A Real-Time PCR Assay for Detection of Low Pneumocystis jirovecii Levels.

Here we report a new real-time PCR assay using SYBR Green which provides higher sensitivity for the specific detection of low levels of Pneumocystis jirovecii. To do so, two primer sets were designed, targeting the family of genes that code for the most abundant surface protein of Pneumocystis spp., namely the major surface glycoproteins (Msg), and the mitochondrial large subunit rRNA (mtLSUrRNA) multicopy gene, simultaneously detecting two regions. PCR methods are instrumental in detecting these low levels; however, current nested-PCR methods are time-consuming and complex. To validate our new real-time Msg-A/mtLSUrRNA PCR protocol, we compared it with nested-PCR based on the detection of Pneumocystis mitochondrial large subunit rRNA (mtLSUrRNA), one of the main targets used to detect this pathogen. All samples identified as positive by the nested-PCR method were found positive using our new real-time PCR protocol, which also detected P. jirovecii in three nasal aspirate samples that were negative for both rounds of nested-PCR. Furthermore, we read both rounds of the nested-PCR results for comparison and found that some samples with no PCR amplification, or with a feeble band in the first round, correlated with higher Ct values in our real-time Msg-A/mtLSUrRNA PCR. This finding demonstrates the ability of this new single-round protocol to detect low Pneumocystis levels. This new assay provides a valuable alternative for P. jirovecii detection, as it is both rapid and sensitive.

Optimized DNA extraction and purification method for characterization of bacterial and fungal communities in lung tissue samples.

Human lungs harbor a scarce microbial community, requiring to develop methods to enhance the recovery of nucleic acids from bacteria and fungi, leading to a more efficient analysis of the lung tissue microbiota. Here we describe five extraction protocols including pre-treatment, bead-beating and/or Phenol:Chloroform:Isoamyl alcohol steps, applied to lung tissue samples from autopsied individuals. The resulting total DNA yield and quality, bacterial and fungal DNA amount and the microbial community structure were analyzed by qPCR and Illumina sequencing of bacterial 16S rRNA and fungal ITS genes. Bioinformatic modeling revealed that a large part of microbiome from lung tissue is composed of microbial contaminants, although our controls clustered separately from biological samples. After removal of contaminant sequences, the effects of extraction protocols on the microbiota were assessed. The major differences among samples could be attributed to inter-individual variations rather than DNA extraction protocols. However, inclusion of the bead-beater and Phenol:Chloroform:Isoamyl alcohol steps resulted in changes in the relative abundance of some bacterial/fungal taxa. Furthermore, inclusion of a pre-treatment step increased microbial DNA concentration but not diversity and it may contribute to eliminate DNA fragments from dead microorganisms in lung tissue samples, making the microbial profile closer to the actual one.

Unusual prevalence of high-risk genotypes of human papillomavirus in a group of women with neoplastic lesions and cervical cancer from Central Mexico.

Human papillomavirus has been identified as a main etiological agent in the development of cervical cancer. HPV 16 and 18 have been reported the most widely prevalent genotypes worldwide. We conducted a study analyzing the prevalence of high and low risk human papillomavirus viral types in the Mexican state of Aguascalientes and neighboring cities in the states of Jalisco and Zacatecas in central Mexico. Specific viral genotype was determined by a PCR and hybridization-based detection test. The presence of 37 high- and low-risk HPV genotypes was evaluated in 883 female participants. Of these, 350 presented low-grade squamous intraepithelial lesions (LGSIL), 176 presented high-grade squamous intraepithelial lesions (HGSIL), 107 suffered from cervical cancer and 250 women with negative cytological report for intraepithelial lesion or malignancy (NILM). HPV 51 was the most prevalent genotype, followed by HPV 16: overall prevalence of HPV 51, including single infections and co-infections was 31.2% in women with LGSIL, whereas prevalence of HPV 16 was 25.1%. Among women with HGSIL, HPV 51 prevalence was 47.2% and HPV 16 was 30.1%. Prevalence of HPV 51 in women with cervical cancer was 49.5% and type 16 was 33.6%. Between single and co-infections, most co-infections were not associated with later stages of the disease, except 51/16 and some others. HPV 51 showed a significant correlation with the progression of the disease (OR = 10.81 for LGSIL, 19.38 for HGSIL and 22.95 for ICC), and when analyzing all other genotypes, five different groups depending on their correlation with all lesion grades were determined. According to our findings, HPV genotype 51 has a higher prevalence than HPV 16 and 18 in the Mexican state of Aguascalientes and neighboring cities in the states of Jalisco and Zacatecas in Central Mexico.

Factores culturales que influirán en el proceso de articulación entre la medicina tradicional y la medicina académica al nuevo modelo de salud en la ciudad de Cochabamba abril-octubre 2009 / Cultural factors that will influence the process of articulation between traditional medicine and academic medicine to the new health model in the city of Cochabamba April-October 2009

Son más de 30 años después de que se aplico la estrategia de la atención primaria de salud, innovando con la proyección de los sanitarios a la comunidad en busca de pacientes y con el fin de promocionar y fomentar la salud. Reforzando este modelo las políticas de salud están destinadas a la implementación aislada de diferentes programas para mejorar los indicadores de salud tan bajos que se tienen en nuestro país y todos estos esfuerzos estaban fundamentalmente dirigidas a disminuir la morbi mortalidad materna y las enfermedades inmuno- prevenibles, la salud sexual y reproductiva. Tenemos que reconocer por mas doloroso que sea que ni aun intentando vacunar casa por casa, ni con las brigadas móviles, ni con los bonos e incentivos económicos no se ha conseguido los efectos esperados de tan arduas estrategias e inversión económica y debemos preguntarnos entonces porque no funcionan estas estrategias y programas en nuestro país. Luego de algunas inquietudes de investigadores antropólogos, estudios etnográficos, sociales, aparentemente los pacientes esconden a sus hijos, mujeres y animales, en un acto de posible de protección de los agentes de salud, médicos porque los consideran de alguna forma riesgosos y no confiables. Finalmente se presume que su forma de entender salud y enfermedad estaban mas relacionadas a sus culturas y tradiciones, los comunarios durante mas de 500 años han estado siendo atendidos en la clandestinidad por sus médicos tradicionales, a los cuales contrariamente al sistema de salud académico son dignos de su confianza y la relación medico tradicional- paciente tiene aparentemente una buena comunicación, con una relación horizontal, que tiene costo económico accesible y que el médico tradicional realiza la mayor parte de su servicio en el domicilio de los pacientes, incluso los visita como un miembro más de su familia.