Development and evaluation of triplex PCR for direct detection of mycobacteria in respiratory specimens

AIMS: To develop a method for the simultaneous detection of Mycobacterium tuberculosis, Mycobacterium avium-intracellularecomplex (MAC), and other mycobacteria in clinical specimens using triplex PCR. METHODS AND RESULTS: The target of amplification was the internal transcribed spacer region between the l6S and 23S rDNA genes. Twenty-two mycobacterial type strains, 118 M. tuberculosis, 87 other mycobacteria, 75 nonmycobacterial pathogens, 115 respiratory specimens from confirmed cases of tuberculous or other mycobacterial diseases, and sputa from 50 patients with nonmycobacterial respiratory diseases were tested. In M. tuberculosis, 322 bp pan-mycobacterial and 133 bp species-specific bands were observed. In MAC, the respective bands were 322 and 84 bp. The other mycobacteria showed single pan-mycobacterial bands of approx. 300-350 bp. Nonspecific amplicons were not found in any of the nonmycobacterial pathogens. In the tuberculosis specimens, 96.4% of smear-positive specimens and 70.2% of smear-negative specimens showed positive reactions. Specimens from two patients with MAC infection were MAC positive. Only 1 of 50 specimens from nonmycobacterial diseases was positive (2.0%). CONCLUSION, SIGNIFICANCE AND IMPACT OF THE STUDY: Triplex PCR enables accurate and rapid diagnosis of tuberculosis and probably is useful for the detection of MAC and other mycobacteria in respiratory specimens.     

Nutritional Characteristics of the Atypical Mycobacteria

The ability of Mycobacterium kansasii and groups II and III of the atypical mycobacteria to utilize oleic acid, as well as selected carbohydrates and other compounds, as sources of carbon for growth was compared with that of the H37Rv and H37Ra strains of M. tuberculosis. The highest rate of growth of all of the mycobacteria examined occurred in the medium containing oleic acid as the carbon source when single substrates were tested. The H37Ra strain of M. tuberculosis and all of the atypical mycobacteria examined, except the P-8 strain of M. kansasii, displayed a deficiency in ability to utilize glucose for growth. The deficiency was manifested as delayed appearance of growth, suboptimal growth, or complete inability to utilize the sugar. Variant substrains of organisms that possessed an enhanced ability to utilize glucose for growth were isolated from representative strains of M. kansasii and groups II and III atypical mycobacteria inoculated on modified Kirchner glucose-agar and incubated for an extended period of time.     

Photochromogenic and scotochromogenic mycobacteria: their clinical significance

In the period 1973--1977, Mycobacterium tuberculosis was isolated by cultivation in 4408 cases from the clinical specimens of patients with positive X-ray findings. On the basis of atypical colony morphology or pigment formation, 263 other mycobacterial strains were identified: of these 23 were photochromogenic and belonged to Mycobacterium kansasii. The strains were cultured on several occasions from the specimens of 4 patients with broncho-pulmonary mycobacteriosis. The strains were resistant to isoniazid and streptomycin, sensitive to ethambutol and rifampicin. A total of 18 scotochromogenic isolates cultured from 14 patients with positive X-ray findings were identified as Mycobacterium aquae (M. gordonae) and its variants: strains showing slow Tween hydrolysis and 1 strain of rapid growth. In 5 cases M. tuberculosis was also obtained, indicating the presence of a mixed mycobacterial population. All scotochromogens were resistant to isoniazid and sensitive to ethambutol, with the exception of two strains sensitive to rifampicin.     

Sensitivity of Mycobacterium tuberculosis to levofloxacin]

Susceptibility of 92 strains of mycobacteria to levofloxacin (5, 10 and 50 mcg/mL) was investigated by indirect method of absolute concentrations on Levenstain-Jensen media. The investigation was performed on 85 strains of Mycobacterium tuberculosis isolated from 83 patients with different types of tuberculosis and also on drug-sensitive laboratory strains of M. tuberculosis H37Rv-M, H37Rv-GISK, Academia, M. bovis-bovinus 8, M. bovis BCG and on two strains of M. fortuitum with total resistance to antimycobacterial agents. 87.8 per cent of clinical isolates were multi-drug resistant. From one patient treated with ciprofloxacin two strains of M. tuberculosis were isolated--one resistant to 5 mcg/mL of levofloxacin and second strain-resistant to 10 mcg/mL of levofloxacin. All other clinical and laboratory strains of mycobacteria (97.8 per cent) were susceptible to all three concentrations of levofloxacin.     

Predictive value of the acid fast smear for detection of Mycobacterium tuberculosis in respiratory specimens in a reference center of HIV/AIDS in Rio de Janeiro, Brazil

In order to evaluate the predictive value of acid fast bacilii (AFB) smear for the diagnosis of Mycobacterium tuberculosis in respiratory specimens in a setting with a high prevalence of AIDS and an unknown prevalence of nontuberculous mycobacteria (NTM), we retrospectively examined specimens cultured for mycobacteria between 1 September 1993 and 30 September 1994 and medical records of patients with positive culture in a General Hospital, AIDS reference in Rio de Janeiro, Brazil. Seventy three per cent (1517/2077) of samples were respiratory specimens and mycobacteria were recovered from 20.6% (313/1517) of these. M. tuberculosis was identified in 94.2% (295/313) and NTM in 5.8% (18/313). The yield of positive AFB smear and of positive culture was 6.1% (93/1517) and 20.6% (313/1517), respectively. The positive predictive value (PPV) of AFB for M. tuberculosis was 98.4% in expectorated sputum and 96.4% in bronchoalveolar lavage. Forty four percent (130/295) of specimens with positive culture for M. tuberculosis and 66.7% (12/18) for NTM were from patients HIV positive. The conclusion was that in our study population, the PPV of AFB for M. tuberculosis in respiratory specimens was high and the prevalence of NTM was low despite the high prevalence of HIV positive.     

利用rpoB基因芯片技术进行快速分枝杆菌菌种鉴定

利用rpoB基因芯片技术快速进行分枝杆菌菌种鉴定。以分枝杆菌rpoB基因编码序列为靶基因, 用基因芯片技术检测21种分枝杆菌标准株;8种其它细菌标准株;126株临床分离株。分枝杆菌与其它细菌标准株经PCR扩增后, 分枝杆菌标准株均扩增出360 bp DNA片段, 在其它细菌中, 除甲型溶血性链球菌和假白喉棒状杆菌出现同样片段外, 其它细菌均未见扩增。21种寡核苷酸探针除海分枝杆菌与偶然分枝杆菌的探针有交叉杂交外, 其余均为特异性杂交。对126株临床分离株进行鉴定, 89株为结核分枝杆菌, 占70.6％(89/126), 非结核分枝杆菌(NTM)占9.2％(9/98)。应用rpoB基因芯片技术鉴定分枝杆菌菌种, 是一种快速、准确的方法, 具有较高的临床应用价值。     

利用rpoB基因芯片技术进行快速分枝杆菌菌种鉴定

利用rpoB基因芯片技术快速进行分枝杆菌菌种鉴定.以分枝杆菌rpoB基因编码序列为靶基因,用基因芯片技术检测21种分枝杆菌标准株;8种其它细菌标准株;126株临床分离株.分枝杆菌与其它细菌标准株经PCR扩增后,分枝杆菌标准株均扩增出360 bp DNA片段,在其它细菌中,除甲型溶血性链球菌和假白喉棒状杆菌出现同样片段外,其它细菌均未见扩增.21种寡核苷酸探针除海分枝杆菌与偶然分枝杆菌的探针有交叉杂交外,其余均为特异性杂交.对126株临床分离株进行鉴定,89株为结核分枝杆菌,占70.6%(89/126),非结核分枝杆菌(NTM)占9.2%(9/98).应用rpoB基因芯片技术鉴定分枝杆菌菌种,是一种快速、准确的方法,具有较高的临床应用价值.     

Non-tuberculous mycobacteria I: one year clinical isolates identification in Tertiary Hospital Aids Reference Center,Rio de Janeiro,Brazil, in pre highly active antiretroviral therapy era

The aim of this study was to determine the prevalence of non-tuberculous mycobacteria (NTM) isolates at University Hospital, Reference Center for Aids in Rio de Janeiro, Brazil, during one year. We used standard biochemical tests for species identification and IS1245 PCR amplification was applied as a Mycobacterium avium specific identification marker. Four hundred and four specimens from 233 patients yielded acid-fast bacilli growth. M. tuberculosis was identified in 85% of the patients and NTM in 15%. NTM disseminated infection was a common event correlated with human immunodeficiency virus (HIV) infected patients and only in HIV negative patients the source of NTM was non sterile site. M. avium complex (MAC) was biochemically identified in 57.8% (49/83) of NTM isolates, most of them from sterile sites (75.5%), and in 94% (46/49) the IS 1245 marker specific for M. avium was present. Twenty NTM strains showed a MAC biochemical pattern with the exception of a urease-positive (99% of MAC are urease-negative), however IS1245 was detected in 96% of the strains leading to their identification as M. avium. In this group differences in NTM source was not significant. The second most frequently isolated NTM was identified as M. scrofulaceum (7.2%), followed by M. terrae (3.6%), M. gordonae (2.4%), M. chelonae (1.2%), M. fortuitum (1.2%) and one strain which could not be identified. All were IS1245 negative except for one strain identified as M. scrofulaceum. It is interesting to note that non-sterile sites were the major source of these isolates (92.8%). Our finding indicated that M. avium is still the major atypical species among in the MAC isolates recovered from Brazilian Aids patients without highty active antiretroviral therapy schema. Some discrepancies were seen between the identification methods and further investigations must be done to better characterize NTM isolates using other phenotypic and genotypic methods.     

Characteristics of the protein complexes of clinical strains of mycobacteria

The protein composition of Mycobacterium tuberculosis and the molecular weight of proteins contained in these organisms have been determined by the method of electrophoresis in the porosity gradient of polyacrylamide gel. Close similarity between the electrophoregrams of M. tuberculosis clinical and laboratory strains has been revealed. The study of 18 strains of different groups of mycobacteria has shown that M. tuberculosis are essentially different from opportunistic mycobacteria and acid-resistant saprophytes. These data may be important for the identification and taxonomy of mycobacteria.     

Comparison of the mycobacteria growth indicator tube with radiometric and solid culture for isolation of mycobacteria from clinical specimens and susceptibility testing of Mycobacterium tuberculosis

We compared the mycobacteria growth indicator tube (MGIT) system with the BACTEC 460 TB and Loewenstein-Jensen (LJ) systems for the recovery of mycobacteria (acid-fast bacilli [AFB]) from 600 clinical specimens. A total of 50 AFB (32 Mycobacterium tuberculosis complex, 10 M. avium complex, 3 M. gordonae, 3 M. xenopi, 1 M. terrae and 1 M. fortuitum) were detected. MGIT recovered 50 isolates of AFB (100% sensitivity), and BACTEC 460 TB and LJ recovered 49 (98% sensitivity) and 19 (38% sensitivity) AFB isolates, respectively. The mean times to detect mycobacteria were 10, 10 and 25 days for MGIT, BACTEC 460, and LJ slants. All isolates of M. tuberculosis complex were tested for susceptibility to streptomycin, isoniazid, rifampin, and ethambutol with the MGIT and BACTEC 460 TB. Both systems yielded identical susceptibility data with different mean times to report (5.38 days for MGIT versus 7.33 days for BACTEC 460 TB, P<0.05). The results suggest that MGIT is equivalent to BACTEC 460 TB in its ability to support the growth of mycobacteria, but significantly more efficient than LJ. MGIT may also be used for susceptibility testing of primary antituberculosis drugs.     

Bactericidal activity of alkaline glutaraldehyde solution against a number of atypical mycobacterial species

The mycobactericidal activity of 2% alkaline glutaraldehyde solution was determined using standardized suspensions of 10 species of atypical mycobacteria and compared with that for virulent Mycobacterium tuberculosis. Suspensions of M. avium, M. intracellulare and M. gordonae were more resistant to disinfection by the glutaraldehyde than were virulent tubercle bacilli while M. kansasii, M. scrofulaceum and M. szulgae were somewhat more susceptible. Mycobacterium marinum, M. smegmatis and M. fortuitum were highly sensitive to the disinfectant action of the alkaline glutaraldehyde solution. This variation in sensitivity shown by apparently closely related strains of mycobacteria to this disinfectant has important practical implications.     

Bactericidal activity of alkaline glutaraldehyde solution against a number of atypical mycobacterial species

The mycobactericidal activity of 2% alkaline glutaraldehyde solution was determined using standardized suspensions of 10 species of atypical mycobacteria and compared with that for virulent Mycobacterium tuberculosis. Suspensions of M. avium, M. intracellulare and M. gordonae were more resistant to disinfection by the glutaraldehyde than were virulent tubercle bacilli while M. kansasii, M. scrofulaceum and M. szulgae were somewhat more susceptible. Mycobacterium marinum, M. smegmatis and M. fortuitum were highly sensitive to the disinfectant action of the alkaline glutaraldehyde solution. This variation in sensitivity shown by apparently closely related strains of mycobacteria to this disinfectant has important practical implications.     

Mycobacterium tuberculosis mammalian cell entry operon (mce) homologs in Mycobacterium other than tuberculosis (MOTT)

The cloned mammalian cell entry gene mce1a from Mycobacterium tuberculosis confers to non-pathogenic Escherichia coli the ability to invade and survive inside macrophages and HeLa cells. The aim of this work was to search for and characterize homologs of the four M. tuberculosis mammalian cell entry operons (mce1, mce2, mce3 and mce4) in mycobacteria other than tuberculosis (MOTT). The dot-blot and polymerase chain reaction (PCR) experiments performed on 24 clinical isolates representing 20 different mycobacterial species indicated that the mce operons were widely distributed throughout the genus Mycobacterium. BLAST search results showed the presence of mce1, mce2 and mce4 homologs in Mycobacterium bovis, Mycobacterium avium and Mycobacterium smegmatis. A homologous region for the mce3 operon was also found in M. avium and M. smegmatis. DNA and protein alignments were done to compare the M. tuberculosis mce operons and the deduced M. bovis, M. avium, and M. smegmatis homologs. The deduced proteins of M. bovis mce1, mce2 and mce4 operons had 99.6-100% homology with the respective M. tuberculosis mce proteins (MTmce). The similarity between M. avium mce proteins and the individual M. tuberculosis homologs ranged from 56.2 to 85.5%. The alignment results between M. smegmatis mce proteins and the respective MTmce proteins ranged from 58.5% to 68.5%. Primer sets were designed from the M. tuberculosis mce4a gene for amplification of 379-bp fragments. Amplification was successful in 14 strains representing 11 different mycobacterial species. The PCR fragments were sequenced from 10 strains representing eight species. Alignment of the sequenced PCR products showed that mce4a homologs are highly conserved in the genus Mycobacterium. In conclusions, the four mce operons in different mycobacterial species are generally organized in the same manner. The phylogenetic tree comparing the different mce operons showed that the mce1 operon was closely related to the mce2 operon and mce3 diverged from the other operons. The wide distribution of the mce operons in pathogenic and non-pathogenic mycobacteria implicates that the presence of these putative virulence genes is not an indicator for the pathogenicity of the bacilli. Instead, the pathogenicity of these factors might be determined by their expression.     

Differentiation between Mycobacterium tuberculosis and Mycobacterium bovis by a multiplex-polymerase chain reaction

A multiplex-polymerase chain reaction (PCR) assay, based on one-step amplification and detection of three different mycobacterial genomic fragments, was designed for differentiation between Mycobacterium bovis and Mycobacterium tuberculosis. The oligonucleotide primers were chosen from the gro EL gene, present in the genus Mycobacterium sp., from the IS 6110 insertion sequence, present in Myco. tuberculosis complex and from the mtp 40 gene, identified as a specificspecies Myco. tuberculosis genomic fragment. This amplification method allowed the detection of two fragments of 576 and 317 base pairs in Myco. bovis and three fragments of 576, 396 and 317 base pairs in Myco. tuberculosis strains, including atypical strains of Myco. tuberculosis where the copy number of the IS 6110 element is low. The multiplex-PCR assay described may be a very useful tool for the rapid and specific differentiation of these related mycobacteria and easy to use in medical and veterinary microbiology laboratories.     

Comparison of a multiplex-PCR assay with mycolic acids analysis and conventional methods for the identification of mycobacteria

A fast, sensitive and cost-effective multiplex-PCR assay for Mycobacterium tuberculosis complex (MTC) and Mycobacterium avium (M. avium) identification for routine diagnosis was evaluated. A total of 158 isolates of mycobacteria from 448 clinical specimens from patients with symptoms of mycobacterial disease were analyzed. By conventional biochemical methods 151 isolates were identified as M. tuberculosis, five as M. avium and two as Mycobacterium chelonae (M. chelonae). Mycolic acid patterns confirmed these results. Multiplex-PCR detected only IS6110 in isolates identified as MTC, and IS1245 was found only in the M. avium isolates. The method applied to isolates from two patients, identified by conventional methods and mycolic acid analysis, one as M. avium and other as M. chelonae, resulted positive for IS6110, suggesting co-infection with M. tuberculosis. These patients were successfully submitted to tuberculosis treatment. The multiplex-PCR method may offer expeditious identification of MTC and M. avium, which may minimize risks for active transmission of these organisms and provide useful treatment information.     

Detection of mycobacteria in clinical specimen using the mycobacteria growth indicator tube (MGIT) and the Lowenstein Jensen medium.

The recovery rates of mycobacteria strains isolated from 1200 clinical specimens using the mycobacteria growth indicator tube (MGIT) system and the conventional Lowenstein Jensen medium (LJ) were assessed. Of the 87 mycobacterial isolates recovered, 54 belonged to the M. tuberculosis complex (MTB) and 33 to the non-tuberculosis complex (NTM). MGIT recovered 78 (89.65%) mycobacteria isolates (51 MTB (94.44%) and 27 NTM (81.81%) and LJ recovered 70 (80.46%) mycobacteria isolates (49 MTB (90.74%) and 21 NTM (63.63%). Sixty one (70.1%) of the total mycobacteria isolates were recovered with both systems (46 (85.2%) MTB and 15 (45.5%) NTM). No significant difference was found between MGIT and LJ (p > 0.05) in both MTB and NTM recoveries. The average detection time for MTB was significantly shorter with MGIT than with LJ, in both the smear-positive specimens (8 vs 30 days: p < 0.0001) and smear-negative specimens (15 vs 30 days: p < 0.001). The average detection time of NTM was also shorter for MGIT (15 vs 30 days: p < 0.0001). However, the contamination rate was higher in MGIT (8.5%) than in LJ (3%). The results suggest that the use of MGIT contributes to a more rapid and effective diagnosis of mycobacterial infections particularly when combined with the classical LJ.     

In Vitro Effect of Rifampin on Mycobacteria

Rifampin inhibited 20 strains of Mycobacterium tuberculosis in concentrations of 0.005 to 0.02 mug/ml in 7H-9 broth with Tween 80 and killed all or nearly all of the inoculum in four to eight times greater concentrations. In the same medium without Tween 80, as well as on 7H-10 agar, about 16 to 64 times these amounts were required to produce the same effect. Rifampin was also active against M. kansasii and some of the nonchromogenic mycobacteria. The incidence of mycobacterial cells resistant to rifampin within the cultures studied was in the range of one to four per 10(8) to 10(9) colony-forming units with concentrations of 4 to 125 mug of rifampin per ml. Only one of the Battey cultures and that of M. fortuitum yielded cells resistant to rifampin at 125 mug/ml but not at 500 mug/ml. The same strains yielded more than double that number of organisms resistant to streptomycin and up to 100 times more organisms resistant to isoniazid. All three drugs stopped the growth or reduced the mycobacterial population in growing cultures after contact for 24 to 48 hr. Complete inhibition of growth was produced by rifampin at 1.0 mug/ml in an average of 6 days and by streptomycin at 5.0 mug/ml in 3 days. After an average contact of 10.7 days with rifampin, five of seven strains resumed growth and all strains began regrowth after exposure to streptomycin for 9.4 days. The marked susceptibility of M. tuberculosis and of atypical mycobacteria to rifampin in vitro and the relatively low incidence of resistant mutants suggests that this agent may have clinical usefulness in the treatment of tuberculosis and some other mycobacterioses.     

Comparison of NaOH-N-acetyl cysteine and sulfuric acid decontamination methods for recovery of mycobacteria from clinical specimens

We compared the NaOH-N-acetyl cysteine (NaOH-NALC) and the sulfuric acid decontamination procedure in the detection of mycobacteria using the Mycobacteria Growth Indicator Tube (MGIT). In total 219 sputum specimens were collected from 142 Zambian patients and subjected to mycobacterial culture. One half of the specimen was decontaminated with NaOH-NALC and the other half was decontaminated with sulfuric acid. From the 438 samples a total of 261 (60%) cultures yielded growth of mycobacteria, consisting of 22 different species. The sulfuric acid method was more successful than the NaOH-NALC method in recovering mycobacteria in MGITs (146 versus 115 respectively, p = 0.001). Of the 146 positive mycobacterial cultures recovered after sulfuric acid decontamination 28 were Mycobacterium tuberculosis, 84 nontuberculous mycobacteria (NTM) and 34 acid fast bacterial isolates which could not be identified to the species level. The 115 mycobacteria recovered by the NaOH-NALC method consisted of 34 M. tuberculosis strains, 55 NTM and 26 acid fast bacteria that could not be identified. The most frequently isolated NTM were Mycobacterium lentiflavum and Mycobacterium intracellulare. Comparing the two decontamination methods the recovery of NTM in the sulfuric acid group was significant higher than in the NaOH-NALC group (p = 0.001). In contrast, no significant difference was found for the recovery of M. tuberculosis. These results show that the decontamination method used affects the recovery of nontuberculous mycobacteria in particular.     

In vitro efficacy of Linezolid on clinical strains of Mycobacterium tuberculosis and other mycobacteria

Linezolid, an oxazolidinone that acts by inhibiting protein synthesis, was evaluated in strains of tuberculosis and non-tubercular mycobacteria resistant to one or more drugs isolated in northern Sardinia. The in vitro activity of Linezolid (Pfizer) was assessed on different isolates of Mycobacterium spp. from clinical samples by the Proportional Method. Linezolid demonstrated an excellent activity against the 24 strains of M. tuberculosis and against M. gordonae, M. marinum, M. aurum, M. phlei, and M. avium, with MIC values ranging from 0.5 to 2 microg/ml. Linezolid can be used in combination with the standard antitubercular medications, or as an effective therapeutic alternative in infections caused by M. tuberculosis or by other species of non-tubercular mycobacteria.     

Zoonotic transmission of tuberculosis between pastoralists and their livestock in South-East Ethiopia

Despite huge global efforts in tuberculosis (TB) control, pastoral areas remain under-investigated. During two years sputum and fine needle aspirate (FNA) specimens were collected from 260 Ethiopian pastoralists of Oromia and Somali Regional States with suspected pulmonary TB and from 32 cases with suspected TB lymphadenitis. In parallel, 207 suspected tuberculous lesions were collected from cattle, camels and goats at abattoirs. All specimens were processed and cultured for mycobacteria; samples with acid-fast stained bacilli (AFB) were further characterized by molecular methods including genus and deletion typing as well as spoligotyping. Non-tuberculous mycobacteria (NTM) were sequenced at the 16S rDNA locus. Culturing of AFB from human sputum and FNA samples gave a yield of 174 (67%) and 9 (28%) isolates, respectively. Molecular typing was performed on 173 of these isolates and 160 were confirmed as Mycobacterium tuberculosis, three as M. bovis, and the remaining 10 were typed as NTMs. Similarly, 48 AFB isolates (23%) yielded from tuberculous lesions of livestock, of which 39 were molecular typed, including 24 M. bovis and 4 NTMs from cattle, 1 M. tuberculosis and 1 NTM from camels and 9 NTMs from goats. Isolation of M. bovis from humans and M. tuberculosis from livestock suggests transmission between livestock and humans in the pastoral areas of South-East Ethiopia.