Tuija Koivula

Global Distribution of Mycobacterium tuberculosis Spoligotypes

We present a short summary of recent observations on the global distribution of the major clades of the Mycobacterium tuberculosis complex, the causative agent of tuberculosis. This global distribution was defined by data-mining of an international spoligotyping database, SpolDB3. This database contains 11,708 patterns from as many clinical isolates originating from more than 90 countries. The 11,708 spoligotypes were clustered into 813 shared types. A total of 1,300 orphan patterns (clinical isolates showing a unique spoligotype) were also detected.

Spread of Drug-Resistant Pulmonary Tuberculosis in Estonia

ABSTRACT Restriction fragment length polymorphism (RFLP) analysis of 209Mycobacterium tuberculosis clinical isolates obtained from newly detected pulmonary tuberculosis patients (151 male and 58 female; mean age, 41 years) in Estonia during 1994 showed that 61 isolates (29%) belonged to a genetically closely related group of isolates, family A, with a predominant IS6110 banding pattern. These strains shared the majority of their IS6110 DNA-containing restriction fragments, representing a predominant banding pattern (similarity, >65%). This family A comprised 12 clusters of identical isolates, and the largest cluster comprised 10 strains. The majority (87.5%) of all multidrug-resistant (MDR) isolates, 67.2% of all isolates with any drug resistance, but only 12% of the fully susceptible isolates of M. tuberculosis belonged to family A. These strains were confirmed by spoligotyping as members of the Beijing genotype family. The spread of Beijing genotype MDR M. tuberculosis strains was also frequently seen in 1997 to 1999. The members of this homogenous group of drug-resistant M. tuberculosis strains have contributed substantially to the continual emergence of drug-resistant tuberculosis all over Estonia.

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 spoligotypes and drug susceptibility pattern of isolates from tuberculosis patients in peri-urban Kampala, Uganda

BackgroundThe poor peri-urban areas of developing countries with inadequate living conditions and a high prevalence of HIV infection have been implicated in the increase of tuberculosis (TB). Presence of different lineages of Mycobacterium tuberculosis has been described in different parts of the world. This study determined the predominant strain lineages that cause TB in Rubaga division, Kampala, Uganda, and the prevalence of resistance to key anti-tuberculosis drugs in this community.MethodsThis was a cross-sectional study of newly diagnosed sputum smear-positive patients aged ≥ 18 years. A total of 344 isolates were genotyped by standard spoligotyping and the strains were compared with those in the international spoligotype database (SpolDB4). HIV testing and anti-tuberculosis drug susceptibility assays for isoniazid and rifampicin were performed and association with the most predominant spoligotypes determined.ResultsA total of 33 clusters were obtained from 57 spoligotype patterns. According to the SpolDB4 database, 241 (70%) of the isolates were of the T2 family, while CAS1-Kili (3.5%), LAM9 (2.6%), CAS1-Delhi (2.6%) were the other significant spoligotypes. Furthermore, a major spoligotype pattern of 17 (4.5%) strains characterized by lack of spacers 15–17 and 19–43 was not identified in SpolDB4. A total of 92 (26.7%) of the patients were HIV sero-positive, 176 (51.2%) sero-negative, while 76 (22.1%) of the patients did not consent to HIV testing. Resistance to isoniazid was found in 8.1% of strains, while all 15 (4.4%) strains resistant to rifampicin were multi-drug resistant. Additionally, there was no association between any strain types in the sample with either drug resistance or HIV sero-status of the patients.ConclusionThe TB epidemic in Kampala is localized, mainly caused by the T2 family of strains. Strain types were neither associated with drug resistance nor HIV sero-status.

Use of Molecular Techniques to Distinguish between Treatment Failure and Exogenous Reinfection with Mycobacterium tuberculosis

We investigated the means by which drug resistance emerges among drug-susceptible Mycobacterium tuberculosis strains during antituberculosis therapy. Patients who experienced failure of treatment for active pulmonary tuberculosis, who initially received diagnoses of infection with drug-susceptible M. tuberculosis, and who had had at least 3 isolates tested for drug susceptibility were selected from a 6-year period in the Estonian National Reference Laboratory archive. Eleven patients from whom 35 sequential isolates of M. tuberculosis had been obtained were recruited into the study. Their clinical data and treatment charts were analyzed and correlated with drug-susceptibility patterns and IS6110 restriction fragment-length polymorphism (RFLP) profiles. Six patients excreted isogenic drug-susceptible M. tuberculosis strains, whereas, in the other 5 patients, the isolated strain shifted from a susceptible to a resistant phenotype. In all cases, this shift correlated to a shift in RFLP pattern, which showed reinfection with a new strain. Exogenous reinfection with drug-resistant M. tuberculosis may be misinterpreted as the emergence of drug resistance if molecular testing techniques are not used.

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.

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

BackgroundMozambique is one of the countries with the highest burden of tuberculosis (TB) in Sub-Saharan Africa, and information on the predominant genotypes of Mycobacterium tuberculosis circulating in the country are important to better understand the epidemic. This study determined the predominant strain lineages that cause TB in Mozambique.ResultsA total of 445 M. tuberculosis isolates from seven different provinces of Mozambique were characterized by spoligotyping and resulting profiles were compared with the international spoligotyping database SITVIT2.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.ConclusionsThe TB epidemic in Mozambique is caused by a wide diversity of spoligotypes with predominance of LAM, EAI, T and Beijing lineages.

Genomic Stability over 9 Years of an Isoniazid Resistant Mycobacterium tuberculosis Outbreak Strain in Sweden

In molecular epidemiological studies of drug resistant Mycobacterium tuberculosis (TB) in Sweden a large outbreak of an isoniazid resistant strain was identified, involving 115 patients, mainly from the Horn of Africa. During the outbreak period, the genomic pattern of the outbreak strain has stayed virtually unchanged with regard to drug resistance, IS6110 restriction fragment length polymorphism and spoligotyping patterns. Here we present the complete genome sequence analyses of the index isolate and two isolates sampled nine years after the index case as well as experimental data on the virulence of this outbreak strain. Even though the strain has been present in the community for nine years and passaged between patients at least five times in-between the isolates, we only found four single nucleotide polymorphisms in one of the later isolates and a small (4 amino acids) deletion in the other compared to the index isolate. In contrast to many other evolutionarily successful outbreak lineages (e.g. the Beijing lineage) this outbreak strain appears to be genetically very stable yet evolutionarily successful in a low endemic country such as Sweden. These findings further illustrate that the rate of genomic variation in TB can be highly strain dependent, something that can have important implications for epidemiological studies as well as development of resistance.

Molecular characterisation of Mycobacterium bovis isolates from cattle carcases at a city slaughterhouse in Uganda.

During a period of eight months, the carcases of 16,800 slaughter cattle were inspected at a city abattoir in Uganda. Eighty-seven of them had tuberculosis-like lesions and tissue samples were cultured. Only 17 cultures yielded acid-fast bacilli; 11 of them were confirmed as Mycobacterium bovis and six as non-tuberculous mycobacteria (NTM). GenoType Mycobacterium assays on the six NTM identified two as Mycobacterium fortuitum and one as Mycobacterium intracellulare, but three were unidentified. Characterisation of the M bovis isolates by spoligotyping and IS6110 restriction fragment length polymorphism (RFLP) revealed that five of the six spoligotype patterns observed in the 11 strains had not been previously reported, and seven of the nine isolates typed by RFLP had multicopy number IS6110 patterns. Six of the 11 infected carcases had multiple sites of infection, but none was condemned as unfit for human consumption.

Molecular epidemiology of drug-resistant tuberculosis in Sweden

Drug-resistant tuberculosis (TB), including the more severe forms of multidrug- and extensively drug-resistant forms, is an increasing public health concern globally. In Sweden the majority of patients with TB are immigrants from countries with a high incidence of TB including the drug-resistant forms. In this study, the spread of resistant TB in Sweden was investigated by molecular fingerprinting. Isolates resistant to at least one of the drugs, isoniazid, rifampicin, ethambutol or streptomycin, from 400 patients collected between 1994 and 2005, were studied by restriction fragment length polymorphism (RFLP) and by spoligotyping. Thirty-five clusters of patients infected with strains with identical RFLP and spoligotyping patterns (2-96 patients per cluster), comprising a total of 203 patients, were found. One large outbreak of isoniazid resistant tuberculosis was identified, involving 96 patients, mainly from the Horn of Africa. To identify chains of transmission, molecular epidemiological characterization of TB isolates should, if possible, be performed on isolates from all new TB patients.