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The immune regulatory mechanisms involved in the acquisition of Mycobacterium tuberculosis infection in children are largely unknown. We investigated the influence of parasitic infections, malnutrition and plasma cytokine profiles on tuberculin skin test (TST) positivity in Warao Amerindians in Venezuela. Pediatric household contacts of sputum smear-positive tuberculosis (TB) cases were enrolled for TST, chest radiograph, plasma cytokine analyses, QuantiFERON-TB Gold In-Tube (QFT-GIT) testing and stool examinations. Factors associated with TST positivity were studied using generalized estimation equations logistic regression models. Of the 141 asymptomatic contacts, 39% was TST-positive. After adjusting for age, gender and nutritional status, TST positivity was associated with Trichuris trichiura infections (OR 3.5, 95% CI 1.1-11.6) and low circulating levels of T helper 1 (Th1) cytokines (OR 0.51, 95% CI 0.33-0.79). Ascaris lumbricoides infections in interaction with Th2- and interleukin (IL)-10-dominated cytokine profiles were positively associated with TST positivity (OR 3.1, 95% CI 1.1-8.9 and OR 2.4, 95% CI 1.04-5.7, respectively). A negative correlation of QFT-GIT mitogen responses with Th1 and Th2 levels and a positive correlation with age were observed (all p < 0.01). We conclude that helminth infections and low Th1 cytokine plasma levels are significantly associated with TST positivity in indigenous Venezuelan pediatric TB contacts., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Background: Tuberculosis (TB) transmission is associated with patient-related risk factors. However, DNA fingerprint analysis has provided anecdotal evidence suggesting a role for bacteriological factors., Methods: To examine the importance of the bacteriological component in TB transmission, we investigated the number of tuberculin skin test-positive (TST induration, ≥ 10 mm) contacts and secondary cases observed in contact investigations around TB cases in relation to the size of the genotype cluster the patient belonged to at the time of diagnosis. We also compared the number of TST-positive contacts and secondary cases of patients with drug-resistant and drug-susceptible TB., Results: Larger clusters were independently associated with an increased number of positive contacts. The mean number of positive contacts ranged from 3.8 for clusters of 2 cases, to 4.7 for clusters of 3-10 cases, to 6.0 for cases in clusters of >10 cases (mean increase in number of positive contacts for every extra case in the cluster, 0.21; 95% confidence interval, 0.09-0.26). The mean number of positive contacts was significantly lower among index cases with isoniazid-monoresistant TB (1.6) than among index cases with pan-susceptible TB (4.6; relative number, 0.45; 95% confidence interval, 0.22-0.92)., Conclusion: These results suggest that spread of tuberculosis also depends on bacteriological factors.

In this study, the currently known typing methods for Mycobacterium tuberculosis isolates were evaluated with regard to reproducibility, discrimination, and specificity. Therefore, 90 M. tuberculosis complex strains, originating from 38 countries, were tested in five restriction fragment length polymorphism (RFLP) typing methods and in seven PCR-based assays. In all methods, one or more repetitive DNA elements were targeted. The strain typing and the DNA fingerprint analysis were performed in the laboratory most experienced in the respective method. To examine intralaboratory reproducibility, blinded duplicate samples were included. The specificities of the various methods were tested by inclusion of 10 non-M. tuberculosis complex strains. All five RFLP typing methods were highly reproducible. The reliability of the PCR-based methods was highest for the mixed-linker PCR, followed by variable numbers of tandem repeat (VNTR) typing and spoligotyping. In contrast, the double repetitive element PCR (DRE-PCR), IS6110 inverse PCR, IS6110 ampliprinting, and arbitrarily primed PCR (APPCR) typing were found to be poorly reproducible. The 90 strains were best discriminated by IS6110 RFLP typing, yielding 84 different banding patterns, followed by mixed-linker PCR (81 patterns), APPCR (71 patterns), RFLP using the polymorphic GC-rich sequence as a probe (70 patterns), DRE-PCR (63 patterns), spoligotyping (61 patterns), and VNTR typing (56 patterns). We conclude that for epidemiological investigations, strain differentiation by IS6110 RFLP or mixed-linker PCR are the methods of choice. A strong association was found between the results of different genetic markers, indicating a clonal population structure of M. tuberculosis strains. Several separate genotype families within the M. tuberculosis complex could be recognized on the basis of the genetic markers used.

In an attempt to characterize an unusual mycobacterial strain isolated from a 2-year-old Somali patient with lymphadenitis, we applied various molecular methods not previously used for the taxonomic classification of mycobacteria. This isolate, designated So93, did not differ from Mycobacterium tuberculosis in the biochemical tests and in its 16S rRNA sequence, but produced smooth and glossy colonies, which is highly exceptional for this species. This smooth phenotype was unstable and switched nonreversibly to a rough colony morphology with a low frequency. The two colony types were equally virulent for the guinea pig, exhibiting characteristic tuberculous disease. Both morphotypes had shorter generation times than the M. tuberculosis reference laboratory strain H37Rv and clinical isolates of M. tuberculosis and Mycobacterium bovis. Furthermore, the So93 isolate differed from all M. tuberculosis complex strains described thus far by having only a single copy of insertion sequence IS1081, an unusual composition of the direct repeat cluster, and a characteristic phenolic glycolipid and lipooligosaccharide. This glycolipid had previously been observed only in a smooth isolate of M. tuberculosis obtained in 1969 by Canetti in France. Analysis of the Canetti strain showed that it shared virtually all genetic properties characteristic of So93, distinguishing these two strains from the known M. tuberculosis complex taxa, M. tuberculosis, Mycobacterium africanum, M. bovis, and Mycobacterium microti. The natural reservoir, host range, and mode of transmission of the group of bacteria described in this paper are presently unknown. This study, partly based on not previously used molecular criteria, supports the idea that the established members within the M. tuberculosis complex and the newly described Canetti grouping should be regarded as a single species, which likely will be designated "M. tuberculosis".

Restriction endonuclease PvuII plays a central role in restriction fragment length polymorphism analysis of Mycobacterium tuberculosis complex isolates with IS6110 as a genetic marker. We have investigated the basis for an apparent dichotomy in PvuII restriction fragment pattersn observed among strains of the M. tuberculosis complex. The chromosomal regions of two modified PvuII restriction sites, located upstream of the katG gene and downstream of an IS1081 insertion sequence, were studied in more detail. An identical 10-bp DNA sequence (CAGCTGGAGC) containing a PvuII site was found in both regions, and site-directed mutagenesis analysis revealed that this sequence was a target for modification. Strain-specific modification of PvuII sites was identified in DNA from over 80% of the nearly 800 isolates examined. Furthermore, the proportion of modifying and nonmodifying strains differs significantly from country to country.

In recent years, deoxyribonucleic acid (DNA) probes have become available to study the epidemiology of tuberculosis by DNA fingerprint techniques. These methods make it possible to distinguish different strains of Mycobacterium tuberculosis. Currently, DNA fingerprinting is integrated with conventional epidemiological approaches to improve understanding of the spread of tuberculosis. This molecular approach has led to the investigation of a wide variety of epidemiological issues, such as adequate identification of outbreaks, tracing of nosocomial infections in hospitals, and investigation of the relative contribution of newly acquired versus reactivated infections in different populations. This paper reviews the potentials of DNA fingerprinting for studying the epidemiology of tuberculosis, and the lessons learned from this strategy. In addition, future prospects for molecular epidemiology will be discussed.

The genetic heterogeneity among Mycobacterium tuberculosis isolates from 501 patients in Ethiopia, Tunisia, and the Netherlands was compared by analysis of DNA polymorphism driven by insertion element IS6110. The percentage of isolates displaying two or more identical patterns differed greatly in the three countries: It was highest among Tunisian isolates and lowest in Dutch isolates. In contrast to isolates from Dutch subjects infected with M. tuberculosis, the majority of strains from Ethiopia and Tunisia were from a few families of genetically highly related strains. Furthermore, little overlap was observed among isolates from the three countries, indicating strict isolation of the bacterial reservoirs in the countries. A few strains from the Netherlands matched strains from Ethiopia and Tunisia. Those strains were invariably isolated from refugees, immigrants, or persons who visited Ethiopia or Tunisia.

Crossed immunoelectrophoresis (CIE) has been used to develop a reference system for classifying mycobacterial antigens. The subsequent use of specific antibodies allowed further determination of antigens by molecular weight. The monoclonal antibody F126-2, originally raised against a 34-kDa antigen of Mycobacterium kansasii, reacted with antigen 84 (Ag84) in the CIE reference system for Mycobacterium bovis BCG and Mycobacterium tuberculosis. To characterize Ag84, we screened a lambda gt11 gene library from M. tuberculosis with antibody F126-2 and identified the encoding gene. The corresponding Mycobacterium leprae Ag84 gene was subsequently selected from a cosmid library, using the M. tuberculosis gene as a probe. Both genes were expressed as 34-kDa proteins in Escherichia coli, and the recombinant proteins indeed corresponded to Ag84 in the CIE reference system. The derived amino acid sequences of the M. tuberculosis and M. leprae proteins showed 85% identity, which indicates that Ag84 constitutes a group of highly conserved mycobacterial antigens. Antibodies of almost 60% of lepromatous leprosy patients responded to Ag84, indicating that the protein is highly immunogenic following infection in multibacillary leprosy.

Five different genetic elements have been found to be associated with genetic rearrangements in Mycobacterium tuberculosis complex strains. Of these elements, the insertion sequence IS6110 is presently the most frequently used genetic marker for strain differentiation of M. tuberculosis. In the present study we compared five genetic elements for their potentials to differentiate a given cluster of M. tuberculosis strains. Because of the presence of only a single copy of IS6110 or two IS6110 copies at the same chromosomal locus, a large number of strains could not be differentiated by IS6110 fingerprinting. Most strains, including the low-copy-number IS6110 strains, could be differentiated by fingerprinting with the 36-bp direct repeat or the polymorphic GC-rich repetitive DNA element. Less discriminative power was obtained with the major polymorphic tandem repeat and the insertion element IS1081. One strain which did not contain IS6110 DNA was encountered. Until now, this element has invariantly been found to be present in all M. tuberculosis complex strains. On the basis of classical taxonomic criteria and sequencing of the 16S rRNA gene, this strain was shown to be a genuine M. tuberculosis strain. Therefore, the use of this element as a target for polymerase chain reaction-facilitated detection of M. tuberculosis should be reconsidered.

A test based on the polymerase chain reaction (PCR) was developed for the detection of the Mycobacterium tuberculosis complex in clinical samples. In this test, a 245-bp sequence of the insertion element IS986 was amplified and detected by agarose gel electrophoresis in the presence of ethidium bromide and by Southern blot and dot blot hybridization by using a 188-bp digoxigenin-labeled probe. We tested clinical specimens from 227 patients suspected of having tuberculosis. These included 102 cerebrospinal fluid, 48 sputum, 18 pleural fluid, 5 bronchoalveolar lavage, 18 blood, 7 pus, 8 bone marrow, and 6 urine samples and 15 tissue biopsy specimens. We also tested sputum samples from 75 patients with diseases other than tuberculosis. Sputum samples were first decontaminated, and all samples were treated with proteinase K-detergent solution to extract the DNA. Part of each sample was spiked with M. tuberculosis to provide a semiquantitative assay and to control for the loss of mycobacteria or interference with the PCR which may cause false-negative results. One femtogram of M. tuberculosis DNA could be detected. PCR was positive for all 32 culture-positive (for M. tuberculosis) and Ziehl-Neelsen staining (ZN)-positive samples, 10 of 12 culture-positive and ZN-negative samples, and all 4 culture-negative and ZN-positive samples. PCR detected M. tuberculosis complex bacteria in 35 of 178 culture- and ZN-negative samples. Clinical data supported the diagnosis of tuberculosis in the majority of the 35 patients from whom those samples were obtained.

Recently, the insertion element IS1081 from Mycobacterium bovis was identified. In this study, the usefulness of IS1081 in the epidemiology of tuberculosis was investigated. The host range of this insertion sequence was found to be restricted exclusively to the group of Mycobacterium tuberculosis complex bacteria, whereas none of the 10 mycobacterial species which do not belong to the M. tuberculosis complex contained IS1081-homologous DNA. All 99 M. tuberculosis complex strains investigated carried five or six copies of IS1081, and very limited IS1081-associated restriction fragment length polymorphisms were observed among the strains. Seven different IS1081-containing bands were distinguished in each strain, and the patterns differed only in one or two insertion sequence-containing bands. The banding pattern of M. bovis BCG differed in the presence of a 8.0-kb IS1081-containing PvuII fragment which was absent from all other M. tuberculosis complex strains.

In this study, the occurrence of repeated DNA sequences in the chromosome of Mycobacterium tuberculosis was investigated systematically. By screening a M. tuberculosis lambda gt-11 gene library with labeled total chromosomal DNA, five strongly hybridizing recombinants were selected, and these contained DNA sequences that were present in multiple copies in the chromosome of M. tuberculosis. These recombinants all contained repeated sequences belonging to a single family of repetitive DNA, which shares homology with a previously described repeated sequence present in recombinant pPH7301. Sequences analysis of pPH7301 showed the presence of a 10-bp sequence that was tandemly repeated and invariably separated by 5-bp unique spacer sequences. Southern blot analysis revealed that the majority of the repeated DNA in M. tuberculosis is composed of this family of repetitive DNA. Because the 10-bp repeats are slightly heterogeneous in sequence, we designated this DNA as a major polymorphic tandem repeat, MPTR. The presence of this repeated sequence in various other mycobacterial species was investigated. Among the MPTR-containing mycobacterial species the chromosomal location of the repetitive DNA is highly variable. The potential use of this polymorphism in the epidemiology of mycobacterioses is discussed.

In this study we established the usefulness of DNA fingerprinting for the epidemiology of tuberculosis on the basis of the DNA polymorphism generated by the insertion sequence (IS) IS986. Although clinical isolates of Mycobacterium tuberculosis displayed a remarkably high degree of restriction fragment length polymorphism, we showed that transposition of this IS element is an extremely rare event in M. tuberculosis complex strains grown either in vitro or in vivo for long periods of time. The M. tuberculosis and Mycobacterium africanum strains tested in this study contained 6 to 17 IS copies. In the Mycobacterium bovis strains, the copy numbers ranged between 1 and 5, and all 27 M. bovis BCG strains investigated invariably contained a single IS copy. This copy was located at a unique chromosomal position, reinforcing the idea that the frequency of IS transposition is very low in M. tuberculosis complex strains. Various microepidemics are described in which each microepidemic corresponds to a particular fingerprint type. The extent of similarity between Dutch and African strains was quantitatively assessed by computer-assisted analysis of DNA fingerprints. The results indicate that M. tuberculosis strains from regions in central Africa, where tuberculosis is highly prevalent, are generally more related to each other than isolates from the Netherlands, where the transmission rate is low and where the majority of the tuberculosis cases are presumed to be the result of reactivation of previously contracted M. tuberculosis infections.

Most strains of the Mycobacterium tuberculosis complex carry multiple copies of an IS3-like element, and these strains are highly polymorphic with regard to the site of integration in the chromosome. In contrast, Mycobacterium bovis BCG contains a single copy of the insertion element, and in all strains this copy is integrated at the same site in the chromosome. In this study, we determined the sequence of the single-copy insertion element from M. bovis BCG, IS987, and its flanking regions. The analysis of IS987 revealed that this element was virtually identical to the sequence of IS986 from M. tuberculosis. IS987 is located in a region containing direct repeats (DRs). The cloned flanking regions contained 20 virtually identical DRs of 36 bp, each separated by 35 to 41 bp of spacer DNA. Analysis of chromosomal DNA by the polymerase chain reaction revealed the presence of a cluster of 49 DRs, and IS987 is inserted in the 30th DR. Furthermore, the DR sequences were found to occur only in species of the M. tuberculosis complex and not in nine other mycobacterial species tested. Analysis of 14 M. tuberculosis strains revealed the presence of one insertion sequence element in the DR-containing region of eight strains, two insertion sequence elements were located in the DR region of five strains, and one strain did not contain an insertion sequence element in this region. Additionally, the DR-containing regions of these 14 M. tuberculosis strains were polymorphic in length and composition. We conclude that the DR cluster is a specific, hot-spot region for integration of insertion elements in the chromosome of M. tuberculosis complex strains.

IS986 of Mycobacterium tuberculosis belongs to the IS3-like family of insertion sequences, and it has previously been shown to be present in multiple copies in the chromosome of M. tuberculosis. In this study we investigated the value of a IS986-based DNA probe in the diagnosis and epidemiology of tuberculosis. IS986 was found only in species belonging to the M. tuberculosis complex. Independent isolates of M. tuberculosis complex strains showed a very high degree of polymorphism of restriction fragments which contained IS986 DNA. In contrast, Mycobacterium bovis BCG vaccine strains as well as clinical isolates of M. bovis BCG contained one copy of IS986, which was present at the same location in the chromosome. Different M. tuberculosis isolates from a recent M. tuberculosis outbreak showed an identical banding pattern. We concluded that IS986 is an extremely suitable tool for the diagnosis and epidemiology of tuberculosis.

A repetitive element (IS986), previously isolated from Mycobacterium tuberculosis and shown to detect multiple restriction fragment-length polymorphisms (RFLPs), has been sequenced. It consists of a potential insertion sequence of 1358bp, with 30-bp inverted repeat ends. IS986 has four potentially significant open reading frames (ORFs): ORFa1, ORFa2 and ORFb on one strand and ORFc on the complementary strand. The sequences of the potential translated products identify IS986 as a member of the IS3 family, with an apparent frameshift between ORFa1 and ORFa2. IS986 has potential as a highly specific probe for detection and typing of M. tuberculosis, as well as for transposon mutagenesis of mycobacteria. The sequence of IS986 is virtually identical to that of another recently described element, IS6110 (Thierry et al., 1990).

During the screening of a Mycobacterium tuberculosis lambda gt-11 gene library with monoclonal antibodies, we detected a recombinant clone, lambda PH7311, which contained a mycobacterial DNA insert that hybridized specifically with DNA of M. tuberculosis complex strains. Part of this insert was sequenced and used for the development of an M. tuberculosis complex-specific polymerase chain reaction (PCR). Only strains belonging to species of the M. tuberculosis complex group contained an amplifiable fragment of 158 base pairs (bp). This fragment was absent in all strains tested belonging to 15 other mycobacterial species. After amplification by PCR and dot blot hybridization with a digoxigenin-labeled oligonucleotide, the limit of detection of purified genomic M. tuberculosis DNA amounted to a quantity corresponding to 20 bacterial cells. By this technique about 10(3) M. tuberculosis bacteria were detectable in sputum. Using PCR, we were also able to detect M. tuberculosis cells in clinical material such as pleural fluid, bronchial washings, and biopsies, and these results were comparable with those obtained by classical bacterial culture. Of 34 M. tuberculosis strains, 5 did not carry the amplifiable 158-bp fragment, which occurs usually as a single copy in the chromosome. Evidence is presented that the 158-bp fragment is located near a repeated sequence in the chromosome. We presume that strains which did not carry the 158-bp fragment have lost a chromosomal segment by a genetic rearrangement induced by the repetitive DNA element.