Effect of Vitamin D Supplementation in Type 2 Diabetes Patients with Tuberculosis: A Systematic Review.

BACKGROUND: Diabetes mellitus (DM) and tuberculosis (TB) have been recognized as reemerging epidemics, especially in developing countries. Among all the risk factors, diabetes causes immunosuppression, increasing the risk of active TB three times. Vitamin D has been found as a link between DM-TB co-morbidity. OBJECTIVE: Vitamin D affects the immune response, suppresses Mycobacterium tuberculosis (Mtb) growth, and affects insulin secretion. The present systematic review determines the effect of vitamin D supplementation on clinical and therapeutic outcomes of DM-TB patients. METHOD: A comprehensive literature search was carried out in PubMed, Cochrane, Web of Science, and Scopus database to determine eligible studies from inception to January 2021. Out of the 639 articles retrieved, three randomized controlled trials (RCTs) were included in the systematic review. RESULT: The effect of vitamin D3 or oral cholecalciferol supplementation was assessed on outcomes, such as duration to sputum smear conversion, TB scores improvement, change in glycemic parameters, including HbA1c, FBS, and PLBS, and laboratory parameters, such as Hb, ESR, and CRP. Duration of sputum smear conversion was decreased by two weeks in the vitamin D3 supplemented group in two studies. TB score improvement and changes in glycemic parameters were inclined towards supplemented group; however, they were not significant. CONCLUSION: The overall effect of vitamin D3 supplementation on TB patients with DM was not significant. Further studies are required in the future examining the effect of supplementation on outcomes in this population.

Antimycobacterial and anti-inflammatory activities of thiourea derivatives focusing on treatment approaches for severe pulmonary tuberculosis.

Tuberculosis (TB) remains a serious public health problem and one of the main concern is the emergence of multidrug-resistant and extensively resistant TB. Hyper-reactive patients develop inflammatory necrotic lung lesions that aggravate the pathology and facilitate transmission of mycobacteria. Treatment of severe TB is a major clinical challenge that has few effective solutions and patients face a poor prognosis, years of treatment and different adverse drug reactions. In this work, fifteen novel and thirty-one unusual thiourea derivatives were synthesized and evaluated in vitro for their antimycobacterial and anti-inflammatory potential and, in silico for ADMET parameters and for structure-activity relationship (SAR). Thioureas derivatives 10, 15, 16, 28 and 29 that had shown low cytotoxicity and high activities were selected for further investigation, after SAR study. These five thioureas derivatives inhibited Mtb H37Rv growth in bacterial culture and in infected macrophages, highlighting thiourea derivative 28 (MIC50 2.0 ± 1.1 and 2.3 ± 1.1 µM, respectively). Moreover, these compounds were active against the hypervirulent clinical Mtb strain M299, in bacterial culture, especially 16, 28 and 29, and in extracellular clumps, highlighting 29, with MIC50 5.6 ± 1.2 µM. Regarding inflammation, they inhibited NO through the suppression of iNOS expression, and also inhibited the production of TNF-α and IL-1ß. In silico studies were carried out suggesting that these five compounds could be administered by oral route and have low toxicological effects when compared to rifampicin. In conclusion, our data show that, at least, thiourea derivatives 16, 28 and 29 are promising antimycobacterial and anti-inflammatory agents, and candidates for further prospective studies aiming new anti-TB drugs, that can be used on a dual approach for the treatment of severe TB cases associated with exacerbated inflammation.

Evaluation of Berberine as an Adjunct to TB Treatment.

Tuberculosis (TB) is the global health problem with the second highest number of deaths from a communicable disease after COVID-19. Although TB is curable, poor health infrastructure, long and grueling TB treatments have led to the spread of TB pandemic with alarmingly increasing multidrug-resistant (MDR)-TB prevalence. Alternative host modulating therapies can be employed to improve TB drug efficacies or dampen the exaggerated inflammatory responses to improve lung function. Here, we investigated the adjunct therapy of natural immune-modulatory compound berberine in C57BL/6 mouse model of pulmonary TB. Berberine treatment did not affect Mtb growth in axenic cultures; however, it showed increased bacterial killing in primary murine bone marrow-derived macrophages and human monocyte-derived macrophages. Ad libitum berberine administration was beneficial to the host in combination with rifampicin and isoniazid. Berberine adjunctive treatment resulted in decreased lung pathology with no additive or synergistic effects on bacterial burdens in mice. Lung immune cell flow cytometry analysis showed that adjunctive berberine treatment decreased neutrophil, CD11b+ dendritic cell and recruited interstitial macrophage numbers. Late onset of adjunctive berberine treatment resulted in a similar phenotype with consistently reduced numbers of neutrophils both in lungs and the spleen. Together, our results suggest that berberine can be supplemented as an immunomodulatory agent depending on the disease stage and inflammatory status of the host.

Retrospective Diagnostic Accuracy Study of Abbott RealTime MTB against Xpert MTB/RIF Ultra and Xpert MTB/RIF for the Diagnosis of Pulmonary Tuberculosis and Susceptibility to Rifampin and Isoniazid Treatment.

High-throughput centralized testing for tuberculosis (TB) and drug resistance is important, but comparative data are limited. In this retrospective cross-sectional study, participants were recruited from Johannesburg, South Africa, and Tbilisi, Georgia. The index tests, Abbott RealTime MTB (RT-MTB) and RealTime MTB RIF/INH (RT-MTB RIF/INH), were performed on specimens stored frozen for an extended period of time (beyond manufacturer-validated specifications) and compared to paired Xpert MTB/RIF Ultra (Xpert Ultra) and Xpert MTB/RIF (Xpert) results obtained with fresh specimens. The detection reference standard was the Mycobacterium tuberculosis complex culture, and for resistance detection, it was phenotypic drug susceptibility testing. The median age of 474 participants was 39 (interquartile range [IQR], 31 to 51) years. On decontaminated sputum, Xpert Ultra had a sensitivity of 91%, compared to 77% for RT-MTB, with a difference of +14% (95% confidence interval [CI], +9.2 to +21%; 18/127). On raw sputum, Xpert Ultra exhibited a sensitivity of 89% and Xpert one of 88%, compared to 80% for RT-MTB, exhibiting differences of +10% (95% CI, +3.3 to +18%; 9/93) and +8.6% (95% CI, +2.4 to +17%; 8/93), respectively. Specificity was ≥98% for all tests. All three tests showed high sensitivity and specificity for detection of rifampin resistance. Abbott assays may have lower sensitivity than Xpert and Xpert Ultra for TB detection but similar performance for detection of resistance. The differences in TB detection may be attributable to differences in testing of frozen (Abbott) versus fresh (Xpert) samples. Studies in compliance with manufacturer's instructions are required to compare performance. IMPORTANCE In 2019, 10 million people fell ill with tuberculosis (TB), of whom 1.4 million died. There are few comparative studies of diagnostic assays, particularly those aiming to be used in high-throughput laboratories. One such assay is the Abbott RealTime MTB (RT-MTB) and RealTime MTB RIF/INH (RT-MTB RIF/INH), which uses the m2000 platform already in use in many settings for HIV load testing and allows the diagnosis of TB and resistance to two first-line drugs, rifampin and isoniazid. Our study compared the RT-MTB and RT-MTB RIF/INH to the WHO-recommended Xpert MTB/RIF Ultra and Xpert MTB/RIF. The study is the largest comparative study to date and was performed independent of the manufacturer. The study results suggest that the Abbott RealTime MTB may have a lower sensitivity, but the study may have placed the Abbott test at a disadvantage by using frozen samples and comparing the results to those for fresh samples for the Xpert.

Nutritional markers and proteome in patients undergoing treatment for pulmonary tuberculosis differ by geographic region.

INTRODUCTION: Contemporary phase 2 TB disease treatment clinical trials have found that microbiologic treatment responses differ between African versus non-African regions, the reasons for which remain unclear. Understanding host and disease phenotypes that may vary by region is important for optimizing curative treatments. METHODS: We characterized clinical features and the serum proteome of phase 2 TB clinical trial participants undergoing treatment for smear positive, culture-confirmed TB, comparing host serum protein expression in clinical trial participants enrolled in African and Non-African regions. Serum samples were collected from 289 participants enrolled in the Centers for Disease Control and Prevention TBTC Study 29 (NCT00694629) at time of enrollment and at the end of the intensive phase (after 40 doses of TB treatment). RESULTS: After a peptide level proteome analysis utilizing a unique liquid chromatography IM-MS platform (LC-IM-MS) and subsequent statistical analysis, a total of 183 core proteins demonstrated significant differences at both baseline and at week 8 timepoints between participants enrolled from African and non-African regions. The majority of the differentially expressed proteins were upregulated in participants from the African region, and included acute phase proteins, mediators of inflammation, as well as coagulation and complement pathways. Downregulated proteins in the African population were primarily linked to nutritional status and lipid metabolism pathways. CONCLUSIONS: We have identified differentially expressed nutrition and lipid pathway proteins by geographic region in TB patients undergoing treatment for pulmonary tuberculosis, which appear to be associated with differential treatment responses. Future TB clinical trials should collect expanded measures of nutritional status and further evaluate the relationship between nutrition and microbiologic treatment response.

Five-year microevolution of a multidrug-resistant Mycobacterium tuberculosis strain within a patient with inadequate compliance to treatment.

BACKGROUND: Whole-genome sequencing has shown that the Mycobacterium tuberculosis infection process can be more heterogeneous than previously thought. Compartmentalized infections, exogenous reinfections, and microevolution are manifestations of this clonal complexity. The analysis of the mechanisms causing the microevolution -the genetic variability of M. tuberculosis at short time scales- of a parental strain into clonal variants with a patient is a relevant issue that has not been yet completely addressed. To our knowledge, a whole genome sequence microevolution analysis in a single patient with inadequate adherence to treatment has not been previously reported. CASE PRESENTATION: In this work, we applied whole genome sequencing analysis for a more in-depth analysis of the microevolution of a parental Mycobacterium tuberculosis strain into clonal variants within a patient with poor treatment compliance in Argentina. We analyzed the whole-genome sequence of 8 consecutive Mycobacterium tuberculosis isolates obtained from a patient within 57-months of intermittent therapy. Nineteen mutations (9 short-term, 10 fixed variants) emerged, most of them associated with drug resistance. The first isolate was already resistant to isoniazid, rifampicin, and streptomycin, thereafter the strain developed resistance to fluoroquinolones and pyrazinamide. Surprisingly, isolates remained susceptible to the pro-drug ethionamide after acquiring a frameshift mutation in ethA, a gene required for its activation. We also found a novel variant, (T-54G), in the 5' untranslated region of whiB7 (T-54G), a region allegedly related to kanamycin resistance. Notably, discrepancies between canonical and phage-based susceptibility testing to kanamycin were previously found for the isolate harboring this mutation. In our patient, microevolution was mainly driven by drug selective pressure. Rare short-term mutations fixed together with resistance-conferring mutations during therapy. CONCLUSIONS: This report highlights the relevance of whole-genome sequencing analysis in the clinic for characterization of pre-XDR and MDR resistance profile, particularly in patients with incomplete and/or intermittent treatment.

The Manipulation of the Lipid Mediator Metabolism as Adjunct Host-Directed Therapy in Tuberculosis.

Host-directed therapies (HDTs) enhance the host response to tuberculosis (TB) infection to reduce disease severity. For instance, the manipulation of lipid mediator production diminishes the hyperactive immune response which is a known pathological feature of TB that generates lung tissue damage. Non-steroidal anti-inflammatory drugs (NSAIDs) and omega-3 long-chain polyunsaturated fatty acids (n-3 LCPUFA) are examples of such HDTs. In this mini-review, we recapitulate the literature available on the effects of NSAIDs and n-3 LCPUFA in TB as well as the immunological pathways underpinning these effects. Many NSAIDs have a great deal of data describing their effects and safety and in many jurisdictions are inexpensive, and sold over the counter in neighborhood convenience stores and supermarkets. The potential benefits of NSAIDs in TB are well-documented in pre-clinical studies. The reduction of pro-inflammatory lipid mediator production by inhibiting cyclooxygenase (COX) pathways with NSAIDs has been found to improve lung histopathology, bacterial control, and survival. Additionally, n-3 LCPUFA and its novel bioactive metabolites produced by COX and lipoxygenase (LOX) have been identified as safe and effective pro-resolving and antibacterial pharmaconutrients. Nevertheless, heterogeneous results have been reported in pre-clinical TB studies. Recently, the importance of the correct timing of NSAIDs and n-3 LCPUFA administration in TB has also been highlighted. This mini-review will provide a better understanding of the potential contribution of these therapies toward reducing inflammatory lung damage and improving bactericidal activity, especially during later stages of TB infection. It further highlights that clinical trials are required to confirm benefit and safety in TB patients.

Accuracy of molecular drug susceptibility testing amongst tuberculosis patients in Karakalpakstan, Uzbekistan.

OBJECTIVES: In this retrospective study, we evaluated the diagnostic accuracy of molecular tests (MT) for the detection of DR-TB, compared to the gold standard liquid-based drug susceptibility testing (DST) in Karakalpakstan. METHODS: A total of 6670 specimens received in the Republican TB No 1 Hospital Laboratory of Karakalpakstan between January and July 2017 from new and retreatment patients were analysed. Samples were tested using Xpert MTB/RIF and line probe assays (LPA) for the detection of mutations associated with resistance. The sensitivity and specificity of MTs were calculated relative to results based on DST. RESULTS: The accuracy of MT for detection of rifampicin resistance was high, with sensitivity and specificity over 98%. However, we observed reduced sensitivity of LPA for detection of resistance; 86% for isoniazid (95% CI 82-90%), 86% for fluoroquinolones (95% CI 68-96%), 70% for capreomycin (95% CI 46-88%) and 23% for kanamycin (95% CI 13-35%). CONCLUSIONS: We show that MTs are a useful tool for rapid and safe diagnosis of DR-TB; however, clinicians should be aware of their limitations. Although detection of rifampicin resistance was highly accurate, our data suggest that resistance mutations circulating in the Republic of Karakalpakstan for other drugs were not detected by the methods used here. This merits further investigation.

Prevalence and associated risk factors of drug-resistant tuberculosis in Thailand: results from the fifth national anti-tuberculosis drug resistance survey.

OBJECTIVE: To assess the prevalence and risk factors of drug-resistant tuberculosis (TB), the fifth national anti-TB drug resistance survey was conducted in Thailand. METHODS: A cross-sectional study was conducted by stratified cluster sampling with probability proportional to size of TB cases from public health facilities in 100 clusters throughout Thailand from August 2017 to August 2018. Susceptibility testing of TB isolates to first- and second-line anti-TB drugs was performed on Löwenstein-Jensen medium using the indirect proportion method. Multiple imputation was done for handling missing data using Stata 16. The proportion of TB cases with drug resistance was determined. The odds ratio was used to evaluate risk factors associated with drug-resistant TB. RESULTS: Among 1501 new TB and 69 previously treated TB cases, 14.0% [95% confidence interval (CI): 12.1-16.1] and 33.4% (95% CI: 23.6-44.8), respectively, had resistance to any anti-TB drug. Multidrug-resistant TB accounted for 0.8% (95% CI: 0.5-1.4) of new TB cases and 13.0% (95% CI: 6.5-24.4) of previously treated TB cases. Drug-resistant TB was associated with prior TB treatment [odds ratio (OR), 2.9; 95% CI: 1.6-5.0], age at 45-54 years (OR, 1.6; 95% CI: 1.0-2.4), male (OR, 1.5; 95% CI: 1.0-2.1) and human immunodeficiency virus (HIV) infection (OR, 1.6; 95% CI: 1.0-2.4). CONCLUSIONS: The burden of drug-resistant TB remains high in Thailand. Intensified prevention and control measures should be implemented to reduce the risks of drug-resistant TB in high-risk groups previously treated, especially individuals of late middle age, males and those with coinfection of TB and HIV.

Efficacy of moxifloxacin combined with levofloxacin in the treatment of drug-resistant tuberculosis.

Drug-resistant tuberculosis is a clinically common respiratory-borne chronic infectious disease. Fluoroquinolone drugs can inhibit the replication and transcription of bacterial DNA and cause bacteria to die, and the antibacterial spectrum of such drugs is broad, especially for Mycobacterium tuberculosis-induced diseases. This article observes and compares the clinical efficacy of levofloxacin and moxifloxacin in the treatment of multidrug-resistant tuberculosis (MDR-TB). At the end of the course of treatment, the treatment success rate was 76.4% in the control group and 68.2% in the treatment group. The difference between the two groups was not statistically significant (P<0.05). The cavity reduction rate was 70.1% in the control group and 62.5% in the treatment group. Adverse reaction rate, the control group was 14.7% and the treatment group was 18.1%. There was no significant difference between the two groups (P >0.05). For multidrug-resistant tuberculosis, levofloxacin tablets and moxifloxacin tablets have similar effects in the treatment of multidrug-resistant tuberculosis, adverse drug reactions, and economically difficult multidrug-resistant patients. Drug sensitivity indicates that they are sensitive to levofloxacin.

Overexpression of the celA1 gene in BCG modifies surface pellicle, glucosamine content in biofilms, and affects in vivo replication.

Biofilm formed in vitro by mycobacteria has been associated with increased antibiotic tolerance as compared with planktonic cells. Cellulose has been identified as a component of DTT-exposed biofilms formed by M. tuberculosis. The celA1 gene of M. tuberculosis encodes a cellulase, which could affect the formation of biofilm by slow-growing mycobacteria. In this work, the celA1 gene of M. tuberculosis was cloned into the integrative pMV361 plasmid and then transformed into M. bovis BCG Pasteur to produce BCG:celA1, to have celA1 expressed from the strong promoter hsp60. We compared planktonic and biofilm growth, possible presence of CelA1 in whole protein extracts, quantitated biofilm, presence of monosaccharides, and bacillary burden in lungs after aerosol infection in BALB/c mice. Differences in the appearance of the surface pellicle and of the biofilm attached to the substrate were observed. In biofilms, we observed a significant decrease of glucosamine in BCG:celA1 compared with BCG:pMV361. Finally, BCG:celA1 had lower viable bacteria than the BCG:pMV361 strain after 24 h and 3 weeks post-infection, but no difference was found at 9 weeks post-infection.

The effect of micro-nutrients on malnutrition, immunity and therapeutic effect in patients with pulmonary tuberculosis: A systematic review and meta-analysis of randomised controlled trials.

OBJECTIVE: Micro-nutrients are closely related to pulmonary tuberculosis (PTB). Most patients with PTB suffer from micro-nutrients deficiency. We aimed to evaluate the efficacy of micro-nutrients support on clinical therapy and chronic inflammation in patients with PTB. METHODS: We searched Pubmed, Springer link, Web of Science, Cochrane, Wan Fang and CNKI databases for randomised controlled trials (RCTs). The patients with anti-TB treatments were divided into two groups, the control group with nutritional advice or placebo, and the experimental group with micro-nutrients support for more than 2 weeks. Two reviewers conducted data extraction and quality assessment of the studies independently, and ReviewManager 5.2 software was used to input and analyse the data. The dichotomous variable was expressed in the risk ratios (RRS) and 95% CI, the continuous data were expressed in the mean difference (MD) and 95% CI, and the heterogeneity of subgroup was evaluated by I (Kerantzas and Jacobs, Jr., 2017) [2] test. RESULTS: A total of 13 trials (2847 participants) were included. First, micro-nutrients improved sputum smears or culture negative conversion rates (OR 0.16 0.03-0.77, 2.29; MD -2.36, -4.72~-0.01, z = 1.97). Meanwhile, micro-nutrients support increased lymphocytes and decreased leukocytes, neutrophils, CRP and ESR (MD 0.20, 0.06-0.35, z = 2.78; MD -0.42, -0.65~-0.18, z = 3.48; MD -0.66, -1.12~-0.20, z = 2.82). However it had not impact on body weight, MUAC, haemoglobin, albumin or monocytes (p > 0.05). CONCLUSION: Micro-nutrients support can reduce chronic inflammation and improve sputum smears or culture conversions to contribute to anti-TB treatment.

Multidrug-resistant Mycobacterium tuberculosis: a report of cosmopolitan microbial migration and an analysis of best management practices.

BACKGROUND: Tuberculosis (TB) control is a primary global health priority but the goal to eliminate TB is being threatened by the increase in incidence of multidrug-resistant tuberculosis (MDR-TB). With this series of seven MDR-TB cases in migrant patients with identical Mycobacterium tuberculosis strains we aim to illustrate the challenges encountered during therapy and follow-up: language barriers, access to care for migrant patients, depression due to isolation, adverse reactions to the treatment, management of pediatric TB, further contact tracing. We also discuss best practices for the management of complex MDR-TB cases in settings with low overall TB incidence focusing on modern diagnostic assays and an individualized and an interdisciplinary therapeutic approach. METHODS: We describe a case series of seven consecutively diagnosed MDR-TB patients, six of them treated at our tertiary care hospital between May 2018 and March 2020. Epidemiologic data was gained by semi-structured patient interviews and reconstruction of the migration route. The origin of the cluster was confirmed by genotyping of the TB-strains. RESULTS: Six related patients were diagnosed with pulmonary MDR-TB between May and August 2018. All had a positive Interferon-Gamma-Release Assay (IGRA), in five patients sputum microscopy was positive for acid-fast bacilli (AFB). The genetic and phenotypical drug susceptibility test did not match with MDR-TB strains from an East-African origin. The index patient was identified through genetical fingerprinting. By changing the therapy to a modern MDR-TB regime and using an interdisciplinary and culture-sensitive approach, all patients improved clinically and radiologically. CONCLUSION: Human migration plays an important role for the global spread of MDR-TB in low incidence countries. Early case detection and adequate treatment are key to prevention of outbreaks. Especially language barriers and complex migration routes make genotyping of TB-strains a crucial tool to identify cases clusters, the potential index patient and transmission dynamics. We are fortunate enough to experience times in which new TB-antibiotics were made available and in which molecular assays revolutionized TB-diagnostics. We need to take advantage of that and develop personalized therapies for patients suffering from drug resistant TB.

Acceptability and Adherence to Peanut-Based Energy-Dense Nutritional Supplement Among Adult Malnourished Pulmonary Tuberculosis Patients in Ballabgarh Block of Haryana, India.

BACKGROUND: Undernutrition is a leading risk factor for tuberculosis and is associated with adverse treatment outcomes. Energy-dense nutritional supplement (EDNS) may be helpful in managing undernutrition in tuberculosis patients. METHODS: A longitudinal study was conducted among 102 newly diagnosed pulmonary tuberculosis patients in the Ballabgarh tuberculosis unit in Haryana, India, between September 2018 and February 2019. Participants were provided daily supplementation with 2 sachets of peanut-based EDNS providing 1000 kcal along with the tuberculosis chemotherapy for 2 months. Acceptability was assessed after one month of supplementation. Reported side effects were assessed every 15 days for 2 months and adherence, in terms of proportion of supplement consumed, was assessed on weekly basis for 2 months. Weight was measured at baseline and after 1 month and 2 months. RESULTS: Mean age of participants was 33 years (standard deviation: 13.8) and 75.5% of the participants were male. Almost 100% of the participants found the taste, smell, consistency, and color of the EDNS acceptable. Three participants rejected it after one month of consuming EDNS and another 7 rejected after 2 months of consuming EDNS. After consuming EDNS for 2 months, 13.9% of the participants complained of nausea, 11.9% complained of vomiting, 10.9% complained of diarrhea, and 9.9% complained of pain in the abdomen and abdominal fullness or bloating anytime during the period of supplementation. The adherence to EDNS among the participants was reported as 92.6%. CONCLUSION: A peanut-based EDNS was highly acceptable and safe, and the adherence to it was high among patients with pulmonary tuberculosis.

From pulmonary susceptible tuberculosis to extensively drug resistant tuberculosis: An interesting case report of a young Indian girl.

Tuberculosis is one of the top ten causes of death and the leading cause from a single infectious agent. Drug-resistant Tuberculosis continues to be a public health crisis. Urgent action is required to improve the coverage and quality of diagnosis, treatment and care for people with drug-resistant Tuberculosis. Patients with pulmonary Tuberculosis can spread the disease by coughing, sneezing, or simply talking. For that reason, it is important to diagnosis Tuberculosis in order to start treatment as soon as possible. In the present manuscript we present the case of a 25-year-old Indian HIV-negative female, no comorbidity, with a history of drug susceptible tuberculosis diagnosed in 2015 which advanced in extensively drug-resistant tuberculosis after two years of treatment. This case report highlights the risk of mismanagement of patient affected by Tuberculosis and the consequences related which could harm the patient's health.

Mycobacterium tuberculosis polyclonal infections through treatment and recurrence.

BACKGROUND: Mixed/polyclonal infections due to different genotypes are reported in Tuberculosis. The current study was designed to understand the fate of mixed infections during the course of treatment and follow-up and its role in disease pathogenesis. METHODS: Sputum samples were collected on 0,1,2,3,6,12 and 24 months from 157 treatment-naïve patients, cultures subjected to Drug-Susceptibility-testing (MGIT 960), spoligotyping, MIRU-VNTR and SNP genotyping. All isolated colonies on thin layer agar (7H11) were subjected to spoligotyping. FINDINGS: One thirty three baseline cultures were positive (133/157, 84.7%), 43(32.3%) had mixture of genotypes. Twenty-four of these patients (55.8%) showed change in genotype while six showed different drug-susceptibility patterns while on treatment. Twenty-three (53.5%) patients with polyclonal infections showed resistance to at least one drug compared to 10/90 (11.1%) monoclonal infections (P<0.0001). Eight patients had recurrent TB, two with a new genotype and two with altered phenotypic DST. CONCLUSIONS: The coexistence of different genotypes and change of genotypes during the same disease episode, while on treatment, confirms constancy of polyclonal infections. The composition of the mixture of genotypes and the relative predominance may be missed by culture due to its limit of detection. Polyclonal infections in TB could be a rule rather than exception and challenges the age-old dogma of reactivation/reinfection.

A model-based analysis identifies differences in phenotypic resistance between in vitro and in vivo: implications for translational medicine within tuberculosis.

Proper characterization of drug effects on Mycobacterium tuberculosis relies on the characterization of phenotypically resistant bacteria to correctly establish exposure-response relationships. The aim of this work was to evaluate the potential difference in phenotypic resistance in in vitro compared to murine in vivo models using CFU data alone or CFU together with most probable number (MPN) data following resuscitation with culture supernatant. Predictions of in vitro and in vivo phenotypic resistance i.e. persisters, using the Multistate Tuberculosis Pharmacometric (MTP) model framework was evaluated based on bacterial cultures grown with and without drug exposure using CFU alone or CFU plus MPN data. Phenotypic resistance and total bacterial number in in vitro natural growth observations, i.e. without drug, was well predicted by the MTP model using only CFU data. Capturing the murine in vivo total bacterial number and persisters during natural growth did however require re-estimation of model parameter using both the CFU and MPN observations implying that the ratio of persisters to total bacterial burden is different in vitro compared to murine in vivo. The evaluation of the in vitro rifampicin drug effect revealed that higher resolution in the persister drug effect was seen using CFU and MPN compared to CFU alone although drug effects on the other bacterial populations were well predicted using only CFU data. The ratio of persistent bacteria to total bacteria was predicted to be different between in vitro and murine in vivo. This difference could have implications for subsequent translational efforts in tuberculosis drug development.

Designing an optimized diagnostic network to improve access to TB diagnosis and treatment in Lesotho.

BACKGROUND: To reach WHO End tuberculosis (TB) targets, countries need a quality-assured laboratory network equipped with rapid diagnostics for tuberculosis diagnosis and drug susceptibility testing. Diagnostic network analysis aims to inform instrument placement, sample referral, staffing, geographical prioritization, integration of testing enabling targeted investments and programming to meet priority needs. METHODS: Supply chain modelling and optimization software was used to map Lesotho's TB diagnostic network using available data sources, including laboratory and programme reports and health and demographic surveys. Various scenarios were analysed, including current network configuration and inclusion of additional GeneXpert and/or point of care instruments. Different levels of estimated demand for testing services were modelled (current [30,000 tests/year], intermediate [41,000 tests/year] and total demand needed to find all TB cases [88,000 tests/year]). RESULTS: Lesotho's GeneXpert capacity is largely well-located but under-utilized (19/24 sites use under 50% capacity). The network has sufficient capacity to meet current and near-future demand and 70% of estimated total demand. Relocation of 13 existing instruments would deliver equivalent access to services, maintain turnaround time and reduce costs compared with planned procurement of 7 more instruments. Gaps exist in linking people with positive symptom screens to testing; closing this gap would require extra 11,000 tests per year and result in 1000 additional TB patients being treated. Closing the gap in linking diagnosed patients to treatment would result in a further 629 patients being treated. Scale up of capacity to meet total demand will be best achieved using a point-of-care platform in addition to the existing GeneXpert footprint. CONCLUSIONS: Analysis of TB diagnostic networks highlighted key gaps and opportunities to optimize services. Network mapping and optimization should be considered an integral part of strategic planning. By building efficient and patient-centred diagnostic networks, countries will be better equipped to meet End TB targets.

Interim treatment outcomes in multidrug-resistant tuberculosis using bedaquiline and/or delamanid in South Korea.

PURPOSE: The Korea Centers for Disease Control & Prevention has implemented a review process for the approval of new drugs used to treat patients with multidrug-resistant tuberculosis (MDR-TB) since September 2016. Therefore, this study aimed to evaluate the efficacy and safety of these new drugs bedaquiline (Bdq) and delamanid (Dlm). METHODS: A total of 318 patients with MDR-TB were reviewed by the committee from September 2016 to February 2018; 282 (88.7%) of them were treated with the new drugs (Bdq, 107 patients; Dlm, 108 patients; and both concurrently or sequentially, 67 patients) and retrospectively evaluated. Culture conversion rates, interim treatment outcomes at 12 months, and predictors of unfavorable outcomes were analyzed. Treatment efficacy was also compared between Bdq and Dlm. RESULTS: The mean age of the patients was 49.3 years, and 197 (69.9%) were male. Three patients were HIV seropositive and 151 (53.5%) were quinolone resistant. The culture conversion rates at 2 and 6 months were 57.4% (81/141) and 89.4% (126/141), respectively. A favorable outcome at 12 months was achieved in 84.8% of patients (239/282). Differences in the culture conversion rate or interim treatment outcomes were not statistically significant among the drug susceptibility test patterns or new drugs used. Multivariable analysis showed that age >60 years and body mass index of <18.5 kg/m2 were significant risk factors for unfavorable outcomes at 12 months. CONCLUSIONS: The use of new drugs resulted in satisfactory interim treatment results, without significant differences between them.

Dynamics of within-host Mycobacterium tuberculosis diversity and heteroresistance during treatment.

BACKGROUND: Studying within-host genetic diversity of Mycobacterium tuberculosis (Mtb) in patients during treatment may identify adaptations to antibiotic and immune pressure. Understanding the significance of genetic heteroresistance, and more specifically heterozygous resistance-associated variants (RAVs), is clinically important given increasing use of rapid molecular tests and whole genome sequencing (WGS). METHODS: We analyse data from six studies in KwaZulu-Natal, South Africa. Most patients (>75%) had baseline rifampicin resistance. Sputum was collected for culture at baseline and at between two and nine intervals until month six. Positive cultures underwent WGS. Mixed infections and reinfections were excluded from analysis. FINDINGS: Baseline Mtb overall genetic diversity (at treatment initiation or major change to regimen) was associated with cavitary disease, not taking antiretroviral therapy if HIV infected, infection with lineage 2 strains and absence of second-line drug resistance on univariate analyses. Baseline genetic diversity was not associated with six-month outcome. Genetic diversity increased from baseline to weeks one and two before returning to previous levels. Baseline genetic heteroresistance was most common for bedaquiline (6/10 [60%] of isolates with RAVs) and fluoroquinolones (9/62 [13%]). Most patients with heterozygous RAVs on WGS with sequential isolates available demonstrated RAV persistence or fixation (17/20, 85%). New RAVs emerged in 9/286 (3%) patients during treatment. We could detect low-frequency RAVs preceding emergent resistance in only one case, although validation of deep sequencing to detect rare variants is required. INTERPRETATION: In this study of single-strain Mtb infections, baseline within-host bacterial genetic diversity did not predict outcome but may reveal adaptations to host and drug pressures. Predicting emergent resistance from low-frequency RAVs requires further work to separate transient from consequential mutations. FUNDING: Wellcome Trust, NIH/NIAID.