High prevalence of asymptomatic malaria in south-eastern Bangladesh

Study setting and procedure

Two cross-sectional surveys were performed: the first during the rainy season from August to October 2007 and the second during the dry season from December 2007 to February 2008. The study was conducted by a team from the Medical University of Vienna, Austria in collaboration with the International Centre for Diarrhoeal Disease Research, Dhaka, Bangladesh (ICDDR, B). All selected communities were visited by members of the study team, prior to sample collection to inform villagers about the ongoing study. Laboratory tests on collected samples and data analysis were carried out at the Malaria Research Initiative Bandarban (MARIB) field research centre in Bandarban town. Written informed consent was obtained from all study participants or their legal representative, the study protocol was approved by the Ethical Review Committee of the International Centre for Diarrhoeal Disease Research, Bangladesh (ICDDR, B).

Sampling

Administratively, Bandarban District consists of seven subdistricts (upazilas), 32 unions, 140 mouzas and 1,482 villages. Geographical multistage cluster sampling techniques in a single domain were employed (Figure 1) using population figures from the 2001 census [22]. For each of the seven subdistricts all mouzas or villages were listed alphabetically and 3 mouzas or villages were randomly selected using a probability proportion to size (PPS) sampling procedure. A list of all households within the mouza or village was prepared and 20 households per mouza or village were randomly selected. All the persons present in the household were invited to participate in the survey.

During the monsoon season 2007 a total of 21 villages in all seven subdistricts were surveyed (Survey I). Eight villages in three subdistricts (Bandarban, Ruma and Rowangchari) were revisited during the second survey period (Survey II). Whenever possible the study team visited the same households as in the previous survey. Household members who declined to participate or had moved away from the area were replaced by their closest neighbouring household.

Data and sample collection

Participants were interviewed and information was recorded on gender, age, ethnicity, occupation, height, weight, history of fever, number of previous episodes of malaria/fever, last date of malaria diagnosis and result of diagnosis, source and drugs used in malaria treatment, and the number of household members. Axillary temperature and pulse were measured in all participants. Three ml of venous blood was collected from participants eight years or older for malaria RDT/microscopy and 100 μl of venous blood transferred on filter paper (903; Schleicher & Schuell, BioScience GmbH, Dassel, Germany) in duplicate. Five drops of blood (one drop of blood for the malaria RDTs, two for microscopy, and two drops of blood for the PCR filter paper) were drawn by finger or heel prick from children below eight years old.

Laboratory methods

Malaria was diagnosed in the field by RDT and later confirmed by microscopy and PCR.

Rapid diagnostic test

RDTs (FalciVax^®, Zephyr Biomedicals, India) based on the detection of P. falciparum-specific histidine-rich protein 2 (HRP2) and P. vivax-specific lactate dehydrogenase (Pv-pLDH) were employed in all participants with malaria-like symptoms [23, 24]. Patients who tested positive were provided with immediate treatment following national guidelines. Febrile patients considered seriously ill were immediately referred to the closest health care facility for further diagnosis and treatment.

Microscopy

Giemsa-stained blood smears were used for microscopic diagnosis of malaria following established standard operating procedures (SOPs). Thick and thin blood films were prepared, stained with Giemsa stain (Merck^®, Darmstadt, Germany) and examined under an oil immersion (Olympus microscope CX21, Tokyo, Japan) for parasite positivity and species determination. Declaring a slide positive or negative and initial species diagnosis was based on the examination of 200 fields in thick films. A slide was considered positive when at least one parasite was found. After finding the first parasite another 200 fields were completed to rule out mixed infections. If no parasite was found in 200 oil fields the slide was considered negative. Parasite density was calculated by counting the number of asexual malaria parasites per 200 white blood cells (WBCs) assuming a WBC count of 8,000/μL [25].

Quality control

A minimum of 5% of all positive and negative slides were randomly selected for internal quality control.

Polymerase chain reaction (PCR)

PCR was performed to assess the malaria prevalence in both, symptomatic and asymptomatic participants. DNA was isolated from filter papers using a modified chelex-based technique [26, 27]. Nested PCR assays and Plasmodium genus-specific primers rPLU1/rPLU5 were used for the Nest1 amplification and rPLU3/rPLU4 for Nest2 reactions as described previously [28–30]. All genus-specific PCR-positive results were analysed to species level using the following species-specific primers as reported previously: rFAL1/rFAL2 (P. falciparum); rVIV1/rVIV2 (P. vivax); rMAL1/rMAL2 (P. malariae); rOVA1WC/rOVA2WC (Plasmodium ovale curtisi and Plasmodium ovale wallikeri); rOVA1/rOVA2 (P. ovale curtisi); rOVA1var/rOVA2var (P. ovale wallikeri) [28–33].

Case definition

Symptomatic clinical malaria cases were defined as PCR-positive individuals with documented fever (axillary temperature ≥37.5°C) and reported clinical symptoms consistent with malaria in the previous seven days. Oligo-symptomatic malaria cases were defined as PCR-positive, afebrile (axillary temperature <37.5°C) cases with a reported history of fever or illness in the previous seven days. Asymptomatic malaria cases were defined as PCR-positive cases without measurable fever (axillary temperature <37.5°C), who reported no malaria-related symptoms, and had not received treatment for malaria in the previous seven days [34].

Splenomegaly

The spleen rate was determined for children aged 14 years and below. Spleen size was measured followingthe method established by Hackett [35] and classified either as negative (Hackett grade 0) or positive (Hackett grades 1–5).

Classification of malaria endemicity

The four classes of endemic malaria were defined as following [35]: hypo-endemic malaria: spleen and/or parasite rate in children two to nine years not exceeding 10%; meso-endemic malaria: spleen and/or parasite rate rate in children two to nine years between 11 and 50%; hyperendemic malaria: spleen and/or parasite rate rate in children two to nine years constantly over 50% and a high spleen and/or parasite rate rate in adults (over 25%); holo-endemic malaria: spleen and/or parasite rate rate in children two to nine years constantly over 75%, but a low spleen and/or parasite rate rate in adults.

Statistical analysis

P < 0.05 was considered significant. Pearson’s Chi-square with Yates’ correction or Fisher’s Exact test as appropriate were used for categorical data, Mann–Whitney U-test was performed for comparing continuous data that did not conform to normal distribution. Student’s t-test was conducted to evaluate differences between quantitative variables that were normally distributed. All analyses were performed using Microsoft Excel^® and VassarStats [36].

Demographic data of the study population

Malaria prevalence and fever in cross-sectional survey I

With 188 cases (36.3%, 95% CI: 32.2-40.6) the malaria prevalence in individuals under 15 years was significant higher (p < 0.01) compared to older participants (N = 247, 27.4%, 95% CI: 24.6-30.5). A significantly higher malaria prevalence (p = 0.015) was found in males (N = 220, 34.0%, 95% CI: 30.4-37.8) as compared to females (N = 215, 27.9%, 95% CI: 24.8-31.2).

The overall geometric mean parasite density determined by microscopy was 707 parasites/μl (95% CI: 565–883) for P. falciparum and 345 parasites/μl (95% CI: 246–484) for P. vivax. Parasite densities in individuals younger than 15 years were significantly higher (p < 0.01).

Out of 416 households in 223 (53.6%, 95% CI: 48.7-58.5) at least one member of the household tested positive for malaria, in 64 households (15.4%, 95% CI: 12.1-19.3) two people tested positive for malaria and in 56 (13.5%, 95% CI: 10.4-17.2) households three or more individuals tested positive by PCR.

Subdistricts/Upazilas

Plasmodium falciparum was the predominant species in all subdistricts. The highest malaria prevalence was found in the eastern subdistricts Rowangchari and Thanchi, followed by Bandarban Sadar, Ruma, Naikhongchhari, and Lama. The lowest malaria prevalence was detected in Ali Kadam, which shares only a short stretch of border with Myanmar. Malaria prevalence among the seven subdistricts is shown in detail in Table 2. Significantly higher malaria prevalence (p < 0.01) was seen in the northern and northeastern subdistricts (Bandarban Sadar, Rowangchhari, Ruma, and Thanchi) located in the foothills close to the Indian/Myanmar boarder compared with the western subdistricts (Lama, Ali Kadam and Naikhongchhari) in or closer to the plains (Figure 2). At village level the highest malaria prevalence by PCR with 71.4% (95% CI: 53.5-84.8) was found in Bagan village in Ruma subdistrict and the lowest with 1.2% (95% CI: 0.06-7.3) in Memberpara village in Lama subdistrict (p < 0.0001).

Spleen rate

Out of a total of 350 children aged two to nine years, 65 had a palpable spleen (spleen rate: 18.6%, 95% CI: 14.7-23.1); 91 of them (26.0%, 95% CI: 21.6-31.0) were slide-positive for malaria either for P. falciparum or P. vivax. A total of 121 (34.6%, 95% CI: 29.6-39.8) children tested positive by PCR for any of the four malaria species. Thirty-eight (10.9%, 95% CI: 7.9-14.7) and 48 (13.7%, 95% CI: 10.4-17.9) children were malaria microscopy-positive or PCR-positive and had an enlarged spleen at the same time. Sensitivity, specificity, positive (PPV) and negative predictive values (NPV) of spleen enlargement for malaria in this population are shown in Table 4 using microscopy or PCR as reference method. The Venn diagram showing relationship between spleen rate, microscopy and PCR positive children aged two to nine years is shown in Figure 3.

PCR/microscopy (%)
	Sensitivity	Specificity	PPV	NPV	Malaria prevalence
Survey I	39.7/41.8	92.6/89.6	73.8/58.5	74.4/81.4	34.6/26.0
Survey II	13.3/25.0	92.5/91.0	44.4/36.4	70.5/85.5	30.9/17.0

Winter survey II

Plasmodium falciparum gametocytes were detected by microscopy in four (3.5%, 95% CI: 1.1-9.1) individuals of whom only one also had asexual parasites. Malaria prevalence within all participants in the winter survey (dry season) was not significantly (p = 0.117) lower compared to the summer survey.

No patient testing positive for malaria was febrile at the time of the survey, therefore 103 (88.8%, 95% CI: 81.3-93.7) were classified as asymptomatic and 13 (11.2%, 95% CI: 6.3-18.7) as oligosymptomatic (Table 3).

The geometric mean parasite densities were 487 asexual parasites/μl (95% CI: 281–845) for P. falciparum and 751 (95% CI: 298–1,894) for P. vivax, respectively. No statistically difference (p > 0.05) was found between the parasite rate in summer and winter survey.

From a total of 172 households, in 77 (44.8%, 95% CI: 37.3-52.5) at least one member tested positive for malaria, in 27 (15.7%, 95% CI: 1.4-7.8) households two individuals and in seven (4.1%, 95% CI: 1.8-8.5) households three or more people tested positive for malaria.

Subdistricts/Upazilas

In the winter survey the highest malaria prevalence by subdistrict was found in Bandarban Sadar (29.7%, 95% CI: 24.4-35.5) followed by Rowangchhari (27.1%, 95% CI: 18.8-37.3), and the lowest malaria prevalence was detected in Ruma (13.4%, 95% CI: 6.7-24.5).

Splenomegaly

Out of 97 children aged two to nine years, nine (9.3%, 95% CI: 4.6-17.3) had a palpable spleen and with 30.9% (N = 30; 95% CI: 22.2-41.2) the parasite prevalence by PCR in survey II was again high but not significantly different from survey I (p = 0.584). Malaria-positive PCR and an enlarged spleen at the same time were found in four (4.1%, 95% CI: 1.3-10.8) children two to nine years old. With 9.3% the spleen rate in winter was also lower than in summer (18.6%; p = 0.043). Sensitivity, specificity, PPV and NPV of spleen enlargement for malaria in the winter survey are shown in Table 4.