Quantification of multidrug-resistant M. tuberculosis bacilli in sputum during the first 8 weeks of treatment.

SETTING: Mulago Hospital, Kampala, Uganda.OBJECTIVE: To quantify Mycobacterium tuberculosis in sputum during the first 8 weeks of pulmonary multidrug-resistant TB (MDR-TB) treatment.DESIGN: We enrolled consecutive adults with pulmonary MDR-TB treated according to national guidelines. We collected overnight sputum samples before treatment and weekly. Sputum samples were cultured on Middlebrook 7H11S agar to measure colony-forming units per mL (cfu/mL) and in MGIT™ 960™ media to measure time to detection (TTD). Linear mixed-effects regression was used to estimate the relational change in log10 cfu/mL and TTD.RESULTS: Twelve adults (median age: 27 years) were enrolled. Half were women, and two-thirds were HIV-positive. At baseline, median log10 cfu/mL was 5.1, decreasing by 0.29 log10 cfu/mL/week. The median TTD was 116.5 h, increasing in TTD by 36.97 h/week. The weekly change was greater in the first 2 weeks (-1.04 log10 cfu/mL/week and 120.02 h/week) than in the remaining 6 weeks (-0.17 log10 cfu/mL/week and 26.11 h/week).CONCLUSION: Serial quantitative culture measures indicate a slow, uneven rate of decline in sputum M. tuberculosis over 8 weeks of standardized pulmonary MDR-TB treatment.

Clinical implications of molecular drug resistance testing for Mycobacterium tuberculosis: a TBNET/RESIST-TB consensus statement.

The emergence of drug-resistant strains of Mycobacterium tuberculosis is a challenge to global tuberculosis (TB) control. Although culture-based methods have been regarded as the gold standard for drug susceptibility testing (DST), molecular methods provide rapid information on mutations in the M. tuberculosis genome associated with resistance to anti-tuberculosis drugs. We ascertained consensus on the use of the results of molecular DST for clinical treatment decisions in TB patients. This document has been developed by TBNET and RESIST-TB groups to reach a consensus about reporting standards in the clinical use of molecular DST results. Review of the available literature and the search for evidence included hand-searching journals and searching electronic databases. The panel identified single nucleotide mutations in genomic regions of M. tuberculosis coding for katG, inhA, rpoB, embB, rrs, rpsL and gyrA that are likely related to drug resistance in vivo. Identification of any of these mutations in clinical isolates of M. tuberculosis has implications for the management of TB patients, pending the results of in vitro DST. However, false-positive and false-negative results in detecting resistance-associated mutations in drugs for which there is poor or unproven correlation between phenotypic and clinical drug resistance complicate the interpretation. Reports of molecular DST results should therefore include specific information on the mutations identified and provide guidance for clinicians on interpretation and on the choice of the appropriate initial drug regimen.

Comparison of time to positive and colony counting in an early bactericidal activity study of anti-tuberculosis treatment.

SETTING: Patients with smear-positive, newly diagnosed pulmonary tuberculosis (TB) presenting to the out-patient TB clinic in Kampala, Uganda. OBJECTIVE: To compare colony-forming unit (cfu) counting and time to positive (TTP) in Mycobacteria Growth Indicator Tube (MGIT) culture as measures of early bactericidal activity (EBA). DESIGN: Patients were enrolled in an EBA feasibility study of standard TB chemotherapy. Sixteen-hour overnight sputum collections were obtained before and on days 2, 4, 7, 10, 12 and 14 of treatment for quantitative culture on selective Middlebrook 7H11 agar media and TTP in the MGIT liquid culture system. RESULTS: Log cfu and TTP were correlated over all time points (r(s) = -0.71, P < 0.001). Within-subject (day to day) variation as a percentage of total variation was very similar between the two measures: 25.7% for cfu and 25% for TTP. Mean EBA 0-14, 0-2 and 2-14 measured by TTP were similar to those previously reported. CONCLUSION: TTP measured by an automated, standardized, commercially available culture system correlates with cfu determinations. EBA measured by TTP provides similar information to cfu counting, and is reproducible across sites and in different patient populations. These findings support replacing cfu counting with TTP as the primary measurement in EBA studies.

Time to detection of Mycobacterium tuberculosis as an alternative to quantitative cultures.

Testing new drugs is critical to improving the treatment of tuberculosis. Quantitative cultures of Mycobacterium tuberculosis on solid media have been used in Phase 1 and 2 trials, but are time and resource intensive. Time to detection (TTD) of growth of M. tuberculosis in automated liquid culture systems is an alternative. TTD has been shown to correlate with CFU in quantitative cultures, and is faster and simpler to perform. We compared TTD in the BACTEC 460 liquid culture system with CFU in a clinical trial that included 110 subjects. Comparing all sputum cultures collected between baseline and 2 months we found a strong negative correlation between log(10) CFU and TTD (rho = -0.91). In addition, when TTD at baseline was compared with 1 and 2 month sputum culture positivity, subjects whose cultures were negative after 1 and 2 months had a significantly longer median baseline TTD compared with subjects whose cultures were positive at 1 and 2 months (5 vs. 3 days and 3 vs. 2 days, respectively). TTD compares closely with CFU and represents a faster, simpler alternative to quantitative cultures.

Sputum Mycobacterium tuberculosis mRNA as a marker of bacteriologic clearance in response to antituberculosis therapy.

mRNA is a marker of cell viability. Quantifying Mycobacterium tuberculosis mRNA in sputum is a promising tool for monitoring response to antituberculosis therapy and evaluating the efficacy of individual drugs. mRNA levels were measured in sputum specimens from patients with tuberculosis (TB) receiving monotherapy in an early bactericidal activity study of fluoroquinolones and in those receiving a standard rifampin-based regimen in an interleukin-2 (IL-2) trial. In the early bactericidal activity study, sputum for quantitative culture and mRNA analysis was collected for 2 days before and daily during 7 days of study drug administration. In the IL-2 trial, sputum was collected for quantitative culture, Bactec 460 liquid culture, and mRNA analysis throughout the intensive treatment phase. RNA was isolated from digested sputum and tested in quantitative reverse transcription-PCR assays for several gene targets. mRNA for the glyoxylate cycle enzyme isocitrate lyase declined at similar rates in patients receiving isoniazid, gatifloxicin, levofloxacin, and moxifloxacin monotherapy. Isocitrate lyase mRNA correlated highly with CFU in sputum prior to therapy and during 7 days of monotherapy in all treatment arms. Isocitrate lyase mRNA was detectable in sputum of culture-positive TB patients receiving a rifampin-based regimen for 1 month. At 2 months, sputum for isocitrate mRNA correlated more closely with growth in liquid culture than did growth on solid culture medium. Data suggest that isocitrate lyase mRNA is a reliable marker of M. tuberculosis viability.

Evaluation of low-colony-number counts of Mycobacterium tuberculosis on solid media as a microbiological marker of cross-contamination.

Low-colony-number counts on solid media are considered characteristic of cross-contamination, although they are normally observed in true-positive cultures from some groups of patients. The aim of this study was to evaluate low-yield growth cultures as a microbiological marker for cross-contamination. We evaluated 106 cultures with <15 colonies from 94 patients, and the proportions of false-positive cultures were 0.9% per sample and 1.1% per patient, which indicates that low-yield growth is not a reliable marker of cross-contamination.

Introduction of an in-house PCR for routine identification of M. tuberculosis in a low-income country.

SETTING: National Tuberculosis (TB) Treatment Centre, Makerere University Medical School and Joint Clinical Research Centre, Kampala, Uganda. OBJECTIVE: To evaluate the introduction of a polymerase chain reaction (PCR) based assay for identification of the Mycobacterium tuberculosis complex (MTC) into routine practice. DESIGN: Routine diagnostic specimens were processed and inoculated into Bactec 12B vials and monitored daily. At a growth index (GI) > or =10, 0.5 ml of the 12B broth was removed and assayed with PCR. The same 12B vial was analyzed using the Bactec NAP method at GI > or =500. Vials at various levels of GI were included. Recurrent cost and time required to perform PCR and NAP were compared. RESULTS: Initially, 71 specimens were analyzed; of these, 68 were NAP-positive while 69 were PCR-positive for MTC. PCR resulted in a 75% reduction in cost for a single test compared with Bactec NAP. PCR has been successfully incorporated into routine practice, and 432 samples have been analyzed. In addition, isolates from solid media were also well identified by PCR. With PCR, more samples can be analyzed at a time, it is faster and is less labor intensive. CONCLUSION: PCR is a reliable and cheaper alternative for the identification of MTC.

Variable-number tandem repeats that are useful in genotyping isolates of Salmonella enterica subsp. enterica serovars Typhimurium and Newport.

The genome of Salmonella enterica subsp. enterica serovar Typhimurium strain LT2 was analyzed for direct repeats, and 54 sequences containing variable-number tandem repeat loci were identified. Ten primer pairs that anneal upstream and downstream of each selected locus were designed and used to amplify PCR targets in isolates of S. enterica serovars Typhimurium and Newport. Four of the 10 loci did not show polymorphism in the length of products. Six loci were selected for analysis. Isolates of S. enterica serovars Typhimurium and Newport that were related to specific outbreaks and showed identical pulsed-field gel electrophoresis patterns were indistinguishable by the length of the six variable-number tandem repeats. Isolates that differed in their pulsed-field gel electrophoresis patterns showed polymorphism in variable-number tandem repeat profiles. Length of the products was confirmed by DNA sequence analysis. Only 2 of the 10 loci contained exact integers of the direct repeat. Eight loci contained partial copies. The partial copies were maintained at the ends of the variable-number tandem repeat loci in all isolates. In spite of having partial copies that were maintained in all isolates, the number of direct repeats at a locus was polymorphic. Six variable-number tandem repeat loci were useful in distinguishing isolates of S. enterica serovars Typhimurium and Newport that had different pulsed-field gel electrophoresis patterns and in identifying outbreak-associated cases that shared a common pulsed-field gel pattern.

Early and extended early bactericidal activity of levofloxacin, gatifloxacin and moxifloxacin in pulmonary tuberculosis.

OBJECTIVE: To evaluate the early bactericidal activity (EBA) of the new fluoroquinolones levofloxacin, gatifloxacin and moxifloxacin in patients with pulmonary tuberculosis (PTB). DESIGN: Randomized, open-label trial. Forty adults with newly diagnosed smear-positive PTB (10 per arm) were assigned to receive isoniazid (INH) 300 mg, levofloxacin 1000 mg, gatifloxacin 400 mg, or moxifloxacin 400 mg daily for 7 days. Sputum for quantitative culture was collected for 2 days before and daily during 7 days of monotherapy. Bactericidal activity was estimated by measuring the decline in bacilli during the first 2 days (EBA 0-2) and last 5 days of monotherapy (extended EBA, EBA 2-7). Laboratory staff were blinded to treatment assignment. RESULTS: The EBA 0-2 of INH (0.67 log10 cfu/ml/day) was greater than that of moxifloxacin and gatifloxacin (0.33 and 0.35 log10 cfu/ml/day, respectively), but not of levofloxacin 1000 mg daily (0.45 log10 cfu/ml/day) (P = 0.14). Bactericidal activity between days 2 and 7 was similar for all three fluoroquinolones. In a pooled comparison, the EBA 2-7 of the fluoroquinolones was greater than for INH. CONCLUSION: Moxifloxacin, gatifloxacin, and high-dose levofloxacin have excellent EBA, only slightly less than for INH, and greater extended EBA. These drugs warrant further study in the treatment of drug-susceptible TB.

Epidemiologic import of tuberculosis cases whose isolates have similar but not identical IS6110 restriction fragment length polymorphism patterns.

Isolates of Mycobacterium tuberculosis from patients with epidemiologic links frequently demonstrate identical IS6110 restriction fragment length polymorphism (RFLP) patterns (i.e., RFLP clustering) because they are infected with the same strain. Uncertainty arises with isolates that differ from one another by a few IS6110 hybridizing bands. During the period from 1 January 1996 to 31 December 1999, isolates from 585 tuberculosis (TB) cases were analyzed by RFLP, representing 98.2% of the 596 culture-positive TB cases reported in Arkansas during the study period. Of the 585 cases for which RFLP was available, 419 (71.6%) had an RFLP pattern with more than five copies of IS6110. Of the total 74 clusters, 48 comprised isolates with more than five copies of IS6110 and included 164 cases. Sixty-nine isolates with more than five copies of IS6110 comprising 16 clusters and 60 unique isolates were found to be similar to at least 1 other isolate (differing from it by one or two hybridizing bands). Among the 129 cases whose isolates were similar to other clustered or unique isolates, 16 cases were discovered with epidemiologic links: 14 (15.2%) were among the 92 cases with IS6110 RFLP patterns similar to those in clusters, and 2 (5.2%) were among the 37 unique cases that were similar to another unique case. The isolates from the epidemiologically linked patients shared common spoligotypes; all except one case shared common polymorphic GC-rich sequence (PGRS) patterns. Of the 129 patients whose isolates differed from another by one or two hybridizing IS6110 bands, 101 (78.3%) shared common spoligotypes and 87 (67.4%) shared common PGRS RFLP patterns.

Activated THP-1 cells: an attractive model for the assessment of intracellular growth rates of Mycobacterium tuberculosis isolates.

Capacity of certain Mycobacterium tuberculosis isolates to grow more rapidly in human macrophages may be indicative of increased virulence. Significant differences were observed in intracellular growth of two isolates from sites of tuberculosis transmission, with an outbreak-associated strain growing faster than a strain causing disease in only one person. Activated THP-1 cells are a suitable alternative to peripheral blood monocyte models.

Microaerophilic induction of the alpha-crystallin chaperone protein homologue (hspX) mRNA of Mycobacterium tuberculosis.

Among the products that are expressed when Mycobacterium tuberculosis undergoes hypoxic shiftdown to nonreplicating persistence (NRP) is the alpha-crystallin chaperone protein homologue (Acr). This expression coincides with the previously reported appearance of a respiratory type of nitrate reductase activity, the increase in glycine dehydrogenase activity, and the production of a unique antigen, URB-1. In a timed sampling study, using a slowly stirred oxygen depletion culture model, we have demonstrated that the hspX mRNA that codes for Acr protein as well as the protein itself is induced just as the bacilli enter the microaerophilic NRP stage 1 (NRP-1). In contrast to the induction observed for hspX mRNA, levels of 16S rRNA, fbpB mRNA (encoding the 85B alpha antigen), and aroB mRNA (encoding dehydroquinate synthase) demonstrate relatively small to no change upon entering NRP-1. Acr protein was shown to be identical to URB-1 by Western analysis with anti-URB-1 antibody. The fact that antibody to Acr is found in a high percentage of tuberculosis patients suggests that the hypoxic shiftdown of tubercle bacilli to the NRP state that occurs in vitro, resulting in production of the alpha-crystallin protein, occurs in vivo as well. Simultaneous abrupt increases in hspX mRNA and Acr protein suggest that Acr protein expression is controlled at the level of transcription.

Quantitative bacillary response to treatment in Mycobacterium tuberculosis infected and M. africanum infected adults with pulmonary tuberculosis.

Data regarding possible differences in microbiological response to therapy of disease caused by Mycobacterium tuberculosis and M. africanum are limited. Presenting clinical characteristics and sputum bacillary load during standard short-course chemotherapy in patients with newly-diagnosed pulmonary tuberculosis due to M. tuberculosis (n = 7) and M. africanum (n = 6) were compared. Changes in sputum bacillary load were measured using quantitative acid-fast bacilli smears, colony forming unit assay, and time until positive culture in the BACTEC radiometric system. Presentation and response to short course chemotherapy were comparable between patients infected with M. tuberculosis and those infected with M. africanum.

A clinic-based molecular epidemiologic study of tuberculosis in Monterrey, Mexico.

SETTING: A tuberculosis clinic associated with a university hospital in Monterrey, Mexico, an urban community with high tuberculosis incidence. OBJECTIVE: To determine the diversity of DNA fingerprint patterns and the extent of drug resistance of Mycobacterium tuberculosis isolates from patients who attended the clinic. DESIGN: Isolates of M. tuberculosis obtained from 186 patients during the period from 31 January 1996 to 31 March 1998 were tested for susceptibility to isoniazid, rifampicin, ethambutol and streptomycin. Demographic data and the social history of each patient were obtained prospectively by interview. The IS6110 DNA fingerprints were obtained for 166 of the 186 isolates. Secondary typing was carried out on isolates with fewer than six copies of IS6110. RESULTS: Thirty-two per cent of the tested isolates (60/ 186) were drug-resistant, and 18% (33/186) were multidrug-resistant. Approximately 55% of the resistant isolates (33/60) were attributed to acquired resistance. A total of 106 different IS6110 fingerprint patterns were observed among the 166 fingerprinted isolates. Based on both IS6110 and pTBN12 fingerprinting, 65 (39%) of the 166 isolates were part of 22 DNA fingerprint clusters. Various drug susceptibility patterns were seen in most clusters. CONCLUSION: Fingerprint clustering indicates extensive recent transmission of tuberculosis in patients attending the clinic. The prevalence of drug-resistant tuberculosis is high.

Secondary typing of Mycobacterium tuberculosis isolates with matching IS6110 fingerprints from different geographic regions of the United States.

Fifty-nine isolates of Mycobacterium tuberculosis obtained from different states in the United States and representing 25 interstate clusters were investigated. These clusters were identified by computer-assisted analysis of DNA fingerprints submitted during 1996 and 1997 by different laboratories participating in the CDC National Genotyping and Surveillance Network. Isolates were fingerprinted with the IS6110 right-hand probe (IS6110-3'), the IS6110 left-hand probe (IS6110-5'), and the probe pTBN12, containing the polymorphic GC-rich sequence (PGRS). Spoligotyping based on the polymorphism in the 36-bp direct-repeat locus was also performed. As a control, 43 M. tuberculosis isolates in 17 clusters obtained from patients in Arkansas during the study period were analyzed. Of the 25 interstate clusters, 19 were confirmed as correctly clustered when all the isolates were analyzed on the same gel using the IS6110-3' probe. Of the 19 true IS6110-3' clusters, 10 (53%) were subdivided by one or more secondary typing methods. Clustering of the control group was virtually identical by all methods. Of the three different secondary typing methods, spoligotyping was the least discriminating. IS6110-5' fingerprinting was as discriminating as PGRS fingerprinting. The data indicate that the IS6110-5' probe not only is a useful secondary typing method but also probably would prove to be a more useful primary typing method for a genotyping network which involves isolates from different geographic regions.

An increase in expression of a Mycobacterium tuberculosis mycolyl transferase gene (fbpB) occurs early after infection of human monocytes.

Changes in the mRNA levels of two Mycobacterium tuberculosis genes (fbpB known as antigen 85B, and hspX known as Acr) were studied in infected human monocytes. Antigen 85B is an enzyme involved in cell wall biosynthesis and is also a major target of the immune response. Acr is a stress protein believed to be involved in the bacillary response to adverse conditions and in non-replicating persistence. During the first 24 h of intracellular infection, the intramonocyte 85B mRNA level increased 54-fold (P = 0.00001) and 14.6 times in comparison with the 16S ribosomal rRNA. In contrast, the Acr mRNA fell 14.3 times. Although monocyte cytokine production was very variable, the 24 h secretion of tumour necrosis factor (TNF)-alpha correlated with the 85B-16S RNA ratio at 24 h (r = 0.77, Pcorr < 0.01). Furthermore, the addition of exogenous TNF-alpha to cultures was associated with a twofold increase in the 85B-16S ratio and, conversely, neutralization of endogenous TNF-alpha reduced the ratio. As antigen 85B also induces TNF-alpha, the positive feedback implied by our findings suggests a previously unsuspected role for this protein in the immunopathogenesis of tuberculosis.

Spoligotyping and polymorphic GC-rich repetitive sequence fingerprinting of mycobacterium tuberculosis strains having few copies of IS6110.

Several genetic loci have been utilized to genotype isolates of Mycobacterium tuberculosis. A shortcoming of the most commonly used method, IS6110 fingerprinting, is that it does not adequately discriminate between isolates having few copies of IS6110. This study was undertaken to compare pTBN12 fingerprinting of polymorphic GC-rich repetitive sequence genes and spoligotyping of the direct repeat locus as secondary typing procedures for M. tuberculosis isolates having fewer than six copies of IS6110. A total of 88 isolates (100% of the isolates with fewer than six copies of IS6110 isolated in Arkansas during 1996 and 1997) were included in this study. Among the 88 isolates, 34 different IS6110 patterns were observed, 10 of which were shared by more than 1 isolate, involving a total of 64 isolates. The 64 isolates were subdivided into 13 clusters (containing 37 isolates) and 27 unique isolates based on a combination of IS6110 and pTBN12 fingerprinting and into 11 clusters (containing 51 isolates) and 13 unique isolates based on a combination of IS6110 fingerprinting and spoligotyping. Identical spoligotypes were found among isolates having different IS6110 patterns, as well as among isolates showing different pTBN12 patterns. In contrast, all isolates that had different IS6110 patterns were found to be unique by pTBN12 typing. The clustering rate was 73, 58, and 42%, respectively, for IS6110 fingerprinting alone, IS6110 fingerprinting and spoligotyping combined, and IS6110 and pTBN12 combined fingerprinting. The data indicate that the pTBN12 method has greater discriminating power among low-copy-number isolates than does spoligotyping.

Mapping of IS6110 insertion sites in two epidemic strains of Mycobacterium tuberculosis.

A widely distributed strain designated 210 was identified in a study of the diversity of Mycobacterium tuberculosis DNA fingerprints from three geographically separate states in the United States. This strain is characterized by a 21-band fingerprint pattern when probed with IS6110, and the pattern is similar to that displayed by strains designated W. Intracellular growth of strain 210 isolates in human macrophages is significantly faster than that of isolates from other clusters or nonclustered isolates. The purpose of this study was to identify the sites of IS6110 insertions in strain 210 and compare these to IS6110 insertion sites in strain W. Our hypothesis is that an IS6110 insertion site(s) could possibly be responsible for a strain's increased capacity for transmission and/or replication. In this report, the insertion sites in strains 210 and W are described and referenced to their location in the M. tuberculosis H37Rv genome sequence. The W and 210 strains have 17 identical sites of IS6110 insertion and additional sequence not found in H37Rv but present in other clinical isolates. The IS6110 insertion site in the 36-bp direct repeat (DR) region of strains 210 and W has 15 spacers in the left flanking region. The DR region on the right side of IS6110 has been deleted. Five sites of insertion in strain 210 not found in strain W are described, as well as two unique sites in strain W. One copy of IS6110 was found to reside 55 bp in the ctpD gene. This gene is expressed, indicating that IS6110 can provide a promoter sequence for the transcription of genes.

Quantitative sputum bacillary load during rifampin-containing short course chemotherapy in human immunodeficiency virus-infected and non-infected adults with pulmonary tuberculosis.

SETTING: National Tuberculosis (TB) Treatment Centre, Mulago Hospital and Joint Clinical Research Centre, Kampala, Uganda. OBJECTIVE: To compare the quantitative sputum bacillary load between TB patients infected with the human immunodeficiency virus (HIV) and those non-infected, during treatment with standard short course chemotherapy (SCC). DESIGN: To compare clinical characteristics and quantitative sputum bacillary load as measured by quantitative acid-fast bacilli (AFB) smears, colony forming unit (cfu) assay and time until positive culture in the BACTEC radiometric liquid system between 14 HIV-infected and 22 non-HIV-infected adults with initial episodes of smear-positive pulmonary TB at baseline and during treatment with standard four-drug SCC. RESULTS: Other than cavitation (P = 0.042) and adenopathy (P = 0.03), which were more common among non-HIV-infected and HIV-infected patients, respectively, there were no significant differences in baseline demographic, clinical, radiological and laboratory characteristics between the groups. Mean pretreatment sputum bacillary burden (6.5+/-0.51 log10 AFB/ml, 5.91+/-0.91 log10 cfu/ml and 1.8+/-1.7 days until positive BACTEC culture for HIV-infected patients and 6.32+/-0.85 log10 AFB/ml, 5.58+/-0.68 log10 cfu/ml and 1+/-1.2 days until positive BACTC culture for non-HIV-infected patients) were comparable between HIV-infected and non-HIV-infected patients. Clinical and bacteriological responses to standard SCC and treatment outcome did not differ between the groups. CONCLUSION: Quantitative sputum bacillary load at baseline and during SCC did not differ significantly between HIV-infected and non-HIV-infected adults with initial episodes of smear-positive TB.

Determination of drug susceptibility and DNA fingerprint patterns of clinical isolates of Mycobacterium tuberculosis from Kampala, Uganda.

OBJECTIVE: To ascertain the rate of initial drug resistance and transmission patterns of Mycobacterium tuberculosis in Kampala, Uganda. SETTING: National Tuberculosis (TB) Treatment Centre, Mulago Hospital, Kampala, Uganda and Case Western Reserve University, Cleveland, Ohio, USA and McClellan Memorial Veterans Hospital, Little Rock, Arkansas, USA. METHODS: Using a radiometric BACTEC 460 TB system, susceptibility of 215 M. tuberculosis isolates from previously untreated patients from Kampala, Uganda (age range, 17-48 years, mean, 28 years; 56% males and 69% human immunodeficiency virus (HIV)-seropositive) was determined for isoniazid, rifampin, streptomycin and ethambutol. Isolates from 73 patients, selected on the basis of geographical location, were tested for strain diversity or relatedness using the IS6110 DNA fingerprinting technique. RESULTS: Resistance rates were as follows: isoniazid, 7.9% streptomycin, 6.1% rifampin, 1.4% and ethambutol 0.9%. Twelve per cent of the strains were resistant to at least one of the first line drugs tested and 4.7% were multiply resistant. There were no significant differences in resistance rates between patients with and without HIV infection. Using the number and size of DNA fragments containing IS6110, only three clusters of isolates with identical RFLP patterns were found out of the 73 isolates tested (8.2%). Each cluster contained two isolates. Three (4.1%) isolates had less than seven copies of IS6110. CONCLUSION: This study shows that in Uganda initial drug resistance rates to anti-tuberculosis agents are low and similar to other sub-Saharan African countries and that multiple strains of M. tuberculosis have been transmitted within the community.

PIP: This study was undertaken to determine the rate of initial drug resistance and transmission patterns of Mycobacterium tuberculosis (TB) in Kampala, Uganda. Using a radiometric BACTEC 460 TB system, 215 M. tuberculosis isolates from previously untreated patients (aged 17-48 years, mean age = 28 years; 56% males and 69% HIV-seropositive) were analyzed for susceptibility to isoniazid, rifampin, streptomycin, and ethambutol. Isolates from 73 patients were examined for strain diversity or relatedness using the insertional sequence 6110 (IS6110) DNA fingerprinting technique. The study revealed the following drug resistance rates: isoniazid, 7.9%; streptomycin, 6.1%; rifampin, 1.4%; and ethambutol, 0.9%. Resistance to at least one of the first line drugs tested were developed by 12% of the strains, while 4.7% showed multiple resistance. However, no significant differences in resistance rates were found between patients with and without HIV infection. Using the number and size of DNA fragments containing IS6110, only three clusters of isolates with identical patterns were found out of the 73 isolates tested (8.2%). Each cluster contained two isolates, and three isolates had less than 7 copies of IS6110. These findings suggest that initial drug resistance to anti-tuberculosis agents in this region is low and similar to other countries in sub-Saharan Africa and that multiple strains of M. tuberculosis have been transmitted within the community.