Pyrazinamide-monoresistant Mycobacterium tuberculosis in the United States

Mycobacterium bovis is naturally resistant to the antituberculosis drug pyrazinamide (PZA). To determine whether all Mycobacterium tuberculosis complex isolates demonstrating PZA monoresistance were truly M. bovis, we examined the phenotype and genotype of isolates reported as PZA monoresistant in five counties in California from January 1996 through June 1999. Isolates reported by local laboratories to be PZA monoresistant were sent to the state reference laboratory for repeat susceptibility testing using the BACTEC radiometric method and to the Centers for Disease Control and Prevention for pncA sequencing and PCR-restriction fragment length polymorphism (RFLP) analysis of the oxyR gene. Of 1,916 isolates, 14 were reported as PZA monoresistant and 11 were available for retesting. On repeat testing, 6 of the 11 isolates were identified as PZA-susceptible M. tuberculosis, 1 was identified as PZA-monoresistant M. bovis, and 1 was identified as M. bovis BCG. The three remaining isolates were identified as PZA-monoresistant M. tuberculosis. Sequencing of the pncA and oxyR genes genotypically confirmed the two M. bovis and the six susceptible M. tuberculosis species. Each of the three PZA-monoresistant M. tuberculosis isolates had different, previously unreported, pncA gene mutations: a 24-bp deletion in frame after codon 88, a base substitution at codon 104 (Ser104Cys), and a base substitution at codon 90 (Ile90Ser). This study demonstrates that PZA monoresistance is not an absolute marker of M. bovis species but may also occur in M. tuberculosis, associated with a number of different mutational events in the pncA gene. It is the first report of PZA-monoresistant M. tuberculosis in the United States.     

Concordance between Molecular and Phenotypic Testing of Mycobacterium tuberculosis Complex Isolates for Resistance to Rifampin and Isoniazid in the United States

Multidrug-resistant (MDR) isolates of Mycobacterium tuberculosis complex (MTBC) are defined by resistance to at least rifampin (RMP) and isoniazid (INH). Rapid and accurate detection of multidrug resistance is essential for effective treatment and interruption of disease transmission of tuberculosis (TB). Overdiagnosis of MDR TB may result in treatment with second-line drugs that are more costly, less effective, and more poorly tolerated than first-line drugs. CDC offers rapid confirmation of MDR TB by the molecular detection of drug resistance (MDDR) for mutations associated with resistance to RMP and INH along with analysis for resistance to other first-line and second-line drugs. Simultaneously, CDC does growth-based phenotypic drug susceptibility testing (DST) by the indirect agar proportion method for a panel of first-line and second-line antituberculosis drugs. We reviewed discordance between molecular and phenotypic DST for INH and RMP for 285 isolates submitted as MTBC to CDC from September 2009 to February 2011. We compared CDC''s results with those from the submitting public health laboratories (PHL). Concordances between molecular and phenotypic testing at CDC were 97.4% for RMP and 92.5% for INH resistance. Concordances between CDC''s molecular testing and PHL DST results were 93.9% for RMP and 90.0% for INH. Overall concordance between CDC molecular and PHL DST results was 91.7% for RMP and INH collectively. Discordance was primarily attributable to the absence of known INH resistance mutations in isolates found to be INH resistant by DST and detection of mutations associated with low-level RMP resistance in isolates that were RMP susceptible by phenotypic DST. Both molecular and phenotypic test results should be considered for the diagnosis of MDR TB.     

Genetic Diversity and Dynamic Distribution of Mycobacterium tuberculosis Isolates Causing Pulmonary and Extrapulmonary Tuberculosis in Thailand

This study examined the genetic diversity and dynamicity of circulating Mycobacterium tuberculosis strains in Thailand using nearly neutral molecular markers. The single nucleotide polymorphism (SNP)-based genotypes of 1,414 culture-positive M. tuberculosis isolates from 1,282 pulmonary tuberculosis (PTB) and 132 extrapulmonary TB (EPTB) patients collected from 1995 to 2011 were characterized. Among the eight SNP cluster groups (SCG), SCG2 (44.1%), which included the Beijing (BJ) genotype, and SCG1 (39.4%), an East African Indian genotype, were dominant. Comparisons between the genotypes of M. tuberculosis isolates causing PTB and EPTB in HIV-negative cases revealed similar prevalence trends although genetic diversity was higher in the PTB patients. The identification of 10 reported sequence types (STs) and three novel STs was hypothesized to indicate preferential expansion of the SCG2 genotype, especially the modern BJ ST10 (15.6%) and ancestral BJ ST19 (13.1%). An association between SCG2 and SCG1 genotypes and particular patient age groups implies the existence of different genetic advantages among the bacterial populations. The results revealed that increasing numbers of young patients were infected with M. tuberculosis SCGs 2 and 5, which contrasts with the reduction of the SCG1 genotype. Our results indicate the selection and dissemination of potent M. tuberculosis genotypes in this population. The determination of heterogeneity and dynamic population changes of circulating M. tuberculosis strains in countries using the Mycobacterium bovis BCG (bacillus Calmette-Guérin) vaccine are beneficial for vaccine development and control strategies.     

Expanded geographical distribution of the N family of Mycobacterium tuberculosis strains within the United States

The N and W-Beijing families of Mycobacterium tuberculosis are phylogenetically closely related. The ability of the W-Beijing family to rapidly cause widespread disease is well described; however, few outbreaks involving the N family have been reported outside the New York City, N.Y., area. During 2002 to 2003, Seattle, Wash., experienced a rapidly expanding tuberculosis outbreak involving 38 persons in a 23-month period. The outbreak strain, SBRI9, exhibited the genotypic properties of the N family. Its IS6110 restriction fragment length polymorphism pattern was identical or nearly identical to those of two N family strains that were responsible for clusters of tuberculosis cases, including a large nosocomial outbreak, in New York City and New Jersey from 1989 to 1990. It was also identical to strains involved in late 1990s tuberculosis cases in Michigan, Maryland, and Arkansas. Further monitoring of the N family may show that it shares with the W-Beijing family the propensity to spread rapidly, suggesting that this characteristic evolved prior to the divergence of the two genetic lineages.     

High Diversity of Mycobacterium tuberculosis Genotypes in South Africa and Preponderance of Mixed Infections among ST53 Isolates

The reemergence of tuberculosis (TB) has become a major health problem worldwide, especially in Asia and Africa. Failure to combat this disease due to nonadherence or inappropriate drug regimens has selected for the emergence of multiple-drug-resistant (MDR) TB. The development of new molecular genotyping techniques has revealed the presence of mixed Mycobacterium tuberculosis infections, which may accelerate the emergence of drug-resistant strains. There are some studies describing the local distribution of circulating strains in South Africa, but to date, reports describing the frequency and distribution of M. tuberculosis genotypes, and specifically MDR genotypes, across the different provinces are limited. Thus, 252 isolates (of which 109 were MDR) from eight of the nine provinces of South Africa were analyzed by spoligotyping. Spoligotyping showed 10 different lineages, and ST53 (11.1%) and ST1 (10.3%) were the most frequent genotypes. Of the 75 different spoligopatterns observed, 20 (7.9%) were previously unreported. Analysis of the mycobacterial interspersed repetitive units of variable-number tandem repeats of the ST53 and ST1 isolates revealed that ∼54% of the ST53 isolates were of mixed M. tuberculosis subpopulations. Drug resistance (defined as resistance to at least isoniazid and/or rifampin) could only be linked to a history of previous anti-TB treatment (adjusted odds ratio, 4.0; 95% confidence interval, 2.27 to 7.10; P = <0.0001). This study describes a high diversity of circulating genotypes in South Africa in addition to a high frequency of mixed M. tuberculosis subpopulations among the ST53 isolates. MDR TB in South Africa could not be attributed to the spread of any single lineage.Tuberculosis (TB) is a major cause of illness and death worldwide but especially in Asia and Africa (42). Twenty-two countries designated high TB burden countries account for 80% of all new cases worldwide (42). As of 2008, South Africa was ranked fourth among these, with an incidence rate of 940 cases per 100,000 persons (42) (up from 536 in 2005 [41]). Due to nonadherence to drug regimens or the use of inappropriate drug regimens, the TB epidemic has been largely exacerbated by the emergence of multidrug-resistant (MDR) TB (7).Traditionally, it has been assumed that TB is caused by an infection with a single strain and that recurrences are the result of reactivation of the strain causing the first episode (6, 24, 39). However, it has recently been shown that patients, both human immunodeficiency virus (HIV) positive and HIV negative, in high-incidence settings may have more than one strain in the same sputum sample (24, 39) and that mixed infections may cause complications in the treatment of the disease if a patient is infected with both a sensitive and a resistant Mycobacterium tuberculosis isolate (39). Extensively drug-resistant (XDR) TB strains (defined as resistant to isoniazid [INH] and rifampin [RIF], in addition to any fluoroquinolones and at least one injectable anti-TB drug) (5) were first reported in South Africa in 2006 and later shown to be present in at least 17 countries and on all of the continents (9). Spoligotyping has revealed that the majority of these cases were caused by M. tuberculosis belonging to the KZN family (ST60), which has been known to be prevalent in this area since 1994 (23). Since then, only one other study has been published (19) providing genotypic information on XDR TB strains in South Africa. That study shows that XDR TB strains in South Africa belong to seven different lineages and are present in four of the nine provinces. Such studies highlight the need for standardized and accurate drug susceptibility testing in combination with high-level molecular genotyping in order to carefully monitor new and emerging MDR and XDR TB strains.The “gold standard” for the typing of M. tuberculosis is currently IS6110-based restriction fragment length polymorphism (RFLP). Combined with spoligotyping, this has proven to be very useful for the study of the transmission, evolution, and phylogeny of M. tuberculosis (18). However, RFLP is laborious, requires a large amount of DNA, and has poor interlaboratory reproducibility. Recently, a new genotyping technique based on PCR amplification of mycobacterial interspersed repetitive units of variable-number tandem repeats (MIRU-VNTR) was introduced (31, 32). This method is much faster than IS6110 RFLP and requires less DNA. Fifteen-locus-based MIRU-VNTR analysis has been shown to have slightly better discriminatory power than IS6110 RFLP, especially when combined with spoligotyping (2, 22). In addition to being a rapid and highly discriminatory genotyping method, MIRU-VNTR can also be used for the detection of mixed subpopulations in a single sputum sample (1, 26). Several studies have investigated the differentiation power of different MIRU-VNTR locus combinations for strains of the Beijing lineage (11, 14, 15). The studies suggest that the choice of appropriate MIRU-VNTR loci requires further investigation in diverse M. tuberculosis lineages in countries with low and high TB endemicity.Thus, the objectives of this study were to assess the distribution and diversity of MDR M. tuberculosis genotypes across the South African provinces and to determine if there is an association between MDR TB and a particular M. tuberculosis genotype. We also determined the general population structure of South African M. tuberculosis isolates, irrespective of the drug susceptibility pattern. Furthermore, we assessed the ability of the MIRU-VNTR method to discriminate the most frequent genotypes observed.     

DNA Fingerprinting of Mycobacterium tuberculosis Complex Culture Isolates Collected in Brazil and Spotted onto Filter Paper

The usefulness of filter paper for preservation of bacterial cells was shown by mixed-linker DNA fingerprint analysis of Mycobacterium tuberculosis isolates from 77 Brazilian patients. DNA fingerprints of samples spotted onto filter paper and conventional culture material were identical. Thus, filter paper specimens analyzed by an amplification-based typing method provide a new resource for epidemiological studies of infectious diseases.     

Genome Sequencing of Mycobacterium abscessus Isolates from Patients in the United States and Comparisons to Globally Diverse Clinical Strains

Nontuberculous mycobacterial infections caused by Mycobacterium abscessus are responsible for a range of disease manifestations from pulmonary to skin infections and are notoriously difficult to treat, due to innate resistance to many antibiotics. Previous population studies of clinical M. abscessus isolates utilized multilocus sequence typing or pulsed-field gel electrophoresis, but high-resolution examinations of genetic diversity at the whole-genome level have not been well characterized, particularly among clinical isolates derived in the United States. We performed whole-genome sequencing of 11 clinical M. abscessus isolates derived from eight U.S. patients with pulmonary nontuberculous mycobacterial infections, compared them to 30 globally diverse clinical isolates, and investigated intrapatient genomic diversity and evolution. Phylogenomic analyses revealed a cluster of closely related U.S. and Western European M. abscessus subsp. abscessus isolates that are genetically distinct from other European isolates and all Asian isolates. Large-scale variation analyses suggested genome content differences of 0.3 to 8.3%, relative to the reference strain ATCC 19977^T. Longitudinally sampled isolates showed very few single-nucleotide polymorphisms and correlated genomic deletion patterns, suggesting homogeneous infection populations. Our study explores the genomic diversity of clinical M. abscessus strains from multiple continents and provides insight into the genome plasticity of an opportunistic pathogen.     

Genetic Diversity and Antimicrobial Susceptibility of Campylobacter jejuni Isolates Associated with Sheep Abortion in the United States and Great Britain

Campylobacter infection is a leading cause of ovine abortion worldwide. Historically, genetically diverse Campylobacter fetus and Campylobacter jejuni strains have been implicated in such infections, but since 2003 a highly pathogenic, tetracycline-resistant C. jejuni clone (named SA) has become the predominant cause of sheep abortions in the United States. Whether clone SA was present in earlier U.S. abortion isolates (before 2000) and is associated with sheep abortions outside the United States are unknown. Here, we analyzed 54 C. jejuni isolates collected from U.S. sheep abortions at different time periods and compared them with 42 C. jejuni isolates associated with sheep abortion during 2002 to 2008 in Great Britain, using multilocus sequence typing (MLST), pulsed-field gel electrophoresis (PFGE), and array-based comparative genomic hybridization (CGH). Although clone SA (ST-8) was present in the early U.S. isolates, it was not as tetracycline resistant (19% versus 100%) or predominant (66% versus 91%) as it was in the late U.S isolates. In contrast, C. jejuni isolates from Great Britain were genetically diverse, comprising 19 STs and lacking ST-8. PFGE and CGH analyses of representative strains further confirmed the population structure of the abortion isolates. Notably, the Great Britain isolates were essentially susceptible to most tested antibiotics, including tetracycline, while the late U.S. isolates were universally resistant to this antibiotic, which could be explained by the common use of tetracyclines for control of sheep abortions in the United States but not in Great Britain. These results suggest that the dominance of clone SA in sheep abortions is unique to the United States, and the use of tetracyclines may have facilitated selection of this highly pathogenic clone.     

Evaluation of a two-step approach for large-scale, prospective genotyping of Mycobacterium tuberculosis isolates in the United States

Genotyping of Mycobacterium tuberculosis isolates is useful in tuberculosis control for confirming suspected transmission links, identifying unsuspected transmission, and detecting or confirming possible false-positive cultures. The value is greatly increased by reducing the turnaround time from positive culture to genotyping result and by increasing the proportion of cases for which results are available. Although IS6110 fingerprinting provides the highest discrimination, amplification-based methods allow rapid, high-throughput processing and yield digital results that can be readily analyzed and thus are better suited for large-scale genotyping. M. tuberculosis isolates (n = 259) representing 99% of culture-positive cases of tuberculosis diagnosed in Wisconsin in the years 2000 to 2003 were genotyped by using spoligotyping, mycobacterial interspersed repetitive unit (MIRU) typing, and IS6110 fingerprinting. Spoligotyping clustered 64.1% of the isolates, MIRU typing clustered 46.7% of the isolates, and IS6110 fingerprinting clustered 29.7% of the isolates. The combination of spoligotyping and MIRU typing yielded 184 unique isolates and 26 clusters containing 75 isolates (29.0%). The addition of IS6110 fingerprinting reduced the number of clustered isolates to 30 (11.6%) if an exact pattern match was required or to 44 (17.0%) if the definition of a matching IS6110 fingerprint was expanded to include patterns that differed by the addition of a single band. Regardless of the genotyping method chosen, the addition of a second or third method decreased clustering. Our results indicate that using spoligotyping and MIRU typing together provides adequate discrimination in most cases. IS6110 fingerprinting can then be used as a secondary typing method to type the clustered isolates when additional discrimination is needed.     

Mixed Infections and Rifampin Heteroresistance among Mycobacterium tuberculosis Clinical Isolates

Mixed infections and heteroresistance of Mycobacterium tuberculosis contribute to the difficulty of diagnosis, treatment, and control of tuberculosis. However, there is still no proper solution for these issues. This study aimed to investigate the potential relationship between mixed infections and heteroresistance and to determine the high-risk groups related to these factors. A total of 499 resistant and susceptible isolates were subjected to spoligotyping and 24-locus variable-number tandem repeat methods to analyze their genotypic lineages and the occurrence of mixed infections. Two hundred ninety-two randomly selected isolates were sequenced on their rpoB gene to examine mutations and heteroresistance. The results showed that 12 patients had mixed infections, and the corresponding isolates belonged to Manu2 (n = 8), Beijing (n = 2), T (n = 1), and unknown (n = 1) lineages. Manu2 was found to be significantly associated with mixed infections (odds ratio, 47.72; confidence interval, 9.68 to 235.23; P < 0.01). Four isolates (1.37%) were confirmed to be heteroresistant, which was caused by mixed infections in three (75%) isolates; these belonged to Manu2. Additionally, 3.8% of the rifampin-resistant isolates showing no mutation in the rpoB gene were significantly associated with mixed infections (χ², 56.78; P < 0.01). This study revealed for the first time that Manu2 was the predominant group in the cases of mixed infections, and this might be the main reason for heteroresistance and a possible mechanism for isolates without any mutation in the rpoB gene to become rifampin resistant. Further studies should focus on this lineage to clarify its relevance to mixed infections.     

Characterization of rpoB Mutations in Rifampin-Resistant Clinical Mycobacterium tuberculosis Isolates from Greece

There is a geographic distribution of Mycobacterium tuberculosis strains with various rpoB gene mutations that account for rifampin resistance. We studied 17 rifampin-resistant clinical isolates from patients in Greece to identify rpoB mutations. The aim of our study was the evaluation of a commercially available line probe assay kit (INNO-LiPA Rif. TB) to detect rpoB mutations and rifampin resistance. The results obtained with the commercially available assay were compared to those obtained by automated DNA sequence analysis of amplified PCR products. Randomly amplified polymorphic DNA (RAPD) analyses of the isolates were also performed. The overall concordance of the line probe assay with phenotypic rifampin susceptibility test was 94%. Three distinct rpoB mutations in codons Ser₅₃₁, His₅₂₆, and Asp₅₁₆ were correctly identified with the kit, but mutations in external regions and insertions were detected only by automated DNA sequence analysis. The changes in codons Ser₅₃₁ and His₅₂₆ accounted for the majority of rifampin resistance, as previously described for isolates from other geographic areas. The results obtained by RAPD analyses of the isolates suggested that clonally related M. tuberculosis strains can have subclones bearing distinct mutant rpoB alleles. We conclude that this line probe assay kit, which is fast and with which tests are easy to perform, can be used for the rapid detection of rifampin resistance in M. tuberculosis before the availability of results by conventional methods and for epidemiological studies but that negative results obtained by this method do not rule out rifampin resistance.     

Rapid Whole-Genome Sequencing of Mycobacterium tuberculosis Isolates Directly from Clinical Samples

The rapid identification of antimicrobial resistance is essential for effective treatment of highly resistant Mycobacterium tuberculosis. Whole-genome sequencing provides comprehensive data on resistance mutations and strain typing for monitoring transmission, but unlike for conventional molecular tests, this has previously been achievable only from cultures of M. tuberculosis. Here we describe a method utilizing biotinylated RNA baits designed specifically for M. tuberculosis DNA to capture full M. tuberculosis genomes directly from infected sputum samples, allowing whole-genome sequencing without the requirement of culture. This was carried out on 24 smear-positive sputum samples, collected from the United Kingdom and Lithuania where a matched culture sample was available, and 2 samples that had failed to grow in culture. M. tuberculosis sequencing data were obtained directly from all 24 smear-positive culture-positive sputa, of which 20 were of high quality (>20× depth and >90% of the genome covered). Results were compared with those of conventional molecular and culture-based methods, and high levels of concordance between phenotypical resistance and predicted resistance based on genotype were observed. High-quality sequence data were obtained from one smear-positive culture-negative case. This study demonstrated for the first time the successful and accurate sequencing of M. tuberculosis genomes directly from uncultured sputa. Identification of known resistance mutations within a week of sample receipt offers the prospect for personalized rather than empirical treatment of drug-resistant tuberculosis, including the use of antimicrobial-sparing regimens, leading to improved outcomes.     

Antibodies against Immunodominant Antigens of Mycobacterium tuberculosis in Subjects with Suspected Tuberculosis in the United States Compared by HIV Status

The immunodominance of Mycobacterium tuberculosis proteins malate synthase (MS) and MPT51 has been demonstrated in case-control studies with patients from countries in which tuberculosis (TB) is endemic. The value of these antigens for the serodiagnosis of TB now is evaluated in a cross-sectional study of pulmonary TB suspects in the United States diagnosed to have TB, HIV-associated TB, or other respiratory diseases (ORD). Serum antibody reactivity to recombinant purified MS and MPT51 was determined by enzyme-linked immunosorbent assays (ELISAs) of samples from TB suspects and well-characterized control groups. TB suspects were diagnosed with TB (n = 87; 49% sputum microscopy negative, 20% HIV⁺) or ORD (n = 63; 58% HIV⁺). Antibody reactivity to MS and MPT51 was significantly higher in U.S. HIV⁺/TB samples than in HIV⁻/TB samples (P < 0.001), and it was significantly higher in both TB groups than in control groups with latent TB infection (P < 0.001). Antibody reactivity to both antigens was higher in U.S. HIV⁺/TB samples than in HIV⁺/ORD samples (P = 0.052 for MS, P = 0.001 for MPT51) but not significantly different between HIV⁻/TB and HIV⁻/ORD. Among U.S. HIV⁺ TB suspects, a positive anti-MPT51 antibody response was strongly and significantly associated with TB (odds ratio, 11.0; 95% confidence interval, 2.3 to 51.2; P = 0.002). These findings have implications for the adjunctive use of TB serodiagnosis with these antigens in HIV⁺ subjects.The detection and treatment of individuals who are at early stages of active pulmonary tuberculosis (TB) is critical for the successful control and elimination of TB (34, 38). Mycobacterium tuberculosis is a slow-growing pathogen, and it takes months to years for an infection (and, presumably, reactivation) to progress to clinical TB. In resource-limited countries, the microscopic examination of smears made directly from unprocessed sputum are used for diagnosis, resulting in the identification of only advanced TB patients with high bacillary burden. In contrast, in industrialized settings the combined use of the fluorescence microscopy of decontaminated and concentrated sputum, mycobacterial culture, and nucleic acid amplification technologies permits the identification of patients with much lower bacillary burden and, thus, in the early stages of TB. Still, only around 50% of TB cases are rapidly diagnosed by optimized microscopy (5, 18). While adjunctive amplification methods increase the yield of confirmed TB, albeit with added cost and delays, around 20% of TB cases remain without microbiologic confirmation (5, 18). Additional tests that can enhance the rapid identification of patients at early stages of TB are required to add to the armamentarium of TB diagnostic tests.The amplification power of immune responses potentially can detect TB at a low antigen threshold and without requiring a specimen from the site of infection. Assays that detect TB infection by measuring the gamma interferon release of circulating lymphocytes in response to M. tuberculosis-specific antigens (IGRAs) cannot distinguish active from latent TB infection (LTBI) (reviewed in reference 15) and have limited utility in patients with advanced HIV infection (3, 32). In contrast, positive antibody (Ab) responses to several new antigens during active TB but not LTBI have been reported. However, most studies have focused on patients with advanced TB, and the utility of these responses appears to be limited in patients with low mycobacterial burden (reviewed in references 29 and 30).Two immunodominant M. tuberculosis proteins, the 81-kDa malate synthase (MS; Rv 1837c, GlcB) and the 27-kDa MPT51 (Rv3803c), are reported to elicit Ab responses during early and advanced stages of TB in both HIV⁻ and HIV⁺ patients (1, 2, 10, 16, 24, 27, 37). This is important because HIV⁺ TB patients appear to develop Ab responses to a smaller repertoire of M. tuberculosis antigens than that of HIV⁻ TB patients (24, 25). In previous case-control studies with HIV⁻ and HIV⁺ patients, pulmonary and extrapulmonary TB patients from settings in which TB is endemic demonstrated the presence of anti-MS and/or anti-MPT51 Abs in about 80% of the TB patients but not in tuberculin skin test (TST)-negative and -positive volunteers (27, 37). Similar studies with U.S. patients demonstrated that while anti-MS and/or anti-MPT51 Abs were present in only ∼40% of the HIV⁻ patients at early stages of TB, ∼80% of the U.S. HIV⁺ TB patients were Ab positive (1). Nevertheless, combining serology with sputum microscopy improved the detection of TB in both groups compared to that of microscopy alone, and it led to the identification of 90% of HIV⁺ TB patients, compared to 60% by microscopy alone (1). These findings are of high clinical relevance, since the rapid identification and treatment of early TB is crucial for HIV⁺ patients, in whom the dual infection leads to the acceleration of both diseases (20, 35).While antigen discovery, selection, and validation initially relies on case-control studies comparing known TB cases to healthy controls, such comparisons result in the overestimation of accuracy (14, 23), and the real value of any antigen needs to be ascertained by cross-sectional studies in clinical settings where the TB suspects include patients with a variety of respiratory diseases. The goals of the current investigations were to (i) identify the range of Ab reactivities to MS and MPT51 in a cross-sectional study of U.S. TB suspects, (ii) compare Ab reactivities between U.S. HIV⁻ and HIV⁺ TB patients and to asymptomatic U.S. non-TB as well as endemic TB controls; and (iii) compare Ab reactivities of HIV⁻ and HIV⁺ TB suspects diagnosed to have respiratory diseases other than TB (ORD) to those of HIV⁻ and HIV⁺ TB patients.     

Specificity and Diversity of Antibodies to Mycobacterium tuberculosis Arabinomannan

Arabinomannan (AM) is a polysaccharide antigen of the mycobacterial capsule. However, it is uncertain whether AM constitutes an immunologically distinct fraction of Mycobacterium tuberculosis. In this study, we analyzed the repertoire and specificity of antibodies to AM by using AM-binding murine monoclonal antibodies (MAbs) and human serum samples. Murine MAbs were found to be diverse in their specificity to AM and cross-reactivity with other arabinose-containing mycobacterial polysaccharides, with MAb 9d8 binding exclusively to AM. Human antibodies to AM were detected in serum samples from patients with pulmonary tuberculosis (TB), as well as in those from healthy, purified protein derivative-negative controls, with significantly higher titers among patients. The binding of human antibodies to AM was inhibited by MAb 9d8 in three patients with TB but not in controls. MAb 5c11, which recognizes other mycobacterial arabinose-containing carbohydrates in addition to AM, inhibited the binding of serum samples from 75% of patients and 76% of controls. Analysis of human antibodies with murine MAbs to human V_H determinants demonstrated diversity among antibodies to AM with qualitative and quantitative differences compared with antibodies to lipoarabinomannan. In summary, our study suggests that antibodies to AM are diverse and heterogeneous with respect to antigen recognition and V_H determinant expression, with human serum samples containing different subsets of antibodies to AM with the specificities of AM-binding murine MAbs. One MAb and a subset of human antibodies bind AM specifically, suggesting that this polysaccharide is antigenically distinct and is expressed in human infection.     

Molecular analysis of Mycobacterium kansasii isolates from the United States

We studied the population genetics of Mycobacterium kansasii isolates from the United States by PCR restriction enzyme analysis (PRA) of the 441-bp Telenti fragment of the hsp-65 gene and pulsed-field gel electrophoresis (PFGE) of genomic DNA with the restriction endonucleases AseI, DraI, and XbaI, and we compared the patterns to those previously reported from France and Japan. By PRA, 78 of 81 clinical isolates (96%) from the United States belonged to subspecies I. With PFGE, 28 AseI patterns, 32 DraI patterns, and 35 XbaI patterns were produced. PFGE showed marked clonality of the U.S. isolates, with differences between genotypes involving only one or two bands. Isolates within Texas showed lower pattern diversity than those from different states. With DraI, 31 of 71 isolates (44%) had the same common PFGE pattern, which matched the predominant pattern in France (pattern Ia), determined by Picardeau et al. (M. Picardeau, G. Prod'hom, L. Raskine, M. P. LePennec, and V. Vincent, J. Clin. Microbiol. 35:25-32, 1997), and in Japan (type M), determined by Iinuma et al. (Y. Iinuma, S. Ichiyama, Y. Hasegawa, K. Shimokata, S. Kawahara, and T. Matsushima, J. Clin. Microbiol. 35:596-599, 1997). With AseI, 42% of isolates produced a common pattern indistinguishable from the common pattern seen in French isolates (Ia) and with only one band difference from the common pattern (type M) in Japan. This study demonstrates that subspecies I is the predominant subspecies of M. kansasii among clinical isolates in the United States, as it is in Europe and Japan, and that genotype I is highly clonal worldwide, with the same major genotype responsible for human infection. The fact that a single clone of M. kansasii is responsible for most cases of human disease suggests that specific virulence factors may be associated with this specific genotype.