Comparison of two rapid tests for anti-phenolic glycolipid-I serology in Brazil and Nepal

The diagnosis of leprosy continues to be based on clinical symptoms and early diagnosis and treatment are critical to preventing disability and transmission. Sensitive and specific laboratory tests are not available for diagnosing leprosy. Despite the limited applicability of anti-phenolic glycolipid-I (PGL-I) serology for diagnosis, it has been suggested as an additional tool to classify leprosy patients (LPs) for treatment purposes. Two formats of rapid tests to detect anti-PGL-I antibodies [ML immunochromatography assay (ICA) and ML Flow] were compared in different groups, multibacillary patients, paucibacillary patients, household contacts and healthy controls in Brazil and Nepal. High ML Flow intra-test concordance was observed and low to moderate agreement between the results of ML ICA and ML Flow tests on the serum of LPs was observed. LPs were "seroclassified" according to the results of these tests and the seroclassification was compared to other currently used classification systems: the World Health Organization operational classification, the bacilloscopic index and the Ridley-Jopling classification. When analysing the usefulness of these tests in the operational classification of PB and MB leprosy for treatment and follow-up purposes, the ML Flow test was the best point-of-care test for subjects in Nepal and despite the need for sample dilution, the ML ICA test yielded better performance among Brazilian subjects. Our results identified possible ways to improve the performance of both tests.

Comparison of two rapid tests for anti-phenolic glycolipid-I serology in Brazil and Nepal.

The diagnosis of leprosy continues to be based on clinical symptoms and early diagnosis and treatment are critical to preventing disability and transmission. Sensitive and specific laboratory tests are not available for diagnosing leprosy. Despite the limited applicability of anti-phenolic glycolipid-I (PGL-I) serology for diagnosis, it has been suggested as an additional tool to classify leprosy patients (LPs) for treatment purposes. Two formats of rapid tests to detect anti-PGL-I antibodies [ML immunochromatography assay (ICA) and ML Flow] were compared in different groups, multibacillary patients, paucibacillary patients, household contacts and healthy controls in Brazil and Nepal. High ML Flow intra-test concordance was observed and low to moderate agreement between the results of ML ICA and ML Flow tests on the serum of LPs was observed. LPs were "seroclassified" according to the results of these tests and the seroclassification was compared to other currently used classification systems: the World Health Organization operational classification, the bacilloscopic index and the Ridley-Jopling classification. When analysing the usefulness of these tests in the operational classification of PB and MB leprosy for treatment and follow-up purposes, the ML Flow test was the best point-of-care test for subjects in Nepal and despite the need for sample dilution, the ML ICA test yielded better performance among Brazilian subjects. Our results identified possible ways to improve the performance of both tests.

Evaluation of a simplified IS6110 PCR for the rapid diagnosis of Mycobacterium tuberculosis in an area with high tuberculosis incidence.

PURPOSE: To assess the diagnostic yield of a simplified IS6110-PCR in an area with high tuberculosis incidence. METHODS: Pulmonary (218) and extrapulmonary (121) samples were collected from 236 patients including smearpositive leprosy patients. All samples were processed to detect acidfast bacilli by microscopy, culture on solid media and PCR. To remove PCR inhibitors, three washing steps of the decontaminated pellet were included before mycobacterial cell lysis by heat treatment. No detergents, enzymes, or chelating agents were used. From the 339 samples, 34 were selected basing on their large volume and were tested by the commercial kit GenoType Mycobacteria Direct (GTMD) (VER 4, Hain Lifescience, Germany) in addition to the tests cited above. RESULTS: The overall sensitivity and specificity of PCR were 93.8 and 98.6% for pulmonary samples, 63.6 and 100% for extrapulmonary samples, respectively. The assay detected MTC in 94.2% of smear positive samples with a positive predictive value of 100%. No inhibition was found among seven samples that were PCR negative but bacteriological confirmed as containing Mycobacterium tuberculosis. No false positive result occurred with samples from leprosy patients. The sensitivities for PCR and GTMD were 81.8 and 75%, respectively. CONCLUSION: PCR could efficiently complement conventional bacteriological tools for the rapid diagnosis of tuberculosis but cannot replace them.

A comparison of DNA extraction procedures for the detection of Mycobacterium ulcerans, the causative agent of Buruli ulcer, in clinical and environmental specimens.

Mycobacterium ulcerans is the causative agent of Buruli ulcer, the third most common mycobacterial disease in humans after tuberculosis and leprosy. Although the disease is associated with aquatic ecosystems, cultivation of the bacillus from the environment is difficult to achieve. Therefore, at the moment, research is based on the detection by PCR of the insertion sequence IS2404 present in M. ulcerans and some closely related mycobacteria. In the present study, we compared four DNA extraction methods for detection of M. ulcerans DNA, namely the one tube cell lysis and DNA extraction procedure (OT), the FastPrep procedure (FP), the modified Boom procedure (MB), and the Maxwell 16 Procedure (M16). The methods were performed on serial dilutions of M. ulcerans, followed by PCR analysis with different PCR targets in M. ulcerans to determine the detection limit (DL) of each method. The purity of the extracted DNA and the time and effort needed were compared as well. All methods were performed on environmental specimens and the two best methods (MB and M16) were tested on clinical specimens for detection of M. ulcerans DNA. When comparing the DLs of the DNA extraction methods, the MB and M16 had a significantly lower DL than the OT and FP. For the different PCR targets, IS2404 showed a significantly lower DL than mlsA, MIRU1, MIRU5 and VNTR6. The FP and M16 were considerably faster than the MB and OT, while the purity of the DNA extracted with the MB was significantly higher than the DNA extracted with the other methods. The MB performed best on the environmental and clinical specimens. This comparative study shows that the modified Boom procedure, although lengthy, provides a better method of DNA extraction than the other methods tested for detection and identification of M. ulcerans in both clinical and environmental specimens.

Prevalence and pattern of cross-reacting antibodies to HIV in patients with tuberculosis.

In many countries, HIV testing among tuberculosis (TB) patients is recommended so that both infections are appropriately treated. Cross-reacting antibodies to HIV antigens have been reported for several conditions, including TB, leprosy, malaria, and rheumatoid arthritis. To study the pattern and prevalence of cross-reacting antibodies to HIV antigens, we examined sera from 153 HIV-negative TB patients and 40 healthy individuals in Chennai, south India. We also studied the differences in cross-reactivity of various HIV antigens using two different Western blot kits. Of the 153 samples studied, 80 were tested using HIV Western blot and 73 were tested using INNOLIA. Most patients in the study had concordantly negative ELISA and rapid tests, and no subject had a positive Western blot. However, seven TB patients had antibodies that cross-reacted with HIV antigens, giving rise to an indeterminate result. While p51/55 was the most frequently recognized antigen in the Western blot assay, antibodies to sgp120 was most frequently identified in INNOLIA. Sequence similarities between the two organisms could be responsible for eliciting cross-reacting antibodies, since a few related epitopes were identified in HIV and Mycobacterium. These findings could have potential implications for the development of diagnostics and vaccines.

The result patterns of ML Flow and ELISA (PGL-I) serologic tests in leprosy-endemic and non-endemic areas.

ML Flow and anti-PGL-I ELISA are serological tests that detect IgM antibodies against the phenolic glycolipid I (PGL-I), specific to Mycobacterium leprae. To evaluate the outcomes of ML Flow and ELISA (PGL-I) serological tests in leprosy-endemic areas in comparison to non-endemic ones, a total of 351 volunteers from Brazil and Chile were examined, including leprosy patients, healthy controls and others affected by other infectious or non-infectious diseases that are common differential diagnoses for leprosy. The ELISA cut-off point was established using the ROC Curve method (>or= 0.157). In endemic areas, 70% of leprosy patients present positive ML Flow results and 53.3% were ELISA-positive. In non-endemic areas, ML Flow was negative in all the subjects tested and ELISA was positive in 4 volunteers. ML Flow is faster and more easily performed and, therefore, a more adequate test for use in basic, primary-level health care centers. ELISA requires trained personnel, in addition to a more complex laboratory infrastructure.

Comportamento dos testes sorológicos ML Flow e ELISA (PGL-I) em áreas endêmica e não endêmica de hanseníase / The result patterns of ML Flow and ELISA (PGL-I) serologic tests in leprosy-endemic and non-endemic areas

O ML Flow e o ELISA PGL-I são testes sorológicos que detectam anticorpos IgM contra o glicolipídio fenólico I específico do Mycobacterium leprae. Para avaliar o comportamento destes testes em áreas endêmica e não endêmica para hanseníase foram estudados 351 voluntários no Brasil e no Chile, incluindo pacientes com hanseníase, controles sadios, portadores de outras doenças infecciosas, não infecciosas e dermatoses que fazem diagnóstico diferencial com hanseníase. O ponto de corte do ELISA foi estabelecido pelo método da Curva ROC (> 0,157). Em área endêmica, o ML Flow apresentou resultados positivos em 70 por cento dos pacientes com hanseníase; o ELISA foi positivo em 53,3%. Em área não endêmica, o ML Flow foi negativo em todos os voluntários testados; o ELISA foi positivo em 4 voluntários. O ML Flow é um ensaio mais rápido, facilmente aplicável e, portanto, mais adequado para ser utilizado na Atenção Básica; o ELISA necessita, alem de uma infra-estrutura de laboratório adequada, pessoal treinado e especializado em sua execução.

ML Flow and anti-PGL-I ELISA are serological tests that detect IgM antibodies against the phenolic glycolipid I (PGL-I), specific to Mycobacterium leprae. To evaluate the outcomes of ML Flow and ELISA (PGL-I) serological tests in leprosy-endemic areas in comparison to non-endemic ones, a total of 351 volunteers from Brazil and Chile were examined, including leprosy patients, healthy controls and others affected by other infectious or non-infectious diseases that are common differential diagnoses for leprosy. The ELISA cut-off point was established using the ROC Curve method (> 0.157). In endemic areas, 70% of leprosy patients present positive ML Flow results and 53.3% were ELISA-positive. In non-endemic areas, ML Flow was negative in all the subjects tested and ELISA was positive in 4 volunteers. ML Flow is faster and more easily performed and, therefore, a more adequate test for use in basic, primary-level health care centers. ELISA requires trained personnel, in addition to a more complex laboratory infrastructure.

QuantiFERON TB Gold: a new method for latent tuberculosis infection.

QuantiFERON-TB Gold obtained approval in 2003 by the Food and Drug Administration as a valid tool for the diagnosis of latent tuberculosis. In this report, we evaluated its potential use in the immunological diagnosis of Mycobacterium tuberculosis infections in different groups of subjects. Our data indicate that QuantiFERON-TB Gold is specific for identifying subjects who have come into contact with M. tuberculosis and its use alongside traditional diagnostic techniques may be an important instrument for controlling tuberculosis.

Anti-neutrophil cytoplasmic antibodies in leprosy.

INTRODUCTION: Anti-Neutrophil Cytoplasmic Antibodies (ANCA) are auto-antibodies directed to intracellular components of neutrophils and used to be considered as present almost exclusively in granulomatous vasculitis. Recently, these auto-antibodies have been found in other autoimmune disorders as well as infectious diseases. MATERIALS AND METHODS: We studied patients with leprosy confirmed by bacilloscopy and/or skin biopsy, in reaction phase from the Ambulatório de Hanseníase do Hospital Universitário Professor Edgar Santos. ANCA and Antinuclear antibodies (ANA) were determined by indirect immunofluorescence using commercially available kits. RESULTS: Twenty patients were enrolled in our study, nine males and 11 females. The mean age was 36.9+/-18.2 years. ANCA were present only in one patient, with a perinuclear staining pattern (p-ANCA), and no patient tested positive for ANA. DISCUSSION: Although other studies have shown the presence of ANCA in leprosy, the low frequency of these antibodies in leprosy sera demonstrated in the present study illustrates the high specificity of ANCA for the diagnosis of Wegener granulomatosis.

Misidentification of Mycobacterium leprae as Mycobacterium intracellulare by the COBAS AMPLICOR M. intracellulare test.

Commercially available nucleic acid probe- and amplification-based systems for detection and differentiation of mycobacteria are widely used in clinical microbiology laboratories. Here we report two cases of human leprosy in which the COBAS AMPLICOR Mycobacterium intracellulare test led to false- positive results. Correct identification of Mycobacterium leprae was possible only by amplification and comparative sequence analysis of the 16S rRNA gene.

Evaluation of commercial enzyme-linked immunosorbent assay kits for detection of tuberculosis in Argentinean population.

Pathozyme-Myco G (Myco G), M, A, and TB complex plus (Omega Diagnostics Ltd., Alloa, Scotland) were evaluated for the serological diagnosis of pulmonary tuberculosis (TB) in an Argentinean population. Sera from 58 patients with pulmonary TB, 24 subjects with pulmonary mycobacteriosis or mycoses (pulmonary MM group), and 45 subjects with other underlying disorders (control group) were analyzed. The sensitivities of the tests ranged from 29% (Myco M) to 82% (Myco G) in smear-positive patients (17 subjects) and from 29% (TB complex plus) to 49% (Myco G) in smear-negative patients (41 subjects). The specificities of the assays varied from 93% (Myco M) to 100% (Myco G and TB complex plus) in controls and from 62% (Myco A) to 96% (TB complex plus) in the pulmonary MM group. Overall, for the diagnosis of smear-negative patients, Myco G had the best characteristics, with a sensitivity of 49% and specificities of 100% for controls and 75% for the pulmonary MM group; after its combination with TB complex plus, its sensitivity improved to 59%. Nevertheless, despite its relatively poor capacity to discriminate between pulmonary TB and pulmonary MM, Myco G, alone or in combination with TB complex plus, would be a useful diagnostic tool for patients with suspected pulmonary TB living in areas where the relative prevalence of pulmonary MM was low.

Rapid method for diagnosis of leprosy by measurements of antibodies to the M. leprae 35-kDa protein: comparison with PGL-I antibodies detected by ELISA and "dipstick" methods.

A new rapid immuno-chromatographic test card for the detection of antibodies to the Mycobacterium leprae 35-kD protein is described. The new assay is compared in the same group of subjects with a direct enzyme ELISA method for 35-kD antibodies and with assays for anti-phenolic glycolipid-I (PGL-I) antibodies using a standard ELISA as well as the recently described "dipstick" method. Good concordance was found between the rapid methods and the corresponding ELISA methods. The detection of untreated paucibacillary leprosy by the 35-kD test card was 59% compared with 27% for the PGL-I dipstick; however, the specificity for the 35-kD test card was 90% compared with 100% for the PGL-I dipstick in an endemic population. The potential application of these new, rapid serologic methods for the diagnosis of leprosy under field conditions is discussed.

Microtiter particle agglutination test for diagnosis of leprosy.

The results of studying the microtiter particle agglutination (MPA) test for detecting anti-Mycobacterium leprae antibodies in blood sera are presented. The serodiagnostic test is based on the agglutination of colored polyacrolein latex microparticles (PAMP) conjugated with 3,6-di-O-methyl-D-glucose (DMG). Sera from 45 leprosy patients (LL, BL), 34 leprosy contacts, and 148 control subjects were investigated by the MPA test. A correlation between the anti-M. leprae antibodies and the bacterial load was found. In many long-treated leprosy patients increased titers of anti-DMG antibodies were observed, which might be due to specific polyneuritis in them. Four contacts of leprosy patients were also positive in the MPA test. "Nonleprosy" sera did not react in the test. The method proposed proved to be of high specificity and sensitivity for the serological diagnosis of leprosy. The rapidity, simplicity, and visual assessment of the results allow the method to be used in the field for epidemiological studies of leprosy contacts and the general population in leprosy-endemic areas.

Novel gelatin particle agglutination test for serodiagnosis of leprosy in the field.

We developed a novel gelatin particle agglutination test (MLPA) for the serodiagnosis of leprosy; this test is especially useful for clinical practice and epidemiological surveys of leprosy in countries in which the disease is endemic. The antigen used in the test is the chemically synthesized trisaccharide moiety of Mycobacterium leprae-specific phenolic glycolipid I. MLPA is a simple and easy technique having sensitivity and specificity comparable to those of the conventional indirect enzyme-linked immunosorbent assay. The new technique was found to be useful for monitoring of chemotherapy and predictive diagnosis of high-risk individuals in contact with persons with leprosy and may be useful for the prediction of relapse. We are now preparing to supply a quality-controlled ready-to-use MLPA kit for leprosy control in countries in which leprosy is endemic.

Use of glutaraldehyde for disinfection in dialysis.

We compared Cidex HD Disinfecting Solution (CHD), a brand of glutaraldehyde disinfectant, alone and with bleach (B) to formaldehyde (F) with bleach for disinfection and cleaning of hemodialysis equipment. Two Drake-Willock central delivery systems (CDS) with 15 accompanying bedside stations and one Drake-Willock 4216 single patient delivery system (SPS) were used. The CDS designated #1, delivered dialysate to bedside stations 1 through 9, while CDS#2 supplied bedside stations 10 through 16. The study was conducted in three stages. Stage I used F six nights per week and B cleaning Wednesday (CDS#1/SPS) versus F Monday, Wednesday, and Friday with B on Wednesday (CDS#2) for four weeks. Stage II compared CHD six nights/week versus CHD Monday, Wednesday, and Friday for eight weeks. Stage III compared CDH six nights/week with B cleaning on Wednesday versus CHD on Monday, Wednesday, and Friday with B cleaning on Wednesday for four weeks. Samples of pretreated water were collected aseptically on Mondays, Wednesdays, and Fridays each week from the following sites: pre/CDS, inflow Hansen-bedside stations #5, #13, and SPS and pre-blood detector on BS #5, #13, and the SPS. During the study, 763 samples were cultured. Microbiological test results were reported as colony forming units per ml (CFU/ml). The CFU/ml were obtained by filtrating a 10 ml aliquot through a 0.45 mu filter, the filter transferred to a petri dish containing standard plate count agar and counted.(ABSTRACT TRUNCATED AT 250 WORDS)

Antibacterial monoclonal antibodies and the dawn of a new era in the control of infection.

Literature reports concerned with monoclonal antibodies against bacteria, or their toxins, which are pathogens for man and animals were surveyed. These antibodies have important potential uses in human and veterinary pathology and medicine. They are likely to become key elements in a fast progression toward a more complete understanding and control of infectious diseases and of toxin poisoning. A new area of bacteriology relevant to sanitary engineering is also being advanced with the help of antibacterial monoclonal antibodies. This area involves bacteria that produce the biofuel methane, along with other molecules of nutritional value, through a process which brings about the recycling of organic wastes and thereby limits or controls microbial contamination of soil and water.