Diversity of DNA Fingerprints of Mycobacterium tuberculosis Isolates in the United States

To investigate the diversity of IS6110 fingerprints of Mycobacterium tuberculosis isolates in the United States and to determine if matching IS6110 fingerprints represent recent interstate tuberculosis transmission, we performed restriction fragment length polymorphism analysis of M. tuberculosis isolates from 1,326 patients in three geographically separated states. Seven hundred ninety-five different IS6110 fingerprint patterns were generated, and pattern diversity was similar in each state. Ninety-six percent of the fingerprint patterns were observed in only one state, demonstrating that most IS6110 fingerprint patterns are confined to a single geographic location. Of the IS6110 fingerprint patterns that were shared by isolates from more than one state, most isolates with 1 to 5 IS6110 copies were separable by pTBN12 fingerprinting whereas those with >15 copies were not. One high-copy-number M. tuberculosis strain had identical IS6110 and pTBN12 fingerprints and included 57 isolates from three states. Epidemiological data demonstrated significant recent transmission of tuberculosis within each city but not among the states. This suggests that identical fingerprints of isolates from geographically separate locations most likely reflect interstate tuberculosis transmission in the past, with subsequent intrastate spread of disease. Further evaluation of M. tuberculosis strains that cause outbreaks in different geographic locations will provide insight into the epidemiological and bacteriological factors that facilitate the spread of tuberculosis.     

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.     

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.     

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.     

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.     

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.     

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.     

Isolation of a Novel Strain of Mycobacterium iranicum from a Woman in the United States

A novel strain of Mycobacterium iranicum, a recently described nontuberculous Mycobacterium species, was isolated from the sputum of a woman. The source of infection was not determined; however, fomite transmission of inhaled aerosolized secretions from her husband''s sleep apnea equipment was historically possible.     

Aspartylglucosaminuria in the United States

Aspartylglucosaminuria (AGU) was diagnosed in two unrelated males with progressive mental retardation, coarse facies and skeletal abnormalities. Until now, this disorder has been described in predominantly Finnish populations with only one previous case reported in the U.S. We conclude that AGU may be more common in nowFinnish populations than the number of reported cases would indicate and should be included in the differential diagnosis in patients with suspected lysosomal storage disorders regardless of their geographical or ethnic backgrounds.     

Rickettsioses in the United States

Rickettsiae are obligate intracellular Gram-negative bacilli with global distribution. In the United States, nine Rickettsia species have been reported to cause disease in humans and are collectively responsible for the thousands of rickettsioses reported each year. Rickettsial infections are transmitted to humans via a variety of arthropod vectors, including ticks, lice, fleas, and mites. The severity of human infection ranges from subclinical and mild to severe and life threatening, dependent upon the infecting Rickettsia species, host factors, and time to appropriate antimicrobial therapy. Clinical presentation is often non-specific, making early recognition a challenge for health care professional. Awareness and appreciation of rickettsial pathogens is key to early diagnosis and prompt treatment.     

Absence of Mycobacterium intracellulare and Presence of Mycobacterium chimaera in Household Water and Biofilm Samples of Patients in the United States with Mycobacterium avium Complex Respiratory Disease

Recent studies have shown that respiratory isolates from pulmonary disease patients and household water/biofilm isolates of Mycobacterium avium could be matched by DNA fingerprinting. To determine if this is true for Mycobacterium intracellulare, household water sources for 36 patients with Mycobacterium avium complex (MAC) lung disease were evaluated. MAC household water isolates from three published studies that included 37 additional MAC respiratory disease patients were also evaluated. Species identification was done initially using nonsequencing methods with confirmation by internal transcribed spacer (ITS) and/or partial 16S rRNA gene sequencing. M. intracellulare was identified by nonsequencing methods in 54 respiratory cultures and 41 household water/biofilm samples. By ITS sequencing, 49 (90.7%) respiratory isolates were M. intracellulare and 4 (7.4%) were Mycobacterium chimaera. In contrast, 30 (73%) household water samples were M. chimaera, 8 (20%) were other MAC X species (i.e., isolates positive with a MAC probe but negative with species-specific M. avium and M. intracellulare probes), and 3 (7%) were M. avium; none were M. intracellulare. In comparison, M. avium was recovered from 141 water/biofilm samples. These results indicate that M. intracellulare lung disease in the United States is acquired from environmental sources other than household water. Nonsequencing methods for identification of nontuberculous mycobacteria (including those of the MAC) might fail to distinguish closely related species (such as M. intracellulare and M. chimaera). This is the first report of M. chimaera recovery from household water. The study underscores the importance of taxonomy and distinguishing the many species and subspecies of the MAC.     

Spoligotype signatures in the Mycobacterium tuberculosis complex

Evolution of the direct repeat region in Mycobacterium tuberculosis has created unique spoligotype signatures specifically associated with IS6110-defined strain families. Spoligotyping signatures may enable the analysis of the strain population structure in different settings and will enable the rapid identification of strain families that acquire drug resistance or escape protective immunity in drug and vaccine trials.     

Mycobacterium tuberculosis complex in atherosclerosis

In recent years, the results of some studies have revealed the possible potential role of several infectious agents in the inflammatory mechanism of atherosclerosis. The detection of specific antibodies against microorganisms such as and as well as Chlamydia pneumoniae and cytomegalovirus as well as antibodies directed to heat shock proteins in the sera of atherosclerotic patients and the presence of genomic material in atheromatous plaques all provide evidence supporting the presumptive role of infectious agents in atherosclerosis. There are some findings that can be accepted as clues for the possible involvement of Mycobacterium tuberculosis in atherosclerosis. These consist of the presence of high levels of mycobacterial heat shock protein 65 in atherosclerotic patients, and in animal studies, the detection of atherosclerotic changes in the vascular wall of animals vaccinated with recombinant heat shock protein 65, and Mycobacterium tuberculosis containing heat shock protein 65. The probable proatherogenic effect of the specific immune response to BCG-associated heat shock protein was also suggested. The mycobacterium cell wall contains a phospholipid, phosphatidylinositol, which was shown to have a procoagulant effect similar to that of a cytomegalovirus possessing phosphatidylserine, another phospholipid showing a procoagulant effect. These data suggest that Mycobacterium tuberculosis may also be involved in the pathogenesis of atherosclerosis.