Comparison of methods available for identification of Mycobacterium chimaera

ObjectivesMycobacterium chimaera is a recently described nontuberculous mycobacterium belonging to the Mycobacterium avium complex (MAC). Because this species is implicated in a worldwide outbreak due to contaminated heater–cooler unit water tanks during open-heart surgery, it has become mandatory for clinical microbiology laboratories to be able to differentiate M. chimaera from the other MAC species, especially M. intracellulare. Such identification has so far been restricted to specialized laboratories because it required the analysis of several gene sequences. The aim of this study was to evaluate commercial methods for identifying M. chimaera with regard to the reference gene sequencing ITS, the internal transcribed spacer 16–23S.MethodsForty-seven clinical and environmental isolates including 41 MAC were identified by (a) PCR sequencing of the ITS and hsp65 genes, (b) three molecular biology kits (INNO-LiPA Mycobacteria, GenoType Mycobacterium CM and GenoType NTM-DR) and (c) matrix-assisted desorption ionization–time of flight mass spectrometry (MALDI-TOF MS) using Microflex LT.ResultsThere was a high concordance for species determination between the reference ITS sequencing and the GenoType NTM-DR test (39/41, 95%), the INNO-LiPA Mycobacteria test (38/41, 93%) and the hsp65 sequencing (38/41, 93%). The GenoType Mycobacterium CM test did not distinguish M. chimaera from M. intracellulare. MALDI-TOF MS distinguished two M. chimaera–M. intracellulare groups separated from M. avium and from the other mycobacterial species on a score-oriented dendrogram, but it also failed to differentiate the two species.ConclusionsINNO-LiPA Mycobacteria and GenoType NTM-DR are efficient assays for M. chimaera identification in clinical microbiology laboratories.     

Impact of different subspecies on disease progression in initially untreated patients with Mycobacterium avium complex lung disease

ObjectiveDisease progression is a strong indicator of treatment for Mycobacterium avium complex lung disease (MAC-LD). The impact of MAC subspecies on the risk of disease progression remains uncertain in MAC-LD patients.MethodsIn this cohort study, we included MAC-LD patients from 2013 to 2018 and classified them into M. intracellulare, M. avium, M. chimaera and other subspecies groups by genotype. We observed the disease progression of MAC-LD, indicated by antibiotic initiation and/or radiographic progression. We used Cox regression analysis to assess predictors for disease progression.ResultsOf 105 MAC isolates from unique MAC-LD patients, 35 (33%) were M. intracellulare, 41 (39%) M. avium, 16 (15%) M. chimaera and 13 (12%) other subspecies. After a mean follow-up time of 1.3 years, 56 (53%) patients developed disease progression: 71% (25/35), 54% (22/41), 31% (4/13) and 31% (5/16) in patients with M. intracellulare, M. avium, others and M. chimaera, respectively. The independent predictors for disease progression were M. chimaera subspecies (HR 0.356, 95% CI (0.134–0.943)), compared with the reference group of M. intracellulare, body mass index ≤20 kg/m² (HR 1.788 (1.022–3.130)) and initial fibrocavitary pattern (HR 2.840 (1.190–6.777)) after adjustment for age, sex and sputum smear positivity. Among patients without fibrocavitary lesions (n = 94), the risk of disease progression significantly decreased in patients with other subspecies (HR 0.217 (0.050–0.945)) and remained low in those with M. chimaera (HR 0.352 (0.131–0.947)).ConclusionsMycobacterium chimaera was not uncommon in this study; unlike M. intracellulare, it was negatively correlated with disease progression of MAC-LD, suggesting a role of MAC subspecies identification in prioritizing patients.     

Association between 16S-23S internal transcribed spacer sequence groups of Mycobacterium avium complex and pulmonary disease

Organisms within the Mycobacterium avium complex (MAC) may have differential virulence. We compared 33 subjects with MAC pulmonary disease to 75 subjects with a single positive culture without disease. M. avium isolates were significantly more likely to be associated with MAC pulmonary disease (odds ratio = 5.14, 95% confidence interval = 1.25 to 22.73) than M. intracellulare.     

In Vitro Activity of Amikacin against Isolates of Mycobacterium avium Complex with Proposed MIC Breakpoints and Finding of a 16S rRNA Gene Mutation in Treated Isolates

Amikacin is a major drug used for the treatment of Mycobacterium avium complex (MAC) disease, but standard laboratory guidelines for susceptibility testing are not available. This study presents in vitro amikacin MICs for 462 consecutive clinical isolates of the MAC using a broth microdilution assay. Approximately 50% of isolates had amikacin MICs of 8 μg/ml, and 86% had MICs of ≤16 μg/ml. Of the eight isolates (1.7%) with MICs of 64 μg/ml, five had an MIC of 32 μg/ml on repeat testing. Ten isolates (2.1%) had an initial amikacin MIC of >64 μg/ml, of which seven (1.5%) had MICs of >64 μg/ml on repeat testing. These seven isolates had a 16S rRNA gene A1408G mutation and included M. avium, Mycobacterium intracellulare, and Mycobacterium chimaera. Clinical data were available for five of these seven isolates, all of which had received prolonged (>6 months) prior therapy, with four that were known to be treated with amikacin. The 16S mutation was not detected in isolates with MICs of ≤64 μg/ml. We recommend primary testing of amikacin against isolates of the MAC and propose MIC guidelines for breakpoints that are identical to the CLSI guidelines for Mycobacterium abscessus: ≤16 μg/ml for susceptible, 32 μg/ml for intermediate, and ≥64 μg/ml for resistant. If considered and approved by the CLSI, this will be only the second drug recommended for primary susceptibility testing against the MAC and should facilitate its use for both intravenous and inhaled drug therapies.     

In vitro activity of aminoglycosides,clofazimine, d-cycloserine and dapsone against 83 Mycobacterium avium complex clinical isolates

Background/Purpose

Treatment success rates for Mycobacterium avium complex (MAC) diseases range from 50% to 55% only. To explore effective antimicrobials against either Mycobacterium intracellulare or M. avium, we determined in vitro activities of five aminoglycosides, clofazimine, dapsone and d-cycloserine compared with primary (clarithromycin) and secondary (moxifloxacin and linezolid) antimycobacterial agents.

Mycobacterium chimaera: Coming out of Nowhere to Dominate 2016 Infection Prevention Discussions

Mycobacterium chimaera is a bacterium of the 21st century. The organism was named in 2004 when molecular assays showed it to belong to the Mycobacterium avium complex. More specifically, it was found to be a close relative of Mycobacterium intracellulare. Merely a decade later, M. chimaera turned out to be the cause of infections associated with heater-cooler units used during open-heart surgery. There are still less than 100 confirmed cases worldwide, but the mycobacterial species has captured the attention of patients and caregivers alike because many of these infections are manifested months to years following surgery. No one currently knows the number of people who have been infected with M. chimaera or how many will ultimately be affected.     

Genetic relatedness of Mycobacterium avium-intracellulare complex isolates from patients with pulmonary MAC disease and their residential soils

Mycobacterium avium-intracellulare complex (MAC) strains were recovered from 48.9% of residential soil samples (agricultural farms (n = 7), residential yards (n = 79), and planting pots (n = 49)) of 100 pulmonary MAC patients and 35 non-infected control patients. The frequency of MAC recovery did not differ among soil types or among patients regardless of the presence of pulmonary MAC disease, infecting MAC species or period of soil exposure. Variable numbers of tandem repeats (VNTR) analysis for MAC clinical and soil isolates revealed 78 different patterns in 47 M. avium clinical isolates and 41 soil isolates, and 53 different patterns in 18 M. intracellulare clinical isolates and 37 soil isolates. Six clinical and corresponding soil isolate pairs with an identical VNTR genotype were from case patients with high soil exposure (≥2 h per week, 37.5% (6/16) with high exposure compared with 0.0% (0/19) with low or no exposure, p <0.01), suggesting that residential soils are a likely source of pulmonary MAC infection.     

Differential drug susceptibility patterns of Mycobacterium chimaera and other members of the Mycobacterium avium-intracellulare complex

Objectives

To determine MIC distributions for Mycobacterium chimaera, Mycobacterium intracellulare, Mycobacterium colombiense and Mycobacterium avium, and to derive tentative epidemiological cut-off (ECOFF) values.

Mycobacterial Interspersed Repetitive-Unit–Variable-Number Tandem-Repeat (MIRU-VNTR) Genotyping of Mycobacterium intracellulare for Strain Comparison with Establishment of a PCR-Based Database

Strain comparison is important to population genetics and to evaluate relapses in patients with Mycobacterium avium complex (MAC) lung disease, but the “gold standard” of pulsed-field gel electrophoresis (PFGE) is time-consuming and complex. We used variable-number tandem repeats (VNTR) for fingerprinting of respiratory isolates of M. intracellulare from patients with underlying bronchiectasis, to establish a nonsequence-based database for population analysis. Different genotypes identified by PFGE underwent species identification using a 16S rRNA gene multiplex PCR. Genotypes of M. intracellulare were confirmed by internal transcribed spacer 1 (ITS1) sequencing and characterized using seven VNTR primers. The pattern of VNTR amplicon sizes and repeat number defined each specific VNTR type. Forty-two VNTR types were identified among 84 genotypes. PFGE revealed most isolates with the same VNTR type to be clonal or exhibit similar grouping of bands. Repetitive sequence-based PCR (rep-PCR) showed minimal pattern diversity between VNTR types compared to PFGE. Fingerprinting of relapse isolates from 31 treated patients using VNTR combined with 16S multiplex PCR unambiguously and reliably distinguished different genotypes from the same patient, with results comparable to those of PFGE. VNTR for strain comparison is easier and faster than PFGE, is as accurate as PFGE, and does not require sequencing. Starting with a collection of 167 M. intracellulare isolates, VNTR distinguished M. intracellulare into 42 clonal groups. Comparison of isolates from different geographic areas, habitats, and clinical settings is now possible.     

Efficient Differentiation of Mycobacterium avium Complex Species and Subspecies by Use of Five-Target Multiplex PCR

Infections caused by the Mycobacterium avium complex (MAC) are on the rise in both human and veterinary medicine. A means of effectively discriminating among closely related yet pathogenetically diverse members of the MAC would enable better diagnosis and treatment as well as further our understanding of the epidemiology of these pathogens. In this study, a five-target multiplex PCR designed to discriminate MAC organisms isolated from liquid culture media was developed. This MAC multiplex was designed to amplify a 16S rRNA gene target common to all Mycobacterium species, a chromosomal target called DT1 that is unique to M. avium subsp. avium serotypes 2 and 3, to M. avium subsp. silvaticum, and to M. intracellulare, and three insertion sequences, IS900, IS901, and IS1311. The pattern of amplification results allowed determination of whether isolates were mycobacteria, whether they were members of the MAC, and whether they belonged to one of three major MAC subspecies, M. avium subsp. paratuberculosis, M. avium subsp. avium, and M. avium subsp. hominissuis. Analytical sensitivity was 10 fg of M. avium subsp. paratuberculosis genomic DNA, 5 to 10 fg of M. avium subsp. avium genomic DNA, and 2 to 5 fg of DNA from other mycobacterial species. Identification accuracy of the MAC multiplex was evaluated by testing 53 bacterial reference strains consisting of 28 different mycobacterial species and 12 nonmycobacterial species. Identification accuracy in a clinical setting was evaluated for 223 clinical MAC isolates independently identified by other methods. Isolate identification agreement between the MAC multiplex and these comparison assays was 100%. The novel MAC multiplex is a rapid, reliable, and simple assay for discrimination of MAC species and subspecies in liquid culture media.Since the early 1980s, there has been an increase in disease caused by organisms broadly categorized as nontuberculous mycobacteria (NTM), a generic term for mycobacteria not in the Mycobacterium tuberculosis complex and other than M. leprae (32). Of these NTM, Mycobacterium avium complex (MAC) species are the most common cause of human and animal disease globally (6, 14, 16, 24). The clinical relevance of the MAC in humans has been amplified in recent decades with the increasing population of immunocompromised individuals resulting from longer life expectancy, immunosuppressive chemotherapy, and the AIDS pandemic (27). The MAC is divided into two main species: M. avium and M. intracellulare. M. avium is further subdivided (per Turenne et al.) into four subspecies: M. avium subsp. avium, M. avium subsp. hominissuis, M. avium subsp. paratuberculosis, and M. avium subsp. silvaticum (39).Members of the family Mycobacteriaceae, comprising the MAC, differ in virulence and ecology. Those designated M. avium subsp. hominissuis are genomically diverse, low-virulence, opportunistic pathogens for both animals and humans. The majority of human M. avium subsp. hominissuis infections occur in HIV-immunocompromised people, immunocompetent persons with underling pulmonary disease, and children with cystic fibrosis (2, 12, 17). Considered ubiquitous in the environment (the most likely source of infection for humans), M. avium subsp. hominissuis has been isolated from water, soil, and dust (9). Domestic water distribution systems have been reported as possible sources of M. avium subsp. hominissuis infections in hospitals, homes, and commercial buildings (26, 27). In animals, M. avium subsp. hominissuis is found as a cause of lymphadenitis of the head and mesenteric lymph nodes of swine recognized at slaughter.Mycobacterium avium subsp. avium has long been recognized as a primary pathogen causing avian tuberculosis in wild and domestic birds (37, 38). Members of this subspecies also sporadically cause disease in other animals (6, 15, 30).For veterinarians, the MAC member of greatest importance is M. avium subsp. paratuberculosis. This MAC member causes a chronic granulomatous enteritis called Johne''s disease or paratuberculosis, most often in ruminants (16, 22, 31). Mycobacterium avium subsp. paratuberculosis is capable of infecting and causing disease a wide array of animal species, including nonhuman primates, without need of immunosuppressive coinfections. The herd-level prevalence of M. avium subsp. paratuberculosis infections in dairy cattle exceeds 50% in most major dairy product-producing countries (29, 31). Two systematic reviews and meta-analyses report a consistent association of M. avium subsp. paratuberculosis with Crohn''s disease, and the zoonotic potential of M. avium subsp. paratuberculosis continues to be a controversial subject discussed in the literature (1, 11). Unlike for most other M. avium subspecies, isolation of M. avium subsp. paratuberculosis requires the addition of the siderophore mycobactin to culture media and prolonged culture incubation for successful isolation from a tissue, soil, or fecal samples (43). After this lengthy incubation period with special media, resultant acid-fast organisms then need to be accurately identified.Unlike the M. avium subspecies, whose type strains were obtained from nonhuman hosts, the type strain of M. intracellulare (ATCC 13950) was isolated from a human, specifically a child who died from disseminated disease. Recently, numerous isolates considered to be M. intracellulare were reclassified as M. chimaera sp. nov. as part of the MAC (35). Few of these isolates were found to be clinically relevant, suggesting that this MAC species has low pathogenicity, and this factor is crucial to therapeutic decision making. Mycobacterium intracellulare appears to have a distinct environmental niche, more prevalent in biofilms and at significantly higher CFU numbers than M. avium (10, 36). It accounts for more documented human infections than M. avium subsp. hominissuis in several countries, including South Korea and Japan (19, 20, 23).Contemporary methods for MAC identification, e.g., high-performance liquid chromatography (HPLC) of cell wall mycolic acids, and genetic probes based on rRNA targets, e.g., AccuProbe, cannot discriminate among M. avium subspecies (2, 9). Given the differences in pathogenicity among M. avium subspecies and the implications regarding the infection source, a practical and accurate method of simply identifying M. avium subspecies is needed (13, 25, 35). In this study, we describe the specificity, discrimination capacity, and sensitivity of a novel five-target PCR, called the MAC multiplex, using a wide array of reference and clinical MAC isolates and numerous nonmycobacterial organisms.     

Variable-Number Tandem-Repeat Analysis of Respiratory and Household Water Biofilm Isolates of “Mycobacterium avium subsp. hominissuis” with Establishment of a PCR Database

“Mycobacterium avium subsp. hominissuis” is an important cause of pulmonary disease. It is acquired from environmental sources, but there is no methodology for large population studies. We evaluated the potential of variable-number tandem-repeat (VNTR) analysis. Clinical and household biofilm M. avium isolates underwent molecular identification. Testing for IS901 was done to separate M. avium subsp. avium from M. avium subsp. hominissuis. VNTR types were defined using VNTR loci, and subtyping was performed using 3′ hsp65 and internal transcribed spacer (ITS) sequencing. Forty-nine VNTR types and eight subtypes of M. avium subsp. hominissuis (IS901 negative) were identified among 416 isolates of M. avium from 121 patients and 80 biofilm sites. Of those types, 67% were found only among patient isolates, 11% only among household water isolates, and 23% among both. Of 13 VNTR types that included ≥4 patients, the majority (61.5%) represented geographic clustering (same city). Most VNTR types with multiple patients belonged to the same 3′ hsp65 sequence code (sequevar). A total of 44 isolates belonging to four M. avium subsp. hominissuis VNTR types (8%), including three with the rare Mav-F ITS sequence and 0/8 subspecies, produced amplicons with IS901 PCR primers. By sequencing, all 44 amplicons were not IS901 but ISMav6, which was recently observed in Japan but had not been previously described among U.S. isolates. VNTR analysis of M. avium subsp. hominissuis isolates is easier and faster than pulsed-field gel electrophoresis. Seven VNTR loci separated 417 isolates into 49 types. No isolates of M. avium subsp. avium were identified. The distributions of the VNTR copy numbers, the allelic diversity, and the low prevalence of ISMav6 differed from the findings for respiratory isolates reported from Japan.     

Mycobacterial bacteraemia in patients infected and not infected with human immunodeficiency virus,Taiwan

The clinical characteristics and outcomes of 71 patients with mycobacterial bacteraemia, infected with human immunodeficiency virus (HIV) (n = 47) and not infected with HIV (n = 24) are described. Mycobacterium avium complex (MAC) (54.9%) constituted the most frequently isolated mycobacterium, followed by Mycobacterium tuberculosis (MTB) (38.0%), Mycobacterium kansasii (4.2%), and Mycobacterium abscessus (2.8%). The Beijing family genotype was the most common type in MTB, and Mycobacterium intracellulare was the most common species in MAC. The overall mortality rate was 33.8%; it was lower in HIV-infected than in non-HIV-infected patients. HIV-infected patients were younger, had fewer underlying diseases and better nutritional status, and were more likely to have MAC bacteraemia than MTB bacteraemia.     

PCR amplification and high-resolution melting curve analysis as a rapid diagnostic method for genotyping members of the Mycobacterium avium–intracellulare complex

Some of the members of the Mycobacterium avium–intracellulare (MAI) complex are recognized as human pathogens in both immunocompromised and immunocompetent patients. The current molecular methods that are available for genotyping the MAI complex members can be both expensive and technically demanding. In this report, we describe for the first time the application of a real-time PCR and high-resolution melt approach to differentiate between the complex members by targeting a member of the Pro-Pro-Glu gene family, MACPPE24. To this end, reference strains of the M. avium subspecies and Mycobacterium intracellulare were used to optimize the technique. Then, this real-time PCR–high-resolution melt approach was used to distinguish ten M. avium ssp. hominissuis field isolates from the M. intracellulare reference strain.     

Identification and Differentiation of Clinically Relevant Mycobacterium Species Directly from Acid-Fast Bacillus-Positive Culture Broth

Mycobacterium species cause a variety of clinical diseases, some of which may be species specific. Therefore, it is clinically desirable to rapidly identify and differentiate mycobacterial isolates to the species level. We developed a rapid and high-throughput system, MycoID, to identify Mycobacterium species directly from acid-fast bacillus (AFB)-positive mycobacterial culture broth. The MycoID system incorporated broad-range PCR followed by suspension array hybridization to identify 17 clinically relevant mycobacterial complexes, groups, and species in one single reaction. We evaluated a total of 271 AFB-positive culture broth specimens, which were identified by reference standard methods in combination with biochemical and molecular tests. The overall identification agreement between the standard and the MycoID system was 89.7% (perfect match) or 97.8% (one match in codetection). In comparison to the standard, the MycoID system possessed an overall sensitivity of 97.1% and specificity of 98.8%. The 159 Mycobacterium avium-M. intracellulare complex isolates were further identified to the species level by MycoID as being M. avium (n = 98; 61.1%), M. intracellulare (n = 57; 35.8%), and mixed M. avium and M. intracellulare (n = 2; 1.3%). M. avium was recovered more frequently from sterile sites than M. intracellulare (odds ratio, 4.6; P = 0.0092). The entire MycoID procedure, including specimen processing, can be completed within 5 h, providing rapid and reliable identification and differentiation of mycobacterium species that is amenable to automation. Additional differentiation of Mycobacterium avium-M. intracellulare complex strains into M. avium and M. intracellulare may provide a tool to better understand the role of Mycobacterium avium-M. intracellulare complex isolates in human disease.The increasing numbers of infections caused by mycobacteria other than tuberculosis have intensified the need for the introduction of rapid methods for isolating and identifying clinically encountered mycobacteria. The detection of pathogenic mycobacterial species continues to depend mainly on mycobacterial culture. The use of liquid culture media with continuous monitoring has significantly improved test sensitivity and test turnaround time. The Bactec mycobacterial growth indicator tube (MGIT) 960 system (Becton Dickinson Microbiology Systems, Sparks, MD), which is a fully automated, continuous-monitoring, nonradiometric, noninvasive device, is sensitive and shortens the turnaround time by 2 to 27 days (13, 25, 31).Current laboratory techniques for the identification of mycobacteria to the species level involve growth in broth, nucleic acid probing, or subculture on solid medium for phenotypic tests, which can take weeks, leading to significant delays in diagnoses. Thus, there is a need for quick and accurate identification of all mycobacteria directly from culture broth. The use of DNA probes (AccuProbe; GenProbe, San Diego, CA) that are specific for the Mycobacterium tuberculosis complex, the M. avium-M. intracellulare complex, M. kansasii, and M. gordonae directly from Bactec TB broth culture has dramatically reduced the time to identification and differentiation of mycobacterial infections (1, 6, 23). This approach, however, can identify only a limited number of mycobacterium species and requires multiple reactions. If the probe results are negative, a battery of biochemical tests must be performed with or without extracted mycolic acid analysis for definitive species identification.Several molecular methods including PCR followed by restriction fragment length polymorphism, melt curve analysis, regular sequencing, and pyrosequencing were previously reported to directly identify and differentiate mycobacterial isolates from acid-fast bacillus (AFB)-producing culture broth (15, 16, 26, 32). In this study, we report the use of a Mycobacterium genus-specific broad-range primer to amplify the partial 16S-23S rRNA gene internal transcribed spacer (ITS) region (24). After amplification, we then designed 25 mycobacterial species-specific capture oligonucleotide probes to identify and differentiate 17 commonly encountered mycobacterial species in the clinical setting by use of an xMAP suspension array system (Luminex, Austin, TX) (9). Results were validated in comparison to a reference standard using a combination of biochemical and molecular methods.(This study was presented in part at the 109th General Meeting of the American Society for Microbiology, Philadelphia, PA, 17 to 21 May 2009.)     

Distribution of Nontuberculous Mycobacteria by Multigene Sequence-Based Typing and Clinical Significance of Isolated Strains

Species identification of nontuberculous mycobacteria (NTM) is challenging due to the increasing number of identified NTM species and the lack of standardized testing strategies. The objectives of this study were to investigate the distribution of NTM species recovered from respiratory specimens by multigene sequence-based typing and to evaluate the clinical significance of identified species. Two hundred thirty-two consecutive clinical NTM isolates were subjected to sequencing of multiple genes, including hsp65, rpoB, and 16S-23S rRNA internal transcribed spacer (ITS) sequence. In addition, clinical data from all patients whose specimens had NTM isolates were analyzed to examine clinical virulence and treatment history. Eighteen strains from 227 isolates from 169 patients were successfully identified at the species level by multigene sequence-based typing. Mycobacterium avium complex and M. abscessus complex made up the majority of isolated NTM (88%; 199/227), followed by M. fortuitum complex (4%; 10/227). The pathogenic potential of NTM differs enormously by species, and M. avium complex and M. abscessus complex revealed especially high levels of virulence compared with findings for other NTM species. The results from our work support M. avium complex and M. abscessus complex being the most common NTM species with highly pathogenic potential isolated from clinical respiratory specimens and could be a good resource for molecular epidemiology of NTM species in South Korea.     

Use of PCR in Detection of Mycobacterium avium Complex (MAC) Bacteremia: Sensitivity of the Assay and Effect of Treatment for MAC Infection on Concentrations of Human Immunodeficiency Virus in Plasma

We evaluated the sensitivity and specificity of a PCR-based qualitative test for the rapid diagnosis of Mycobacterium avium-M. intracellulare complex (MAC) bacteremia in patients with AIDS disease. Eleven subjects with newly culture-proven MAC bacteremia had the following tests performed at biweekly intervals during the first 8 weeks of therapy: blood culture, Mycobacterium-specific PCR, and quantitative human immunodeficiency virus (HIV) viral-load testing. Mycobacterium genus-specific biotinylated primers were used to amplify a sequence of approximately 582 nucleotides within the 16S rRNA genes of M. avium and M. intracellulare. Detection of the amplified product was performed with an oligonucleotide probe-coated microwell plate combined with an avidin-horseradish peroxidase–tetramethylbenzidine conjugate-substrate system. While not as sensitive as BACTEC culture, PCR detected 17 of 18 specimens which grew ≥40 organisms/ml (94.4% sensitivity) and 9 of 16 specimens which grew ≤40 organisms/ml (56.3% sensitivity). No clear change in HIV viremia occurred in response to successful treatment of patients’ MAC bacteremia. Use of the PCR test allowed detection of MAC bacteremia in 1 day, with a sensitivity similar to those of quantitative blood culture techniques, and it may prove useful for rapid screening of suspected cases. HIV viremia was unaffected by 8 weeks of MAC therapy.     

Cellular Immune Responses to ESAT-6 Discriminate between Patients with Pulmonary Disease Due to Mycobacterium avium Complex and Those with Pulmonary Disease Due to Mycobacterium tuberculosis

ESAT-6 (for 6-kDa early secreted antigenic target) is a secreted antigen found almost exclusively in organisms of the Mycobacterium tuberculosis complex. We compared in vitro gamma interferon (IFN-γ) responses by peripheral blood mononuclear cells to this antigen in patients with pulmonary disease due to either Mycobacterium avium complex (MAC) or Mycobacterium tuberculosis with those in healthy, skin test-negative, control subjects. Significant IFN-γ responses to ESAT-6 were detected in 16 (59%) of 27 M. tuberculosis pulmonary disease patients, 0 (0%) of 8 MAC disease patients, and 0 (0%) of 8 controls. Significant IFN-γ responses to M. tuberculosis purified protein derivative were detected in 23 (85%) of 27 M. tuberculosis disease patients, 2 (25%) of 8 MAC disease patients, and 5 (63%) of 8 healthy controls. M. avium sensitin was recognized in 24 (89%) of 27 M. tuberculosis disease patients, 4 (50%) of 8 MAC disease patients, and 1 (13%) of 8 controls. IFN-γ responses to ESAT-6 are specific for disease due to M. tuberculosis and are not observed in patients with MAC disease or in healthy controls.     

Virulence and Immune Response Induced by Mycobacterium avium Complex Strains in a Model of Progressive Pulmonary Tuberculosis and Subcutaneous Infection in BALB/c Mice

The genus Mycobacterium comprises more than 150 species, including important pathogens for humans which cause major public health problems. The vast majority of efforts to understand the genus have been addressed in studies with Mycobacterium tuberculosis. The biological differentiation between M. tuberculosis and nontuberculous mycobacteria (NTM) is important because there are distinctions in the sources of infection, treatments, and the course of disease. Likewise, the importance of studying NTM is not only due to its clinical significance but also due to the mechanisms by which some species are pathogenic while others are not. Mycobacterium avium complex (MAC) is the most important group of NTM opportunistic pathogens, since it is the second largest medical complex in the genus after the M. tuberculosis complex. Here, we evaluated the virulence and immune response of M. avium subsp. avium and Mycobacterium colombiense, using experimental models of progressive pulmonary tuberculosis and subcutaneous infection in BALB/c mice. Mice infected intratracheally with a high dose of MAC strains showed high expression of tumor necrosis factor alpha (TNF-α) and inducible nitric oxide synthase with rapid bacillus elimination and numerous granulomas, but without lung consolidation during late infection in coexistence with high expression of anti-inflammatory cytokines. In contrast, subcutaneous infection showed high production of the proinflammatory cytokines TNF-α and gamma interferon with relatively low production of anti-inflammatory cytokines such as interleukin-10 (IL-10) or IL-4, which efficiently eliminate the bacilli but maintain extensive inflammation and fibrosis. Thus, MAC infection evokes different immune and inflammatory responses depending on the MAC species and affected tissue.     

Continued Upward Trend in Non-Tuberculous Mycobacteria Isolation over 13 Years in a Tertiary Care Hospital in Korea

PurposeDespite decreased prevalence of tuberculosis, the incidence of the diseases associated with nontuberculous mycobacteria (NTM) has been increasing in South Korea and around the world. The present retrospective study was conducted to determine longitudinal changes in the epidemiology and distribution of NTM over 13 years at a tertiary care hospital in Korea.Materials and MethodsWe retrospectively analyzed data on Mycobacterium species over 13 years (January 2007 to December 2019) by utilizing the laboratory information system. Mycobacterium species were identified using biochemical tests and PCR-restriction fragment length polymorphism and Mycobacteria GenoBlot assays.ResultsAfter excluding duplicates from the initial pool of 17996 mycobacterial isolates, 7674 strains were analyzed and 2984 (38.9%) NTM were isolated. The proportion of NTM continuously increased over the 13-year period, from 17.0% in 2007 to 57.5% in 2019. Among the NTM isolates, the most common species were Mycobacterium intracellulare (50.6%), M. avium (18.3%), M. fortuitumcomplex (4.9%), M. abscessus (4.5%), M. gordonae (3.3%), M. kansasii (1.1%), M. chelonae (1.0%), and M. massiliense (0.9%). In patients over the age of 70 years, the proportion of NTM among the isolates increased from 26.6% in 2007 to 62.0% in 2019, and that of M. intracellulare isolates among the NTM increased from 13.9% (11/79) in 2007 to 37.4% (175/468) in 2019.ConclusionThe number of NTM isolates continuously increased over the study period, and the increase in the proportion of M.intracellulare in patients aged over 70 years was notable.     

Hemolysin as a Virulence Factor for Systemic Infection with Isolates of Mycobacterium avium Complex

Isolates of the Mycobacterium avium complex were examined for hemolysin expression. Only invasive isolates of M. avium were observed to be hemolytic (P < 0.001), with activity the greatest for isolates of serovars 4 and 8. Thus, M. avium hemolysin appears to represent a virulence factor necessary for invasive disease.