Quasispecies tropism and compartmentalization in gut and peripheral blood during early and chronic phases of HIV-1 infection: possible correlation with immune activation markers.

HIV quasispecies was analysed in plasma and proviral genomes hosted by duodenal mucosa and peripheral blood cells (PBMC) from patients with early or chronic infection, with respect to viral heterogeneity, tropism compartmentalization and extent of immune activation. Seventeen HIV-1-infected combined antiretroviral therapy naive patients were enrolled (11 early infection and six chronic infection). V3 and nef genomic regions were analysed by ultra-deep pyrosequencing. Sequences were used to infer co-receptor usage and to construct phylogenetic trees. As markers of immune activation, plasma sCD14 and soluble tumour necrosis factor receptor II (sTNFRII) levels were measured. Median diversity of HIV RNA was lower in patients with early infection versus chronic infection patients. Overall, direct correlation was observed between V3 diversity and X4 frequency; V3 diversity of HIV RNA was inversely correlated with CD4 T-cell count; median sCD14 and sTNFRII values were similar in early and chronic patients, but X4 frequency of HIV RNA was directly correlated with plasma sCD14. The proportion of patients harbouring X4 variants and median intra-patient X4 frequency of proviral genomes tended to be higher in chronic infection than early infection patients. More pronounced compartmentalization of proviral quasispecies in gut compared with PBMC samples was observed in patients with early infection compared with chronic patients. The loss of gut/PBMC compartmentalization in more advanced stages of HIV infection was confirmed by longitudinal observation. More studies are needed to understand the pathogenetic significance of early HIV quasispecies compartmentalization and progressive intermixing of viral variants in subsequent phases of the infection, as well as the role of immune activation in tropism switch.

Next-generation sequencing technology in clinical virology.

Recent advances in nucleic acid sequencing technologies, referred to as 'next-generation' sequencing (NGS), have produced a true revolution and opened new perspectives for research and diagnostic applications, owing to the high speed and throughput of data generation. So far, NGS has been applied to metagenomics-based strategies for the discovery of novel viruses and the characterization of viral communities. Additional applications include whole viral genome sequencing, detection of viral genome variability, and the study of viral dynamics. These applications are particularly suitable for viruses such as human immunodeficiency virus, hepatitis B virus, and hepatitis C virus, whose error-prone replication machinery, combined with the high replication rate, results, in each infected individual, in the formation of many genetically related viral variants referred to as quasi-species. The viral quasi-species, in turn, represents the substrate for the selective pressure exerted by the immune system or by antiviral drugs. With traditional approaches, it is difficult to detect and quantify minority genomes present in viral quasi-species that, in fact, may have biological and clinical relevance. NGS provides, for each patient, a dataset of clonal sequences that is some order of magnitude higher than those obtained with conventional approaches. Hence, NGS is an extremely powerful tool with which to investigate previously inaccessible aspects of viral dynamics, such as the contribution of different viral reservoirs to replicating virus in the course of the natural history of the infection, co-receptor usage in minority viral populations harboured by different cell lineages, the dynamics of development of drug resistance, and the re-emergence of hidden genomes after treatment interruptions. The diagnostic application of NGS is just around the corner.

Detection of haemagglutinin D222 polymorphisms in influenza A(H1N1)pdm09-infected patients by ultra-deep pyrosequencing.

This study was aimed at establishing the genetic heterogeneity of influenza virus haemagglutinin (HA) gene quasi-species and the polymorphisms at codon 222, by application of ultra-deep pyrosequencing (UDPS) to respiratory samples from patients hospitalized for influenza A(H1N1)pdm09 infection, presenting with severe or moderate-mild disease. HA diversity was significantly higher in samples collected from patients with severe manifestations than in those from patients with moderate-mild manifestations (p 0.02). D222 polymorphism was detected in 40.7% of patients by UDPS, and in only 7.1% by Sanger sequencing. D222E, D222G, D222N and D222A were observed in 37.0%, 11.1%, 7.4% and 3.7% of patients, respectively; 10.7% of samples harboured more than two variants. The relative frequency of each single variant showed a wide range of intrapatient variation. D222G/N/A were detected, as either minor or predominant variants, only in severe cases, whereas D222E was equally represented in severe and moderate-mild infections. Other amino acid variants were observed at different positions within the analysed HA fragment. Consistent with higher heterogeneity, non-D222 variants were more frequently detected in severe cases than in moderate-mild cases. In addition, seven non-D222 mutations carried by minority variants, not previously described, were observed.

Analysis of p73 expression pattern in acute myeloid leukemias: lack of DeltaN-p73 expression is a frequent feature of acute promyelocytic leukemia.

p73, the homologue of p53, is a nuclear protein whose ectopic expression, in p53+/+ and p53-/- cells, recapitulates the most well-characterized p53 effects, such as growth arrest, apoptosis and differentiation. Unlike p53, which is mutated in half of human cancers, p73 is rarely mutated. However, altered expression of the p73 gene has been reported in neuroblastoma, lung cancer, prostate cancer and renal cell carcinoma. To investigate the potential involvement of p73 in acute myeloid leukemias (AMLs), we analyzed 71 samples from AML patients for the expression pattern of N-terminal transactivation-p73alpha (TA-p73alpha), its spliced isoforms and N-terminal-deleted-p73 transcripts (DeltaN-p73). We detected p73 gene expression in AML irrespective of FAB (French-American-British) subtypes. Notably, the analysis of DeltaN-p73 expression, which has been reported to inactivate both p53 and p73 antitumor effects, revealed a rather peculiar pattern. In fact, DeltaN-p73 transcript and protein were detectable in 27/28 (96.4%) cases of M0, M1, M2, M4, M5 and M6 AML and in 13/41 (31.7%) cases of PML-RARalpha-positive M3 AML (P<0.01). Thus, the distinct gene expression profile of p73 further supports the notion that acute promyelocytic leukemia is a biologically different subset of AML.

Users' perceptions of the quality of maternal and child health (MCH) services in Grenada

What do users think about the Maternal and Child Health (MCH) Services provided at government run community health facilities in Grenada? This study was done to analyse the quality of MCH services delivered, according to the perception of the users. Data were collected using a pre-tested structured questionnaire, focus group discussions and natural group interviews. 237 users of MCH services were interviewed at randomly selected community health facilities. Ninety-six percent of users were of the opinion that their health workers showed interest and understanding and were friendly and courteous. Ninety-six percent of users were comfortable with the health workers who attended to them. Ninety-two percent of users were questioned by health workers. However, 26 percent received no explanations and instructions. Forty-nine percent of users perceived consultations with health workers to be short. The hours of service delivery were inconvenient for employed persons using the service. Statistically significant differences existed in user satisfaction by location of facility and type of facility at which care was received. Areas identified for improvement include health workers' inter-personal relations and infrastructure, including the availability of sanitary facilities and supplies for users. 65 percent of users of MCH services were not gainfully employed (37 percent unemployed, 28 percent housewives). Occupational status and educational attainment levels of respondents reflected a disproportionaly higher number from lower socioeconomic groups among users of the MCH services. Rescheduling of hours for service delivery is recommended to encourage more employed persons to use the services.(AU)

Users perceptions of the quality of MCH services in Grenada

This study was done to analyse the quality of Maternal and Child Health (MCH) Services delivered according to the perception of users. Data was collected using a pre-tested structured questionnaire, focus group discussions and natural group interviews. 237 users of MCH services were interviewed at randomly selected community health facilities. Ninety-six percent of users were of the opinion that their health workers showed interest and understanding and were friendly and courteous. Ninety-six percent of users were comfortable with the health workers who attended to them. Ninety-two percent of users were questioned by health workers. However, 26 percent received no explanations and instructions. Forty-nine percent of users perceived consultations with health workers to be short. The hours of service delivery were inconvenient for employed persons using the service. Statistically significant differences existed in user satisfaction by location of facility and type of facility at which care was received. Areas identified for improvement include health workers' interpersonal relations and infrastructure including the availability of sanitary facilities and supplies for users. 65 percent of users of MCH services were not gainfully employed (37 percent - unemployed, 28 percent - housewives). Occupational status and educational attainment levels of respondents reflected a disproportionately higher number from lower socioeconomic groups among users of the MCH services. Rescheduling of hours for service delivery is recommended to encouraged more employed persons to use the services.

Users perceptions of the quality of MCH services in Grenada

This study was done to analyse the quality of Maternal and Child Health (MCH) Services delivered according to the perception of users. Data was collected using a pre-tested structured questionnaire, focus group discussions and natural group interviews. 237 users of MCH services were interviewed at randomly selected community health facilities. Ninety-six percent of users were of the opinion that their health workers showed interest and understanding and were friendly and courteous. Ninety-six percent of users were comfortable with the health workers who attended to them. Ninety-two percent of users were questioned by health workers. However, 26 percent received no explanations and instructions. Forty-nine percent of users perceived consultations with health workers to be short. The hours of service delivery were inconvenient for employed persons using the service. Statistically significant differences existed in user satisfaction by location of facility and type of facility at which care was received. Areas identified for improvement include health workers' interpersonal relations and infrastructure including the availability of sanitary facilities and supplies for users. 65 percent of users of MCH services were not gainfully employed (37 percent - unemployed, 28 percent - housewives). Occupational status and educational attainment levels of respondents reflected a disproportionately higher number from lower socioeconomic groups among users of the MCH services. Rescheduling of hours for service delivery is recommended to encouraged more employed persons to use the services. (AU)

Radioisotopic method for nasal mucociliary function evaluation.

In 13 healthy young adults, nasal mucociliary transport velocity was measured by means of albumine microsphere labeled with Tc99m as a tracer. M + ISD of clearance velocity values and of the half time radioactivity (T1/2) resulted to be respectively 1.02 + 0.22 cm/min and 1.07 + 0.24 min. A significant correlation (P less than 0.05) between half time clearance (T1/2) values of the radioactive particles and the mucociliary velocity ones could be detected, making T1/2 a reliable and rapidly obtainable parameter for determining nasal mucociliary function. Advantages and disadvantages of the different techniques are discussed.