Molecular typing of Mycobacterium bovis isolates in Argentina: first description of a person-to-person transmission case.

Bovine tuberculosis is caused by Mycobacterium bovis, a mycobacterium highly similar to M. tuberculosis that belongs to the M. tuberculosis complex. The main host of M. bovis is cattle but it also affects many other mammalians including humans. Tuberculosis in humans caused by either M. bovis or M. tuberculosis is clinically hard to distinguish. During 2004-2005, samples from 448 patients with diagnosis of TB were collected from different regions of Argentina. The PRA technique identified 400 isolates with representative patterns of mycobacterium. The predominant ones were the M. tuberculosis complex, the M. avium-M. intracellulare complex and M. gordonae. Samples with M. tuberculosis complex PRA restriction profiles were analyzed with a multiplex PCR to differentiate between M. tuberculosis and M. bovis. Multiplex PCR identified nine M. bovis. The results allowed the possibility to establish that 2% of pulmonary tuberculosis was due to M. bovis. Isolates of M. bovis from humans were examined using spoligotyping. These isolates presented five different spoligotypes. The main spoligotype was also the most frequently one found in cattle. The remaining human spoligotypes (grouped in clusters) are occasionally found in cattle. Variable number tandem repeat (VNTR) analysis identified five different patterns. By combining the results of spoligotyping and VNTR analysis, we were able to differentiate seven M. bovis isolates. The remaining two M. bovis samples showed the same spoligotype and VNTR profile and belonged to household contacts. An MDR-M. bovis was isolated from the samples of these household contacts. The identification of two epidemiologically linked cases of human M. bovis infection suggests person-to-person transmission of an MDR-M. bovis.

[In-vitro activity of two hybrid synthetic peptides having antimicrobial activity against mycobacteria]. / Sensibilidad in vitro de micobacterias a dos péptidos sintéticos híbridos con actividad antimicrobiana.

The increase in both Mycobacterium tuberculosis human clinical isolates resistant to the essential drugs and cases of disseminated micobacteriosis due to Mycobacterium avium Complex, underlines the need to investigate new antimicobacterial agents. The antimicrobial peptides are a new group of active antibiotics with a particular mechanism of action. Some of them, like cecropin and melittin, isolated from insects, have demonstrated good in vitro activity against Gram-positive and Gram-negative bacteria. Synthetic hybrids of those peptides have been more active than individual peptides. In this study, the in vitro activity of two hybrid synthetic peptides from melittin and cecropin against M. tuberculosis, M. avium Complex, Mycobacterium fortuitum and Mycobacterium smegmatis has been evaluated. The minimal inhibitory concentration was determined by using the broth macrodilution technique. The minimal bactericide concentration in Lowenstein Jensen medium was then obtained. The peptides studied were active, in vitro, against M. smegmatis, but they did not show any activity against the other mycobacteria analyzed.

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.

Associations between leptospiral infection and seropositivity in rodents and environmental characteristics in Argentina.

Our objective was to look for associations between leptospiral infection in rodents and selected environmental and rodent characteristics in Santa Fe, Argentina. Rodents (n = 214) were trapped alive from January 1998 to December 1999 in three environmental settings. Kidneys from 118 rodents were cultured and serum samples from 201 were processed by enzyme-linked immunosorbent assay (ELISA). Logistic regression was performed with ELISA seropositivity as the dependent variable and rodent characteristics were offered as independent variables. Overall prevalence of positive ELISA reactions was 42% (84/201). In urban areas, leptospiral isolations belonged to the Ballum serogroup; in natural corridors, they belonged to the Icterohaemorragiae serogroup. M. musculus (house mouse) was the most-frequently captured species and the predominant one in urban areas. Most isolates and seropositivity results were obtained on this species. Adults and subadults had higher seroprevalences than juvenile rodents. Oligoryzomys flavescens had higher seroprevalence than Akodon azarae, Mus musculus, Rattus rattus and Rattus norvegicus.

[Outbreak of human leptospirosis after a flood in Reconquista, Santa Fe, 1998]. / Brote de leptospirosis humana luego de una inundación en Reconquista (Santa Fe), 1998.

Since April of 1998 a high number of leptospirosis cases were detected, coming from the area of Reconquista Central Hospital in Santa Fe province. Since January of that year a notable increase in rainfall and river levels was observed causing inundation. As screening test, macroscopic agglutination (MAT) using 10 serotypes of L. interrogans. Among the 122 patients studied 71 were TR positive and 52 were also ELISA positive, leptospirosis diagnosis being confirmed in 40 of them. Five infecting serogroups were identified: Icterohaemorrhagiae (7/40), Ballum (5/40), Sejroe (3/40), Pomona (3/40) and Canicola (2/40). In the remaining cases (20/40), co-agglutinins were found at the same titer against two or more serotypes of leptospires. Infection prevalence was higher in men and productive age (21 to 40 years). The clinical symptoms more frequently observed were headache, fever and myalgias. All cases occurred after the rains and in the period when the area was flooded. Their clinical presentation, time distribution, geographical localization and high frequency of contact with the risk factor inundation could indicate that, independently of search activities, there was an outbreak.

Brote de leptospirosis humana luego de una inundación en Reconquista (Santa Fe), 1998 / Outbreak of human leptospirosis after a flood in Reconquista, Santa Fe, 1998

Since April of 1998 a high number of leptospirosis cases were detected, coming from the area of Reconquista Central Hospital in Santa Fe province. Since January of that year a notable increase in rainfall and river levels was observed causing inundation. As screening test, macroscopic agglutination (MAT) using 10 serotypes of L. interrogans. Among the 122 patients studied 71 were TR positive and 52 were also ELISA positive, leptospirosis diagnosis being confirmed in 40 of them. Five infecting serogroups were identified: Icterohaemorrhagiae (7/40), Ballum (5/40), Sejroe (3/40), Pomona (3/40) and Canicola (2/40). In the remaining cases (20/40), co-agglutinins were found at the same titer against two or more serotypes of leptospires. Infection prevalence was higher in men and productive age (21 to 40 years). The clinical symptoms more frequently observed were headache, fever and myalgias. All cases occurred after the rains and in the period when the area was flooded. Their clinical presentation, time distribution, geographical localization and high frequency of contact with the risk factor inundation could indicate that, independently of search activities, there was an outbreak.

Evaluation of the diagnostic value of measuring IgG, IgM and IgA antibodies to the recombinant 16-kilodalton antigen of mycobacterium tuberculosis in childhood tuberculosis.

OBJECTIVE: To evaluate the usefulness of the recombinant 16-kDa antigen (re-Ag16) of Mycobacterium tuberculosis in the serodiagnosis of tuberculosis (TB) in children. MATERIALS: Seventy-four children with active TB, 49 apparently healthy contact children and 149 children suffering from non-mycobacterial diseases were evaluated. Detection of anti 16-kDa antigen IgG, IgM and IgA was performed by enzyme-immunoassay. RESULTS: An increased mean antibody response to re-Ag16 was observed in contact children compared with non-mycobacterial disease patients (IgG assay: 89.1 enzymatic units [eu] vs. 40.8 eu; IgM assay: 64.7 eu vs. 38.1 eu; IgA assay: 138.2 eu vs. 78.2 eu for contact children and non-mycobacterial disease patients, respectively), indicating that anti-16-kDa antibodies could be elevated in response to infections even without clinically apparent TB. Setting the specificity as the 95th percentile of the contact group's ELISA units, the sensitivity of the IgG, IgA and IgM assays were 34%, 19% and 3% respectively; combining results of the IgG and IgA assays led to 43% positivity in children with active TB. CONCLUSION: The detection of anti 16-kDa IgG and IgA may be useful as a complementary technique for the diagnosis of childhood TB. Recognition of this antigen seems to be heterogeneous; combining responses against other antigens may be a good strategy to improve the performance of this assay.

Development and validation of an ELISA for the detection of leptospire-specific antibodies in rodents.

An enzyme-linked immunosorbent assay (ELISA) for the detection of IgG antibodies in rodents was developed and validated with the microscopic agglutination test (MAT) and leptospiral cultures. Sonicated antigen from cultures of serovars tarassovi and pyrogenes was used. As conjugate, a combination of anti-rat and anti-hamster IgG labeled with peroxidase was used. The optimal cut-off point was determined by plotting the sensitivity and specificity for various cut-off point values by means of receiver operating characteristic (ROC) curve. Concordance between ELISA and each of the MAT titers was measured by kappa (kappa). Proportions of positive results were compared by means of McNemar's test. Total 214 rodents were trapped, but only 117 could be processed by the three techniques (culture, ELISA, MAT; 1:20, 1:40, 1:50) and used for statistical analysis. Although, MAT titers in rodents infected with the serogroup Ballum tended to be lower than those infected with the serogroup Icterohaemorrhagiae, all (20/20) were ELISA-positive and almost all (19/20) were MAT-positive.The percentage of positive results obtained by ELISA, 47.0% exceeded significantly the 40.2% obtained by MAT (1:50). Difference between ELISA and MAT (1:40) was not significant and no differences were observed between ELISA and MAT (1:20). Agreement, specificity, sensitivity and the consequent area under the ROC curve between ELISA and MAT were higher as MAT cut-off points were lowered, being optimal at 1:20. The fact that differences between ELISA and MAT were significant at 1:50, but not at 1:40 or 1:20, supports the suggestion that lower MAT titers should be considered positive in rodents. The ELISA developed to detect leptospire-specific antibodies had optimal sensitivity and specificity in relation to MAT and it is concluded that it may constitute a very useful indicator for epidemiological purposes of past or present leptospiral infection in rodents.

Multicentered study of viral acute lower respiratory infections in children from four cities of Argentina, 1993-1994.

This study describes the first multicentered study of acute lower respiratory infection viral etiology in young children from four different geographical areas of Argentina. A total of 1,278 children under 5 years of age, hospitalized in primary care centers from Buenos Aires, Córdoba, Santa Fé and Mar del Plata cities during a 2-year period were studied (1993-1994). Nasopharyngeal aspirates were investigated for respiratory syncytial virus (RSV), adenovirus, parainfluenza, and influenza A and B viruses by indirect immunofluorescence. Out of the patients studied, 946 (74%) were under 1 year of age. Viruses were detected in 399 patients (32%). RSV was observed in 25.3% of the samples, representing 78.2% of all viral positive cases. Adenoviruses were detected in 2.5% of the cases, parainfluenza in 2.2%, influenza A in 2.1%, and influenza B in 0.2%. Compared with other viruses, the higher RSV frequency was statistically significant (P < 0.000). Most RSV cases were detected between May and September with a significant peak in July (P < 0.000). Pneumonia was observed in 46% of the patients, bronchiolitis in 41% and other entities in 13%. The case fatality rate observed during the 2 year study was 0.73%. Most of the above respiratory viruses were detected in the four cities, however, the frequency of RSV and influenza were different in the southern city.

[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.

Respiratory syncytial virus: changes in prevalence of subgroups A and B among Argentinian children, 1990-1996.

The frequency of respiratory syncytial virus (RSV) and the distribution of subgroups A and B strains during 7 consecutive years (1990-1996) were examined in two cities of Argentina. Nasopharyngeal aspirates from 1,304 children less than 2 years of age hospitalized with acute lower respiratory infection were studied by indirect immunofluorescence. RSV was detected in 352 cases (26.9%), and the peak activity was observed in midwinter. Subgroup characterization was performed with two monoclonal antibodies against the F protein on nasopharyngeal aspirate smears. Of 195 samples, 174 (89.2%) were identified as subgroup A strains and 21 (10.8%) as subgroup B. Both strains cocirculated during 5 of 7 years studied with subgroup A predominating. Subgroup A occurred at least 8 times as often in all years except for 1994-1995. Children infected by subgroup A were younger than those infected by subgroup B (P < 0.05). The association of subgroup A infection with bronchiolitis and subgroup B with pneumonia was statistically significant (P < 0.03).

Clinical and epidemiologic characteristics of respiratory syncytial virus subgroups A and B infections in Santa Fe, Argentina.

Respiratory Syncytial Virus (RSV) has two major antigenic groups, A and B. The implications of these variants in the epidemiology and pathogenesis of RSV infection are not well defined. This study was undertaken to compare the two RSV subgroups in patients admitted to hospital. Clinical and epidemiologic features of RSV subgroups in children under 30 months of age with proven RSV acute lower respiratory infections were examined during 4 winters from 1993 to 1996 in Santa Fe, Argentina. RSV typing was carried out with monoclonal antibodies in nasopharyngeal cells by indirect immunofluorescence. Of the 177 RSV positive nasopharyngeal aspirates obtained from 1993 to 1996, 85 (48%) were available for typing. Seventy-three (85.9%) specimens were identified as Subgroup A and 12 (14.1%) as Subgroup B. Except in 1993, in which only Subgroup A was detected, both variants circulated throughout the epidemic season. Subgroup A infections produced more severe disease than Subgroup B infections, as assessed by the length of the hospital stay and the use of respiratory support. This difference was age related, being evident in infants 0-6 months old. Patients with Subgroup B infections were also significantly less frequently breast-fed (95% vs. 75% for A and B subgroups, respectively; P = 0.04). It is concluded that the severity of disease in Argentinian patients admitted with acute RSV infections may be associated with Subgroup A strains as determined by a serogrouping method.

[Report on a leptospirosis outbreak in the city of Santa Fe, Argentina, March-April 1998]. / Descripción de un brote de leptospirosis en la ciudad de Santa Fe, Argentina, marzo-abril de 1998.

In March-April 1998 in a neighborhood in the city of Santa Fe, Argentina, there was an outbreak of an acute disease characterized by fever, headaches, and intense myalgias. This article presents the studies surrounding this outbreak and the attempts to identify the source and the mode of transmission. The epidemiological, serological, and clinical findings indicated that the causative agent was Leptospira interrogans. As a screening test, macroscopic agglutination with heat-resistant antigen was applied, followed by the ELISA test, and, as a confirmatory test, microscopic agglutination for 10 serotypes of L. interrogans. The study covered 32 persons, 8 dogs, and 8 water samples. Among the 32 persons, 12 cases were confirmed, 2 were suspicious, and 18 were negative. Six dogs were found to be infected, and motile spirochetes were found in the water samples. The human sera reacted with the ballum, canicola, icterohaemorrhagiae, and pyrogenes serotypes; the canine sera reacted with the ballum, canicola, and pomona serotypes. The coagglutination found in all the confirmed cases indicates that they were acute cases of leptospirosis, but it was impossible to identify the causal serotype. Except for the index case, the disease was not recognized clinically. Several facts suggest that the outbreak was caused by rain that had flooded the study area. The results of this study emphasize the need for active surveillance of leptospirosis when there are floods and other natural disasters.

[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.

[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.