Phylogeography and transmission of M. tuberculosis in Moldova: A prospective genomic analysis.

BACKGROUND: The incidence of multidrug-resistant tuberculosis (MDR-TB) remains critically high in countries of the former Soviet Union, where >20% of new cases and >50% of previously treated cases have resistance to rifampin and isoniazid. Transmission of resistant strains, as opposed to resistance selected through inadequate treatment of drug-susceptible tuberculosis (TB), is the main driver of incident MDR-TB in these countries. METHODS AND FINDINGS: We conducted a prospective, genomic analysis of all culture-positive TB cases diagnosed in 2018 and 2019 in the Republic of Moldova. We used phylogenetic methods to identify putative transmission clusters; spatial and demographic data were analyzed to further describe local transmission of Mycobacterium tuberculosis. Of 2,236 participants, 779 (36%) had MDR-TB, of whom 386 (50%) had never been treated previously for TB. Moreover, 92% of multidrug-resistant M. tuberculosis strains belonged to putative transmission clusters. Phylogenetic reconstruction identified 3 large clades that were comprised nearly uniformly of MDR-TB: 2 of these clades were of Beijing lineage, and 1 of Ural lineage, and each had additional distinct clade-specific second-line drug resistance mutations and geographic distributions. Spatial and temporal proximity between pairs of cases within a cluster was associated with greater genomic similarity. Our study lasted for only 2 years, a relatively short duration compared with the natural history of TB, and, thus, the ability to infer the full extent of transmission is limited. CONCLUSIONS: The MDR-TB epidemic in Moldova is associated with the local transmission of multiple M. tuberculosis strains, including distinct clades of highly drug-resistant M. tuberculosis with varying geographic distributions and drug resistance profiles. This study demonstrates the role of comprehensive genomic surveillance for understanding the transmission of M. tuberculosis and highlights the urgency of interventions to interrupt transmission of highly drug-resistant M. tuberculosis.

Whole-Genome Sequencing Has the Potential To Improve Treatment for Rifampicin-Resistant Tuberculosis in High-Burden Settings: a Retrospective Cohort Study.

Treatment of multidrug-resistant or rifampicin-resistant tuberculosis (MDR/RR-TB), although improved in recent years with shorter, more tolerable regimens, remains largely standardized and based on limited drug susceptibility testing (DST). More individualized treatment with expanded DST access is likely to improve patient outcomes. To assess the potential of TB drug resistance prediction based on whole-genome sequencing (WGS) to provide more effective treatment regimens, we applied current South African treatment recommendations to a retrospective cohort of MDR/RR-TB patients from Khayelitsha, Cape Town. Routine DST and clinical data were used to retrospectively categorize patients into a recommended regimen, either a standardized short regimen or a longer individualized regimen. Potential regimen changes were then described with the addition of WGS-derived DST. WGS data were available for 1274 MDR/RR-TB patient treatment episodes across 2008 to 2017. Among 834 patients initially eligible for the shorter regimen, 385 (46%) may have benefited from reduced drug dosage or removing ineffective drugs when WGS data were considered. A further 187 (22%) patients may have benefited from more effective adjusted regimens. Among 440 patients initially eligible for a longer individualized regimen, 153 (35%) could have been switched to the short regimen. Overall, 305 (24%) patients had MDR/RR-TB with second-line TB drug resistance, where the availability of WGS-derived DST would have allowed more effective treatment individualization. These data suggest considerable benefits could accrue from routine access to WGS-derived resistance prediction. Advances in culture-free sequencing and expansion of the reference resistance mutation catalogue will increase the utility of WGS resistance prediction.

Rifampicin-Monoresistant Tuberculosis Is Not the Same as Multidrug-Resistant Tuberculosis: a Descriptive Study from Khayelitsha, South Africa.

Rifampin monoresistance (RMR; rifampin resistance and isoniazid susceptibility) accounts for 38% of all rifampin-resistant tuberculosis (RR-TB) in South Africa and is increasing. We aimed to compare RMR-TB with multidrug-resistant TB (MDR-TB) in a setting with high TB, RR-TB, and HIV burdens. Patient-level clinical data and stored RR Mycobacterium tuberculosis isolates from 2008 to 2017 with available whole-genome sequencing (WGS) data were used to describe risk factors associated with RMR-TB and to compare RR-conferring mutations between RMR-TB and MDR-TB. A subset of isolates with particular RR-conferring mutations were subjected to semiquantitative rifampin phenotypic drug susceptibility testing. Among 2,041 routinely diagnosed RR-TB patients, 463 (22.7%) had RMR-TB. HIV-positive individuals (adjusted odds ratio [aOR], 1.4; 95% confidence interval [CI], 1.1 to 1.9) and diagnosis between 2013 and 2017 versus between 2008 and 2012 (aOR, 1.3; 95% CI, 1.1 to 1.7) were associated with RMR-TB. Among 1,119 (54.8%) patients with available WGS data showing RR-TB, significant differences in the distribution of rpoB RR-conferring mutations between RMR and MDR isolates were observed. Mutations associated with high-level RR were more commonly found among MDR isolates (811/889 [90.2%] versus 162/230 [70.4%] among RMR isolates; P < 0.0001). In particular, the rpoB L430P mutation, conferring low-level RR, was identified in 32/230 (13.9%) RMR isolates versus 10/889 (1.1%) in MDR isolates (P < 0.0001). Among 10 isolates with an rpoB L430P mutation, 7 were phenotypically susceptible using the critical concentration of 0.5 µg/ml (range, 0.125 to 1 µg/ml). The majority (215/230 [93.5%]) of RMR isolates showed susceptibility to all other TB drugs, highlighting the potential benefits of WGS for simplified treatment. These data suggest that the evolution of RMR-TB differs from MDR-TB with a potential contribution from HIV infection.

Risk factors for recurrent tuberculosis after successful treatment in a high burden setting: a cohort study.

BACKGROUND: People successfully completing treatment for tuberculosis remain at elevated risk for recurrent disease, either from relapse or reinfection. Identifying risk factors for recurrent tuberculosis may help target post-tuberculosis screening and care. METHODS: We enrolled 500 patients with smear-positive pulmonary tuberculosis in South Africa and collected baseline data on demographics, clinical presentation and sputum mycobacterial cultures for 24-loci mycobacterial interspersed repetitive unit-variable number tandem repeat (MIRU-VNTR) typing. We used routinely-collected administrative data to identify recurrent episodes of tuberculosis occurring over a median of six years after successful treatment completion. RESULTS: Of 500 patients initially enrolled, 333 (79%) successfully completed treatment for tuberculosis. During the follow-up period 35 patients with successful treatment (11%) experienced a bacteriologically confirmed tuberculosis recurrence. In our Cox proportional hazards model, a 3+ AFB sputum smear grade was significantly associated with recurrent tuberculosis with a hazard ratio of 3.33 (95% CI 1.44-7.7). The presence of polyclonal M. tuberculosis infection at baseline had a hazard ratio for recurrence of 1.96 (95% CI 0.86-4.48). CONCLUSION: Our results indicate that AFB smear grade is independently associated with tuberculosis recurrence after successful treatment for an initial episode while the association between polyclonal M. tuberculosis infection and increased risk of recurrence appears possible.

Accuracy of line probe assays for the diagnosis of pulmonary and multidrug-resistant tuberculosis: a systematic review and meta-analysis.

Only 25% of multidrug-resistant tuberculosis (MDR-TB) cases are currently diagnosed. Line probe assays (LPAs) enable rapid drug-susceptibility testing for rifampicin (RIF) and isoniazid (INH) resistance and Mycobacterium tuberculosis detection. Genotype MTBDRplusV1 was WHO-endorsed in 2008 but newer LPAs have since been developed.This systematic review evaluated three LPAs: Hain Genotype MTBDRplusV1, MTBDRplusV2 and Nipro NTM+MDRTB. Study quality was assessed with QUADAS-2. Bivariate random-effects meta-analyses were performed for direct and indirect testing. Results for RIF and INH resistance were compared to phenotypic and composite (incorporating sequencing) reference standards. M. tuberculosis detection results were compared to culture.74 unique studies were included. For RIF resistance (21 225 samples), pooled sensitivity and specificity (with 95% confidence intervals) were 96.7% (95.6-97.5%) and 98.8% (98.2-99.2%). For INH resistance (20 954 samples), pooled sensitivity and specificity were 90.2% (88.2-91.9%) and 99.2% (98.7-99.5%). Results were similar for direct and indirect testing and across LPAs. Using a composite reference standard, specificity increased marginally. For M. tuberculosis detection (3451 samples), pooled sensitivity was 94% (89.4-99.4%) for smear-positive specimens and 44% (20.2-71.7%) for smear-negative specimens.In patients with pulmonary TB, LPAs have high sensitivity and specificity for RIF resistance and high specificity and good sensitivity for INH resistance. This meta-analysis provides evidence for policy and practice.

Diagnostics for pulmonary tuberculosis.

Tuberculosis (TB) remains a leading cause of human suffering and mortality despite decades of effective treatment being available. Accurate and timely diagnosis remains an unmet goal. The HIV epidemic has also led to new challenges in the diagnosis of TB. Several new developments in TB diagnostics have the potential to positively influence the global campaign against TB. We aim to review the performance of both established as well as new diagnostics for pulmonary TB in adults, and discuss the ongoing challenges.

Potential contribution of HIV during first-line tuberculosis treatment to subsequent rifampicin-monoresistant tuberculosis and acquired tuberculosis drug resistance in South Africa: a retrospective molecular epidemiology study.

BACKGROUND: South Africa has a high burden of rifampicin-resistant tuberculosis (including multidrug-resistant [MDR] tuberculosis), with increasing rifampicin-monoresistant (RMR) tuberculosis over time. Resistance acquisition during first-line tuberculosis treatment could be a key contributor to this burden, and HIV might increase the risk of acquiring rifampicin resistance. We assessed whether HIV during previous treatment was associated with RMR tuberculosis and resistance acquisition among a retrospective cohort of patients with MDR or rifampicin-resistant tuberculosis. METHODS: In this retrospective cohort study, we included all patients routinely diagnosed with MDR or rifampicin-resistant tuberculosis in Khayelitsha, Cape Town, South Africa, between Jan 1, 2008, and Dec 31, 2017. Patient-level data were obtained from a prospective database, complemented by data on previous tuberculosis treatment and HIV from a provincial health data exchange. Stored MDR or rifampicin-resistant tuberculosis isolates from patients underwent whole-genome sequencing (WGS). WGS data were used to infer resistance acquisition versus transmission, by identifying genomically unique isolates (single nucleotide polymorphism threshold of five). Logistic regression analyses were used to assess factors associated with RMR tuberculosis and genomic uniqueness. FINDINGS: The cohort included 2041 patients diagnosed with MDR or rifampicin-resistant tuberculosis between Jan 1, 2008, and Dec 31, 2017; of those, 463 (22·7%) with RMR tuberculosis and 1354 (66·3%) with previous tuberculosis treatment. In previously treated patients, HIV positivity during previous tuberculosis treatment versus HIV negativity (adjusted odds ratio [OR] 2·07, 95% CI 1·35-3·18), and three or more previous tuberculosis treatment episodes versus one (1·96, 1·21-3·17) were associated with RMR tuberculosis. WGS data showing MDR or rifampicin-resistant tuberculosis were available for 1169 patients; 360 (30·8%) isolates were identified as unique. In previously treated patients, RMR tuberculosis versus MDR tuberculosis (adjusted OR 4·96, 3·40-7·23), HIV positivity during previous tuberculosis treatment (1·71, 1·03-2·84), and diagnosis in 2013-17 (1·42, 1·02-1·99) versus 2008-12, were associated with uniqueness. In previously treated patients with RMR tuberculosis, HIV positivity during previous treatment (adjusted OR 5·13, 1·61-16·32) was associated with uniqueness as was female sex (2·50 [1·18-5·26]). INTERPRETATION: These data suggest that HIV contributes to rifampicin-resistance acquisition during first-line tuberculosis treatment and that this might be driving increasing RMR tuberculosis over time. Large-scale prospective cohort studies are required to further quantify this risk. FUNDING: Swiss National Science Foundation, South African National Research Foundation, and Wellcome Trust.

Spatially targeted screening to reduce tuberculosis transmission in high-incidence settings.

As the leading infectious cause of death worldwide and the primary proximal cause of death in individuals living with HIV, tuberculosis remains a global concern. Existing tuberculosis control strategies that rely on passive case-finding appear insufficient to achieve targets for reductions in tuberculosis incidence and mortality. Active case-finding strategies aim to detect infectious individuals earlier in their infectious period to reduce onward transmission and improve treatment outcomes. Empirical studies of active case-finding have produced mixed results and determining how to direct active screening to those most at risk remains a topic of intense research. Our systematic review of literature evaluating the effects of geographically targeted tuberculosis screening interventions found three studies in low tuberculosis incidence settings, but none conducted in high tuberculosis incidence countries. We discuss open questions related to the use of spatially targeted approaches for active screening in countries where tuberculosis incidence is highest.

Trends in C-Reactive Protein, D-Dimer, and Fibrinogen during Therapy for HIV-Associated Multidrug-Resistant Tuberculosis.

HIV-positive adults on treatment for multi drug-resistant tuberculosis (MDR-TB) experience high mortality. Biomarkers of HIV/MDR-TB treatment response may enable earlier treatment modifications that improve outcomes. To determine whether changes in C-reactive protein (CRP), D-dimer, and fibrinogen were associated with treatment outcome among those with HIV/MDR-TB coinfection, we studied 20 HIV-positive participants for the first 16 weeks of MDR-TB therapy. Serum CRP, fibrinogen, and D-dimer were measured at baseline and serially while on treatment. At baseline, all biomarkers were elevated above normal levels, with median CRP 86.15 mg/L (interquartile range [IQR] 29.25-149.32), D-dimer 0.85 µg/mL (IQR 0.34-1.80), and fibrinogen 4.11 g/L (IQR 3.75-6.31). C-reactive protein decreased significantly within 10 days of treatment initiation and fibrinogen within 28 days; D-dimer did not change significantly. Five (25%) participants died after a median of 32 days. Older age (median age of 38y among survivors and 54y among deceased, P = 0.008) and higher baseline fibrinogen (3.86 g/L among survivors and 6.37 g/L among deceased, P = 0.02) were significantly associated with death. After adjusting for other measured variables, higher CRP concentrations at the beginning of each measurement interval were significantly associated with a higher risk of death during that interval. Trends in fibrinogen and CRP may be useful for evaluating early response to treatment among individuals with HIV/MDR-TB coinfection.

Evaluation of Tuberculosis Treatment Response With Serial C-Reactive Protein Measurements.

BACKGROUND: Novel biomarkers are needed to assess response to antituberculosis therapy in smear-negative patients. METHODS: To evaluate the utility of C-reactive protein (CRP) in monitoring response to antituberculosis therapy, we conducted a post hoc analysis on a cohort of adults with symptoms of tuberculosis and negative sputum smears in a high-tuberculosis and HIV prevalence setting in KwaZulu-Natal, South Africa. Serial changes in CRP, weight, and hemoglobin were evaluated over 8 weeks. RESULTS: Four hundred twenty-one participants being evaluated for smear-negative tuberculosis were enrolled, and 33 were excluded. Two hundred ninety-five were treated for tuberculosis (137 confirmed, 158 possible), and 93 did not have tuberculosis. One hundred and eighty-three of 213 (86%) participants who agreed to HIV testing were HIV positive. At week 8, the on-treatment median CRP reduction in the tuberculosis group (interquartile range [IQR]) was 79.5% (25.4% to 91.7%), the median weight gain was 2.3% (-1.0% to 5.6%), and the median hemoglobin increase was 7.0% (0.8% to 18.9%); P < .0001 for baseline to week 8 comparison of absolute median values. Only CRP changed significantly at week 2 (median reduction [IQR], 75.1% [46.9% to 89.2%]) in the group with confirmed tuberculosis and in the possible tuberculosis group (median reduction [IQR], 49.0% [-0.4% to 80.9%]). Failure of CRP to reduce to ≤55% of the baseline value at week 2 predicted hospitalization or death in both tuberculosis groups, with 99% negative predictive value. CONCLUSIONS: Change in CRP may have utility in early evaluation of response to antituberculosis treatment and to identify those at increased risk of adverse outcomes.

Diagnostic Outcomes After Chest Radiograph Interpretation in Patients With Suspected Tuberculosis and Negative Sputum Smears in a High-Burden Human Immunodeficiency Virus and Tuberculosis Setting.

BACKGROUND: Evaluation of patients with suspected tuberculosis and negative sputum smears for acid-fast bacilli (AFB) is challenging, especially in high human immunodeficiency virus coinfection settings where sputum smears have lower sensitivity for detecting AFB. METHODS: We examined the utility of chest radiographs for detecting smear-negative pulmonary tuberculosis. Three hundred sixty sputum smear-negative patients who were referred from primary care clinics in the KwaZulu-Natal province of South Africa were evaluated. Chest radiographs were read by experienced pulmonologists using a previously validated Chest X-Ray Reading and Recording System (CRRS). RESULTS: Agreement between observers using CRRS was high at 91% with a Cohen's kappa of 0.64 (95% confidence interval [CI] = 0.52-0.76). Against a reference standard of sputum culture, sensitivity was 93% (95% CI = 86%-97%), whereas specificity was 14% (95% CI = 10%-19%). Performance against clinical diagnosis (following World Health Organization guidelines) was similar with sensitivity of 92% (95% CI = 88%-95%) and specificity of 20% (95% CI = 13%-28%). CONCLUSION: The low specificity of CRRS in this setting indicates poor diagnostic utility for detecting pulmonary tuberculosis.