Diagnosis Of Mycobacterial Infections Research Articles

Rapid lateral flow test for Mycobacterium tuberculosis complex and non-tuberculous mycobacteria differentiation

The diagnosis of mycobacterial infections, including both the Mycobacterium tuberculosis complex (MTBC) and non-tuberculous mycobacteria (NTM), poses a significant global medical challenge. This study proposes a novel approach using immunochromatographic (IC) strip tests for the simultaneous detection of MTBC and NTM. Traditional methods for identifying mycobacteria, such as culture techniques, are hindered by delays in distinguishing between MTBC and NTM, which can affect patient care and disease control. Molecular methods, while sensitive, are resource-intensive and unable to differentiate between live and dead bacteria. In this research, we developed unique monoclonal antibodies (mAbs) against Ag85B, a mycobacterial secretory protein, and successfully implemented IC strip tests named 8B and 9B. These strips demonstrated high concordance rates with conventional methods for detecting MTBC, with positivity rates of 93.9% and 85.9%, respectively. For NTM detection, the IC strip tests achieved a 63.2% detection rate compared to culture methods, considering variations in growth rates among different NTM species. Furthermore, this study highlights a significant finding regarding the potential of MPT64 and Ag85B proteins as markers for MTBC detection. In conclusion, our breakthrough method enables rapid and accurate detection of both MTBC and NTM bacteria within the BACTEC MGIT system. This approach represents a valuable tool in clinical settings for distinguishing between MTBC and NTM infections, thereby enhancing the management and control of mycobacterial diseases.Key points• Panel of mAbs for differentiating MTB versus NTM• IC strips for diagnosing MTBC and NTM after the BACTEC MGIT• Combined detection of MTP64 and Ag85B enhances diagnostic accuracy

Pulmonary Granulomas and Mycobacterial Infection: Concordance between the Results of Special Stains Performed on Lung Tissue Sections and Tissue Cultures.

The most common cause of infectious pulmonary granulomas worldwide is Mycobacterium tuberculosis. The diagnosis is based on clinical presentation, histopathologic findings, detection of acid-fast bacilli (AFB) in tissue or sputum using special stains, and/or isolation of mycobacteria in cultures or via PCR-based methods. Different studies have shown that high levels of discrepancy exist between these diagnostic approaches in lung tissue specimens. To assess the degree of concordance between the results of special stains and cultures on lung tissue specimens in the diagnosis of mycobacterial infections. Eighty-seven patients with a diagnosis of granulomas (necrotizing and non-necrotizing) on lung tissue specimens were identified. Cohen's kappa was used to measure the general concordance between the results of the histopathological examination (special stains) and bacteriological tissue cultures. With Kinyoun acid-fast stains, 8/48 (16.7%) cases were positive for AFB. With FITE stains, 10/57 (17.5%) cases were positive for AFB. There was strong agreement between Kinyoun acid-fast and FITE stains (Kappa = 0.806; p-value < 0.001). Tissue cultures were performed on 38/87 cases (43.7%), and 10/38 (26.3%) of the cultures were positive for mycobacteria. There was no concordance between Kinyoun acid-fast stains or FITE stains and tissue cultures results. Our observations represent an initial step in the process of reviewing the two methods used at our institution to diagnose mycobacterial infections on lung tissue specimens and highlight the need of incorporating more advanced diagnostic methods such as PCR to confirm mycobacterial infections and improve patient management. Importantly, species-level identification of mycobacteria is necessary to guide treatment.

P6‐32: Clinical efficacy of endobronchial ultrasonography in the diagnosis of mycobacterial infections

P6‐32: Clinical efficacy of endobronchial ultrasonography in the diagnosis of mycobacterial infections

Phage Amplification Assay for Detection of Mycobacterial Infection: A Review.

An important prerequisite for the effective control, timely diagnosis, and successful treatment of mycobacterial infections in both humans and animals is a rapid, specific, and sensitive detection technique. Culture is still considered the gold standard in the detection of viable mycobacteria; however, mycobacteria are extremely fastidious and slow-growing microorganisms, and therefore cultivation requires a very long incubation period to obtain results. Polymerase Chain Reaction (PCR) methods are also frequently used in the diagnosis of mycobacterial infections, providing faster and more accurate results, but are unable to distinguish between a viable and non-viable microorganism, which results in an inability to determine the success of tuberculosis patient treatment or to differentiate between an active and passive infection of animals. One suitable technique that overcomes these shortcomings mentioned is the phage amplification assay (PA). PA specifically detects viable mycobacteria present in a sample within 48 h using a lytic bacteriophage isolated from the environment. Nowadays, an alternative approach to PA, a commercial kit called Actiphage™, is also employed, providing the result within 6–8 h. In this approach, the bacteriophage is used to lyse mycobacterial cells present in the sample, and the released DNA is subsequently detected by PCR. The objective of this review is to summarize information based on the PA used for detection of mycobacteria significant in both human and veterinary medicine from various kinds of matrices.

A case report of suspicious lung cancer or atypical mycobacteria amidst the pandemic era

Background: Starting 2020, the medical guidelines were shacked by COVID-19 pandemic. The threshold for hospital admission increased in order to limit Severe Acute Respiratory Syndrome coronavirus 2 spread. All this affected screening such as for lung cancer which remained largely undiagnosed. On the other hand, it became challenging to differentiate easily between COVID-19 and other diseases such as atypical infections and simple community acquired pneumonia. Moreover, immunocompromised patients are at higher risk of COVID-19 infection overshadowing any other infection such as tuberculosis and non-tuberculosis infections. Case Presentation: We present the case of an 85-year-old female with a long history of scleroderma treated with methotrexate. Patient had non-resolving pneumonia and after two negative RT-PCR, bronchoalveolar lavage showed positive Real Time Polymerase Chain Reaction. Imaging showed persistent 2.6 cm solid nodule in left upper lobe worrisome for an underlying neoplasm. However, culture of Bronchoalveolar lavage grew with few colonies of acid fast bacilli making the diagnosis atypical mycobacteria highly probable especially that patient is chronically immunosuppressed. Unfortunately, she refused further genotyping. Conclusion: To authors’ knowledge that are no, or few reported cases of associated COVID-19 with atypical mycobacterial infections and the treatment modalities are unclear. The diagnosis of mycobacterial infections is usually difficult and in the setting of COVID-19 this becomes more challenging. Hence, a more thorough clinical approach is needed for the future to help clinicians diagnose and treat complicated cases of COVID-19 and concomitant other infections such as TB or Nontuberculous Mycobacteria. Furthermore, amidst the pandemic screening of lung cancer should continue while maintaining safety precautions.

Fluorescent methods in morphological diagnostics of respiratory tuberculosis in HIV-infected patients

Background. Among the spectrum of respiratory diseases in HIV-infected patients, the leading place is taken by tuberculosis (TB). First of all, difficulties in the morphological diagnostics of TB are associated with the smooth over of its typical histological signs under condition of severe immunodeficiency. To objectify the histological conclusion about the presence of tuberculosis lesion in a morphology a number of specific histochemical and fluorescent methods have been developed, which are based on the direct or indirect detection of M. tuberculosis (MBT) in the affected tissues. Objective – to determine the advisability and effectiveness of using of fluorescent methods in the morphological diagnostics of respiratory tuberculosis in HIV-infected patients. Methods. The morphological study included 37 cases. Two methods were used: the traditional fluorescent method (FM) and immunohistochemical study with a polyclonal antibody to M. Tuberculosis with a fluorescent label (FITC). In the study group, the histological conclusions were compared with the final clinical diagnosis and the results of microbiological investigations. The sensitivity, specificity and effectiveness have been established for each of the methods. Results. At hospitalization only in one third of cases, based on the results of clinical and radiological data and laboratory tests, various forms of respiratory TB were suspected. In 26 cases (70,3 %), with traditional staining of tissue samples with hematoxylin and eosin, a histological diagnosis of tuberculosis infection was established. When performing FM, the largest number of positive results was obtained (16 cases, 84,2 %), whereas with FITC, the MBT was detected only in 11 cases (57,9 %). The specificity of the FITC method was 90,5 %, and FM – 84,6 %. The overall efficiency of the parallel application of 2 fluorescent methods was 69,1 %. The coincidence of the results of both methods was noted in 22 cases of 31 (70,9 %). In total, of the using of morphological fluorescent methods, mycobacterium infection in HIV-infected patients was diagnosed in 29 cases (78,4 %), and in 4 cases (10,8 %) mycobacterium infection was established as a background pathology. Conclusion. At the time of hospitalization, in more than half of patients with HIV infection (59,5 %), the results of clinical laboratory and radiological data do not allow determining the true nature of the pathological process. The traditional study of histological samples stained with hematoxylin and eosin made it possible to diagnose mycobacterial infection in 70,3 % of cases. The use of fluorescent methods increased the efficiency of morphological diagnosis of mycobacterial infection by 10,8 %. The fluorescence method showed a high sensitivity (88,0 %), while indicator of specificity was higher with the immunofluorescence method (90,5 %). The overall efficiency of using fluorescent methods for the histological detection of M. tuberculosis was 69,1 %, which indicates the advisability of using these methods in cases of diagnostics mycobacterial infection.

Clinical Performance of Mycobacterial Immunohistochemistry in Anatomic Pathology Specimens.

Diagnosis of mycobacterial infections poses significant challenges in anatomic pathology. We recently described the use of antimycobacteria immunohistochemistry (IHC) as a sensitive, efficient diagnostic tool and now report the clinical performance of this assay among general, noninfectious disease pathology-trained anatomic pathologists. Over a 2-year period, all cases were retrospectively identified in which mycobacterial IHC was performed during routine diagnostic workup. From October 2017 to September 2019, mycobacterial IHC was evaluated for 267 cases, resulting in 58 (22%) positive stains. Compared with culture and molecular results, the sensitivity and specificity of IHC were 52% and 80%, respectively. IHC performed significantly better than acid-fast bacilli (AFB) staining (Ziehl-Neelsen) (P < .0001; sensitivity 21%, specificity 92%) but similarly to modified AFB staining (mAFB; Fite-Faraco) (P = .9; sensitivity 61%, specificity 84%). In cases with discordant IHC and mAFB staining, there were no differences in rates of culture or polymerase chain reaction-confirmed positivity. Mycobacterial IHC was well adopted with superior clinical performance to AFB and comparable performance to mAFB. These results support the use of IHC as an adjunctive tool in the diagnosis of mycobacterial infections and suggests its potential role as a rapid screening test for molecular testing.

Morphological diagnostics of tuberculosis in HIV-­infected patients with thoracic pathology

Objective — to determine the diagnostic indicators of various histochemical and immunohistochemical methods for the detection of M. tuberculosis and their antigens in lung tissue in HIV-infected patients.Materials and methods. The morphological study included 37 cases of biopsy, surgical and autopsy material of fragments of the lungs, lymph nodes and parietal pleura in HIV-infected patients. Serial sections were stained with hematoxylin and eosin, method of Ziehl—Neelsen (ZN), the traditional fluorescence method (FM), and immunohistochemical studies, without (IHC) and with a fluorescent label (FITC). The sensitivity, specificity and effectiveness of each method were calculated taking into account the final clinical diagnosis and the results of the microbiological detection of M. tuberculosis.Results and discussion. At the time of hospitalization of HIV-infected patients in 56.8 % of cases the results of clinical, laboratory and radiological data did not allow to determine the nature of the pathological process. According to the results of our study, the highest sensitivity was established for IHC and FM methods — 100.0 and 88.5 % respectively. The ZN method showed the highest specificity index (100.0 %), while the specificity of the FITC and FM method was 75.0 and 60.0 % respectively. The highest diagnostic efficacy indicators were established for IHC and FM methods — 83.8 and 72.2 %. The overall effectiveness of all 4 special histological examination methods was 72.1 %. As a result of an extended histological examination, the main diagnosis of mycobacterial infection was confirmed in 64.8 % cases, including an association with fungal infection in 29.7 % cases. In 10.8 % cases, the diagnosis of mycobacterial infection was withdrawn, and in these cases a fungal infection was diagnosed as the main pulmonary pathology. In 10.8 % cases the presence of mycobacterial infection as a background pathology with other diseases of the chest cavity was proved. The result of a comprehensive histological examination allowed to establish a final clinical diagnosis in 94.6 % cases, and to confirm the presence of mycobacterial infection in 81.1 % cases. In 13.5 % cases there was discrepancy in the histological picture at traditional staining and the results of additional histological methods of staining the preparations.Conclusions. Taking into account the variety of respiratory pathology in HIV-infected patients, including infectious ones, in order to establish a final clinical diagnosis during the diagnostic process, if there is a suspicion of mycobacterial infection, it is advisable to conduct a comprehensive morphological study using various histological methods for diagnosing infections.

Spectroscopic Characterization of Bovine, Avian and Johnin Purified Protein Derivative (PPD) with High-Throughput Fourier Transform InfraRed-Based Method

Tuberculins purified protein derivatives (PPDs) are obtained by precipitation from heat treated mycobacteria. PPDs are used in diagnosis of mycobacterial infections in humans and animals. Bovine PPD (PPDB) is obtained from Mycobacterium bovis (Mycobacterium tuberculosis complex), while Avian PPD (PPDA) and Johnin PPD (PPDJ) are extracted, respectively, from Mycobacterium avium and M. avium subsp. paratuberculosis (M. avium complex). PPDB and PPDA are used for bovine tuberculosis diagnosis, while PPDJ is experimentally used in the immunodiagnosis of paratuberculosis. Although PPDs date back to the 19th Century, limited knowledge about their composition is currently available. The goal of our study was to evaluate Fourier Transform InfraRed (FTIR) spectroscopy as a tool to differentiate PPDB, PPDA, and three PPDJs. The results highlighted that the three PPDs have specific profiles, correlated with phylogenetic characteristics of mycobacteria used for their production. This analysis is eligible as a specific tool for different PPDs batches characterization and for the assessment of their composition. The entire PPD production may be efficiently controlled, since the N content of each preparation is related to IR spectra, with a reference spectrum for each PPD and a standardized analysis protocol.

Added diagnostic value of 16S rRNA gene pan-mycobacterial PCR for nontuberculous mycobacterial infections: a 10-year retrospective study

The diagnosis of mycobacterial infections has been dramatically improved by the introduction of molecular methods aimed to reduce the time to diagnosis as compared with culture. The broad range pan-mycobacterial PCR can detect all the mycobacterial species directly from clinical specimens. We aimed to evaluate its usefulness and its clinical added value for the diagnosis of nontuberculous mycobacterial (NTM) infections. We performed a retrospective study (2003–2013) including 952 samples taken from 639 patients with clinical suspicion of NTM infection. The performance of smear microscopy, PCR and culture was established using clinical data to investigate discrepant results. We also compared the time to microbial diagnosis between the direct PCR and culture. The sensitivity, specificity, positive and negative predictive values of the PCR were 61.6% (53.5–69.1), 99.1% (98.2–99.6), 92.8% (85.8–96.5) and 93.4% (91.6–94.9), respectively, when considering all specimens. When considering smear-positive specimens and smear-negative specimens, the sensitivity was 81.6% and 40%, respectively. The sensitivity for pulmonary and extra-pulmonary smear-positive specimens was 85.2% versus 72.7%. The median time to identification at species level was 35 days (SD, 17.67) for culture and 6 days (SD, 2.67) for the PCR (when positive), which represents a 29-day shorter time to results (p < 0.0001). The 16S rRNA gene pan-mycobacterial PCR displays a substantial benefit in terms of time to diagnose NTM infections when compared with culture. Despite an excellent specificity, its sensitivity is yet limited in particular for smear-negative specimens, which might be improved by relying onto real-time PCRs.

Mycobacterium marinum lymphocutaneous infection

Mycobacterium marinum is a nontuberculous mycobacteria with worldwide distribution that lives in fresh or salt water and is responsible for infections in fish, and sometimes in humans. Human disease consists mainly of cutaneous nodules, but deep structure involvement may also occur. Diagnosis of M. marinum infection remains a challenge, with a considerable time delay between onset of symptoms and diagnosis. We present a 33-year-old man with no immunosuppressive history who was seen in our department with skin nodules over his hand and forearm, distributed in a sporotrichoid pattern. His hobbies included maintaining an aquarium of tropical fish. Histological examination of the patient's skin biopsy was compatible with the diagnosis of mycobacterial infection, and the Ziehl-Neelsen staining revealed acid-fast bacilli. Molecular techniques confirmed the suspicion of M. marinum infection. A necropsy was performed on one of the patient's fish, more specifically, a Poecilia reticulata, and resulted in identification of M. marinum from its gut. The patient was treated with clarithromycin, ethambutol, and rifampicin for 9 months, with clearance of infection.

Frequency of Mycobacterium bovis and mycobacteria in primary immunodeficiencies.

Susceptibility to mycobacterial diseases is observed in some primary immunodeficiency diseases. In this study, we aimed to evaluate mycobacterial infections in primary immunodeficiency diseases. Patients under follow-up by Ege University Pediatric Immunology Department for severe combined and combined immunodeficiencies, interleukin 12/ interferon gamma receptor deficiency, nuclear factor kappa-beta essential modulator deficiency and chronic granulomatosis disease were evaluated retrospectively in terms of the frequency and characteristics of mycobacterial infections using a questionnaire form for demographic properties, clinical features and laboratory tests. A diagnosis of mycobacterial infection was made clinically in a total of 25 patients including five (11.3%) of 45 patients who had severe combined immune deficiency, 12 (52.3%) of 21 patients who had chronic granulomatous disease, four patients (100%) who had interferon gamma receptor 2 partical deficiency, two patients (100%) who had interleukin 12 receptor beta 1 deficiency and one patient (100%) who had nuclear factor kapa-beta essential modulator deficiency. Mycobacterium strain could be typed in 14 (33%) of these 25 patients including Mycobacterium bovis, Mycobacterium chelonea, Mycobacterium elephantis, Mycobacterium fortuitum, and Mycobacterium tuberculosis. All patients were treated with anti-tuberculosis therapy. Thirty-six percent of these 25 patients underwent hematopoietic stem cell transplantation. Eight patients (five before, three after transplantation) died. Non-tuberculosis mycobacteria including mainly Mycobacterium bovis were observed with a higher rate compared to Mycobacterium tuberculosis in primary immunodeficiencies, especially in those affecting the interleukin 12/interferon gamma pathway. Early diagnosis of primary immunodeficiencies with neonatal screening program and preventing administration of the Bacille Calmette-Guerin vaccine in these patients is important.

Verification of Bact / Alert 3D System Results for Detection of Mycobacterium Tuberculosis

Background: The rapid diagnosis of mycobacterial infections is essential to implement the adequate antimicrobial therapy. Objective : To verify BacT/ALERT 3D system results by PCR and evaluate its performance against Lowenstein Jensen medium as regards the recovery rate, the time to detection, and contamination rate. Methodology: Sixty sputum specimens were inoculated in BacT/ALERT MP bottles and on LJ slants. Ziehl-Neelsen [ZN], subculture and PCR was done to confirm the positive signal MP bottles. Results : Thirty two [53.3%] mycobacterial isolates [31 M. tuberculosis and one non-tuberculous mycobacterium] were detected. The recovery rate of 32 mycobacterial isolates for the BacT/ALERT 3D system was 100% and that of LJ medium was 71.9%. Of the 32 signal positive / PCR positive bottles, 27[84.4%] bottles were ZN smear positive [from the bottle] and 5[15.6%] were ZN negative. The mean times to detection of mycobacteria by BacT/ALERT 3D system and LJ medium were 14.2 and 24.3 days, respectively, while overall contamination rates were 6.7% and 8.3%, respectively. Conclusion: Sensitivity and time to detection were significantly better with BacT/ALERT 3D system than with solid LJ medium. The PCR assay allows the fast and exact identification of Mycobacterium tuberculosis directly from positive liquid medium. LJ culture still plays an important role in isolation of mycobacteria from clinical samples, it provide visible colonies that allow identification and susceptibility testing of the isolate. Signal positive /ZN negative bottles should be confirmed by other methods [PCR and/or subculture].

Clinical and radiographic differentiation of lung nodules caused by mycobacteria and lung cancer: a case-control study.

BackgroundLung nodules caused by mycobacteria can resemble lung cancer on chest imaging. The advent of lung cancer screening with low-dose Computed Tomography is accompanied by high false-positive rates, making it necessary to establish criteria to differentiate malignant from benign nodules.MethodsWe conducted a retrospective case–control study of 52 patients with mycobacterial lung nodules and 139 patients with lung cancer, diagnosed between 2010 and 2012. We compared clinical and radiographic characteristics to identify predictors of disease by univariate and multivariate analysis. The discriminatory power of maximum Standardized Uptake Values from Positron-Emission-Tomography was also evaluated.ResultsSeveral variables were correlated with a diagnosis of mycobacterial infection or lung cancer on univariate analysis. Such variable include smoking status and history, lesion size and imaging evidence of tree-in-bud opacities, lymphadenopathy or emphysema on computed tomography. Upon author consensus, the most clinically-relevant variables were selected to undergo multivariate analysis. A history of current or former smoking [OR 4.4 (95 % CI 1.2–15.6) and 2.7 (95 % CI 1.1–6.8), respectively P = 0.04] was correlated with diagnoses of lung cancer. Contrarily, the presence of tree-in-bud opacities was less likely to be correlated with a diagnosis of malignancy [OR 0.04 (95 % CI 0.0–1.0), P = 0.05]. Additionally, higher maximum standardized uptake values from positron emission tomography were associated with malignancy on multivariate analysis [OR 1.1 (95 % CI 1.0–1.2), P = 0.04]; but the accuracy of the values in differentiating between diseases was only 0.67 as measured by the area under the curve. Lesion size was not independently associated with diagnosis [OR 0.5 (95 % CI 0.2–1.2), (P = 0.12)].ConclusionsEstablishing the likelihood of malignancy for lung nodules based on isolated clinical or radiographic criteria is difficult. Using the variables found in this study may allow clinicians to stratify patients into groups of high and low risk for malignancy, and therefore establish efficient diagnostic strategies.Electronic supplementary materialThe online version of this article (doi:10.1186/s12879-015-1185-4) contains supplementary material, which is available to authorized users.

Diagnosis of Mycobacterium bovis in Captive Sloth Bears (Melursus ursinus) by Polymerase Chain reaction

Diagnosis of mycobacterial infection is paramount important from the public health perspective since treatment and control measures are very significant, particularly in captive animals. In this diagnostic study of Mycobacterium bovis infection in sloth bears (Melursus ursinus), polymerase chain reaction (PCR) had been used with the primer sequence of pncA-8 (5′-GGTTGGGTGGCCGCCGGTCAG-3′) and pncA-11 (5′-GCTTTGCGGCGAGCGCTCCA-3′) that were specific for M. bovis pncA gene. Forty-two fresh faecal samples were collected randomly from the apparently healthy sloth bears maintained in captive conditions. The DNA extraction procedure was done as per the manufacturer’s protocol and further subjected to amplification. The amplification profile includes respectively: initial heating of the samples for 5 min at 94 °C, annealing at 55 °C for 1 min, primer extension at 72 °C for 1 min and final elongation step for 10 min at 72 °C. Out of 57 samples, 5 samples were yielded on expected amplified PCR product size of 744 bp when electrophoresed in 1.5 % agarose gel. A positive control of M. bovis DNA procured from Tuberculosis Research Centre and a negative control from a healthy bovine sample were used. These results demonstrated that PCR test will increase the effectiveness of laboratory diagnosis to detect and identifying the M. bovis in captive wild animals.

Evaluation of BacT/ALERT 3D System for Mycobacteria Isolates

Background: The rapid diagnosis of mycobacterial infections is essential to implement the adequate antimicrobial therapy. This study evaluates the performance of the BacT/ALERT 3D system for isolates and identification of mycobacteria from clinical samples. Methods: 1011 clinical specimens from nonsterile and sterile body sites were studied from August 2010 to December 2012 at the National Reference Laboratory of Tuberculosis, IPK, Cuba. The results obtained were compared with respect to time detection of mycobacteria and contamination rates, and performance indicators of BacT/ALERT 3D were calculated. Results: The time detection of growth (TDG) for Mycobacterium tuberculosis (Mtb) and nontuberculous mycobacteria (NTM) by BacT/ALERT 3D was 16,435 and 10,956, respectively; by LJ the TDG was 33.577 for Mtb and 35.952 for NTM. By culture method used the TDG for LJ was 33,577 and 6.435 by BacT/ALERT 3D, this difference being statistically significant. The overall contamination rate (CR) for BacT/ALERT 3D was 4.6% and 7.8% for LJ. Conclusions: BacT/ALERT 3D were a suitable method for recovering mycobacteria from clinical samples. It demonstrated a shorter time to detection of mycobacteria growth; it was very useful to provide faster treatment and a better prognosis in patients AFB smear negative with HIV. The use of LJ culture and BacT/ALERT 3D System was useful to assure a total mycobacterial recovery.

MYCOBACTERIUM KANSASIIISOLATION FROM CAPTIVE SOUTH AMERICAN COATI (NASUA NASUA)

Three of six captive South American coatis (Nasua nasua) presented with respiratory distress and died despite treatment. Postmortem examination performed on two of these animals revealed granulomatous pleuropneumonia associated with acid-fast bacilli. Because of the possible diagnosis of mycobacterial infection, the three remaining asymptomatic coatis were anesthetized. Tracheal washes were sampled and submitted for microbiology, and the animals were euthanatized and postmortem examinations performed. One of these asymptomatic adult males had whitish granulomas in multiple organs and tissues. Additionally, the isolate from this male's tracheal wash was identified as Mycobacterium kansasii by molecular analysis. To the authors' knowledge, this is the first report of M. kansasii infection in Nasua nasua.

Systematic Use of Universal 16S rRNA Gene Polymerase Chain Reaction (PCR) and Sequencing for Processing Pleural Effusions Improves Conventional Culture Techniques

Conventional culture of pleural fluid samples frequently provides false-negative results. Universal polymerase chain reaction (PCR) of the 16S ribosomal ribonucleic acid (rRNA) gene (16S PCR) has proven useful in the diagnosis of various bacterial infections. We conducted a prospective study to assess the value of 16S PCR in the etiologic diagnosis of pleural effusion. All pleural fluid samples received for culture were also studied using 16S PCR. Positive samples were sequenced for identification. Clinical records and conventional culture results were analyzed to classify pleural fluid samples as infected or not infected. We studied 723 samples. We excluded 188 samples because they were obtained from a long-term chest tube, there was a diagnosis of mycobacterial infection, or there were insufficient data to classify the episode. Finally, 535 pleural fluid samples were analyzed. According to our criteria, 82 (15.3%) were infected and 453 (84.7%) were not infected. In the infected samples, 16S PCR was positive in 67 samples (81.7%) while conventional culture was positive in 45 (54.9%). There were 4 false positives with 16S PCR (0.9%) and 12 with culture (2.6%). The values for the etiologic diagnosis of bacterial pleural effusion of conventional culture compared with 16S PCR were as follows: sensitivity, 54.9%/81.7%; specificity, 97.4%/99.1%; positive predictive value, 76.3%/94.4%; negative predictive value, 92.6%/96.8%; and accuracy, 90.8%/96.5%.When compared with conventional culture, 16S PCR plus sequencing substantially improves the etiologic diagnosis of infectious pleural effusion. In our opinion, this technique should be added to the routine diagnostic armamentarium of clinical microbiology laboratories.

Standardization of laboratory tests for tuberculosis and their proficiency testing

Explanations of proper collection procedures are imperative for accurate laboratory analysis. The quality of specimens collected and the proper transport of those specimens to the laboratory are critical to successful isolation of etiological agents. Most mycobacteria grow at a relatively slow rate. Therefore, the acid-fast smear plays an important role in the early diagnosis of mycobacterial infection. There are several methods of determining the acid-fast nature of an organism. In the carbolfuchsin procedures (Ziehl-Neelsen, Kinyoun), acid-fast organisms appear red, and in the fluorochrome procedures (auramine O, auramine-rhodamine, acridine orange), the acid-fast organisms fluoresce yellow to orange. Fluorochrome-stained slides may be directly restained with any of the carbolfuchsin staining procedures. This may be done to confirm a positive fluorochrome slide and to study organism morphology. In the last 10 years, there were many advances in the culture examinations of mycobacteria. The newly developed Mycobacteria Growth Indicator Tube (MGIT), BacT/Alert, ESP Myco, MB Redox and KRD "Nichi B", biphasic Septi-Chek AFB and Myco-Acid, and radiometric BACTEC 460TB systems based on liquid media, proved to be significantly better than the egg-based solid media for the isolation of mycobacteria from clinical specimens. In addition to liquid-based medium, agar (Middlebrook 7H10 or 7H11)- or egg (Ogawa or Löwenstein-Jensen)-based media should be used in the primary isolation of mycobacteria. To identify mycobacteria, conventional biochemical tests are traditionally used. Key test can be used to identify species, or further preliminary grouping may be used. Other approaches to identifying some species of mycobacteria are available. They include niacin accumulation, p-nitrobenzoic acid and p-nitro-alpha-acetylamino-beta-hydroxypropiophenone tests for discrimination of the Mycobacterium tuberculosis complex from mycobacteria other than M. tuberculosis (MOTT); DNA probe methods for identification or confirmation of the M. tuberculosis complex, M. avium complex, M. kansasii, and M. gordonae; DNA-DNA hybridization method for identification of 22 Mycobacterium species; and gas-liquid chromatography or high performance liquid chromatography analyses for recognizing the patterns of the mycobacterial cell wall fatty acids or mycolic acids. The advantages of the last four methods are that they are capable of providing definitive identification within 2 to 5 h after adequate growth. Capilia TB is the newly developed immunochromatographic assay for rapid discrimination between the M. tuberculosis complex and MOTT bacilli. The kit can be easily used for rapid identification of the M. tuberculosis complex in combination with the culture systems based on liquid media. In addition, Capilia TB could correctly detect the M. tuberculosis complex from mixed cultures with the M. tuberculosis complex and MOTT bacilli. The WHO/IUATLD supranational reference laboratory (SRL) network was created in 1994, to ascertain the accuracy of the susceptibility test methods used in different laboratories across the world, and to allow comparability of the surveillance data gathered in countries participating in the Global Project on Anti-tuberculosis Drug Resistance Surveillance. Today, the network has evolved and 23 SRLs actively participate. Results of five rounds of proficiency testing in the SRL network suggest that performance of the network has improved substantially through the years. This progress has been particularly evident for streptomycin and ethambutol sensitivity, which was very low in the first rounds of proficiency testing. In 1998 sensitivity for these two drugs was higher than 95%. For isoniazid and rifampin, sensitivity has been consistently high since the beginning of the Global Project. This indeed reflects the enhanced efforts made by the SRLs to improve their individual performance.

Detection of mycobacteria by polymerase chain reaction

The polymerase chain reaction (PCR) was used to detect mycobacterial DNA sequences in the cultured or the clinical specimens. Four oligonucleotide primers derived from the sequence of a gene coding 65-kilodalton antigen of Mycobacterium tuberculosis amplified DNA samples of all the 11 species of mycobacteria tested. Serial dilution of M. bovis BCG showed that DNA extracted from only 12 bacilli was enough for the detection by PCR method. However, mycobacteria in sputum were detected by PCR when more than 10(3) bacilli were present. The PCR method may become a useful tool for the rapid diagnosis of mycobacterial infections.