Molecular diversity of Mycobacterium tuberculosis isolates from patients with pulmonary tuberculosis in Mozambique

<p>Abstract</p> <p>Background</p> <p>Mozambique is one of the countries with the highest burden of tuberculosis (TB) in Sub-Saharan Africa, and information on the predominant genotypes of <it>Mycobacterium tuberculosis </it>circulating in the country are important to better understand the epidemic. This study determined the predominant strain lineages that cause TB in Mozambique.</p> <p>Results</p> <p>A total of 445 <it>M. tuberculosis </it>isolates from seven different provinces of Mozambique were characterized by spoligotyping and resulting profiles were compared with the international spoligotyping database SITVIT2.</p> <p>The four most predominant lineages observed were: the Latin-American Mediterranean (LAM, n = 165 or 37%); the East African-Indian (EAI, n = 132 or 29.7%); an evolutionary recent but yet ill-defined T clade, (n = 52 or 11.6%); and the globally-emerging Beijing clone, (n = 31 or 7%). A high spoligotype diversity was found for the EAI, LAM and T lineages.</p> <p>Conclusions</p> <p>The TB epidemic in Mozambique is caused by a wide diversity of spoligotypes with predominance of LAM, EAI, T and Beijing lineages.</p

DNA restriction fragment length polymorphism analysis of Mycobacterium tuberculosis isolates from HIV-seropositive and HIV-seronegative patients in Kampala, Uganda

<p>Abstract</p> <p>Background</p> <p>The identification and differentiation of strains of <it>Mycobacterium tuberculosis </it>by DNA fingerprinting has provided a better understanding of the epidemiology and tracing the transmission of tuberculosis. We set out to determine if there was a relationship between the risk of belonging to a group of tuberculosis patients with identical mycobacterial DNA fingerprint patterns and the HIV sero-status of the individuals in a high TB incidence peri-urban setting of Kampala, Uganda.</p> <p>Methods</p> <p>One hundred eighty three isolates of <it>Mycobacterium tuberculosis </it>from 80 HIV seropositive and 103 HIV seronegative patients were fingerprinted by standard IS<it>6110</it>-RFLP. Using the BioNumerics software, strains were considered to be clustered if at least one other patient had an isolate with identical RFLP pattern.</p> <p>Results</p> <p>One hundred and eighteen different fingerprint patterns were obtained from the 183 isolates. There were 34 clusters containing 54% (99/183) of the patients (average cluster size of 2.9), and a majority (96.2%) of the strains possessed a high copy number (≥ 5 copies) of the IS<it>6110 </it>element. When strains with <5 bands were excluded from the analysis, 50.3% (92/183) were clustered, and there was no difference in the level of diversity of DNA fingerprints observed in the two sero-groups (adjusted odds ratio [aOR] 0.85, 95%CI 0.46–1.56, <it>P </it>= 0.615), patients aged <40 years (aOR 0.53, 95%CI 0.25–1.12, <it>P </it>= 0.100), and sex (aOR 1.12, 95%CI 0.60–2.06, <it>P </it>= 0.715).</p> <p>Conclusion</p> <p>The sample showed evidence of a high prevalence of recent transmission with a high average cluster size, but infection with an isolate with a fingerprint found to be part of a cluster was not associated with any demographic or clinical characteristics, including HIV status.</p

Genetic diversity and potential routes of transmission of Mycobacterium bovis in Mozambique

CITATION: Machado, A., et al. 2018. Genetic diversity and potential routes of transmission of Mycobacterium bovis in Mozambique. PLOS Neglected Tropical Diseases, 12(1):e0006147, doi:10.1371/journal.pntd.0006147.The original publication is available at https://journals.plos.org/plosntdsBovine tuberculosis is a zoonotic disease with largely unknown impact in Africa, with risk factors such as HIV and direct contact with animals or consumption of Mycobacterium bovis infected animal products. In order to understand and quantify this risk and design intervention strategies, good epidemiological studies are needed. Such studies can include molecular typing of M. bovis isolates. The aim of this study was to apply these tools to provide novel information concerning the distribution of bovine tuberculosis in cattle in Mozambique and thereby provide relevant information to guide policy development and strategies to contain the disease in livestock, and reduce the risk associated with transmission to humans. A collection of 178 M. bovis isolates was obtained from cattle in Mozambique. Using spoligotyping and regions of difference analysis, we classified the isolates into clonal complexes, thus reporting the first characterisation of M. bovis strains in this region. Data from MIRU-VNTR typing was used to compare isolates from a number of African countries, revealing a deeply geographically structured diversity of M. bovis. Eastern Africa appears to show high diversity, suggesting deep evolution in that region. The diversity of M. bovis in Africa does not seem to be a function of recent importation of animals, but is probably maintained within each particular region by constant reinfection from reservoir animals. Understanding the transmission routes of M. bovis in Mozambique and elsewhere is essential in order to focus public health and veterinary resources to contain bovine tuberculosis.https://journals.plos.org/plosntds/article?id=10.1371/journal.pntd.0006147Publisher's versio

The Guinea-Bissau Family of Mycobacterium tuberculosis Complex Revisited

The Guinea-Bissau family of strains is a unique group of the Mycobacterium tuberculosis complex that, although genotypically closely related, phenotypically demonstrates considerable heterogeneity. We have investigated 414 M. tuberculosis complex strains collected in Guinea-Bissau between 1989 and 2008 in order to further characterize the Guinea-Bissau family of strains. To determine the strain lineages present in the study sample, binary outcomes of spoligotyping were compared with spoligotypes existing in the international database SITVIT2. The major circulating M. tuberculosis clades ranked in the following order: AFRI (n = 195, 47.10%), Latin-American-Mediterranean (LAM) (n = 75, 18.12%), ill-defined T clade (n = 53, 12.8%), Haarlem (n = 37, 8.85%), East-African-Indian (EAI) (n = 25, 6.04%), Unknown (n = 12, 2.87%), Beijing (n = 7, 1.68%), X clade (n = 4, 0.96%), Manu (n = 4, 0.97%), CAS (n = 2, 0.48%). Two strains of the LAM clade isolated in 2007 belonged to the Cameroon family (SIT61). All AFRI isolates except one belonged to the Guinea-Bissau family, i.e. they have an AFRI_1 spoligotype pattern, they have a distinct RFLP pattern with low numbers of IS6110 insertions, and they lack the regions of difference RD7, RD8, RD9 and RD10, RD701 and RD702. This profile classifies the Guinea-Bissau family, irrespective of phenotypic biovar, as part of the M. africanum West African 2 lineage, or the AFRI_1 sublineage according to the spoligtyping nomenclature. Guinea-Bissau family strains display a variation of biochemical traits classically used to differentiate M. tuberculosis from M. bovis. Yet, the differential expression of these biochemical traits was not related to any genes so far investigated (narGHJI and pncA). Guinea-Bissau has the highest prevalence of M. africanum recorded in the African continent, and the Guinea-Bissau family shows a high phylogeographical specificity for Western Africa, with Guinea-Bissau being the epicenter. Trends over time however indicate that this family of strains is waning in most parts of Western Africa, including Guinea-Bissau (p = 0.048)

Mycobacterium tuberculosis causing tuberculous lymphadenitis in Maputo, Mozambique

BACKGROUND: The zoonosis bovine tuberculosis (TB) is known to be responsible for a considerable proportion of extrapulmonary TB. In Mozambique, bovine TB is a recognised problem in cattle, but little has been done to evaluate how Mycobacterium bovis has contributed to human TB. We here explore the public health risk for bovine TB in Maputo, by characterizing the isolates from tuberculous lymphadenitis (TBLN) cases, a common manifestation of bovine TB in humans, in the Pathology Service of Maputo Central Hospital, in Mozambique, during one year. RESULTS: Among 110 patients suspected of having TBLN, 49 had a positive culture result. Of those, 48 (98 %) were positive for Mycobacterium tuberculosis complex and one for nontuberculous mycobacteria. Of the 45 isolates analysed by spoligotyping and Mycobacterial Interspersed Repetitive Unit - Variable Number Tandem Repeat (MIRU-VNTR), all were M. tuberculosis. No M. bovis was found. Cervical TBLN, corresponding to 39 (86.7 %) cases, was the main cause of TBLN and 66.7 % of those where from HIV positive patients. We found that TBLN in Maputo was caused by a variety of M. tuberculosis strains. The most prevalent lineage was the EAI (n?=?19; 43.2 %). Particular common spoligotypes were SIT 48 (EAI1_SOM sublineage), SIT 42 (LAM 9), SIT 1 (Beijing) and SIT53 (T1), similar to findings among pulmonary cases. CONCLUSIONS: M. tuberculosis was the main etiological agent of TBLN in Maputo. M. tuberculosis genotypes were similar to the ones causing pulmonary TB, suggesting that in Maputo, cases of TBLN arise from the same source as pulmonary TB, rather than from an external zoonotic source. Further research is needed on other forms of extrapulmonary TB and in rural areas where there is high prevalence of bovine TB in cattle, to evaluate the risk of transmission of M. bovis from cattle to humans.Swedish International Development Cooperation Agency / Department for Research Cooperation (Sida/SAREC) through Eduardo Mondlane University and Karolinska Institutet Research and Training (KIRT) collaboratio

Drug Resistant Mycobacterium tuberculosis of the Beijing Genotype Does Not Spread in Sweden

BACKGROUND: Drug resistant (DR) and multi-drug resistant (MDR) tuberculosis (TB) is increasing worldwide. In some parts of the world 10% or more of new TB cases are MDR. The Beijing genotype is a distinct genetic lineage of Mycobacterium tuberculosis, which is distributed worldwide, and has caused large outbreaks of MDR-TB. It has been proposed that certain lineages of M. tuberculosis, such as the Beijing lineage, may have specific adaptive advantages. We have investigated the presence and transmission of DR Beijing strains in the Swedish population. METHODOLOGY/PRINCIPAL FINDINGS: All DR M. tuberculosis complex isolates between 1994 and 2008 were studied. Isolates that were of Beijing genotype were investigated for specific resistance mutations and phylogenetic markers. Seventy (13%) of 536 DR strains were of Beijing genotype. The majority of the patients with Beijing strains were foreign born, and their country of origin reflects the countries where the Beijing genotype is most prevalent. Multidrug-resistance was significantly more common in Beijing strains than in non-Beijing strains. There was a correlation between the Beijing genotype and specific resistance mutations in the katG gene, the mabA-inhA-promotor and the rpoB gene. By a combined use of RD deletions, spoligotyping, IS1547, mutT gene polymorphism and Rv3135 gene analysis the Beijing strains could be divided into 11 genomic sublineages. Of the patients with Beijing strains 28 (41%) were found in altogether 10 clusters (2-5 per cluster), as defined by RFLP IS6110, while 52% of the patients with non-Beijing strains were in clusters. By 24 loci MIRU-VNTR 31 (45%) of the patients with Beijing strains were found in altogether 7 clusters (2-11 per cluster). Contact tracing established possible epidemiological linkage between only two patients with Beijing strains. CONCLUSIONS/SIGNIFICANCE: Although extensive outbreaks with non-Beijing TB strains have occurred in Sweden, Beijing strains have not taken hold, in spite of the proximity to high prevalence countries such as Russia and the Baltic countries. The Beijing sublineages so far introduced in Sweden may not be adapted to spread in the Scandinavian population

Clinically important mycobacteria in Guinea-Bissau, West Africa : Phenotypic and genetic diversity

Tuberculosis (TB) is a major and still increasing health problem in West Africa. In Guinea-Bissau, TB has an estimated annual incidence of 1501100.000. Knowledge on infections caused by nontuberculous mycobacteria, such as Mycobacterium avium complex (MAC), is very limited in Africa, including Guinea-Bissau. M tuberculosis complex isolates (n=229) and MAC isolates (n=28) collected in Guinea-Bissau during 1989 to 1996, from sputum samples from approximately 1000 patients with clinical diagnosis of pulmonary TB, were analysed for phenotypical and genotypical characteristics. There was a high degree of heterogeneity in the M tuberculosis complex isolates in terms of biochemical properties as compared to what is normal in European isolates. Phenotypically, these isolates were assigned to one of five biovars, ranging within a spectrum of classical M. tuberculosis (biovar 5) to classical M. bovis (biovar 1). Genotypically, the strains could be divided into three groups (A-C), of which group A isolates are proposed to be imported and of European descent, while groups B and C isolates are unique and proposed to originate from West Africa (the Guinea-Bissau family of strains). A detailed genotypic analysis was carried out on 35 isolates from the Guinea-Bissau family. Based on the data obtained, and by comparing corresponding genes in mycobacteria outside the M. tuberculosis complex, it is proposed that the Guinea-Bissau family of strains is a unique branch of the M tuberculosis complex tree in between classical M. tuberculosis and classical M. bovis. Drug susceptibility testing demonstrated a low rate of resistance to drugs used for TB treatment in Guinea-Bissau. However, studies in vitro of thiacetazone showed that the minimum inhibitory concentration of this drug on M africanum subtype 11 strains (biovar 4) was significantly higher than that for other strains studied. It is concluded that in areas where M africanum subtype II is a prevalent cause of TB, thiacetazone should not be considered for treatment. M. bovis is known to lack the mtp40 gene. However, other investigators earlier reported two strains of M. bovis possessing the mtp40 gene. A detailed genotypic re-examination of these strains was performed showing that the two isolates, in fact, should be classified as M. africanum rather than M. bovis. This finding further strengthens the proposed evolutionary scenario of the M tuberculosis complex. The finding of MAC in sputum samples is one of the first reports of MAC in patients from Africa. The results demonstrate the importance of adequate laboratory diagnosis of mycobacteria. In geographic areas where MAC pulmonary infections are common, it is of significance to identify MAC, especially so in patients often incorrectly thought to suffer from drug-resistant TB. The MAC isolates were studied by several molecular methods. The highest discriminatory power was obtained with 16S rRNA sequencing. By this method most of the Guinea-Bissau strains were found to belong to the M. intracellulare branch of the phylogenetic tree of MAC. In summary, the phenotypic heterogeneity, and the genetic clustering revealed three major clusters allowing the identification of the new Guinea-Bissau family of M tuberculosis complex strains. This will contribute to a better understanding of the epidemiology of these organisms and also their evolution. The incidence of pulmonary MAC infections in patients in Guinea-Bissau, underlines the importance of correct laboratory diagnostic methods, not only for correct treatment, but also for epidemiological surveillance - frequently such MAC infections are incorrectly judged to be treatment failures caused by drug resistant TB complex organisms

Higher mortality in HIV-2/HTLV-1 co-infected patients with pulmonary tuberculosis in Guinea-Bissau, West Africa, compared to HIV-2-positive HTLV-1-negative patients.

OBJECTIVES: To investigate the effect of human T-lymphotropic virus type 1 (HTLV-1) on CD4 counts and mortality in tuberculosis (TB) patients with or without human immunodeficiency virus (HIV). METHODS: A prospective study on 280 hospitalized patients with pulmonary TB was performed in Guinea-Bissau, 1994-1997, including HIV, CD4 counts and clinical outcome. We compared the CD4 count levels at the time of inclusion between HIV-negative and HIV-positive patients, with or without HTLV-1. Mortality was determined while patients were on treatment for TB. RESULTS: Median CD4% was significantly higher in HIV-positive subjects co-infected with HTLV-1 compared to HTLV-1-negative patients. Two hundred thirty-three individuals were included in the analysis of mortality, and among HIV-negative subjects the mortality was 18.6/100 person-years . In HIV-2-positive HTLV-1-negative subjects the mortality was 39.5/100 person-years and in HIV-2/HTLV-1 co-infected patients it was 113.6/100 person-years (adjusted mortality rate ratio 4.7, 95% CI 1.5-14.4; p < 0.01). When all HIV-positive patients were analyzed together, corresponding mortality rates were 53.5/100 person-years and 104.8/100 person-years , respectively (not significant). CONCLUSIONS: HIV/HTLV-1 co-infected patients hospitalized for pulmonary TB had a high mortality and had significantly higher CD4% compared to only HIV-positive subjects. This may imply that HTLV-1 has an adverse effect on the immune system in HIV-infected subjects, independently of the CD4 count, that makes co-infected subjects more vulnerable to TB

Increased Prevalence of HTLV-1 in Patients With Pulmonary Tuberculosis Coinfected With HIV, but Not in HIV-Negative Patients With Tuberculosis.

BACKGROUND:: Few and inconclusive results have been presented regarding the influence of human T-lymphotropic virus 1 (HTLV-1) infection on the risk of acquiring tuberculosis (TB). METHODS:: In 1994-1997, we performed a prospective study on hospitalized adult patients with pulmonary TB in Guinea-Bissau and compared the clinical outcome in HIV-2 and HIV-negative patients. We determined the prevalence of HTLV-1 in all patients screened and diagnosed with TB in that study and compared the infection rate with a serosurvey of HTLV-1 in a population sample from a community-based study conducted at the same time and in the same city. RESULTS:: In the TB group, a total of 32 (11.4%) of 280 patients were positive for HTLV-1. This was significantly higher compared with the population-based group in which 74 (3.5%) of 2117 were HTLV-1 positive [crude odds ratio (OR) = 3.6; 95% confidence interval (CI) 2.2 to 5.6, P < 0.001]. However, in a logistic regression analysis controlling for age, gender, and HIV result, the difference was no longer significant (OR = 1.61; 95% CI 0.95 to 2.70, P = 0.074). In HIV-negative patients, no association was found between HTLV-1 and TB (OR = 1.18; 95% CI 0.48 to 2.89, P = 0.71), whereas a significant association was found in HIV-positive patients (OR = 2.41; 95% CI 1.26 to 4.61, P = 0.008). CONCLUSIONS:: The immunosuppressive effect of HTLV-1 alone was not enough to increase the risk of TB in a highly endemic country, but HTLV-1 increased the risk of TB among HIV-infected individuals