Kwadwo A. Koram

Increased pfmdr1 gene copy number and the decline in pfcrt and pfmdr1 resistance alleles in Ghanaian Plasmodium falciparum isolates after the change of anti-malarial drug treatment policy

BackgroundWith the introduction of artemisinin-based combination therapy (ACT) in 2005, monitoring of anti-malarial drug efficacy, which includes the use of molecular tools to detect known genetic markers of parasite resistance, is important for first-hand information on the changes in parasite susceptibility to drugs in Ghana. This study investigated the Plasmodium falciparum multidrug resistance gene (pfmdr1) copy number, mutations and the chloroquine resistance transporter gene (pfcrt) mutations in Ghanaian isolates collected in seven years to detect the trends in prevalence of mutations.MethodsArchived filter paper blood blots collected from children aged below five years with uncomplicated malaria in 2003–2010 at sentinel sites were used. Using quantitative real-time polymerase chain reaction (qRT-PCR), 756 samples were assessed for pfmdr1 gene copy number. PCR and restriction fragment length polymorphism (RFLP) were used to detect alleles of pfmdr1 86 in 1,102 samples, pfmdr1 184, 1034, 1042 and 1246 in 832 samples and pfcrt 76 in 1,063 samples. Merozoite surface protein 2 (msp2) genotyping was done to select monoclonal infections for copy number analysis.ResultsThe percentage of isolates with increased pfmdr1 copy number were 4, 27, 9, and 18% for 2003–04, 2005–06, 2007–08 and 2010, respectively. Significant increasing trends for prevalence of pfmdr1 N86 (×2u2009=u200996.31, p <0.001) and pfcrt K76 (×2 = 64.50, p <0.001) and decreasing trends in pfmdr1 Y86 (×2u2009=u200938.52, p <0.001) and pfcrt T76 (×2u2009=u200943.49, p <0.001) were observed from 2003–2010. The pfmdr1 F184 and Y184 prevalence showed an increasing and decreasing trends respectively but were not significant (×2u2009=u20097.39,p=0.060; ×2u2009=u20097.49, pu2009=u20090.057 respectively). The pfmdr1 N86-F184-D1246 haplotype, which is alleged to be selected by artemether-lumefantrine showed a significant increasing trend (×2u2009=u200920.75, p <u20090.001).ConclusionIncreased pfmdr1 gene copy number was observed in the isolates analysed and this finding has implications for the use of ACT in the country although no resistance has been reported. The decreasing trend in the prevalence of chloroquine resistance markers after change of treatment policy presents the possibility for future introduction of chloroquine as prophylaxis for malaria risk groups such as children and pregnant women in Ghana.

Anti-sporozoite antibodies as alternative markers for malaria transmission intensity estimation

BackgroundReported malaria cases continue to decline globally, and this has been attributed to strategic implementation of multiple malaria control tools. Gains made would however need to be sustained through continuous monitoring to ensure malaria elimination and eradication. Entomological inoculation rate (EIR) is currently the standard tool for transmission monitoring but this is not sensitive enough, especially in areas of very low transmission. Transmission estimation models based on seroconversion rates (λ) of antibodies to Plasmodium falciparum blood stage antigens are gaining relevance. Estimates of λ, which is the measure of transmission intensity, correlate with EIR but are limited by long-term persistence of antibodies to blood stage antigens. Seroprevalence of antibodies to sporozoite antigens may be better alternatives since these antigens usually have shorter immune exposure times. The aim of this study was to develop transmission estimation models based on the seroprevalence of antibodies to two P. falciparum sporozoite antigens (CSP, CelTOS) and compare with models based on the classical blood stage antigen AMA1.MethodsAntibody levels in archived plasma from three cross-sectional surveys conducted in 2009 in a low transmission area of Southern Ghana were assessed by indirect ELISA. Seroprevalence of antibodies against CSP, CelTOS and AMA1 were fitted to reversible catalytic models to estimate λ and corresponding seroreversion rates (ρ) for each antibody.ResultsOf the three models developed, the anti-CSP model predicted a 13-fold decrease in λ four years prior to the time of sampling (2009). Anti-AMA1 antibodies formed at a four-fold greater rate compared to that of anti-CelTOS antibodies, and anti-CSP antibodies during the period of decreased λ. In contrast, anti-AMA1 antibodies decayed at a five-fold slower rate relative to that of anti-CSP antibodies while anti-AMA1 and anti-CelTOS antibody decay rates were not significantly different. Anti-CSP antibodies were relatively short-lived as they formed at an 11.6-fold slower rate relative to their decay during the period of decreased λ.ConclusionsThese features of anti-CSP antibodies can be exploited for the development of models for predicting seasonal, short-term changes in transmission intensity in malaria-endemic areas, especially as the elimination phase of malaria control is approached.

Therapeutic efficacy of artemether-lumefantrine combination in the treatment of uncomplicated malaria among children under five years of age in three ecological zones in Ghana

BackgroundIn 2008, artemether - lumefantrine (AL) and dihydroartemisinin - piperaquine (DHAP) were added to artesunate - amodiaquine (AS-AQ) as first-line drugs for uncomplicated malaria in Ghana. The introduction of new drugs calls for continuous monitoring of these drugs to provide timely information on trends of their efficacy and safety to enhance timely evidence-based decision making by the National Malaria Control Programme. In this regard, the therapeutic efficacy of AL was monitored from September 2010 to April 2011 in four sentinel sites representing the three main ecological zones of the country.MethodsThe study was a one-arm prospective evaluation of clinical and parasitological responses to directly observed treatment for uncomplicated malaria among children aged 6u2009months to 59u2009months using the 2009 WHO protocol for surveillance of anti-malarial drug efficacy. Children recruited into the study received weight-based 20/120u2009mg AL at 0, 8, 24, 36, 48, and 60u2009hrs. Parasitaemia levels were assessed on days 2, 3, 7, 14, 21, 28, and at any time a study child was brought to the clinic with fever.ResultsA total of 175 children were enrolled into the study: 56 in the savanna zone, 78 in the forest zone and 41 in the coastal zone. Per-protocol analysis showed that the overall PCR-corrected cure rates on day 14 and day 28 were 96.5% (95% CI: 92.1, 98.6) and 95.4% (95% CI: 90.3, 98.0), respectively, with statistically significant differences between the ecological zones. The 90.4%u2009day-28 cure rate observed in the savannah zone (95% CI: 78.2, 96.4) was significantly the lowest compared with 100% (95% CI: 93.2, 99.9) in the forest zone and 93.8% (95% CI: 77.8, 98.9) in the coastal zone (Pu2009=u20090.017). Fever and parasite clearance were slower among children enrolled in the savannah zone. Gametocytaemia after day-3 post-treatment was rare in all the zones.ConclusionsThe study has shown that AL remains efficacious in Ghana with significant ecologic zonal differences. The savannah zone may be a potential zone for any emergence of resistant alleles as a result of the slower parasite clearance observed in the zone.

Genetic diversity of Plasmodium falciparum isolates from uncomplicated malaria cases in Ghana over a decade

BackgroundGenotyping malaria parasites to assess their diversity in different geographic settings have become necessary for the selection of antigenic epitopes for vaccine development and for antimalarial drug efficacy or resistance investigations. This study describes the genetic diversity of Plasmodium falciparum isolates from uncomplicated malaria cases over a ten year period (2003–2013) in Ghana using the polymorphic antigenic marker, merozoite surface protein 2 (msp2).MethodsArchived filter paper blood blots from children aged nine years and below with uncomplicated malaria collected from nine sites in Ghana were typed for the presence of the markers. A total of 880 samples were genotyped for msp2 for the two major allelic families, FC27 and 3D7, using nested polymerase chain reaction (PCR). The allele frequencies and the multiplicity of infection were determined for the nine sites for five time points over a period of ten years, 2003–2004, 2005–2006, 2007–2008, 2010 and 2012–2013 malaria transmission seasons.ResultsThe number of different alleles detected for the msp2 gene by resolving PCR products on agarose gels was 14. Both of the major allelic families, 3D7 and FC27 were common in all population samples. The highest multiplicity of infection (MOI) was observed in isolates from Begoro (forest zone, rural site): 3.31 for the time point 2007–2008. A significant variation was observed among the sites in the MOIs detected per infection (Fishers exact test, Pu2009<u20090.001) for the 2007 isolates and also at each of the three sites with data for three different years, Hohoe, Pu2009=u20090.03; Navrongo, Pu2009<u20090.001; Cape Coast, Pu2009<u20090.001. Overall, there was no significant difference between the MOIs of the three ecological zones over the years (Pu2009=u20090.37) and between the time points when data from all sites were pooled (Pu2009=u20090.40).ConclusionsThe diversity and variation between isolates detected using the msp2 gene in Ghanaian isolates were observed to be profound; however, there was homogeneity throughout the three ecological zones studied. This is indicative of gene flow between the parasite populations across the country probably due to human population movements (HPM).

Therapeutic efficacy of artesunate-amodiaquine and artemether-lumefantrine combinations in the treatment of uncomplicated malaria in two ecological zones in Ghana

BackgroundCase management based on prompt diagnosis and adequate treatment using artemisinin-based combination therapy (ACT) remains the main focus of malaria control in Ghana. As part of routine surveillance on the therapeutic efficacy of ACT in Ghana, the efficacy of amodiaquine-artesunate (AS-AQ) and artemether-lumefantrine (AL) were studied in six sentinel sites representing the forest and savannah zones of the country.MethodsThree sites representing the two ecological zones studied AS-AQ whilst the other three sites studied AL. In each site, the study was a one-arm prospective evaluation of the clinical, parasitological, and haematological responses to directly observed therapy for uncomplicated malaria with either AS-AQ or AL among children aged 6xa0months and 9xa0years. The WHO 2009 protocol for monitoring anti-malarial drug efficacy was used for the study between July 2013 and March 2014.ResultsPer-protocol analyses on day 28 showed an overall PCR-corrected cure rate of 100xa0% for AS-AQ and 97.6xa0% (95xa0% CI 93.1, 99.5) for AL: 97.2xa0% (95xa0% CI 92.0, 99.4) in the forest zone and 100xa0% in the savannah zone. Kaplan–Meier survival analysis showed similar outcomes. Prevalence of fever decreased by about 75xa0% after the first day of treatment with each ACT in the two ecological zones. No child studied was parasitaemic on day 3, and gametocytaemia was generally maintained at low levels (<5xa0%). Post-treatment mean haemoglobin concentrations significantly increased in the two ecological zones.ConclusionsTherapeutic efficacy of AS-AQ and AL remains over 90xa0% in the forest and savannah zones of Ghana. Additionally, post-treatment parasitaemia on day 3 is rare suggesting that artemisinin is still efficacious in Ghana.

Antitrypanosomal Activities and Mechanisms of Action of Novel Tetracyclic Iridoids from Morinda lucida Benth.

ABSTRACT Trypanosoma brucei parasites are kinetoplastid protozoa that devastate the health and economic well-being of millions of people in Africa through the disease human African trypanosomiasis (HAT). New chemotherapy has been eagerly awaited due to severe side effects and the drug resistance issues plaguing current drugs. Recently, there has been an emphasis on the use of medicinal plants worldwide. Morinda lucida Benth. is a popular medicinal plant widely distributed in Africa, and several research groups have reported on the antiprotozoal activities of this plant. In this study, we identified three novel tetracyclic iridoids, molucidin, ML-2-3, and ML-F52, from the CHCl3 fraction of M. lucida leaves, which possess activity against the GUTat 3.1 strain of T. brucei brucei. The 50% inhibitory concentrations (IC50) of molucidin, ML-2-3, and ML-F52 were 1.27 μM, 3.75 μM, and 0.43 μM, respectively. ML-2-3 and ML-F52 suppressed the expression of paraflagellum rod protein subunit 2, PFR-2, and caused cell cycle alteration, which preceded apoptosis induction in the bloodstream form of Trypanosoma parasites. Novel tetracyclic iridoids may be promising lead compounds for the development of new chemotherapies for African trypanosomal infections in humans and animals.

Seroprevalence of antibodies against plasmodium falciparum sporozoite antigens as predictive disease transmission markers in an area of Ghana with seasonal malaria transmission

Introduction As an increasing number of malaria-endemic countries approach the disease elimination phase, sustenance of control efforts and effective monitoring are necessary to ensure success. Mathematical models that estimate anti-parasite antibody seroconversion rates are gaining relevance as more sensitive transmission intensity estimation tools. Models however estimate yearly seroconversion and seroreversion rates and usually predict long term changes in transmission, occurring years before the time of sampling. Another challenge is the identification of appropriate antigen targets since specific antibody levels must directly reflect changes in transmission patterns. We therefore investigated the potential of antibodies to sporozoite and blood stage antigens for detecting short term differences in malaria transmission in two communities in Northern Ghana with marked, seasonal transmission. Methods Cross-sectional surveys were conducted during the rainy and dry seasons in two communities, one in close proximity to an irrigation dam and the other at least 20 Km away from the dam. Antibodies against the sporozoite-specific antigens circumsporozoite protein (CSP) and Cell traversal for ookinetes and sporozoites (CelTOS) and the classical blood stage antigen apical membrane antigen 1 (AMA1) were measured by indirect ELISA. Antibody levels and seroprevalence were compared between surveys and between study communities. Antibody seroprevalence data were fitted to a modified reversible catalytic model to estimate the seroconversion and seroreversion rates. Results Changes in sporozoite-specific antibody levels and seroprevalence directly reflected differences in parasite prevalence between the rainy and dry seasons and hence the extent of malaria transmission. Seroconversion rate estimates from modelled seroprevalence data did not however support the above observation. Conclusions The data confirms the potential utility of sporozoite-specific antigens as useful markers for monitoring short term/seasonal changes in malaria transmission. It may however be essential to update models to allow for assessment of seasonal changes in malaria transmission, which usually occur within four to six months.

Clonal types of Toxoplasma gondii among immune compromised and immune competent individuals in Accra, Ghana

There are three major clonal lineages, types I, II, and III, of Toxoplasma gondii known to cause human toxoplasmosis worldwide. Toxoplasma gondii infections have, however, not been genotyped in Ghana. This study detected the clonal types infecting immune compromised and immune competent individuals in Accra, Ghana. Blood samples were obtained from 148 HIV seropositive pre-antiretroviral therapy individuals (0 ≤ CD4(+) T-cell count/μl blood ≤ 200) at the Fevers Unit and 149 HIV seronegative apparently healthy blood donors at the blood bank, all of the Korle-Bu Teaching Hospital. Genomic DNA was extracted and multilocus genotyping conducted by nested PCR-RFLP analysis using GRA6, SAG3, and BTUB gene markers. Among the HIV seropositive participants, 54.7% (81/148) were T. gondii DNA positive for any of the markers. Out of the 81, 42.0% (34) were positive for SAG3 only, 30.9% (25) for GRA6 only, 24.7% (20) for both SAG3 and GRA6, and 2.5% (2) for SAG3, GRA6, and BTUB. Overall, 93.8% of the positives were of clonal type II, 1.2% type I, while 4.9% (4) were atypical or mixed types (I and II). In the healthy blood donors, prevalence of T. gondii DNA positivity was 3.4% (5/149) by SAG3 and/or GRA6; among them, 60.0% (3/5) were type I, and the remaining 40.0%, type II. This study showed a relatively high prevalence of active T. gondii infections in immune compromised patients and low prevalence in immune competent individuals in Accra. Type II was highly prevalent. Detection of T. gondii in blood donors raises public health concerns and screening for T. gondii should be considered.

Measuring regional and district variations in the incidence of pregnancy‐induced hypertension in Ghana: challenges, opportunities and implications for maternal and newborn health policy and programmes

The objectives were to assess the quality of health management information system (HMIS) data needed for assessment of local area variation in pregnancy‐induced hypertension (PIH) incidence and to describe district and regional variations in PIH incidence.

Improved prediction of gestational hypertension by inclusion of placental growth factor and pregnancy associated plasma protein-a in a sample of Ghanaian women

BackgroundWe assessed whether adding the biomarkers Pregnancy Associated Plasma Protein-A (PAPP-A) and Placental Growth Factor (PlGF) to maternal clinical characteristics improved the prediction of a previously developed model for gestational hypertension in a cohort of Ghanaian pregnant women.MethodsThis study was nested in a prospective cohort of 1010 pregnant women attending antenatal clinics in two public hospitals in Accra, Ghana. Pregnant women who were normotensive, at a gestational age at recruitment of between 8 and 13xa0weeks and provided a blood sample for biomarker analysis were eligible for inclusion. From serum, biomarkers PAPP-A and PlGF concentrations were measured by the AutoDELFIA immunoassay method and multiple of the median (MoM) values corrected for gestational age (PAPP-A and PlGF) and maternal weight (PAPP-A) were calculated. To obtain prediction models, these biomarkers were included with clinical predictors maternal weight, height, diastolic blood pressure, a previous history of gestational hypertension, history of hypertension in parents and parity in a logistic regression to obtain prediction models. The Area Under the Receiver Operating Characteristic Curve (AUC) was used to assess the predictive ability of the models.ResultsThree hundred and seventy three women participated in this study. The area under the curve (AUC) of the model with only maternal clinical characteristics was 0.75 (0.64–0.86) and 0.89(0.73–1.00) for multiparous and primigravid women respectively. The AUCs after inclusion of both PAPP-A and PlGF were 0.82 (0.74–0.89) and 0.95 (0.87–1.00) for multiparous and primigravid women respectively.ConclusionAdding the biomarkers PAPP-A and PlGF to maternal characteristics to a prediction model for gestational hypertension in a cohort of Ghanaian pregnant women improved predictive ability. Further research using larger sample sizes in similar settings to validate these findings is recommended.