A. M. Jordaan

Beijing and Haarlem genotypes are overrepresented among children with drug-resistant tuberculosis in the Western Cape province of South Africa

ABSTRACT Drug resistance among children with culture-confirmed tuberculosis (TB) provides an accurate measure of transmitted drug resistance within the community. We describe the genotype diversity in children with culture-confirmed TB and investigate the relationship between genotype and drug resistance. A prospective study was conducted from March 2003 through August 2005 at Tygerberg Childrens Hospital, in the Western Cape Province of South Africa. All children (<13 years of age) diagnosed with culture-confirmed TB were included. Genotype analysis and phenotypic drug susceptibility testing were performed on the first culture-positive isolate from each patient. Mutation analysis was performed on all drug-resistant isolates. Spoligotyping was successfully performed on isolates from 391/399 (98%) children diagnosed with culture-confirmed TB. Drug susceptibility testing was also performed on 391 isolates; 49 (12.5%) were resistant to isoniazid, and 20 (5.1%) of these were resistant to both isoniazid and rifampin. Beijing was the most common genotype family, identified in 130/391 (33.2%) cases, followed by LAM in 114/391 (29.2%) cases. The presence of both Beijing and Haarlem genotype families was significantly associated with drug resistance (26/49 [53.1%] versus 113/342 [33.0%]; odds ratio, 1.7; 95% confidence interval, 1.0 to 2.9). The high prevalence of Beijing and LAM in children with culture-confirmed TB reflects considerable transmission of these genotype families within the community. The overrepresentation of Beijing and Haarlem genotype families in children with drug-resistant TB demonstrates their contribution to transmitted drug resistance and their potential importance in the emergent drug-resistant TB epidemic.

Papulonecrotic tuberculid : identification of Mycobacterium tuberculosis DNA by polymerase chain reaction

Sections from 22 formalin-fixed, paraffin-embedded skin biopsies from 12 patients with papulonecrotic tuberculid (PNT) were examined for the presence of Mycobacterium tuberculosis DNA with use of the polymerase chain reaction. All patients had a positive tuberculin skin test and a compatible clinical picture and responded to antituberculous therapy. Histological examination showed the typical morphology of PNT lesions with dermal necrosis surrounded by an ill-formed granulomatous infiltrate. Mycobacterial DNA was detected in 11 of the 22 biopsies. None of the negative controls showed positive DNA identification by amplification. Great care was taken in avoiding false-positive results due to contamination. After reviewing the literature, we believe this is the first time that PNT lesions have been investigated by PCR for the presence of mycobacterial DNA. These findings provide direct proof that mycobacterial products are present in PNT lesions and support the theory that this organism is in some way responsible for the development of PNT.

Discordance between Mycobacterial Interspersed Repetitive-Unit-Variable-Number Tandem-Repeat Typing and IS6110 Restriction Fragment Length Polymorphism Genotyping for Analysis of Mycobacterium tuberculosis Beijing Strains in a Setting of High Incidence of Tuberculosis

ABSTRACT IS6110 restriction fragment length polymorphism (RFLP) genotyping is the most widely used genotyping method to study the epidemiology of Mycobacterium tuberculosis. However, due to the complexity of the IS6110 RFLP genotyping technique, and the interpretation of RFLP data, mycobacterial interspersed repetitive-unit-variable-number tandem-repeat (MIRU-VNTR) genotyping has been proposed as the new genotyping standard. This study aimed to determine the discriminatory power of different MIRU-VNTR locus combinations relative to IS6110 RFLP genotyping, using a collection of Beijing genotype M. tuberculosis strains with a well-established phylogenetic history. Clustering, diversity index, clustering concordance, concordance among unique genotypes, and divergent and convergent evolution were calculated for seven combinations of 27 different MIRU-VNTR loci and compared to IS6110 RFLP results. Our results confirmed previous findings that MIRU-VNTR genotyping can be used to estimate the extent of recent or ongoing transmission. However, molecular epidemiological linking of cases varied significantly depending on the genotyping method used. We conclude that IS6110 RFLP and MIRU-VNTR loci evolve independently and at different rates, which leads to discordance between transmission chains predicted by the respective genotyping methods. Concordance between the two genotyping methods could be improved by the inclusion of genetic distance (GD) into the clustering formulae for some of the MIRU-VNTR loci combinations. In summary, our findings differ from previous reports, which may be explained by the fact that in settings of low tuberculosis incidence, the genetic distance between epidemiologically unrelated isolates was sufficient to define a strain using either marker, whereas in settings of high incidence, continuous evolution and persistence of strains revealed the weaknesses inherent to these markers.

Polymerase Chain Reaction in the Diagnosis of Tuberculous Meningitis

43 cerebrospinal fluid (CSF) specimens obtained from 20 children with tuberculous meningitis (TBM) at varying times during the first month of treatment were examined by polymerase chain reaction (PCR) for the presence of M. tuberculosis DNA. Overall 27 CSF specimens (63%) from 16 patients (80%) gave > or = 1 positive results and positive results were obtained from CSF specimens throughout the first 4 weeks of therapy. Nine CSF specimens (21%) gave a doubtful result (only 1 of duplicate determinations positive) and 7 (16%) a negative result. CSF from patients with suspected TBM should be submitted for PCR evaluation and positive results may be obtained up to at least 4 weeks after the start of treatment.

Spread of a Low-Fitness Drug-Resistant Mycobacterium tuberculosis Strain in a Setting of High Human Immunodeficiency Virus Prevalence

ABSTRACT The fitness cost associated with the evolution of resistance to rifampin in Mycobacterium tuberculosis may be different in clinical isolates compared to in vitro-generated mutants. An atypical Beijing strain (attenuated phenotype) demonstrated the ability to spread despite acquiring resistance to rifampin. Transmission was linked to human immunodeficiency virus coinfection (P = 0.029), raising concern for the spread of drug resistance in vulnerable populations.

Fluorometric Assay for Testing Rifampin Susceptibility of Mycobacterium tuberculosis Complex

ABSTRACT The emergence and transmission of multidrug-resistant tuberculosis (MDR-TB) and extensively drug-resistant tuberculosis (XDR-TB) have raised concern about diagnostic delay associated with culture-based drug susceptibility testing methods. The association between rifampin resistance and MDR-TB or XDR-TB makes it an important genetic marker for genotypic drug susceptibility testing. In this article, we describe the analysis of the physical properties of the rifampin resistance-determining region (RRDR) in the rpoB gene by high-resolution thermal melt analysis as a method for detecting rifampin resistance in Mycobacterium tuberculosis complex. The RRDR from the M. tuberculosis complex was amplified by PCR from DNA templates extracted from sputum cultures of M. tuberculosis or the laboratory strain (H37Rv) in the presence of a fluorescent DNA binding dye. Subsequent mixing of the amplification products from the respective sputum cultures and the laboratory strain and thermocycling allowed the formation of DNA duplexes. The thermal denaturation properties of these DNA duplexes were determined by measuring the derivative of the intensity of fluorescence at different temperatures. Analysis of DNA extracted from 153 sputum cultures showed a sensitivity of 98% and a specificity of 100% for the detection of rifampin resistance compared to the “gold standard” culture-based phenotyping method. No statistical difference was detected in the performance of the method when applied to crude DNA from 134 boiled cultures. This method, named “FAST-Rif” (“fluorometric assay for susceptibility testing of rifampin”), allowed the rapid, reliable, and easy detection of genotypic rifampin resistance as a marker for MDR-TB and XDR-TB.

Genome and MIC stability in Mycobacterium tuberculosis and indications for continuation of use of isoniazid in multidrug-resistant tuberculosis

Mycobacterium tuberculosis strains resistant to two or more of the first line antituberculosis drugs (MDR) are a serious threat to successful tuberculosis control programmes. For this retrospective study, 85 follow-up drug resistant isolates from 23 patients residing in a community with a high incidence of tuberculosis were collected and the level of in-vitro resistance to antibiotics determined quantitatively. PCR-SSCP and sequencing techniques were used to screen for gene mutations associated with resistance in 31 follow-up samples from a smaller group of eight patients. DNA fingerprint analysis was done on sequential isolates to confirm identity. Although treatment had a profound effect on changes in drug resistance patterns, the MIC for a particular agent remained constant in follow-up isolates. DNA fingerprinting and mutational analysis (14 different loci) showed that the genome of MDR strains of M. tuberculosis is relatively stable during the course of therapy. The rpoB gene was the most frequently mutated structural gene involved in drug resistance and a novel C to T mutation upstream of open reading frame (ORF)1 of the inhA operon was detected. No evidence was found of the presence of strain W (New York) in this group of MDR strains. The results stress the importance of confirming individuality of strains for the accurate calculation of frequencies of particular mutations associated with drug resistance, particularly in a high incidence area. Approximately one-half (47.8%) of the patients had isolates resistant to concentrations just above the critical concentration for isoniazid (MICs of 0.2-5 mg/L). Therefore, these patients and their contacts who develop primary drug-resistant tuberculosis may respond to higher dosages of treatment which could have a considerable impact on the cost and the ease of management of resistant tuberculosis.

Sequence Polymorphism in the rrs Gene of Mycobacterium tuberculosis Is Deeply Rooted within an Evolutionary Clade and Is Not Associated with Streptomycin Resistance

ABSTRACT A mutation (C-to-T transition) at position 491 of therrs gene was identified in a Mycobacterium tuberculosis strain family (n = 208 isolates) that was predominant in a suburb of Cape Town, South Africa. This nucleotide change is not involved in streptomycin resistance, and we suggest caution in assuming that all mutations in genes targeted by antituberculosis drugs confer drug resistance.

IS6110-mediated deletion polymorphism in the direct repeat region of clinical isolates of Mycobacterium tuberculosis

This study investigates the phenomenon of IS6110-mediated deletion polymorphism in the direct repeat (DR) region of the genome of Mycobacterium tuberculosis. Clinical isolates and their putative predecessors were compared using a combination of DR region restriction fragment length polymorphism, IS6110 DNA fingerprinting, spoligotyping, and DNA sequencing, which allowed the mapping of chromosome structure and deletion junctions. The data suggest that adjacently situated IS6110 elements mediate genome deletion. However, in contrast to previous reports, deletions appear to be mediated by inversely oriented IS6110 elements. This suggests that these events may occur via mechanisms other than RecA-mediated homologous recombination. The results underscore the important role of IS6110-associated deletion hypervariability in driving M. tuberculosis genome evolution.

The evolving epidemic of drug-resistant tuberculosis among children in Cape Town, South Africa

SETTING Tygerberg Childrens Hospital, Cape Town, South Africa. OBJECTIVE To determine the prevalence and trend of drug resistance and human immunodeficiency virus (HIV) co-infection among children with culture-confirmed tuberculosis (TB). METHOD Prospective surveillance from March 2007 to February 2009, compared to three previous surveys (1994-1998, 2003-2005, 2005-2007). Drug susceptibility testing (DST) against isoniazid (INH) and rifampicin (RMP) was performed using genotypic and phenotypic testing. If multidrug-resistant TB (MDR-TB) was detected, further DST against ethambutol (EMB) and second-line drugs was performed. RESULTS A total of 294 children with a median age of 26 months (range 3 days-13 years) were diagnosed with culture-confirmed TB. DST results were available for 292 (99.3%); 41 (14%) were INH-resistant, including 26 (8.9%) with MDR-TB. Four children (1.4%) had RMP monoresistance. EMB resistance was present in 12/24 (50%) MDR-TB cases tested. Two isolates were resistant to ofloxacin; none had extensively drug-resistant TB. Of those tested, 29% (63/217) were HIV-infected. Any resistance to RMP increased between 1994 and 2009 (P < 0.001), as did RMP monoresistance (P = 0.009) and MDR-TB (P < 0.001). Sensitivity was 87.5% and specificity 100% for genotypic compared to phenotypic testing for INH resistance. CONCLUSIONS RMP, and consequently multidrug, resistance is increasing among children with TB in this setting. EMB resistance is common among children with resistance to RMP and INH.