Genetic diversity of Mycobacterium tuberculosis complex strains isolated from patients with pulmonary tuberculosis in Anambra State, Nigeria

In this study, we analyzed Mycobacterium tuberculosis complex [MTC] genetic diversity in Anambra State, Nigeria based on spoligotyping followed by 5-loci exact tandem repeats [ETRs]. Spoligotyping of 180 MTC strains isolated in 2009-2011 from pulmonary tuberculosis [TB] patients led to a total of 31 distinct patterns. A comparison with the SITVIT2 international database showed that all the 31 patterns could be classified as Shared-types [SITs] in this database; briefly, 26/31 SITs [n = 174 isolates] matched a preexisting shared-type in the database, whereas 5/31 SITs [n = 6 isolates] were newly created due to 2 or more strains belonging to an identical new pattern within this study [S1T3396] or after a match with an orphan in the database [S1T3397, S1T3398, S1T3399 and S1T3400]. A total of 18/31 SITs containing 167 or 92.8% isolates were clustered within this study [2-89 isolates per cluster] while 13/31 SITs contained unique strains. Using VNTR typing, a total of 36 distinct patterns were identified; 27 patterns [n = 157 isolates] matched a pattern already reported in the SITVIT2 database. Combination of both the methods generated 47 combined patterns for the 180 strains: 17 belonged to clustered isolates [n = 127 isolates or 70.5%] while 30 corresponded to as many unique strains [note 23 strains could not be typed using 5-loci ETRs]. No correlation was found between the spoligotyping pattern and the HIV status of the patient or drug sensitivity of the strain. This study showed that the LAM10-CAM prototype SIT61 accounted for highest number of isolates [n = 89] in Anambra State, showing its relative contribution to the TB burden in the study

Prevalence of Latin-American-Mediterranean genetic family in population structure of Mycobacterium tuberculosis in Bulgaria

Tuberculosis [TB] control remains an important public health priority for Bulgaria. The population structure of Mycobacterium tuberculosis is clonal and certain genetic families of this species [e.g., Latin-American-Mediterranean [LAM]] have attracted more attention due to their global dissemination and/or particular pathogenic properties, e.g., association with multidrug resistance [MDR]. The aim of this study was to evaluate the prevalence of the M. tuberculosis LAM family in Bulgaria based on the use of different molecular markers. A total of 101 previously spoligotyped M. tuberculosis strains were studied by LAM-specific PCR assay to detect an insertion of IS6110 in the specific genome region. On the whole, clear-cut results were obtained for most strains; spoligotype-based family was reassigned in some of them. At the same time, double bands were amplified in some cases and warrant further validation studies of this method. The higher MDR rate among LAM versus other genotype isolates was observed [P = 0.04]. In conclusion, these results suggest a low [<4%] prevalence rate of LAM in Bulgaria [that is similar to its Balkan neighbors] and highlight the importance of using robust markers for correct detection of the LAM family

Mycobacterium tuberculosis polyclonal infections and microevolution identified by MIRU-VNTRs in an epidemiological study

The advent of molecular typing using MIRU-VNTR mini-satellites has largely facilitated tuberculosis [TB] molecular epidemiological studies. Apart from detecting the chains of transmission and risk factors, these markers have also allowed to study the phenomena of mixed strain infections versus microevolutionary events. An initial set of Mycobacterium tuberculosis strains [n = 161] genotyped using spoligotyping and MIRU-VNTRs in Guyana and Suriname was evaluated for indications mixed strain infections [characterized by the detection of double alleles in 2 or more MIRU loci] versus "in-patient" microevolutionary events [characterized by the detection of double alleles in a single locus]. The present study hereby reports evidence of microevolution in 3.7% [n = 6/161] of the studied population, vs. 0.6% [n = 1/161] for mixed infection. The strains belonged to three different spoligotyping-based lineages, namely the T [SITs 44, 53, and 1081], Haarlem [SIT47], and EAI [SITs 72 and 349] lineages, while 1 isolate [SIT237] could not be assigned to any lineage. By comparing these results on microevolutionary cases [n = 6] to 112,000 strains present in the SITVIT2 database, evidence is presented that in 2/6 cases [each case corresponding to 2 patterns due to MIRU double bands], one of the patterns corresponded to a shared type found exclusively in Suriname or Guyana. Phylogenetic analysis showed that no spoligotyping lineage in particular was more prone to microevolutionary events in this study's sample. Overall, the observations fortify the awareness regarding the existence of microevolution and polyclonal TB infections which has important implications for patient care

Efficient discrimination by MIRU-VNTRs of Mycobacterium tuberculosis clinical isolates belonging to the predominant SIT11/EAI3-IND ancestral genotypic lineage in Kerala, India

The present study evaluated the ability of MIRU-VNTRs to discriminate Mycobacterium tuberculosis [MTB] clinical isolates belonging to the SIT11/EAI3-IND ancestral genotypic lineage, which is highly prevalent in Kerala, India. Starting from 168 MTB clinical isolates, spoligotyping [discriminatory index of 0.9113] differentiated the strains into 68 distinct patterns, the biggest cluster being SIT11/48 SIT11 [n = 48]. The present study shows that 12-loci MIRUs and 3 ETRs allowed an efficient discrimination of these isolates [discriminatory indexes of 0.7819 and 0.5523, respectively]

Utility and diagnostic performance of Mycobacterium tuberculosis complex by two immunochromatographic assays as compared with the molecular Genotype assay in Nigeria

Among the disadvantages of smear microscopy for detection of tuberculosis cases is its inability to differentiate between Mycobacterium tuberculosis [MTB] and non-tuberculous mycobacteria [NTM]. This study evaluated two, new immunochromatographic assays - Capilia TB-Neo and SD Bioline - on unheated and heated cultures at 80C for 30 min respectively for their ability to discriminate between MTB complex and NTM as compared with the molecular Genotype assay. Mycobacteria used in the study were obtained from smear-positive specimens collected from patients at four major hospitals in Cross River State, Nigeria. Capilia TB-Neo and SD Bioline showed sensitivities of 98.8% and 93.8% respectively and 100% specificity for both assays. Heating the isolates did not significantly impact the test performance. Both tests are recommended for use in rapid differentiation of strains isolated in Nigeria

Multidrug resistance and demography of newly diagnosed tuberculosis patients in Cross River state, Nigeria

Nigeria has the world's fourth largest tuberculosis burden, and multidrug resistant tuberculosis [MDR-TB] represents a serious public health problem. To describe the demography of TB patients and determine the susceptibility of Mycobacterium tuberculosis isolates to the major TB drugs. One hundred and thirty-seven newly diagnosed TB patients [26 [19%] being HIV positive] from all age groups were recruited into the study. Each specimen was cultured using BACTEC MGIT960, followed by inoculation and growth on Lowenstein-Jensen [LJ] medium. Primary identification was carried out using an immunochromatographic technique [Capilia TB-Neo], and further confirmed by genotyping. Drug susceptibility testing [DST] was carried out by the agar proportion method. Of the 97 pure mycobacterial cultures on LJ medium, 81 [83.5%] isolates were identified as M. tuberculosis complex, while 16 [16.5%] were Capilia negative. DST was carried out on 58 isolates. The drug susceptibility pattern showed that resistance occurred in 16 [27.6%] for streptomycin, 11 [19%] for isoniazid, 9 [16%] for rifampicin, and 10 [17.2%] for ethambutol. Rifampicin monoresistance occurred in 2 [3.4%] cases. MDR [combined resistance to isoniazid and rifampicin], also involving resistance to streptomycin and ethambutol, occurred in 6/58 [10.3%] isolates; although laboratory cross-contamination could not be excluded in 4/6 MDR strains with identical MIRU patterns characterized by consecutive strain numbers. Considering that first out of these 4 isolates was not due to laboratory carryover, the results of this study still report a minimal MDR-TB rate of 3/58 [5.2%] among newly diagnosed TB patients in Cross River State, Nigeria. An increase in drug resistance was observed in this study as compared with previous studies in the country. Hence, introduction of culture in routine diagnostic mycobacteriology laboratories will prevent the emergence and dissemination of MDR-TB, while improved quality control strategies would in parallel prevent laboratory cross-contamination, thereby reducing mislabeling, unnecessary treatment, and drug toxicity for patients

Predominance of Beijing genotype in extensively drug resistant Mycobacterium tuberculosis isolates from a tertiary care hospital in New Delhi, India

Out of a total of 311 Mycobacterium tuberculosis isolates from sputum specimens subjected to first- and second-line drug-susceptibility testing [DST] at a hospital serving as a referral center for chronic tuberculosis [TB] cases in New Delhi, 232/311 [74.6%] isolates were found to be resistant to isoniazid and rifampicin. Among multidrug-resistant [MDR] isolates, 119/232 [51.3%] were resistant to four first-line drugs [streptomycin, isoniazid, rifampicin and ethambutol]. Mono-resistance to isoniazid was observed in 18 [5.7%] isolates, while none of the isolates tested showed mono-resistance to rifampicin. 50/232 [21.5%] isolates met the definition of extensively drug resistant [XDR] TB, i.e., additional resistance to a fluoroquinolone and at least one of the three injectable second-line drugs: kanamycin, capreomycin, or amikacin. Spoligotyping of the XDR-TB isolates revealed 14 patterns; 39/50 [78%] isolates being grouped in three clusters vs. 11/50 [22%] isolates being unique. SIT1/Beijing represented the largest cluster [n = 21, 42%], followed by SIT26/CAS1-Delhi [n = 10, 20%] and SIT 53/T1 [n = 8 isolates; 16%]. This study corroborates recent observations from North India suggesting that both Beijing and CAS1-Delhi lineages constitute the bulk of XDR-TB isolates that are disseminating rapidly across a large geographical region in and around the capital city of India

emergence of beijing genotype of mycobacterium tuberculosis in the Kingdom of Saudi Arabia

To investigate the prevalence of Beijing genotype strains of Mycobacterium tuberculosis in the Kingdom of Saudi Arabia. We analyzed the available data on a total of 1505 strains isolated during 2002-2005. Spoligotyping results revealed that Beijing family isolates represented 4.5% of all the isolates. Existence of Beijing clade is alarming as this family is known to be multi-drug resistant and transmissible. This study showed that the occurrence of Beijing genotype is associated with young age and drug resistance. The Beijing strains affected both Saudi nationals as well as migrants originating in Asia. The Beijing clade could be responsible for the ongoing transmission of tuberculosis within the community