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Background: It is unknown whether immunosuppression influences the physiologic state of Mycobacterium tuberculosis in vivo. We evaluated the impact of host immunity by comparing M. tuberculosis and human gene transcription in sputum between human immunodeficiency virus (HIV)-infected and uninfected patients with tuberculosis.Methods: We collected sputum specimens before treatment from Gambians and Ugandans with pulmonary tuberculosis, revealed by positive results of acid-fast bacillus smears. We quantified expression of 2179 M. tuberculosis genes and 234 human immune genes via quantitative reverse transcription-polymerase chain reaction. We summarized genes from key functional categories with significantly increased or decreased expression.Results: A total of 24 of 65 patients with tuberculosis were HIV infected. M. tuberculosis DosR regulon genes were less highly expressed among HIV-infected patients with tuberculosis than among HIV-uninfected patients with tuberculosis (Gambia, P < .0001; Uganda, P = .037). In profiling of human genes from the same sputa, HIV-infected patients had 3.4-fold lower expression of IFNG (P = .005), 4.9-fold higher expression of ARG1 (P = .0006), and 3.4-fold higher expression of IL10 (P = .0002) than in HIV-uninfected patients with tuberculosis.Conclusions: M. tuberculosis in HIV-infected patients had lower expression of the DosR regulon, a critical metabolic and immunomodulatory switch induced by NO, carbon monoxide, and hypoxia. Our human data suggest that decreased DosR expression may result from alternative pathway activation of macrophages, with consequent decreased NO expression and/or by poor granuloma formation with consequent decreased hypoxic stress. [ABSTRACT FROM AUTHOR]

Background: In the context of advanced immunosuppression, M. tuberculosis is known to cause detectable mycobacteremia. However, little is known about the intra-patient mycobacterial microevolution and the direction of seeding between the sputum and blood compartments.Methods: From a diagnostic study of HIV-infected TB patients, 51 pairs of concurrent blood and sputum M. tuberculosis isolates from the same patient were available. In a previous analysis, we identified a subset with genotypic concordance, based on spoligotyping and 24 locus MIRU-VNTR. These paired isolates with identical genotypes were analyzed by whole genome sequencing and phylogenetic analysis.Results: Of the 25 concordant pairs (49 % of the 51 paired isolates), 15 (60 %) remained viable for extraction of high quality DNA for whole genome sequencing. Two patient pairs were excluded due to poor quality sequence reads. The median CD4 cell count was 32 (IQR; 16-101)/mm(3) and ten (77 %) patients were on ART. No drug resistance mutations were identified in any of the sequences analyzed. Three (23.1 %) of 13 patients had SNPs separating paired isolates from blood and sputum compartments, indicating evidence of microevolution. Using a phylogenetic approach to identify the ancestral compartment, in two (15 %) patients the blood isolate was ancestral to the sputum isolate, in one (8 %) it was the opposite, and ten (77 %) of the pairs were identical.Conclusions: Among HIV-infected patients with poor cellular immunity, infection with multiple strains of M. tuberculosis was found in half of the patients. In those patients with identical strains, whole genome sequencing indicated that M. tuberculosis intra-patient microevolution does occur in a few patients, yet did not reveal a consistent direction of spread between sputum and blood. This suggests that these compartments are highly connected and potentially seed each other repeatedly. [ABSTRACT FROM AUTHOR]

Background: Molecular resistance testing fails to explain all fluoroquinolone resistance, with a continued need for a suitable rapid phenotypic drug susceptibility testing method.Objective: To evaluate the optimal method for phenotypic fluoroquinolone susceptibility testing.Methods: Using Löwenstein-Jensen medium, Middlebrook 7H11 agar, BACTEC-MGIT 960 and the resazurin microtitre plate assay, we determined susceptibility to fluoroquinolones in Mycobacterium tuberculosis and investigated cross-resistance between ofloxacin, levofloxacin, moxifloxacin and gatifloxacin. We compared MICs of all four fluoroquinolones for 91 strains on Löwenstein-Jensen (as the gold standard) with their MICs in resazurin plates, and with ofloxacin susceptibility at a single concentration in MGIT and on 7H11 agar, in addition to sequencing of the gyrAB genes.Results and Conclusions: Applying a cut-off of 2 mg/L ofloxacin, 1 mg/L levofloxacin and 0.5 mg/L moxifloxacin and gatifloxacin in all methods, some discordance between solid medium and MGIT methods was observed, yet this tended to be explained by MICs around the cut-off. The high discordance between Löwenstein-Jensen (LJ) and resazurin plates suggests that the currently applied cut-offs for all fluoroquinolones in the resazurin method should decrease and minor changes in colour (from blue to purple) be considered as meaningful. High-level resistance in all assays to all drugs correlated well with the presence of gyrA mutations, in support of recent findings that fluoroquinolone resistance should be tested at different concentrations, as patients with lower levels of resistance may continue to benefit from high-dose fluoroquinolone-based therapy. [ABSTRACT FROM AUTHOR]

Responses to tuberculosis antigens were analyzed in a longitudinal cohort of patients with immune reconstitution inflammatory syndrome (IRIS). IRIS patients had a slow recovery of general T-cell responses (both CD4+ and CD8+) but specific increases in monofunctional CD4+IFN-γ+ responses to tuberculosis antigens during IRIS.Background. A common complication of starting antiretroviral therapy (ART) for human immunodeficiency virus (HIV) is the development of immune reconstitution inflammatory syndrome (IRIS) in approximately 25% of patients. Despite similarities with paradoxical reactions to tuberculosis and reversal reactions in leprosy, the exact mechanisms, and therefore potential determinants, of IRIS are still unknown.Methods. In this longitudinal cohort study, we analyzed 20 patients who developed IRIS following initiation of ART and 16 patients who did not, matched for ART time point. Peripheral blood mononuclear cells were stimulated overnight with a positive control antigen and 2 tuberculosis-specific antigens (purified protein derivative [PPD] and ESAT-6/CFP10), followed by polychromatic flow cytometry for analysis of cytokine production from CD4+ and CD8+ T cells.Results. Responses to PPD were significantly higher in IRIS patients compared to controls during the IRIS time point, but CD4+ and CD8+ T-cell responses to the positive control stimulation were significantly lower in IRIS patients at all time points. Furthermore, whereas control patients had rejuvenated polyfunctional T-cell responses by 3 months after ART, IRIS patients were strikingly monofunctional (generally interferon γ alone), even up to 6 months of ART in response to all stimulations.Conclusions. Our findings suggest that the peripheral T-cell responses to the underlying pathogen are exaggerated in IRIS patients but that the overall quality of the peripheral T-cell pool is significantly reduced compared to non-IRIS patients. Furthermore, these effects are apparent at least up to 3 months after cessation of IRIS. [ABSTRACT FROM PUBLISHER]

Mycobacterium africanum consists of two phylogenetically distinct lineages within the Mycobacterium tuberculosis complex, known as M. africanum West African 1 and M. africanum West African 2. These lineages are restricted to West Africa, where they cause up to half of human pulmonary tuberculosis. In this review we discuss the definition of M. africanum, describe the prevalence and restricted geographical distribution of M. africanum West African 1 and 2, review the occurrence of M. africanum in animals, and summarize the phenotypic differences described thus far between M. africanum and M. tuberculosis sensu stricto. [ABSTRACT FROM AUTHOR]

Mycobacterium africanum consists of two phylogenetically distinct lineages within the Mycobacterium tuberculosis complex, known as M. africanum West African 1 and M. africanum West African 2. These lineages are restricted to West Africa, where they cause up to half of human pulmonary tuberculosis. In this review we discuss the definition of M. africanum, describe the prevalence and restricted geographical distribution of M. africanum West African 1 and 2, review the occurrence of M. africanum in animals, and summarize the phenotypic differences described thus far between M. africanum and M. tuberculosis sensu stricto. [ABSTRACT FROM AUTHOR]

Mycobacterium africanum (MAF) is a common cause of human pulmonary tuberculosis in West Africa. We previously described phenotypic differences between MAF and Mycobacterium tuberculosis (MTB) among 290 patients. In the present analysis, we compared 692 tuberculosis patients infected with the two most common lineages within the (MTB) complex found in the Gambia, namely MAF West African type 2 (39% prevalence) and Euro-American MTB (55% prevalence). We identified additional phenotypic differences between infections with these two organisms. MAF patients were more likely to be older and HIV infected. In addition, they had worse disease on chest X-ray, despite complaining of cough for an equal duration, and were more likely severely malnourished. In this cohort, the prevalence of MAF did not change significantly over a 7-year period. [ABSTRACT FROM AUTHOR]

Background: Currently available tools cannot be used to distinguish between sub-species of the M. tuberculosis complex causing latent tuberculosis (TB) infection. M. africanum causes up to half of TB in West- Africa and its relatively lower progression to disease suggests the presence of a large reservoir of latent infection relative to M. tuberculosis. Methods: We assessed the immunogenicity of the TbD1 region, present in M. africanum and absent from "modern" M. tuberculosis, in an ELISPOT assay using cells from confirmed M. africanum or M. tuberculosis infected TB patients without HIV infection in the Gambia. Results: Antigens from the TbD1 region induced IFNγ responses in only 35% patients and did not discriminate between patients infected with M. africanum vs. M. tuberculosis, while PPD induced universally high responses. Conclusions: Further studies will need to assess other antigens unique to M. africanum that may induce discriminatory immune responses. [ABSTRACT FROM AUTHOR]

Background: Helicobacter pylori, a lifelong and typically asymptomatic infection of the stomach, profoundly alters gastric immune responses, and may benefit the host in protection against other pathogens. We explored the hypothesis that H. pylori contributes to the control of infection with Mycobacterium tuberculosis. Methodology/Principal Findings: We first examined M. tuberculosis-specific IFN-γ and H. pylori antibody responses in 339 healthy Northern Californians undergoing routine tuberculin skin testing. Of 97 subjects (29%) meeting criteria for latent tuberculosis (TB) infection (LTBI), 45 (46%) were H. pylori seropositive. Subjects with LTBI who were H. pylori-seropositive had 1.5-fold higher TB antigen-induced IFN-γ responses (p = 0.04, ANOVA), and a more Th-1 like cytokine profile in peripheral blood mononuclear cells, compared to those who were H. pylori seronegative. To explore an association between H. pylori infection and clinical outcome of TB exposure, we evaluated H. pylori seroprevalence in baseline samples from two high risk TB case-contact cohorts, and from cynomolgus macaques experimentally challenged with M. tuberculosis. Compared to 513 household contacts who did not progress to active disease during a median 24 months follow-up, 120 prevalent TB cases were significantly less likely to be H. pylori infected (AOR: 0.55, 95% CI 0.0.36-0.83, p = 0.005), though seroprevalence was not significantly different from non-progressors in 37 incident TB cases (AOR: 1.35 [95% CI 0.63-2.9] p = 0.44). Cynomolgus macaques with natural H. pylori infection were significantly less likely to progress to TB 6 to 8 months after M. tuberculosis challenge (RR: 0.31 [95% CI 0.12-0.80], p = 0.04). Conclusions/Significance: H. pylori infection may induce bystander effects that modify the risk of active TB in humans and non-human primates. That immunity to TB may be enhanced by exposure to other microbial agents may have important implications for vaccine development and disease control. [ABSTRACT FROM AUTHOR]

Background. There is considerable variability in the outcome of Mycobacterium tuberculosis infection. We hypothesized that Mycobacterium africanum was less likely than M. tuberculosis to transmit and progress to tuberculosis disease. Methods. In a cohort study of patients with tuberculosis and their household contacts in The Gambia, we categorized 1808 HIV-negative tuberculosis contacts according to exposure to M. tuberculosis or M. africanum. Positive skin test results indicated transmission, and development of tuberculosis during 2 years of follow-up indicated progression to disease. Results. Transmission rates were similar, but rates of progression to disease were significantly lower in contacts exposed to M. africanum than in those exposed to M. tuberculosis (1.0% vs. 2.9%; hazard ratio [HR], 3.1 [95% confidence interval {CI}, 1.1- 8.7]). Within M. tuberculosis sensu stricto, contacts exposed to a Beijing family strain were most likely to progress to disease (5.6%; HR relative to M. africanum, 6.7 [95% CI, 2.0 -22]). Conclusions. M. africanum and M. tuberculosis transmit equally well to household contacts, but contacts exposed to M. africanum are less likely to progress to tuberculosis disease than those exposed to M. tuberculosis. The variable rate of progression by lineage suggests that tuberculosis variability matters in clinical settings and should be accounted for in studies evaluating tuberculosis vaccines and treatment regimens for latent tuberculosis infection. [ABSTRACT FROM AUTHOR]

Background: Mycobacterium africanum, a member of the M. tuberculosis complex that is infrequently found outside of western Africa, is the cause of up to half of the tuberculosis cases there.Methods: We genotyped mycobacterial isolates obtained from a study of patients with tuberculosis and their household contacts and compared T cell responses and tuberculin skin test results by infecting genotype.Results: The T cell response to early secreted antigenic target, 6 kDa (ESAT-6), was attenuated in patients with tuberculosis (odds ratio [OR], 0.41 [95% confidence interval {CI}, 0.19-0.89]; P = .024) and household contacts (OR, 0.56 [95% CI, 0.38-0.83]; P = .004) infected with M. africanum, compared with the response in those infected with M. tuberculosis. In these same groups, responses to culture filtrate protein, 10 kDa (CFP-10), were nonsignificantly attenuated (P = .22 and P = .16, respectively), as were tuberculin skin test results (P = .30 and P = .46, respectively). Sequencing of region of difference 1 of M. africanum revealed that Rv3879c is a pseudogene in M. africanum; however, this finding does not provide an obvious mechanism for the attenuated ESAT-6 response.Conclusions: This is the first evidence, to our knowledge, that strain differences affect interferon- gamma -based T cell responses. Our findings highlight the need to test new diagnostic candidates against different strains of mycobacteria. Integrating additional immunologic and genomic comparisons of M. tuberculosis and M. africanum into further studies may provide fundamental insights into the interactions between humans and mycobacteria. [ABSTRACT FROM AUTHOR]

Staphylococcus aureus is one of the most common causes of hospital-acquired infections. At the same time, 25% of healthy persons are symptom-free S. aureus carriers, and they have an increased risk of developing nosocomial S. aureus septicemia. Paradoxically, their prognosis is much better than that of noncarriers. We compared the antibody profiles for carriers and noncarriers toward S. aureus superantigens. In carriers, we found high titers of neutralizing antibodies specific for those superantigens that are expressed by their colonizing strain. The results show that carriage status confers strain-specific humoral immunity, which may contribute to protection during S. aureus septicemia. [ABSTRACT FROM AUTHOR]

Discusses the negative correlation between marijuana use and adherence to antiretroviral therapy (ART). Use of ART for the treatment of HIV infection; Details on the medicine properties of marijuana; Effects of marijuana on adherence to ART.

Globally, the HIV and tuberculosis epidemics are stoking each other, creating a public health crisis of enormous proportions. At the level of individuals, contemporaneous infection with M. tuberculosis and HIV poses great challenges to clinical management. This chapter provides an overview of active and latent tuberculosis treatment in HIV-infected and -uninfected individuals. The discussion focuses on medication issues, including interactions between antitubercular drugs, antiretroviral drugs, and medicines used for opportunistic infections and treatment in the face of comorbidities. Clinical questions specific to coinfection are discussed, including duration and timing initiation of therapy and immune reconstitution. Most of the data presented were generated in industrialized settings and are presented to assist patient management in such settings. However, given the disproportionate amount of TB/HIV in less-developed nations and the increasing availability of antiretroviral therapy in resource-limited settings, the issues presented will become increasingly relevant globally. [ABSTRACT FROM AUTHOR]

Outcomes of treatment of tuberculosis patients with regimens including pretomanid have not yet been systematically reviewed. To appraise existing evidence on efficacy and safety of pretomanid in tuberculosis. Pubmed, clinicaltrials.gov. and Cochrane library. Quantitative studies presenting original data on clinical efficacy or safety of pretomanid. Patients with tuberculosis. Treatment with pretomanid or pretomanid-containing regimens in minimum one study group. Two authors independently extracted data and assessed risk of bias. Data on efficacy (early bactericidal activity, bactericidal activity, end-of-treatment outcomes and acquired resistance) and safety were summarized in tables. Mean differences in efficacy outcomes between regimens across studies were calculated. Eight studies were included; four randomized controlled trials on 2-week early bactericidal activity in rifampicin-susceptible tuberculosis, three trials with randomized rifampicin-susceptible tuberculosis arms and a single rifampicin-resistant tuberculosis arm (two on 8-week bactericidal activity, one on end-of-treatment outcomes), one single-arm trial with end-of-treatment outcomes in highly resistant tuberculosis. Activity of pretomanid–moxifloxacin–pyrazinamide was superior to standard treatment on daily change in colony-forming units at days 0–2, 0–56 and 7–56 and time to culture conversion in rifampicin-susceptible tuberculosis (hazard ratio: 1.7; 95% CI 1.1–2.7), but not at end of treatment in one study. This study was stopped due to serious hepatotoxic adverse events, including three deaths, in 4% (95% CI 2–8) patients on pretomanid–moxifloxacin–pyrazinamide and none in controls. In patients with uncomplicated rifampicin-resistant tuberculosis on pretomanid-moxifloxacin-pyrazinamide treatment, 91% (95% CI 59–100) had favourable end-of-treatment outcomes. In patients with highly resistant tuberculosis, 90% (95% CI 83–95) on pretomanid–bedaquiline–linezolid had favourable outcomes six months after treatment, but linezolid-related toxicity was frequent. No acquired resistance to pretomanid was reported. Evidence suggests an important role for pretomanid in rifampicin-resistant and highly resistant tuberculosis. Trials comparing pretomanid to existing core and companion drugs are needed to further define that role. [ABSTRACT FROM AUTHOR]

Background: Mycobacterium tuberculosis culturing remains the gold standard for laboratory diagnosis of tuberculosis. Tuberculosis remains a great public health problem in developing countries like The Gambia, as most of the methods currently used for bacterial isolation are either time-consuming or costly. Objective: To evaluate the Kudoh swab method in a West African setting in Gambia, with a particular focus on the method's performance when culturing Mycobacterium africanum West Africa 2 (MAF2) isolates. Method: 75 sputum samples were collected in the Greater Banjul Area and decontaminated in parallel with both the standard N-acetyl-L-Cysteine-NaOH (NALC-NaOH) and the Kudoh swab method in the TB diagnostics laboratory in the Medical Research Council Unit The Gambia between 30th December 2017 and 25th February 2018. These samples were subsequently cultured on standard Löwenstein-Jensen and Modified Ogawa media respectively and incubated at 37°C for mycobacterial growth. Spoligotyping was done to determine if the decontamination and culture methods compared could equally detect Mycobacterium tuberculosis, Mycobacterium africanum West Africa 1 and Mycobacterium africanum West Africa 2. Result: Among the 50 smear positives, 35 (70%) were culture-positive with Kudoh and 32 (64%) were culture positive with NALC-NaOH, whilst 7(28%) of the 25 smear negative samples were culture positive with both methods (Table 2). There was no significant difference in recovery between both methods (McNemar's test, p-value = 0.7003), suggesting that the overall positivity rate between the two methods is comparable. There were no differences in time-to-positivity or contamination rate between the methods. However, Kudoh yielded positive cultures that were negative on LJ and vice versa. All findings were irrespective of mycobacterial lineages. Conclusion: The Kudoh method has comparable sensitivity to the NALC-NaOH method for detecting Mycobacterium tuberculosis complex isolates. It is easy to perform and could be an add on option for mycobacterial culture in the field in The Gambia, since it requires less biosafety equipment. [ABSTRACT FROM AUTHOR]

Traditional public health methods for detecting infectious disease transmission, such as contact tracing and molecular epidemiology, are time-consuming and costly. Information and communication technologies, such as global positioning systems, smartphones, and mobile phones, offer opportunities for novel approaches to identifying transmission hotspots. However, mapping the movements of potentially infected persons comes with ethical challenges. During an interdisciplinary meeting of researchers, ethicists, data security specialists, information and communication technology experts, epidemiologists, microbiologists, and others, we arrived at suggestions to mitigate the ethical concerns of movement mapping. These suggestions include a template Data Protection Impact Assessment that follows European Union General Data Protection Regulations. [ABSTRACT FROM AUTHOR]

Background: Leprosy is an ancient infectious disease with an annual global incidence of around 200,000 over the past decade. Since 2018, the World Health Organization (WHO) recommends single-dose rifampicin as post-exposure prophylaxis (SDR-PEP) for contacts of leprosy patients. The Post ExpOsure Prophylaxis for Leprosy (PEOPLE) trial evaluated PEP with a double dose of rifampicin in Comoros and Madagascar. Preliminary results of this trial show some reduction in leprosy incidence in intervention villages but a stronger regimen may be beneficial. The objective of the current Bedaquiline Enhanced ExpOsure Prophylaxis for LEprosy trial (BE-PEOPLE) is to explore effectiveness of a combination of bedaquiline and rifampicin as PEP. Methods: BE-PEOPLE is a cluster-randomized trial in which 44 clusters in Comoros will be randomized to two study arms. Door-to-door screening will be conducted annually during four years, leprosy patients identified will be offered standard of care treatment. Based on study arm, contacts aged five years and above and living within a 100-meter radius of an index case will either receive bedaquiline (400-800 mg) and rifampicin (150-600 mg) or only rifampicin (150–600 mg). Contacts aged two to four years will receive rifampicin only. Household contacts randomized to the bedaquiline plus rifampicin arm will receive a second dose four weeks later. Incidence rate ratios of leprosy comparing contacts who received either of the PEP regimens will be the primary outcome. We will monitor resistance to rifampicin and/or bedaquiline through molecular surveillance in all incident tuberculosis and leprosy patients nationwide. At the end of the study, we will assess anti-M. leprae PGL-I IgM seropositivity as a proxy for the population burden of M. leprae infection in 8 villages (17,000 individuals) that were surveyed earlier as part of the PEOPLE trial. Discussion: The COLEP trial on PEP in Bangladesh documented a reduction of 57% in incidence of leprosy among contacts treated with SDR-PEP after two years, which led to the WHO recommendation of SDR-PEP. Preliminary results of the PEOPLE trial show a lesser reduction in incidence. The BE-PEOPLE trial will explore whether reinforcing SDR-PEP with bedaquiline increases effectiveness and more rapidly reduces the incidence of leprosy, compared to SDR-PEP alone. Trial registration: NCT05597280. Protocol version 5.0 on 28 October 2022. [ABSTRACT FROM AUTHOR]

In settings with high tuberculosis (TB) endemicity, distinct genotypes of the Mycobacterium tuberculosis complex (MTBC) often differ in prevalence. However, the factors leading to these differences remain poorly understood. Here we studied the MTBC population in Dar es Salaam, Tanzania over a six-year period, using 1,082 unique patient-derived MTBC whole-genome sequences (WGS) and associated clinical data. We show that the TB epidemic in Dar es Salaam is dominated by multiple MTBC genotypes introduced to Tanzania from different parts of the world during the last 300 years. The most common MTBC genotypes deriving from these introductions exhibited differences in transmission rates and in the duration of the infectious period, but little differences in overall fitness, as measured by the effective reproductive number. Moreover, measures of disease severity and bacterial load indicated no differences in virulence between these genotypes during active TB. Instead, the combination of an early introduction and a high transmission rate accounted for the high prevalence of L3.1.1, the most dominant MTBC genotype in this setting. Yet, a longer co-existence with the host population did not always result in a higher transmission rate, suggesting that distinct life-history traits have evolved in the different MTBC genotypes. Taken together, our results point to bacterial factors as important determinants of the TB epidemic in Dar es Salaam. Author summary: Tuberculosis (TB) is among the deadliest human infectious diseases caused by one single agent, Mycobacterium tuberculosis (Mtb). The origins of Mtb have been traced to East Africa millennia ago, where it likely became adapted to infect and transmit in humans. Here, we show that in Dar es Salaam, Tanzania, an East African setting with a high burden of TB, infections are caused by distinct Mtb genotypes introduced in recent evolutionary times from different parts of the world. These genotypes differed in traits important to Mtb transmission; while some Mtb genotypes transmitted more efficiently during a given period of time, patients infected by other genotypes remained infectious for longer. These traits evolved independently in the different Mtb genotypes and could not be explained by the time of co-existence between the host population and the pathogen. This suggests that bacterial factors are important determinants of the TB epidemic. More generally, we demonstrate that distinct pathogenic life history characteristics can co-exist in one host population. [ABSTRACT FROM AUTHOR]

Background: In India, leprosy clusters at hamlet level but detailed information is lacking. We aim to identify high-incidence hamlets to be targeted for active screening and post-exposure prophylaxis. Methodology: We paid home visits to a cohort of leprosy patients registered between April 1st, 2020, and March 31st, 2022. Patients were interviewed and household members were screened for leprosy. We used an open-source app(ODK) to collect data on patients' mobility, screening results of household members, and geographic coordinates of their households. Clustering was analysed with Kulldorff's spatial scan statistic(SaTScan). Outlines of hamlets and population estimates were obtained through an open-source high-resolution population density map(https://data.humdata.org), using kernel density estimation in QGIS, an open-source software. Results: We enrolled 169 patients and screened 1,044 household contacts in Bisfi and Benipatti blocks of Bihar. Median number of years of residing in the village was 17, interquartile range(IQR)12-30. There were 11 new leprosy cases among 658 household contacts examined(167 per 10,000), of which seven had paucibacillary leprosy, one was a child under 14 years, and none had visible disabilities. We identified 739 hamlets with a total population of 802,788(median 163, IQR 65–774). There were five high incidence clusters including 12% of the population and 46%(78/169) of the leprosy cases. One highly significant cluster with a relative risk (RR) of 4.7(p<0.0001) included 32 hamlets and 27 cases in 33,609 population. A second highly significant cluster included 32 hamlets and 24 cases in 33,809 population with a RR of 4.1(p<0.001). The third highly significant cluster included 16 hamlets and 17 cases in 19,659 population with a RR of 4.8(p<0.001). High-risk clusters still need to be screened door-to-door. Conclusions: We found a high yield of active household contact screening. Our tools for identifying high-incidence hamlets appear effective. Focusing labour-intensive interventions such as door-to-door screening on such hamlets could increase efficiency. Author summary: India is the highest-burden country in the world where leprosy is known to cluster in hamlets. As no geographical data about hamlets is available, we develop a system to outline them using a high-resolution population density map. Then, using the household coordinates of 169 new leprosy cases enrolled in two hyperendemic blocks of Bihar, we screened household contacts and assessed clustering at hamlet level. The patients interviewed had lived in their current villages for a median of seventeen years at the moment of the survey. We found 11 new cases among 658 contacts examined equivalent to 16.7 per 1,000 population. There were three statistically significant clusters among five at the hamlet level and three including 78 cases in 98,623 population. Our results can be used to guide targeted and more efficient active case finding and post-exposure prophylaxis. [ABSTRACT FROM AUTHOR]

Cavaliero and colleagues [[1]] describe a high yield of retrospective active case finding for leprosy in Cambodia, which has a fairly low level of endemicity. On Anjouan, in addition to mini campaigns, household contacts of leprosy patients diagnosed are either visited in their homes by nurses trained in leprosy or invited to present to Primary Health Care (PHC) facilities for leprosy screening. [Extracted from the article]

• We report leprosy prevalence and clustering from an extensive population dataset. • Of 455 leprosy patients, 44% belonged to high prevalent clusters. • In new leprosy patients, 38% resided ≤25 m of another leprosy patient. • Leprosy risk decreased progressively with distance to the nearest leprosy patient. • Risk ratios varied from 7.3 in residents at the same household and 1.7 at 75–<100 m. To identify patterns of spatial clustering of leprosy. We performed a baseline survey for a trial on post-exposure prophylaxis for leprosy in Comoros and Madagascar. We screened 64 villages, door-to-door, and recorded results of screening, demographic data and geographic coordinates. To identify clusters, we fitted a purely spatial Poisson model using Kulldorff's spatial scan statistic. We used a regular Poisson model to assess the risk of contracting leprosy at the individual level as a function of distance to the nearest known leprosy patient. We identified 455 leprosy patients; 200 (44.0%) belonged to 2735 households included in a cluster. Thirty-eight percent of leprosy patients versus 10% of the total population live ≤25 m from another leprosy patient. Risk ratios for being diagnosed with leprosy were 7.3, 2.4, 1.8, 1.4 and 1.7, for those at the same household, at 1–<25 m, 25–<50 m, 50–<75 m and 75–<100 m as/from a leprosy patient, respectively, compared to those living at ≥100 m. We documented significant clustering of leprosy beyond household level, although 56% of cases were not part of a cluster. Control measures need to be extended beyond the household, and social networks should be further explored. [ABSTRACT FROM AUTHOR]

In its 2020 guidelines for the treatment of rifampicin-resistant TB (RR-TB), the WHO recommends all-oral fluoroquinolone-based regimens, with bedaquiline replacing the second-line injectable drugs (SLIDs). SLIDs were used for their strong acquired resistance-preventing activity. Data from three cohorts showed acquired bedaquiline resistance ranging between 2.5% and 30.8%, with no protection from a SLID in most cases. If bedaquiline resistance is that easily acquired, it will fail to protect fluoroquinolones and other drugs from acquiring resistance. Until evidence on resistance-preventing activity shows that SLIDs can safely be replaced, we call for more prudent use of the few potent second-line TB drugs available. Studies on new treatment regimens need to prioritize the prevention of acquired resistance along with treatment success. Meanwhile, reducing the dosing of SLIDs to thrice weekly from Day 1, and their replacement for any degree of audiometry abnormalities before or during treatment will largely avoid serious ototoxicity. [ABSTRACT FROM AUTHOR]

• Northern Ethiopia hosts a wide diversity of Mycobacterium tuberculosis complex (MTBc) lineages: L1–L4 and L7. • East-African-Indian (L3) is the most frequent lineage, followed by Euro-American (L4). • MTBc population structure was similar in genotyping of sputa and cultured isolates. • Most MTBc strains (94.5%) were susceptible to both rifampicin and isoniazid. This study described the population structure of M. tuberculosis complex (MTBc) strains among patients with pulmonary or lymph node tuberculosis (TB) in Northwest Ethiopia and tested the performance of culture isolation and MPT64-based speciation for Lineage 7 (L7). Patients were recruited between April 2017 and June 2019 in North Gondar, Ethiopia. The MPT64 assay was used to confirm MTBc, and spoligotyping was used to characterize mycobacterial lineages. Line probe assay (LPA) was used to detect resistance to rifampicin and isoniazid. Among 274 MTBc genotyped isolates, there were five MTBc lineages: L1–L4 and L7 were identified, with predominant East-African-Indian (L3) (53.6%) and Euro-American (L4) (40.1%) strains, and low prevalence (2.6%) of Ethiopia L7. The genotypes were similarly distributed between pulmonary and lymph node TB, and all lineages were equally isolated by culture and recognized as MTBc by the MPT64 assay. Additionally, LPA showed that 259 (94.5%) MTBc were susceptible to both rifampicin and isoniazid, and one (0.4%) was multi-drug resistant (resistant to both rifampicin and isoniazid). These findings show that TB in North Gondar, Ethiopia, is mainly caused by L3 and L4 strains, with low rates of L7, confirmed as MTBc by MPT64 assay and with limited resistance to rifampicin and isoniazid. [ABSTRACT FROM AUTHOR]

• Shorter treatment regimen for multidrug-resistant tuberculosis. • Effect of companion drug resistance on adverse outcomes. • Gatifloxacin and isoniazid minimum inhibitory concentration. • Whole-genome sequencing. • Non-eligibility for the regimen not associated with adverse outcome. • Exclusions justified only in the case of fluoroquinolone or kanamycin resistance. We investigated whether companion drug resistance was associated with adverse outcomes of the shorter multidrug-resistant tuberculosis (MDR-TB) treatment regimen in Bangladesh after adjustment for fluoroquinolone resistance. MDR-TB/rifampicin-resistant tuberculosis patients registered for treatment with a standardized gatifloxacin-based shorter MDR-TB treatment regimen were selected for the study. Drug resistance was determined by the proportion method, gatifloxacin and isoniazid minimum inhibitory concentration testing for selected isolates, and whole-genome sequencing. Low-level fluoroquinolone resistance and high-level fluoroquinolone resistance were the most important predictors of adverse outcomes, with pyrazinamide resistance having a significant yet lower impact. In patients with fluoroquinolone-/second-line-injectable-susceptible tuberculosis, non-eligibility for the shorter MDR-TB treatment regimen (initial resistance to pyrazinamide, ethionamide, or ethambutol) was not associated with adverse outcome (adjusted odds ratio 1.01; 95% confidence interval 0.4–2.8). Kanamycin resistance was uncommon (1.3%). Increasing levels of resistance to isoniazid predicted treatment failure, also in a subgroup of patients with high-level fluoroquinolone-resistant tuberculosis. Our results suggest that resistance to companion drugs in the shorter MDR-TB treatment regimen, except kanamycin resistance, is of no clinical importance as long as fluoroquinolone susceptibility is preserved. Hence, contrary to current WHO guidelines, exclusions to the standard regimen are justified only in the case of fluoroquinolone resistance. and possibly kanamycin resistance. [ABSTRACT FROM AUTHOR]

Buruli ulcer (BU) is an emerging ulcerative skin disease caused by infection with Mycobacterium ulcerans. Efforts to control its spread have been hampered by our limited understanding of M. ulcerans reservoirs and transmission, and the factors leading to the emergence of BU disease in a particular region. In this report we investigate an anecdotal link between damming the Mapé River in Cameroon and the emergence of BU in the Health Districts bordering Lake Bankim, the impoundment created by the Mapé dam. We used bacterial population genomics and molecular dating to find compelling support for a 2000 M. ulcerans introduction event that followed about 10 years after the filling of the newly created impoundment in 1988. We compared the genomic reconstructions with high-resolution satellite imagery to investigate what major environmental alterations might have driven the emergence of the new focus. Author summary: Buruli ulcer is a destructive skin and soft tissue infection caused by Mycobacterium ulcerans. Although the disease is not fatal, the infection can often leave patients with significant cosmetic and functional damage to limbs. Currently, one of the major hurdles facing Buruli ulcer control is our incomplete understanding of the factors leading to the emergence of disease in a particular region. In this report we investigate an anecdotal link between the damming of the Mapé River in Cameroon and the emergence of Buruli ulcer in the Health Districts bordering the impoundment created by the Mapé dam. We compare the genome sequences of M. ulcerans isolates recovered from regional Buruli ulcer patients that were identified in a previous molecular epidemiology study. Additionally, we investigate historic satellite imagery to quantify changes in land cover use that followed damming the river. The appearance of Buruli ulcer in the region was found to follow about ten years after the 1988 damming of the Mapé River, supporting the idea that alterations to landscape hydrology can increase BU incidence. While this temporal association does not infer causation, this research helps define the ecological risk factors linked to the spread of BU. [ABSTRACT FROM AUTHOR]

Background: Since a meta-analysis showed little or no effect of second-line injectables on treatment success, and using injectables may induce ototoxicity, injectable-free rifampicin-resistant tuberculosis (RR-TB) treatment regimens are recommended. However, acquired resistance preventing activity was overlooked. No previous study assessed the effect of shortening the duration of kanamycin administration to 2 months during the intensive phase of the RR-TB shorter treatment regimen (STR). Methods: Retrospective cohort study of the effect of using 2 months of kanamycin instead of the standard 4(+) months (extension if lack of smear conversion at 4 months) on recurrence (either treatment failure or relapse) and fluoroquinolone acquired drug resistance, in patients treated with a gatifloxacin-based STR in Damien Foundation supported clinics in Bangladesh. Logistic regression was used to estimate associations. Results: Five of 52 (9.6%) treated with a STR containing two months of kanamycin had recurrence, compared to 21 of 738 (2.8%) patients treated with the standard STR containing 4(+) months of kanamycin (OR 3.7; 95%CI:1.5–10.3). In those with initially fluoroquinolone-susceptible TB, acquired resistance to fluoroquinolone was detected in none of 639 patients treated with 4(+) months of kanamycin and two (4.5%) of 44 treated with two months of kanamycin (OR 75.2; 95%CI:3.6–1592.1). Conclusion: Two months of kanamycin was insufficient to prevent recurrence with acquired resistance to gatifloxacin, the core drug of the most effective RR-TB STR. Injectable mediated resistance prevention is important to reach high effectiveness, to safeguard all treatment options after recurrence, and to prevent the spread of resistant TB. Studies on all-oral regimens should also assess the effect of regimen composition on resistance acquisition. Until evidence shows that other drugs can assure at least the same strong resistance preventing activity of the injectables, it seems wise to continue using this group of drugs, and adapt the regimen if any ototoxicity is detected. [ABSTRACT FROM AUTHOR]

The surveillance of drug resistance among tuberculosis (TB) patients is central to preventing the spread of antimicrobial resistance. The Democratic Republic of the Congo (DR Congo) is classified by the World Health Organization (WHO) as a country with a high burden of TB and multidrug-resistant TB (MDR-TB), but there are no nationally representative data on drug resistance. In 2016–2017, a national survey of TB patients was conducted in 108 microscopy centres across all 11 provinces of the country using innovative molecular approaches. Sputum samples were collected from 1,545 new and 163 previously treated patients. These were tested by the Xpert MTB/RIF assay, followed by targeted next-generation sequencing performed directly on sputum. The prevalence of rifampicin resistance was low, at 1.8% (95% CI: 1.0–3.2) among new and 17.3% (95% CI: 11.9–24.4) among previously treated patients. Resistance to pyrazinamide, fluoroquinolones and second-line injectables was also low. The prevalence of resistance to isoniazid among rifampicin-susceptible patients was higher, at 6.6% (95% CI: 4.4–9.8) among new and 8.7% (95% : 3.2–21.2) among previously treated patients. Diagnosing and treating isoniazid-resistant patients remains a challenge, given that many will be missed by the current national diagnostic algorithm that is driven by detecting rifampicin resistance by Xpert MTB/RIF. This is the first nationwide survey incorporating targeted sequencing directly on sputum. It serves as a proof-of-concept for other settings that do yet have rapid specimen transport networks or capacity to conduct culture. [ABSTRACT FROM AUTHOR]

Background: Meta-analyses on impact of isoniazid-resistant tuberculosis informed the World Health Organization recommendation of a levofloxacin-strengthened rifampicin-based regimen. We estimated the effect of initial rifampicin resistance (Rr) and/or isoniazid resistance (Hr) on treatment failure or relapse. We also determined the frequency of missed initial and acquired Rr to estimate the impact of true Hr. Methods: Retrospective analysis of 7291 treatment episodes with known initial isoniazid and rifampicin status obtained from individual patient databases maintained by the Damien Foundation Bangladesh over 20 years. Drug susceptibility test results were confirmed by the programme's designated supra-national tuberculosis laboratory. To detect missed Rr among isolates routinely classified as Hr, rpoB gene sequencing was done randomly and on a sample selected for suspected missed Rr. Results: Initial Hr caused a large recurrence excess after the 8-month regimen for new cases (rifampicin for two months), but had little impact on rifampicin-throughout regimens: (6 months, new cases; 3.8%; OR 0.8, 95%CI:0.3,2.8; 8 months, retreatment cases: 7.3%, OR 1.8; 95%CI:1.3,2.6). Rr was missed in 7.6% of randomly selected "Hr" strains. Acquired Rr was frequent among recurrences on rifampicin-throughout regimens, particularly after the retreatment regimen (31.9%). It was higher in mono-Hr (29.3%; aOR 3.5, 95%CI:1.5,8.5) and poly-Hr (53.3%; aOR 10.2, 95%CI 4.4,23.7) than in susceptible tuberculosis, but virtually absent after the 8-month new case regimen. Comparing Bangladesh (low Rr prevalence) with a high Rr prevalence setting,true Hr corrected for missed Rr caused only 2–3 treatment failures per 1000 TB cases (of whom 27% were retreatments) in both. Conclusions: Our analysis reveals a non-negligible extent of misclassifying as isoniazid resistance of what is actually missed multidrug-resistant tuberculosis. Recommending for such cases a "strengthened" regimen containing a fluoroquinolone provokes a direct route to extensive resistance while offering little benefit against the minor role of true Hr tuberculosis in rifampicin-throughout first-line regimen. [ABSTRACT FROM AUTHOR]

In recent years Bamako has been faced with an emerging threat from multidrug resistant TB (MDR-TB). Whole genome sequence analysis was performed on a subset of 76 isolates from a total of 208 isolates recovered from tuberculosis patients in Bamako, Mali between 2006 and 2012. Among the 76 patients, 61(80.3%) new cases and 15(19.7%) retreatment cases, 12 (16%) were infected by MDR-TB. The dominant lineage was the Euro-American lineage, Lineage 4. Within Lineage 4, the Cameroon genotype was the most prevalent genotype (n = 20, 26%), followed by the Ghana genotype (n = 16, 21%). A sub-clade of the Cameroon genotype, which emerged ~22 years ago was likely to be involved in community transmission. A sub-clade of the Ghana genotype that arose approximately 30 years ago was an important cause of MDR-TB in Bamako. The Ghana genotype isolates appeared more likely to be MDR than other genotypes after controlling for treatment history. We identified a clade of four related Beijing isolates that included one MDR-TB isolate. It is a major concern to find the Cameroon and Ghana genotypes involved in community transmission and MDR-TB respectively. The presence of the Beijing genotype in Bamako remains worrying, given its high transmissibility and virulence. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

After an alert regarding ≈31 tuberculosis (TB) cases, 3 of which were rifampin-resistant TB cases, in Mbuji-Mayi Central Prison, Democratic Republic of the Congo, we conducted an outbreak investigation in January 2015. We analyzed sputum of presumptive TB patients by using the Xpert MTB/RIF assay. We also assessed the Mycobacterium tuberculosis isolates' drug-susceptibility patterns and risk factors for TB infection. Among a prison population of 918 inmates, 29 TB case-patients were already undergoing treatment. We found an additional 475 presumptive TB case-patients and confirmed TB in 170 of them. In March 2015, the prevalence rate of confirmed TB was 21.7% (199/918 inmates). We detected an additional 14 cases of rifampin-resistant TB and initiated treatment in all 14 of these case-patients. Overcrowded living conditions and poor nutrition appeared to be the driving factors behind the high TB incidence in this prison. [ABSTRACT FROM AUTHOR]

Background: Buruli ulcer (BU) is a chronic necrotizing infectious skin disease caused by Mycobacterium ulcerans. The treatment with BU-specific antibiotics is initiated after clinical suspicion based on the WHO clinical and epidemiological criteria. This study aimed to estimate the predictive values of these criteria and how they could be improved. Methodology/Principal findings: A total of 224 consecutive patients presenting with skin and soft tissue lesions that could be compatible with BU, including those recognized as unlikely BU by experienced clinicians, were recruited in two BU treatment centers in southern Benin between March 2012 and March 2015. For every participant, the WHO and four additional epidemiological and clinical diagnostic criteria were recorded. For microbiological confirmation, direct smear examination and IS2404 PCR were performed. We fitted a logistic regression model with PCR positivity for BU confirmation as outcome variable. On univariate analysis, most of the clinical and epidemiological WHO criteria were associated with a positive PCR result. However, lesions on the lower limbs and WHO category 3 lesions were rather associated with a negative PCR result (respectively OR: 0.4, 95%CI: 0.3–0.8; OR: 0.5, 95%IC: 0.3–0.9). Among the additional characteristics studied, the characteristic smell of BU was strongest associated with a positive PCR result (OR = 16.4; 95%CI = 7.5–35.6). Conclusion/Significance: The WHO diagnostic criteria could be improved upon by differentiating between lesions on the upper and lower limbs and by including lesion size and the characteristic smell recognized by experienced clinicians. [ABSTRACT FROM AUTHOR]

Since 2000, cases of the neglected tropical disease Buruli ulcer, caused by infection with Mycobacterium ulcerans, have increased 100-fold around Melbourne (population 4.4 million), the capital of Victoria, in temperate southeastern Australia. The reasons for this increase are unclear. Here, we used whole-genome sequence comparisons of 178 M. ulcerans isolates obtained primarily from human clinical specimens, spanning 70 years, to model the population dynamics of this pathogen from this region. Using phylogeographic and advanced Bayesian phylogenetic approaches, we found that there has been a migration of the pathogen from the east end of the state, beginning in the 1980s, 300 km west to the major human population center around Melbourne. This move was then followed by a signifi- cant increase in M. ulcerans population size. These analyses inform our thinking around Buruli ulcer transmission and control, indicating that M. ulcerans is introduced to a new environment and then expands, rather than it being from the awakening of a quiescent pathogen reservoir. IMPORTANCE Buruli ulcer is a destructive skin and soft tissue infection caused by Mycobacterium ulcerans and is characterized by progressive skin ulceration, which can lead to permanent disfigurement and long-term disability. Despite the majority of disease burden occurring in regions of West and central Africa, Buruli ulcer is also becoming increasingly common in southeastern Australia. Major impediments to controlling disease spread are incomplete understandings of the environmental reservoirs and modes of transmission of M. ulcerans. The significance of our research is that we used genomics to assess the population structure of this pathogen at the Australian continental scale. We have then reconstructed a historical bacterial spread and modeled demographic dynamics to reveal bacterial population expansion across southeastern Australia. These findings provide explanations for the observed epidemiological trends with Buruli ulcer and suggest possible management to control disease spread. [ABSTRACT FROM AUTHOR]

Objective: Paragonimiasis is a foodborne trematode infection of the lungs caused by Paragonimus spp., presenting clinically with similar symptoms to active tuberculosis (TB). Worldwide, an estimated 20.7 million people are infected with paragonimiasis, but relatively little epidemiological data exists for Africa. Given a recently reported case, we sought to establish whether paragonimiasis should be considered as an important differential diagnosis for human TB in The Gambia, West Africa. Results: We developed a novel PCR-based diagnostic test for Paragonimus species known to be found in West Africa, which we used to examine archived TB negative sputum samples from a cross-sectional study of volunteers with tuberculosis-like symptoms from communities in the Western coastal region of The Gambia. Based on a "zero patient" design for detection of rare diseases, 300 anonymised AFB smear negative sputum samples, randomly selected from 25 villages, were screened for active paragonimiasis by molecular detection of Paragonimus spp. DNA. No parasite DNA was found in any of the sputa of our patient group. Despite the recent case report, we found no evidence of active paragonimiasis infection masking as TB in the Western region of The Gambia. [ABSTRACT FROM AUTHOR]

Background. Basidiobolomycosis is a rare subcutaneous mycosis, which can be mistaken for several other diseases, such as soft tissue tumors, lymphoma, or Buruli ulcer in the preulcerative stage. Microbiological confirmation by PCR for Basidiobolus ranarum and culture yield the most specific diagnosis, yet they are not widely available in endemic areas and with varying sensitivity. A combination of histopathological findings, namely, granulomatous inflammation with giant cells, septate hyphal fragments, and the Splendore-Hoeppli phenomenon, can confirm basidiobolomycosis in patients presenting with painless, hard induration of soft tissue. Case Presentations. We report on three patients misdiagnosed as suffering from Buruli ulcer, who did not respond to Buruli treatment. Histopathological review of the tissue sections from these patients suggests basidiobolomycosis. All patients had been lost to follow-up, and none received antifungal therapy. On visiting the patients at their homes, two were reported to have died of unknown causes. The third patient was found alive and well and had experienced local spontaneous healing. Conclusion. Basidiobolomycosis is a rare subcutaneous fungal disease mimicking preulcerative Buruli ulcer. We stress the importance of the early recognition by clinicians and pathologists of this treatable disease, so patients can timely receive antifungal therapy. [ABSTRACT FROM AUTHOR]

Background: Drug resistant tuberculosis presents a major public health challenge. Case presentation: We present here the first two patients diagnosed with extensively drug resistant tuberculosis in Bamako, Mali. Genotypic findings suggest possible nosocomial transmission from the first patient to the second one, resulting in superinfection of the second patient. After being diagnosed with extensively drug resistant tuberculosis in August 2016, the patients only started receiving appropriate treatment 10 months later. Conclusion: The identification of these patients highlights the need for improved diagnostic and treatment algorithms for better surveillance and management of drug resistance in Mali. In the interest of these as well as future patients suffering from resistant tuberculosis, all steps recommended for programmatic management of drug resistant tuberculosis must be urgently prioritized in order to strengthen the multidrug resistant tuberculosis program. [ABSTRACT FROM AUTHOR]

Background Ethiopia has an extremely high rate of extrapulmonary tuberculosis, dominated by tuberculous lymphadenitis (TBLN). However, little is known about Mycobacterium tuberculosis complex (MTBc) lineages responsible for TBLN in Southwest Ethiopia. Methods A total of 304 MTBc isolates from TBLN patients in Southwest Ethiopia were genotyped primarily by spoligotyping. Isolates of selected spoligotypes were further analyzed by 15-loci mycobacterial interspersed repetitive unit–variable number tandem repeat (MIRU-VNTR) (n = 167) and qPCR-based single nucleotide polymorphism (n = 38). Isolates were classified into main phylogenetic lineages and families by using the reference strain collections and identification tools available at MIRU - VNTRplus data base. Resistance to rifampicin was determined by Xpert MTB/RIF. Results The majority of isolates (248; 81.6%) belonged to the Euro-American lineage (Lineage 4), with the ill-defined T and Haarlem as largest families comprising 116 (38.2%) and 43 (14.1%) isolates respectively. Of the T family, 108 isolates were classified as being part of the newly described Ethiopian families, namely Ethiopia_2 (n = 44), Ethiopia_3 (n = 34) and Ethiopia_H 37 Rv-like (n = 30). Other sub-lineages included URAL (n = 18), S (n = 17), Uganda I (n = 16), LAM (n = 13), X (n = 5), TUR (n = 5), Uganda II (n = 4) and unknown (n = 19). Lineage 3 (Delhi/CAS) was the second most common lineage comprising 44 (14.5%) isolates. Interestingly, six isolates (2%) were belonged to Lineage 7, unique to Ethiopia. Lineage 1 (East-African Indian) and Lineage 2 (Beijing) were represented by 3 and 1 isolates respectively. M. bovis was identified in only two (0.7%) TBLN cases. The cluster rate was highest for Ethiopia_3 isolates showing clonal similarity with isolates from North Ethiopia. Lineage 3 was significantly associated with rifampicin resistance. Conclusions In TBLN in Southwest Ethiopia, the recently described Ethiopia specific Lineage 4 families were predominant, followed by Lineage 3 and Lineage 4-Haarlem. The contribution of M. bovis in TBLN infection is minimal. [ABSTRACT FROM AUTHOR]

Background: Emergence of Multidrug-resistant (MDR) strains constitutes a significant public health problem worldwide. Prevalence of MDR tuberculosis from Chad is unavailable to date.Methods: We collected samples from consecutive TB patients nationwide in the seven major cities of Chad between 2007 and 2012 to characterize drug resistance and the population structure of circulating Mycobacterium tuberculosis complex (MTBC) strains. We tested drug sensitivity using Line Probe Assays and phenotypic drug susceptibility testing (DST) were used for second line drugs. We genotyped the isolates using spoligotype analysis and MIRU-VNTR.Results: A total of 311 cultures were isolated from 593 patients. The MDR prevalence was 0.9% among new patients and 3.5% among retreatment patients, and no second line drug resistance was identified. The distribution of genotypes suggests a dissemination of MDR strains in the Southern city of Moundou, bordering Cameroon and Central African Republic.Conclusion: Emerging MDR isolates pose a public health threat to Southern Chad, with risk to neighboring countries. This study informs public health practitioners, justifying the implementation of continuous surveillance with DST for all retreatment cases as well as contacts of MDR patients, in parallel with provision of adequate 2nd line regimens in the region. [ABSTRACT FROM AUTHOR]

Background: Buruli ulcer (BU) is an infectious disease caused by Mycobacterium ulcerans and considered the third most prevalent mycobacterial disease in humans. Secondary bacterial infections in open BU lesions are the main cause of pain, delayed healing and systemic illness, resulting in prolonged hospital stay. Thus, understanding the diversity of bacteria, termed the microbiome, in these open lesions is important for proper treatment. However, adequately studying the human microbiome in a clinical setting can prove difficult when investigating a neglected tropical skin disease due to its rarity and the setting. Methodology/Principal findings: Using 16S rRNA sequencing, we determined the microbial composition of 5 BU lesions, 3 non-BU lesions and 3 healthy skin samples. Although no significant differences in diversity were found between BU and non-BU lesions, the former were characterized by an increase of Bacteroidetes compared to the non-BU wounds and the BU lesions also contained significantly more obligate anaerobes. With this molecular-based study, we were also able to detect bacteria that were missed by culture-based methods in previous BU studies. Conclusions/Significance: Our study suggests that BU may lead to changes in the skin bacterial community within the lesions. However, in order to determine if such changes hold true across all BU cases and are either a cause or consequence of a specific wound environment, further microbiome studies are necessary. Such skin microbiome analysis requires large sample sizes and lesions from the same body site in many patients, both of which can be difficult for a rare disease. Our study proposes a pipeline for such studies and highlights several drawbacks that must be considered if microbiome analysis is to be utilized for neglected tropical diseases. [ABSTRACT FROM AUTHOR]

Buruli ulcer (BU) is an insidious neglected tropical disease. Cases are reported around the world but the rural regions of West and Central Africa are most affected. How BU is transmitted and spreads has remained a mystery, even though the causative agent, Mycobacterium ulcerans, has been known for more than 70 years. Here, using the tools of population genomics, we reconstruct the evolutionary history of M. ulcerans by comparing 165 isolates spanning 48 years and representing 11 endemic countries across Africa. The genetic diversity of African M. ulcerans was found to be restricted due to the bacterium's slow substitution rate coupled with its relatively recent origin. We identified two specific M. ulcerans lineages within the African continent, and inferred that M. ulcerans lineage Mu_A1 existed in Africa for several hundreds of years, unlike lineage Mu_A2, which was introduced much more recently, approximately during the 19th century. Additionally, we observed that specific M. ulcerans epidemic Mu_A1 clones were introduced during the same time period in the three hydrological basins that were well covered in our panel. The estimated time span of the introduction events coincides with the Neo-imperialism period, during which time the European colonial powers divided the African continent among themselves. Using this temporal association, and in the absence of a known BU reservoir or -vector on the continent, we postulate that the so-called "Scramble for Africa" played a significant role in the spread of the disease across the continent. [ABSTRACT FROM AUTHOR]

Introduction: Early diagnosis and initiation to appropriate treatment is vital for tuberculosis (TB) control. The XpertMTB/RIF (Xpert) assay offers rapid TB diagnosis and quantitative estimation of bacterial burden through Cycle threshold (Ct) values. We assessed whether the Xpert Ct value is associated with delayed TB diagnosis as a potential monitoring tool for TB control programme performance. Materials and Methods: This analysis was nested in a prospective study under the routine TB surveillance procedures of the National TB Control Program in Manhiça district, Maputo province, Mozambique. Presumptive TB patients were tested using smear microscopy and Xpert. We explored the association between Xpert Ct values and self-reported delay of Xpert-positive TB patients as recorded at the time of diagnosis enrolment. Patients with >60 days of TB symptoms were considered to have long delays. Results: Of 1,483 presumptive TB cases, 580 were diagnosed as TB of whom 505 (87.0%) were due to pulmonary TB and 302 (94.1%) were Xpert positive. Ct values (range, 9.7–46.4) showed a multimodal distribution. The median (IQR) delay was 30 (30–45) days. Ct values showed no correlation with delay (R2 = 0.001, p = 0.621), nor any association with long delays: adjusted odds ratios (AOR) (95% confidence interval [CI]) comparing to >28 cycles 0.99 (0.50–1.96; p = 0.987) for 23–28 cycles, 0.93 (0.50–1.74; p = 0.828) for 16–22 cycles; and 1.05 (0.47–2.36; p = 0.897) for <16 cycles. Being HIV-negative (AOR [95% CI]), 2.05 (1.19–3.51, p = 0.009) and rural residence 1.74 (1.08–2.81, p = 0.023), were independent predictors of long delays. Conclusion: Xpert Ct values were not associated with patient delay for TB diagnosis and cannot be used as an indicator of TB control program performance. [ABSTRACT FROM AUTHOR]

Background: MPT64 rapid speciation tests are increasingly being used in diagnosis of tuberculosis (TB). Mycobacterium africanum West Africa 2 (Maf 2) remains an important cause of TB in West Africa and causes one third of disease in The Gambia. Since the introduction of MPT64 antigen tests, a higher than expected rate of suspected non-tuberculous mycobacteria (NTM) was seen among AFB smear positive TB suspects, which led us to prospectively assess sensitivity of the MPT64 antigen test in our setting. Methodology/Principal Findings: We compared the abundance of mRNA encoded by the mpt64 gene in sputa of patients with untreated pulmonary TB caused by Maf 2 and Mycobacterium tuberculosis (Mtb). Subsequently, prospectively collected sputum samples from presumptive TB patients were inoculated in the BACTEC MGIT 960 System. One hundred and seventy-three acid fast bacilli (AFB)-positive and blood agar negative MGIT cultures were included in the study. Cultures were tested on the day of MGIT positivity with the BD MGIT TBc Identification Test. A random set of positives and all negatives were additionally tested with the SD Bioline Ag MPT64 Rapid. MPT64 negative cultures were further incubated at 37°C and retested until positive. Bacteria were spoligotyped and assigned to different lineages. Maf 2 isolates were 2.52-fold less likely to produce a positive test result and sensitivity ranged from 78.4% to 84.3% at the beginning and end of the recommended 10 day testing window, respectively. There was no significant difference between the tests. We further showed that the decreased rapid test sensitivity was attributable to variations in mycobacterial growth behavior and the smear grades of the patient. Conclusions/Significance: In areas where Maf 2 is endemic MPT64 tests should be cautiously used and MPT64 negative results confirmed by a second technique, such as nucleic acid amplification tests, to avoid their misclassification as NTMs. [ABSTRACT FROM AUTHOR]

Understanding the circulating Mycobacterium tuberculosis resistance mutations is vital for better TB control strategies, especially to inform a new MDR-TB treatment programme. We complemented the phenotypic drug susceptibility testing (DST) based drug resistance surveys (DRSs) conducted in Uganda between 2008 and 2011 with Whole Genome Sequencing (WGS) of 90 Mycobacterium tuberculosis isolates phenotypically resistant to rifampicin and/or isoniazid to better understand the extent of drug resistance. A total of 31 (34.4 %) patients had MDR-TB, 5 (5.6 %) mono-rifampicin resistance and 54 (60.0 %) mono-isoniazid resistance by phenotypic DST. Pyrazinamide resistance mutations were identified in 32.3% of the MDR-TB patients. Resistance to injectable agents was detected in 4/90 (4.4%), and none to fluoroquinolones or novel drugs. Compensatory mutations in rpoC were identified in two patients. The sensitivity and specificity of drug resistance mutations compared to phenotypic DST were for rpoB 88.6% and 98.1%, katG 60.0% and 100%, fabG1 16.5% and 100%, katG and/or fabG1 71.8% and 100%, embCAB 63.0% and 82.5%, rrs 11.4% and 100%, rpsL 20.5% and 95.7% and rrs and/or rpsL 31.8% and 95.7%. Phylogenetic analysis showed dispersed MDR-TB isolate, with only one cluster of three Beijing family from South West Uganda. Among tuberculosis patients in Uganda, resistance beyond first-line drugs as well as compensatory mutations remain low, and MDR-TB isolates did not arise from a dominant clone. Our findings show the potential use of sequencing for complementing DRSs or surveillance in this setting, with good specificity compared to phenotypic DST. The reported high confidence mutations can be included in molecular assays, and population-based studies can track transmission of MDR-TB including the Beijing family strains in the South West of the country. [ABSTRACT FROM AUTHOR]

Objective To estimate the population prevalence of active pulmonary tuberculosis in Gambia. Methods Between December 2011 and January 2013, people aged ≥ 15 years participating in a nationwide, multistage cluster survey were screened for active pulmonary tuberculosis with chest radiography and for tuberculosis symptoms. For diagnostic confirmation, sputum samples were collected from those whose screening were positive and subjected to fluorescence microscopy and liquid tuberculosis cultures. Multiple imputation and inverse probability weighting were used to estimate tuberculosis prevalence. Findings Of 100 678 people enumerated, 55 832 were eligible to participate and 43 100 (77.2%) of those participated. A majority of participants (42 942; 99.6%) were successfully screened for symptoms and by chest X-ray. Only 5948 (13.8%) were eligible for sputum examination, yielding 43 bacteriologically confirmed, 28 definite smear-positive and six probable smear-positive tuberculosis cases. Chest X-ray identified more tuberculosis cases (58/69) than did symptoms alone (43/71). The estimated prevalence of smear-positive and bacteriologically confirmed pulmonary tuberculosis were 90 (95% confidence interval, CI: 53-127) and 212 (95% CI: 152-272) per 100 000 population, respectively. Tuberculosis prevalence was higher in males (333; 95% CI: 233-433) and in the 35-54 year age group (355; 95% CI: 219-490). Conclusion The burden of tuberculosis remains high in Gambia but lower than earlier estimates of 490 per 100 000 population in 2010. Less than half of all cases would have been identified based on smear microscopy results alone. Successful control efforts will require interventions targeting men, increased access to radiography and more accurate, rapid diagnostic tests. [ABSTRACT FROM AUTHOR]

Background: Phylogenetically distinct Mycobacterium tuberculosis lineages differ in their phenotypes and pathogenicity. Consequently, understanding mycobacterial population structures phylogeographically is essential for design, interpretation and generalizability of clinical trials. Comprehensive efforts are lacking to date to establish the West African mycobacterial population structure on a sub-continental scale, which has diagnostic implications and can inform the design of clinical TB trials. Methodology/Principal Findings: We collated novel and published genotyping (spoligotyping) data and classified spoligotypes into mycobacterial lineages/families using TBLineage and Spotclust, followed by phylogeographic analyses using statistics (logistic regression) and lineage axis plot analysis in GenGIS, in which a phylogenetic tree constructed in MIRU-VNTRplus was analysed. Combining spoligotyping data from 16 previously published studies with novel data from The Gambia, we obtained a total of 3580 isolates from 12 countries and identified 6 lineages comprising 32 families. By using stringent analytical tools we demonstrate for the first time a significant phylogeographic separation between western and eastern West Africa not only of the two M. africanum (West Africa 1 and 2) but also of several major M. tuberculosis sensu stricto families, such as LAM10 and Haarlem 3. Moreover, in a longitudinal logistic regression analysis for grouped data we showed that M. africanum West Africa 2 remains a persistent health concern. Conclusions/Significance: Because of the geographical divide of the mycobacterial populations in West Africa, individual research findings from one country cannot be generalized across the whole region. The unequal geographical family distribution should be considered in placement and design of future clinical trials in West Africa. [ABSTRACT FROM AUTHOR]

Background: Increased availability of Next Generation Sequencing (NGS) techniques allows, for the first time, to distinguish relapses from reinfections in patients with multiple Buruli ulcer (BU) episodes. Methodology: We compared the number and location of single nucleotide polymorphisms (SNPs) identified by genomic screening between four pairs of Mycobacterium ulcerans isolates collected at the time of first diagnosis and at recurrence, derived from a collection of almost 5000 well characterized clinical samples from one BU treatment center in Benin. Principal Findings: The findings suggest that after surgical treatment—without antibiotics—the second episodes were due to relapse rather than reinfection. Since specific antibiotics were introduced for the treatment of BU, the one patient with a culture available from both disease episodes had M. ulcerans isolates with a genomic distance of 20 SNPs, suggesting the patient was most likely reinfected rather than having a relapse. Conclusions: To our knowledge, this study is the first to study recurrences in M. ulcerans using NGS, and to identify exogenous reinfection as causing a recurrence of BU. The occurrence of reinfection highlights the contribution of ongoing exposure to M. ulcerans to disease recurrence, and has implications for vaccine development. [ABSTRACT FROM AUTHOR]

Plasmodium vivax infections often recur due to relapse of hypnozoites from the liver. In malaria-endemic areas, tools to distinguish relapse from reinfection are needed. We applied amplicon deep sequencing to P. vivax isolates from 78 Cambodian volunteers, nearly one-third of whom suffered recurrence at a median of 68 days. Deep sequencing at a highly variable region of the P. vivax merozoite surface protein 1 gene revealed impressive diversity-- generating 67 unique haplotypes and detecting on average 3.6 cocirculating parasite clones within individuals, compared to 2.1 clones detected by a combination of 3 microsatellite markers. This diversity enabled a scheme to classify over half of recurrences as probable relapses based on the low probability of reinfection by multiple recurring variants. In areas of high P. vivax diversity, targeted deep sequencing can help detect genetic signatures of relapse, key to evaluating antivivax interventions and achieving a better understanding of relapsereinfection epidemiology. [ABSTRACT FROM AUTHOR]