A molecular epidemiological and genetic diversity study of tuberculosis in Ibadan, Nnewi and Abuja, Nigeria

BACKGROUND: Nigeria has the tenth highest burden of tuberculosis (TB) among the 22 TB high-burden countries in the world. This study describes the biodiversity and epidemiology of drug-susceptible and drug-resistant TB in Ibadan, Nnewi and Abuja, using 409 DNAs extracted from culture positive TB isolates. METHODOLOGY/PRINCIPAL FINDINGS: DNAs extracted from clinical isolates of Mycobacterium tuberculosis complex were studied by spoligotyping and 24 VNTR typing. The Cameroon clade (CAM) was predominant followed by the M. africanum (West African 1) and T (mainly T2) clades. By using a smooth definition of clusters, 32 likely epi-linked clusters related to the Cameroon genotype family and 15 likely epi-linked clusters related to other "modern" genotypes were detected. Eight clusters concerned M. africanum West African 1. The recent transmission rate of TB was 38%. This large study shows that the recent transmission of TB in Nigeria is high, without major regional differences, with MDR-TB clusters. Improvement in the TB control programme is imperative to address the TB control problem in Nigeria.     

Role of mannose binding lectin gene variants on its protein levels and macrophage phagocytosis with live Mycobacterium tuberculosis in pulmonary tuberculosis

Mannose-binding lectin (MBL) plays an important role in innate immunity. Functional mutant homozygotes of the MBL gene affect the serum MBL levels and have been correlated with disease susceptibility. We have studied the regulatory role of variant MBL genotypes on serum MBL level and macrophage phagocytosis with live Mycobacterium tuberculosis, and the lymphoproliferative response to M. tuberculosis culture filtrate antigen in pulmonary tuberculosis (PTB) patients (n = 48) and normal healthy subjects (NHS) (n = 58). The total serum MBL level was higher in PTB patients than in NHS (P = 0.0085). Patients and NHS with AA genotype (homozygotes of MBL - common alleles) showed a very high serum MBL level, and those with OO genotype (functional mutant homozygotes of MBL - less frequent alleles) showed a very low MBL level (AA vs. OO: NHS, P = 3.3 x 10(-9); PTB, P = 3.1 x 10(-9)). A significantly lower phagocytosis was observed in NHS with AA genotype than in NHS with AO (heterozygotes) genotype (P = 0.046). In PTB patients, no such difference was observed. A negative correlation of macrophage phagocytosis with MBL level was seen in patients and NHS (P = 0.019). Antigen-induced lymphoproliferative response was significantly decreased in PTB patients with AA genotype as compared with NHS with AA genotype (P = 0.036). The present study suggests that AA genotype with its associated higher serum MBL levels plays a regulatory role in immunity to tuberculosis than functional mutant homozygotes (OO genotype) with its associated lower level of MBL.     

Molecular identification and characterization of rifampicin-resistant Mycobacterium tuberculosis isolates by line probe assay: an approach for rapid diagnosis of multidrug-resistant tuberculosis

Aim:  Early identification and characterization of rifampicin-resistant (R^r) Mycobacterium tuberculosis isolates recovered from the samples of tuberculosis (TB) patients in the Aegean (West Anatolian) Region was intended.
Methods and Results:  Sixty isolates [47 (78·3%) multidrug-resistant (MDR)], which were identified as M. tuberculosis complex and phenotypically resistant to rifampicin by both BACTEC mycobacteria growth indicator tube (MGIT) 960 and 460 systems were analysed by a commercial line probe assay (INNO-LiPA Rif TB). The concordance of LiPA with the in vitro susceptibility test was found as 98·3%. Among the isolates, S531L (R5 pattern; 46·7%) and L511P/R, S512T, Q513L/K (ΔS1 pattern; 11·7%) were the most frequent mutation patterns. As compared with the BACTEC systems and conventional techniques for cultivation, identification and in vitro susceptibility testing, INNO-LiPA Rif TB after cultivation in BACTEC MGIT 960 system provided an average of 20 days early diagnosis of R^r M. tuberculosis isolates.
Conclusions:  Rapid molecular identification and characterization of R^r M. tuberculosis isolates after BACTEC MGIT 960 cultivation would be useful for faster diagnosis, infection control and planning of accurate treatment in MDR-TB patients.
Significance and Impact of the Study:  Patients with MDR-TB need a specified treatment and efficient follow-up strategies. Rapid and practical methodologies to diagnose and follow these patients should be applied in routine use.     

Mycobacterium tuberculosis complex differentiation using gyrB-restriction fragment length polymorphism analysis

Mycobacterium tuberculosis complex (MTBC) members are causative agents of human and animal tuberculosis. Differentiation of MTBC members is required for appropriate treatment of individual patients and for epidemiological purposes. Strains from six MTBC species -- M. tuberculosis, M. bovis subsp. bovis, M. bovis BCG, M. africanum, M. pinnipedii, and "M. canetti" -- were studied using gyrB-restriction fragment length polymorphism (gyrB-RFLP) analysis. A table was elaborated, based on observed restriction patterns and published gyrB sequences. To evaluate applicability of gyrB-RFLP at Instituto Adolfo Lutz, Sao Paulo, Mycobacterial Reference Laboratory, 311 MTBC clinical isolates, previously identified using traditional methods as M. tuberculosis (306), M. bovis (3), and M. bovis BCG (2), were analyzed by gyrB-RFLP. All isolates were correctly identified by the molecular method, but no distinction between M. bovis and M. bovis BCG was obtained. Differentiation of M. tuberculosis and M. bovis is of utmost importance, because they require different treatment schedules. In conclusion, gyrB-RFLP is accurate and easy-to-perform, with potential to reduce time needed for conventional differentiation methods. However, application for epidemiological studies remains limited, because it cannot differentiate M. tuberculosis from M. africanum subtype II, and "M. canetti", M. africanum subtype I from M. pinnipedii, and. M. bovis from M. bovis BCG.     

Taxonomic studies on the Mycobacterium tuberculosis series

Numerical classification of slowly growing mycobacteria, including 159 strains received as Mycobacterium tuberculosis, M. bovis, M. africanum, and M. microti, was carried out using 88 characters, and the following results were obtained. 1) All 159 strains received as M. tuberculosis, M. bovis, M. africanum, and M. microti formed one cluster, and no clear-cut differentiation among four species was achieved. These four species should be reduced to one species, M. tuberculosis. 2) Within the cluster, two subclusters appeared, with a number of strains located outside the subclusters. One subcluster was composed, except for two strains, of only M. tuberculosis strains, and another of M. bovis and M. africanum strains only. The subclusters were regarded as subspecies tuberculosis and subspecies bovis, respectively. M. africanum was regarded as a synonym of M. bovis, i.e., a niacin-positive variety of M. bovis (subsp. bovis). 3) An intermediate subcluster was observed outside the M. tuberculosis and M. bovis subclusters. This may be regarded as intermediate between the two subspecies, tuberculosis and bovis. 4) In the last two decades, the characteristics of M. tuberculosis strains isolated from patients seemed to be approaching those of M. bovis strains. The recently isolated strains showed stronger arylsulfatase activity and lower resistance to thiophene-2-carboxylic acid hydrazide than the strains isolated 16 to 19 years ago.     

The polyketide Pks1 contributes to biofilm formation in Mycobacterium tuberculosis

Infections caused by biofilms are abundant and highly persistent, displaying phenotypic resistance to high concentrations of antimicrobials and modulating host immune systems. Tuberculosis (TB), caused by Mycobacterium tuberculosis, shares these qualities with biofilm infections. To identify genetic determinants of biofilm formation in M. tuberculosis, we performed a small-scale transposon screen using an in vitro pellicle biofilm assay. We identified five M. tuberculosis mutants that were reproducibly attenuated for biofilm production relative to that of the parent strain H37Rv. One of the most attenuated mutants is interrupted in pks1, a polyketide synthase gene. When fused with pks15, as in some M. tuberculosis isolates, pks1 contributes to synthesis of the immunomodulatory phenolic glycolipids (PGLs). However, in strains such as H37Rv with split pks15 and pks1 loci, PGL is not produced and pks1 has no previously defined role. We showed that pks1 complementation restores biofilm production independently of the known role of pks1 in PGL synthesis. We also assessed the relationship among biofilm formation, the pks15/1 genotype, and M. tuberculosis phylogeography. A global survey of M. tuberculosis clinical isolates revealed surprising sequence variability in the pks15/1 locus and substantial variation in biofilm phenotypes. Our studies identify novel M. tuberculosis genes that contribute to biofilm production, including pks1. In addition, we find that the ability to make pellicle biofilms is common among M. tuberculosis isolates from throughout the world, suggesting that this trait is relevant to TB propagation or persistence.     

Decreased plasma granulysin and increased interferon-gamma concentrations in patients with newly diagnosed and relapsed tuberculosis

Granulysin and interferon-gamma (IFN-γ) have broad antimicrobial activity which controls Mycobacterium tuberculosis (M. tuberculosis) infection. Circulating granulysin and IFN-γ concentrations were measured and correlated with clinical disease in Thai patients with newly diagnosed, relapsed and chronic tuberculosis (TB). Compared to controls, patients with newly diagnosed, relapsed and chronic TB had lower circulating granulysin concentrations, these differences being significant only in newly diagnosed and relapsed TB (P < 0.001 and 0.004, respectively). Granulysin concentrations in patients with newly diagnosed and relapsed TB were significantly lower than in those with chronic TB (P= 0.003 and P= 0.022, respectively). In contrast, significantly higher circulating IFN-γ concentrations were found in patients with newly diagnosed and relapsed TB compared to controls (P < 0.001). The IFN-γ concentrations in newly diagnosed and relapsed patients were not significantly different from those of patients with chronic TB. However, in vitro stimulation of peripheral blood mononuclear cells (PBMCs) from patients with newly diagnosed, relapsed and chronic TB with purified protein derivative (PPD) or heat killed M. tuberculosis (H37Ra) enhanced production of granulysin by PBMCs. In vitro, stimulation of PBMCs of newly diagnosed TB patients with PPD produced greater amounts of IFN-γ than did controls, while those stimulated with H37Ra did not. The results demonstrate that patients with active pulmonary TB have low circulating granulysin but high IFN-γ concentrations, suggesting possible roles in host defense against M. tuberculosis for these agents.     

Rifampicin exposure reveals within-host Mycobacterium tuberculosis diversity in patients with delayed culture conversion

Mycobacterium tuberculosis (Mtb) genetic micro-diversity in clinical isolates may underline mycobacterial adaptation to tuberculosis (TB) infection and provide insights to anti-TB treatment response and emergence of resistance. Herein we followed within-host evolution of Mtb clinical isolates in two cohorts of TB patients, either with delayed Mtb culture conversion (> 2 months), or with fast culture conversion (< 2 months). We captured the genetic diversity of Mtb isolates obtained in each patient, by focusing on minor variants detected as unfixed single nucleotide polymorphisms (SNPs). To unmask antibiotic tolerant sub-populations, we exposed these isolates to rifampicin (RIF) prior to whole genome sequencing (WGS) analysis. Thanks to WGS, we detected at least 1 unfixed SNP within the Mtb isolates for 9/15 patients with delayed culture conversion, and non-synonymous (ns) SNPs for 8/15 patients. Furthermore, RIF exposure revealed 9 additional unfixed nsSNP from 6/15 isolates unlinked to drug resistance. By contrast, in the fast culture conversion cohort, RIF exposure only revealed 2 unfixed nsSNP from 2/20 patients. To better understand the dynamics of Mtb micro-diversity, we investigated the variant composition of a persistent Mtb clinical isolate before and after controlled stress experiments mimicking the course of TB disease. A minor variant, featuring a particular mycocerosates profile, became enriched during both RIF exposure and macrophage infection. The variant was associated with drug tolerance and intracellular persistence, consistent with the pharmacological modeling predicting increased risk of treatment failure. A thorough study of such variants not necessarily linked to canonical drug-resistance, but which are prone to promote anti-TB drug tolerance, may be crucial to prevent the subsequent emergence of resistance. Taken together, the present findings support the further exploration of Mtb micro-diversity as a promising tool to detect patients at risk of poorly responding to anti-TB treatment, ultimately allowing improved and personalized TB management.     

Molecular, kinetic and thermodynamic characterization of Mycobacterium tuberculosis orotate phosphoribosyltransferase

Tuberculosis (TB) is a chronic infectious disease caused mainly by Mycobacterium tuberculosis. The worldwide emergence of drug-resistant strains, the increasing number of infected patients among immune compromised populations, and the large number of latent infected individuals that are reservoir to the disease have underscored the urgent need of new strategies to treat TB. The nucleotide metabolism pathways provide promising molecular targets for the development of novel drugs against active TB and may, hopefully, also be effective against latent forms of the pathogen. The orotate phosphoribosyltransferase (OPRT) enzyme of the de novo pyrimidine synthesis pathway catalyzes the reversible phosphoribosyl transfer from 5'-phospho-α-D-ribose 1'-diphosphate (PRPP) to orotic acid (OA), forming pyrophosphate and orotidine 5'-monophosphate (OMP). Here we describe cloning and characterization of pyrE-encoded protein of M. tuberculosis H37Rv strain as a homodimeric functional OPRT enzyme. The M. tuberculosis OPRT true kinetic constants for forward reaction and product inhibition results suggest a Mono-Iso Ordered Bi-Bi kinetic mechanism, which has not been previously described for this enzyme family. Absence of detection of half reaction and isothermal titration calorimetry (ITC) data support the proposed mechanism. ITC data also provided thermodynamic signatures of non-covalent interactions between substrate/product and M. tuberculosis OPRT. These data provide a solid foundation on which to base target-based rational design of anti-TB agents and should inform us how to better design inhibitors of M. tuberculosis OPRT.     

Evaluation of reasons of the MDR M. tuberculosis strains dissemination by analysis of the rifampicin and/or isoniazid resistant isolates

During the last years in Novosibirsk region of Russia the rate of TB patients infected by MDR strains of M. tuberculosis has been constantly increasing. This increase may occur as a result of the spontaneously mutated mycobacterium selection during treatment of patients or as a result of primary infection by the resistant M. tuberculosis, or also, as a result of both reasons in combination. If the main reason of MDR strain dissemination is selection of resistant bacterium during patient treatment, the equal apportionment of the dominated mutation into the mycobacterium genotypes would be observed. If the main reason is the primary infection by resistant M. tuberculosis, the unequal apportionment would be revealed. For deeper understanding of the main reasons of the fast MDR strains spreading in the region, the distribution of the main mutations over genotypes of strains in Novosibirsk (170 isolates) and Tomsk prison (51 isolates) was investigated. Mutations in rpoB gene associated with the rifampicin resistance and in katG (isoniazid resistance) were detected by biochips. M. tuberculosis genotypings were carried out by IS6110 PCR typing or MIRU typing, in the last method the twelve loci (MIRU 2, 4, 10, 16, 20, 23, 24, 26, 27, 31, 39, 40) have been used. The most frequent mutation in the rpoB gene was Ser531-->Leu (60-70% of the rifampicin resistant strains) and Ser315-->Thr in gene katG (80% of the isoniazid resistant M. tuberculosis). Both in Novosibirsk and in Tomsk prison the rates of clustered cases transmissions were high (69 and 63% respectively). Analysis of the distribution of the dominated mutations Ser531-->Leu (rpoB) and Ser315-->Thr (katG) revealed that all of them were detected in each clusters, but in Novosibirsk there were only two clusters, in which the percentage of strains, containing mutation Ser531-->Leu (rpoB) were higher (85.7% and 77.7% respectively, P < 0.05), then in others. Among the Tomsk prison's clusters it was revealed one in which the proportion of the Ser3 15-->Thr mutation in katGwas higher (96.4%, P < 0.05). The nonuniform distribution of the dominated mutations highlighted that the epidemic spread of drug-resistant strains of M. tuberculosis in region resulted from the selection of them during patient treatment and the subsequent transmission by TB patients.     

Mycobacterium tuberculosis strains of the Beijing genotype are rarely observed in tuberculosis patients in South America

The frequency of the Beijing genotype of Mycobacterium tuberculosis as a cause of tuberculosis (TB) in South America was determined by analyzing genotypes of strains isolated from patients that had been diagnosed with the disease between 1997 and 2003 in seven countries of the subcontinent. In total, 19 of the 1,202 (1.6%) TB cases carried Beijing isolates, including 11 of the 185 patients from Peru (5.9%), five of the 512 patients from Argentina (1.0%), two of the 252 Brazilian cases (0.8%), one of the 166 patients from Paraguay (0.6%) and none of the samples obtained from Chile (35), Colombia (36) and Ecuador (16). Except for two patients that were East Asian immigrants, all cases with Beijing strains were native South Americans. No association was found between carrying a strain with the Beijing genotype and having drug or multi-drug resistant disease. Our data show that presently transmission of M. tuberculosis strains of the Beijing genotype is not frequent in Latin America. In addition, the lack of association of drug resistant TB and infection with M. tuberculosis of the Beijing genotype observed presently demands efforts to define better the contribution of the virulence and lack of response to treatment to the growing spread of Beijing strains observed in other parts of the world.     

Mycobacterium africanum--review of an important cause of human tuberculosis in West Africa

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.     

Modern and ancestral genotypes of Mycobacterium tuberculosis from Andhra Pradesh, India

Traditionally, the distribution of the Mycobacterium tuberculosis genotypes in India has been characterized by widespread prevalence of ancestral lineages (TbD1+ strains and variants) in the south and the modern forms (TbD1(-) CAS and variants) predominating in the north of India. The pattern was, however, not clearly known in the south-central region such as Hyderabad and the rest of the state of Andhra Pradesh where the prevalence of both tuberculosis (TB) and human immunodeficiency virus (HIV) infection is one of the highest in the country; this area has been the hotspot of TB vaccine trials. Spoligotyping of 101 clinical isolates obtained from Hyderabad and rural Andhra Pradesh confirmed the occurrence of major genogroups such as the ancestral (or the TbD1+ type or the East African Indian (EAI) type), the Central Asian (CAS) or Delhi type and the Beijing lineage in Andhra Pradesh. Sixty five different spoligotype patterns were observed for the isolates included in this study; these were further analyzed based on specific genetic signatures/mutations. It was found that the major genogroups, CAS and "ancestral," were almost equally prevalent in our collection but followed a north-south compartmentalization as was also reported previously. However, we observed a significant presence of MANU lineage in south Andhra Pradesh, which was earlier reported to be overwhelmingly present in Mumbai. This study portrays genotypic diversity of M. tuberculosis from the Indian state of Andhra Pradesh and provides a much needed snapshot of the strain diversity that will be helpful in devising effective TB control programs in this part of the world.     

IS6110 restriction fragment length polymorphism of Mycobacterium tuberculosis isolated from patients with pulmonary tuberculosis in Campinas,Brazil: evidence of intercontinental distribution of strains

Tuberculosis (TB) is a major concern in developing countries. In Brazil, few genotyping studies have been conducted to verify the number of IS6110 copies present in local prevalent strains of Mycobacterium tuberculosis, the distribution and clustering of strains. IS6110 DNA fingerprinting was performed on a sample of M. tuberculosis isolates from patients with AFB smear-positive pulmonary TB, at a hospital in Brazil. The IS6110 profiles were analyzed and compared to a M. tuberculosis database of the Houston Tuberculosis Initiative, Houston, US. Seventy-six fingerprints were obtained from 98 patients. All M. tuberculosis strains had an IS6110 copy number between 5-21 allowing for differentiation of the isolates. Human immunodeficiency virus infection was confirmed in nearly half the patients of whom data was available. Fifty-eight strains had unique patterns, while 17 strains were grouped in 7 clusters (2 to 6 strains). When compared to the HTI database, 6 strains matched isolates from El Paso, Ciudad de Juarez, Houston, and New York. Recently acquired infections were documented in 19% of cases. The community transmission of infection is intense, since some clustered strains were recovered during the four-year study period. The intercontinental dissemination of M. tuberculosis strains is suspected by demonstration of identical fingerprints in a distant country.     

Lack of correlation between colony morphology and lipooligosaccharide content in the Mycobacterium tuberculosis complex.

Rough and smooth colony variants of the Mycobacterium tuberculosis complex were compared with respect to their composition in trehalose-containing glycolipid antigens in view of the results of a recent investigation suggesting that the chemical basis of rough and smooth colony morphology in mycobacteria may reside in the occurrence of lipooligosaccharides. A careful chemical characterization of the individual glycolipids of the selected strains allowed the identification of the major glycolipids. The comparative study of the glycolipid content of the smooth Canetti strain, its spontaneous rough variant, and 16 additional strains of M. tuberculosis, M. bovis and M. africanum showed that the presence of lipooligosaccharides was not related to the morphology of the colonies.     

Differentiation of the Mycobacterium tuberculosis W-Beijing family strains from the Russian Federation by the VNTR-typing

Recent phylogenetic studies allowed the Mycobacterium tuberculosis complex to be divided into a number of the strain families. The W-Beijing family is one of most widespread M. tuberculosis variants frequently causing epidemic outbreaks. This family is genetically homogenous and conserved so that ETR A, B, C, D, E - typing is insufficient for the W-Beijing differentiation. All W-Beijing isolates have common profile (42435). This led to the false clustering in the molecular epidemiology study, especially in the region of predominance of the W-Beijing family. In this investigation we searched for the VNTR loci with high evolution rate, which were polymorphic in the W-Beijing genome. Eleven VNTR-loci were assayed in the DNA panel of 99 M. tuberculosis isolates from the tuberculosis patients in North-West and West-Siberian regions of Russia during the period from 2000 to 2001. Ninety nine strains of M. tuberculosis were divided into 74 VNTR-types, 51 isolates of the W-Beijing family were subdivided into 30 VNTR-types. The Hunter-Gudson index (HGDI) for all studied loci (ETR-A, ETR-C, ETR-E, V, V2, V3, V4, V5, V6, V10, V11) was close to one of the IS6110 RFLP indices being "the gold standard" of the M. tuberculosis complex genotyping. The V2, V3 loci located in the sequences of the PPE gene family, were highly polymorphic and more discriminative then others (HGDI is about 0.8). The congruence between the IS6110 RFLP-typing and 11 loci VNTR-typing was measured during genotyping for 23 isolates of the W-Beijing family. The isolates were divided into 9 genotypes by the IS6110 RFLP and into 13 variants by the VNTR-typing. The profiles correlation coefficient was 0.767689 that reflected the differences in the rate and type of the given genome target evolution.     

Functional and phenotypic changes in monocytes from patients with tuberculosis are reversed with treatment

Alterations of monocyte/macrophages have been reported in patients with tuberculosis (TB), but their significance is poorly understood. Blood mononuclear cells from patients with different clinical forms of TB, at various times of anti-TB treatment, and healthy tuberculin positive individuals, were double-stained for CD14 plus CD206, TLR-2, IFN-gammaR1, CD40, HLA-DR, CD36 and CD163, and analyzed by flow cytometry. Monocytes were infected with Mycobacterium tuberculosis H37Rv and 24h later the phenotype, induction of necrosis and apoptosis and production of tumor necrosis factor TNFalpha, interleukin (IL)-10 and IL-12p40 were determined. TB patients presented higher percentage of CD14+ cells but lower percentage of CD14+DR+ and CD14+CD36+ cells. Expression of CD14, HLA-DR and CD36 was decreased in TB patients. Normal percentages and expression were restored during anti-TB treatment. Monocytes from TB patients underwent necrosis and apoptosis after M. tuberculosis infection, whereas monocytes from healthy controls exhibited only apoptosis. Anti-TB treatment reverted necrosis. There were no differences between the various clinical forms of TB. In vitro M. tuberculosis infection decreased expression of the membrane molecules studied. HLA-DR and CD36 inhibition correlated with induction of apoptosis. Restoration of monocyte alterations during anti-TB treatment suggests that such alterations may be caused by the high M. tuberculosis load present during active disease.     

Comparison of the mycobacteria growth indicator tube with radiometric and solid culture for isolation of mycobacteria from clinical specimens and susceptibility testing of Mycobacterium tuberculosis

We compared the mycobacteria growth indicator tube (MGIT) system with the BACTEC 460 TB and Loewenstein-Jensen (LJ) systems for the recovery of mycobacteria (acid-fast bacilli [AFB]) from 600 clinical specimens. A total of 50 AFB (32 Mycobacterium tuberculosis complex, 10 M. avium complex, 3 M. gordonae, 3 M. xenopi, 1 M. terrae and 1 M. fortuitum) were detected. MGIT recovered 50 isolates of AFB (100% sensitivity), and BACTEC 460 TB and LJ recovered 49 (98% sensitivity) and 19 (38% sensitivity) AFB isolates, respectively. The mean times to detect mycobacteria were 10, 10 and 25 days for MGIT, BACTEC 460, and LJ slants. All isolates of M. tuberculosis complex were tested for susceptibility to streptomycin, isoniazid, rifampin, and ethambutol with the MGIT and BACTEC 460 TB. Both systems yielded identical susceptibility data with different mean times to report (5.38 days for MGIT versus 7.33 days for BACTEC 460 TB, P<0.05). The results suggest that MGIT is equivalent to BACTEC 460 TB in its ability to support the growth of mycobacteria, but significantly more efficient than LJ. MGIT may also be used for susceptibility testing of primary antituberculosis drugs.     

Relationship between single nucleotide polymorphism of interleukin-18 and susceptibility to pulmonary tuberculosis in the Chinese Han population

Interleukin-18 (IL-18) is a multi-functional cytokine capable of inducing either Th1 or Th2 polarization depending on the immunologic milieu. IL-18 may influence the host response to Mycobacterium tuberculosis (M.tb) infection. To investigate the relationship between single nucleotide polymorphisms of the IL-18 and susceptibility to pulmonary tuberculosis in the Chinese Han population, the IL-18 gene was sequenced to detect polymorphisms and to examine the genotype frequencies in 300 patients and 702 healthy controls. DNA sequencing revealed three IL-18 variants: rs1946518, rs5744247, and rs549908. It also revealed that allele A of rs1946518 confers a 1.47-fold increased risk of developing tuberculosis (TB) (P = 0.0001, OR [95%CI] = 1.47 [1.21-1.78]), and that the C allele of rs5744247 confers a 0.77-fold decreased risk of disease (P = 0.01, R [95%CI] = 0.77 [0.632-0.937]). The genotypes rs1946518, rs5744247 and rs549908 were found to be significantly associated with TB. Estimation of the frequencies of haplotypes revealed a potential risk haplotype AGA (P = 0.01, OR [95%CI] = 1.41 [1.15-1.72]) and a protective haplotype CCA (P = 0.01, OR [95%CI] = 0.70 [0.57-0.85]) for TB. The present findings suggest that polymorphisms in the IL-18 gene may affect susceptibility to TB and increase the risk of developing the disease in the Chinese Han population.