Exploring the "Latin American Mediterranean" family and the RDRio lineage in Mycobacterium tuberculosis isolates from Paraguay, Argentina and Venezuela.

BACKGROUND: The Latin American & Mediterranean (LAM) spoligotype family is one of the most successful genotype of Mycobacterium tuberculosis worldwide and particularly prevalent in South-America. Within this family, a sublineage named Region of Difference Rio (RDRio) was reported initially in Brazil and is characterized by a genomic deletion of about 26.3 kb. This lineage seems to show a specific adaptation to the Euro-Latin American population. In this context, we sought to evaluate the LAM family and the presence of the RDRio genotype in samples from three Latin American countries including Paraguay, Venezuela and Argentina. To detect LAM strains reliably we applied a typing scheme using spoligotyping, 12 loci MIRU-VNTR, the Ag85C103 SNP and the regions of difference RDRio and RD174. IS6110-RFLP results were also used when available. RESULTS: Genotyping of 413 M. tuberculosis isolates from three Latin-American countries detected LAM (46%) and the ill-defined T clade (16%) as the most frequent families. The highest clustering rate was detected in the sample population from the city of Caracas in Venezuela. We observed considerable differences in the presence of the RDRio lineage, with high frequency in Caracas-Venezuela (55%) and low frequency in Buenos Aires-Argentina (11%) and Paraguay (10%). The molecular markers (RD174, Ag85C103, MIRU02-MIRU40 signature) of the RDRio lineage were essentially confirmed. For the LAM family, the most polymorphic loci were MIRU40, MIRU31, MIRU10, MIRU26, MIRU16 and the least polymorphic MIRU24, MIRU20, MIRU04, MIRU23. CONCLUSIONS: Our results suggest a differential adaptation of LAM-sublineages in neighboring populations and that RDRio strains spread regionally with different rates of distribution. The Ag85C SNP and RDs (RD174, RDRio) tested in this study can in fact facilitate molecular epidemiological studies of LAM strains in endemic settings and low-income countries.

Mycobacterium tuberculosis strains of the Beijing genotype are rarely observed in tuberculosis patients in South America.

The frequency of the Beijing genotype of Mycobacterium tuberculosis as a cause of tuberculosis (TB) in South America was determined by analyzing genotypes of strains isolated from patients that had been diagnosed with the disease between 1997 and 2003 in seven countries of the subcontinent. In total, 19 of the 1,202 (1.6%) TB cases carried Beijing isolates, including 11 of the 185 patients from Peru (5.9%), five of the 512 patients from Argentina (1.0%), two of the 252 Brazilian cases (0.8%), one of the 166 patients from Paraguay (0.6%) and none of the samples obtained from Chile (35), Colombia (36) and Ecuador (16). Except for two patients that were East Asian immigrants, all cases with Beijing strains were native South Americans. No association was found between carrying a strain with the Beijing genotype and having drug or multi-drug resistant disease. Our data show that presently transmission of M. tuberculosis strains of the Beijing genotype is not frequent in Latin America. In addition, the lack of association of drug resistant TB and infection with M. tuberculosis of the Beijing genotype observed presently demands efforts to define better the contribution of the virulence and lack of response to treatment to the growing spread of Beijing strains observed in other parts of the world.

Mycobacterium tuberculosis strains of the Beijing genotype are rarely observed in tuberculosis patients in South America

The frequency of the Beijing genotype of Mycobacterium tuberculosis as a cause of tuberculosis (TB) in South America was determined by analyzing genotypes of strains isolated from patients that had been diagnosed with the disease between 1997 and 2003 in seven countries of the subcontinent. In total, 19 of the 1,202 (1.6 percent) TB cases carried Beijing isolates, including 11 of the 185 patients from Peru (5.9 percent), five of the 512 patients from Argentina (1.0 percent), two of the 252 Brazilian cases (0.8 percent), one of the 166 patients from Paraguay (0.6 percent) and none of the samples obtained from Chile (35), Colombia (36) and Ecuador (16). Except for two patients that were East Asian immigrants, all cases with Beijing strains were native South Americans. No association was found between carrying a strain with the Beijing genotype and having drug or multi-drug resistant disease. Our data show that presently transmission of M. tuberculosis strains of the Beijing genotype is not frequent in Latin America. In addition, the lack of association of drug resistant TB and infection with M. tuberculosis of the Beijing genotype observed presently demands efforts to define better the contribution of the virulence and lack of response to treatment to the growing spread of Beijing strains observed in other parts of the world.

First insight into Mycobacterium tuberculosis genetic diversity in Paraguay.

BACKGROUND: We present a picture of the biodiversity of Mycobacterium tuberculosis in Paraguay, an inland South American country harboring 5 million inhabitants with a tuberculosis notification rate of 38/100,000. RESULTS: A total of 220 strains collected throughout the country in 2003 were classified by spoligotyping into 79 different patterns. Spoligopatterns of 173 strains matched 51 shared international types (SITs) already present in an updated version of SpolDB4, the global spoligotype database at Pasteur Institute, Guadeloupe. Our study contributed to the database 13 new SITs and 15 orphan spoligopatterns. Frequencies of major M. tuberculosis spoligotype lineages in our sample were as follows: Latin-American & Mediterranean (LAM) 52.3%, Haarlem 18.2%, S clade 9.5%, T superfamily 8.6%, X clade 0.9% and Beijing clade 0.5%. Concordant clustering by IS6110 restriction fragment length polymorphism (RFLP) and spoligotyping identified transmission in specific settings such as the Tacumbu jail in Asuncion and aboriginal communities in the Chaco. LAM genotypes were ubiquitous and predominated among both RFLP clusters and new patterns, suggesting ongoing transmission and adaptative evolution in Paraguay. We describe a new and successfully evolving clone of the Haarlem 3 sub-lineage, SIT2643, which is thus far restricted to Paraguay. We confirmed its clonality by RFLP and mycobacterial interspersed repetitive unit (MIRU) typing; we named it "Tacumbu" after the jail where it was found to be spreading. One-fifth of the spoligopatterns in our study are rarely or never seen outside Paraguay and one-tenth do not fit within any of the major phylogenetic clades in SpolDB4. CONCLUSION: Lineages currently thriving in Paraguay may reflect local host-pathogen adaptation of strains introduced during past migrations from Europe.

[Implementation and evaluation of a locally sustainable system of prenatal diagnosis to detect cases of congenital Chagas disease in endemic areas of Paraguay]. / Implementación y evaluación de un sistema localmente sustentable de diagnostico prenatal que permite detectar casos de transmisión congénita de la enfermedad de Chagas en zonas endémicas del Paraguay.

A locally sustainable system of prenatal screening of Trypanosoma cruzi infection has been implemented in rural health care centers of endemic areas in Paraguay A total of 61.091 women from Paraguari and Cordillera Departments were serologically evaluated, where 7.802 (12,7%) resulted to be anti-T. cruzi IgG positive. A total of 1,865 infants born to seropositive mothers were examined by parasitological techniques, such as direct microscopic observation and polymerase chain reaction, and serologically by ELISA, ELISA-SAPA and IFI. 104 infected babies were detected and treated with benznidazole. The recovery of babies born to seropositive mothers performing a single examination at the age of 6 months was significantly higher, as compared with the recommended method involving two examinations, both at birth and after 6 months of age. Although at 6 months of age in 7% of the infants maternal IgG was still detected. PCR was the most sensitive technique for early detection of T. cruzi infection in babies, but we do not recommend it use for diagnosis in high endemic areas, considering that for the screening of 815 babies, 2000 reactions were needed. We propose a strategy to detect congenital transmission of Chagas disease, based on a large-scale study, where the shortcomings of the different serological and parasitological techniques are discussed.

High frequency of MIC null haplotype (HLA-B48-MICA-del-MICB*0107 N) in the Angaite Amerindian community in Paraguay.

We describe a high frequency of the MIC null haplotype, HLA-B48-MICA-del-MICB*0107 N, in the Angaite Amerindian community in Paraguay. Of the 16 unrelated subjects, 9 (56.5%) had this haplotype. The structural analyses revealed this haplotype was similar to the previously reported Asian haplotype in that they had a large-scale deletion including the entire MICA gene and linked to MICB*0107 N and HLA-B*48. The novel recombination haplotype between this MIC null haplotype and HLA-B15, HLA-B15-MICA-del-MICB*0107 N, was also found in this community.

Diarrea por Rotavirus: Detencion del Genoma Viral en heces por tecnicas Moleculares / Annual Reprts 1997 / Diarrea by Rotavirus: Detection of Viral Genome in feces by Molecular techniques

A continuación presentamos los resultados preliminares sobre la detección de genomas de rotavirus en 27 de 121 (22 por ciento) muestras de heces de niños con edades compredidas desde 0 y 5 años con cuadros de diarrea aguda de más de dos días. Las muestras analizadas fueron comparadas con los estudios convencionales, frotis y/o coprocultivo, y en algunos casos se ha comparado la sensibilidad con la técnica inmunológica conocidas como aglutinación de partículas de latex. El producto de la extracción del acido ribonucleico (ARN) total presente en 40 microlitos de heces permitió detectar en seis muestras ARN viral por medio de una electroforesis en gel de agarosa al 3 por ciento y visualizado con bromuro de etidio. Las 6 muestras positiva fueron comparadas con patrones de referencia en geles de poliacrilamida teñidos con argéntica. Los resultados preliminares indican que las ténicas moleculares empleadas han sido fáciles, rápidas y sensibles teniendo como ventaja las bajas exigencias en cuanto a la conservación y trasporte de materiales

Detención de Adenovirus en cultivos celulares a partir de Aspirados Nasofaringeos / Annual Reports 1997 / Detection of Adenovirus in cell culture from Nesopharyngeal Aspirates

Se investigó la etiología viral (adenovirus repiratorio sincicial) por aislamiento de cultivo celular e inmunofluorescencia (IF) de los ANF. El aislamiento en cultivo celular permitió detectar 5 muestras positivas para adenovirus por su efecto citopático característico, formación de racimos en células Hep2. El tiempo en dar el ECP en la 5 muestras positivas varió de 1 a 10 días y los mismos fueron confirmados por IF en las células infectadas. Al comparar la sensibilidad del cultivo celular frente a la inmunofluorescencia directa del aspirado nasofaringeo, el primero fue mucho mayor, debido a que sólo 1 de las 5 muestras positivas para la adenovirus fue visualizado en forma directa por IF. No se detectó la precencia de virus respiratorio sincicial en las 12 muestras analizadas, muestra que el estudio bacteriológico permitió detectar tres casos de coinfección por S. pneumonidea y adenovirus. Estos estudios preliminares sobre cultivo y aislamiento de los agentes virales en casos de niños con IRA nos permitirá guardar congelados los aislados y proceder a la carecterización antigénica y genética de los mismos

Amplificación por PCR de una secuencia genomica de 272-pb que permite la diferenciación entre Trypanosoma cruzi y Trypanosoma rangeli / Annual reports 1994 / PCR amplification of a 272-bp genome sequence differentiates between Trypanosoma cruzi and Trypanosoma rangeli

La reacción en cadena de la polimerasa (PCR) fue empleada para amplificar un fragmento de 272-pb presente en el genoma del Trypanosoma cruzi, pero no detectado en el genoma del Trypanosoma rangeli. Para la amplificación por PCR se emplearon los oligonucleótidos TC4-1 y TC4-2 de 25-pb cada uno. Con el fin de aumentar la sensibilidad de la detección de los productos de amplificación en muestras biológicas, se preparó una sonda clonada en el vector pCR II y luego secuenciando el fragmento de 272-pb obtenido por amplificación del ADN de la cepa Tulahuen. Hemos confirmado por hibridación con la técnica Southern blot la no amplificación de ADN de T. rangeli con los cebadores TC4-1 y TC4-2. Estos resultados sugieren que esta técnica podría ser empleada para diferenciar infecciones por T. rangeli y T. cruzi