Public health impact of isoniazid-resistant Mycobacterium tuberculosis strains with a mutation at amino-acid position 315 of katG: a decade of experience in The Netherlands.

A previous limited study demonstrated that Mycobacterium tuberculosis isolates with a mutation at amino-acid position 315 of katG (Delta315) exhibited high-level resistance to isoniazid and were more frequently resistant to streptomycin. In the present study, isoniazid-resistant M. tuberculosis isolates from 8,332 patients in The Netherlands (1993-2002) were screened for the Delta315 mutation. Isoniazid resistance was found in 592 (7%) isolates, of which 323 (55%) carried Delta315. IS6110 restriction fragment length polymorphism analysis showed that Delta315 isolates occurred in clusters, suggesting recent transmission, at the same frequency as isoniazid-susceptible isolates. In contrast, other isoniazid-resistant isolates clustered significantly less frequently. Delta315 isolates were high-level isoniazid-resistant, streptomycin-resistant and multidrug-resistant significantly more often, and may have a greater impact on public health, than other isoniazid-resistant isolates.

Molecular epidemiology of tuberculosis in Cuba outside of Havana, July 1994-June 1995: utility of spoligotyping versus IS6110 restriction fragment length polymorphism.

SETTING: Molecular typing has become an important tool for examining the extent of active transmission of tuberculosis. OBJECTIVES: To examine transmission of tuberculosis in Cuba using IS6110 restriction fragment length polymorphism (RFLP) typing and to evaluate the utility of spoligotyping. DESIGN: One hundred and sixty Mycobacterium tuberculosis strains isolated over a one year period in Cuba were subjected to RFLP and spoligotyping. RESULTS: Forty-eight percent of the isolates were found in 19 clusters of strains with identical RFLP patterns. In general, cluster sizes were limited, except for two large institutional outbreaks. Age was strongly inversely correlated to clustering. Most streptomycin-resistant isolates were found in clusters. Fifteen spoligotype clusters comprised 78% of the isolates. Significantly different IS6110 RFLP types subdivided 11 spoligotype clusters, whereas none of the IS6110 clusters were subdivided by spoligotyping. CONCLUSIONS: Considering the short study period, 48% clustering is high, indicating that recent transmission plays an important role in Cuba. Although resistance is still a minor problem, transmission of streptomycin-resistant strains occurs. The high polymorphism observed with IS6110 RFLP indicates that this marker is useful for future molecular epidemiological studies in Cuba. Spoligotyping appeared less suitable for population-based studies.

Investigation of cross contamination in a Mycobacterium tuberculosis laboratory using IS6110 DNA fingerprinting.

SETTING: A laboratory for routine culturing of Mycobacterium tuberculosis. OBJECTIVE: Investigation of an episode of laboratory cross contamination using IS6110 restriction fragment length polymorphism (RFLP) typing. Improvement of laboratory protocols to prevent contaminations in the future. To stress the importance of 'good laboratory practice', and interaction with clinicians about laboratory results. DESIGN: Fingerprinting of mycobacterial isolates from 1) cultures suspected of being contaminated and 2) strains suspected of being the source of the cross-contamination. RESULTS: RFLP typing results indicated that clinical samples were contaminated by strains which had been processed in species identification procedures one day earlier in the same safety cabinet. This cross contamination also resulted in exceptional RFLP typing results--mixed banding patterns. Three patients were treated on the basis of false-positive laboratory results. Because the laboratory results were confusing for the clinicians, the treatment of one true tuberculosis patient was severely delayed. CONCLUSION: 'Good laboratory practice' is very important to prevent cross contamination. RFLP typing proved to be a useful tool to trace the source of contamination. Interaction with clinicians receiving doubtful results is of the utmost importance.

Use of IS901 and IS1245 in RFLP typing of Mycobacterium avium complex: relatedness among serovar reference strains, human and animal isolates.

SETTING: Mycobacterium avium complex (MAC) includes major acquired immune deficiency syndrome (AIDS)-associated pathogens. Formerly, MAC serotyping was used for epidemiological purposes. Recently, restriction fragment length polymorphism (RFLP) typing has become available. OBJECTIVE: Examination of the usefulness of insertion sequence IS1245 in RFLP typing of MAC isolates and the association with IS901 RFLP. DESIGN: Ninety-four serovar reference strains were compared with 144 clinical and animal MAC isolates in RFLP typing. RESULTS: All but four strains containing M. avium-specific-rRNA possessed IS1245. Most human isolates showed polymorphic multiband IS1245 patterns, which were associated with serovars 4, 6 and 8. Sequential clinical isolates obtained at up to five years' distance displayed indistinguishable/closely related patterns. Eleven M. paratuberculosis isolates showed indistinguishable six-band patterns. All 29 MAC isolates from 23 bird species, 7/23 from mammals and 1/81 clinical isolates showed an IS1245 three-band pattern, associated with serovars 1, 2 and 3. All these IS1245 'bird' type strains showed closely related IS901 RFLPs. Only three IS1245 'non-bird' type strains contained IS901, but exhibited completely different RFLP patterns. CONCLUSION: IS1245-RFLP typing is useful for the classification of M. avium and epidemiology of most human isolates. The highly conserved IS901 and IS1245 RFLPs among 'bird' type isolates provide proof that these strains constitute a separate taxon within the MAC.

Restriction fragment length polymorphism typing of mycobacteria.

In principle, restriction fragment length polymorphism (RFLP) typing can be applied to strains of all mycobacterial species for which suitable probes have been identified. International consensus has been achieved regarding the methodology of IS6110 RFLP typing of Mycobacterium tuberculosis complex isolates (1) and IS1245 RFLP typing of Mycobacterium avium strains (2). This chapter describes the technical details of these standardized methods regarding the isolation of DNA, restriction enzymes, electrophoresis conditions, internal- and external-size markers, Southern blotting, and several probes used for hybridization. Furthermore, RFLP typing of isolates of some other mycobacterial species is described.

[Exogenous reinfection with Mycobacterium tuberculosis caused by nosocomial transmission by a patient with AIDS]. / Exogene reïnfectie met Mycobacterium tuberculosis door nosocomiale transmissie bij een patiënt met AIDS.

A 55-year-old AIDS patient relapsed with tuberculosis as a result of exogenous reinfection, 1.5 years after a prior diagnosis of tuberculosis, for which he had been treated. He was reinfected after exposure to another AIDS patient, a 25-year-old man with tuberculosis, when they were hospitalized together during 5 days. The diagnosis of tuberculosis in the latter patient was delayed because the clinical picture was obscured by another infection. Reinfection and nosocomial transmission were demonstrated by analysis of the restriction-fragment-length polymorphism patterns on serial isolates of Mycobacterium tuberculosis. Increased alertness to tuberculosis, especially among HIV-infected persons, and implementation of effective infection control precautions are important in the prevention of nosocomial transmission.

Multiple strains of Mycobacterium avium subspecies hominissuis infections associated with aborted fetuses and wasting in pigs.

A herd of pigs being reared for breeding and fattening, in which there had been incidences of abortion and wasting, reduced growth rates and an increase in mortality for the past year, were tested for Mycobacterium infection by pathological examinations, skin test, serology and Mycobacterium culture. In one placenta, and also in the lung tissues of fetuses, Ziehl-Neelsen staining revealed acid-fast bacilli in combination with infiltrations of neutrophils, macrophages and multinucleated giant cells. Acid-fast bacilli were also found in the mesenteric lymph nodes, liver and/or spleen and jejunum of pigs with wasting and in slaughtered animals. The specimen cultures were identified as Mycobacterium avium subspecies hominissuis using IS1245-specific PCR and IS1245 restriction fragment length polymorphism (RFLP). IS1245 RFLP revealed that the herd was infected with multiple M avium subspecies hominissuis strains belonging to at least two different clades. It is suggested that this infection may have played a more important role in the economic losses of the pig farm than had been assumed previously.

Lymphadenitis in children is caused by Mycobacterium avium hominissuis and not related to 'bird tuberculosis'.

Mycobacterium avium is the most commonly encountered mycobacterium species among non-Mycobacterium tuberculosis complex (nontuberculous mycobacteria) isolates worldwide and frequently causes lymphadenitis in children. During a multi-centre study in The Netherlands that was performed to determine the optimal treatment for mycobacterial lymphadenitis, concern was expressed in the media about the possible role of birds as sources of these M. avium infections, referred to as 'bird tuberculosis.' To examine the involvement of birds in mycobacterial lymphadenitis, 34 M. avium isolates from lymphadenitis cases were subjected to IS1245 restriction fragment length polymorphism (RFLP) typing. This genotyping method enables the distinction of the subspecies M. avium subsp. hominissuis and the 'bird-type' M. avium spp. avium. Highly variable RFLP patterns were found among the lymphadenitis M. avium isolates, and all belonged to the M. avium hominissuis subspecies. A relation to pet birds in the etiology of mycobacterial lymphadenitis could not be established, and the source of the infections may be environmental.

Completeness of notification of tuberculosis in The Netherlands: how reliable is record-linkage and capture-recapture analysis?

The aim of this study was to describe a systematic process of record-linkage, cross-validation, case-ascertainment and capture-recapture analysis to assess the quality of tuberculosis registers and to estimate the completeness of notification of incident tuberculosis cases in The Netherlands in 1998. After record-linkage and cross-validation 1499 tuberculosis patients were identified, of whom 1298 were notified, resulting in an observed under-notification of 13.4%. After adjustment for possible imperfect record-linkage and remaining false-positive hospital cases observed under-notification was 7.3%. Log-linear capture-recapture analysis initially estimated a total number of 2053 (95% CI 1871-2443) tuberculosis cases, resulting in an estimated under-notification of 36.8%. After adjustment for possible imperfect record-linkage and remaining false-positive hospital cases various capture-recapture models estimated under-notification at 13.6%. One of the reasons for the higher than expected estimated under-notification in a country with a well-organized system of tuberculosis control might be that some tuberculosis cases, e.g. extrapulmonary tuberculosis, are managed by clinicians less familiar with notification of infectious diseases. This study demonstrates the possible impact of violation of assumptions underlying capture-recapture analysis, especially the perfect record-linkage, perfect positive predictive value and absent three-way interaction assumptions.

Analysis of genetic polymorphisms affecting the four phospholipase C (plc) genes in Mycobacterium tuberculosis complex clinical isolates.

The Mycobacterium tuberculosis genome contains four highly related genes which present significant similarity to Pseudomonas aeruginosa genes encoding phospholipase C enzymes. Three of these genes, plcA, plcB and plcC, are organized in tandem (locus plcABC). The fourth gene, plcD, is located in a different region. This study investigates variations in plcABC and plcD genes in clinical isolates of M. tuberculosis, Mycobacterium africanum and 'Mycobacterium canettii'. Genetic polymorphisms were examined by PCR, Southern blot hybridization, sequence analysis and RT-PCR. Seven M. tuberculosis isolates contain insertions of IS6110 elements within plcA, plcC or plcD. In 19 of 25 M. tuberculosis isolates examined, genomic deletions were identified, resulting in loss of parts of genes or complete genes from the plcABC and/or plcD loci. Partial plcD deletion was observed in one M. africanum isolate. In each case, deletions were associated with the presence of a copy of the IS6110 element and in all occurrences IS6110 was transposed in the same orientation. A mechanism of deletion resulting from homologous recombination of two copies of IS6110 was recognized in a group of genetically related M. tuberculosis isolates. Five M. tuberculosis isolates presented major polymorphisms in the plcABC and plcD regions, along with loss of expression competence that affected all four plc genes. Phospholipase C is a well-known bacterial virulence factor. The precise role of phospholipase C in the pathogenicity of M. tuberculosis is unknown, but considering the potential importance that the plc genes may have in the virulence of the tubercle bacillus, the study of isolates cultured from patients with active tuberculosis bearing genetic variations affecting these genes may provide insights into the significance of phospholipase C enzymes for tuberculosis pathogenicity.

Insertion element IS1081-associated restriction fragment length polymorphisms in Mycobacterium tuberculosis complex species: a reliable tool for recognizing Mycobacterium bovis BCG.

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.

Transmission of Mycobacterium tuberculosis depending on the age and sex of source cases.

This study estimated to what extent tuberculosis transmission in the Netherlands depends on the age and sex of source cases. DNA fingerprints of Mycobacterium tuberculosis isolates were matched to patient information in the Netherlands Tuberculosis Register for 1993-1998. Clusters were defined as groups of patients with pulmonary tuberculosis whose isolates had identical DNA fingerprints. Source cases were assigned by using two models. The first-case model assumed that the first diagnosed case was the source case. The incidence rate model estimated source case probabilities from the incidence rates of potential source cases and the time of diagnosis. DNA fingerprints of 6,102 isolates were matched to patient information on 5,080 (83%) cases, 3,479 of whom had pulmonary disease. According to both models, the number of infectious cases generated per source case was lower for female than for male source cases and decreased with increasing age of the source case. The authors concluded that transmission of tuberculosis is associated with the age and sex of source cases as well as the age of secondary cases. Increased transmission among immigrant groups in the Netherlands is largely attributable to the relatively young age of immigrant source cases.

Mutations at amino acid position 315 of the katG gene are associated with high-level resistance to isoniazid, other drug resistance, and successful transmission of Mycobacterium tuberculosis in the Netherlands.

The prevalence of mutations at amino acid (aa) position 315 in the katG gene of isoniazid (INH)-resistant Mycobacterium tuberculosis isolates in The Netherlands and the mutation's association with the level of INH resistance, multidrug resistance, and transmission were determined. Of 4288 M. tuberculosis isolates with available laboratory results, 295 (7%) exhibited INH resistance. Of 148 aa 315 mutants, 89% had MICs of 5-10 microg/mL, whereas 75% of the other 130 INH-resistant strains had MICs of 0.5-1 microg/mL. Of the aa 315 mutants, 33% exhibited monodrug resistance, compared with 69% of other INH-resistant strains (P<.0001). Multidrug resistance was found among 14% of the aa 315 mutants and 7% of the other INH-resistant strains (P>.05). The probability of being in an IS6110 DNA restriction fragment length polymorphism cluster was similar for aa 315 mutants and INH-susceptible strains, but the probability was reduced in other INH-resistant strains. Thus, aa 315 mutants lead to secondary cases of tuberculosis as often as INH-susceptible strains do.

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.

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.

Analysis of tuberculosis transmission between nationalities in the Netherlands in the period 1993-1995 using DNA fingerprinting.

Immigration from high prevalence areas may contribute to an increased risk of tuberculosis in Europe. This study aimed at quantifying transmission of tuberculosis between and within nationalities among residents of the Netherlands. DNA "fingerprints," on the basis of restriction fragment length polymorphism using marker IS6110, were made of all Mycobacterium tuberculosis isolates in the Netherlands from January 1993 through June 1995. Clusters were defined as groups of patients that had isolates with identical fingerprints. It was assumed that the probability of a patient being the source of a cluster was proportional to the incidence rate of potential sources times the probability that a potential source would give rise to a cluster. The transmission index was defined as the average number of secondary cases of infectious tuberculosis caused directly or indirectly through recent transmission by a single potential source case and was used to estimate the effective reproductive rate associated with recent transmission, ReFAST. Among a total of 623 Dutch tuberculosis cases, 17% (95% confidence interval 9-25%) of cases were attributable to recent transmission from a non-Dutch source. The transmission index varied strongly by nationality, and was highest among the Surinamese (1.3), Moroccan (0.8), and Turkish (0.8) populations; ReFAST was 0.26. Aggregation of tuberculosis cases of given nationalities within clusters was most pronounced among recent immigrants from Somalia and (ex-)Yugoslavia. The authors conclude that differences in transmission between subpopulations can be quantified and may be used to evaluate and direct tuberculosis control.

Occurrence and stability of insertion sequences in Mycobacterium tuberculosis complex strains: evaluation of an insertion sequence-dependent DNA polymorphism as a tool in the epidemiology of tuberculosis.

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.

Genetic heterogeneity in Mycobacterium tuberculosis isolates reflected in IS6110 restriction fragment length polymorphism patterns as low-intensity bands.

Mycobacterium tuberculosis isolates with identical IS6110 restriction fragment length polymorphism (RFLP) patterns are considered to originate from the same ancestral strain and thus to reflect ongoing transmission. In this study, we investigated 1,277 IS6110 RFLP patterns for the presence of multiple low-intensity bands (LIBs), which may indicate infections with multiple M. tuberculosis strains. We did not find any multiple LIBs, suggesting that multiple infections are rare in the Netherlands. However, we did observe a few LIBs in 94 patterns (7.4%) and examined the nature of this phenomenon. With single-colony cultures it was found that LIBs mostly represent mixed bacterial populations with slightly different RFLP patterns. Mixtures were expressed in RFLP patterns as LIBs when 10 to 30% of the DNA analyzed originated from a bacterial population with another RFLP pattern. Presumably, a part of the LIBs did not represent mixed bacterial populations, as in some clusters all strains exhibited LIBs in their RFLP patterns. The occurrence of LIBs was associated with increased age in patients. This may reflect either a gradual change of the bacterial population in the human body over time or IS6110-mediated genetic adaptation of M. tuberculosis to changes in the environmental conditions during the dormant state or reactivation thereafter.

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.