Disseminated infection due to multidrug-resistant Mycobacterium bovis in a patient who was seropositive for human immunodeficiency virus.
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Antibiotics, Antitubercular↗
Biomedical subjects
D van Soolingen
Publications and source records attributed to D van Soolingen.
Characterization of the catalase-peroxidase gene (katG) and inhA locus in isoniazid-resistant and -susceptible strains of Mycobacterium tuberculosis by automated DNA sequencing: restricted array of mutations associated with drug resistance.
The catalase-peroxidase gene (katG) and a two-gene locus (inhA) containing mutations associated with resistance to isoniazid in Mycobacterium tuberculosis were sequenced in 34 resistant and 12 susceptible strains. Virtually all resistant organisms had amino acid changes in KatG or nucleotide substitutions upstream of inhA. A region of katG encoding two amino acids frequently altered in resistant strains (residues Ser315 and Arg463) and the inhA locus were sequenced in 10 susceptible and 51 isoniazid-resistant isolates from the Netherlands. Most (84%) of the resistant isolates had mutations in katG or the inhA locus or lacked katG. Together, approximately 75% of isoniazid-resistant isolates had replacements at amino acids 315 or 463 in KatG or nucleotide substitutions upstream of inhA. All 16 strains of Mycobacterium bovis and Mycobacterium microti studied had Leu463 rather than Arg463 in KatG, an observation consistent with the hypothesis that Leu463 is the ancestral condition in M. tuberculosis.
Amino Acid Sequence↗
Host-mediated modification of PvuII restriction in Mycobacterium 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.
Bacterial Proteins↗
Use of IS6110 DNA fingerprinting in tracing man-to-man transmission of Mycobacterium tuberculosis in the Czech Republic.
The epidemiological relationship between tuberculosis cases in a prison and between cases within five families was investigated. Therefore, the isolated Mycobacterium tuberculosis strains were subjected to restriction fragment length polymorphism (RFLP) analysis using insertion sequence IS6110 as a probe. In case of 11 patients, the expected links of transmission were confirmed by RFLP typing. In contrast, in case of 4 patients the conclusion of classical contact tracing were not in agreement with the DNA fingerprinting results. These findings reinforce the usefulness of this recently developed technique as an additional tool in contact tracing. The IS6110DNA fingerprints of all strains investigated consisted of 7 to 13 bands and showed a high degree of polymorphism. Comparison of these fingerprints with those recorded in the Czech Republic previously, revealed the presence of a predominant DNA fingerprint type, without a known connection between the cases. Furthermore, other patterns found in the present study showed a high degree of similarity with the previously obtained fingerprints. Part of the patients were sampled twice. All of these double isolates showed identical fingerprints, confirming the previously described stability of IS6110DNA fingerprints. In contrast, from one couple of strains, isolated from husband and wife both suffering from tuberculosis, a slight change in one of the two patterns was observed. The patterns shared 10 band positions, confirming the expected relationship between these cases, but one of the patterns contained one additional band.
Contact Tracing↗
[Osteomyelitis as a complication following BCG vaccination].
Osteomyelitis of the proximal femur was diagnosed in a 5-month-old girl after BCG vaccination at the age of 2 weeks. The diagnosis was confirmed by biochemical and molecular techniques (DNA fingerprinting). The girl responded well to antituberculous treatment.
Antitubercular Agents↗
Laboratory-acquired tuberculosis.
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DNA Fingerprinting↗
Analysis of the population structure of Mycobacterium tuberculosis in Ethiopia, Tunisia, and The Netherlands: usefulness of DNA typing for global tuberculosis epidemiology.
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.
Cluster Analysis↗
Unique gene organization of thioredoxin and thioredoxin reductase in Mycobacterium leprae.
The thioredoxin system comprising thioredoxin (Trx), thioredoxin reductase (TR) and NADPH operates via redox-active disulphides and provides electrons for a wide variety of different metabolic processes in prokaryotic and eukaryotic cells. Thioredoxin is also a general protein disulphide reductase involved in redox regulation. In bacteria, the Trx and TR proteins previously identified were encoded by separate genes (trxA and trxB). In this study, we report a novel genomic organization of TR and Trx in mycobacteria and show that at least three modes of organization of TR and Trx genes can exist within a single bacterial genus: (i) in the majority of mycobacterial strains the genes coding for TR and Trx are located on separate sites of the genome; (ii) interestingly, in all pathogenic Mycobacterium tuberculosis complex mycobacteria both genes are found on the same locus, overlapping in one nucleotide; (iii) in the pathogen Mycobacterium leprae, TR and Trx are encoded by a single gene. Sequence analysis of the M. leprae gene demonstrated that the N-terminal part of the protein corresponds to TR and the C-terminal part to Trx. A corresponding single protein product of approximately 49 kDa was detected in cell extracts of M. leprae. These findings demonstrate the very unusual phenomenon of a single gene coding for both the substrate (thioredoxin) and the enzyme (thioredoxin reductase), which seems to be unique to M. leprae.
Amino Acid Sequence↗
Predominance of a single genotype of Mycobacterium tuberculosis in countries of east Asia.
Analysis of the population structure of Mycobacterium tuberculosis strains from the People's Republic of China showed that the vast majority belong to a genetically closely related group. These strains shared the majority of their IS6110 DNA-containing restriction fragments, and also, the DNA polymorphism associated with other repetitive DNA elements, like the polymorphic GC-rich sequence and the direct repeat, was very limited. Because the majority of these strains originated from the province of Beijing, we designated this grouping the "Beijing family" of M. tuberculosis strains. Strains of this family were also found to dominate in neighboring countries such as Mongolia, South Korea, and Thailand, whereas a low prevalence of such strains was observed in countries on other continents. These data indicate that strains of the Beijing family recently expanded from a single ancestor which had a selective advantage. It is speculated that long-term Mycobacterium bovis BCG vaccination may be one of the selective forces implicated in the successful spread of the Beijing genotype.
China↗
DNA fingerprinting and phenotyping of Mycobacterium tuberculosis isolates from human immunodeficiency virus (HIV)-seropositive and HIV-seronegative patients in Tanzania.
With the purpose of determining whether the risk of infection with a particular clone of Mycobacterium tuberculosis is influenced by the human immunodeficiency virus (HIV) status of the host, we analyzed and compared 68 mycobacterial isolates obtained from HIV-seropositive patients with tuberculosis (TB) in Dar es Salaam, Tanzania, with 66 mycobacterial isolates obtained from HIV-seronegative patients with TB in the same geographical region by using both DNA fingerprinting and classical phenotyping methods. One hundred one different IS6110 fingerprinting patterns were observed in the 134 isolates. The level of diversity of the DNA fingerprints observed in the HIV-seropositive group was comparable to the level of the diversity observed in the HIV-seronegative group. Resistance to a single anti-TB drug was found in 8.8% of the tested isolates, and 3.2% of the isolates were resistant to more than one anti-TB drug. The drug susceptibility profiles were not significantly difference between the two groups of isolates compared in the present study. Phenotypic characteristics which classify M. tuberculosis strains as belonging to the Asian subgroup correlated with a low IS6110 copy number per isolate. However, the occurrence of Asian subgroup strains was not associated with the HIV status of the patients. The results of the study suggested an equal risk of infection with a defined M. tuberculosis clone for HIV-seropositive and HIV-seronegative individuals.
AIDS-Related Opportunistic Infections↗
Molecular epidemiology of tuberculosis in Denmark in 1992.
The incidence of tuberculosis (TB) is increasing all over the world, including in countries with a high standard of living and good social security. Denmark represents such a region. Furthermore, it is a small country (5 million inhabitants) with a long tradition in TB control, including a centralization of the bacteriological diagnostic facility. The present study was intended to analyze the transmission of Mycobacterium tuberculosis in a country in which TB has low endemicity by a combination of conventional epidemiological approaches and DNA fingerprinting techniques, whereby individual bacterial strains can be traced. M. tuberculosis isolates from 92% of all new cases of bacteriologically verified TB in Denmark during 1992 were subjected to IS6110 DNA fingerprinting to visualize the DNA restriction fragment length polymorphism (RFLP) patterns of the isolated strains. The data obtained from the RFLP analyses were interpreted by using demographic data, such as age, sex, ethnicity, and residence, for the patients. The risk factors among the patients for being part of an active chain of transmission, as opposed to demonstrating reactivation of a previously acquired latent infection, were estimated by statistical analyses. The magnitude of TB transmission in 1992 in Denmark was determined, and transmitted infections were shown to comprise at least one quarter of the total number of cases. Almost half of the TB cases involved patients of foreign origin. However, most of these isolates showed unique DNA fingerprint patterns and were rarely part of an active chain of transmission. The major chains of recent transmission were localized to distinct geographical regions in the country. TB is frequent among immigrants, especially from Asia and Africa, but it is apparently readily suspected, diagnosed, and treated by the health care system. Danish patients with pulmonary symptoms are not primarily suspected to have TB and, therefore, play an important role in recent TB transmission in Denmark.
Adolescent↗
Epidemiology of tuberculosis by DNA fingerprinting.
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.
Contact Tracing↗
DNA fingerprinting of Mycobacterium tuberculosis.
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Base Sequence↗
Use of various genetic markers in differentiation of Mycobacterium bovis strains from animals and humans and for studying epidemiology of bovine tuberculosis.
One hundred fifty-three Mycobacterium bovis strains from cattle, various animal species from zoos and wild parks, and humans were analyzed for three different genetic markers for use in the epidemiology of bovine tuberculosis. M. bovis strains isolated from cattle were found to carry a single IS6110 element, whereas the majority of strains from other animals such as antelopes, monkeys, and seals harbored multiple IS6110 elements, suggesting that the reservoirs in cattle and wild animals are separated. Because the single IS6110 element in cattle strains is located at the same chromosomal position, strain differentiation by insertion sequence fingerprinting was hampered. Therefore, we investigated the usefulness of the direct repeat and polymorphic GC-rich repeat elements for strain differentiation. Both markers allowed sufficient strain discrimination for epidemiological purposes. Evidence is presented that in Argentina, most human M. bovis infections are due to transmission from cattle, whereas M. bovis infections among humans in the Netherlands are mainly contracted from animals other than cattle. Various outbreaks of M. bovis among animals and humans are described, including a small one which likely involved transmission from human to human.
Animals↗
Restriction fragment length polymorphism Mycobacterium tuberculosis strains isolated from Greenland during 1992: evidence of tuberculosis transmission between Greenland and Denmark.
In order to describe the transmission of tuberculosis (TB) at the clonal level in a defined geographic region during a certain period of time, all isolates of Mycobacterium tuberculosis collected during 1992 from Greenland were subjected to analyses of DNA restriction fragment length polymorphism (RFLP). The RFLP patterns obtained by probing the genomic DNA with the repetitive insertion segment IS6110 revealed a high degree of similarity among the isolates, indicating a relatively high transmission rate and a close relationship between the individual M. tuberculosis clones. This was further confirmed by reprobing the Southern blots with two more-stable genetic markers, IS1081 and the DR sequence. The RFLP patterns were compared with those of 245 M. tuberculosis strains collected from Denmark during the same period (representing 91% of all new, bacteriologically verified cases of TB in Denmark in 1992). One of the three prevalent IS6110-defined clusters was traced to a group of immigrants from Greenland living in a small, defined geographical region in Denmark and to a group of Danish citizens either with known contact with these immigrants or, in other cases, with a record of previous travel or working activities in Greenland. The study showed that the present technique is extremely helpful in monitoring the spread of TB and thereby also contributing to improved disease control.
Cross-Sectional Studies↗
Nature of DNA polymorphism in the direct repeat cluster of Mycobacterium tuberculosis; application for strain differentiation by a novel typing method.
Mycobacterium tuberculosis complex strains contain a unique chromosomal region, which consists of multiple 36bp direct repeats (DRs), which are interspersed by unique spacers 35 to 41 bp in length. In this study we investigated the nature of the DNA polymorphism of this DR cluster by sequencing part of this region in a large number of M. tuberculosis complex strains. Two types of genetic rearrangements were observed. One type consists of the variation in one or a few discrete, contiguous DRs plus spacer sequences. This variation is probably driven by homologous recombination between adjacent or distant DRs. The other type of polymorphism is probably driven by transpositional events of the insertion sequence, IS6110, which is almost invariably present in the DR cluster of M. tuberculosis complex strains. Based on the nature of the DNA polymorphism in the DR cluster, we developed a novel method of strain differentiation, direct variable repeat polymer chain reaction (DVR-PCR), which enables typing of individual M. tuberculosis strains in a single PCR. The method allows an excellent differentiation of epidemiologically unrelated isolates and, in principle, the DVR-PCR allows the detection of M. tuberculosis and strain differentiation at the same time.
Bacterial Typing Techniques↗
Comparison of various repetitive DNA elements as genetic markers for strain differentiation and epidemiology of Mycobacterium tuberculosis.
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.
Bacterial Typing Techniques↗
Sero-diagnosis of tuberculosis with A60 antigen enzyme-linked immunosorbent assay: failure in HIV-infected individuals in Ghana.
In order to assess the diagnostic usefulness of the A60 (ANDA Biological, Strassbourg, France) sero-diagnostic enzyme-linked immunosorbent assay (ELISA) kit for tuberculosis in Africa, sera of 53 pulmonary smear-positive tuberculosis (TB) patients, 30 apparently healthy control subjects and 6 AIDS suspects were sampled in Agogo Hospital in the forest area of Ghana. These sera were analyzed for antibodies to HIV-1 and HIV-2, and IgG-antibodies to the A60 BCG-antigen, while the non-HIV individuals were tested for total IgG levels. One healthy control subject, all of 6 AIDS suspects and 7 of the TB patients has HIV infections. In the non-HIV TB group, the sensitivity and specifity of the A60 ELISA was 78% and 86%, respectively, which was much poorer than expected from published reports about the A60 test. The A60 test failed, completely however, to discriminate between TB and non-TB in the HIV-positive group. In the non-HIV groups, total IgG levels were significantly higher in TB patients than in controls. It seems that the usefulness of the A60 ELISA test to diagnose tuberculosis is very limited in this high-incidence area, and that it seems to be of no value in patients infected with HIV.
Adult↗