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author:("hassan, Andy")
1.  Babesia: Impact of Cold Storage on the Survival and the Viability of Parasites in Blood Bags 
Transfusion  2013;54(3):585-591.
Babesia represents one of the major infectious threats to the blood supply since clinically silent infections in humans are common and these can be life-threatening in certain recipients. It is important to understand the effect of blood storage conditions on the viability of babesia as this will impact the occurrence and severity of transfusion transmitted babesiosis.
Babesia divergens was introduced into blood bags containing leukoreduced RBC and stored at 4°C for 0–31 days. Samples were withdrawn for assessment of the presence, morphology and viability of parasites. Blood smears were made immediately on removal from blood bags at different time intervals and evaluated blood film microscopy. RBCs withdrawn from the bags were also cultured for 8 days using conditions optimal for parasite reproduction and growth to allow assessment of parasite viability.
After 24 h of storage at 4°C, there was a substantial reduction of parasitemia in the blood-bags, which was maintained throughout storage. This decrease was accompanied by a change in morphology of parasites, with the number of altered parasites increasing through the period of storage. However, viability was maintained through 31 days of cold storage with a lag in achieving exponential growth seen in the parasites subjected to longer periods of refrigeration.
Refrigeration of B. divergens leads to an alteration of parasite morphology and a decrease in parasite numbers. However, there are sufficient parasites that are robust enough to survive 31 days of storage at 4°C and yield high end-point parasitemia.
PMCID: PMC3823633  PMID: 23888882
2.  A Simple Isothermal DNA Amplification Method to Screen Black Flies for Onchocerca volvulus Infection 
PLoS ONE  2014;9(10):e108927.
Onchocerciasis is a debilitating neglected tropical disease caused by infection with the filarial parasite Onchocerca volvulus. Adult worms live in subcutaneous tissues and produce large numbers of microfilariae that migrate to the skin and eyes. The disease is spread by black flies of the genus Simulium following ingestion of microfilariae that develop into infective stage larvae in the insect. Currently, transmission is monitored by capture and dissection of black flies and microscopic examination of parasites, or using the polymerase chain reaction to determine the presence of parasite DNA in pools of black flies. In this study we identified a new DNA biomarker, encoding O. volvulus glutathione S-transferase 1a (OvGST1a), to detect O. volvulus infection in vector black flies. We developed an OvGST1a-based loop-mediated isothermal amplification (LAMP) assay where amplification of specific target DNA is detectable using turbidity or by a hydroxy naphthol blue color change. The results indicated that the assay is sensitive and rapid, capable of detecting DNA equivalent to less than one microfilaria within 60 minutes. The test is highly specific for the human parasite, as no cross-reaction was detected using DNA from the closely related and sympatric cattle parasite Onchocerca ochengi. The test has the potential to be developed further as a field tool for use in the surveillance of transmission before and after implementation of mass drug administration programs for onchocerciasis.
PMCID: PMC4191976  PMID: 25299656
3.  Identification and Characterization of the RouenBd1987 Babesia divergens Rhopty-Associated Protein 1 
PLoS ONE  2014;9(9):e107727.
Human babesiosis is caused by one of several babesial species transmitted by ixodid ticks that have distinct geographical distributions based on the presence of competent animal hosts. The pathology of babesiosis, like malaria, is a consequence of the parasitaemia which develops through the cyclical replication of Babesia parasites in a patient's red blood cells, though symptoms typically are nonspecific. We have identified the gene encoding Rhoptry-Associated Protein −1 (RAP-1) from a human isolate of B. divergens, Rouen1987 and characterized its protein product at the molecular and cellular level. Consistent with other Babesia RAP-1 homologues, BdRAP-1 is expressed as a 46 kDa protein in the parasite rhoptries, suggesting a possible role in red cell invasion. Native BdRAP-1 binds to an unidentified red cell receptor(s) that appears to be non-sialylated and non-proteinacious in nature, but we do not find significant reduction in growth with anti-rRAP1 antibodies in vitro, highlighting the possibility the B. divergens is able to use alternative pathways for invasion, or there is an alternative, complementary, role for BdRAP-1 during the invasion process. As it is the parasite's ability to recognize and then invade host cells which is central to clinical disease, characterising and understanding the role of Babesia-derived proteins involved in these steps are of great interest for the development of an effective prophylaxis.
PMCID: PMC4166668  PMID: 25226276
4.  The Genetic Diversity of Merozoite Surface Antigen 1 (MSA-1) among Babesia bovis Detected from Cattle Populations in Thailand, Brazil and Ghana 
In the present study, we screened blood DNA samples obtained from cattle bred in Brazil (n=164) and Ghana (n=80) for Babesia bovis using a diagnostic PCR assay and found prevalences of 14.6% and 46.3%, respectively. Subsequently, the genetic diversity of B. bovis in Thailand, Brazil and Ghana was analyzed, based on the DNA sequence of merozoite surface antigen-1 (MSA-1). In Thailand, MSA-1 sequences were relatively conserved and found in a single clade of the phylogram, while Brazilian MSA-1 sequences showed high genetic diversity and were dispersed across three different clades. In contrast, the sequences from Ghanaian samples were detected in two different clades, one of which contained only a single Ghanaian sequence. The identities among the MSA-1 sequences from Thailand, Brazil and Ghana were 99.0–100%, 57.5–99.4% and 60.3–100%, respectively, while the similarities among the deduced MSA-1 amino acid sequences within the respective countries were 98.4–100%, 59.4–99.7% and 58.7–100%, respectively. These observations suggested that the genetic diversity of B. bovis based on MSA-1 sequences was higher in Brazil and Ghana than in Thailand. The current data highlight the importance of conducting extensive studies on the genetic diversity of B. bovis before designing immune control strategies in each surveyed country.
PMCID: PMC3942984  PMID: 23856760
Babesia bovis; Brazil; Ghana; MSA-1; Thailand
5.  Babesia: An Emerging Infectious Threat in Transfusion Medicine 
PLoS Pathogens  2013;9(7):e1003387.
PMCID: PMC3708872  PMID: 23853577
6.  Genetic diversity among Trypanosoma (Duttonella) vivax strains from Zambia and Ghana, based on cathepsin L-like gene 
Parasite  2013;20:24.
Understanding the evolutionary relationships of Trypanosoma (Duttonella) vivax genotypes between West Africa and Southern Africa can provide information on the epidemiology and control of trypanosomosis. Cattle blood samples from Zambia and Ghana were screened for T. vivax infection using specie-specific PCR and sequencing analysis. Substantial polymorphism was obtained from phylogenetic analysis of sequences of cathepsin L-like catalytic domains. T. vivax from Ghana clustered together with West African and South American sequences, while T. vivax from Zambia formed one distinct clade and clustered with East African and Southern African sequences. This study suggests existence of distinct genetic diversity between T. vivax genotypes from West Africa and Zambia as per their geographical origins.
PMCID: PMC3718526  PMID: 23815966
Trypanosomiasis; Trypanosoma vivax; Zambia; Ghana
7.  Molecular epidemiological studies on animal trypanosomiases in Ghana 
Parasites & Vectors  2012;5:217.
African trypanosomes are extracellular protozoan parasites that are transmitted between mammalian hosts by the bite of an infected tsetse fly. Human African Trypanosomiasis (HAT) or sleeping sickness is caused by Trypanosoma brucei rhodesiense or T. brucei gambiense, while African Animal Trypanosomiasis (AAT) is caused mainly by T. vivax, T. congolense, T. simiae,T. evansi and T. brucei brucei. Trypanosomiasis is of public health importance in humans and is also the major constraint for livestock productivity in sub-Saharan African countries. Scanty information exists about the trypanosomiasis status in Ghana especially regarding molecular epidemiology. Therefore, this study intended to apply molecular tools to identify and characterize trypanosomes in Ghana.
A total of 219 tsetse flies, 248 pigs and 146 cattle blood samples were collected from Adidome and Koforidua regions in Ghana in 2010. Initial PCR assays were conducted using the internal transcribed spacer one (ITS1) of ribosomal DNA (rDNA) primers, which can detect most of the pathogenic trypanosome species and T. vivax-specific cathepsin L-like gene primers. In addition, species- or subgroup-specific PCRs were performed for T. b. rhodesiense, T. b. gambiense, T. evansi and three subgroups of T. congolense.
The overall prevalence of trypanosomes were 17.4% (38/219), 57.5% (84/146) and 28.6% (71/248) in tsetse flies, cattle and pigs, respectively. T. congolense subgroup-specific PCR revealed that T. congolense Savannah (52.6%) and T. congolense Forest (66.0%) were the endemic subgroups in Ghana with 18.6% being mixed infections. T. evansi was detected in a single tsetse fly. Human infective trypanosomes were not detected in the tested samples.
Our results showed that there is a high prevalence of parasites in both tsetse flies and livestock in the study areas in Ghana. This enhances the need to strengthen control policies and institute measures that help prevent the spread of the parasites.
PMCID: PMC3480844  PMID: 23025330
Trypanosomiasis; Human African Trypanosomiasis; Ghana; PCR
8.  Evolutionary History of Rabies in Ghana 
Rabies virus (RABV) is enzootic throughout Africa, with the domestic dog (Canis familiaris) being the principal vector. Dog rabies is estimated to cause 24,000 human deaths per year in Africa, however, this estimate is still considered to be conservative. Two sub-Saharan African RABV lineages have been detected in West Africa. Lineage 2 is present throughout West Africa, whereas Africa 1a dominates in northern and eastern Africa, but has been detected in Nigeria and Gabon, and Africa 1b was previously absent from West Africa. We confirmed the presence of RABV in a cohort of 76 brain samples obtained from rabid animals in Ghana collected over an eighteen-month period (2007–2009). Phylogenetic analysis of the sequences obtained confirmed all viruses to be RABV, belonging to lineages previously detected in sub-Saharan Africa. However, unlike earlier reported studies that suggested a single lineage (Africa 2) circulates in West Africa, we identified viruses belonging to the Africa 2 lineage and both Africa 1 (a and b) sub-lineages. Phylogeographic Bayesian Markov chain Monte Carlo analysis of a 405 bp fragment of the RABV nucleoprotein gene from the 76 new sequences derived from Ghanaian animals suggest that within the Africa 2 lineage three clades co-circulate with their origins in other West African countries. Africa 1a is probably a western extension of a clade circulating in central Africa and the Africa 1b virus a probable recent introduction from eastern Africa. We also developed and tested a novel reverse-transcription loop-mediated isothermal amplification (RT-LAMP) assay for the detection of RABV in African laboratories. This RT-LAMP was shown to detect both Africa 1 and 2 viruses, including its adaptation to a lateral flow device format for product visualization. These data suggest that RABV epidemiology is more complex than previously thought in West Africa and that there have been repeated introductions of RABV into Ghana. This analysis highlights the potential problems of individual developing nations implementing rabies control programmes in the absence of a regional programme.
Author Summary
Rabies virus (RABV) is widespread throughout Africa, with the domestic dog being the principal vector. Dog rabies is estimated to cause 24,000 human deaths per year in Africa, however, this estimate is still considered to be conservative. Two sub-Saharan African RABV lineages (Africa 1 and 2) are thought to circulate in western and central Africa. We confirmed the presence of RABV in a cohort of 76 brain samples obtained from rabid animals in Ghana collected from 2007 to 2009. In addition we developed and tested a novel molecular diagnostic assay for the detection of RABV, which offers an alternative RABV diagnostic tool for African laboratories. Our analysis of the genetic sequences obtained confirmed all viruses to be RABV, however, unlike previous studies we detected two sub-Saharan African RABV viruses (Africa 1 and 2) in this cohort, which included a single virus previously undetected in West Africa. We suggest that there has been repeated introduction of new RABVs into Ghana over a prolonged period from other West African countries and more recently from eastern Africa. These observations further highlight the problems of individual developing nations implementing rabies control programmes at a local, rather than regional level.
PMCID: PMC3071360  PMID: 21483707

Results 1-8 (8)