Potential biomarkers for identification of mycobacterial cultures by proton transfer reaction mass spectrometry analysis.

RATIONALE: Several mycobacterial species can produce serious infections in humans, and the treatment required depends on the infecting species. Fast identification, ideally with minimal manipulation of the infecting species, is therefore critical; here, we propose a method potentially allowing cultures to be identified by headspace analysis and use it to screen for differences between mycobacterial species based on the volatiles released during growth. METHODS: Short-chain volatile organic compound emissions from two non-tuberculosis slow growing mycobacterial species, Mycobacterium avium and Mycobacterium kansasii, and a non-pathogenic fast growing species, Mycobacterium smegmatis, in Middlebrook M7H9 culturing media were followed online with a proton transfer reaction quadrupole mass spectrometer. RESULTS: Measurable differences between the headspace of the two slow growing mycobacteria M. kansasii and M. avium were found, as well as differences with respect to the faster growing mycobacteria M. smegmatis. Three compounds, attributed to sulfur-containing volatiles--dimethyl sulfide, propanethiol and dimethyl disulfide--were found to be specific to M. avium. CONCLUSIONS: Clear differences were detected in the low molecular weight volatile emissions compounds of the mycobacterial species under study, without the need for sample manipulation. Further studies with other mycobacterial species will reveal if the differences observed are specific to the species studied here. Furthermore, the use of an ion trap as a mass analyzer with the same ionization technique, allowing molecular detection over a wider molecular range, could allow the detection of additional biomarkers thus capturing a wider molecular range.

Light emitting diodes for auramine O fluorescence microscopic screening of Mycobacterium tuberculosis.

We describe the simple adaptation of a standard fluorescent microscope for illumination using a 'Royal Blue' Luxeon light emitting diode (LED) and demonstrate that this form of illumination is suitable for the detection of auramine O stained Mycobacterium spp. The low cost, low power consumption, safety and reliability of LEDs makes them attractive alternatives to mercury vapour lamps.

Isolation, chemical and immunological characterization of two strongly basic nuclear proteins from human spermatozoa.

Human spermatozoa were solubilized by treatment with urea/guanidine and fractionated by ion-exchange chromatography with Bio-Rex 70 resin. Four fractions were obtained. The acidic proteins pass unretarded, whereas the moderately basic and two strongly basic protein fractions are eluted by means of guanidine gradients. The main protein components of the strongly basic protein fractions have been isolated by gel filtration on Sephadex G-50. The purified proteins have been named human sperm protamine 1 and 2. They contain 47 and 51 aminoacid residues (mol. wt 6280 and 6840), of which 22 and 24 are arginine and 5 and 4 are half cystine residues, respectively. The electrophoretic mobility in urea/polyacrylamide gels is between that of calf thymus histone and salmon protamine. Human protamine 1 and 2 are both auto-antigens as has been detected by a quantitative immunofluorescence inhibition test.

PIP: Chemical and immunological characterization of 2 strongly basic nuclear proteins isolated from human spermatozoa are described. Spermatozoa proteins were solubilized by treatment with urea/ guanidine and fractionated by ion-exchange chromatography with Bio-Rex 70 resin. The initial peak (Fraction 1) was acidic nonabsorbed protein, Fraction 2 was moderately basic, and Fractions 3 and 4 were strongly basic. The acidic proteins passed unretarded whereas the basic proteins were eluted by guanidine gradients. The main protein components of Fractions 3 and 4 were isolated by gel filtration on Sephadex G-50. The purified proteins, named human sperm protamine 1 and 2, contain 47 and 51 aminoacid residues (molecular weight 6277 and 6844) of which 22 and 24 are arginine and 5 and 4 are half cystine residues, respectively. The electrophoretic mobility in urea/polyacrylamide gels is between that of calf thymus histone and salmon protamine. Quantitative immunofluorescence inhibition tests revealed that protamine 1 and 2 are both autoantigens.

Anti-Mycobacterium leprae monoclonal antibodies cross-react with human skin: an alternative explanation for the immune responses in leprosy.

A panel of 17 mouse monoclonal antibodies (MoAb) raised against Mycobacterium leprae (M. leprae) antigens was used to detect antigenic determinants in normal human skin. An indirect immunoperoxidase technique was used. Eight of the MoAb detected epidermal antigens similar to patterns well known for human sera. Five of these MoAb detected determinants in the dermis, too. These observations may indicate a certain degree of similarity between the antigenic determinants occurring in M. leprae and in the human host. We propose that such a similarity on the one hand may facilitate the survival of M. leprae in the human host when the antigens are not recognized as "non-self," a situation which seems to occur in lepromatous leprosy, when the patients' tissues are loaded with bacteria virtually without any immune response. On the other hand, M. leprae antigens which mimic host antigens may induce an auto-immune reaction against the host's own antigens, which could explain the immune reaction in tuberculoid leprosy and during a "reversal reaction" when M. leprae is not observed in the host tissues, but extensive granuloma formation occurs.

The potential contribution of the polymerase chain reaction to the diagnosis of tuberculous meningitis.

OBJECTIVE: To examine diagnostic utility of polymerase chain reaction (PCR) on cerebrospinal fluid (CSF) in tuberculous meningitis (TBM). DESIGN: Comparison study. SETTING: Referral center for tuberculosis diagnosis and treatment in Ho Chi Minh City, Vietnam, and research laboratory in Amsterdam, the Netherlands. PATIENTS: One hundred thirty-six consecutive patients, aged 4 months to 85 years, with features compatible with TBM seen during a 12-month period. MEASUREMENTS: Clinical examination; cytology; Gram, india ink, and Ziehl-Neelsen staining; culture of CSF for bacteria, mycobacteria, fungi, and viruses; and CSF chloride, protein, and glucose. All these tests were performed in Vietnam. The PCR on CSF was performed in the Netherlands. RESULTS: Patients were managed in Vietnam without knowledge of PCR results. Based on clinical grounds and the results of initial CSF microscopy, antituberculous treatment was given to 104 patients, 66 of whom had evidence of extraneural tuberculosis. Among the 39 patients with confirmed TBM (ie, positive Ziehl-Neelsen staining or culture or PCR results for Mycobacterium tuberculosis), PCR detected 32 patients (82%), 1 case was proven positive through microscopy and 17 (44%) had positive culture results. There were no false-positive PCR results. In 99 patients with a final diagnosis of confirmed or probable TBM (ie, clinical features of TBM and response to antituberculous treatment), PCR had a sensitivity of 32%; culture, 17% and microscopy, 1%. CONCLUSIONS: Many patients who respond to treatment for TBM do not have M tuberculosis in the CSF identifiable by microscopy, PCR, or culture. Polymerase chain reaction on CSF is the best method for the laboratory diagnosis of TBM. Polymerase chain reaction is especially useful for the early diagnosis of TBM in those without active extraneural tuberculosis.

Early diagnosis of tuberculous meningitis by polymerase chain reaction.

BACKGROUND: Rapid detection of Mycobacterium tuberculosis is of vital importance for patients with tuberculous meningitis. We evaluated an improved polymerase chain reaction (PCR) technique for rapid and specific identification of M tuberculosis in CSF. METHODS: The technique was used on CSF samples from 42 patients (3 of whom were human immunodeficiency virus seropositive) with clinical symptoms, signs and laboratory findings suggestive of tuberculous meningitis. The target for amplification was a nucleotide sequence located within IS6110. A small amount of DNA from M smegmatis strain 1008, containing a modified IS6110, was added in the PCR as a control for inhibitors and to quantitate the PCR for M tuberculosis. RESULTS: On the basis of symptoms and clinical findings, antituberculous treatment was started in 35 patients, but was later stopped in 11 because of lack of response. From 12 patients responding to treatment and with a positive diagnostic test, 11 cases were detected by PCR, nine cases were culture positive, and two cases microscopy positive. Of the remaining 12 patients who had negative CSF by microscopy, PCR, and culture, 11 were diagnosed as having tuberculous meningitis on the basis of the response to treatment (three had active pulmonary tuberculosis) and one had mycobacteria other than M tuberculosis in sputum and urine. The sensitivity of the PCR was 48% in those with a final diagnosis of tuberculous meningitis (culture or PCR confirmed cases, plus those with clinical evidence and who responded to antituberculous treatment), which is much higher than the 9% sensitivity of microscopy. There were no false-positive PCR results. CONCLUSIONS: PCR on CSF is a rapid method for the accurate diagnosis of tuberculous meningitis.

Measurement of antigen specific lymphocyte proliferation using 5-bromo-deoxyuridine incorporation. An easy and low cost alternative to radioactive thymidine incorporation.

The classical in vitro assay for the determination of cell mediated immune responses is the lymphocyte transformation test (LTT) in which cell proliferation is measured by incorporation of radioactive labeled thymidine (3H-TdR). The LTT assay using 3H-TdR is less suited for modestly equipped laboratories as it is costly, laborious and involves the need to handle radioactive isotopes and specialized equipment. Here we describe an improved alternative LTT method which is capable of detecting specific cellular immune reactions (CMI) against (mycobacterial) antigens in vitro. This assay, the bromodeoxyuridine-ELISA LTT test, is simple, less expensive, reproducible and is as sensitive as the 3H-TdR test. The specific advantages of the test are a simple denaturation step and the fact that no radioactive isotopes are needed. The test is specifically suited for research laboratories in tropical countries which study CMI in those human infectious diseases where this arm of the immune response plays a pivotal role in the generation of immunity, e.g., in tuberculosis, leprosy and leishmaniasis.

Rapid identification of cultured Mycobacterium tuberculosis with a panel of monoclonal antibodies in western blot and immunofluorescence.

This paper describes the identification of cultured mycobacteria with a panel of monoclonal antibodies directed against species-specific epitopes in a Western blot (WB) test and in an immunofluorescence test (IFT). In WB, we identified mycobacteria of the Mycobacterium tuberculosis complex (M. tuberculosis, M. africanum, M. bovis, M. bovis BCG, and M. microti) with 10(8) bacteria. In the IFT, we identified mycobacteria of the M. tuberculosis complex, the M. avium complex (M. avium, M. intracellulare and M. scrofulaceum) and M. kansasii with 10(7) bacteria. Using a panel of 105 mycobacterial patient isolates, we compared identification by WB and IFT with conventional, culture and biochemical identification. Identification of the M. tuberculosis complex in WB had a specificity and sensitivity of 96.3 and 98.3%, respectively. Identification in IFT of the M. tuberculosis complex had a specificity and sensitivity of 94.6% and 89.7%, respectively. Since the identification of mycobacteria with mAb requires only a small number of bacteria, these tests will reduce by several weeks the time necessary for microbiological identification.

Routine application of the polymerase chain reaction for detection of Mycobacterium tuberculosis in clinical samples.

AIM: To investigate the use of the polymerase chain reaction (PCR) in the routine laboratory for the detection of Mycobacterium tuberculosis in clinical samples. METHODS: Samples were divided and processed separately for the detection of M tuberculosis by microscopy, culture and PCR. After DNA extraction, PCR was performed with primers specific for the insertion element IS6110 and the product was analysed by agarose gel electrophoresis, Southern blotting or dot blotting and hybridisation with a digoxigenin labelled internal probe. Each sample was tested for inhibitors of Taq polymerase with the aid of an internal control. Multiple negative and positive controls were used to monitor each step of the procedure. RESULTS: The data from two laboratories, using the same operating procedures, were combined. Of 1957 specimens, 79 (4%) were culture and PCR positive, while 1839 (93.9%) were negative in both tests. Thirty specimens (1.5%) were PCR positive only and nine (0.5%) were culture positive but PCR negative. CONCLUSION: Using culture and clinical history as the gold standard, sensitivity and specificity for PCR were 92.1% and 99.8%, respectively. With elaborate precautions, PCR is a suitable and reliable method for the detection of M tuberculosis in clinical samples in a routine microbiology laboratory.

Trypsin-treated and coomassie blue-stained epimastigote antigen in a microagglutination test for Chagas' disease.

A microagglutination test using trypsin-treated and Coomassie blue-stained Trypanosoma cruzi epimastigote antigen was adapted for the diagnosis of Chagas' disease. When incorporated in the test, 2-mercaptoethanol treatment of chagasic sera had no influence on antibody titer. In contrast, titers in sera from patients with visceral leishmaniasis, African trypanosomiasis, and autoimmune disorders, subjected to similar treatment, showed remarkable decline. Accordingly, a lower cut-off point for Chagas' disease serological negativity could be taken resulting in a higher sensitivity (95.6%); the specificity was 94.7%. Similar specificities were obtained with Leishmania donovani chagasi and L. d. donovani antigens applied to homologous visceral leishmaniasis and heterologous Chagas' sera. Of 316 nonchagasic sera, only 3 with leptospirosis and 1 with leprosy showed seropositive titers prior to and after 2-mercaptoethanol treatment.

A simple and economical direct agglutination test for serodiagnosis and sero-epidemiological studies of visceral leishmaniasis.

A simple and economical direct agglutination test for the detection of visceral leishmaniasis is described. Trypsin-treated, Coomassie Brilliant Blue-stained, formalin-preserved promastigotes were used as antigen in re-usable V-well microtitre plates. In 21 patients with recent kala-azar, titres of 1:51200 or higher were found. Cured kala-azar patients treated 4 to 14 months before testing, showed titres in the range of 1:3,200 to greater than 1:51,200. Healthy and diseased controls had titres below 1:1,600 with the exception of African trypanosomiasis patients who showed titres of 1:200 to 1:12,800, overlapping with the titres of cured kala-azar patients. Where trypanosomiasis is not a consideration, a titre of 1:1,600 could be considered indicative of visceral leishmaniasis, the sensitivity and specificity were then 100%. The test was applied to sera of 280 inhabitants of Baringo District, a known focus of visceral leishmaniasis in Kenya. When treated cases were included, the test showed a sensitivity of 100% and specificity of 99.3%. This test could be used in district hospitals and health centres in endemic areas as an aid in diagnosis of kala-azar and in the field for sero-epidemiological studies.

First reported case of Mycobacterium ulcerans infection in a patient from China.

Buruli ulcers have not been previously described in China, and only once at higher latitudes on the northern hemisphere. A patient who travelled in the Shan Dong Province in the People's Republic of China developed an ulcer which was proven to be a Buruli ulcer. The clinical picture and histopathological findings from biopsy specimens are characteristic for a Buruli ulcer, and also the growth in culture (Coletsos medium) at a restricted temperature of 30 degrees C. A multiplex polymerase chain reaction (PCR) based on the amplification of the gene encoding for 16S ribosomal RNA and a nested PCR based on the Mycobacterium ulcerans specific repeated sequence 2404 were performed. These PCR investigations identified the bacteria as M. ulcerans, subspecies shinshuense. The patient was initially treated with clarithromycin and rifampicin, which was changed to ciprofloxacin and rifabutin when rifampicin resistance of the first isolate was established. There were no signs of reactivation of the disease 6 months after the end of treatment. M. ulcerans infection occurs above 30 degrees latitude on the northern hemisphere in China and is caused by M. ulcerans, subspecies shinshuense. This case appears to be cured by chemotherapy alone, in contrast to the general experience that surgical treatment is indicated. The granulomatous reaction with only fragments of acid-fast bacteria in the biopsy at the end of treatment many indicate the development of an adequate cell-mediated immune response leading to resistance to the infection.

Evaluation of a newly developed direct agglutination test (DAT) for serodiagnosis and sero-epidemiological studies of visceral leishmaniasis: comparison with IFAT and ELISA.

A newly developed direct agglutination test (DAT) for visceral leishmaniasis, IFAT and ELISA were applied to sera of patients with visceral leishmaniasis, African and American trypanosomiasis, other parasitic infections and healthy controls. The sensitivities of the 3 tests were comparable (96.3% to 100%); excluding patients with African and American trypanosomiasis, the specificities of DAT and IFAT were 100% and ELISA 87.3%. When trypanosomiasis sera were included, the specificities were 72.6%, 94.3% and 79.4% in DAT, IFAT and ELISA respectively. In 273 sera from a leishmaniasis endemic area (Baringo District, Kenya), the sensitivity was 80% in DAT and IFAT and 60% in ELISA, specificities being 99.6% (DAT), 98.5% (IFAT) and 62.5% (ELISA). As the new DAT is economical and easy to perform, it is recommended for sero-epidemiological field work on visceral leishmaniasis.

Aetiology of uveitis in Sierra Leone, west Africa.

BACKGROUND: In 1992, non-onchocercal uveitis caused 9% of blindness, 8% of visual impairment, and 11% of uniocular blindness among patients visiting an eye hospital in Sierra Leone, west Africa. The aim of this study was to determine the aetiology of uveitis in this population. METHODS: General and ophthalmic examination complemented by serum and aqueous humour analyses for various infectious agents was performed for 93 uveitis patients and compared with serum (n = 100) and aqueous humour (n = 9) analysis of endemic controls. RESULTS: At the initial examination, 45 patients (48%) proved to be severely visually handicapped. After clinical and laboratory analyses, an aetiological diagnosis was established for 49 patients (52%). Toxoplasma gondii was the most important cause of uveitis (40/93; 43%). Anti-toxoplasma IgM antibodies were detected in serum samples of seven of 93 patients (8%) compared with one of 100 controls (1%, p < 0.05). At least six patients (15%) with ocular toxoplasmosis had acquired the disease postnatally. Antibodies against Treponema pallidum were detected in 18 of 92 patients (20%) and in 21 controls (21%). Other causes of uveitis were varicella zoster virus (one patient), herpes simplex virus (two patients), and HLA-B27 positive acute anterior uveitis with ankylosing spondylitis (one patient), while one patient had presumed HTLV-I uveitis. CONCLUSIONS: In a hospital population in Sierra Leone, west Africa, uveitis was associated with severe visual handicap and infectious diseases. Toxoplasmosis proved to be the most important cause of the uveitis. Although the distribution of congenital versus acquired toxoplasmosis in this population could not be determined, the results indicate an important role of postnatally acquired disease. The results further suggested minor roles for HIV, tuberculosis, toxocariasis, and sarcoidosis as causes of uveitis in this population.

Rapid species identification of wild mycobacterial isolates by monoclonal antibodies in ELISA.

An enzyme-linked immunosorbent assay (ELISA) for the identification of cultured mycobacteria belonging to the Mycobacterium tuberculosis complex and Mycobacterium kansasii originally described by R. Schöningh, C. P. J. H. Verstijnen, S. Kuijper and A. H. J. Kolk (1) was used for identification of mycobacteria from three week old primary cultures. A panel of six monoclonal antibodies (MoAbs) was used: two were specific for Mycobacterium tuberculosis (M. tuberculosis) complex, one for M. kansasii, one was directed against M. avium complex and two were broadly reacting with all mycobacterial species. The ELISA was introduced to a microbiology laboratory located in an area where M. kansasii infections are endemic. All acid-fast bacteria isolated from sputum samples over one month period were identified by ELISA and culture. All fifteen M. tuberculosis isolates and all seventeen M. kansasii were correctly identified by ELISA before culture results were known. Two of three M. avium complex strains could be identified in ELISA as belonging to the M. avium complex using the M. avium complex specific monoclonal antibody.

[Development of a serological test for tuberculosis]. / Ontwikkeling van een serologische test voor tuberculose.

Antigens secreted by M. tuberculosis in the culture medium and antigens obtained from sonicated M. tuberculosis were characterized, at the laboratory of Tropical Hygiene of the Royal Tropical Institute and the division of Pulmonary Medicine at the Academic Medical Centre, in order to explore which antigens could be valuable in the development of a serological test for tuberculosis. Using murine monoclonal antibodies in immunoblot, a pattern of protein bands distinct from that of the corresponding M. tuberculosis sonicate was found in the culture medium. The major protein bands of the culture medium, of 24 and 12 kD, and the major protein band of 16 kD in sonicate were purified. These antigens were tested in ELISA with sera from 20 patients with tuberculosis, diagnosed by a positive culture of M. tuberculosis, and from 21 control subjects. The ELISA results obtained with these 3 antigens were combined and the mean value obtained in the control group plus 2 times the standard deviation was chosen as the cut-off level. Sixteen of the 20 patients with tuberculosis had antibodies against 1 of the purified antigens, while none of the control subjects had. By combining the results, obtained with these 3 antigens, 17 of the 20 patients with tuberculosis were positive in this serological test and none of the control subjects. The 24, 12 and 16 kD antigens may be valuable for the development of a serological test for tuberculosis.

[Mycobacterium genavense infection in 2 HIV seropositive patients in Amsterdam]. / Infectie met Mycobacterium genavense bij 2 HIV-seropositieve patiënten in Amsterdam.

Two patients with an HIV-I infection, a man aged 47 with confusion, aphasia and diarrhoea, and a man aged 32 with dysphagia, a non-productive cough and diarrhoea, were diagnosed as having a disseminated Mycobacterium genavense infection. Both had low counts of CD4+ T lymphocytes. They responded to antimycobacterial treatment. M. genavense was recognized in Geneva in the early nineties as a causative agent of disseminated mycobacterial infections in HIV-seropositive patients with poor cellular immunity. The clinical picture resembles that of a generalized infection with M. avium-intracellulare. M. genavense is a slowly growing mycobacterium which can be isolated and identified using enriched nutrient media and molecular-biological techniques. The infection probably begins in the gastrointestinal tract after oral contamination. DNA of M. genavense can be demonstrated in 25% of the intestinal biopsy samples of non-HIV-seropositive patients.

Breath analysis as a potential diagnostic tool for tuberculosis.

SETTING: Cape Town, South Africa. OBJECTIVES: We investigated the potential of breath analysis by gas chromatography-mass spectrometry (GC-MS) to discriminate between samples collected prospectively from patients with suspected tuberculosis (TB). DESIGN: Samples were obtained in a TB-endemic setting in South Africa, where 28% of culture-proven TB patients had Ziehl-Neelsen (ZN) negative sputum smear. A training set of breath samples from 50 sputum culture-proven TB patients and 50 culture-negative non-TB patients was analysed using GC-MS. We used support vector machine analysis for classification of the patient samples into TB and non-TB. RESULTS: A classification model with seven compounds had a sensitivity of 72%, a specificity of 86% and an accuracy of 79% compared with culture. The classification model was validated with breath samples from a different set of 21 TB and 50 non-TB patients from the same area, giving a sensitivity of 62%, a specificity of 84% and an accuracy of 77%. CONCLUSION: This study shows that GC-MS breath analysis is able to differentiate between TB and non-TB breath samples even among patients with a negative ZN sputum smear but a positive culture for Mycobacterium tuberculosis. We conclude that breath analysis by GC-MS merits further research.

Proton Transfer Reaction Mass Spectrometry detects rapid changes in volatile metabolite emission by Mycobacterium smegmatis after the addition of specific antimicrobial agents.

The metabolic activity of plants, animals or microbes can be monitored by gas headspace analysis. This can be achieved using Proton Transfer Reaction Mass Spectrometry (PTR-MS), a highly sensitive detection method for trace gas analysis. PTR-MS is rapid and can detect metabolic responses on-line as they occur. Here, we study the headspace of actively growing cultures of paired ciprofloxacin sensitive and resistant bacterial strains (Mycobacterium smegmatis in Middlebrook M7H9 liquid media) after the addition of the antibiotics ciprofloxacin and gentamicin in real time. Following the emission patterns of the mycobacteria over time allowed volatile markers specific for the bacterial response to each antibiotic to be detected. A proportion of the measured responses were very rapid, occurring within three hours after the addition of the compounds and varied between isolates with different resistance phenotypes. Specifically, we observed a two fold increase of m73 (unidentified C4 compound) within 10h after the addition of ciprofloxacin and a threefold increase of m45 (acetaldehyde) within 4h after the addition of gentamicin as compared to values before the addition. Monitoring the emission of specific volatiles into the culture headspace thus has the potential for rapid drug susceptibility testing. Moreover, these and other differences in the measured responses to the two tested compounds provide evidence that monitoring multiple compounds may also give an indication of the mechanism of action of the compound added.