HIV-1 drug resistance surveillance using dried whole blood spots.

BACKGROUND: Field-friendly methods for HIV drug resistance (HIVDR) surveillance in resource-limited regions are urgently needed. Despite evidence that dried blood spots (DBS) are suitable for HIV serology, viral load and CD4+ T-cell enumeration, no study has evaluated DBS for HIVDR genotyping. We assessed the feasibility of genotyping HIV-1 from field-collected DBS stored under challenging environmental conditions. METHODS: We prospectively collected specimens from newly diagnosed, treatment-naive HIV-positive subjects in Mexico. Whole blood was spotted onto filter cards, air dried at ambient temperature and stored with desiccant at 37 degrees C and 85% humidity for 3 months. Genotypes obtained from DBS-extracted nucleic acids using an in-house nested reverse transcription-PCR method were compared to genotypes derived from matched plasma. RESULTS: Genotypes from 103 phylogenetically matched plasma and DBS were compared. In total, 90.1% of all DBS specimens could be amplified in either the region of HIV protease or the region of reverse transcriptase. Failure to amplify from DBS did not correlate with low plasma viral loads. Between paired specimens, the median nucleotide similarity was 99.95%. In the nine specimens with drug resistance mutations, all differences between pairs were partial discordances. Mutations identified in plasma were found in the majority of replicate DBS amplifications. CONCLUSION: The results suggest that genotypes obtained from DBS are equivalent to those from plasma. DBS are a promising public health tool for HIVDR surveillance of treatment-naive subjects, especially in regions where specimens might be exposed to severe environmental conditions and where logistical difficulties could prevent timely specimen processing. More studies are needed to validate DBS for patient monitoring.

Antigenic properties of phage displayed peptides comprising disulfide-bonded loop of the immunodominant region of HIV-1 gp41.

The HIV-1 envelope glycoprotein gp41 contains Cys(X)5Cys motif, which has been shown to elicit a strong antibody response in almost all HIV-1 infected individuals. This disulfide-bonded loop region is conserved in most retroviruses suggesting the existence of an essential function in virus life cycle. In this study, we displayed the peptides comprising 12 amino acids of the immunodominant loop of gp41 on the surface of M13 phage as N-terminal fusions to the minor coat protein pIII and major coat protein pVIII of the phage and demonstrated that cysteine loop containing peptide expressed on phage recognized 62 out of 63 (98.4%) HIV-1 positive samples but not control negative sera while phage bearing linear peptides detected 4-30% of HIV-1-positive sera. The main advantage of phage-based ELISA or other antibody detection-based diagnostic tests of HIV-infection to be used for massive screening in developing countries is the reproducible, simple, rapid and low-cost production of recombinant antigens.

Enhancing Latinas' participation in research on sexual assault: cultural considerations in the design and implementation of research in the Latino community.

To provide a more complete picture of the prevalence, impact, and treatment of sexual assault among Latinas, researchers must begin to develop and employ culturally competent recruitment and data-collection procedures that can facilitate participation and disclosure. In this article, we sought to synthesize recommendations from research with Latino populations, community-based and participatory action research, and research on sexual assault to provide a list of suggestions for conducting culturally competent research about sexual assault with Latino populations. Innovative examples from our own focus group study with Latinas are provided throughout.

Variable epitope libraries: new vaccine immunogens capable of inducing broad human immunodeficiency virus type 1-neutralizing antibody response.

The extreme antigenic variability of human immunodeficiency virus (HIV) leads to immune escape of the virus, representing a major challenge in the design of effective vaccine. We have developed a novel concept for immunogen construction based on introduction of massive mutations within the epitopes targeting antigenically variable pathogens and diseases. Previously, we showed that these immunogens carrying large combinatorial libraries of mutated epitope variants, termed as variable epitope libraries (VELs), induce potent, broad and long lasting CD8+IFN-γ+ T-cell response. Moreover, we demonstrated that these T cells recognize more than 50% of heavily mutated variants (5 out of 10 amino acid positions were mutated in each epitope variant) of HIV-1 gp120 V3 loop-derived cytotoxic T lymphocyte epitope (RGPGRAFVTI) in mice. The constructed VELs had complexities of 10000 and 12500 individual members, generated as plasmid DNA or as M13 phage display combinatorial libraries, respectively, and with structural composition RGPGXAXXXX or XGXGXAXVXI, where X is any of 20 natural amino acids. Here, we demonstrated that sera from mice immunized with these VELs are capable of neutralizing 5 out of 10 viral isolates from Tier 2 reference panel of subtype B envelope clones, including HIV-1 isolates which are known to be resistant to neutralization by several potent monoclonal antibodies, described previously. These data indicate the feasibility of the application of immunogens based on VEL concept as an alternative approach for the development of molecular vaccines against antigenically variable pathogens.

[Resistance to non-beta-lactam antibiotics in the clinical isolates of Streptococcus pneumoniae of children in Latin America. SIREVA II, 2000-2005]. / Resistencia a antibióticos no betalactámicos de aislamientos invasores de Streptococcus pneumoniae en niños latinoamericanos. SIREVA II, 2000-2005.

OBJECTIVE: To examine the development of resistance to erythromycin, chloramphenicol, trimethoprim-sulfamethoxazole (TMP-SMZ), and vancomycin of the invasive isolates of Streptococcus pneumoniae obtained from children in 10 Latin American/Caribbean countries during six years of surveillance. METHODS: Analysis of 8 993 isolates of S. pneumoniae recovered in 2000-2005 from children with invasive infections, who were less than 6 years of age, and from Argentina, Brazil, Chile, Colombia, Cuba, Dominican Republic, Mexico, Paraguay, Uruguay, or Venezuela. Antibiotic susceptibility was determined through the methods established and standardized by the SIREVA project. Multidrug resistance was defined as: resistance to three or more antibiotics of the same class; to the non-beta-lactams analyzed by this study; or, to the beta-lactams evaluated by a previous study, in which 37.8% of these isolates showed decreased susceptibility to penicillin. RESULTS: Some degree of resistance was found to TMP-SMZ and erythromycin (56.4% and 15.4% of the isolates studied, respectively), with 4.6% highly resistant to chloramphenicol. All isolates were susceptible to vancomycin. The highest prevalence of TMP-SMZ resistance was observed in the pneumonia isolates; and that of erythromycin, in cases of sepsis (61.6% and 25.5%, respectively; P < 0.01). The highest prevalence of TMP-SMZ resistance was found in Brazil (71.9%), and that of erythromycin, in Mexico (38.2%) and Venezuela (32.9%). The 14, 6B, 19F, and 23F serotypes were most often associated with resistance to the antibiotics in the study. CONCLUSIONS: High and increasing rates of isolates resistant to TMP-SMZ and erythromycin were observed, as well as a decreasing percentage of isolates resistant to chloramphenicol. These trends highlight differences among the countries studied.

Resistencia a antibióticos no betalactámicos de aislamientos invasores de Streptococcus pneumoniae en niños latinoamericanos: SIREVA II, 2000-2005 / Resistance to non-beta-lactam antibiotics in the clinical isolates of Streptococcus pneumoniae of children in Latin America: SIREVA II, 2000-2005

OBJETIVO. Determinar la evolución de la resistencia a la eritromicina, el cloranfenicol, el trimetoprim-sulfametozaxol (SXT) y la vancomicina de aislamientos invasores de Streptococcus pneumoniae obtenidos de niños de 10 países de América Latina y del Caribe en seis años de vigilancia. MÉTODOS. Se analizaron 8 993 aislamientos de S. pneumoniae recuperados entre 2000 y 2005 de niños menores de 6 años con infecciones invasoras, procedentes de Argentina, Brasil, Chile, Colombia, Cuba, México, Paraguay, República Dominicana, Uruguay y Venezuela. La sensibilidad a los antibióticos se determinó mediante los métodos establecidos y estandarizados en el proyecto SIREVA. La resistencia a múltiples antibióticos se definió como la resistencia a tres o más familias de antibióticos, de los no betalactámicos analizados en este estudio o de los betalactámicos evaluados en un estudio previo en el que 37,8% de estos aislamientos presentaron sensibilidad disminuida a la penicilina. RESULTADOS. Se encontró algún grado de resistencia al SXT y la eritromicina (56,4% y 15,4% de los aislamientos estudiados, respectivamente) y 4,6% presentó alta resistencia al cloranfenicol. Todos los aislamientos fueron sensibles a la vancomicina. Se observó la mayor frecuencia de resistencia al SXT en los aislamientos de neumonía y a la eritromicina en los casos de sepsis (61,6% y 25,5%, respectivamente; P < 0,01). La mayor frecuencia de resistencia al SXT se observó en Brasil (71,9%) y a la eritromicina en México (38,2%) y Venezuela (32,9%). Los serotipos 14, 6B, 19F y 23F fueron los que más frecuentemente se asociaron con la resistencia a los antibióticos estudiados. CONCLUSIONES. Se observó una elevada y creciente frecuencia de aislamientos resistentes al SXT y la eritromicina, y una disminución en la proporción de aislamientos resistentes al cloranfenicol. Estas tendencias mostraron diferencias entre los países estudiados.

OBJECTIVE. To examine the development of resistance to erythromycin, chloramphenicol, trimethoprim-sulfamethoxazole (TMP-SMZ), and vancomycin of the invasive isolates of Streptococcus pneumoniae obtained from children in 10 Latin American/Caribbean countries during six years of surveillance. METHODS. Analysis of 8 993 isolates of S. pneumoniae recovered in 2000­2005 from children with invasive infections, who were less than 6 years of age, and from Argentina, Brazil, Chile, Colombia, Cuba, Dominican Republic, Mexico, Paraguay, Uruguay, or Venezuela. Antibiotic susceptibility was determined through the methods established and standardized by the SIREVA project. Multidrug resistance was defined as: resistance to three or more antibiotics of the same class; to the non-beta-lactams analyzed by this study; or, to the beta-lactams evaluated by a previous study, in which 37.8% of these isolates showed decreased susceptibility to penicillin. RESULTS. Some degree of resistance was found to TMP-SMZ and erythromycin (56.4% and 15.4% of the isolates studied, respectively), with 4.6% highly resistant to chloramphenicol. All isolates were susceptible to vancomycin. The highest prevalence of TMP-SMZ resistance was observed in the pneumonia isolates; and that of erythromycin, in cases of sepsis (61.6% and 25.5%, respectively; P < 0.01). The highest prevalence of TMP-SMZ resistance was found in Brazil (71.9%), and that of erythromycin, in Mexico (38.2%) and Venezuela (32.9%). The 14, 6B, 19F, and 23F serotypes were most often associated with resistance to the antibiotics in the study. CONCLUSIONS. High and increasing rates of isolates resistant to TMP-SMZ and erythromycin were observed, as well as a decreasing percentage of isolates resistant to chloramphenicol. These trends highlight differences among the countries studied

Análisis numérico de imágenes digitalizadas de western blot. El caso del VIH / Numeric analysis of digitalized images of western blot. The case of VIH

El presente trabajo explora la digitalización de imágenes de "western blot" (WB) para extraer mayor información acerca de la respuesta inmune humoral del paciente infectado por el virus de la inmunodeficiencia adquirida humana (VIH) y analizar de manara multivariada los datos obtenidos. Se realizó la digitalización y análisis de las imágenes de WB de 115 sueros. Con estos datos se hicieron análisis tanto cualitativos: dendograma y análisis de componentes principales (ACP), como cuantitativos: ACP con el total de bandas tomando sólo los antígenos propios del virus o utilizando los antígenos que no pertenecen al virus. Los resultados demuestran la factibilidad de diagnosticar de forma mecánica un número grande de imágenes de WB. Tanto el dendograma como el ACP cualitativo separaron, de manera adecuada, imágenes blancas, imágenes con menos de cuatro bandas e imágenes con patrones más complejos. El análisis cuantitativo, que conserva más información, separa perfectamente imágenes de diagnóstico negativo, indeterminado y positivo. Además, se encontró que las imágenes con patrones complejos correlacionan más con individuos asintomáticos. Este análisis reveló también la existencia de banda que no parecen corresponder a proteínas virales, mismas que pudieran corresponder a autoantígenos o antígenos cruzados entre el VIH y el ser humano, dando cauce a autoinmunidad. El análisis digital de imágenes de WB en el caso del VIH, prueba así su gran utilidad en el diagnóstico de caso y en el seguimiento de la evolución y patogenia de la enfermedad