The Genetic Background Modulates the Evolution of Fluoroquinolone-Resistance in Mycobacterium tuberculosis.

Fluoroquinolones (FQ) form the backbone in experimental treatment regimens against drug-susceptible tuberculosis. However, little is known on whether the genetic variation present in natural populations of Mycobacterium tuberculosis (Mtb) affects the evolution of FQ-resistance (FQ-R). To investigate this question, we used nine genetically distinct drug-susceptible clinical isolates of Mtb and measured their frequency of resistance to the FQ ofloxacin (OFX) in vitro. We found that the Mtb genetic background led to differences in the frequency of OFX-resistance (OFX-R) that spanned two orders of magnitude and substantially modulated the observed mutational profiles for OFX-R. Further, in vitro assays showed that the genetic background also influenced the minimum inhibitory concentration and the fitness effect conferred by a given OFX-R mutation. To test the clinical relevance of our in vitro work, we surveyed the mutational profile for FQ-R in publicly available genomic sequences from clinical Mtb isolates, and found substantial Mtb lineage-dependent variability. Comparison of the clinical and the in vitro mutational profiles for FQ-R showed that 51% and 39% of the variability in the clinical frequency of FQ-R gyrA mutation events in Lineage 2 and Lineage 4 strains, respectively, can be attributed to how Mtb evolves FQ-R in vitro. As the Mtb genetic background strongly influenced the evolution of FQ-R in vitro, we conclude that the genetic background of Mtb also impacts the evolution of FQ-R in the clinic.

HIV Infection Functionally Impairs Mycobacterium tuberculosis-Specific CD4 and CD8 T-Cell Responses.

Human immunodeficiency virus (HIV) infection is the major risk factor predisposing for Mycobacterium tuberculosis progression from latent tuberculosis infection (LTBI) to tuberculosis disease (TB). Since long-term-treated aviremic HIV-infected individuals remained at higher risk of developing TB than HIV-uninfected individuals, we hypothesized that progression from LTBI to pulmonary TB (PTB) might be due not only to CD4 T-cell depletion but also to M. tuberculosis-specific CD4 T-cell functional impairment. To test this hypothesis, M. tuberculosis-specific T-cell frequencies and cytokine profiles were investigated in untreated Tanzanian individuals suffering from LTBI (n = 20) or PTB (n = 67) and compared to those of untreated M. tuberculosis/HIV-coinfected individuals suffering from LTBI (n = 15) or PTB (n = 10). We showed that HIV infection significantly reduced the proportion of Th2 (interleukin 4 [IL-4]/IL-5/IL-13) producing M. tuberculosis-specific CD4 T cells and IL-2-producing M. tuberculosis-specific CD4 and CD8 T cells in individuals with LTBI or PTB (P < 0.05). Interestingly, the loss of IL-2 production was associated with a significant increase of PD-1 expression on M. tuberculosis-specific CD4 and CD8 T cells (P < 0.05), while the loss of Th2 cytokine production was associated with a significant reduction of Gata-3 expression in memory CD4 T cells (P < 0.05). Finally, we showed that the serum levels of IL-1α, IL-6, C-reactive protein (CRP), IL-23, and IP-10 were significantly reduced in M. tuberculosis/HIV-coinfected individuals with PTB compared to those in HIV-negative individuals with PTB (P < 0.05), suggesting that HIV infection significantly suppresses M. tuberculosis-induced systemic proinflammatory cytokine responses. Taken together, this study suggests that in addition to depleting M. tuberculosis-specific CD4 T cells, HIV infection significantly impairs functionally favorable M. tuberculosis-specific CD4 T-cell responses in Tanzanian individuals with LTBI or PTB.IMPORTANCEMycobacterium tuberculosis and human immunodeficiency virus (HIV) infections are coendemic in several regions of the world, and M. tuberculosis/HIV-coinfected individuals are more susceptible to progression to tuberculosis disease. We therefore hypothesized that HIV infection would potentially impair M. tuberculosis-specific protective immunity in individuals suffering from latent tuberculosis infection (LTBI) or active pulmonary tuberculosis (PTB). In this study, we demonstrated that M. tuberculosis/HIV-coinfected individuals have fewer circulating M. tuberculosis-specific CD4 T cells and that those that remained were functionally impaired in both LTBI and PTB settings. In addition, we showed that HIV infection significantly interferes with M. tuberculosis-induced systemic proinflammatory cytokine/chemokine responses. Taken together, these data suggest that HIV infection impairs functionally favorable M. tuberculosis-specific immunity.

Preservation of sputum samples with cetylpyridinium chloride (CPC) for tuberculosis cultures and Xpert MTB/RIF in a low-income country.

BACKGROUND: Culture contamination with environmental bacteria is a major challenge in tuberculosis (TB) laboratories in hot and humid climate zones. We studied the effect of cetylpyridinium chloride (CPC) preservation on culture results and performance of Xpert MTB/RIF. METHODS: Consecutive sputum samples from microscopy smear-positive TB patients were collected. Two-hundred samples were equally split in two aliquots, one aliquot was treated with CPC and stored at ambient temperature for 7 days. The second aliquot was immediately processed. Samples were decontaminated for 20, 15 or 10 min, and subsequently cultured on Löwenstein-Jensen medium. Furthermore, 50 samples were stored for 7, 14 and 21 days, and 100 CPC-pretreated samples tested by Xpert MTB/RIF. RESULTS: CPC pretreated samples showed a higher culture yield compared to non-treated sputum samples across all decontamination times: 94% vs. 73% at 10 min (p = 0.01), 94% vs. 64% at 15 min (p = 0.004), and 90% vs. 52% at 20 min (p < 0.001). The quantitative culture grading was consistently higher in CPC treated compared to non-CPC treated samples. The proportion of contaminated cultures was lower in CPC pretreated samples across all decontamination times (range 2-6%) compared to non-CPC treated samples (15-16%). For storage times of CPC treated samples of 7, 14, and 21 days, 84, 86, and 84% of the respective cultures were positive. Of 91 CPC treated samples with a positive culture, 90 were also Xpert MTB/RIF positive. CONCLUSIONS: CPC increases culture yield, decreases the proportion of contamination, and does not alter the performance of Xpert MTB/RIF.

Local adaptation in populations of Mycobacterium tuberculosis endemic to the Indian Ocean Rim.

Background: Lineage 1 (L1) and 3 (L3) are two lineages of the Mycobacterium tuberculosis complex (MTBC) causing tuberculosis (TB) in humans. L1 and L3 are prevalent around the rim of the Indian Ocean, the region that accounts for most of the world's new TB cases. Despite their relevance for this region, L1 and L3 remain understudied. Methods: We analyzed 2,938 L1 and 2,030 L3 whole genome sequences originating from 69 countries. We reconstructed the evolutionary history of these two lineages and identified genes under positive selection. Results: We found a strongly asymmetric pattern of migration from South Asia toward neighboring regions, highlighting the historical role of South Asia in the dispersion of L1 and L3. Moreover, we found that several genes were under positive selection, including genes involved in virulence and resistance to antibiotics. For L1 we identified signatures of local adaptation at the esxH locus, a gene coding for a secreted effector that targets the human endosomal sorting complex, and is included in several vaccine candidates. Conclusions: Our study highlights the importance of genetic diversity in the MTBC, and sheds new light on two of the most important MTBC lineages affecting humans.

Sonography to Rule Out Tuberculosis in Sub-Saharan Africa: A Prospective Observational Study.

BACKGROUND: Patients with suspected tuberculosis are often overtreated with antituberculosis drugs. We evaluated the diagnostic value of the focused assessment with sonography for HIV-associated tuberculosis (FASH) in rural Tanzania. METHODS: In a prospective cohort study, the frequency of FASH signs was compared between patients with confirmed tuberculosis and those without tuberculosis. Clinical and laboratory examination, chest x-ray, Xpert MTB/RIF assay, and culture from sputum, sterile body fluids, lymph node aspirates, and Xpert MTB/RIF urine assay was done. RESULTS: Of 191 analyzed patients with a 6-month follow-up, 52.4% tested positive for human immunodeficiency virus, 21.5% had clinically suspected pulmonary tuberculosis, 3.7% had extrapulmonary tuberculosis, and 74.9% had extrapulmonary and pulmonary tuberculosis. Tuberculosis was microbiologically confirmed in 57.6%, probable in 13.1%, and excluded in 29.3%. Ten of eleven patients with splenic or hepatic hypoechogenic lesions had confirmed tuberculosis. In a univariate model, abdominal lymphadenopathy was significantly associated with confirmed tuberculosis. Pleural- and pericardial effusion, ascites, and thickened ileum wall lacked significant association. In a multiple regression model, abnormal chest x-ray (odds ratio [OR] = 6.19; 95% confidence interval [CI], 1.96-19.6; P < .002), ≥1 FASH-sign (OR = 3.33; 95% CI, 1.21-9.12; P = .019), and body temperature (OR = 2.48; 95% CI, 1.52-5.03; P = .001 per °C increase) remained associated with tuberculosis. A combination of ≥1 FASH sign, abnormal chest x-ray, and temperature ≥37.5°C had 99.1% sensitivity (95% CI, 94.9-99.9), 35.2% specificity (95% CI, 22.7-49.4), and a positive and negative predictive value of 75.2% (95% CI, 71.3-78.7) and 95.0% (95% CI, 72.3-99.3). CONCLUSIONS: The absence of FASH signs combined with a normal chest x-ray and body temperature <37.5°C might exclude tuberculosis.

Insights into the genetic diversity of Mycobacterium tuberculosis in Tanzania.

BACKGROUND: Human tuberculosis (TB) is caused by seven phylogenetic lineages of the Mycobacterium tuberculosis complex (MTBC), Lineage 1-7. Recent advances in rapid genotyping of MTBC based on single nucleotide polymorphisms (SNP), allow for phylogenetically robust strain classification, paving the way for defining genotype-phenotype relationships in clinical settings. Such studies have revealed that, in addition to host and environmental factors, strain variation in the MTBC influences the outcome of TB infection and disease. In Tanzania, such molecular epidemiological studies of TB however are scarce in spite of a high TB burden. METHODS AND FINDINGS: Here we used SNP-typing to characterize a nationwide collection of 2,039 MTBC clinical isolates representative of 1.6% of all new and retreatment TB cases notified in Tanzania during 2012 and 2013. Four lineages, namely Lineage 1-4 were identified within the study population. The distribution and frequency of these lineages varied across regions but overall, Lineage 4 was the most frequent (n = 866, 42.5%), followed by Lineage 3 (n = 681, 33.4%) and 1 (n = 336, 16.5%), with Lineage 2 being the least frequent (n = 92, 4.5%). We found Lineage 2 to be independently associated with female sex (adjusted odds ratio [aOR] 2.14; 95% confidence interval [95% CI] 1.31 - 3.50, p = 0.002) and retreatment cases (aOR 1.67; 95% CI 0.95 - 2.84, p = 0. 065) in the study population. We found no associations between MTBC lineage and patient age or HIV status. Our sublineage typing based on spacer oligotyping on a subset of Lineage 1, 3 and 4 strains revealed the presence of mainly EAI, CAS and LAM families. Finally, we detected low levels of multidrug resistant isolates among a subset of 144 retreatment cases. CONCLUSIONS: This study provides novel insights into the MTBC lineages and the possible influence of pathogen-related factors on the TB epidemic in Tanzania.

Xpert MTB/RIF Ultra assay for the diagnosis of pulmonary tuberculosis in children: a multicentre comparative accuracy study.

OBJECTIVES: We evaluated the diagnostic performance of the novel next-generation Xpert MTB/RIF Ultra (Xpert Ultra) in comparison to Xpert MTB/RIF (Xpert) assay for the detection of paediatric pulmonary tuberculosis in high burden settings. METHODS: From May 2011 to September 2012, children with suspected pulmonary tuberculosis were enrolled at two Tanzanian sites and sputum samples were examined using sputum smear, Xpert and culture. Xpert Ultra was tested between January and June 2017 using sputum pellets, which had been stored at -80°C. The diagnostic accuracy of Ultra versus Xpert was determined using well-defined case definitions as reference standard. RESULTS: In total, 215 children were included. The median age was 5.4 years, the HIV prevalence was 52% and 13% had culture-confirmed pulmonary tuberculosis. When only the first available sample of each patient was analysed, the sensitivity of Xpert Ultra was 64.3 % (95% CI: 44.1 to 81.4) while that of Xpert was 53.6% (95%CI: 33.9 to 72.5). The specificity of Xpert Ultra based on analysis of all available samples was 98.1% (95%CI: 93.4 to 99.7), that of Xpert was 100%. CONCLUSIONS: Xpert Ultra was found to have a higher sensitivity, but slightly reduced specificity compared to Xpert in detecting pulmonary tuberculosis in children.

Distinct clinical characteristics and helminth co-infections in adult tuberculosis patients from urban compared to rural Tanzania.

BACKGROUND: Differences in rural and urban settings could account for distinct characteristics in the epidemiology of tuberculosis (TB). We comparatively studied epidemiological features of TB and helminth co-infections in adult patients from rural and urban settings of Tanzania. METHODS: Adult patients (≥ 18 years) with microbiologically confirmed pulmonary TB were consecutively enrolled into two cohorts in Dar es Salaam, with ~ 4.4 million inhabitants (urban), and Ifakara in the sparsely populated Kilombero District with ~ 400 000 inhabitants (rural). Clinical data were obtained at recruitment. Stool and urine samples were subjected to diagnose helminthiases using Kato-Katz, Baermann, urine filtration, and circulating cathodic antigen tests. Differences between groups were assessed by χ2, Fisher's exact, and Wilcoxon rank sum tests. Logistic regression models were used to determine associations. RESULTS: Between August 2015 and February 2017, 668 patients were enrolled, 460 (68.9%) at the urban and 208 (31.1%) at the rural site. Median patient age was 35 years (interquartile range [IQR]: 27-41.5 years), and 454 (68%) were males. Patients from the rural setting were older (median age 37 years vs. 34 years, P = 0.003), had a lower median body mass index (17.5 kg/m2 vs. 18.5 kg/m2, P <  0.001), a higher proportion of recurrent TB cases (9% vs. 1%, P <  0.001), and in HIV/TB co-infected patients a lower median CD4 cell counts (147 cells/µl vs. 249 cells/µl, P = 0.02) compared to those from urban Tanzania. There was no significant difference in frequencies of HIV infection, diabetes mellitus, and haemoglobin concentration levels between the two settings. The overall prevalence of helminth co-infections was 22.9% (95% confidence interval [CI]: 20.4-27.0%). The significantly higher prevalence of helminth infections at the urban site (25.7% vs. 17.3%, P = 0.018) was predominantly driven by Strongyloides stercoralis (17.0% vs. 4.8%, P <  0.001) and Schistosoma mansoni infection (4.1% vs. 16.4%, P <  0.001). Recurrent TB was associated with living in a rural setting (adjusted odds ratio [aOR]: 3.97, 95% CI: 1.16-13.67) and increasing age (aOR: 1.06, 95% CI: 1.02-1.10). CONCLUSIONS: Clinical characteristics and helminth co-infections pattern differ in TB patients in urban and rural Tanzania. The differences underline the need for setting-specific, tailored public health interventions to improve clinical management of TB and comorbidities.

Prevalence and clinical relevance of helminth co-infections among tuberculosis patients in urban Tanzania.

BACKGROUND: Helminth infections can negatively affect the immunologic host control, which may increase the risk of progression from latent Mycobacterium tuberculosis infection to tuberculosis (TB) disease and alter the clinical presentation of TB. We assessed the prevalence and determined the clinical relevance of helminth co-infection among TB patients and household contact controls in urban Tanzania. METHODOLOGY: Between November 2013 and October 2015, we enrolled adult (≥18 years) sputum smear-positive TB patients and household contact controls without TB during an ongoing TB cohort study in Dar es Salaam, Tanzania. We used Baermann, FLOTAC, Kato-Katz, point-of-care circulating cathodic antigen, and urine filtration to diagnose helminth infections. Multivariable logistic regression models with and without random effects for households were used to assess for associations between helminth infection and TB. PRINCIPAL FINDINGS: A total of 597 TB patients and 375 household contact controls were included. The median age was 33 years and 60.2% (585/972) were men. The prevalence of any helminth infection among TB patients was 31.8% (190/597) and 25.9% (97/375) among controls. Strongyloides stercoralis was the predominant helminth species (16.6%, 161), followed by hookworm (9.0%, 87) and Schistosoma mansoni (5.7%, 55). An infection with any helminth was not associated with TB (adjusted odds ratio (aOR) 1.26, 95% confidence interval (CI): 0.88-1.80, p = 0.22), but S. mansoni infection was (aOR 2.15, 95% CI: 1.03-4.45, p = 0.040). Moreover, S. mansoni infection was associated with lower sputum bacterial load (aOR 2.63, 95% CI: 1.38-5.26, p = 0.004) and tended to have fewer lung cavitations (aOR 0.41, 95% CI: 0.12-1.16, p = 0.088). CONCLUSIONS/SIGNIFICANCE: S. mansoni infection was an independent risk factor for active TB and altered the clinical presentation in TB patients. These findings suggest a role for schistosomiasis in modulating the pathogenesis of human TB. Treatment of helminths should be considered in clinical management of TB and TB control programs.