Search

Specimens submitted for mycobacterial culture should be collected in clean, sterile, wide-mouthed containers. If there is a delay in transport or processing, the specimen should be refrigerated, since this discourages the multiplication of contaminating organisms, which can make decontamination of the specimen difficult.

The performance of the Septi-Chek AFB system was compared with that of the BACTEC radiometric system and that of Lowenstein-Jensen agar slants (LJ) for detection of mycobacteria in clinical specimens. A total of 642 specimens were cultured; 61 (9.5%) yielded mycobacteria. Mycobacterium tuberculosis (34 isolates) and Mycobacterium avium complex (25 isolates) were the predominant species isolated. Of the 61 culture-positive specimens, 30 were smear positive and 31 were smear negative. Overall, 95% of the positive specimens were detected by Septi-Chek and BACTEC (100% of M. tuberculosis isolates) and 75% by LJ (82% of M. tuberculosis isolates). The mean times to detection were 15 days for BACTEC, 23 days for Septi-Chek, and 27 days for LJ. Of the 30 smear-positive specimens, 100% were recovered by Septi-Chek and BACTEC and 90% were recovered by LJ. Of the 31 smear-negative specimens, 90% were detected by Septi-Chek and BACTEC and 61% were detected by LJ. The Septi-Chek and BACTEC systems are superior to the conventional (LJ) mycobacterial culture method. Although Septi-Chek requires more time for the detection of mycobacteria than BACTEC, it is comparable in terms of overall recovery.

The Infectious Diseases Clinic (IDC) discussed serves adults who are seropositive for human immunodeficiency virus. The authors reviewed the outpatient and inpatient microbiology tests of a three-month period during 1989 for a systematic sample of IDC patients. The 249 patients in the sample had 682 microbiology tests performed during the period (mean 2.7 tests per patient). Tests most frequently requested were mycobacterial culture, routine blood culture, and cryptococcal antigen determination. Patients with acquired immunodeficiency syndrome (43% of IDC patients) accounted for 63% of the requested IDC tests. IDC patients comprised about 2.4% of patients served but accounted for 3.9% of the requested microbiology tests and 6.6% of the microbiology work load for reported tests. Using Centers for Disease Control case projections, the authors estimated that services to IDC patients in 1993 would comprise 6.6% of all microbiology tests and 10.6% of the microbiology work load. The implications of these data for microbiology probably also apply to other laboratory testing and emphasize the need for more efficient ways to use and perform diagnostic studies required by patients with HIV infection.

Although it is not rare to find sputum that is positive acid-fast bacilli (AFB) smear but subsequent culture fails to isolate mycobacteria in clinical practice, the incidence and clinical implication of those sputa from new patients has not been clearly elucidated. The aim of this study was to determine the incidence and clinical implication of sputum with positive AFB smear but negative in mycobacterial culture. All sputa that were positive AFB smear requested during diagnostic work up for new patients visiting Seoul National University Hospital from 1 January 2005 through 31 December 2006 were included. Sputa producing a positive AFB smear but negative mycobacterial culture were classified into one of four categories: laboratory failure to isolate mycobacteria, false positive AFB smear, pathogen may show a positive AFB smear other than mycobacteria, and indeterminate results. Out of 447 sputa with a positive AFB smear, 29 (6.5%) failed to culture any organism. Among these 29 sputa, 18 were caused by laboratory failure to isolate mycobacteria, six were false positive smears, and five indeterminate. Although most sputum with a positive AFB smear but negative culture could be classified as a laboratory failure, clinicians should consider the possibility of false positive AFB smear.

Background : Mycobacterial culture is a confirmatory test to detect. M. tuberculosis, but it takes at least 6 weeks to diagnose. PCR is a rapid and sensitive method, but it is known that PCR has a high false positive rate due to contamination, and a high false negative rate due to inhibitors. It is also known that LCR and PCR-Hybridization, recently developed methods, are more specific methods than PCR in terms of detecting M. tuberculosis. In this study, we estimated the clinical utility of in house PCR, LCR and PCR-Hybridization for the detection of M. tuberculosis. Methods : We evaluated 75 specimens, upon which M. tuberculosis culture based testing was requested, by PCR LCR, and PCR-Hybridization and compared results. Mycobacterial culture was performed on 3% Ogawa media for 8 weeks, and an in house PCR, LCx Mycobacterium tuberculosis assay kit (Abbott Laboratories, North Chicago, III) and the AMPLICOR M. tuberculosis test kit (Roche Molecular Systems, Inc. Branchburg, NJ. USA). Results : In the view of the culture results, the sensitivities of the three tests were 40%, 80%, and 100% and their specificities were 98.6%, 94.3%, and 94.3%. Conclusion : LCR and PCR-Hybridization are rapid and sensitive methods for detecting M. tuberculosis in clinical laboratories.

Figure 1. A 22-year-old man lacerated the back of his left index finger while cleaning his fish tank. When seen three months later, he had a verrucous, crusted plaque at the site of the injury and two subcutaneous nodules along the line of lymphatic drainage. He was afebrile and asymptomatic but was concerned about the nodules. A punch biopsy of the border of the plaque was obtained. Half was sent for routine histologic analysis and staining for acid-fast bacilli; the results of both were nondiagnostic. The other half was sent for mycobacterial culture at 30 to 33°C, which revealed Mycobacterium . . .

Tuberculosis poses a serious diagnostic and therapeutic problem, even in developed countries. As originally described, 1 tuberculids are cutaneous eruptions that result from an abnormally marked host response to the presence of mycobacteria. One such tuberculid is erythema induratum of Bazin (EIB), in which Mycobacterium tuberculosis has recently been detected. 2 Patients and Methods. Sixteen formaldehyde solution— fixed, paraffin-embedded blocks of EIB were collected from 1988 to 1995 ( Table ). 3 As a positive control in all procedures, a paraffin-embedded sample of M tuberculosis as confirmed by mycobacterial culture was included. As a negative control, an acrochordon specimen was included. Reagent controls were also included. DNA was extracted from paraffin-embedded blocks as previously described 4 and analyzed for M tuberculosis DNA by polymerase chain reaction (PCR) using a primer specific for M tuberculosis and M bovis . 5 The methods outlined by Eisenach et al 5 were adhered to stringently with 1

Automated radiometric technic (BACTEC Johnston Laboratories, Towson, MD) was compared with conventional mycobacterial culture procedure (Lowenstein-Jensen plus Gruft modification of Lowenstein-Jensen) in this study of 1,000 clinical specimens. In addition, 8-azaguanine inhibition was tested by radiometric technic as a rapid procedure for the differentiation of Mycobacterium tuberculosis from other mycobacterial species. A total of 59 mycobacteria was recovered. Of 28 clinically significant isolates (M. tuberculosis, M. kansasii, M. avium, M. fortuitum), the BACTEC system detected 26 (93%). Conventional methods recovered 23 (82%). The BACTEC system required an average of seven days to recover M. tuberculosis from smear-positive specimens compared with 18 days required by Lowenstein-Jensen or Gruft slants. From smear-negative specimens, the BACTEC detected M. tuberculosis in an average of 20 days versus 28 days by conventional procedure. All 20 isolates of M. tuberculosis were inhibited by 8-azaguanine, whereas 39 isolates of mycobacteria other than M. tuberculosis were not inhibited. The BACTEC system accomplishes more rapid recovery of mycobacteria and provides a higher yield than conventional methods.

Under contract with the National Institute of Allergy and Infectious Diseases, the Trudeau Mycobacterial Culture Collection has been greatly expanded to provide for the scientific community a collection of representative strains of mycobacteria of biomedical importance. Problems concerned with the preparation, bottling, and distribution of such organisms have been dealt with and are detailed in this paper. Examination of collected data revealed that the temperature of storage and not the suspending menstruum was more important for prolonged survival of mycobacteria stored at subzero temperatures. For optimum results, mycobacteria may be suspended either in Dubos Tween-albumin broth or in Middlebrook 7H-9 liquid medium supplemented with ADC enrichment (commercial sources used) and stored at −70 C. Either of these suspending fluids supplied a growth-supporting medium for cultures which must be shipped long distances, often without refrigeration. To avoid sublimation of suspending medium during prolonged storage at −70 C (a problem inherent in many screw-capped containers), we have chosen to use vaccine-stoppered serum bottles sealed with aluminum crimp caps. The methods described have provided suspensions with (i) excellent viability over prolonged periods of storage, (ii) stable metabolic activities, and (iii) highly reproducible inocula for animal experiments.

Two series of experiments were made to develop experimental cavernous tuberculosis of the lungs in guinea-pigs. For preliminary sensitization, the Freund adjuvant was used in the first series of experiments, whereas the BCG vaccine was employed in the second series. The cavity was formed after the culture of H37Rv mycobacteria was inoculated into the lungs of the sensitized animals. The animals vaccinated with BCG developed cavernous tuberculosis with a three-layer cavity wall and with the multiplication of the bacterial population. The animals sensitized with the Freund adjuvant after the mycobacterial culture was inoculated into the lungs demonstrated changes of the caseous pneumonia type to form lung tissue destruction. Attempts to develop the three-layer wall with the latter method ended in failure. The application of BCG vaccine enabled the optimal model of cavernous tuberculosis to be obtained.

A 72-year-old man was admitted with tenosynovitis of the left forearm and hand. Histopathologic examination of the excised tissue showed noncaseating granulomas. Mycobacterial culture produced Mycobacterium terrae complex. M. terrae infection of any site is rare. Analysis of four documented cases and our present case indicates that this organism causes infection in the forearm and hand after probable direct inoculation. It has not disseminated systemically from the site of local infection and may infect otherwise healthy hosts.

In summary, rapidly growing mycobacteria, M. fortuitum and M. chelonei, are pathogens of increasing importance which are often hospital-acquired and can infect patients with iatrogenic immunosuppression. They readily grow on routine mycobacterial culture media and must be distinguished from non-pathogenic rapidly growing species and slowly growing mycobacteria. Widely distributed in nature, they are often present in hospital environments, especially in water. Compared to M. tuberculosis they are weak pathogens, and infected patients are not considered contagious. Disease is probably acquired from environmental sources by direct entry of the organisms through traumatized skin or mucous membranes or by aspiration into previously abnormal lungs. They are usually resistant to antituberculous agents but are susceptible to several commonly used antibacterial agents. Treatment generally requires one or more active antibiotics plus adjunctive surgery in many cases. Prevention of nosocomial infection lies in proper disinfection of potentially contaminated medical devices and elimination of contaminated water.

A survey of 26 Czechoslovakian children diagnosed with BCG osteitis during 1981-1986 is presented. Mycobacterial culture was attempted in 19 cases with confirmation of bacillus Calmette-Guerin (BCG) Mycobacterium bovis strain in nine cases. Symptoms appeared approximately 17 months after vaccination; the proximal tibial end, distal femur, and proximal humerus were most affected. Although vaccination has been obligatory since 1953, a different vaccine was introduced in 1980, which led to the diagnosis of BCG osteitis in 1981. The vaccination doses, symptomatology, and methods of treatment are described. The risk of complications and a project for vaccination at later age are discussed.

Acridine orange staining for the detection of mycobacteria was compared with staining by auramine 0 and with mycobacterial culture in a series of 1071 clinical specimens. A total of 78 (7 %) specimens were positive by staining. No false positive or negative findings were recorded by the acridine orange method. The two fluorochromes proved equal in their ability to detect mycobacteria in specimens from culture positive cases of tuberculosis. In the rapid bacteriological diagnosis of tuberculosis, acridine orange offers a good alternative to auramine 0 which is considered carcinogenic.

A group of 34 mycobacteria, consisting of 25 Mycobacterium tuberculosis and nine strains of three other species, was isolated from 400 expectorated sputum specimens submitted on 148 patients from county-wide sources. Eight strains (24% of the total) were isolated from specimens evaluated by Gram stain to be oropharyngeal fluids. The remaining 26 strains were isolated from ungradable specimens and those primarily of lower respiratory origin. It was concluded that the random examination of sputum by Gram stain to determine the specimen's quality for mycobacterial isolation is not necessary.

On a utilise une technique microspectrofluorometrique quantitative afin de pouvoir determiner la multiplication initiale d'un tres faible inoculum de la souche H37Rv deMycobacterium tuberculosis. Cette technique a permis de mettre en evidence l'effet fortement stimulant d'un filtrat de culture de la meme souche sur la multiplication bacillaire precoce, a condition que ce filtrat soit ajoute au milieu de culture apres deux jours d'incubation des mycobacteries.

LI)~ohartrriitrn trd~crrulosis and certain atypical niycobacteria may cause a spectrum of cutaneous disease and m,iy sometimes pose difficult diagnostic problems (Table 1). This is especially true in irrimunocompromised patients, where even niycobacteria not previously thought to c u s e disease niay d o so. A high index of suspicion is required and unusual skin lesions presenting in these circ~unst~inces should always be biopsied and sent for mycobacterial culture and staining and PCR where possible.

Objective/background To evaluate the Kudoh swab method for improving laboratory diagnosis of tuberculosis (TB) in Gambia. Methods A total of 75 sputa (50 smear positive and 25 smear negative) were examined. Sputum samples were collected from leftover routine samples from the Medical Research Council Unit, Gambia TB Diagnostic Laboratory. The samples were processed using the standard N-acetyl- l -cysteine-NaOH (NALC-NaOH) methods currently used and Kudoh swab method. These were cultured on standard Lowenstein Jensen (LJ) and Modified Ogawa media, respectively, and incubated aerobically at 36 ± 1 °C for mycobacterial growth. To determine if the decontamination and culture methods compared could equally detect the Mycobacterium tuberculosis complex (MTBC) highly commonly isolated in Gambia, spoligotyping was done. Results In total, 72% (54/75) of MTBC were recovered by both LJ and Modified Ogawa methods. The LJ method recovered 52% (39/75) and Modified Ogawa recovered 56% (42/75) of the MTBC, respectively. Spoligotyping showed Euro-American 35% (19/54), Indo-Oceanic 35% (19/54), Mycobacterium africanum (West African type 2) 26% (14/54), Beijing 2% (1/54), and M. africanum (West African type 1) 2% (1/54). Conclusion The Kudoh method is simpler and cheaper than the NALC-NaOH method. There was no significant difference in recovery between the methods. The Kudoh method is ideal in overburdened TB laboratories with poor resources in developing countries. The predominant lineages were Euro-American and Indo-Oceanic, followed by M. africanum (West African type 2).

Nocardia isolations increased from 0.7 to 11.7/1,000 acid-fast bacillus and mycological cultures (P less than 0.000001). Only three isolations from one patient represented infection. Pseudoepidemic strain identity was confirmed by DNA fingerprinting; the isolate causing infection was distinct. The end of the pseudoepidemic was associated with changing the needle sterilizer and prolonging needle sterilization time on the BACTEC 460 machine. To our knowledge, this is the first reported Nocardia asteroides pseudoepidemic.

The pathology, frequency and diagnostic implications of ‘no visible lesion’ (NVL) tuberculosis (Tb), i.e. infection with Mycobacterium bovis in the absence of macroscopic lesions, are described in a wide taxonomic range of wildlife hosts. Information collected and evaluated on the definition and occurrence of NVL Tb, histopathological characteristics, post-mortem techniques to detect minimal lesions, and diagnostic difficulties revealed most Tb-infected individuals with NVL had minute tuberculous lesions, which were difficult to see by eye. Acidfast organisms (AFO) were sometimes detected in the lesions. Ideally, mycobacterial culture of pools of lymph nodes and/or oropharyngeal tonsils is necessary for the accurate diagnosis of Tb in the absence of macroscopic lesions. At a very minimum, the diagnostic methods applied for studying the prevalence of Tb in the population should be clearly described, to allow comparison between studies., The authors acknowledge financial support from Santander and Fundación Marcelino Botín, and the Department for Environment, Food and Rural Affairs, UK. The advice and input of TR Crawshaw, Veterinary Laboratories Agency, and LAL Corner and S Gordon, University College Dublin, Ireland, are gratefully acknowledged.

The Xpert® MTB/RIF (Xpert) assay is a rapid PCR-based assay for the detection of Mycobacterium tuberculosis complex DNA (MTBc) and mutations associated with rifampin resistance (RIF). An updated version introduced in 2011, the G4 Xpert, included modifications to probe B and updated analytic software. An analytical study was performed to assess Xpert detection of mutations associated with rifampin resistance in rifampin-susceptible and -resistant isolates. A clinical study was performed in which specimens from US and non-US persons suspected of tuberculosis (TB) were tested to determine Xpert performance characteristics. All specimens underwent smear microscopy, mycobacterial culture, conventional drug-susceptibility testing and Xpert testing; DNA from isolates with discordant rifampin resistance results was sequenced. Among 191 laboratory-prepared isolates in the analytical study, Xpert sensitivity for detection of rifampin resistance associated mutations was 97.7% and specificity was 90.8%, which increased to 99.0% after DNA sequencing analysis of the discordant samples. Of the 1,096 subjects in the four clinical studies, 49% were from the US. Overall, Xpert detected MTBc in 439 of 468 culture-positive specimens for a sensitivity of 93.8% (95% confidence interval [CI]: 91.2%–95.7%) and did not detect MTBc in 620 of 628 culture-negative specimens for a specificity of 98.7% (95% CI: 97.5%–99.4%). Sensitivity was 99.7% among smear-positive cases, and 76.1% among smear-negative cases. Non-determinate MTBc detection and false-positive RIF resistance results were low (1.2 and 0.9%, respectively). The updated Xpert assay retained the high sensitivity and specificity of the previous assay versions and demonstrated low rates of non-determinate and RIF resistance false positive results.

Background The present study was conducted to assess the efficiency of using TK SLC-L (Salubris, Inc.) for the primary isolation of mycobacteria from clinical samples by comparing it to the MGIT detection system (Becton Dickinson Diagnostic Instrument Systems). Although TK SLC, a biphasic medium, has been evaluated previously, this is the first study to evaluate TK SLC-L, a liquid medium. Methods Clinical specimens from a total of 146 clinically suspected cases of tuberculosis were studied. Each processed sample was evaluated by ZN staining and inoculated into TK SLC-L and MGIT tubes. The TK SLC tubes were incubated in a MYCOLOR TK while the MGIT tubes were incubated in a MGIT system. Growth, indicated by automated systems, was confirmed through production of a smear and microscopic evaluation after ZN staining. Results Mycobacterial growth was positive in 35 TK SLC-L and in 34 MGIT samples. Although the growth detection time was approximately 3 to 5 days shorter, on average, with the MGIT system, the contamination rate was significantly lower using TK SLC-L. The total time spent for the repetition of cultures for contaminated samples in MGIT make the total return time for culture results equal to or longer than the time required by TK SLC-L. Conclusions The TK Culture System using TK SLC-L is an efficient system and possible alternative to other rapid mycobacterial culture systems.

Excerpt To the editor: Since July 1980 we have seen four patients with acquired immunodeficiency syndrome who developed disseminatedMycobacterium avium intracellulareinfection during their illness....