Altered error specificity of RNase H-deficient HIV-1 reverse transcriptases during DNA-dependent DNA synthesis

Asp443 and Glu478 are essential active site residues in the RNase H domain of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT). We have investigated the effects of substituting Asn for Asp443 or Gln for Glu478 on the fidelity of DNA-dependent DNA synthesis of phylogenetically diverse HIV-1 RTs. In M13mp2 lacZα-based forward mutation assays, HIV-1 group M (BH10) and group O RTs bearing substitutions D443N, E478Q, V75I/D443N or V75I/E478Q showed 2.0-to 6.6-fold increased accuracy in comparison with the corresponding wild-type enzymes. This was a consequence of their lower base substitution error rates. One-nucleotide deletions and insertions represented between 30 and 68% of all errors identified in the mutational spectra of RNase H-deficient HIV-1 group O RTs. In comparison with the wild-type RT, these enzymes showed higher frameshift error rates and higher dissociation rate constants (koff) for DNA/DNA template-primers. The effects on frameshift fidelity were similar to those reported for mutation E89G and suggest that in HIV-1 group O RT, RNase H inactivation could affect template/primer slippage. Our results support a role for the RNase H domain during plus-strand DNA polymerization and suggest that mutations affecting RNase H function could also contribute to retrovirus variability during the later steps of reverse transcriptionMinistry of Economy and Competitiveness (Spain) [BIO2010/15542]; Ministry of Health, Social Services and Equality (Spain) [EC11-025]; Fondo de Investigación Sanitaria (through the ‘Red Temática de Investigación Cooperativa en SIDA’) [RD06/0006]; Fundació n Ramón Areces (institutional grant to Centro de Biología Molecular ‘Severo Ochoa’). Funding for open access charge: Research grant [BIO2010/15542

Haemoglobin levels for population from Gambo, a rural area of Ethiopia, and their association with anaemia and malaria

BACKGROUND: Knowledge of appropriate reference intervals is critical not only to provide optimal clinical care, but also to enrol populations in medical research. The aim of this study was to generate normal ranges of laboratory values for haemoglobin among healthy Ethiopian adults and children and to determine if anaemia is a possible indicator of malaria in women and children in this area of Ethiopia. METHODS: This study was carried out from January 2008 to May 2010. The reference sample population with malaria-negative consisted of 454 individuals, divided women, men and children. The malaria-infected sample population consisted of 117 individuals. The reference ranges were based on the guidelines from the Clinical and Laboratory Standards Institute. Haemoglobin concentration was determined by Hemo-Control EKF Diagnostic Analyser on whole blood. Testing for malaria-positive and negative infection was done by microscopy and by PCR. RESULTS: The lower limits for adult haemoglobin range obtained from this population were slightly higher than those derived from other African populations, but were equal to those established by other studies in Ethiopia and the World Health Organization (WHO). Regarding children, the minimum values were lower than those obtained from different African populations and those established by WHO. The malaria-negative group had anaemia in 35.6% of cases and in the malaria-positive group in 70.9%. There was a stronger, statistically significant association between anaemia and malaria-positive samples than between anaemia and malaria-negative samples in women and both groups of children. CONCLUSIONS: The results from this study are a contribution in the definition of the haemoglobin parameters in African populations, which could be taken as standards for interpretation of laboratory results. The haemoglobin indices in adults from Gambo tended to be higher than other African populations and in children were lower than other studies in Africa. The results also suggest that anaemia is not useful as a supportive diagnostic criterion to monitor and evaluate malaria in women and children from Ethiopia, because a 29.1% of malaria cases will be not detected, because of not having anaemia

Microscopy and molecular biology for the diagnosis and evaluation of malaria in a hospital in a rural area of Ethiopia

Abstract Background Malaria is a leading public health problem in Ethiopia. Accurate diagnosis of Plasmodium infections is crucial for the reduction of malaria in tropical areas and for epidemiological studies. The role of light microscopy (LM) as gold standard has been questioned and, therefore, new molecular methods have been developed for the detection of Plasmodium species. The aim of the present work was to compare different malaria diagnostic methods in order to detect the most common species of Plasmodium and to broaden the knowledge of malaria prevalence in a hospital in a rural area in Ethiopia. Methods A cross-sectional survey of 471 individuals was carried out in a hospital in the rural area of Gambo (Ethiopia). Blood samples were prepared for microscopic observation and collected in filter paper for Seminested-Multiplex PCR (SnM-PCR) and real time PCR (qPCR) testing. The SnM-PCR was considered as the gold standard technique and compared with the rest. Thus, agreement between SnM-PCR and LM was determined by calculating Kappa Statistics and correlation between LM and qPCR quantification was calculated by pair-wise correlation co-efficient. Results Samples analysed by LM and SnM-PCR were positive for Plasmodium sp. 5.5% and 10.5%, respectively. Sensitivity was 52.2% by LM and 70% by qPCR. Correlation co-efficient between microscopy counts and qPCR densities for Plasmodium vivax was R2 = 0.586. Prevalence was estimated at 7% (95% CI: 4.7–9.3). Plasmodium vivax was the dominant species detected and the difference was statistically significant (χ2 = 5.121 p  Conclusion Accurate malaria diagnostic methods have a great effect in the reduction of the number of malaria-infected individuals. SnM-PCR detection of malaria parasites may be a very useful complement to microscopic examination in order to obtain the real prevalence of each Plasmodium species. Although SnM-PCR shows that it is a good tool for the determination of Plasmodium species, today light microscopy remains the only viabletool for malaria diagnosis in developing countries. Therefore, re-inforcement in the training of microscopists is essential for making the correct diagnosis of malaria. Plasmodium vivax was the predominant species in Gambo, a meso-endemic area for this species.</p

Therapeutic Potential of a Combination of Two Gene-Specific Small Interfering RNAs against Clinical Strains of Acanthamoeba▿

Pathogenic strains of the genus Acanthamoeba are causative agents of severe infections, such as fatal encephalitis and a sight-threatening amoebic keratitis. Antimicrobial therapy for these infections is generally empirical, and patient recovery is often problematic, due to the existence of a highly resistant cyst stage in these amoebae. In previous studies, small interfering RNAs (siRNAs) against the catalytic domains of extracellular serine proteases and glycogen phosphorylase from Acanthamoeba were designed and evaluated for future therapeutic use. The silencing of proteases resulted in Acanthamoeba failing to degrade human corneal cells, and silencing of glycogen phosphorylase caused amoebae to be unable to form mature cysts. After the siRNA design and concentration were optimized in order to avoid toxicity problems, cultures of Acanthamoeba were treated with a combination of both siRNAs, and cells were evaluated under an inverted microscope. This siRNA-based treatment dramatically affected the growth rate and cellular survival of the amoebae. These results were observed less than 48 h after the initiation of the treatment. In order to check possible toxic effects of the siRNA combination, three eukaryotic cell lines (HeLa, murine macrophages, and osteosarcoma cells) were treated with the same molecules, and cytotoxicity was examined by measuring lactate dehydrogenase release. The future use of the combination of these siRNAs is proposed as a potential therapeutic approach against pathogenic strains of Acanthamoeba

Microscopy and molecular biology for the diagnosis and evaluation of malaria in a hospital in a rural area of Ethiopia

BACKGROUND: Malaria is a leading public health problem in Ethiopia. Accurate diagnosis of Plasmodium infections is crucial for the reduction of malaria in tropical areas and for epidemiological studies. The role of light microscopy (LM) as gold standard has been questioned and, therefore, new molecular methods have been developed for the detection of Plasmodium species. The aim of the present work was to compare different malaria diagnostic methods in order to detect the most common species of Plasmodium and to broaden the knowledge of malaria prevalence in a hospital in a rural area in Ethiopia. METHODS: A cross-sectional survey of 471 individuals was carried out in a hospital in the rural area of Gambo (Ethiopia). Blood samples were prepared for microscopic observation and collected in filter paper for Seminested-Multiplex PCR (SnM-PCR) and real time PCR (qPCR) testing. The SnM-PCR was considered as the gold standard technique and compared with the rest. Thus, agreement between SnM-PCR and LM was determined by calculating Kappa Statistics and correlation between LM and qPCR quantification was calculated by pair-wise correlation co-efficient. RESULTS: Samples analysed by LM and SnM-PCR were positive for Plasmodium sp. 5.5% and 10.5%, respectively. Sensitivity was 52.2% by LM and 70% by qPCR. Correlation co-efficient between microscopy counts and qPCR densities for Plasmodium vivax was R2 = 0.586. Prevalence was estimated at 7% (95% CI: 4.7-9.3). Plasmodium vivax was the dominant species detected and the difference was statistically significant (χ2 = 5.121 p < 0.05). The highest prevalence of the parasite (10.9%) was observed in age groups under 15 years old. CONCLUSION: Accurate malaria diagnostic methods have a great effect in the reduction of the number of malaria-infected individuals. SnM-PCR detection of malaria parasites may be a very useful complement to microscopic examination in order to obtain the real prevalence of each Plasmodium species. Although SnM-PCR shows that it is a good tool for the determination of Plasmodium species, today light microscopy remains the only viabletool for malaria diagnosis in developing countries. Therefore, re-inforcement in the training of microscopists is essential for making the correct diagnosis of malaria. Plasmodium vivax was the predominant species in Gambo, a meso-endemic area for this species.The authors would like to acknowledge the Spanish Society of Tropical Medicine and International Health (SEMTSI) for supporting an International SEMTSI 2009 fellowship and the laboratory staff of the Gambo General Rural Hospital for their support in collecting the data. This survey was funded by the Tropical Disease Cooperative Research Network (RICET) and the University Institute of Tropical Diseases and Public Health of the Canary Islands.S