Quality assessment of smear microscopy for acid-fast bacilli in the Argentine tuberculosis laboratory network, 1983-2001.

SETTING: Tuberculosis Laboratory Network, Argentina. OBJECTIVES: 1) To evaluate the technical quality of smear microscopy for acid-fast bacilli (AFB) supervised in 1983-2001, and 2) to analyse the effect of procedural errors on the results. DESIGN: Registers of technical evaluation results for AFB microscopy were analysed. The quality of specimens, smears, staining and readings were evaluated, as was the relationship between these parameters. RESULTS: The proportion of good quality specimens was considered acceptable. A direct relationship was demonstrated between positivity in results and mucopurulent sputum. The proportion of thin smears was relatively high. Positivity and bacillary count were lower in thin smears. Staining quality was considered good. The average agreement in readings throughout the country was 98%. Nevertheless, the false-positive rate was considered significant, and 46% of false-positive results were associated with defective staining. CONCLUSIONS: The technical quality and agreement in the laboratory network were satisfactory. Nevertheless, improvements need to be made in the following: the quality of the smears, staining and reading, coverage, decentralisation of supervision, the slide selection method and data registration. Operational research on the storage and preservation of slides is also necessary.

Molecular epidemiology of Mycobacterium bovis isolates from South America.

One hundred seventy-eight isolates of Mycobacterium bovis were subjected to DNA restriction fragment length polymorphism (RFLP) analysis, using the direct repeat element (DR) and the polymorphic GC-rich repeat sequence (PGRS) as probes. By combining the patterns generated by the two repeat DNA elements, 93 different patterns were observed. One hundred-one isolates were grouped in clusters, which include 25 different clusters. One pattern was the most frequently observed, clustering 18.5% of isolates. It was only found in the Center and northeast regions of Argentina and in one isolate from Paraguay. The isolates from Brazil analyzed here presented exclusive patterns (only found in a particular region). The number of exclusive patterns was high in all argentine regions: northeast 78%, center 81%, and Buenos Aires 81%.

Immigration and health: the Italian National Focal Point experience.

This report is a description of the situation of migrant populations in Italy. It was written by a committed team of experts from public institutions, non-governmental organisations (NGO) and volunteer associations that for three years have been part of the Italian National Focal Point (NFP) within the European Project "AIDS & Mobility", which is being co-ordinated by the Netherlands Institute for Health Promotion and Disease Prevention (the Netherlands) and financed by the European Commission DG/V. This year the Italian National Focal Point, co-ordinated by the Telefono Verde AIDS of the Istituto Superiore di Sanità, has produced a second report (the first one was published in 1998) on health issues related to migrant populations. Besides providing an updated picture on the presence of foreigners in Italy, such report illustrates the present legislative situation within the sanitary area and some interventions regarding prevention, treatment and rehabilitation provided by each structure in the Italian NFP to foreign citizens. The initiatives carried out during the year 1999 by public institutions, NGO and volunteer associations are also reported in order to detect the psycho-social-sanitary needs of immigrants and target prevention programmes to their particular and specific needs.

[Resistance and multiresistance to antitubercular drugs in Argentina and in other Latin American countries]. / La resistencia y multirresistencia a los medicamentos antituberculosos en la Argentina y en otros países de América Latina.

Resistance of Mycobacterium tuberculosis to both isoniazid (INH) and rifampicin (RPM), the most important antituberculosis drugs, with or without simultaneous resistance to other drugs, is known as multidrug resistance (MDR). It is the main obstacle to attain the cure of patients by the specific treatment, and a threat to the tuberculosis control. Between 1994 and 1997, several Latin American countries undertook countrywide surveys or surveillance programs to determine their primary and acquired drug resistance prevalence rates. These studies followed the WHO/International Union Against Tuberculosis and Lung Diseases (IUATLD) guidelines. Percentages of not previously treated patients with tuberculosis due to MDR strains ranged from null or very small (Uruguay, Cuba, Chile) to 4% or higher (Dominican Republic, Argentina). In Argentina, a remarkable correlation between MDR tuberculosis, AIDS and the assistance in urban reference hospitals for infections diseases was observed. Coincidentally with the survey, nosocomial spread of HIV-related MDR tuberculosis occurred in two of these hospitals situated in Buenos Aires and Rosario. But, at the same time, an alarming emergence of MDR was evidenced among non HIV-infected patients with history of previous antituberculosis treatment. Directly observed treatment (DOT) should be increasingly applied, and drug supply guaranteed. Treatment as well as microscopy services for diagnosis and follow up of patients, should be decentralized from the big specialized hospitals in urban areas to the peripheral health centers, in order to make easier for the patients to attend regularly and receive their medications. These strategies will contribute to increase cure rates and to reduce the tuberculosis transmission.

[Etiology of acute lower respiratory tract infections among children younger than 5 years old in Santa Fe]. / Etiología de las infecciones agudas del tracto respiratório inferior en niños menores de cinco años en Santa Fe.

The etiology of acute lower respiratory tract infections (ARI) and nasopharyngeal bacterial carriage in children less than 5 years old living in Santa Fe city, Argentina, was studied. A total of 518 children were included in the study: 450 suffering from ARI and 68 asymptomatic children. Blood samples, pleural effusions and nasopharyngeal secretions (NS) were obtained from children for bacterial isolations. NS was also used for fluorescent antibody techniques, and serum samples were employed for detecting IgM anti Chlamydia trachomatis. A bacterial pathogen was isolated from blood in 6.2% (14/224) of the children with ARI. A total of 11 Streptococcus pneumoniae (five of them oxacillin resistant), two Haemophilus influenzae and one Staphylococcus aureus strains were isolated. The most frequently detected pathogen in the ARI group was respiratory syncytial virus (RSV). It was found in 23.3% (105/450) of the children with ARI. Among children with risk of Chlamydia trachomatis infection, 24% presented high titters of specific IgM antibodies. Main bacteria carried in NS in the ARI group were H.influenzae (31.6%) and S. pneumoniae (23.4%) while viridans streptococci (26.5%), H.influenzae (23.5%) and Moraxella catarrhalis (22.1%) were more frequently isolated from controls. The most common pneumococcal types were 14 and 7 and the main type of H.influenzae was b biotype I. During the period of this study, the susceptibility of the pneumococcal isolates to oxacillin decreased from 60% to 50.8%, and the H.influenzae susceptibility to ampicillin fell from 92.3% to 79%. All the H.influenzae type b isolations were susceptible to ampicillin.

[Diagnosis of tuberculosis in children in provinces of Argentina]. / Diagnóstico de la tuberculosis infantil en provincias de la Argentina.

Confirming the diagnosis of pediatric tuberculosis is cumbersome, due to the clinical features (generally paucibacillary forms) of the disease. This national study was undertaken in order to establish: the features of childhood tuberculosis at the time of diagnosis, the criteria on which the pediatricians based the diagnosis, the bacteriologic contribution to the diagnosis and the quality of notifications to the National Programme. Medical and laboratory records were reviewed for children under 15 years of age who were diagnosed with tuberculosis disease or primary infection during 1995. The study included children cared for at health centres from Argentinean provinces (capital city excluded) where pediatricians accepted to participate. Four hundred cases (17% of childhood notifications to the National Programme) and 81 primary infections were studied. The percentage of children studied by means of chest radiology, presence of symptoms, Mantoux test, case contact investigation and bacteriology were 95.3%, 79.6%, 90.1%, 92.7% and 35% for pulmonary cases, respectively, and 87.7%, 100%, 87.7%, 85.9% and 78.9% for extra-pulmonary cases, respectively. Of the evaluated pulmonary cases, 99.1% had abnormal x-rays, 79.0% had a tuberculin test > or = 10 mm, 79.8% had symptoms and 80.2% had a history of close contact. All extra-pulmonary patients had symptoms at the time of diagnosis; 63.0% had abnormal chest radiograph at diagnosis. Bacteriologic confirmation was achieved in 10.7% of the cases (20.8% and 40.0% of the investigated pulmonary and extra-pulmonary cases, respectively). This study would indicate that the diagnosis is made at relatively early stages of the disease. In general, recommendations of the Argentine Society of Pediatrics were followed. A low rate of bacteriological proof of diagnosis was observed, probably due to the scarce bacteriologic investigation and the low yield achieved in culturing pulmonary specimens. The study found under-register of cases and lack of precision in the information reaching the National Programme.

[Utility of gas chromatography for the identification of mycobacterial species]. / Utilidad de la cromatografia gaseosa para la identificación de micobacterias.

The human immunodeficiency virus (HIV) epidemic has altered the epidemiological profile of tuberculosis in both industrialized and developing countries. Serious diseases caused by mycobacteria other than Mycobacterium tuberculosis, mostly belonging to the M. avium-intracellulare complex (MAC), have become very common in association with severe immunosuppression. The increase in mycobacterial disease complexity has stimulated the development of more rapid and efficient methods for diagnosis. In the present study, we investigated and assessed the suitability of a gas-liquid chromatography technique for diagnosis of clinically important mycobacteria in Argentina. An identification scheme was developed from the results obtained in a previous study where we characterized the cellular fatty acids and the mycolic acid cleavage products from most frequent species in Argentina. Of 183 isolates tested, 69% were correctly identified to species level and 5% were incorrectly classified. If we only take into account the isolates that could be identified, 93% were correctly identified. Although all of the isolates of M. tuberculosis were correctly identified, four isolates of MAC incorrectly matched by M. tuberculosis. Gas chromatography provides a rapid technique of highly predictive value for mycobacteria identification; it could be used in reference laboratories as a rapid presumptive identification until the biochemical tests are completed.

[Characterization of fatty acids and mycolic acid degradation products in mycobacterial species of major incidence in Argentina]. / Caracterización de ácidos grasos y productos de degradación de ácidos micólicos en especies micobacterianas de mayor incidenica en Argentina.

The human immunodeficiency virus (HIV) epidemic has altered the epidemiological profile of tuberculosis in both industrialized and developing countries. Serious diseases caused by mycobacteria other that M. tuberculosis, mostly belonging to the M. avium-intracellulare complex (MAC), have become very common in association with severe immunosuppression. The increase in mycobacterial disease complexity has stimulated the development of more rapid and efficient methods of diagnosis. In the present study we characterized the cellular fatty acids and the mycolic acid cleavage product from most frequent mycobacteria species in Argentina using gas chromatography in order to develop a rapid technique for their identification. Fatty acids and mycolic acids extracted from saponified mycobacterial cells were examined as methyl esters by capillary has chromatography. The major constituent fatty acids in all species, with the exception of M. smegmatis, were octadecenoic (18:1) and hexadecanoic (16:1) acids. The fatty acids and mycolic acid cleavage product profiles from the studied species were quantitatively but not qualitatively different. Tuberculostearic acid was found in all species. Significantly different amounts of some fatty acids (p < 0.01) were observed among clinical isolates of M. tuberculosis, M. bovis and MAC. Traces of 2-eicosanol were detected in the M. tuberculosis H37Rv strain. Although a limited number of strains and species were tested, preliminary results indicate that this method could be used to characterize mycobacterial cultures.

Serodiagnosis of pulmonary tuberculosis in Argentina by enzyme-linked immunosorbent assay (ELISA) of IgG antibody to Mycobacterium tuberculosis antigen 5 and tuberculin purified protein derivative.

IgG antibody to Mycobacterium tuberculosis antigen 5 and tuberculin purified protein derivative (PPD) was measured, by enzyme-linked immunosorbent assay (ELISA), in serum samples from 86 patients with active pulmonary tuberculosis and 91 non-tuberculous control subjects from Santa Fé, Argentina. The geometric mean titre for the tuberculosis patients was 74.6 with antigen 5 and 99.5 with PPD. In 91 control subjects the geometric mean titres were 3.6 and 15.6 respectively. Titres were not related to tuberculin reactor status or prior BCG vaccination. At a serum dilution end-point of 1:40, ELISA with antigen 5 had a sensitivity of 81.4% and a specificity of 93.4% for tuberculosis. At 1:40, ELISA with PPD showed a sensitivity of 82.6% and a specificity of 54.9% for tuberculosis. Applied at a serum dilution of 1:40 to a hypothetical model population with a tuberculosis prevalence of 2%, ELISA using antigen 5 would correctly classify 93.2% of persons and ELISA with PPD, 55.5%. At a dilution of 1:80, accuracy is increased to 99.3% with antigen 5 and 83.3% with PPD, but sensitivity decreases to 64.0% with antigen 5 and 72.1% with PPD. Thus, antigen 5 is more accurate than PPD for the diagnosis of tuberculosis using ELISA.

Usefulness of spoligotyping in molecular epidemiology of Mycobacterium bovis-related infections in South America.

Two hundred twenty-four Mycobacterium bovis isolates, mainly from South American countries, were typed by spoligotyping, and 41 different spoligotypes were identified. A total of 202 M. bovis isolates (90%) were grouped into 19 different clusters. The largest cluster contained 96 isolates (42.8%) on the basis of the most frequently observed spoligotype, spoligotype 34. Nineteen M. bovis isolates from humans in Argentina had spoligotypes and polymorphic GC-rich repetitive sequence (PGRS) types that represented the most common types found among isolates from cattle. All five isolates from Uruguay and three of the six isolates from Paraguay had spoligotypes that were also detected for isolates from Argentina. The spoligotypes of isolates from Brazil, Costa Rica, and Mexico and of some of the isolates from Paraguay could not be found in Argentina. A total of 154 M. bovis isolates were selected in order to compare the discriminative power of spoligotyping and restriction fragment length polymorphism (RFLP) analysis with direct repeat (DR) and PGRS probes. By spoligotyping, 31 different types were found, while AluI-digested DR probe-associated RFLP analysis identified 42 types, and RFLP analysis with the PGRS probe also detected 42 types; these were partly independent of the DR types. By combining the results obtained by spoligotyping and by RFLP analysis with the DR and PGRS probes, 88 different types were obtained. Although the differentiation of M. bovis by spoligotyping was less discriminatory than differentiation by RFLP analysis with the DR and PGRS probes, spoligotyping is easier to perform and its results are easier to interpret. Therefore, for the purpose of typing of M. bovis isolates, spoligotyping could be performed first and the isolates could be grouped into clusters and then analyzed by RFLP analysis with the DR and PGRS probes.

Caracterización de ácidos grasos y productos de degradación de ácidos micólicos en especies micobacterianas de mayor incidencia en Argentina / Characterization of fatty acids and mycolic acid cleavage products in mycobacteria of major incidence in Argentina

El virus de la inmunodeficiencia humana (HIV) causa un profundo impacto sobre el problema de la tuberculosis tanto en los países industrializados como en los en vías de desarrollo. Enfermedades graves causadas por micobacterias no tuberculosas, la mayoría correspondiente al complejo Mycobacterium avium-intracellulare (MAC), se han vuelto muy comunes en asociación con la inmunosupresión severa. El aumento de la complejidad de las enfermedades micobacterianas ha estimulado el desarrollo de métodos de diagnóstico más rápidos y eficientes. En el presente estudio se caracterizaron los ácidos grasos y los productos de degradación de los ácidos micólicos celulares de las especies micobacterianas más frecuentes en la Argentina empleando cromatografía gaseosa (CG), para luego poder desarrollar una técnica rápida de identificación de especies. Los ácidos grasos y los ácidos micólicos de las células micobacterianas saponificadas fueron analizados como ésteres metílicos por CG capilar. Los principales ácidos grasos detectados en todas las especies estudiadas, con excepción de M. smegmatis, fueron los ácidos octadecenoico (18:1) y hexadecanoico (16:0). Los perfiles cromatográficos presentaron diferencias cuantitativas y no cualitativas entre las distintas especies. El ácido tuberculoesteárico se detectó en todas las micobacteias analizadas. Se observaron diferencias significativas (p<0,01) en las medias de las cantidades relativas de algunos ácidos grasos entre aislamientos clínicos de M. tuberculosis, M. bovis y MAC. Se detectaron trazas de 2-elcosanol en cepas de M. tuberculosis H37Rv. Aunque se estudió un número limitado de cepas y de especies, los resultados preliminares indican que este método podría ser usado para caracterizar cultivos micobacterianos