Trends in multiplicity of Plasmodium falciparum infections among asymptomatic residents in the middle belt of Ghana.

BACKGROUND: Malaria is the most important cause of mortality and morbidity in children living in the Kintampo districts in the middle part of Ghana. This study has investigated the multiplicity of infection (MOI) within asymptomatic residents of the Kintampo districts, and the influence of age and seasonality on MOI, by studying the distribution of the polymorphic Plasmodium falciparum antigen merozoite surface protein 2 (MSP2). METHODS: DNA was extracted from an asymptomatic cohort of children and adults infected with P. falciparum during the period November 2003 to October 2004. Polymerase chain reaction was carried out and multiplicity of infection (MOI) was determined. RESULTS: Children under 10 years of age had an average MOI of 2.3 while adults 18 years and above had an average MOI of 1.4. Children below five years had high and low average MOIs of 2.8 in the March/April survey and 0.9 in the May/June survey respectively. A similar trend in the monthly distribution of MOI was observed for the entire cohort. IC/3D7 strains outnumbered the FC27 strains throughout the year by a ratio of about 4:1 with the difference between the prevalence of the two strains being least marked in the March/April survey, at the beginning of the rainy season. MOI was not linked to the level of malaria transmission as measured by the entomological inoculation rate. DISCUSSION/CONCLUSION: The impact of interventions, introduced since this baseline study was carried out on the parasite diversity of asymptomatic residents will be the subject of further investigations.

Misclassification of Plasmodium infections by conventional microscopy and the impact of remedial training on the proficiency of laboratory technicians in species identification.

BACKGROUND: Malaria diagnosis is largely dependent on the demonstration of parasites in stained blood films by conventional microscopy. Accurate identification of the infecting Plasmodium species relies on detailed examination of parasite morphological characteristics, such as size, shape, pigment granules, besides the size and shape of the parasitized red blood cells and presence of cell inclusions. This work explores misclassifications of four Plasmodium species by conventional microscopy relative to the proficiency of microscopists and morphological characteristics of the parasites on Giemsa-stained blood films. CASE DESCRIPTION: Ten-day malaria microscopy remedial courses on parasite detection, species identification and parasite counting were conducted for public health and research laboratory personnel. Proficiency in species identification was assessed at the start (pre) and the end (post) of each course using known blood films of Plasmodium falciparum, Plasmodium malariae, Plasmodium ovale and Plasmodium vivax infections with densities ranging from 1,000 to 30,000 parasites/µL. Outcomes were categorized as false negative, positive without speciation, P. falciparum, P. malariae, P. ovale, P. vivax and mixed infections. DISCUSSION AND EVALUATION: Reported findings are based on 1,878 P. falciparum, 483 P. malariae, 581 P. ovale and 438 P. vivax cumulative results collated from 2008 to 2010 remedial courses. Pre-training false negative and positive misclassifications without speciation were significantly lower on P. falciparum infections compared to non-falciparum infections (p < 0.0001). Post-training misclassifications decreased significantly compared to pre- training misclassifications which in turn led to significant improvements in the identification of the four species. However, P. falciparum infections were highly misclassified as mixed infections, P. ovale misclassified as P. vivax and P. vivax similarly misclassified as P. ovale (p < 0.05). CONCLUSION: These findings suggest that the misclassification of malaria species could be a common occurrence especially where non-falciparum infections are involved due to lack of requisite skills in microscopic diagnosis and variations in morphological characteristics within and between Plasmodium species. Remedial training might improve reliability of conventional light microscopy with respect to differentiation of Plasmodium infections.

Estimating malaria parasite density: assumed white blood cell count of 10,000/µl of blood is appropriate measure in Central Ghana.

BACKGROUND: White blood cells count (WBCc) is a bedrock in the estimation of malaria parasite density in malaria field trials, interventions and patient management. White blood cells are indirectly and relatively used in microscopy to estimate the density of malaria parasite infections. Due to frequent lack of facilities in some malaria-endemic countries, in order to quantify WBCc of patients, an assumed WBCc of 8.0 X 10(9)/L has been set by the World Health Organization to help in estimating malaria parasite densities. METHODS: This comparative analysis study, in Central Ghana, compiled laboratory data of 5,902 Plasmodium falciparum malaria parasite positive samples. Samples were obtained from consented participants of age groups less than five years. Full blood counts (FBC) of participants' samples were analysed using the ABX Micros 60 Haematology Analyzer. Blood slides were read by two competent microscopists to produce concordant results. All internal and external quality control measures were carried out appropriately. Parasite densities were calculated using participants' absolute WBCc and assumed WBCc of 5,000 to 10,000 per microlitre of blood. RESULTS: From the 5,902 Pf malaria positive samples, the mean (SD) WBCc and geometric mean parasite density were 10.4 (4.6) × 10(9)/L and 7,557/µL (95% CI 7,144/µL to 7,994/µL) respectively. The difference in the geometric mean parasite densities calculated using absolute WBCs and compared to densities with assumed WBCs counts were significantly lower for 5.0 × 10(9)/L; 3,937/µL, 6.0 × 10(9)/L; 4,725/µL and 8.0 × 10(9)/L; 6,300/µL. However, the difference in geometric mean parasite density, 7,874/µL (95 % CI, 7,445/µL to 8,328/µL), with assumed WBCc of 10.0 × 10(9)/L was not significant. CONCLUSION: Using the assumed WBCc of 8.0 X 10(9)/L or lower to estimate malaria parasite densities in Pf infected children less than five years old could result in significant underestimation of parasite burden. Assumed WBCc of 10.0 × 10(9)/L at 95 % CI of geometric mean of parasite density statistically agreed with the parasite densities produce by the absolute WBCc of participants. The study suggests where resources are limited, use of assumed WBCc of 10.0 × 10(9)/L of blood to estimate malaria parasite density in central Ghana. Preferably, absolute WBCc should be used in drug efficacy and vaccine trials.

Patterns and seasonality of malaria transmission in the forest-savannah transitional zones of Ghana.

BACKGROUND: Knowledge of the local pattern of malaria transmission and the effect of season on transmission is essential for the planning and evaluation of malaria interventions. Therefore, entomological surveys were carried out in the forest-savannah transitional belt of Ghana (Kintampo) from November 2003 to November 2005 in preparation for drug and vaccine trials. RESULTS: A total of 23,406 mosquitoes were caught from 919 traps over the two-year period (November 2003 to November 2005): 54.3% were Culicines, 36.2% Anopheles funestus, and 9.4% Anopheles gambiae. Infection rates with Plasmodium falciparum were 4.7% and 1.5% for Anopheles gambiae and Anopheles funestus, respectively. Entomological inoculation rates (EIRs) were 269 infective bites per person per year in the first year (November 2003-October 2004) and 231 the following year (November 2004-November 2005). Polymerase Chain Reaction (PCR) analysis detected only Anopheles gambiae s.s. Nineteen mosquitoes were tested by PCR in the wet season; 16 were S-molecular form, 2 M-molecular form and 1 hybrid (S/M). In the dry season, sixteen mosquitoes were tested; 11 S-molecular form, 2 M-molecular form and 3 S/M hybrids. The frequency of knock down resistance (kdr) genotypes F(R) was 0.60. CONCLUSION: The dynamics and seasonal abundance of malaria vectors in the Kintampo area was influenced by micro-ecology, rainfall and temperature patterns. Transmission patterns did not differ significantly between the two years (2004 and 2005) and both Anopheles gambiae and Anopheles funestus were identified as effective vectors. EIR estimates in 2004/2005 were between 231 and 269 infective bites per person per year. The information provided by the study will help in planning intensified malaria control activities as well as evaluating the impact of malaria interventions in the middle belt of Ghana.

Epidemiology of malaria in the forest-savanna transitional zone of Ghana.

BACKGROUND: Information on the epidemiology of malaria is essential for designing and interpreting results of clinical trials of drugs, vaccines and other interventions. As a background to the establishment of a site for anti-malarial drugs and vaccine trials, the epidemiology of malaria in a rural site in central Ghana was investigated. METHODS: Active surveillance of clinical malaria was carried out in a cohort of children below five years of age (n = 335) and the prevalence of malaria was estimated in a cohort of subjects of all ages (n = 1484) over a 12-month period. Participants were sampled from clusters drawn around sixteen index houses randomly selected from a total of about 22,000 houses within the study area. The child cohort was visited thrice weekly to screen for any illness and a blood slide was taken if a child had a history of fever or a temperature greater than or equal to 37.5 degree Celsius. The all-age cohort was screened for malaria once every eight weeks over a 12-month period. Estimation of Entomological Inoculation Rate (EIR) and characterization of Anopheline malaria vectors in the study area were also carried out. RESULTS: The average parasite prevalence in the all age cohort was 58% (95% CI: 56.9, 59.4). In children below five years of age, the average prevalence was 64% (95% CI: 61.9, 66.0). Geometric mean parasite densities decreased significantly with increasing age. More than 50% of all children less than 10 years of age were anaemic. Children less than 5 years of age had as many as seven malaria attacks per child per year. The attack rates decreased significantly with increasing cut-offs of parasite density. The average Multiplicity of Infection (MOI) was of 6.1. All three pyrimethamine resistance mutant alleles of the Plasmodium falciparum dhfr gene were prevalent in this population and 25% of infections had a fourth mutant of pfdhps-A437G. The main vectors were Anopheles funestus and Anopheles gambiae and the EIR was 269 infective bites per person per year. CONCLUSION: The transmission of malaria in the forest-savanna region of central Ghana is high and perennial and this is an appropriate site for conducting clinical trials of anti-malarial drugs and vaccines.