Kwame Kumi Asare

High prevalence of PfCRT K76T mutation in Plasmodium falciparum isolates in Ghana.

Plasmodium falciparum has successfully developed resistance to almost all currently used antimalarials. A single nucleotide polymorphism in the P. falciparum chloroquine resistance transporter (Pfcrt) gene at position 76 resulting in a change in coding from lysine to threonine (K76T) has been implicated to be the corner stone of chloroquine resistance. Widespread resistance to chloroquine in endemic regions led to its replacement with other antimalarials. In some areas this replacement resulted in a reversion of the mutant T76 allele to the wild-type K76 allele. This study was conducted to determine the prevalence of the K76T mutation of the Pfcrt gene eight years after the ban on chloroquine sales and use. A cross-sectional study was conducted in 6 regional hospitals in Ghana. PCR-RFLP was used to analyse samples collected to determine the prevalence of Pfcrt K76T mutation. Of the 1318 participants recruited for this study, 246 were found to harbour the P. falciparum parasites, of which 60.98% (150/246) showed symptoms for malaria. The prevalence of the Pfcrt T76 mutant allele was 58.54% (144/246) and that of the K76 wild-type allele was 41.46% (102/246). No difference of statistical significance was observed in the distribution of the alleles in the symptomatic and asymptomatic participants (P=0.632). No significant association was, again, observed between the alleles and parasite density (P=0.314), as well as between the alleles and Hb levels of the participants (P=0.254). Notwithstanding the decline in the prevalence of the Pfcrt T76 mutation since the antimalarial policy change in 2004, the 58.54% prevalence recorded in this study is considered high after eight years of the abolishment of chloroquine usage in Ghana. This is in contrast to findings from other endemic areas where the mutant allele significantly reduced in the population after a reduction chloroquine use.

Use of proscribed chloroquine is associated with an increased risk of pfcrt T76 mutation in some parts of Ghana

BackgroundAfter years of disuse of chloroquine (CQ) as first-line anti-malarial drug in Ghana, reports from molecular studies conducted in parts of the country indicate varying prevalence of T76 mutation in the pfcrt gene. This situation has several health implications, one being that mutations that confer resistance to CQ have been reported to show substantial cross-resistance to other anti-malarial drugs. It is important to identify some of the factors contributing to the continuous presence of CQ resistance markers in the country. This study determined the prevalence of T76 mutation in pfcrt gene of Plasmodium falciparum isolates collected from selected areas of the Central region of Ghana and correlated with the level of CQ use in these areas.MethodsPlasmodium falciparum DNA was extracted from collected blood-blot filter paper samples in the study sites. The prevalence of T76 point mutation in pfcrt gene was assessed using nested PCR followed by RFLP. CQ from pharmacy and chemical shops was obtained using mystery buying method. The extent of CQ use by the participants was determined by measuring the level of the drug in their urine samples using the Saker-Solomon method.ResultsOf the 214 P. falciparum isolates analysed, 71.9% were found to have T76 mutation of pfcrt gene. The study revealed that 14.49% of community pharmacies and chemical shops had stocks of CQ for sale while 16.9% of the participants had CQ in their urine samples. There is five times more risks of becoming infected with CQ resistant strain for staying in an area where CQ is stocked for sale [RR = 0.20, p < 0.0001] and thirteen times more risks of having CQ-resistant mutant from those who still use CQ than non-users [OR = 0.08, p < 0.0001].ConclusionThis study has shown that high variation in the prevalence of T76 mutations of P. falciparum is linked with the level of CQ stocking and usage within study area.

Natural antibody responses to Plasmodium falciparum MSP3 and GLURP(R0) antigens are associated with low parasite densities in malaria patients living in the Central Region of Ghana

BackgroundPlasmodium falciparum genetic diversity and multiplicity of infection (MOI) are parasite features that have been suggested to influence the acquisition of protective immunity against malaria. This study sought to assess the relationship between MOI and parasite density (PD) in malaria patients living in the Central Region of Ghana and to determine whether naturally occurring antibody levels against P. falciparum GLURP (PF3D7_1035300) and MSP3 (PF3D7_1035400) antigens are associated with decreased parasite load.MethodsDried filter paper blood blots were obtained from children and adults diagnosed with uncomplicated P. falciparum malaria. Microscopy was used to estimate P. falciparum parasite density and polymerase chain reaction (PCR) amplification of the polymorphic regions of msp1 (PF3D7_0930300) and msp2 (PF3D7_0206800) was used for parasite genotyping and MOI determination. ELISA was used to measure the serum IgG concentration of R0 fragment of GLURP (GLURP(R0)) and MSP3 antibodies.ResultsAll 115 samples were positive for P. falciparum by PCR using either the msp1 or msp2 genotyping primer sets. The most prevalent msp1 and msp2 alleles were KI and 3D7, respectively. The geometric mean (GM) for MOI determined by both msp1 and msp2 genotyping was 1.3 for the entire population and was generally higher in children than in adults. Seropositivity was estimated at 67 and 63% for GLURP(R0) and MSP3 antibodies, respectively, and antibody titers were negatively correlated with parasite density.ConclusionsThe negative correlation between naturally occurring GLURP(R0) and MSP3 antibody levels and parasite density observed in this study suggest that augmenting the antibody response with the GMZ2 vaccine could enhance protection in the Central Region of Ghana.

Antibody responses to two new Lactococcus lactis -produced recombinant Pfs48/45 and Pfs230 proteins increase with age in malaria patients living in the Central Region of Ghana

BackgroundRecent advances in malaria control efforts have led to an increased number of national malaria control programmes implementing pre-elimination measures and demonstrated the need to develop new tools to track and control malaria transmission. Key to understanding transmission is monitoring the prevalence and immune response against the sexual stages of the parasite, known as gametocytes, which are responsible for transmission. Sexual-stage specific antigens, Pfs230 and Pfs48/45, have been identified and shown to be targets for transmission blocking antibodies, but they have been difficult to produce recombinantly in the absence of a fusion partner.MethodsRegions of Pfs48/45 and Pfs230 known to contain transmission blocking epitopes, 6C and C0, respectively, were produced in a Lactococcus lactis expression system and used in enzyme linked immunosorbent assays to determine the seroreactivity of 95 malaria patients living in the Central Region of Ghana.ResultsPfs48/45.6C and Pfs230.C0 were successfully produced in L. lactis in the absence of a fusion partner using a simplified purification scheme. Seroprevalence for L. lactis-produced Pfs48/45.6C and Pfs230.C0 in the study population was 74.7 and 72.8%, respectively.ConclusionsA significant age-dependent increase in antibody titers was observed, which suggests a vaccine targeting these antigens could be boosted during a natural infection in the field.

In vivo antimalarial activity of stem bark extracts of Plumeria alba against Plasmodium berghei in imprinting control region mice

License. The full terms of the License are available at http://creativecommons.org/licenses/by-nc/3.0/. Non-commercial uses of the work are permitted without any further permission from Dove Medical Press Ltd, provided the work is properly attributed. Permissions beyond the scope of the License are administered by Dove Medical Press Ltd. Information on how to request permission may be found at: http://www.dovepress.com/permissions.php Reports in Parasitology 2013:3 19–25 Reports in Parasitology Dovepress

Cryptolepine, an indoloquinoline alkaloid, in the management of diabetes mellitus and its associated complications.

Background: Effective long-term management is the key to treatment of diabetes mellitus (DM) and its complications. Aim: To ascertain the ability of cryptolepine (CRP) in managing DM and some associated complications. Materials and Methods: Changes in fasting blood sugar (FBS), body weight, response to thermally-induced pain, and semen quality were assessed in normal and alloxan-induced diabetic rats treated with CRP (10, 30, or 100 mg/kg), glibenclamide (10 mg/kg), or normal saline (2 ml/kg) per os. Hematological profile, liver and kidney function tests, lipid profile, as well as liver, kidney, and pancreas histopathological examinations were also conducted to establish possible effects of CRP treatment. Results: CRP treatment reduced (P ≤ 0.001) FBS and body weight, inhibited (P ≤ 0.05 - 0.001) the latency to tail flick or withdrawal from pain stimulus. It did not alter (P > 0.05): Hematological parameters, elevated (P ≤ 0.05 - 0.001) plasma aspartate transaminase, alanine transaminase, and gamma-glutamyl transferase, reduced (P ≤ 0.01) plasma urea, and elevated (P ≤ 0.001) plasma creatinine associated with DM. CRP, however, reversed (P ≤ 0.05 - 0.001) DM-associated elevation (P ≤ 0.05 - 0.001) of plasma cholesterol, triglycerides, and low-density lipoproteins, and the reduction in high-density lipoproteins. CRP (10-30 mg/kg) showed dose-dependent regeneration of β-islet cells but could not repair degenerated liver and kidney tissue. CRP worsens dose-dependently (P ≤ 0.001) reduced sperm quality associated with DM. Conclusion: CRP abolishes hyperglycemia, weight loss, cold allodynia, neuropathic pain, and hyperlipidemia as well as pancreatic β-islet cell damage associated with DM. It, however, does not improve liver and kidney damage and lowered semen quality.

Modulation of cyptosporidiosis by CD4 levels in chronic diarrhoea HIV/AIDS individuals visiting Tarkwa Municipal hospital, Ghana

Abstract Objective To investigate the role CD4+ levels play in controlling diarrhea conditions caused by intestinal coccidian infections among HIV/AIDS infected individuals visiting Tarkwa Municipal Hospital. Methods Fifty HIV/AIDS infected subjects with diarrhea conditions were enrolled into the study. Stool and blood samples were collected from each in two or three consecutive times to examine intestinal coccidian and microsporidian infections using microscopy and also estimate CD4+ cells using BD FACSCount TM. Results Fourteen of the participants had intestinal coccidian or microsporidian representing 28% while 72% of the participants had diarrhea of unknown origin. Cryptosporidium recorded the highest prevalence of 42.86% whilst Cyclospora and Microsporidia equally recorded a prevalence of 28.57%. A significant protection against cryptosporidiosis was observed for CD4+ count above 200 cells/µL (χ2 = 6.522, P = 0.038), but not cyclosporiasis (P = 0.233) or microsporidiosis (P = 0.060). Conclusions This study has shown that CD4+ cells above 200 cell/µL of blood protect HIV-infected patients from cryptosporidiosis. Standardization of the association between CD4+ cells and diarrhea condition caused by Cryptosporidium species is therefore suggested to serve as an indicator for prompt diagnosis and treatment of HIV-infected individuals with cryptosporidiosis.

Microscopic identification of possible Clonorchis/Opisthorchis infection in two Ghanaian women with undiagnosed abdominal discomfort: two case reports

IntroductionThe impact of foodborne trematode infections is gaining recognition worldwide. Clonorchiasis and opisthorchiasis are some of the most neglected tropical foodborne diseases that pose a significant threat to human health. Persistent or chronic infection of Clonorchis/Opisthorchis often leads to hepatobiliary diseases including cholangitis, cholelithiasis, cholecystitis, pancreatitis, hepatic fibrosis, cholangiocarcinoma and liver cancer. Two cases of Clonorchis/Opisthorchis infection in humans in the Central Region of Ghana are reported.Case presentationEggs suspected to be from Clonorchis sinensis or Opisthorchis species were detected in the stools of a 29-year-old Ghanaian woman and an 18-year-old Ghanaian woman in two clinics in the Central Region of Ghana. The diagnosis was based on clinical symptoms as well as detection of the eggs of the trematode in the faeces of the patients using light microscopy after staining with Giemsa or Ziehl–Neelsen stains.ConclusionsTo the best of our knowledge these are the first documented cases of Clonorchis sinensis or Opisthorchis species infection in Ghana. The detection of this infection in these patients in Ghana should be of concern to clinicians because the infection can be easily misdiagnosed since the accompanying clinical symptoms are malaria-like. Consideration should therefore be given to Clonorchis sinensis and Opisthorchis species when diagnosing patients presenting with malaria-like symptoms.

Socio-Behavioral Risk Factors Associated with Cryptosporidiosis in HIV/AIDS Patients Visiting the HIV Referral Clinic at Cape Coast Teaching Hospital, Ghana

Objective: To identify the socio-behavioral risk factors associated with cryptosporidiosis among HIV/AIDS patients with chronic diarrhea symptoms visiting the HIV referral clinic at Cape Coast Teaching Hospital, Ghana. Methods: A cross-sectional study was conducted among 50 HIV/AIDS patients with recurrent diarrhea. Questionnaires were administered to collect social and behavioral risk factors associated with Cryptosporidium and other opportunistic protozoan parasitic infections in HIV patients. Stool samples were collected for the diagnosis of enteric protozoan pathogens using modified Ziehl-Neelsen and acid-fast staining methods. CD4+ cells counts of study subjects were obtained from patients clinical records. The data obtained were analyzed using Pearson chi-square and multivariate-adjusted statistics tool on SPSS 16 for Windows. Results: Twenty-seven (54%) of the subjects were infected with enteric protozoan pathogens. The prevalences of Cryptosporidium, Cyclospora and Microsporidium infections were 46%, 32% and 16%, respectively. Cryptosporidium infection was significantly associated with drinking water (×2=13.528, p<0.001), Cyclospora was associated with the type of drinking water (×2=14.931, p<0.001) and toilet facilities used by the study subjects (×2=12.463, p<0.01), whiles Microsporidium infection was associated with hand washing behavior (×2=12.463, p<0.01). Enteric protozoans were frequently encountered among subjects with CD4+ T-cell count <200 cells/mm3. However, coinfection of Cyclospora spp & Cryptosporidium spp was not observed in CD4+ cell count <200 and >500 cells/mm3. Multivariate analysis showed that the risk factor for Cryptosporidium infection among HIV/AIDS patients was the source of drinking water (pipe borne water 76.2% prevalence: sachet water 25%; OR=0.10, 95%CI: 0.03-0.39, p<0.001). Conclusion: We report the risk factor for exposure of Cryptosporidium infection among HIV/AIDS patients for the first time in Ghana. The contamination of drinking water by protozoan parasites should be a public health concern. These results provide the stepping block to understand the transmission dynamics of Cryptosporidium and other opportunistic pathogens in HIV/AIDS infected patients in Ghana.

Synergism between Pfcrt and Pfmdr1 genes could account for the slow recovery of chloroquine sensitive Plasmodium falciparum strains in Ghana after chloroquine withdrawal

Unlike other countries, the chloroquine resistant marker Pfcrt T76 mutant has remained fairly stable in Ghana several years after official disuse of chloroquine. Certain mutations in Pfmdr1 may potentiate Pfcrt T76, offering a possible explanation for this observation. To understand the phenomenon, the co-existence of mutations in Pfmdr1 with Pfcrt T76 in Ghanaian Plasmodium falciparum isolates was studied. The reported presence of parasites with reduced sensitivity to amodiaquine and quinine in the country was also studied. Blood samples collected from confirmed malaria patients presenting at health facilities in two distinct ecological zones were analyzed. The prevalence of Pfcrt K76T and the five point mutations in Pfmdr1 were determined using nested PCR followed by RFLP analysis. The association between genes was determined by chi square analysis, and synergism between the two genes was ascertained using the Jonckheere-Terptra (J-T) test followed by Monte Carlo simulation (MCS). Nearly fifty-four percent (53.7%) of the P. falciparum isolates examined had the Pfcrt T76 gene, out of which 18.3% had both K76 and T76 alleles. Mutations at codon 86, 184, 1034, 1042 and 1246 of the Pfmdr1 gene were detected in 36.0%, 87.9%, 71.0%, 91.6% and 8.4% of the isolates, respectively. The haplotypes of Pfmdr1 present were NFCDD (43.46%), YFCDD (27.57%), NFSDD (7.48%), NYSNY (5.14%) and YFSDD (4.67%). Pfcrt T76 was significantly associated with a double mutation at codon 86 and 184 of Pfmdr1 (YF; χ2=18.045, p=0.006). Associations were observed between Pfcrt K76T and Pfmdr1 triple mutation at codons 86, 184 and 1034 (NFC; χ2=13.770, p=0.032 and YFC; χ2=16.489, p=0.011). The J-T test showed significant synergism between Pfcrt 76 and Pfmdr1 polymorphisms (p<0.0001), which was confirmed by MCS at 99% CI. Synergism between Pfcrt and Pfmdr1 mutant genes could account for the slow recovery of chloroquine sensitive P. falciparum in Ghana. The same phenomenon could explain resistance to amodiaquine and quinine. The outcomes of this study also indicated a possible emergence of artemether-lumefantrine resistance in Ghana.