Detection of lipoarabinomannan antibodies in patients with newly acquired tuberculosis and patients with relapse tuberculosis

A commercially available dot immunoassay that employs the lipoarabinomannan antigen was evaluated for the serologic diagnosis of tuberculosis. The test showed a high specificity (100%); however, its sensitivity was low (18.5%). Antibodies to lipoarabinomannan were detected in the sera of 7 of 71 patients with newly acquired tuberculosis and in sera of 10 of 21 patients with relapse tuberculosis. It has been shown by others that sera from patients with relapse tuberculosis had a higher concentration of antibodies and reacted with a greater variety of antigens (native culture filtrates of Mycobacterium tuberculosis H37Rv) than did sera from patients with newly acquired tuberculosis. Our data confirm the results of these previous studies as far as lipoarabinomannan is concerned. We conclude that the differences in the production of antibodies shown by the two groups of tuberculous patients (new and relapse) must be taken into account when assessing the usefulness of serologic tests for the diagnosis of tuberculosis.     

Biopsy for diagnosis of tuberculosis

Recent advances of molecular techniques make diagnosis of pulmonary tuberculosis rapid and easier comparing to conventional techniques. However, diagnosis for tuberculosis, especially tuberculous pleuritis, tuberculous lymphadenitis and non-pulmonary tuberculosis, is not easy. Transbroncheal lung biopsy (TBLB) is a helpful examination for not only pulmonary tuberculosis but also malignant diseases. The pleural biopsy using Cope needle was commonly used as a method for diagnosis of tuberculous pleuritis, and the sensitivity of pleural biopsy is about 50% or more. Furthermore, the pleural biopsy using thoracoscopy was reported to show a higher sensitivity for the diagnosis of tuberculous pleuritis. A lymph node biopsy is also a useful method for diagnosis of tuberculous lymphadenitis. Thus, a biopsy method is the reliable tool for diagnosis of not only pulmonary tuberculosis, but also other organ tuberculosis.     

Detection of specific antibodies to an antigenic mannoprotein for diagnosis of Penicillium marneffei penicilliosis

The disseminated and progressive fungal disease Penicillium marneffei penicilliosis is one of the most common infectious diseases in AIDS patients in Southeast Asia. To diagnose systemic penicilliosis, we developed an enzyme-linked immunosorbent assay (ELISA)-based antibody test with Mp1p, a purified recombinant antigenic mannoprotein of P. marneffei. Evaluation of the test with guinea pig sera against P. marneffei and other pathogenic fungi indicated that this assay was specific for P. marneffei. Clinical evaluation revealed that high levels of specific antibody were detected in two immunocompetent penicilliosis patients. Furthermore, approximately 80% (14 of 17) of the documented penicilliosis patients with human immunodeficiency virus tested positive for the specific antibody. No false-positive results were found for serum samples from 90 healthy blood donors, 20 patients with typhoid fever, and 55 patients with tuberculosis, indicating a high specificity of the test. Thus, this ELISA-based test for the detection of anti-Mp1p antibody can be of significant value as a diagnostic for penicilliosis.     

Role of endoscopy for the diagnosis of tuberculosis

The usefulness of bronchoscopy in diagnosis of pulmonary tuberculosis (PT) has been demonstrated with many workers after 1980's. The indication for bronchoscopy is in patients suspected of having active PT with negative sputum smear and polymerase chain reaction (PCR). The combination of bronchial aspiration, brushing, bronchoalveolar lavage, biopsy and postbronchoscopy sputum give a high yield of early diagnosis, ranging from 40-70% of cases. Furthermore, early diagnostic rate improves by these procedure adding PCR in 85%. The bacteriologic study of pleural effusion in diagnosis of tuberculous pleurisy has shown positive results in less than 30 percent of cases. Although the diagnostic rate improves in 60 percent by blind needle biopsy, the diagnostic accuracy is much greater when the thoracoscopy is used, because the pathologist is provided with multiple, selected biopsy. So not only surgeon but also physician should learn thoracoscopy for the diagnosis of pleural effusion.     

Clinical efficacy of the amplified Mycobacterium tuberculosis direct test for the diagnosis of pulmonary tuberculosis

The amplified Mycobacterium tuberculosis direct test (MTD) is a rapid diagnostic test based on a nucleic acid amplification technique, which can be used directly on processed clinical specimens. We evaluated the clinical utility of the MTD for diagnosing pulmonary tuberculosis by comparing the sensitivity and specificity of the test with acid-fast smear, mycobacterial culture, and clinical evaluation. The study included 844 respiratory tract specimens from 421 patients, which were submitted to the microbiology laboratory of our urban teaching hospital over a 6-mo period. Compared with culture, MTD had a sensitivity of 93.6% and specificity of 97.8%. MTD was more sensitive in detecting pulmonary tuberculosis in patients with previously undiagnosed disease (74.7%) than in those with established disease receiving chemotherapy (29.2%), and in smear-positive (95.5%) than in smear-negative (70.0%) disease. There were two false positive MTD results in patients with nontuberculous mycobacteria, for a specificity in this population of 97.3%. We conclude that MTD, when used in conjunction with routine smear and culture, is a useful rapid diagnostic test for suspected pulmonary tuberculosis.     

The possibilities for improving the serological diagnosis of active tuberculosis by using new mycobacterial antigens and immunoblot and ELISA technics

The study concerns 264 cases among which: 119 active lung tb. eliminating and 11 cases not-eliminating M. Tuberculosis; 17 cases of extrarespiratory tb. confirmed by bacteriology and/or by anatomopathology; 18 cases of bone-joint non-tb disease; 38 cases of chronic lung disease other than tb; 61 healthy persons (controls). Sera from these cases were collected before treatment and submitted concomitantly to two different methods: (1) Mycodot test (immunoblot) with lipoarabinomannan (LAM) as antigen, on nitrocellulose discs (Dynagen, Cambridge, MA, USA); (2) ELISA test with antigen 60 (A60) (ANDA-Biologicals, Strasbourg, France) and with antigen I.C. (Cantacuzino Institute, Bucharest). The results were estimated on terms of sensitivity and specificity. As for sensitivity the results show 74-90%. the highest values were reached in ELISA with A60 IgA. The specificity of the Mycodot test was highest: 90-100% in the two successive experiments. The active tb diagnosis discrimination capacity of the studied methods allows the following classification: 1. Mycodot test with LAM antigen 2. ELISA with A60-Ig G complex 3. ELISA with I.C. antigen The Mycodot test is more advantageous being more rapid and more simple to perform.     

Detection of low-avidity IgG antibodies in the diagnosis of primary acute infection by hepatitis C virus

The hydrophobicity and strength of attachment of several lactic acid bacteria with antimicrobial activity were studied. Hydrophobicity was determined by bacterial adherence to hydrocarbons (BATH; octane or xylene), adhesion to nitrocellulose filters (NCF), salt aggregation test (SAT) and adherence to phenyl-Sepharose beads (PSB). The relative hydrophobicity of lactic acid bacteria depended markedly on the method used. No correlation between either SAT or BATH (octane) and strength of attachment (Sr value) existed. However, a significant relationship between strength of attachment and BATH (xylene), NCF and PSB, respectively, was observed, showing the highest correlation coefficient (r = 0.778) for BATH (xylene).     

Microbiological diagnosis of tuberculosis

The increased incidence of tuberculosis as well as the availability of new diagnostic testing methods clearly have various implications for the routine microbiology laboratory: samples must be sent to the microbiology lab for testing immediately after being taken and microscopically investigated the same day. In other countries, difficult to treat, multi-resistant Mycobacterium tuberculosis strains have occurred. Thus decisive hygienic measures must be taken early on in cases of highly infectious patients (i.e. patients with microscopically positive sputum). Liquid media (MB Check Roche, Bactec) as well as L?wenstein Jensen media must be inoculated in the lab. Liquid media allow both faster detection of certain atypical mycobacteria and increased accuracy. Classification of culturally established agents through commercial genetic probes (AccuProbe Mycobacterien) or with high pressure liquid chromatography is possible within hours when acid-fast rods are present. Time consuming identification by determination of biochemical and culture morphological characteristics should be reserved for reference labs. Today, rapid tests like analysis of tuberculostearic acid or polymerase chain reaction are already useful for special questions like ruling out tuberculous meningitis. In most cases, however, these rapid tests cannot replace identification of microbes with culture techniques.     

Bacteriological diagnosis of tuberculosis

Microscopic examination and culture are still today essential elements of the bacteriological diagnosis of tuberculosis. Microscopic examination of a Ziehl fuchsin or auramine stained specimen allows detection of most strains in less than an hour. Culture on L?wenstein-Jensen medium is more sensitive than the microscopic examination and is required for identification and to measure sensitivity to antibiotics. Mycobacterium colonies, generally the causal agent in tuberculosis, usually grow within 28 days and are easily recognized by their "cauliflower" aspect. The niacin test is used for formal identification. Currently, radiometric respirometry allows detection of M. tuberculosis growth and provides antibiotic sensitivity results more rapidly, usually within 10 days. Use of this technique is however limited because the culture medium contains radioactive carbon. Genetic probes are on the other hand quite easy to use and allow identification of cultured bacteria in only a few hours. After polymerization chain reaction gene amplification, M. tuberculosis strains can be detected directly in the specimen within 2 or 3 hours, but in practice, this method has not become a routine laboratory technique, particularly due to lack of sufficient specificity and sensitivity. No other serologic tests are currently reliable enough for the diagnosis of tuberculosis. For cases with low-count specimens, there still is no reliable "on-the-spot" diagnostic test.     

Slide agglutination test for the diagnosis of pulmonary and extra-pulmonary tuberculosis

Nasal polyposis can be defined as a chronic inflammatory disease of the paranasal sinus mucosa, leading to a protrusion of benign edematous polyps from the meatus into the nasal cavities. Nasal polyps are histologically characterized by massive edema and accumulation of eosinophils. IgE-mediated allergy seems to play only a minor role in eosinophil accumulation, leaving the place for a new concept of non-allergic rhinitis with eosinophilia. The central question still remains, however, why eosinophils accumulate into nasal polyposis tissue. Some initial data show that tissue structural cells, i.e. epithelial cells or fibroblasts, could produce cytokines (GM-CSF) and play a role in eosinophil accumulation (micro-environmental theory). However, further studies showed, that GM-CSF was mainly produced by eosinophils themselves (autocrine theory), leading to the hypothesis of an intrinsic eosinophilic inflammatory process. Eosinophils may contribute to nasal polyp formation and growth not only through inflammation but also by exerting their effects on extracellular matrix including stimulation of collagen synthesis. Another feature associated with nasal polyposis is aspirin sensitivity. Some preliminary data indicate that eosinophils could also be involved in aspirin-sensitivity mechanisms.     

Differential diagnosis of pulmonary tuberculosis

Differential diagnosis of pulmonary tuberculosis is discussed. Chest X-ray findings of pulmonary tuberculosis may be greatly varied, because tuberculosis may cause three different lesions: an exudative lesion, a proliferative lesion, and a fibrotic lesion, and because it may invade all the structure. Thus, the differential diagnosis of pulmonary tuberculosis includes very many diseases. The most important differential diagnosis of nodule is tuberculoma and lung cancer. The clue of the diagnosis is the feature of the nodule and surrounding structure, such as pleural indentation, or knotching. There is, however, the limitation of the diagnosis by imaging: some tuberculoma may show the identical feature with the pulmonary adenocarcinoma. It is important to gather the pathological or bacteriological evidences by means of suitable procedures.     

Interaction of catalytically active antibodies with oligoribonucleotides

The interaction of antibodies from blood sera of patients with autoimmune pathology, systemic lupus erythematosus with oligoribonucleotides was studied. The RNA-hydrolyzing activity was shown to be an intrinsic property of autoantibodies. Enzymic activity of antibodies in hydrolysis of poly(U) was estimated at 20-40% of that of RNase A. In contrast to known eukaryotic RNases, the autoantibodies possess a specific RNA-hydrolyzing activity for oligo r(A). The RNA-nicking activity of antibodies in hydrolysis of oligoadenylates was more higher than with hydrolysis of oligo d(A). Optimal conditions of r(pA)13 hydrolysis were selected, including the optimal of pH = 8.7.     

Encephalocytozoon intestinalis-specific monoclonal antibodies for laboratory diagnosis of microsporidiosis

Two monoclonal antibodies which can be used for the unambiguous identification by fluorescence microscopy of Encephalitozoon intestinalis spores in clinical specimens are described. Monoclonal antibody Si91 is specific for the extruded polar filament, and Si13 recognizes the surfaces of E. intestinalis spores. No cross-reaction with spores of Encephalitozoon hellem was observed. Immunogold electron microscopy confirmed the specific reactivities of both antibodies. Combined in an indirect immunofluorescence assay, these antibodies are used to identify spores in feces. Although there was some cross-reaction with fecal bacteria and fungi, the typical morphology of the extruded polar filaments enabled proper identification of the E. intestinalis spores. Parasites could also be demonstrated to be present in urine, nasal swabs, lung brush biopsy specimens, and bronchoalveolar lavage fluid from a patient with disseminated infection with E. intestinalis. The use of these monoclonal antibodies facilitates the detection and species determination of E. intestinalis in clinical specimens.     

New tools for the diagnosis of tuberculosis: the perspective of developing countries

New diagnostics for tuberculosis are urgently needed to replace or facilitate acid-fast bacilli (AFB) microscopy for the identification of smear-positive cases, and to improve the diagnosis of AFB smear-negative cases. These need to be appropriate for use in low income countries. Tests to replace or facilitate AFB microscopy must offer improvements to this test, including increased sensitivity, speed, ease of use, and safety. Products to improve the identification of smear-negative cases should focus on the diagnosis of patients with paucibacillary pulmonary disease, including children and human immunodeficiency virus (HIV) infected persons, and those with extrapulmonary forms of tuberculosis.     

Rapid diagnosis of tuberculosis using the amplification method

BACKGROUND: Amplification methods identify Microbacterium tuberculosis on the basis of genus- or species-specific sequence of bases in nucleic acids which they replete exponentially. The objective of the work was comparison of results of biological samples by the method Gen-Probe Mycobacterium tuberculosis Direct Test (MTD) using amplification of ribosomal RNA and BK by microscopy and cultivation, assessment of standard indicators of their efficiency of the method and analysis of diverging results. METHODS AND RESULTS: During the period between April 19, 1994 and October 19, 1995 a total of 650 specimens from 316 patients examined for suspected tuberculosis were processed. After decontamination the specimens were divided into two portions and examined by the Gen-Probe MTD and by classical BK microscopy and cultivation. 95 specimens were Gen-Probe MTD positive. As compared with BK cultivation the sensitivity of Gen-Probe MTD is 94.7%, the specificity 93.1%, the positive predictive value 56.8% and the negative predictive value 99.5%. Falsely positive were the specimens from 33 patients, most frequently with a diagnosis of tumours, non-specific bronchopneumonias and interstitial pulmonary fibroses. CONCLUSIONS: Gen-Probe MTD is a rapid examination with an equal or higher sensitivity than BK cultivation. The disadvantage is a somewhat lower specificity and higher cost.     

Bacterial diagnosis of tuberculosis using the PCR technique

At the beginning of 80-ties new system BACTEC for mycobacteria culture has been introduced. So time of this culture has been shortened to 1-3 weeks. At the end 80-ties Polymerase Chain Reaction (PCR) technique was started and at present it is used in many laboratories in order to get information about genetic material of mycobacteria in different biological samples. Most often insertive factor IS 6110, located in chromosome Myc. tub. complex, is used. DNA amplification occurs in three stages: 1) DNA denaturation. 2) addition of starters (primers): 3) elongation of starter with d NTP synthesis. PCR technique identifies genetic material (DNA) of mycobacterium which is presented in a given biological sample. Positive PCR result not necessarily means that active disease takes place.     

Limits of commercial molecular tests for diagnosis of pulmonary tuberculosis

Several studies report high specificity, but variable sensitivity, of Amplified Mycobacterium tuberculosis Direct Test (AMTDT, Gen-Probe) based on ribosomal ribonucleic acid (rRNA) amplification and Amplicor Mycobacterium tuberculosis test (Amplicor, Roche) based on deoxyribonucleic acid (DNA) amplification for diagnosis of pulmonary tuberculosis. We have retrospectively evaluated these assays on selected acid-fast bacilli (AFB)-positive and -negative smear specimens and compared the results obtained from nucleic acid amplification with those of AFB staining of semi-quantitative cultures as determined by radiometric Bactec and L?wenstein-Jensen cultures. In comparison to cultures, Amplicor and AMTDT assays exhibited identical overall sensitivities of 80%, while the staining had a lower sensitivity of 62%. The sensitivities of Amplicor and AMTDT were 98% and 100%, respectively, for the AFB-positive specimens, and 50 and 46%, respectively for the AFB-negative specimens in comparison to cultures. The sensitivities of both assays appeared similar, and were directly related to the number of bacilli in the specimens studied. The low sensitivity (50%) for smear-negative specimens showed that current amplification assays may be unsuitable to replace cultures for diagnosis of tuberculosis. The decision to perform these molecular techniques should result from close co-operation between clinicians and microbiologists, taking into account the sensitivity results reported here, as well as the expense of the assays.     

Genital herpes. Type-specific antibodies for diagnosis and management

The high incidence of unrecognized genital herpes infections and the role of such undiagnosed infections in continuing the spread of genital herpes has been due, in part, to the limited availability of accurate serologic assays for herpes simplex virus types 1 (HSV-1) and 2 (HSV-2). Type-specific serology test kits for HSV diagnosis have been developed and are expected to be widely available in 1998. Clinicians should understand the proper application of these new test and in addition, should be aware that older, inaccurate test will remain on the market for the foreseeable future.