Performance of a stool-based quantitative PCR assay for the diagnosis of tuberculosis in adolescents and adults: a multinational, prospective diagnostic accuracy study.

BACKGROUND: Despite increasing availability of rapid molecular tests for the diagnosis of tuberculosis in high-burden settings, many people with tuberculosis are undiagnosed. Reliance on sputum as the primary specimen for tuberculosis diagnostics contributes to this diagnostic gap. We evaluated the diagnostic accuracy and additive yield of a novel stool quantitative PCR (qPCR) assay for the diagnosis of tuberculosis in three countries in Africa with high tuberculosis burdens. METHODS: We undertook a prospective diagnostic accuracy study in Eswatini, Mozambique, and Tanzania from Sept 21, 2020, to Feb 2, 2023, to compare the diagnostic accuracy for tuberculosis of a novel stool qPCR test with the current diagnostic standard for Mycobacterium tuberculosis DNA detection from sputum and stool, Xpert-MTB/RIF Ultra (Xpert Ultra). Sputum, stool, and urine samples were provided by a cohort of participants, aged 10 years or older, diagnosed with tuberculosis. Participants with tuberculosis (cases) were enrolled within 72 h of treatment initiation for tuberculosis diagnosed clinically or following laboratory confirmation. Participants without tuberculosis (controls) consisted of household contacts of the cases who did not develop tuberculosis during a 6-month follow-up. The performance was compared with a robust composite microbiological reference standard (CMRS). FINDINGS: The cohort of adolescents and adults (n=408) included 268 participants with confirmed or clinical tuberculosis (cases), 147 (55%) of whom were living with HIV, and 140 participants (controls) without tuberculosis. The sensitivity of the novel stool qPCR was 93·7% (95% CI 87·4-97·4) compared with participants with detectable growth on M tuberculosis culture, and 88·1% (81·3-93·0) compared with sputum Xpert Ultra. The stool qPCR had an equivalent sensitivity as sputum Xpert Ultra (94·8%, 89·1-98·1) compared with culture. Compared with the CMRS, the sensitivity of the stool qPCR was higher than the current standard for tuberculosis diagnostics on stool, Xpert Ultra (80·4%, 73·4-86·2 vs 73·5%, 66·0-80·1; p=0·025 on paired comparison). The qPCR also identified 17-21% additional tuberculosis cases compared to sputum Xpert Ultra or sputum culture. In controls without tuberculosis, the specificity of the stool qPCR was 96·9% (92·2-99·1). INTERPRETATION: In this study, a novel qPCR for the diagnosis of tuberculosis from stool specimens had a higher accuracy in adolescents and adults than the current diagnostic PCR gold standard on stool, Xpert-MTB/RIF Ultra, and equivalent sensitivity to Xpert-MTB/RIF Ultra on sputum. FUNDING: National Institutes of Health (NIH) Allergy and Infectious Diseases, and NIH Fogarty International Center.

Contribution of PEPFAR-Supported HIV and TB Molecular Diagnostic Networks to COVID-19 Testing Preparedness in 16 Countries.

The US President's Emergency Plan for AIDS Relief (PEPFAR) supports molecular HIV and tuberculosis diagnostic networks and information management systems in low- and middle-income countries. We describe how national programs leveraged these PEPFAR-supported laboratory resources for SARS-CoV-2 testing during the COVID-19 pandemic. We sent a spreadsheet template consisting of 46 indicators for assessing the use of PEPFAR-supported diagnostic networks for COVID-19 pandemic response activities during April 1, 2020, to March 31, 2021, to 27 PEPFAR-supported countries or regions. A total of 109 PEPFAR-supported centralized HIV viral load and early infant diagnosis laboratories and 138 decentralized HIV and TB sites reported performing SARS-CoV-2 testing in 16 countries. Together, these sites contributed to >3.4 million SARS-CoV-2 tests during the 1-year period. Our findings illustrate that PEPFAR-supported diagnostic networks provided a wide range of resources to respond to emergency COVID-19 diagnostic testing in 16 low- and middle-income countries.

Epidemiologic Investigation of Intestinal Parasite Infection and Associated Risk Factors among Primary Schoolchildren in the Manzini and Lubombo Provinces, the Kingdom of Eswatini.

Although the deworming program has been executed since 2000, the intestinal parasitic infection (IPI) rates among primary schoolchildren (PSC) in the two provinces of the Kingdom of Eswatini investigated in 2010 remained high, reaching 32.2%. In this study, we monitored the IPI status along with the associated risk factors for PSC in two provinces-Manzini and Lubombo. After consent from their parents/guardians, a total of 316 samples collected from PSC with grades 1 to 3 from four primary schools in Manzini and Lubombo were examined by the Merthiolate-Iodine-Formaldehyde (MIF) method. In addition, demographic characteristics and risk factors acquired by questionnaire surveys were included to be statistically analyzed. The overall prevalence was 40.5% (128/316), of which the infection rate in Manzini and Lubombo was 28.8% (19/66) and 58.3% (74/140), respectively. Pathogenic protozoa had the highest infection rate of 20.6% (65/316), including Entamoeba histolytica/dispar (8.5%, 27/316), Giardia duodenalis (14.6%, 46/316), and Blastocystis hominis (9.8%, 31/316). In terms of helminth infection, the infection rate was quite low, 1.6% only, and these five infected cases included four cases of Hymenolepis nana and one case of Enterobius vermicularis infection. Present study showed that 27.8% (88/316) of PSC were infected by more than one pathogenic parasite. Personal hygiene like washing hands before a meal has a significant protection effect (OR = 0.32, 95% CI = 0.14-0.75, p=0.009). Rain or well water and the type of water supply from which they drank also showed a considerable risk factor (OR = 2.44, 95% CI = 1.25-4.79, p=0.04). The IPI rate in PSC seems unlikely changed compared to that of the previous survey conducted in 2010, especially when the pathogenic protozoan infection rate remains high. Treatment of infected PSC with appropriate medication to reduce intestinal pathogenic protozoan infection should be seriously considered by Eswatini Health Authority.

The impact of financial incentives on HIV incidence among adolescent girls and young women in Eswatini: Sitakhela Likusasa, a cluster randomised trial.

INTRODUCTION: Incentives conditional on school attendance or on remaining free of sexually transmitted infections have produced mixed results in reducing HIV incidence. METHODS: HIV-negative adolescent girls and young women aged 15-22%-50% of whom were out of school-were recruited from 293 clusters in Eswatini from urban (30%) and rural areas (70%).Financial incentives conditional on education attendance were randomly allocated at the cluster level. All participants were further individually randomised into eligibility for a raffle incentive conditional on random selection into the raffle, on negative tests for syphilis and Trichomonas vaginalis and on being a raffle winner, creating four subarms in a 2×2 factorial design: no-intervention, raffle incentive, education incentive and raffle & education incentive. Randomisation was unblinded to participants.Logistic regressions were used in intention-to-treat analysis of HIV incidence over 3 years to estimate the impact of incentives conditional on school attendance and raffle incentives conditional on remaining sexually transmitted infection free. RESULTS: The study recruited 4389 HIV-negative participants, who were distributed into four subarms: no intervention (n=1068), raffle incentive (n=1162), education incentive (n=1088) and raffle and education incentive (n=1071).At endline, 272 participants from 3772 for whom endline data were collected, tested positive for HIV. HIV incidence among participants in education treatment arm was significantly lower than in the education control arm, 6.34% (119/1878) versus 8.08% (153/1894) (p=0.041); OR: 0.766 (0.598 to 0.981); adjusted OR (aOR): 0.754 (0.585 to 0.972). Compared with the no intervention subarm, HIV incidence in the raffle and education incentive subarm was significantly lower, 5.79% (54/878) versus 8.84% (80/905); OR: 0.634 (0.443 to 0.907); aOR: 0.622 (0.433 to 0.893), while it was not significantly lower in the raffle incentive subarm. CONCLUSION: Financial incentives conditional on education participation significantly reduced HIV infection among adolescent girls and young women in Eswatini and appear to be a promising tool for prevention in high HIV prevalence settings. TRIAL REGISTRATION NUMBER: Western Institutional Review Board-protocol number 20 141 630.Eswatini National Health Research Review Board-FWA00026661.Pan African Clinical Trials Registry-PACTR201811609257043.

Rapid molecular diagnostics of tuberculosis resistance by targeted stool sequencing.

BACKGROUND: Stool is an important diagnostic specimen for tuberculosis in populations who struggle to provide sputum, such as children or people living with HIV. However, the culture of Mycobacterium tuberculosis (M. tuberculosis) complex strains from stool perform poorly. This limits the opportunity for phenotypic drug resistance testing with this specimen. Therefore, reliable molecular methods are urgently needed for comprehensive drug resistance testing on stool specimens. METHODS: We evaluated the performance of targeted next-generation sequencing (tNGS, Deeplex® Myc-TB) for the detection of mutations associated with M. tuberculosis complex drug resistance on DNA isolated from stool specimens provided by participants from a prospective cohort of patients treated for tuberculosis in Eswatini (n = 66; 56 with and 10 participants without M. tuberculosis complex DNA detected in stool by real-time quantitative PCR), and an independent German validation cohort of participants with culture-confirmed tuberculosis (n = 21). RESULTS: The tNGS assay detected M. tuberculosis complex DNA in 38 of 56 (68%) samples; for 28 of 38 (74%) samples, a full M. tuberculosis complex drug resistance prediction report was obtained. There was a high degree of concordance with sputum phenotypic drug susceptibility results (κ = 0.82). The ability to predict resistance was concentration-dependent and successful in 7/10 (70%), 18/25 (72%), and 3/21 (14%) of samples with stool PCR concentration thresholds of > 100 femtogram per microliter (fg/µl), 1 to 100 fg/µl, and < 1 fg/µl, respectively (p = 0.0004). The German cohort confirmed these results and demonstrated a similarly high concordance between stool tNGS and sputum phenotypic drug susceptibility results (κ = 0.84). CONCLUSIONS: tNGS can identify drug resistance from stool provided by tuberculosis patients. This affords the opportunity to obtain critical diagnostic information for tuberculosis patients who struggle to provide respiratory specimens.

Predicting, Diagnosing, and Treating Acute and Early HIV Infection in a Public Sector Facility in Eswatini.

BACKGROUND: The lack of acute and early HIV infection (AEHI) diagnosis and care contributes to high HIV incidence in resource-limited settings. We aimed to assess the yield of AEHI, predict and diagnose AEHI, and describe AEHI care outcomes in a public sector setting in Eswatini. SETTING: This study was conducted in Nhlangano outpatient department from March 2019 to March 2020. METHODS: Adults at risk of AEHI underwent diagnostic testing for AEHI with the quantitative Xpert HIV-1 viral load (VL) assay. AEHI was defined as the detection of HIV-1 VL on Xpert and either an HIV-seronegative or HIV-serodiscordant third-generation antibody-based rapid diagnostic test (RDT) result. First, the cross-sectional analysis obtained the yield of AEHI and established a predictor risk score for the prediction of AEHI using Lasso logistic regression. Second, diagnostic accuracy statistics described the ability of the fourth-generation antibody/p24 antigen-based Alere HIV-Combo RDT to diagnose AEHI (vs Xpert VL testing). Third, we described acute HIV infection care outcomes of AEHI-positive patients using survival analysis. RESULTS: Of 795 HIV-seronegative/HIV-serodiscordant outpatients recruited, 30 (3.8%, 95% confidence interval: 2.6% to 5.3%) had AEHI. The predictor risk score contained several factors (HIV-serodiscordant RDT, women, feeling at risk of HIV, swollen glands, and fatigue) and had sensitivity and specificity of 83.3% and 65.8%, respectively, to predict AEHI. The HIV-Combo RDT had sensitivity and specificity of 86.2% and 99.9%, respectively, to diagnose AEHI. Of 30 AEHI-positive patients, the 1-month cumulative treatment initiation was 74% (95% confidence interval: 57% to 88%), and the 3-month viral suppression (<1000 copies/mL) was 87% (67% to 98%). CONCLUSION: AEHI diagnosis and care seem possible in resource-limited settings.

The Role of Molecular Testing in Pediatric Meningitis Surveillance in Southern and East African Countries, 2008-2017.

BACKGROUND: As part of the global Invasive Bacterial Vaccine-Preventable Diseases Surveillance Network, 12 African countries referred cerebrospinal fluid (CSF) samples to South Africa's regional reference laboratory. We evaluated the utility of real-time polymerase chain reaction (PCR) in detecting and serotyping/grouping Haemophilus influenzae, Neisseria meningitidis, and Streptococcus pneumoniae (HNS). METHODS: From 2008 to 2017, CSF samples collected from children <5 years old with suspected meningitis underwent routine microbiology testing in-country, and 11 680 samples were submitted for HNS PCR at the regional reference laboratory. Unconditional logistic regression, with adjustment for geographic location, was performed to identify factors associated with PCR positivity. RESULTS: The overall HNS PCR positivity rate for all countries was 10% (1195 of 11 626 samples). In samples with both PCR and culture results, HNS PCR positivity was 11% (744 of 6747 samples), and HNS culture positivity was 3% (207 of 6747). Molecular serotype/serogroup was assigned in 75% of PCR-positive specimens (762 of 1016). Compared with PCR-negative CSF samples, PCR-positive samples were more often turbid (adjusted odds ratio, 6.80; 95% confidence interval, 5.67-8.17) and xanthochromic (1.72; 1.29-2.28), had elevated white blood cell counts (6.13; 4.71-7.99) and high protein concentrations (5.80; 4.34-7.75), and were more often HNS culture positive (32.70; 23.18-46.12). CONCLUSION: PCR increased detection of vaccine-preventable bacterial meningitis in countries where confirmation of suspected meningitis cases is impeded by limited culture capacity.

Effectiveness and safety of reactive focal mass drug administration (rfMDA) using dihydroartemisinin-piperaquine to reduce malaria transmission in the very low-endemic setting of Eswatini: a pragmatic cluster randomised controlled trial.

INTRODUCTION: To reduce malaria transmission in very low-endemic settings, screening and treatment near index cases (reactive case detection (RACD)), is widely practised, but the rapid diagnostic tests (RDTs) used miss low-density infections. Reactive focal mass drug administration (rfMDA) may be safe and more effective. METHODS: We conducted a pragmatic cluster randomised controlled trial in Eswatini, a very low-endemic setting. 77 clusters were randomised to rfMDA using dihydroartemisin-piperaquine (DP) or RACD involving RDTs and artemether-lumefantrine. Interventions were delivered by the local programme. An intention-to-treat analysis was used to compare cluster-level cumulative confirmed malaria incidence among clusters with cases. Secondary outcomes included safety and adherence. RESULTS: From September 2015 to August 2017, 222 index cases from 47 clusters triggered 46 RACD events and 64 rfMDA events. RACD and rfMDA were delivered to 1455 and 1776 individuals, respectively. Index case coverage was 69.5% and 62.4% for RACD and rfMDA, respectively. Adherence to DP was 98.7%. No serious adverse events occurred. For rfMDA versus RACD, cumulative incidences (per 1000 person-years) of all malaria were 2.11 (95% CI 1.73 to 2.59) and 1.97 (95% CI 1.57 to 2.47), respectively; and of locally acquired malaria, they were 1.29 (95% CI 1.00 to 1.67) and 0.97 (95% CI 0.71 to 1.34), respectively. Adjusting for imbalance in baseline incidence, incidence rate ratio for rfMDA versus RACD was 0.95 (95% CI 0.55 to 1.65) for all malaria and 0.82 (95% CI 0.40 to 1.71) for locally acquired malaria. Similar results were obtained in a per-protocol analysis that excluded clusters with <80% index case coverage. CONCLUSION: In a very low-endemic, real-world setting, rfMDA using DP was safe, but did not lower incidence compared with RACD, potentially due to insufficient coverage and/or power. To assess impact of interventions in very low-endemic settings, improved coverage, complementary interventions and adaptive ring trial designs may be needed. TRIAL REGISTRATION NUMBER: NCT02315690.

MDR M. tuberculosis outbreak clone in Eswatini missed by Xpert has elevated bedaquiline resistance dated to the pre-treatment era.

BACKGROUND: Multidrug-resistant (MDR) Mycobacterium tuberculosis complex strains not detected by commercial molecular drug susceptibility testing (mDST) assays due to the RpoB I491F resistance mutation are threatening the control of MDR tuberculosis (MDR-TB) in Eswatini. METHODS: We investigate the evolution and spread of MDR strains in Eswatini with a focus on bedaquiline (BDQ) and clofazimine (CFZ) resistance using whole-genome sequencing in two collections ((1) national drug resistance survey, 2009-2010; (2) MDR strains from the Nhlangano region, 2014-2017). RESULTS: MDR strains in collection 1 had a high cluster rate (95%, 117/123 MDR strains) with 55% grouped into the two largest clusters (gCL3, n = 28; gCL10, n = 40). All gCL10 isolates, which likely emerged around 1993 (95% highest posterior density 1987-1998), carried the mutation RpoB I491F that is missed by commercial mDST assays. In addition, 21 (53%) gCL10 isolates shared a Rv0678 M146T mutation that correlated with elevated minimum inhibitory concentrations (MICs) to BDQ and CFZ compared to wild type isolates. gCL10 isolates with the Rv0678 M146T mutation were also detected in collection 2. CONCLUSION: The high clustering rate suggests that transmission has been driving the MDR-TB epidemic in Eswatini for three decades. The presence of MDR strains in Eswatini that are not detected by commercial mDST assays and have elevated MICs to BDQ and CFZ potentially jeopardizes the successful implementation of new MDR-TB treatment guidelines. Measures to limit the spread of these outbreak isolates need to be implemented urgently.

Detecting tuberculosis in pregnant and postpartum women in Eswatini.

BACKGROUND: Tuberculosis diagnosis in pregnancy is complex because tuberculosis symptoms are often masked by physiological symptoms of pregnancy. Untreated tuberculosis in pregnant and postpartum women may lead to maternal morbidity and low birth weight. Tuberculosis in HIV-positive pregnant women increases the risk of maternal and infant mortality. OBJECTIVE: This study aimed to determine tuberculosis prevalence stratified by HIV status and identify screening algorithms that maximise detection of active tuberculosis among pregnant and postpartum women in Eswatini. METHODS: Women were enrolled at antenatal and postnatal clinics in Eswatini for tuberculosis screening and diagnostic investigations from 01 April to 30 November 2015 in a cross-sectional study. Sputum samples were collected from all participants for tuberculosis diagnostic tests (smear microscopy, GeneXpert, MGIT culture). Blood and urine samples were collected from HIV-positive women for cluster-of-differentiation-4 cell count, interferon gamma release assay and tuberculosis lateral flow urine lipoarabinomannan tests. RESULTS: We enrolled 990 women; 52% were pregnant and 47% were HIV-positive. The prevalence of tuberculosis among HIV-positive pregnant women was 5% (95% confidence interval [CI]: 2-7) and among postpartum women it was 1% (95%CI: -1-3). Tuberculosis prevalence was 2% (95%CI: 0-3) in HIV-negative pregnant women and 1% (95%CI: -1-2) in HIV-negative postpartum women. The national tuberculosis symptom screening tool failed to identify women who tested tuberculosis-culture positive. CONCLUSION: Routine tuberculosis symptom screening alone is insufficient to rule out tuberculosis in pregnant and postpartum women. Only sputum culture maximised the detection of tuberculosis, indicating a need to balance access and cost in developing countries.

DNA hypermethylation during tuberculosis dampens host immune responsiveness.

Mycobacterium tuberculosis (M. tuberculosis) has coevolved with humans for millennia and developed multiple mechanisms to evade host immunity. Restoring host immunity in order to improve outcomes and potentially shorten existing therapy will require identification of the full complement by which host immunity is inhibited. Perturbation of host DNA methylation is a mechanism induced by chronic infections such as HIV, HPV, lymphocytic choriomeningitis virus (LCMV), and schistosomiasis to evade host immunity. Here, we evaluated the DNA methylation status of patients with tuberculosis (TB) and their asymptomatic household contacts and found that the patients with TB have DNA hypermethylation of the IL-2/STAT5, TNF/NF-κB, and IFN-γ signaling pathways. We performed methylation-sensitive restriction enzyme-quantitative PCR (MSRE-qPCR) and observed that multiple genes of the IL-12/IFN-γ signaling pathway (IL12B, IL12RB2, TYK2, IFNGR1, JAK1, and JAK2) were hypermethylated in patients with TB. The DNA hypermethylation of these pathways was associated with decreased immune responsiveness with decreased mitogen-induced upregulation of IFN-γ, TNF, IL-6, CXCL9, CXCL10, and IL-1ß production. The DNA hypermethylation of the IL-12/IFN-γ pathway was associated with decreased IFN-γ-induced gene expression and decreased IL-12-inducible upregulation of IFN-γ. This study demonstrates that immune cells from patients with TB are characterized by DNA hypermethylation of genes critical to mycobacterial immunity resulting in decreased mycobacteria-specific and nonspecific immune responsiveness.

Active Case Finding for Malaria: A 3-Year National Evaluation of Optimal Approaches to Detect Infections and Hotspots Through Reactive Case Detection in the Low-transmission Setting of Eswatini.

BACKGROUND: Reactive case detection (RACD) is a widely practiced malaria elimination intervention whereby close contacts of index cases receive malaria testing to inform treatment and other interventions. However, the optimal diagnostic and operational approaches for this resource-intensive strategy are not clear. METHODS: We conducted a 3-year prospective national evaluation of RACD in Eswatini, a malaria elimination setting. Loop-mediated isothermal amplification (LAMP) was compared to traditional rapid diagnostic testing (RDT) for the improved detection of infections and for hotspots (RACD events yielding ≥1 additional infection). The potential for index case-, RACD-, and individual-level factors to improve efficiencies was also evaluated. RESULTS: Among 377 RACD events, 10 890 participants residing within 500 m of index cases were tested. Compared to RDT, LAMP provided a 3-fold and 2.3-fold higher yield to detect infections (1.7% vs 0.6%) and hotspots (29.7% vs 12.7%), respectively. Hotspot detection improved with ≥80% target population coverage and response times within 7 days. Proximity to the index case was associated with a dose-dependent increased infection risk (up to 4-fold). Individual-, index case-, and other RACD-level factors were considered but the simple approach of restricting RACD to a 200-m radius maximized yield and efficiency. CONCLUSIONS: We present the first large-scale national evaluation of optimal RACD approaches from a malaria elimination setting. To inform delivery of antimalarial drugs or other interventions, RACD, when conducted, should utilize more sensitive diagnostics and clear context-specific operational parameters. Future studies of RACD's impact on transmission may still be needed.

Field Suitability and Diagnostic Accuracy of the Biocentric Open Real-Time PCR Platform for Dried Blood Spot-Based HIV Viral Load Quantification in Eswatini.

BACKGROUND: To assess the performance and suitability of dried blood spot (DBS) sampling using filter paper to collect blood for viral load (VL) quantification under routine conditions. METHODS: We compared performance of DBS VL quantification using the Biocentric method with plasma VL quantification using Roche and Biocentric as reference methods. Adults (≥18 years) were enrolled at 2 health facilities in Eswatini from October 12, 2016 to March 1, 2017. DBS samples were prepared through finger-prick by a phlebotomist (DBS-1), and through the pipetting of whole venous blood by a phlebotomist (DBS-2) and by a laboratory technologist (DBS-3). We calculated the VL-testing completion rate, correlation, and agreement, as well as diagnostic accuracy estimates at the clinical threshold of 1000 copies/mL. RESULTS: Of 362 patients enrolled, 1066 DBS cards (DBS-1: 347; DBS-2: 359; DBS-3: 360) were tested. Overall, test characteristics were comparable between DBS-sampling methods, irrespective of the reference method. The Pearson correlation coefficients ranged from 0.67 to 0.82 (P < 0.001) for different types of DBS sampling using both reference methods, and the Bland-Altman difference ranged from 0.15 to 0.30 log10 copies/mL. Sensitivity estimates were from 85.3% to 89.2% and specificity estimates were from 94.5% to 98.6%. The positive predictive values were between 87.0% and 96.5% at a prevalence of 30% VL elevations, and negative predictive values were between 93.7% and 95.4%. CONCLUSIONS: DBS VL quantification using the newly configured Biocentric method can be part of contextualized VL-testing strategies, particularly for remote settings and populations with higher viral failure rates.

High Genetic Diversity of Plasmodium falciparum in the Low-Transmission Setting of the Kingdom of Eswatini.

BACKGROUND: To better understand transmission dynamics, we characterized Plasmodium falciparum genetic diversity in Eswatini, where transmission is low and sustained by importation. METHODS: Twenty-six P. falciparum microsatellites were genotyped in 66% of confirmed cases (2014-2016; N = 582). Population and within-host diversity were used to characterize differences between imported and locally acquired infections. Logistic regression was used to assess the added value of diversity metrics to classify imported and local infections beyond epidemiology data alone. RESULTS: Parasite population in Eswatini was highly diverse (expected heterozygosity [HE] = 0.75) and complex: 67% polyclonal infections, mean multiplicity of infection (MOI) 2.2, and mean within-host infection fixation index (FWS) 0.84. Imported cases had comparable diversity to local cases but exhibited higher MOI (2.4 vs 2.0; P = .004) and lower mean FWS (0.82 vs 0.85; P = .03). Addition of MOI and FWS to multivariate analyses did not increase discrimination between imported and local infections. CONCLUSIONS: In contrast to the common perception that P. falciparum diversity declines with decreasing transmission intensity, Eswatini isolates exhibited high parasite diversity consistent with high rates of malaria importation and limited local transmission. Estimates of malaria transmission intensity from genetic data need to consider the effect of importation, especially as countries near elimination.

SNP-IT Tool for Identifying Subspecies and Associated Lineages of Mycobacterium tuberculosis Complex.

The clinical phenotype of zoonotic tuberculosis and its contribution to the global burden of disease are poorly understood and probably underestimated. This shortcoming is partly because of the inability of currently available laboratory and in silico tools to accurately identify all subspecies of the Mycobacterium tuberculosis complex (MTBC). We present SNPs to Identify TB (SNP-IT), a single-nucleotide polymorphism-based tool to identify all members of MTBC, including animal clades. By applying SNP-IT to a collection of clinical genomes from a UK reference laboratory, we detected an unexpectedly high number of M. orygis isolates. M. orygis is seen at a similar rate to M. bovis, yet M. orygis cases have not been previously described in the United Kingdom. From an international perspective, it is possible that M. orygis is an underestimated zoonosis. Accurate identification will enable study of the clinical phenotype, host range, and transmission mechanisms of all subspecies of MTBC in greater detail.

Evaluation of the QuantiFERON-Tuberculosis Gold Plus Assay in Children with Tuberculosis Disease or Following Household Exposure to Tuberculosis.

Interferon-gamma release assays are increasingly used in children to establish evidence of tuberculosis (TB) infection and to assist in the diagnosis of TB disease. The QuantiFERON-TB Gold In-Tube assay is being phased out in favor of a next-generation test, the QuantiFERON-TB Gold Plus (QFT-Plus) assay. The QFT-Plus assay is designed with two antigen tubes to differentially stimulate CD4+ and CD8+ T cells. The performance of this assay has been documented extensively in adults but has not yet been evaluated in children. Here, we compare the performance of the two assays in a cohort of 46 children exposed to TB and 12 children diagnosed with TB disease in Eswatini. The tests demonstrated excellent concordance in both TB disease (100% agreement, Cohen's kappa = 1) and TB infection (96% agreement, Cohen's kappa = 0.91). Most of the children with household exposure tested negative for TB infection by both tests, indicating the ongoing need for new tests for TB infection that can be easily implemented in TB high-burden settings at minimal cost.

Field suitability and diagnostic accuracy of the Biocentric® open real-time PCR platform for plasma-based HIV viral load quantification in Swaziland.

BACKGROUND: Viral load (VL) testing is being scaled up in resource-limited settings. However, not all commercially available VL testing methods have been evaluated under field conditions. This study is one of a few to evaluate the Biocentric platform for VL quantification in routine practice in Sub-Saharan Africa. METHODS: Venous blood specimens were obtained from patients eligible for VL testing at two health facilities in Swaziland from October 2016 to March 2017. Samples were centrifuged at two laboratories (LAB-1, LAB-2) to obtain paired plasma specimens for VL quantification with the national reference method and on the Biocentric platform. Agreement (correlation, Bland-Altman) and accuracy (sensitivity, specificity) indicators were calculated at the VL thresholds of 416 (2.62 log10) and 1000 (3.0 log10) copies/mL. Leftover samples from patients with discordant VL results were re-quantified and accuracy indicators recalculated. Logistic regression was used to compare laboratory performance. RESULTS: A total of 364 paired plasma samples (LAB-1: n = 198; LAB-2: n = 166) were successfully tested using both methods. The correlation was high (R = 0.82, p < 0.01), and the Bland-Altman analysis showed a minimal mean difference (- 0.03 log10 copies/mL; 95% CI: -1.15 to 1.08). At the clinical threshold level of 3.0 log10 copies/mL, the sensitivity was 88.6% (95% CI: 78.7 to 94.9) and the specificity was 98.3% (95% CI: 96.1 to 99.4). Sensitivity was higher in LAB-1 (100%; 95% CI: 71.5 to 100) than in LAB-2 (86.4%; 95% CI: 75.0 to 94.0). Most upward (n = 8, 2.2%) and downward (n = 11, 3.0%) misclassifications occurred at the 2.62 log threshold, with LAB-2 having a 16 (95% CI: 2.26 to 113.27; p = 0.006) times higher odds of downward misclassification. After retesting of discordant leftover samples (n = 17), overall sensitivity increased to 93.5% (95% CI: 85.5 to 97.9) and 97.1% (95% CI: 90.1 to 99.7) at the 2.62 and 3.0 thresholds, and specificity increased to 98.6% (95% CI: 96.5 to 99.6) and 99.0% (95% CI: 97.0 to 99.8) respectively. CONCLUSIONS: The test characteristics of the Biocentric platform were overall comparable to the national reference method for VL quantification. One laboratory tended to misclassify VL results downwards, likely owing to unmet training needs and lack of previous hands-on practice.

Outbreak of multidrug-resistant tuberculosis in South Africa undetected by WHO-endorsed commercial tests: an observational study.

BACKGROUND: Global roll-out of rapid molecular assays is revolutionising the diagnosis of rifampicin resistance, predictive of multidrug-resistance, in tuberculosis. However, 30% of the multidrug-resistant (MDR) strains in an eSwatini study harboured the Ile491Phe mutation in the rpoB gene, which is associated with poor rifampicin-based treatment outcomes but is missed by commercial molecular assays or scored as susceptible by phenotypic drug-susceptibility testing deployed in South Africa. We evaluated the presence of Ile491Phe among South African tuberculosis isolates reported as isoniazid-monoresistant according to current national testing algorithms. METHODS: We screened records of 37 644 Mycobacterium tuberculosis positive cultures from four South African provinces, diagnosed at the National Health Laboratory Service-Dr George Mukhari Tertiary Laboratory, to identify isolates with rifampicin sensitivity and isoniazid resistance according to Xpert MTB/RIF, GenoType MTBDRplus, and BACTEC MGIT 960. Of 1823 isolates that met these criteria, 277 were randomly selected and screened for Ile491Phe with multiplex allele-specific PCR and Sanger sequencing of rpoB. Ile491Phe-positive strains (as well as 17 Ile491Phe-bearing isolates from the eSwatini study) were then tested by Deeplex-MycTB deep sequencing and whole-genome sequencing to evaluate their patterns of extensive resistance, transmission, and evolution. FINDINGS: Ile491Phe was identified in 37 (15%) of 249 samples with valid multiplex allele-specific PCR and sequencing results, thus reclassifying them as MDR. All 37 isolates were additionally identified as genotypically resistant to all first-line drugs by Deeplex-MycTB. Six of the South African isolates harboured four distinct mutations potentially associated with decreased bedaquiline sensitivity. Consistent with Deeplex-MycTB genotypic profiles, whole-genome sequencing revealed concurrent silent spread in South Africa of a MDR tuberculosis strain lineage extending from the eSwatini outbreak and at least another independently emerged Ile491Phe-bearing lineage. Whole-genome sequencing further suggested acquisition of mechanisms compensating for the Ile491Phe fitness cost, and of additional bedaquiline resistance following the introduction of this drug in South Africa. INTERPRETATION: A substantial number of MDR tuberculosis cases harbouring the Ile491Phe mutation in the rpoB gene in South Africa are missed by current diagnostic strategies, resulting in ineffective first-line treatment, continued amplification of drug resistance, and concurrent silent spread in the community. FUNDING: VLIR-UOS, National Research Foundation (South Africa), and INNOVIRIS.

Prediction of Susceptibility to First-Line Tuberculosis Drugs by DNA Sequencing.

BACKGROUND: The World Health Organization recommends drug-susceptibility testing of Mycobacterium tuberculosis complex for all patients with tuberculosis to guide treatment decisions and improve outcomes. Whether DNA sequencing can be used to accurately predict profiles of susceptibility to first-line antituberculosis drugs has not been clear. METHODS: We obtained whole-genome sequences and associated phenotypes of resistance or susceptibility to the first-line antituberculosis drugs isoniazid, rifampin, ethambutol, and pyrazinamide for isolates from 16 countries across six continents. For each isolate, mutations associated with drug resistance and drug susceptibility were identified across nine genes, and individual phenotypes were predicted unless mutations of unknown association were also present. To identify how whole-genome sequencing might direct first-line drug therapy, complete susceptibility profiles were predicted. These profiles were predicted to be susceptible to all four drugs (i.e., pansusceptible) if they were predicted to be susceptible to isoniazid and to the other drugs or if they contained mutations of unknown association in genes that affect susceptibility to the other drugs. We simulated the way in which the negative predictive value changed with the prevalence of drug resistance. RESULTS: A total of 10,209 isolates were analyzed. The largest proportion of phenotypes was predicted for rifampin (9660 [95.4%] of 10,130) and the smallest was predicted for ethambutol (8794 [89.8%] of 9794). Resistance to isoniazid, rifampin, ethambutol, and pyrazinamide was correctly predicted with 97.1%, 97.5%, 94.6%, and 91.3% sensitivity, respectively, and susceptibility to these drugs was correctly predicted with 99.0%, 98.8%, 93.6%, and 96.8% specificity. Of the 7516 isolates with complete phenotypic drug-susceptibility profiles, 5865 (78.0%) had complete genotypic predictions, among which 5250 profiles (89.5%) were correctly predicted. Among the 4037 phenotypic profiles that were predicted to be pansusceptible, 3952 (97.9%) were correctly predicted. CONCLUSIONS: Genotypic predictions of the susceptibility of M. tuberculosis to first-line drugs were found to be correlated with phenotypic susceptibility to these drugs. (Funded by the Bill and Melinda Gates Foundation and others.).