Diagnosis Of Mycobacterial Infections Research Articles

DIAGNOSTIC VALUE OF DIFFERENT PCR ASSAYS FOR THE DETECTION OF MYCOBACTERIAL DNA IN GRANULOMATOUS LYMPHADENOPATHY

Diagnosis of mycobacterial infection is made by assessment of characteristic histological features, staining of acid-fast bacilli, or agar culture. Recent advances in molecular biology have provided alternative approaches for the detection of mycobacteria, but only limited data are available dealing with the comparative evaluation of these methods. In order to determine the diagnostic applicability of polymerase chain reaction (PCR)-based assays, 20 formalin-fixed and paraffin-embedded lymph nodes with bacille Calmette-Guérin (BCG) lymphadenitis were investigated which in Löwenstein Jensen agar culture were either positive or negative (ten cases each); ten lymph nodes with non-specific lymphadenitis served as negative controls. Ziehl-Neelsen staining as well as three different PCR assays (including nested PCR), amplifying a specific sequence of the Mycobacterium tuberculosis complex or sequences of the 65 kD antigen gene, were performed. Positive culture was only obtained from lymph nodes which had been surgically removed within 20 weeks after vaccination (P < 0.001). In contrast to microscopic examination, which yielded no more information than agar culture, PCR detection of mycobacterial DNA was unrelated to culture findings. Combined use of different assays, as well as DNA extraction from at least three paraffin sections from each specimen, resulted in the detection of mycobacterial DNA in all lymph nodes with amplifiable DNA (18 out of 20 cases). Controls remained consistently negative. Thus, the combined use of different PCR assays is proposed as a rapid and sensitive technique for the detection of mycobacterial DNA in formalin-fixed and paraffin-embedded tissue.

Current and future applications of mycobacterial amplification assays

Introduct ion During the last 2 to 3 yr, numerous articles have been published concerning the reemergence of tuberculosis in the U.S. This resurgence was a result of a number of complex factors, among them homelessness and other socioeconomic factors, the human immunodeficiency virus (HIV) epidemic, and hospital and other group-setting outbreaks of tuberculosis. Further complicating these outbreaks were frequent delays in laboratory repotting of smear, culture, and/or susceptibility test resuits. As a consequence, an important question was raised as to how one could provide a more rapid diagnosis for tuberculosis given the urgent need. Because of the obvious demand for a reliable and rapid means of diagnosing tuberculosis for public health and therapeutic reasons, the discovery of the polymerase chain reaction (PCR) about 10 yr ago, with its potential ability to exponentially copy virtually any targeted sequence of DNA in an afternoon, is particularly attractive. Since PCR's discovery, a number of other approaches to amplification have also been introduced to detect Mycobacterium tuberculosis (MTB) directly in clinical specimens; some of these amplification formats are listed in Table 1. Regardless of the format, these amplification assays show promise as rapid, sensitive, and specific methods to diagnose tuberculosis. Despite the significant promise these assays hold for improved laboratory diagnosis of tuberculosis, a number of issues must be considered by clinical microbiologists before these assays are introduced into the laboratory routine. Key issues include expense, the assays' exact role in the diagnosis of mycobacterial infections, and their clinical utility. In this article, I would like to discuss important issues concerning these assays and then suggest some possible immediate and future applications for them. However, if these issues are to be fully appreciated, a brief summary of data published to date is prerequisite.

The role of polymerase chain reaction in the diagnosis of mycobacterial infections

The role of polymerase chain reaction in the diagnosis of mycobacterial infections

The diagnostic mycobacteriology laboratory of the 1990s.

The diagnostic mycobacterial laboratory of the 1990s must respond to a change in the clinical spectrum of mycobacterial infections brought about by an increase in the number of patients who are immunocompromised, are indigent, or have temporary or permanent implanted devices. Emerging pathogens such as Mycobacterium haemophilum and Mycobacterium genavense, multidrug-resistant strains of Mycobacterium tuberculosis, and catheter infections with rapidly growing mycobacteria are examples of new issues. Fortunately, new methods for detection and identification of microbes have been or are being developed. Procedures that, when applied directly to clinical specimens or actively growing cultures, dramatically reduce the time to diagnosis of mycobacterial infections include radiometric broth, lysis-centrifugation, and biphasic systems for specimen culture, and DNA probes, high-performance liquid chromatography, DNA hybridization, restriction fragment length polymorphism, and gene amplification for organism detection and identification. At present, in vitro antimicrobial susceptibility tests are most helpful in guiding treatment of infections caused by M. tuberculosis and rapidly growing mycobacteria.

Use of serum antibody and lysozyme levels for diagnosis of leprosy and tuberculosis.

Active tuberculosis (TB) and leprosy are difficult to diagnose early because there are few organisms to detect and the specific immune response does not distinguish between active and inactive disease. We developed an immunoassay for lysozyme to see whether serum lysozyme levels could be used to identify individuals with clinical leprosy or TB. The immunoassay for lysozyme proved superior to standard enzyme assays that were less sensitive and reliable. The lysozyme assay was compared with assays for antibodies to Mycobacterium tuberculosis lipoarabinomannan (LAM) and M. leprae phenolic glycolipid-1. The sera tested were from Ethiopian leprosy (paucibacillary and multibacillary) and TB patients and from healthy Ethiopian and U.S. controls. The lysozyme assay was able to detect more of the individuals with TB (sensitivity, 100% for 19 patients) or leprosy (sensitivity, 86% for 36 patients) than either antibody assay. In particular, lysozyme levels were raised in a higher proportion of the paucibacillary leprosy patients (83% of 17), for whom the antibody assays were less sensitive; the LAM IgG and the phenolic glycolipid-1 IgM levels were raised in only 62 and 44% of 16 patients, respectively. The data suggest that lysozyme measurements may be useful in the diagnosis of mycobacterial infections and other chronic infectious granulomatoses.

Mycobacterial infection is an important infective complication in British Asian dialysis patients

To define the extent and nature of mycobacterial infection in patients on an adult dialysis unit whose catchment population contains a large proportion of non-Caucasian subjects, a retrospective survey of all new patients accepted onto our maintenance dialysis programme between January 1987 and December 1989 was carried out. Twenty-six Asian, 13 Afro-Caribbean, two Oriental and 170 Caucasian patients were accepted onto the dialysis programme in the three-year recruitment period. Eight of the 26 Asian patients, but none of the others, had developed mycobacterial infection by the end of December 1990. One patient had a cerebral tuberculoma with miliary mottling on chest X-ray, one pulmonary tuberculosis, one tuberculous adenitis and 5 tuberculous peritonitis (four due to Mycobacterium tuberculosis and one Mycobacterium kansasii). All the patients had been living in the UK for an average of 15 (range 6–24) years, with no known recent exposure to tuberculosis. Five patients are now alive and well, one developed malabsorption following M. kansasii peritonitis, but two with tuberculous peritonitis died before treatment could be instituted. Mycobacterial infections were associated with a high level of mortality and morbidity. No Asian patient developed mycobacterial infection during post-transplant immunosuppressive therapy in the study period, probably because of the routine antituberculous chemoprophylaxis employed in this group of patients. The diagnosis of mycobacterial infection should be suspected when an Asian dialysis patient develops a pyrexia of unknown origin. It is likely, though not proven, that anti-tuberculous chemoprophylaxis might reduce this high incidence of tuberculous infection in Asian dialysis patients.

Assay of specific antibody response to mycobacterial antigen for the diagnosis of a pleural effusion in a patient with aids

A diagnosis of mycobacterial infection was supported by a serological assay in a patient with AIDS. Specific antibody levels were not above the threshold of positivity determined in non-immunodeficient patients, but sera obtained previously were available, and a significant rise in titre was observed.

Mycobacterial infection in goats: Diagnosis and pathogenicity of the organism

Abstract Counterimmunoelectrophoresis of serum samples and intradermal tuberculin testing were compared with cultural results for the diagnosis of mycobacterial infection in a goat herd. Only eight of 47 culture-positive animals were not identified by a combination of the other two methods. Typical isolates resembled Mycobacterium paratuberculosis in cultural characteristics and mycobactin dependence but serologically resembled Mycobacterium avium . One isolate was inoculated into guinea-pigs, mice, chickens and calves and, although the infection in guinea-pigs, mice and chickens resembled that of M. paratuberculosis , it did not cause Johne's-like disease in calves.

The Role of Nontuberculous Mycobacterial Skin Test Antigens in the Diagnosis of Mycobacterial Infections

A retrospective study of 212 patients with mycobacterial infection was conducted to determine intradermal reactivity to five tuberculin units (TU) of purified protein derivative of Mycobacterium tuberculosis (PPD-S), and purified protein derivatives (PPDs) derived from non-tuberculous mycobacteria. PPDs B, Y, A, G, and F were used. The study included 138 patients with Mycobacterium tuberculosis infection, and 74 with proved nontuberculous mycobacterial infection. Eight possible patterns of skin test reactivity were discerned using PPD-S, PPD-B, and PPD-Y. In this population, selection of the largest skin test reaction within any one pattern correlated with the infecting organism in 87 per cent of the cases. Use of PPD-A, PPD-G, and PPD-F did not increase diagnostic capability. We conclude that differential skin testing with PPD-S, PPD-B and PPD-Y, is useful in the diagnosis of mycobacterial disease.