Malaria epidemiology in central Myanmar: identification of a multi-species asymptomatic reservoir of infection

The last decade has seen global progress in malaria control [1, 2]. This is threatened by the emergence of Plasmodium falciparum resistant to artemisinin, the world’s front-line anti-malarial [3]. So far, P. falciparum with reduced susceptibility to artemisinin has been described in Cambodia, Thailand, Vietnam, Laos and Myanmar [4‐7]. The World Health Organization (WHO) recently set its sights on malaria elimination in Southeast Asia in order to contain this threat [2]. The emergence of the zoonotic primate malaria Plasmodium knowlesi as a parasite of significant public health importance in Malaysian Borneo complicates elimination [8, 9]; the distribution and prevalence of this species in other countries in the region remains uncertain.

Myanmar is reported to account for the vast majority of malaria cases and deaths within Southeast Asia [10]. Better understanding the epidemiology of malaria here would likely contribute to successful elimination. Malaria distribution in Myanmar is heterogeneous; forests are a major environmental factor driving patterns of disease [11, 12] and international and internal migration are also risk factors [13, 14]. Whilst transmission is relatively well documented in border areas [13], few data exist from central Myanmar and the burden and drivers of infection in this area remain poorly understood.

Light microscopy and rapid diagnostic tests (RDTs) underestimate the prevalence of infection with Plasmodium spp, particularly in low-endemic settings [15]. Polymerase chain reaction (PCR) is more sensitive in detecting current infection and allows further testing such as for drug resistance-associated mutations [16]. Serological methods have been shown to reliably estimate malaria transmission and can identify risk factors with achievable sample sizes in low prevalence areas [17]. Prevalence of antibodies to specific malaria antigens, which indicate malaria exposure, can be used to calculate the seroconversion rate (SCR, equivalent to the force of infection) [18]. Changes in SCR over time are of particular utility in monitoring the success of public health campaigns.

This study combined highly sensitive PCR testing and serological evidence of transmission intensity in a household survey in central Myanmar with collection of detailed epidemiological data to investigate the prevalence of, and risk factors for, malaria exposure.

A total of 701 houses in 27 clusters (villages) across 14 administrative wards were visited, of which 500 houses had at least one resident present. Of the 2112 recorded residents of consenting households, 474 (22.4%) were absent (Table 1). 1638 were present and all consented to take part in the study.

The median age was 30 years (range 6 months-88 years) and 55% were female. 43% (57% of men and 27% of women) were classified as having had forest exposure (Table 1). 80% of households were classified as having universal bed net coverage, but very few households were reported as having been sprayed with insecticide in the past year (3%). The majority of participants were of Burmese origin (63%) whilst people of Indian origin represented 33% of the sample and Shan origin 3%. Absent household residents were more likely to be male and less likely to be Indian compared to those included in the study.

This household study reveals low-level malaria transmission among the general population in central Myanmar. The WHO estimates that P. falciparum causes 75% of clinical malaria in Myanmar and P. vivax 25% [28], whilst in this study P. falciparum mono-infection comprised 45% of infections and P. vivax 12% (Fig. 2). This study found P. knowlesi [29] in more than 35% of positive samples. Clinical diagnosis of malaria and most epidemiological studies in Myanmar use microscopy or RDT positivity to identify infection and both RDT and microscopy have poor sensitivity and specificity for P. knowlesi [30, 31]. In this study, reliable microscopy also proved technically challenging. This may explain the underrepresentation of P. knowlesi in national statistics and population-level data when compared to the findings presented here. In Malaysia, P. knowlesi is responsible for a similar proportion of cases as found in this study and can cause fatal illness [32]. The epidemiology of P. knowlesi in Myanmar requires further study, utilizing sensitive diagnostic tools.

One quarter of P. falciparum samples harboured K13 mutations, all from afebrile, asymptomatic participants, none of whom reported anti-malarial treatment within the previous three months and none were followed up—it is therefore difficult to comment on the importance of these alleles as determinants of artemisinin susceptibility. Of the five K13 mutations detected, four have never previously been reported. One of the novel mutations, Arg539Val, is similar to previously described variants at codon 539 from Cambodia, Laos and Myanmar, though these all encode threonine rather than valine as the variant amino acid [27]. K13 mutations have recently been mapped in convenience samples in Myanmar and appear prevalent in Upper Myanmar and eastern border areas; no K13 mutations were found in a clinical cohort of 52 patients in Bago Region in 2013–2014 [33], although the prevalence of K13 mutations amongst clinical cohorts in the high-risk township of Shwe Kyin, Bago, was similar to that found in our population [5].

Most participants (n = 38; 93%) with current infection were afebrile and none reported symptoms. The three individuals with raised temperatures tested negative on RDT, possibly signifying a low-density parasitaemia (or alternative diagnoses) which may be associated with mild clinical symptoms [34]. However, even sub-clinical infections are important during elimination campaigns [35]; accurate identification and treatment of asymptomatic carriers—who might transmit infections and will tend not to seek treatment—proved crucial to eliminate malaria in a similar low-endemic setting [36].

The serological data support the PCR data and suggest that there has been ongoing low-level transmission of P. falciparum and P. vivax in the general population in Bago region. There was a notable increase in exposure to infection in men aged over 23, who experience a SCR 16-fold higher than the general population. Because the antibodies detected by these methods persist for an unknown period of time, there are two explanations indistinguishable by serology alone: malaria epidemiology may have changed 23 years ago such that men now aged over 23 were historically exposed to more malaria, or there may be ongoing behavioural risk factors specific to those aged over 23. The best working hypothesis is that the change in SCR is behavioural; it is confined to a well-defined epidemiological group (men of working age) and this population is overrepresented in contemporary clinic-based studies suggesting they remain at increased risk of malaria [16].

Forest work is an established risk factor for malaria transmission [37]. Plasmodium knowlesi is particularly associated with forest malaria, due to the outdoor biting habits of P. knowlesi vectors and the natural primate reservoirs living in forests [38]. The questionnaire captured self-reported information relating to occupational exposure and forest visits. Individuals reporting forest exposure were three times more likely to have evidence of previous exposure to malaria in the unadjusted analysis. However, forest workers were significantly more likely to be men and aged over 23 and less likely to be Indian. After adjusting for other factors including age, ethnicity and SES the association with forest exposure was no longer significant, which is consistent with forest exposure being on the causal pathway (although it cannot be excluded that it is a confounder). Illicit forest-related activities such as illegal logging are often unreliably reported in surveys, and qualitative studies may be needed to obtain more information on the spectrum of forest exposure as a risk behaviour for malaria in this region.

The Myanmar Ministry of Health offers malaria prevention, diagnosis and treatment to the general population without distinction. This approach is reflected in the high level of bed-net ownership and might contribute to the low levels of transmission observed. However, these data suggest that a strategy tailored to those at highest risk and with asymptomatic infection might be required to eliminate malaria since bed-nets will not protect those working in forest plantations at dawn and dusk [14]. Targeted interventions have been trialed in other areas with varying degrees of success, including: insecticide-treated clothing and hammocks (for use in the forest); toxic mosquito baits; and personal insect-repellents [39]. Targeted mass administration of anti-malarials to specific demographic groups is another option [40]. As yet, none have been evaluated in Myanmar.

This is the first study in Myanmar to combine detailed population-level epidemiological data with highly sensitive molecular techniques. The strength and novelty of this study also lie in the size of this probability sample from an important but unstudied population, with generalizability to Bago Region and central Myanmar. However, higher diagnostic sensitivity has been achieved with the technique of high volume ultra-sensitive qPCR [16] though this requires collection of larger blood volumes by venepuncture, and an unbroken cold-chain for transport of packed red cells to a specialist molecular biology laboratory. Point prevalence of current infection is sensitive to seasonal variation, and this study was conducted towards the end of the rains—regarded as the malaria high season—to sample at the peak of malaria transmission (though different seasonal peaks have been reported for some areas nearby [41, 42]). Moreover, others report little by way of seasonal variation [43], and by correlating PCR findings with serology, which is less sensitive to seasonal changes, robust hypotheses can be developed. The study aimed to minimize bias in this population-based study by weighting the sample so that it was broadly representative of national demographic data in Myanmar in terms of gender and age [44], but the sampling strategy excluded the population living closest to the flooded Sittang River due to adverse weather conditions, which made these areas inaccessible, and where malaria transmission might be higher. In addition, the ethnic composition of this sample differs from national averages because ethnicity in Myanmar is clustered [44]; the sample over-represented Indian participants (33% compared to 2% in national statistics) and underrepresented Shan (3% compared to 9%). One-fifth of residents were absent during study visits, and these individuals were more likely to be male and less likely to be of Indian origin, and this household survey did not include hospital in-patients being treated for malaria. We were only able to provide population weighting at the ward level due to a lack of available population statistics for individual villages. In our experience, village sizes did vary, though it is unlike that this would have affected our prevalence estimates, given the ward-level weighting applied and the small number of infections detected. The overall effect of our diagnostic techniques and sampling strategy is that the study design might have underestimated rather than overestimated malaria prevalence.

Malaria transmission in this area was low. However, there were low numbers of asymptomatic carriers who probably play an important role in maintaining local transmission, and many were infected with P. knowlesi. A higher proportion of K13 mutations among P. falciparum cases were observed than has previously documented in this region, and working age men appear at significantly higher risk of malaria exposure when compared to the general population, which needs further research. However, the public health implications are clear; the WHO goal of malaria elimination in this region will require identification and treatment of asymptomatic carriers among high-risk populations in order to control artemisinin resistance and successfully eliminate malaria in this setting.