Valeriu Crudu

Multidrug resistant tuberculosis (MDR-TB) epidemics are sustained by transmission of reproductively fit MDR M. tuberculosis ( Mtb ) strains. We search a large publicly available dataset of ~200,000 Mtb whole genome sequences to identify strains related to a highly successful MDR clade circulating in Moldova belonging to lineage 4.2.1/Ural. We characterize a clade of 1604 drug-resistant Mtb sequences harboring conserved resistance-conferring mutations. We identify the Russian Federation as the most likely country of origin for this clade and infer several independent migration events from Russia and Moldova to other European and Asian countries. We estimate that this clade is expanding more rapidly than comparable clades of lineage 4.2.1/Ural. The broad dispersal of this highly successful clade is an urgent global health threat. Genomic surveillance is essential to track the evolution and spread of this and other strains of concern.

Over the past three decades, molecular epidemiological studies have provided new opportunities to investigate the transmission dynamics of M. tuberculosis. In most studies, a sizable fraction of individuals with notified tuberculosis cannot be included, either because they do not have culture-positive disease (and thus do not have specimens available for molecular typing) or because resources for conducting sequencing are limited. A recent study introduced a regression-based approach for inferring the membership of unsequenced tuberculosis cases into transmission clusters based on host demographic and epidemiological data. This method was able to identify the most likely cluster to which an unsequenced strain belonged with an accuracy of 35%, though in a low burden setting where a large fraction of cases occurred among foreign-born migrants. Here, we apply a similar model to M. tuberculosis WGS data from the Republic of Moldova, a setting of relatively high local transmission. Using a maximum cluster span of ~40 SNPs and a cluster size cutoff of n ≥ 10, we could best predict the specific cluster to which each clustered case was most likely to be a member with an accuracy of 17.2%. In sensitivity analyses, we found that a more restrictive (~20 SNPs threshold) or permissive (~80 SNPs) threshold did not improve performance. We found that increasing the minimum cluster size improved prediction accuracy. These findings highlight the challenges of transmission interference in high burden settings like Moldova.

Objectives: This study aims to determine the progression rate, risk factors and timeline for the progression from exposure to active tuberculosis (TB) in a high-risk population. Using a prospective cohort in the Republic of Moldova, we investigated pulmonary TB disease progression among close contacts of patients with TB in a low-burden country with high rates of drug-resistant TB. Methods: Close contacts of patients with newly diagnosed TB were recruited and monitored to evaluate for progression rates to active TB. Data collected included demographic information, medical and exposure history, and clinical samples. Follow-up clinical evaluations of close contacts were conducted at regular intervals over at least 24 months to monitor for progression to TB disease. Results: The overall incidence rate of TB disease among close contacts was 3.7%. Among the close contacts, 2.3% were identified as progressor cases, developing TB disease more than 30 days after index case treatment initiation. Thirteen (1.3%) were co-prevalent cases, diagnosed within 30 days of index case treatment initiation. Identified risk factors for progression included male sex, active tobacco use, prior TB infection, and frequent, prolonged exposure to index cases. Close contacts with daily exposure of more than eight hours had a significantly higher risk of disease progression (adjusted OR: 4.28, 95% CI: 1.79–10.23). Conclusion: The incidence of TB disease among close contacts was consistent with global findings, highlighting the need for enhanced diagnostic tools and targeted interventions to manage TB transmission and progression. These results underscore the importance of contact tracing and progression monitoring in low-burden, high drug-resistant TB settings. Future research should focus on developing a better understanding of factors contributing to the risk for and timeline of TB disease progression, and more precise methods, including biomarkers, to identify individuals at the highest risk for progression from TB exposure to active disease.

Over the past three decades, molecular epidemiological studies have provided new opportunities to investigate the transmission dynamics of M. tuberculosis. In most studies, a sizable fraction of individuals with notified tuberculosis cannot be included, either because they do not have culture-positive disease (and thus do not have specimens available for molecular typing) or because resources for conducting sequencing are limited. A recent study introduced a regression-based approach for inferring the membership of unsequenced tuberculosis cases into transmission clusters based on host demographic and epidemiological data. This method was able to identify the most likely cluster to which an unsequenced strain belonged with an accuracy of 35%, though in a low burden setting where a large fraction of cases occurred among foreign-born migrants. Here, we apply a similar model to M. tuberculosis WGS data from the Republic of Moldova, a setting of relatively high local transmission. Using a maximum cluster span of ~40 SNPs and a cluster size cutoff of n ≥ 10, we could best predict the specific cluster to which each clustered case was most likely to be a member with an accuracy of 17.2%. In sensitivity analyses, we found that a more restrictive (~20 SNPs threshold) or permissive (~80 SNPs) threshold did not improve performance. We found that increasing the minimum cluster size improved prediction accuracy. These findings highlight the challenges of transmission interference in high burden settings like Moldova.

Background: Emerging evidence suggests that shortened, simplified treatment regimens for rifampicin-resistant tuberculosis (RR-TB) can achieve comparable end-of-treatment (EOT) outcomes to longer regimens. We compared a 6-month regimen containing bedaquiline, pretomanid, linezolid, and moxifloxacin (BPaLM) to a standard of care strategy using a 9- or 18-month regimen depending on whether fluoroquinolone resistance (FQ-R) was detected on drug susceptibility testing (DST).Methods and findings: The primary objective was to determine whether 6 months of BPaLM is a cost-effective treatment strategy for RR-TB. We used genomic and demographic data to parameterize a mathematical model estimating long-term health outcomes measured in quality-adjusted life years (QALYs) and lifetime costs in 2022 USD ($) for each treatment strategy for patients 15 years and older diagnosed with pulmonary RR-TB in Moldova, a country with a high burden of TB drug resistance. For each individual, we simulated the natural history of TB and associated treatment outcomes, as well as the process of acquiring resistance to each of 12 anti-TB drugs. Compared to the standard of care, 6 months of BPaLM was cost-effective. This strategy was estimated to reduce lifetime costs by $3,366 (95% UI: [1,465, 5,742]p< 0.001) per individual, with a nonsignificant change in QALYs (−0.06; 95% UI: [−0.49, 0.03]p= 0.790). For those stopping moxifloxacin under the BPaLM regimen, continuing with BPaL plus clofazimine (BPaLC) provided more QALYs at lower cost than continuing with BPaL alone. Strategies based on 6 months of BPaLM had at least a 93% chance of being cost-effective, so long as BPaLC was continued in the event of stopping moxifloxacin. BPaLM for 6 months also reduced the average time spent with TB resistant to amikacin, bedaquiline, clofazimine, cycloserine, moxifloxacin, and pyrazinamide, while it increased the average time spent with TB resistant to delamanid and pretomanid. Sensitivity analyses showed 6 months of BPaLM to be cost-effective across a broad range of values for the relative effectiveness of BPaLM, and the proportion of the cohort with FQ-R. Compared to the standard of care, 6 months of BPaLM would be expected to save Moldova’s national TB program budget $7.1 million (95% UI: [1.3 million, 15.4 million]p= 0.002) over the 5-year period from implementation. Our analysis did not account for all possible interactions between specific drugs with regard to treatment outcomes, resistance acquisition, or the consequences of specific types of severe adverse events, nor did we model how the intervention may affect TB transmission dynamics.Conclusions: Compared to standard of care, longer regimens, the implementation of the 6-month BPaLM regimen could improve the cost-effectiveness of care for individuals diagnosed with RR-TB, particularly in settings with a high burden of drug-resistant TB. Further research may be warranted to explore the impact and cost-effectiveness of shorter RR-TB regimens across settings with varied drug-resistant TB burdens and national income levels.

Background: Mixed infection with multiple strains of the same pathogen in a single host can present clinical and analytical challenges. Whole genome sequence (WGS) data can identify signals of multiple strains in samples, though the precision of previous methods can be improved. Here, we present MixInfect2, a new tool to accurately detect mixed samples from Mycobacterium tuberculosis WGS data. We then evaluate three approaches for reconstructing the underlying mixed constituent strain sequences. This allows these samples to be included in downstream analysis to gain insights into the epidemiology and transmission of mixed infections. Methods: We employed a Gaussian mixture model to cluster allele frequencies at mixed sites (hSNPs) in each sample to identify signals of multiple strains. Building upon our previous tool, MixInfect, we increased the accuracy of classifying in vitro mixed samples through multiple improvements to the bioinformatic pipeline. Major and minor proportion constituent strains were reconstructed using three approaches and assessed by comparing the estimated sequence to the known constituent strain sequence. Lastly, mixed infections in a real-world Mycobacterium tuberculosis population from Moldova were detected with MixInfect2 and clusters of recent transmission that included major and minor constituent strains were built. Results: All 36/36 in vitro mixed and 12/12 non-mixed samples were correctly classified with MixInfect2, and major strain proportions estimated with high accuracy, outperforming previous tools. Reconstructed major strain sequences closely matched the true constituent sequence by taking the allele at the highest frequency at hSNPs, while the best performing approach to reconstruct the minor proportion strain sequence was identifying the closest non-mixed isolate in the same population, though no approach was effective when the minor strain proportion was at 5%. Finally, fewer mixed infections were identified in Moldova than previous estimates (6.6% vs 17.4%) and we found multiple instances where the constituent strains of mixed samples were present in transmission clusters. Conclusions: MixInfect2 accurately detects samples with evidence of mixed infection from WGS data and provides an excellent estimate of the mixture proportions. While there are limitations in reconstructing the constituent strain sequences of mixed samples, we present recommendations for the best approach to include these isolates in further analyses.

The projected trajectory of multidrug resistant tuberculosis (MDR-TB) epidemics depends on the reproductive fitness of circulating strains of MDR M. tuberculosis (Mtb). Previous efforts to characterize the fitness of MDR Mtb have found that Mtb strains of the Beijing sublineage (Lineage 2.2.1) may be more prone to develop resistance and retain fitness in the presence of resistance-conferring mutations than other lineages. Using Mtb genome sequences from all culture-positive cases collected over two years in Moldova, we estimate the fitness of Ural (Lineage 4.2) and Beijing strains, the two lineages in which MDR is concentrated in the country. We estimate that the fitness of MDR Ural strains substantially exceeds that of other susceptible and MDR strains, and we identify several mutations specific to these MDR Ural strains. Our findings suggest that MDR Ural Mtb has been transmitting efficiently in Moldova and poses a substantial risk of spreading further in the region.