The baseline distribution of malaria in the initial phase of elimination in Sabang Municipality, Aceh Province, Indonesia

Sabang Municipality is administratively included in Aceh Province, Indonesia. It is located at latitude 5° 49' 30" north and longitude 95° 18' 28" east (Figure1). It occupies an area of 4,051 sq km, and in 2009 its total population was 24,815. The rainy season usually occurs from December to April, during which time the temperature centres around 18–20°C. Dry season temperatures are around 25-33°C.

Malariometric and G6PD surveys were conducted from May to October 2010, in 14 villages with known malaria transmission. Urban areas with no recorded transmission were excluded from the survey. Malariometric surveys include interview, physical examination of malaria symptoms and signs, body weight and axillary temperature. For each individual, thick and thin blood smears were collected along with a blood spot for PCR on filter paper and whole blood drops for G6PD test and haemoglobin (Hb) tests. All slides were reviewed by an expert microscopist. Slide positive cases were confirmed with PCR. In addition, 10% of negative cases were screened with PCR, and all blood samples from villages with microscopically positive cases. Selection of the 10% negative samples was done through computer programme. All negative samples in each village were listed sequentially in microsoft excel. A Random option was used to generate sample sequence and the samples were selected in ten-fold fashion. At the end, 1,725 samples were proportionally selected. Locations of confirmed positive cases were geocoded with GPS (Figure2). For G6PD test, blood were directly dropped onto a 1.5 ml microtube containing 760 μl of water, 20ul of substrate mixture and 20 μl of dye mixture of G6PD assay kit. The tube was then vigorously shaked for 5 sec and incubated at 25-37°C for 20–30 minutes. The reaction was stopped by adding 10 μl of 1 mol/l HCL and compared the developed color with those of positive and negative controls. This method used water-soluble tetrazolium salt, WST-8, that also produces water-soluble formazan (Dojindo Laboratories, Tokyo, Japan). The G6PD enzyme activity was divided into five categories: A: 0% activity (G6PD-deficient), B: 25% activity, C: 50% activity, D: 75% activity and E: 100% activity (normal activity). The sample selection for G6PD screening was based on households in urban village of Cot Bau where the population of the entire municipality was well represented. In each selected household, chief of the household and his wife were enrolled. The total subjects enrolled in G6PD survey was 937 subjects and represented over 50% of the total households in this village. To determine the Hb status, a portable digital haemocytometer (Haemocue AB Hb201+, Angelholm, Sweden) was used.

Thick and thin blood films were stained with Giemsa and examined using 1,000X oil immersion light microscopy. At least 200 ocular fields were examined before considering a slide negative, and parasite densities were counted as parasites per 200 leukocytes and reported as parasites/mm³ assuming a white blood cell count of 8,000/mm³[6].

Parasite and human host DNA was extracted from blood samples using chelex-100 ion exchange (Biorad Laboratories, Hercules, CA, USA) according to a procedure described previously[7]. The DNA was either used immediately for PCR amplification or stored at −20°C for later analysis.

Molecular analyses were performed using PCR amplification, restriction fragment length polymorphisms (RFLP), and sequencing on several genes of the parasite such as Pfdhfr, Pfdhps, pfmdr1, pfcrt[8‐10], Pfmsp1, Pfmsp2[11, 12], Pvdhfr, Pvdhps[13, 14], Pvmdr1[15] and host gene such as G6PD gene[16]. The PCR reactions were carried out in a condition similar to the previously published report[8‐16]. PCR diagnosis to further confirm the parasite rate on the entire positive cases was performed using 18S rRNA gene primer in a condition similar to the previously published report[17].

In all, 16,229 individuals were surveyed from 14 villages. The surveys covered 83.47% of the total inhabitants of the targeted survey areas and by microscopy 10 subjects were found with Plasmodium falciparum infection: seven from the village of Batee Shok, two from Paya Keunekai and one from Ie Meuleu. Plasmodium vivax malaria was found in one subject from Iboih and one from Rondo island. PCR testing was performed on seven malaria falciparum slide positive cases and one malaria vivax slide positive case (blood blots were unavailable for three malaria falciparum slide positive cases and one malaria vivax slide positive case). PCR (10% of negative cases and all blood samples from villages with microscopically positive) revealed an additional 11 malaria-positive subjects: one mixed P. falciparum/P. vivax infection from Batee Shok, one P. falciparum from Paya Keunekai village, and nine P. vivax from Batee Shok village (Table1 and2). The slide positive rate was 0.074% (CI 95%: 0.070 – 0.078) and PCR corrected 0.590% (CI 95%: 0.582 – 0.597). Of those that were positive for malaria by microscopy, 9 (75%) had clinical symptoms such as fever and/or history of headache. The remaining 14 subjects, all from the village of Batee Shok, had neither symptoms nor history of recent fever. Overall, of the total 23 positive cases by microscopy and PCR, 17 (73.9%) cases were found at Batee Shok and the remainder were found in Iboi, Paya Keunekai, Ie Meulee and Rondo island. However, malaria prevalence peaked in the village of Batee Shok, which harboured 17 malaria-positive subjects among a total 1,109 subjects examined (1.53%).

The head of each household and his wife were enrolled in the G6PD survey. Of the total 937 subjects enrolled, two (0.2%) tested positive for G6PD deficiency. Analysis for the presence of the most commonly found mutations: Mediterranean (563C/T), Coimbra (592 C/T), Vianchang Canton (1376 G/T), Vanua Lava (383 T/C), Mahidol (487 G/T), Chatam (1003 G/A), Kaiping (1388 G/A) and Vianchang (871 G/A, 1311 C/T) found none of these in the two positive subjects.

A total of 16,229 subjects was analysed for weight and Hb levels. The mean of Hb level was relatively high in each age group except for the below five years, which had a Hb level slightly above or below the borderline (anaemia cut-off for Hb level is 11 g/dL). Distribution of age, children, sex and mean of Hb level per village also indicated relatively high in each group. However, due to the low prevalence of malaria, the relationship between anaemia and malaria could not be determined. Malnutrition was found in one village Kreung Raya (Additional files1,2,3,4, and5).

Molecular analysis of the parasites collected throughout the survey found the 76 T allele of the pfcrt gene, a molecular marker for parasite resistance to chloroquine (Table3). The proportion of the isolates carrying the 86Y polymorphism of the pfmdr1 gene was 37.5%. The 1042D polymorphism of pfmdr1 was detected in one isolate. No polymorphisms at codons 1032 and 1246 of the pfmdr 1 gene were observed in any of the isolates examined. Amplification of the dhfr gene indicated that the majority of the isolates carried the mutant 108 N allele. 108T, 59R, 16V, 50R, 51I, 164L, 436A 437G, 540E, 581G and 613 S/T polymorphisms were not detected in any of the isolates examined. Analysis of the parasite genotype indicated infection with multiple parasite strains (Table3). All samples carried the 3D7 type of which three samples were mixed with the FC27 type. Analysis on MSP1 gene revealed the K1, MAD20 and RO33 types but three samples could not be determined.

Molecular analysis of the parasites collected throughout the survey indicated that the 976 F allele of the pvmdr1 gene, a molecular marker for resistance to chloroquine, was found in 10 of 11 isolates examined. Amplification of the Pvdhfr gene indicated that 63.6% of the isolates carried the wild type 117S allele and the reminder carried the 117 T. Mixed allelic infection of 117 T/N was found in one isolate. Analysis of codon 57 and 58 of the Pvdhfr gene revealed three isolates carried the 57 F allele and five isolates carried the 58S allele, three of which are mixed allelic infection with 58R. No polymorphisms were observed at codons 13 or 16 in any of the isolates examined. Furthermore, no polymorphisms were observed at codons 383 and 553 of Pvdhps gene in any of the isolates examined in this study (Table4).