric TB cases, the MODS test was shown to be significantly more sensitive than both smear (46.0% vs. 8.8%) and LJ (46.0% vs. 38.9%), and significantly faster than LJ with a median time difference of 28 days in favour of MODS (7 days vs. 35 days). The findings suggest that MODS is a rapid low-cost diagnostic tool for TB diagnosis in the paediatric population. The additional yield of mycobacterial blood culture was assessed in comparison to routine detection methods for TB diagnosis in children in two hospitals in Vietnam. The findings show that mycobacterial blood culture detected an additional six TB cases of which 5 cases were negative with other tests and in the remaining case no other tests were done. All six cases were susceptible to rifampicin and isoniazid. The overall performance of TB blood culture was poor as compared to routine culture with regard to detection rate (2.9%, 16/554 vs. 16.6%, 92/554) and turnaround time (26 days vs. 14 days). The incremental cost for adding one additional TB case is substantial. Therefore, addition of mycobacterial blood culture into routine diagnostics has limited utility in resource limited settings. To assess the molecular epidemiology of paediatric TB in northern Vietnam, a collection of 125 MTB isolates from children with TB admitted to NHP during 2009 to 2013 was analysed. Drug susceptibility testing results from 121 isolates and genotypes from 120 isolates were generated. The phenotypic drug susceptibility testing showed that 20.7% was resistant to isoniazid (25/121), 3.3% resistant to rifampicin (4/121), 28.1% resistant to streptomycin (34/121) and 2.5% resistant to ethambutol (1/121). There were 4 cases with multidrug resistant TB. The high frequency of resistance to isoniazid and streptomycin are consistent with data from adults in Vietnam, suggesting the ongoing transmission of drug resistant MTB in the community. MIRU typing patterns showed that the Beijing genotype was predominant in this population (63.3%, 76/120), which is in agreement with various prior studies in adults in Vietnam. These findings provide more evidence to support the hypothesis of the epidemic spread of the Beijing genotype in Vietnam. In this study, an association between Beijing genotype and drug resistance to streptomycin and isoniazid was observed. The number of MDR isolates was too small to draw conclusions regarding association of MDR and Beijing genotype. Collectively, these results demonstrate that liquid culture can improve the yield of TB diagnosis in Vietnam and mycobacterial blood culture should not be routinely performed for paediatric cases. The molecular epidemiology study also showed that the Beijing genotype is the predominant lineage among actively transmitted strains in Vietnam and that it is associated with both isoniazid and streptomycin resistance. Paediatric TB remains a significant cause of morbidity and mortality in Vietnamese children and sustained political and social commitment from all stakeholders, including governments, funders, academics and the medical community will be needed to improve diagnosis, treatment and prevention of TB in children globally

Evaluation of Diagnostic Tests for HIV Detection in Children in Malawi

Annually >40,000 Malawian babies are exposed to HIV, and >1800 get infected. Maternal antibodies limit the utility of HIV antibody testing in children <18 months, and HIV-DNA-PCR is preferred. However, HIV-PCR testing sites are few, PCR is expensive and PCR result turn-around-times (TAT) average 3 months leading to ~ 1/3rd of tested infants being lost to follow-up. A point-of-care HIV Test (POCT) may improve testing uptake and infant retention. This study aimed to identify an appropriate POCT for HIV diagnosis in children <18 months of age and to estimate its impact on infant diagnosis and linkage to HIV care. We conducted mixed methods study in two hospitals in Southern Malawi and evaluated: (1) performance, TAT, usability, acceptability, cost and cost-effectiveness for XpertHIV and compared it with HIV-DNA-PCR, and (2) performance and accuracy of plasma IP-10 in screening for HIV diagnosis and treatment failure among children aged 0-14 years; and (3) explored the potential role of HIV-LAMP assay in the diagnosis of HIV. XpertHIV had high sensitivity (97.8%) and specificity (98.1%). TAT was reduced from 24 days to 5.3 hours: 85% of the children-initiated ART on the day of testing. Compared with qPCR cost ($66.66), XpertHIV was cheaper at$42.34 per diagnostic test. With the difference in test costs, if XpertHIV is adopted nationwide for testing of 40,000 HIV exposed babies, Malawi could save ~USD2,193,538.88 annually. XpertHIV was acceptable to care givers, parents, laboratory technologists and nurses. Although IP-10 levels were higher in those with high viral load and under two years of age, it could not distinguish HIV from other infections. The HIV LAMP optimisation was unsuccessful. XpertHIV, if deployed in Malawi, could improve the diagnosis of paediatric HIV and its treatment uptake, and reduce costs of early infant diagnosis (EID). IP-10 is not an accurate screening marker for HIV in children. Further studies are needed to evaluate whether HIV LAMP is a suitable test for EI

Molekylær epidemiologi blant pasienter med multiresistent tuberkulose i Tigray, Etiopia

Tuberculosis (TB), which is caused by closely related Mycobacterium tuberculosis complex (MTBC) species, is an ancient human disease that gravely affects millions of people every year. TB is a preventable and treatable infectious disease. The continuing emergence and spread of multidrug-resistant tuberculosis (MDR-TB ) threaten the global TB control efforts. TB is the first killer among infectious diseases worldwide. According to the WHO report, there were an estimated 10.0 million incident cases, 1.4 million TB deaths, with more than 95% of these deaths in developing countries in 2019. Ethiopia is among the three high TB, TB/HIV, and MDR-TB burden countries. In 2019 in Ethiopia, there were 157,000 new TB cases, 1,400 MDR/RR-TB cases and 23,800 death from TB. This thesis aimed to describe the molecular epidemiology of multidrugresistant Mycobacterium tuberculosis among pulmonary TB patients in Tigray Region, Ethiopia. Three hundred sputum samples were collected from six hospitals of the Tigray Region between July 2018 and August 2019. The 227 samples culture positive for MTBC were subjected to drug susceptibility test to 1st- and 2nd- line anti-TB drugs using line probe assay. Among the 227 positive cultures, 74 samples were sequenced using whole-genome sequencing (WGS). WGS analysis showed diversified Mycobacterium tuberculosis genotypes circulating in the region, with L4 as the predominant genotype. The overall proportion of MDR-TB was high. The high proportion of MDR-TB among new and previously treated patients is alarming and calls for an urgent intervention to improve patient management. The high proportion of MDR-TB among newly diagnosed cases and the high level of recent transmission index indicates an ongoing transmission, which suggests the need for an enhanced TB control program performance to interrupt transmission. The study highlighted the usefulness of mutations at rpoB, katG, embB, rpsL, pncA, ethA, gyrA and rrs genes as a molecular marker for the rapid detection of resistance to RIF, INH, ETB, SM, PZA, ETH, FLQs and injectable 2nd-line anti-TB drugs, respectively. Besides the canonical mutations, a significant number of disputed rpoB mutations were also reported. Overall, the regional TB control program should be strengthened to detect and provide appropriate early treatment and follow-up for drug-resistant TB (DR-TB) cases. Abiding by the five WHOrecommended priority actions for DR-TB management is necessary to reduce the current high MDR-TB burden in the study region. Periodic surveillance of drug-resistance conferring mutations, early diagnosis and treatment of TB, and scaling up of drug susceptibility testing facilities to prevent and control the transmission of DR-TB in the community is recommended.Tuberkulose (TB) forårsakes av nært beslektede arter innen Mycobacterium tuberculosiskomplekset (MTBC), og er en eldgammel, alvorlig sykdom hos mennesker som rammer millioner hvert år. TB er en smittsom sykdom som kan forebygges og behandles, men fremveksten og spredningen av multiresistent tuberkulose (MDR-TB) truer den globale bekjempelsesinnsatsen. TB er den smittsomme sykdommen som gir størst antall dødsfall i verden. WHO anslår at det i 2019 var anslagsvis 10,0 millioner tilfeller, 1,4 millioner TB dødsfall, med mer enn 95% av disse dødsfallene i utviklingsland. Etiopia er blant de tre land som er mest belastet, med høye tall av TB, TB / HIV og MDR-TB. I Etiopia var det i 2019 157.000 nye TB-tilfeller, 1400 MDR tilfeller og 23 800 dødsfall fra TB. Denne doktorgradens overordnede mål var å beskrive den molekylære epidemiologien til sjukdom forårsaket av multiresistent Mycobacterium tuberculosis blant lungepasienter i Tigray-regionen, Etiopia. Tre hundre sputumprøver ble samlet inn fra seks sykehus i Tigray-regionen mellom juli 2018 og august 2019. Av disse ble de 227 prøvene som var positive for MTBC, undersøkt med en følsomhetstest mot 1. og 2. linje anti-TB medisiner ved bruk av Line Probe Assay. Av de 227 positive kulturene ble 74 prøver sekvensert ved bruk av helgenomsekvensering (WGS). WGS-analysene viste at forskjellige MTBC-genotyper som sirkulerte i regionen, med L4 som den dominerende genotypen. Den høye andelen MDR-TB blant nye og tidligere behandlede pasienter er alarmerende og stiller store krav til en forbedret pasienthåndtering. Den høye andelen MDR-TB blant nylig diagnostiserte tilfeller og det høye nivået av nylig overførte infeksjoner indikerer en pågående overføring, og antyder behovet for en forbedret ytelse av TB-kontrollprogrammet for å avbryte overføringen. Studien fremhevet nytten av mutasjoner ved rpoB, katG, embB, rpsL, pncA, ethA, gyrA og rrs gener som en molekylær markør for rask påvisning av resistens mot RIF, INH, ETB, SM, PZA, ETH, FLQ og injiserbare andrelinje anti-TB medisiner. Samlet sett bør det regionale TB-kontrollprogrammet styrkes for å oppdage og gi tilpasset passende behandling og oppfølging av TB-tilfeller. Å identifisere TB-kontrollprogrammets begrensninger i regionen, og følge de fem WHO-anbefalte prioriterte tiltakene for styring av DRTB, er nødvendig for å redusere den nåværende høye MDR-TB-belastningen i studieområdet. Det anbefales periodisk overvåking av mutasjoner som gir antibiotikaresistens, tidlig diagnose og behandling av TB, og oppskalering av fasiliteter for testing av isolater for antibiotikafølsomhet for å forhindre og kontrollere overføring av DR-TB i samfunnet.Mekelle Universit

Drug Resistance in Mycobacterium Tuberculosis: An Investigation of Drug Transporter P-glycoprotein and an Evaluation of Improved Diagnosis of Drug Resistant Tuber- culosis in Cameroonian Pulmonary Tuberculosis Patients.

Ph.D. Thesis. University of Hawaiʻi at Mānoa 2017

Advancing animal tuberculosis surveillance using culture-independent long-read whole-genome sequencing

Acknowledgments Some of the figures (Figures 4–6 and Supplementary Material S1) were generated using BioRender and draw.io, respectively. Funding The author(s) declare financial support was received for the research, authorship, and/or publication of this article. This research was funded by the Wellcome Foundation (grant #222941/Z/21/Z), the South African Medical Research Council, American Association of Zoo Veterinarians Wild Animal Health Fund [S005651 and S007355], the National Research Foundation South African Research Chair Initiative [grant #86949], and MHM was supported by Wellcome Trust (grant #216634/Z/19/Z). AGL is supported by the EDCTP TESA III network (CSA2020NoE-3104).Peer reviewedPublisher PD

Advancing animal tuberculosis surveillance using culture-independent long-read whole-genome sequencing

Animal tuberculosis is a significant infectious disease affecting both livestock and wildlife populations worldwide. Effective disease surveillance and characterization of Mycobacterium bovis (M. bovis) strains are essential for understanding transmission dynamics and implementing control measures. Currently, sequencing of genomic information has relied on culture-based methods, which are time-consuming, resource-demanding, and concerning in terms of biosafety. This study explores the use of culture-independent long-read whole-genome sequencing (WGS) for a better understanding of M. bovis epidemiology in African buffaloes (Syncerus caffer). By comparing two sequencing approaches, we evaluated the efficacy of Illumina WGS performed on culture extracts and culture-independent Oxford Nanopore adaptive sampling (NAS). Our objective was to assess the potential of NAS to detect genomic variants without sample culture. In addition, culture-independent amplicon sequencing, targeting mycobacterial-specific housekeeping and full-length 16S rRNA genes, was applied to investigate the presence of microorganisms, including nontuberculous mycobacteria. The sequencing quality obtained from DNA extracted directly from tissues using NAS is comparable to the sequencing quality of reads generated from culture-derived DNA using both NAS and Illumina technologies. We present a new approach that provides complete and accurate genome sequence reconstruction, culture independently, and using an economically affordable technique

Technical and health governance aspects of the External Quality Assessment Scheme for the SARS-CoV-2 molecular tests: Institutional experience performed in all clinical laboratories of a Regional Health Service

Objectives: Since December 2019, the worldwide public health has been threatened by a severe acute respiratory syndrome caused by Coronavirus-2. From the beginning, a turning point has been the identification of new cases of infection, in order to minimize the virus spreading among the population. For this reason, it was necessary introducing a panel of tests able to identify positive cases, which became crucial for all countries. Methods: As a Regional Reference Centre, the CRQ Laboratory (Regional Laboratory for the Quality Control) developed and conducted an External Quality Assessment (EQA) panel of assay, so as to evaluate the quality of real-time reverse transcription polymerase chain reaction (PCR), which were used by 62 Sicilian laboratories, previously authorized to issue certificates for the COVID-19 diagnosis, on behalf of the Public Health Service. Results: The qualitative performance test was based on pooled samples with different viral loads of SARS-CoV-2 or human Coronavirus OC43. 75% of the participating laboratories tested all core samples correctly, while the remaining 25% interpreted incorrectly the EQA exercise samples matching negatively the standards required. Conclusions: Subsequent inspection visits confirmed the issue of incorrect positive and negative certifications for COVID-19 by private and public laboratories, despite the possession of the authorization requirements currently provided for by current regulations, with a significant impact on the SSR