Richard H. Asmah

New oligonucleotide primers for P-typing of rotavirus strains: Strategies for typing previously untypeable strains

BACKGROUND The use of molecular methods for rotavirus characterisation provides increased sensitivity for typing, and allows the identification of putative reassortant strains. However, due to the constant accumulation of point mutations through genetic drift; and to the emergence of novel genotypes; and possibly zoonotic transmission and subsequent reassortment, the reagents and methods used for genotyping require close monitoring and updating. OBJECTIVES To design and evaluate a new VP4 consensus oligonucleotide primer pair that provides increased sensitivity and allows typing of strains that were untypeable using available methods. STUDY DESIGN A total of 489 rotavirus-positive faecal specimens from studies conducted between 1996 and 2006 were used for the evaluation of the new VP4 primers which was performed in the WHO Rotavirus Collaborating and Reference centres in the US, Australia, South Africa and the UK. RESULTS The new primer pair allowed P-typing of rotavirus strains and provided increased sensitivity, allowing typing of a significant number of strains that previously could not be P-typed. CONCLUSIONS This study highlights the importance of a constant reconsideration of primer sequences employed for the molecular typing of rotaviruses.

Changing Patterns of Rotavirus Genotypes in Ghana: Emergence of Human Rotavirus G9 as a Major Cause of Diarrhea in Children

ABSTRACT Genotyping of human rotaviruses was performed on 312 rotavirus-positive samples collected from 2,205 young children with diarrhea in the Upper East District of Ghana, a rural community. Of the 271 (86.9%) rotavirus strains that could be VP7 (G) or VP4 (P) characterized, 73 (26.9%) were of G9 specificity. The predominant G9 genotype was G9P[8], which constituted 79.5% of all G9 strains detected, followed by G9P[6] (12.3%), G9P[10] (2.7%), and G9P[4] (1.3%). G9 strains with mixed P types constituted 2.7% of all G9 strains found in the study. All the G9P[8] strains had a long RNA electrophoretic pattern with VP6 subgroup II specificity. Four G9 isolates, GH1319, GH1416, GH3550, and GH3574, which were selected based on the abundance of stool material and were representative of the three electropherotypes observed, were cloned and sequenced. The Ghanaian isolates shared more than 98% sequence nucleotide homology with other G9 strains from the United States (US1205), Malawi (MW69), Brazil (R160), Japan (95H115), and Nigeria (Bulumkutu). However, they showed only 95% nucleotide homology with the Thai G9 strain Mc345. Phylogenetic analysis of the nucleic acid sequence revealed the existence of at least three clusters, with Ghanaian strains forming one cluster, Nigerian and Brazilian strains forming a second cluster, and U.S., Malawian, and Japanese strains forming a third.

Prevalence of unusual human rotavirus strains in Ghanaian children

Sixty‐seven rotavirus‐positive fecal samples, collected between January and April 1999, from children with diarrhea in the Upper East Region of Ghana were examined for rotavirus VP7 and VP4 types. Sufficient viral RNA could be obtained from 46 (68.7%) of the samples and all the isolates had short electrophoretic pattern and typed as subgroup I rotaviruses by subgroup ELISA. Three rotavirus strains with G8 specificity were identified for the first time in Ghana. G and P typing by PCR identified two distinct strains, P[6]G2 (50%) and P[6]G8 (4.3%). Eighty‐two percent of the isolates (n = 38) were of the “putative” neonatal P[6] genotype. Two of these G8 isolates carried the VP4 P[6] genotype whereas the third could not be assigned a P type. Mixed infections of G1, G2, G3 and G8 were detected amongst the stool samples. The presence of these unusual strains, especially the high incidence of G2 rotavirus strains in Ghana, reinforces the need to put in place a surveillance system for the detection of new and exotic rotavirus strains, that will provide information on the spread of these strains in West Africa as well as useful data for the formulation of the next generation of rotavirus vaccines. J. Med. Virol. 63:67–71, 2001.

Rotavirus G and P genotypes in rural Ghana.

ABSTRACT An epidemiological study of rotavirus infection was conducted on specimens collected from patients with gastroenteritis and domiciled in the rural Upper Eastern Region of Ghana during 1998. Fifty isolates, randomly selected from 165 human group A rotavirus-positive samples, were G and P characterized by a reverse transcription (RT)-PCR assay using a seminested multiplex method. Rotaviruses of the G3 genotype were found to be the predominant strain (78%), followed by G2 (14%) and G1 (2%). Mixed infections, as shown by combinations of G3 and G2 (4%) and G3 and G1 (2%), were also observed. P typing showed P[4] (72.34%) to be the prevalent strain, followed by P[6] (21.3%), P[8] (2.13%), and a combination of P[4] and P[6] (4.3%).

First isolation of a new species of Leishmania responsible for human cutaneous leishmaniasis in Ghana and classification in the Leishmania enriettii complex.

An active case detection approach with PCR diagnosis was used in the Ho District of the Volta Region, Ghana that identified individuals with active cutaneous leishmaniasis. Three isolates were successfully cultured and DNA sequences from these were analysed (ribosomal RNA internal transcribed spacer 1; ribosomal protein L23a intergenic spacer; RNA polymerase II large subunit), showing them to be Leishmania, identical to each other but different from all other known Leishmania spp. Phylogenetic analysis showed the parasites to be new members of the Leishmania enriettii complex, which is emerging as a possible new subgenus of Leishmania parasites containing human pathogens.

Accuracy of point-of-care testing for circulatory cathodic antigen in the detection of schistosome infection: Systematic review and meta-analysis

Abstract Objective To assess the accuracy of point-of-care testing for circulatory cathodic antigen in the diagnosis of schistosome infection. Methods We searched MEDLINE, EMBASE, LILACS and other bibliographic databases for studies published until 30 September 2015 that described circulatory cathodic antigen testing compared against one to three Kato–Katz tests per subject – for Schistosoma mansoni – or the filtration of one 10-ml urine sample per subject – for S. haematobium. We extracted the numbers of true positives, false positives, true negatives and false negatives for the antigen testing and performed meta-analyses using a bivariate hierarchical regression model. Findings Twenty-six studies published between 1994 and 2014 met the inclusion criteria. In the detection of S. mansoni, a single antigen test gave a pooled sensitivity of 0.90 (95% confidence interval, CI: 0.84–0.94) and a pooled specificity of 0.56 (95% CI: 0.39–0.71; n = 7) when compared against a single Kato–Katz test. The corresponding values from comparisons with two to three Kato–Katz tests per subject were 0.85 (95% CI: 0.80–0.88) and 0.66 (95% CI: 0.53–0.76; n = 14), respectively. There appeared to be no advantage in using three antigen tests per subject instead of one. When compared against the results of urine filtration, antigen testing for S. haematobium showed poor sensitivity and poor specificity. The performance of antigen testing was better in areas of high endemicity than in settings with low endemicity. Conclusion Antigen testing may represent an effective tool for monitoring programmes for the control of S. mansoni.

A survey of pathogens associated with Cyperus esculentus L (tiger nuts) tubers sold in a Ghanaian city.

BackgroundCyperus esculentus L, is a minor but important crop in Ghana. They are noted mostly by their aphrodisiac properties among others. The nuts are often eaten raw as an unprocessed snack due to its rich flavour and texture. Though eaten raw, the nuts are sometimes handled unhygienically, posing a public health threat. This study therefore aimed at determining the level and distribution of parasitic and bacterial contaminants associated with the crop as it is sold.ResultsFour types of intestinal parasites were identified, and the most prevalent was Cryptosporidium parvum (30.0%). Nuts contaminated with parasites were found only among street vendors. Bacteriological examination showed three different groups of bacterial isolates with the most prevalent being coliforms (54.2%). Unlike parasites, bacteria isolates were found among samples from both street vendors and market places. Multiple drug resistance was displayed by Proteus vulgaris.ConclusionsBuying and eating nuts as well as other fruits taken raw from street vendors and market places could pose a significant public health threat. There is a need for efficient monitoring systems for food borne pathogens in Ghana.

Rotavirus Infection in Children with Diarrhea at Korle-Bu Teaching Hospital, Ghana.

Human rotavirus infection was studied over a 13-month period (January 2004 to January 2005) in children <5 years of age admitted with severe diarrhea at the Korle-Bu Teaching Hospital in Accra, Ghana. During this period, 206 hospitalizations for diarrhea were recorded, with 34.0% (70/206) being positive for rotavirus infection. Infection occurred throughout the year, with peak rotavirus infection occurring during the month of March. Hospitalization associated with rotaviruses was most common in the 6-8 month age group. The case fatality rate of rotavirus infection was 2.9% (2/70) and occurred in children <12 months of age. Four rotavirus VP7 genotypes (G1, G2, G3, and G9) were detected. The predominant genotypes were G2 (22.9%), G1 (17.1%), G9 (17.1%) and G3 (12.9%). Mixed G types were also detected. The predominant VP4 genotypes (P types) were P[6] (38.6%), P[8] (21.4%), P[4] (4.3%) and P[9] (1.4%). The predominant rotavirus strains infecting children in Accra were G9P[6] (10.0%) and G1P[8] (8.6%). Strains with unusual genotypes such as G2P[8] and G(2/3)P[6] were also detected.

Epidemiology of Helicobacter pylori infection in dyspeptic Ghanaian patients

Introduction Helicobacter pylori is a gram-negative urease-producing bacterium causally linked with gastritis, peptic ulcer disease and gastric adenocarcinoma. Infection is more frequent and acquired at an earlier age in developing countries compared to European populations. The incidence of Helicobacter pylori infection in dyspeptic Ghanaian patients was 75.4%. However, epidemiological factors associated with infection vary across populations. Methods This study used a cross-sectional design to consecutively sample dyspeptic patients at the Endoscopy Unit of the Korle-Bu Teaching Hospital, Accra between 2010 and 2012. The study questionnaire elicited their epidemiological clinical characteristics. Helicobacter pylori infection was confirmed by rapid-urease examination of antral biopsies at upper Gastro-intestinal endoscopy. Results The sample population of dyspeptic patients attending the Endoscopy Unit for upper GI endoscopy yielded 242 patients of which 47.5% were females. The age distribution of H. pylori-infection was even across most age – groups, ranging from 69.2% (61 – 70) years to 80% (21 – 30) years. Helicobacter pylori prevalence decreased across areas mapping to the three residential classes in accordance with increasing affluence with rural areas having the highest prevalence. The unemployed and patients in farming had relatively high Helicobacter pylori infection rates of 92.3% and 91.7% respectively. Conclusion Helicobacter pylori is endemic in Ghana but the persistently high prevalence across age groups despite significant community anti-microbial use suggests likely re-crudescence or re-infection from multiple sources in a developing country. Socio-cultural factors such as residential class and farming may be facilitating factors for its continued prevalence.

Severity of malaria in relation to a complement receptor 1 polymorphism: a case–control study

Abstract Background: Studies on the relationship between complement receptor 1 (CR1) polymorphisms in exon 29 encoding the Knops blood group antigens (Swain–Langley (Sl) and McCoy (McC)) and outcome of clinical malaria have produced inconsistent results. Methods: Blood samples from Ghanaian children (n = 150) aged 1–12 years with complicated and uncomplicated malaria were genotyped for the Sl and McC blood group alleles by polymerase chain reaction and restriction fragment length polymorphism. Effect of Sl and McC genotypes on the clinical outcome of malaria was evaluated using logistic regression. Results: McCa/b genotype was significantly associated with more than two-fold increased susceptibility for severe malaria (OR = 2.31; 95% CI: 1.03–5.20, P = 0.043). However, McCb/b was associated with an 88% reduced risk of severe malaria (OR = 0.12; 95% CI: 0.02–0.64, P = 0.013). In contrast, there was no significant association between severe malaria and Sl1/1, Sl1/2, Sl2/and McCa/a genotypes. There was a trend towards decreased susceptibility to both cerebral malaria (CM) (OR = 0.13; 95% CI: 0.02–1.15, P = 0.07) and severe malarial anaemia (SA) (OR = 0.14; 95% CI: 0.02–1.19, P = 0.07) for McCb/b genotype when compared with the McCa/a genotype. There were no significant associations between Sl1/2 or Sl2/2 genotype and CM or SA when compared with Sl1/1 genotype. Conclusions: McCa/b was associated with increased susceptibility to severe malaria and McCb/b associated with reduced risk of severe malaria. Further studies with large sample size in other malaria endemic regions in Africa are warranted to confirm these findings.