Diagnostic capacity, and predictive values of rapid diagnostic tests for accurate diagnosis of Plasmodium falciparum in febrile children in Asante-Akim, Ghana.

BACKGROUND: This study seeks to compare the performance of HRP2 (First Response) and pLDH/HRP2 (Combo) RDTs for falciparum malaria against microscopy and PCR in acutely ill febrile children at presentation and follow-up. METHODS: This is an interventional study that recruited children < 5 years who reported to health facilities with a history of fever within the past 72 h or a documented axillary temperature of 37.5 °C. Using a longitudinal approach, recruitment and follow-up of participants was done between January and May 2012. Based on results of HRP2-RDT screening, the children were grouped into one of the following three categories: (1) tested positive for malaria using RDT and received anti-malarial treatment (group 1, n = 85); (2) tested negative for malaria using RDT and were given anti-malarial treatment by the admitting physician (group 2, n = 74); or, (3) tested negative for malaria using RDT and did not receive any anti-malarial treatment (group 3, n = 101). Independent microscopy, PCR and Combo-RDT tests were done for each sample on day 0 and all follow-up days. RESULTS: Mean age of the study participants was 22 months and females accounted for nearly 50%. At the time of diagnosis, the mean body temperature was 37.9 °C (range 35-40.1 °C). Microscopic parasite density ranged between 300 and 99,500 parasites/µL. With microscopy as gold standard, the sensitivity of HRP2 and Combo-RDTs were 95.1 and 96.3%, respectively. The sensitivities, specificities and predictive values for RDTs were relatively higher in microscopy-defined malaria cases than in PCR positive-defined cases. On day 0, participants who initially tested negative for HRP2 were positive by microscopy (n = 2), Combo (n = 1) and PCR (n = 17). On days 1 and 2, five of the children in this group (initially HRP2-negative) tested positive by PCR alone. On day 28, four patients who were originally HRP2-negative tested positive for microscopy (n = 2), Combo (n = 2) and PCR (n = 4). CONCLUSION: The HRP2/pLDH RDTs showed comparable diagnostic accuracy in children presenting with an acute febrile illness to health facilities in a hard-to-reach rural area in Ghana. Nevertheless, discordant results recorded on day 0 and follow-up visits using the recommended RDTs means improved malaria diagnostic capability in malaria-endemic regions is necessary.

A Novel Gametocyte Biomarker for Superior Molecular Detection of the Plasmodium falciparum Infectious Reservoirs.

Background: Complete malaria eradication and optimal use of transmission-reducing interventions require knowledge of submicroscopic infectious reservoirs among asymptomatic individuals. Even submicroscopic levels of Plasmodium falciparum gametocytes can infect mosquitoes and promote onward transmission. Most efforts to identify gametocyte carriers use polymerase chain reaction amplification of the gametocyte-specific transcript Pfs25. Methods: To expand the repertoire of biomarkers available for superior gametocyte detection, we compared the gene expression profiles of gametocytes and asynchronous blood-stage P. falciparum parasites by microarray technology. This allowed the identification of 56 molecules abundantly expressed in the gametocyte stage of the parasite. The analytical sensitivity for gametocyte detection was evaluated for 25 genes with the highest expression levels. Results: One candidate, Pfg17, exhibited superior analytical sensitivity against a panel of gametocyte-spiked whole blood, detecting 10 gametocytes/mL; in comparison, Pfs25 detected only 25.3 gametocytes/mL. Pfg17 also exhibited superior clinical sensitivity, identifying 19.1% more samples from blood-film microscopy-negative Ghanaian children and 40% more samples from asymptomatic adults as gametocyte positive. Conclusions: Cumulatively, our results suggest Pfg17 is an excellent biomarker for detecting asymptomatic infectious reservoirs otherwise missed by the most sensitive molecular method available. Our study has also improved the repertoire of transmission-stage antigens available for evaluation as candidate vaccines.

Insights into the Affordable Medicines Facility-malaria in Ghana: the role of caregivers and licensed chemical sellers in four regions.

BACKGROUND: The Affordable Medicine Facility-malaria (AMFm) was an innovative global financing mechanism for the provision of quality-assured artemisinin-based combination therapy (ACT) across both the private and public health sectors in eight countries in sub-Saharan Africa. This study evaluated the effectiveness of AMFm subsidies in increasing access to ACT in Ghana and documented malaria management practices at the household and community levels during the implementation of the AMFm. METHODS: This study, conducted in four regions in Ghana between January, 2011 to December, 2012, employed cross-sectional mixed-methods design that included qualitative and quantitative elements, specifically household surveys, focus group discussions (FGD) and in-depth interviews. RESULTS: The study indicated high ACT availability, adequate provider knowledge and reasonably low quality-assured ACT use in the study areas, all of which are a reflection of a high market share of ACT in these hard-to-reach areas of the country. Adequate recognition of childhood malaria symptoms by licensed chemical seller (LCS) attendants was observed. A preference by caregivers for LCS over health facilities for seeking treatment solutions to childhood malaria was found. CONCLUSIONS: Artemisinin-based combination therapy with the AMFm logo was accessible and affordable for most people seeking treatment from health facilities and LCS shops in rural areas. Caregivers and LCS were seen to play key roles in the health of the community especially with children under 5 years of age.

Polymerase chain reaction-based tests for pan-species and species-specific detection of human Plasmodium parasites.

BACKGROUND: There is still a need to improve the sensitivity of polymerase chain reaction (PCR) tests for malaria to detect submicroscopic asexual stage Plasmodium infections during the early phase and chronic, asymptomatic phase of infection when the parasite burden is very low. STUDY DESIGN AND METHODS: The inhibitory effect of hemoglobin (Hb) on PCR limits the volume of blood that can be used in the PCR-based detection of intraerythrocytic Plasmodium parasites. We lysed red blood cells with saponin to reduce the Hb concentration in extracted nucleic acid and, as a result, significantly increased the volume of blood that can be tested by PCR. The analytical sensitivity of the PCR was determined using whole blood spiked with ring-stage Plasmodium falciparum parasites, and its clinical sensitivity by testing blood film-positive and blood film-negative samples from individuals living in an endemic area in Ghana. RESULTS: We have developed a pan-Plasmodium PCR that detects all five human Plasmodium species with the highest analytical sensitivity of two P. falciparum parasites/mL of whole blood and species-specific PCR tests that distinguished between the five human Plasmodium species. Pan-Plasmodium PCR detected 78 of 78 (100%) blood film-positive and 19 of 101 (18.81%) blood film-negative samples from asymptomatic individuals living in Ghana. Pan-Plasmodium PCR was equally sensitive with samples collected as anticoagulated whole blood and clotted blood and in blood collected by finger stick into capillaries. CONCLUSION: We have developed PCR tests with the highest reported sensitivity to date for pan-Plasmodium diagnosis and species-specific diagnosis and detected blood film-negative asymptomatic infections in individuals living in malaria-endemic countries.

Relationship of hepcidin with parasitemia and anemia among patients with uncomplicated Plasmodium falciparum malaria in Ghana.

The pathogenesis of malarial anemia is incompletely understood. Hepcidin, a recently discovered peptide hormone, is a major regulator of iron metabolism and is thought to play a central role in the anemia of chronic inflammation. The specific aim of the study was to characterize the association between urinary hepcidin, hemoglobin, and parasitemia in 199 patients presenting for evaluation of Plasmodium falciparum malaria in Ghana. Urinary hepcidin was semi-quantitatively assessed using surface-enhanced laser desorption/ionization time-of-flight mass spectrometry. Urinary hepcidin (intensity/mmol creatinine) was associated with log parasitemia in 86 children (beta = 0.086, standard error [SE] = 0.035, P < 0.017), 31 pregnant women (beta = 0.218, SE = 0.085, P < 0.016), and 82 adults (beta = 0.184, SE =0.043, P < 0.0001). Urinary hepcidin was not significantly associated with hemoglobin or anemia. Urinary hepcidin is more strongly associated with parasitemia than hemoglobin or anemia among patients with acute P. falciparum malaria in Ghana.

Targeted community based interventions improved malaria management competencies in rural Ghana.

BACKGROUND: Malaria is one of the most challenging public health concerns in the developing world. To address its impact in endemic regions, several interventions are implemented by stakeholders. The Affordable Medicine Facility-malaria (AMFm) is an example of such interventions. Its activities include communication interventions to enhance the knowledge of caregivers of children under five years, licensed chemical sellers (LCS) and prescribers on malaria management with artemisinin-based combination therapy (ACT). This study was conducted to evaluate the effectiveness of the AMFm activities on malaria among targeted groups in two rural communities in Ghana. METHODS: A communication intervention study was conducted in the Asante-Akim North and South Districts of Ghana. Repeated cross-sectional pre and post surveys were deployed. Relevant malaria messages were designed and used to develop the information, education and communication (IEC) tools for the intervention. With the aid of posters and flipcharts developed by our study, community health workers (CHWs), prescribers, and licenced chemical sellers provided proper counselling to clients on malaria management. Trained CHWs and community based volunteers educated caregivers of children under five years on malaria management at their homes and at public gatherings such as churches, mosques, schools. Chi-square tests and logistic regression were run to determine associations and control for demographic differences respectively. RESULTS: There was significantly high exposure to malaria/ACT interventions in the intervention district than in the comparison district (OR = 16.02; 95% CI = 7.88-32.55) and same for malaria/ACT-related knowledge (OR = 3.63; 95% CI = 2.52-5.23). The participants in the intervention district were also more knowledgeable about correct administration of dispersible drug for children <5 years than their counterparts in the unexposed district. CONCLUSION: Our data show that targeted interventions improve malaria based competences in rural community settings. The availability of subsidized ACTs and the intensity of the communication campaigns contributed to the AMFm-related awareness, improved knowledge on malaria/ACTs and management practices.

Characterization of malaria transmission by vector populations for improved interventions during the dry season in the Kpone-on-Sea area of coastal Ghana.

BACKGROUND: Malaria is a major public health problem in Ghana. We present a site-specific entomological study of malaria vectors and transmission indices as part of an effort to develop a site for the testing of improved control strategies including possible vaccine trials. METHODS: Pyrethrum spray catches (PSC), and indoor and outdoor human landing collections of adult female anopheline mosquitoes were carried out over a six-month period (November 2005 - April 2006) at Kpone-on-Sea, a fishing village in southern Ghana. These were morphologically identified to species level and sibling species of the Anopheles gambiae complex further characterized by the polymerase chain reaction (PCR). Enzyme-linked immunosorbent assay was used to detect Plasmodium falciparum mosquito infectivity and host blood meal sources. Parity rate was examined based on dilatation of ovarian tracheoles following dissection. RESULTS: Of the 1233 Anopheles mosquitoes collected, An. gambiae s.l. was predominant (99.5%), followed by An. funestus (0.4%) and An. pharoensis (0.1%). All An. gambiae s.l. examined (480) were identified as An. gambiae s.s. with a majority of M molecular form (98.2%) and only 1.8% S form with no record of M/S hybrid. A significantly higher proportion of anophelines were observed outdoors relative to indoors (χ2 = 159.34, df = 1, p < 0.0000). Only An. gambiae M molecular form contributed to transmission with a high degree of anthropophily, parity rate and an estimated entomological inoculation rate (EIR) of 62.1 infective bites/person/year. The Majority of the infective bites occurred outdoors after 09.00 pm reaching peaks between 12.00-01.00 am and 03.00-04.00 am. CONCLUSION: Anopheles gambiae M molecular form is responsible for maintaining the status quo of malaria in the surveyed site during the study period. The findings provide a baseline for evidence-based planning and implementation of improved malaria interventions. The plasticity observed in biting patterns especially the combined outdoor and early biting behavior of the vector may undermine the success of insecticide-based strategies using insecticide treated nets (ITN) and indoor residual spray (IRS). As such, novel or improved vector interventions should be informed by the local malaria epidemiology data as it relates to vector behavior.