Absence of asymptomatic malaria in a cohort of 133 individuals in a malaria endemic area of Assam, India.

BACKGROUND: Malaria in northeast India affects children and adults annually. The number of malaria cases reported has declined over the past few years. Nevertheless, it is not clear whether there is an actual decline in parasitaemia or whether asymptomatic malaria infections are on the rise, especially in forested and forest-fringed areas. Asymptomatic malaria forms a parasite reservoir that acts as an epicentre for malaria spread during high-transmission season. Therefore it is important to understand the quantum of asymptomatic malaria infections among the vulnerable population. METHOD: Four forest fringed historically malaria endemic villages were selected for the study. A total of 133 individuals without a fever history in the past four weeks were tested for malaria parasite using rapid diagnostic test (RDT), microscopy and polymerase chain reaction (PCR) assay during January - February 2014. Indoor resting Anopheles vectors were collected, identified and tested for sporozoite using VectorTest™ panel assay during October 2013 to March 2014, which is a low transmission season for malaria. Social and demographic data were recorded during the study. RESULTS: Mean age (± SEM) of the participants was 16.1 ± 1.2 years (95 % CI: 13.8-18.4). All participants (100 %) reported to use mosquito nets. Altogether, 43.6 % of participants had education below primary level and only 9 % reported a travel history during the past four weeks. All RDT, microscopy and PCR assays were found negative indicating no asymptomatic malaria parasitaemia. Seven known malaria vector species namely, Anopheles nivipes, An. minimus, An. annularis, An. vagus, An. aconitus, An. philippinensis and An. culicifacies, were recorded in the present study. VectorTest™ sporozoite panel assay conducted on 45 pools (N = 224) of vector mosquitoes were found negative for Plasmodium sporozoite. DISCUSSION: Northeastern states of India report asymptomatic malaria parasitemia along with high malaria transmission. An. minimus and An. dirus are recognised as efficient vectors, but An. culicifacies, An. philippinensis and An. annularis also play role in malaria transmission. Currently all participants were found negative for asymptomatic malaria, however the small sample size may restrict the scope of present results to the population living in more remote areas. CONCLUSION: No cases of asymptomatic malaria infections parasitaemia was found in the present study conducted during a low transmission season indicating that asymptomatic malaria parasitaemia may not be prevalent in the region. Mosquito specimens were tested negative for the malaria sporozoites. Study findings encourage the ongoing malaria intervention efforts and recommends similar investigations in different ecological areas involving large populations.

Genetic polymorphism and amino acid sequence variation in Plasmodium falciparum GLURP R2 repeat region in Assam, India, at an interval of five years.

BACKGROUND: The R2 repeat region of GLURP has been reported as a good genetic marker for Plasmodium falciparum genotyping. Proper knowledge of the extent and nature of P. falciparum genetic diversity using highly immunogenic R2 repeat region in malaria-endemic areas is a crucial element to understand various aspects related to immunity acquisition and disease pathogenesis. METHODS: Population diversity of P. falciparum GLURP and amino acid sequence repeats in GLURP R2 region was studied in malaria-endemic Assam state, northeast India and compared at an interval of five years during 2005 (Group-A) and 2011 (Group-B). RESULTS: Of the 66 samples, a total of 55 samples showed positive PCR bands for GLURP R2 region and altogether ten types of alleles with size ranging from 501 bp to 1,050 bp (50 bp bin) were observed and coded as genotypes I-X. In Group-A (n = 29), 24 samples were found infected with single, four with double and one with triple P. falciparum genotype, while in Group-B (n = 26), single genotype was found in 23 samples, double in two samples and triple in one sample. Genotype IV showed significant increase (p = 0.002) during 2011 (Group-B). Genotypes I to V were more common in Group-B (62%), however genotypes VI to X were more frequently distributed in Group-A. The expected heterozygosity was found slightly higher in Group-A (HE = 0.87) than Group-B (HE = 0.85), whereas multiplicity of infection (MOI) in Group-A (MOI = 1.21 ± 0.49) and Group-B (MOI = 1.12 ± 0.43) did not display significant variation. The amino acid repeat sequence unit (AAU) DKNEKGQHEIVEVEEILPE (called 'a') was more frequent in the well-conserved part of R2 repeat region. CONCLUSION: The present study is the first extensive study in India which has generated substantial data for understanding the type and distribution of naturally evolved genetic polymorphism at amino acid sequence level in GLURP R2 repeat region in P. falciparum. There was decrease in the PCR amplicon size as well as the number of AAU [amino acid repeat unit] in Group-B displaying the bottleneck effect. The present study described a new type of AAU 'd' which varied from the other previous known AAUs.

Nested PCR detection of Plasmodium malariae from microscopy confirmed P. falciparum samples in endemic area of NE India.

The present study evaluates the performance of OptiMAL-IT test and nested PCR assay in detection of malaria parasites. A total of 76 randomly selected blood samples collected from two malaria endemic areas were tested for malaria parasites using microscopy and OptiMAL-IT test in the field. PCR assays were performed in the laboratory using DNA extracted from blood spots of the same samples collected on the FTA classic cards. Of the total of 61 field confirmed malaria positive samples, only 58 (95%) were detected positive using microscopy in the laboratory. Sensitivity, specificity, positive predictive value, negative predictive value and false discovery rate of OptiMal-IT in comparison to the microscopy were 93%, 83%, 95%, 79% and 5%, respectively. On the other hand, the sensitivity and specificity of PCR assay were 97% and 100%, respectively, whereas positive predictive value, negative predictive value and false discovery rate were 100%, 90% and 0%, respectively. The overall performance of OptiMal-IT and PCR assays for malaria diagnosis was 76% and 97%, respectively. PCR assay enabled the identification of infection with Plasmodium malariae Laveran, 1881 in four samples misidentified by microscopy and Plasmodium-specific antigen (PAN) identified by the OptiMAL-IT test. In addition to the standard methods, such PCR assay could be useful to obtain the real incidence of each malaria parasite species for epidemiological perspectives.

Malaria incidence among paramilitary personnel in an endemic area of Tripura.

BACKGROUND & OBJECTIVES: Paramilitary operations along the Indo-Bangladesh border are adversely affected by malaria induced morbidity and mortality. Villages surrounding the paramilitary installations often serve as disease reservoirs. Malaria incidence in Tripura State Rifles (TSR) units in Dhalai District of Tripura was studied and the role of the village population in disease transmission was also assessed. METHODS: Mass blood surveys were carried out among TSR personnel and villagers during 2007 to 2009. Malaria diagnosis through blood smear examination and rapid detection kits was done, and percentage parasitaemia was determined. Activity of malaria vectors was monitored using CDC light traps. RESULTS: Slide positivity rates (SPR) in the neighbouring villages (51.4%) was significantly higher than that in TSR (27.7%) (P<0.0001). Malaria incidence in villages did not show seasonal variability while it was lowest during post-monsoon season in TSR (P<0.325; OR = 0.74). Per cent Pf parasitaemia was high in TSR (0.29) as compared to villagers (0.20) (P<0.0001). Anopheles minimus and An. dirus were the major malaria vectors observed. INTERPRETATION & CONCLUSIONS: Paramilitary and public health authorities should adopt targeted measures to reduce the malaria incidence in the villages surrounding the paramilitary installations as the village populations play a major role in disease transmission.

Malaria epidemiology along Indo-Bangladesh border in Tripura State, India.

Malaria epidemiological surveys were conducted in 16 villages along the Indo-Bangladesh border in Tripura, northeastern India. Insecticide resistance among malaria vectors and chloroquine resistance in the parasite were also studied along with monitoring of vector density using light traps. The epidemiological data indicated that malaria incidence was highest during June-July and lowest during November. Examination of blood smears collected through door to door surveys indicated slide positivity rate (SPR) of 25.2% and that Plasmodium falciparum was the predominant parasite (slide falciparum rate of 22.3%). The incidence rates of falciparum malaria varied significantly among the age groups (p<0.001) and 2-4 year olds were the most affected. Major malaria vectors recorded in light trap collections were An. dirus, An. minimus and An. philippinensis/nivipes. Chloroquine resistance studies indicated that treatment failure occurred in 35% of the cases and hence the use of artesunate combination therapy (ACT) was recommended for treatment of malaria in the area.

Bio-nanoparticle assembly: a potent on-site biolarvicidal agent against mosquito vectors.

Background: Vector-borne diseases such as malaria, dengue, yellow fever, encephalitis and filariasis are considered serious human health concerns in the field of medical entomology. Controlling the population of mosquito vectors is one of the best strategies for combating such vector-borne diseases. However, the use of synthetic insecticides for longer periods of time increases mosquito resistance to the insecticides. Recently, the search for new environmentally friendly and efficient insecticides has attracted major attention globally. With the evolution of material sciences, researchers have reported the effective control of such diseases using various sustainable resources. The present investigation demonstrates a potent on-site biolarvicidal agent against different mosquito vectors such as Aedes albopictus, Anopheles stephensi and Culex quinquefasciatus. Methods: Stable and photo-induced colloidal silver nanoparticles were generated via the surface functionalization of the root extract of Cyprus rotundas. Characterizations of the nanoparticles were performed using assorted techniques, such as UV-visible spectroscopy, FTIR spectroscopy, DLS and HRTEM. The bioefficacy of the synthesized nanoparticles was investigated against different species of mosquito larvae through the evaluation of their life history trait studies, fecundity and hatchability rate of the treated larvae. Histopathological and polymerase chain reaction-random amplified polymorphic DNA (RAPD) analyses of the treated larvae were also examined to establish the cellular damage. Results: The synthesized nanoparticles showed remarkable larvicidal activity against mosquito larvae in a very low concentration range (0.001-1.00) mg L-1. The histopathological study confirmed that the present nanoparticles could easily enter the cuticle membrane of mosquito larvae and subsequently obliterate their complete intestinal system. Furthermore, RAPD analysis of the treated larvae could assess the damage of the DNA banding pattern. Conclusion: The present work demonstrates a potent biolarvicidal agent using sustainable bioresources of the aqueous Cyprus rotundas root extract. The results showed that the synthesized nanoparticles were stable under different physiological conditions such as temperature and photo-induced oxidation. The effectiveness of these materials against mosquito larvae was quantified at very low dose concentrations. The present biolarvicidal agent can be considered as an environmentally benign material to control the mosquito vectors with an immense potential for on-site field applications.

Disappearance of Anopheles minimus and Anopheles dirus from Certain Malaria Endemic Areas of Assam, India.

BACKGROUND: Orang Primary Health Centre (OPHC) and Balipara Primary Health Centre (BPHC) of Assam (India) report mosquito borne diseases annually. Current study was performed to ascertain the prevalence of known malaria and Japanese Encephalitis (JE) vectors and their possible role in disease transmission. METHODS: Malaria epidemiological data for 2006-2010 and JE data for 2008-2013 of Assam, India were obtained from the health authority. Mosquitoes were collected using CDC light traps and identified morpho-taxonomically. RESULTS: Plasmodium falciparum cases (81.5%, 95% CI= 72.0-91.1) were statistically higher in OPHC (P< 0.0001, t= 8.0) during the recent years. There was 4.4 folds rise in the confirmed acute encephalitis syndrome (AES) and 3.2 folds increase in the confirmed JE cases during 2013 as compared to 2008. Altogether 9,218 mosquito specimens (PTND= 153.6), comprising of 44.1% anophelines (PTND= 67.7), 42.3% culicines (PTND= 65.0) and 9.5% mansonia (PTND= 14.6) were recorded. In BPHC, Anopheles vagus was recorded in high density (P< 0.0001), whereas Culex quinquefasciatus was the predominant JE vector (P= 0.04). In OPHC, among the known malaria vectors, the density of Anopheles annularis was significantly high (P< 0.0001). However Culex bitaeniorhynchus was the predominant known JE vector (P< 0.0001) followed by Cx. quinquefasciatus. CONCLUSION: Even in the absence of known efficient vectors, many Anopheles species are still involved in malaria transmission. There was disappearance of Anopheles minimus and Anopheles dirus and establishment of An. annularis, An. vagus and An. philippinensis/nivipes mosquitoes in study area.

Evaluation of Insecticides Susceptibility and Malaria Vector Potential of Anopheles annularis s.l. and Anopheles vagus in Assam, India.

During the recent past, development of DDT resistance and reduction to pyrethroid susceptibility among the malaria vectors has posed a serious challenge in many Southeast Asian countries including India. Current study presents the insecticide susceptibility and knock-down data of field collected Anopheles annularis sensu lato and An. vagus mosquito species from endemic areas of Assam in northeast India. Anopheles annularis s.l. and An. vagus adult females were collected from four randomly selected sentinel sites in Orang primary health centre (OPHC) and Balipara primary health centre (BPHC) areas, and used for testing susceptibility to DDT, malathion, deltamethrin and lambda-cyhalothrin. After insecticide susceptibility tests, mosquitoes were subjected to VectorTest™ assay kits to detect the presence of malaria sporozoite in the mosquitoes. An. annularis s.l. was completely susceptible to deltamethrin, lambda-cyhalothrin and malathion in both the study areas. An. vagus was highly susceptible to deltamethrin in both the areas, but exhibited reduced susceptibility to lambda-cyhalothrin in BPHC. Both the species were resistant to DDT and showed very high KDT50 and KDT99 values for DDT. Probit model used to calculate the KDT50 and KDT99 values did not display normal distribution of percent knock-down with time for malathion in both the mosquito species in OPHC (p<0.05) and An. vagus in BPHC (χ2 = 25.3; p = 0.0), and also for deltamethrin to An. vagus in BPHC area (χ2 = 15.4; p = 0.004). Minimum infection rate (MIR) of Plasmodium sporozoite for An. vagus was 0.56 in OPHC and 0.13 in BPHC, while for An. annularis MIR was found to be 0.22 in OPHC. Resistance management strategies should be identified to delay the expansion of resistance. Testing of field caught Anopheles vectors from different endemic areas for the presence of malaria sporozoite may be useful to ensure their role in malaria transmission.

Preliminary in vitro antiplasmodial activity of Aristolochia griffithii and Thalictrum foliolosum DC extracts against malaria parasite Plasmodium falciparum.

BACKGROUND: Resistance development in human malaria parasites against commonly used antimalarial drugs has necessitated the scientific exploration of traditionally used antimalarial plants. Plant derivatives have been used for curing malaria historically. The present study involves in vitro evaluation of two medicinally important plants Aristolochia griffithii and Thalictrum foliolosum DC used in antimalarial chemotherapy by the tribes of northeast India. METHOD: Chloroform, ethyl acetate and n-butanol extracts of Aristolochia griffithii and Thalictrum foliolosum DC were evaluated in vitro against chloroquine sensitive (SS) and chloroquine resistance strains (RS) of P. falciparum. The tests were conducted following WHO standard method and the inhibition of parasite (IC50) was calculated. RESULTS: In A. griffithii, the IC50 value for ethyl acetate extracts against SS was 6.2 ± 0.02 µg/ml and found to be lower than chloroform extracts, which exhibited an IC50 value of 14.1 ± 0.1 µg/ml (t = 191.1; p < 0.0001). The IC50 values of both chloroform and ethyl acetate extracts for RS were higher as compared to the SS (p < 0.0001). In T. foliolosum DC the IC50 concentration of chloroform extracts for SS and RS were 0.5 ± 0.0 and 1.1 ± 0.0 µg/ml respectively (t = 54.2; p < 0.0001). CONCLUSION: The present findings, although preliminary, but scientifically demonstrate that identification and isolation of active compounds of these two plant materials and testing against different Plasmodium species could lead to the development of potential antimalarial drugs for future.

Role of asymptomatic carriers and weather variables in persistent transmission of malaria in an endemic district of Assam, India.

BACKGROUND: Malaria transmission is perennial in the Assam-Arunachal Pradesh interstate border areas in the Sonitpur district of Assam, India. A yearlong study was carried out on the incidence of symptomatic and asymptomatic malaria and the role of asymptomatic malaria carriers in persistent transmission of the disease. The relationships between malaria incidence and weather parameters were also investigated. METHODS: Active and mass blood surveys were conducted on a monthly basis in Bengenajuli, Sapairaumari Pathar, and Nigam villages near the Assam-Arunachal Pradesh border. Epidemiological indices were estimated for malaria-positive cases. Multiple linear regression between monthly malaria incidence and monthly average temperature, and relative humidity along with monthly total rainfall was carried out. The known malaria vectors collected in CDC light traps were identified and recorded. RESULTS: Slide positivity rate (SPR) and Plasmodium falciparum percent (Pf%) for symptomatic malaria were 26.1 and 79.8, respectively. Prevalence of malaria vectors was observed throughout the year with varying density. Anopheles philippinensis/nivipes and A. annularis were predominant among the seven known vector species recorded currently. Asymptomatic parasitemia was detected throughout the year with SPR ranging from 4.8 to 5.3. Monthly rainfall with 1-month lag had the highest correlation (r=0.92) with SPR. The relationship between SPR and weather factors was established as SPR=-114.22+0.58 Tmin+1.38 RH+0.03 RF (R2=0.89; p=0.00). CONCLUSION: Low and relatively constant levels of asymptomatic parasitemia was present in the study area. High malaria vector density and presence of asymptomatic malaria parasite carriers were responsible for persistent malaria transmission in the region. This study concludes that passive detection and prompt treatment of asymptomatic carriers is essential for preventing persistent disease transmission. Rainfall along with some other weather variables may be used for predicting the malaria epidemics in the region. The predictive information could be useful to target resources more effectively.

Synergistic mosquito-repellent activity of Curcuma longa, Pogostemon heyneanus and Zanthoxylum limonella essential oils.

Mosquito repellents play an important role in preventing man-mosquito contact. In the present study, we evaluated the synergistic mosquito-repellent activity of Curcuma longa, Pogostemon heyneanus and Zanthoxylum limonella essential oils. The mosquito repellent efficacies of three essential oils were evaluated separately and in combination under laboratory and field conditions. N,N-Diethylphenylacetamide (DEPA) and dimethylphthalate (DMP) were used for comparison of the protection time of the mixture of essential oils. At an optimum concentration of 20%, the essential oils of C. longa, Z. limonella and P. heyneanus provided complete protection times (CPTs) of 96.2, 91.4 and 123.4 min, respectively, against Aedes albopictus mosquitoes in the laboratory. The 1:1:2 mixture of the essential oils provided 329.4 and 391.0 min of CPT in the laboratory and field trials, respectively. The percent increases in CPTs for the essential oil mixture were 30 for DMP and 55 for N,N-diethylphenylacetamide (DEPA). The synergistic repellent activity of the essential oils used in the present study might be useful for developing safer alternatives to synthetic repellents for personal protection against mosquitoes.

Pfcrt mutant haplotypes may not correspond with chloroquine resistance.

INTRODUCTION: Chloroquine resistance in Plasmodium falciparum is associated with mutations in pfcrt and pfmdr1 genes. The frequency distribution of pfcrt K76T and pfmdr1 N86Y mutations and their association with chloroquine susceptibility was studied in an endemic area along the Indo-Bangladesh border. METHODOLOGY: A single-arm prospective study of clinical and parasitological responses in P. falciparum malaria patients to chloroquine was conducted in vivo. PCR-RFLP assay was used to detect pfcrt K76T and pfmdr1 N86Y mutations in P. falciparum. The PCR products of pfcrt gene were sequenced,  translated and aligned for haplotyping. RESULTS: Out of 63 cases, 44 (69.8%) responded adequately to chloroquine treatment. Pfcrt K76T mutation was recorded in 100% of the treatment failure cases, whereas pfmdr1 N86Y mutation was found in 52.6% of the cases only. Early treatment failure (84.2%) occurred more frequently than late treatment failure (15.8%). Kaplan-Meier survival analysis showed that the probability estimate for treatment success after 7 and 15 days was 0.84 (95% CI = 0.72-0.92) and 0.70 (95% CI = 0.57-0.80), respectively. Sequence analysis of 72 to 76 pfcrt gene codons revealed the presence of two mutant (CVMNT, CVIET) and two wild (CVMNK, CVIEK) haplotypes. The mutant CVIET haplotype was predominantly distributed (42.1%). CONCLUSIONS: The presence of mutations in pfcrt K76T and pfmdr1 N86Y genes is not sufficient to explain the therapeutic efficacy of chloroquine to P. falciparum. Study suggests that pfcrt K76T mutant haplotypes are widely distributed and are spreading diligently, which needs to be taken into account in devising an antimalarial policy.

High prevalence of pfcrt K76T and mdr1 N86Y mutations in Sonitpur district of Assam, India.

Present study reports the prevalence and distribution of pfcrt K76T and mdr1 N86Y mutations in malaria endemic areas of Sonitpur district of Assam. Out of 163 individuals tested for malaria, 67 (SPR = 41.1 %) were detected positive for malaria infection using rapid detection kit in the field and PCR assay in the laboratory. Nested PCR-RFLP assay was used to amplify pfcrt K76T and mdr1 N86Y genes flanking the K76T and N86Y mutations. P. falciparum was most abundant (91.04 %) among the three Plasmodium species reported and its prevalence was significantly higher as compared to P. vivax and P. malariae (χ(2) = 150.76; p ≤ 0.0001; df = 2). Malaria was equally distributed among all the age groups and both the sexes. Hemoglobin contents in severe anaemic patients had a significant linear decreasing trend among patients with the increase in age (χ(2) = 4.33; p = 0.03), whereas non severe anaemic patients exhibited significant linear increasing trend among the patients with the increase in age (χ(2) = 18.38; p ≤ 0.0001). Pfcrt K76T mutation was recorded in 44 (72.13 %) isolates, whereas mdr1 N86Y mutation could be detected in 28 (41.79 %) isolates only. Only 32.7 % of the samples had both pfcrt K76T and mdr N86Y mutations. Number of pfcrt K76T mutant isolates was significantly higher than the wild type. However no significant difference was observed among the number of isolates with mdr1 N86Y mutant and wild isolates.

Chemotherapy and drug resistance status of malaria parasite in northeast India.

India reports the highest number of malaria cases in Southeast Asia, of which Plasmodium falciparum contribute more than half of the cases every year. North eastern states of India contribute only 3.96% of country's population but account for >10% of total reported malaria cases, 11% of Plasmodium falciparum cases and 20% of malaria related deaths annually. In India, chloroquine resistance was reported for the first time from northeast region and since then chloroquine treatment failure is being reported from many parts of the region. Increased chloroquine treatment failure has led to change of the drug policy to artemisinin combination therapy as first line of malaria treatment in the region. However, replacing chloroquine to artemisinin combination therapy has not shown significant difference in the overall malaria incidence in the region. The present review addresses the current malaria situation of northeastern region of India in the light of antimalarials drug resistance.

Polymerase chain reaction detection of human host preference and Plasmodium parasite infections in field collected potential malaria vectors.

This study was carried out to determine the human host preference and presence of Plasmodium parasite in field collected Anopheles mosquitoes among four villages around a military cantonment located in malaria endemic Sonitpur district of Assam, India. Encountered malaria vector mosquitoes were identified and tested for host preference and Plasmodium presence using PCR method. Human host preference was detected using simple PCR, whereas vectorial status for Plasmodium parasite was confirmed using first round PCR with genus specific primers and thereafter nested PCR with three Plasmodium species specific primers. Out of 1874 blood fed vector mosquitoes collected, 187 (10%) were processed for PCR, which revealed that 40·6% had fed on human blood; 9·2% of human blood fed mosquito were harbouring Plasmodium parasites, 71·4% of which were confirmed to Plasmodium falciparum. In addition to An. minimus, An. annularis and An. culicifacies were also found positive for malaria parasites. The present study exhibits the human feeding tendency of Anopheles vectors highlighting their malaria parasite transmission potential. The present study may serve as a model for understanding the human host preference of malaria vectors and detection of malaria parasite inside the anopheline vector mosquitoes in order to update their vectorial status for estimating the possible role of these mosquitoes in malaria transmission. The study has used PCR method and suggests that PCR-based method should be used in this entire malarious region to correctly report the vectorial position of different malaria vectors.

Temporal and spatial variation in MSP1 clonal composition of Plasmodium falciparum in districts of Assam, Northeast India.

Polymorphism in MSP1 gene generated by insertion/deletion of repeats causing repeat length polymorphisms is widely used as a marker for parasite genotyping. Elucidating Plasmodium falciparum clonal composition in relation to transmission intensity and other epidemiological factors in endemic areas is crucial to understanding the dynamics of host-parasite relationship and the development of immunity in malaria. We have examined here the allelic diversity of P. falciparum and attempted to understand the polymorphism and distribution of alleles of MSP1 with transition in transmission season and with differences in malaria epidemiology between sites. MSP1 diversity expressed as mean number of distinct alleles per isolate was 0.68 at Dimakusi and was much higher (p=0.007) than seen at Guabari (0.336) and Kondoli (0.45) as was multiplicity of infection at 4.12, indicating the highest diversity at this site. Size polymorphism of the allelic families at Guabari was distinctly different from Kondoli but shared similarity with Dimakusi. Infections in high transmission summer season tended to be more complex with higher number of alleles. The frequency of alleles of RO33 and MAD20 allelic families at Guabari was found to be different between the two transmission periods. A 380 base pair allele of RO33 was over represented in high transmission summer season and seen frequently in isolates with high parasitaemia. At Kondoli allele distribution of only MAD20 was found to be different in each study year. Study site and ethnicity but not age of the study population were identified as risk factors in infection complexity. The present study demonstrates that allelic composition of P. falciparum varied with study site and between periods of high and low transmission as well as in different years of study.