Clinical and molecular surveillance of drug resistant vivax malaria in Myanmar (2009–2016)

One of the major challenges for control and elimination of malaria is ongoing spread and emergence of drug resistance. While epidemiology and surveillance of the drug resistance in falciparum malaria is being explored globally, there are few studies on drug resistance vivax malaria. To assess the spread of drug-resistant vivax malaria in Myanmar, a multisite, prospective, longitudinal study with retrospective analysis of previous therapeutic efficacy studies, was conducted. A total of 906 from nine study sites were included in retrospective analysis and 208 from three study sites in prospective study. Uncomplicated vivax mono-infected patients were recruited and monitored with longitudinal follow-up until day 28 after treatment with chloroquine. Amplification and sequence analysis of molecular markers, such as mutations in pvcrt-O, pvmdr1, pvdhps and pvdhfr, were done in day-0 samples in prospective study. Clinical failure cases were found only in Kawthaung, southern Myanmar and western Myanmar sites within 2009–2016. Chloroquine resistance markers, pvcrt-O ‘AAG’ insertion and pvmdr1 mutation (Y976F) showed higher mutant rate in southern and central Myanmar than western site: 66.7, 72.7 vs 48.3% and 26.7, 17.0 vs 1.7%, respectively. A similar pattern of significantly higher mutant rate of antifolate resistance markers, pvdhps (S382A, K512M, A553G) and pvdhfr (F57L/I, S58R, T61M, S117T/N) were noted. Although clinical failure rate was low, widespread distribution of chloroquine and antifolate resistance molecular makers alert to the emergence and spread of drug resistance vivax malaria in Myanmar. Proper strategy and action plan to eliminate and contain the resistant strain strengthened together with clinical and molecular surveillance on drug resistance vivax is recommended.


Background
Plasmodium vivax is the most globally widespread malaria parasite, causing significant high public health issues in many countries. World Health Organization (WHO) estimated 13.8 million vivax cases globally in 2015. Although vivax was believed to be a benign In the era of pre-elimination, relatively increased prevalence on vivax was observed [1]. In Myanmar, vivax malaria was the second most common malaria species composed of approximately 30 percent of all malaria cases until 2014 [1]. Afterward, the relative prevalence of the vivax has been increasing. Chloroquine (Chloroquine Phosphate Tablet BP, Remedica Ltd-Cyprus) 25 mg/kg for 3 days followed by primaquine (Remedica Ltd-Cyprus) 0.25 mg/kg for 14 days is the recommended treatment for vivax malaria in Myanmar while ACT (artemisinin-based combinational therapy) has been using to treat falciparum malaria since 2003. Although artemisinin resistance falciparum malaria was confirmed by clinically and molecular approaches, very few document on drug resistance vivax malaria was reported in Myanmar.
Chloroquine-resistant vivax malaria was first reported in Papua New Guinea in 1989 [2]. Chloroquine-resistant vivax has been confirmed in ten countries, including Myanmar [1], and treatment failure within day 28 or chemoprophylaxis failure with chloroquine was reported in 21 countries [3]. Unfortunately, there are no accepted and validated molecular markers for chloroquine or other anti-malarial for resistant vivax malaria. However, potential molecular makers for chloroquine-resistant vivax, such as mutations in pvcrt-O (P. vivax chloroquine resistance transporter-O) and pvmdr1 (P. vivax multidrug resistance protein 1) and antifolate resistant vivax such as pvdhps (P. vivax dihydropteroate synthetase) and pvdhfr (P. vivax dihydrofolate reductase) were used for molecular detection to estimate the underlying drug resistance. As there is no documented study on molecular markers analysis on vivax malaria, clinical and molecular markers analysis was conducted in multi-sentinel sites study in Myanmar.

Study site and participants
The study included the retrospective analysis of previously conducted therapeutic efficacy studies (TES) of chloroquine in uncomplicated vivax malaria in Myanmar and prospective multi-site, longitudinal study by clinical and molecular markers analysis. From 2009 to 2012, TES on vivax malaria was conducted in nine sentinel sites ( Fig. 1) which covered most of the malaria-endemic areas in Myanmar. In 2012, Shwegyin, in the southern part of central Myanmar, was selected for TES of chloroquine as this study site has a high burden of both falciparum and vivax malaria among migrant goldmine workers. In 2015-2016, TES of chloroquine on vivax malaria was conducted in Buthidaung, in the western border area and Kawthaung, southern Myanmar.

Procedure
All procedures on case selection, recruitment and followup were carried out according to the standardized protocol recommended by WHO [4]. Briefly, uncomplicated vivax mono-infected patients over 6 years old with fever or history of fever within previous 48 h, were recruited and treated with chloroquine standard dose calculated by body weight, followed by observation with follow-up schedule, i.e., day 3, 7, 21 and 28 after treatment. Clinical and parasitological assessment was done on each followup day. All anti-malarials used in this study were provided by the national malaria control programme.
Blood samples were taken on day 0 and all follow-up days for microscopic examination and molecular analysis. Blood film examination on peripheral blood smear was carried out after 3% Giemsa stain for 45 min. Blood film examination was done as described [4] and calculated as parasite count per µL of blood. For molecular analysis, finger-prick blood samples were collected on filter paper (Whatman©), dried and stored in plastic bags with desiccant until analysis. Molecular markers analysis was conducted only in prospective study sites such as Kawthaung, Shwegyin and Buthidaung.

Molecular analysis
All day-0 samples were subjected to analysis for distribution and spread of chloroquine and antifolate drug resistance markers. Filter papers were prepared and extracted for parasite genomic DNA using QIAamp DNA Blood Mini Kit (QIAGEN) according to manufacturer's instruction. The target genes were amplified by using the specific pair of primers (Table 1). In this study, chloroquine resistance marker, ' AAG' insert in pvcrt-O; multidrug resistance marker, mutations in pvmdr1; antifolate resistance makers, mutations in pvdhps and pvdhfr were amplified and analysed.
For target gene amplifications, initial denaturation at 95 °C for 10 min was followed by 35 cycles of 95 °C for 30 s, 58 °C (pvdhps) or 60 °C (pvcrt-O) or 62 °C (pvdhps and pvdhfr) for 45 s, 72 °C for 1 min, and a final extension of 72 °C for 10 min. Amplified products were checked by 1% agarose gel electrophoresis stained with Red safe © (iNtRON, Seongnam, Republic of Korea). The PCR clean-up was proceeded by MEGAquick-spin DNA fragment purification Kit (iNtRON, Republic of Korea) and sequencing. The nucleotide and amino acid sequences were aligned and analysed by Lasergene ® software (DNASTAR, Madison, WI, USA) using the reference strain of Sal-1 retrieved from Plasmodium data base [5]. The nucleotide sequences were submitted to GenBank under accession numbers KX000945-KX000959.

Statistical analysis
For clinical data, efficacy outcomes were classified as adequate clinical and parasitological response (ACPR) for successful cure cases until day 28 after treatment or treatment failure (TF) that may be early treatment failure (ETF): failure until day 3 after treatment; or late treatment failure (LTF): failure within days 3 to 28. Data were counter-checked and analysed by MS Excel and SPSS

Prospective longitudinal study
Kawthaung (southern Myanmar), Shwegyin (central Myanmar) and Buthidaung (western Myanmar) were selected for prospective longitudinal study after treatment with chloroquine regimens. A total of 208 uncomplicated vivax cases were included in this study with mean age of 24.2 (±9.03) years and geometric mean of the parasite density of 4160 with 95% CI (3567-4851) ( Table 2). There was no clinical failure case in Shwegyin, but one LTF case in Kawthaung at day 28 (1/60, 1.7%) and two in Buthidaung at days 21 and 28 (2/60, 3.3%).

Discussion
This study explores the clinical and molecular pattern of candidate drug resistance markers in Myanmar. Retrospective and prospective analysis on TES of chloroquine on vivax malaria (2009-2016) showed that clinical failures were detected only in southern and western Myanmar. Molecular markers analysis augments additional information on the pattern of drug resistance. In this study, molecular maker analysis indicated widespread distribution of chloroquine, antifolate and multidrug resistance markers, with the highest mutant rate in southern Myanmar. Surveillance on drug resistance malaria is of great concern for global control and elimination of malaria. As drug-resistant vivax was reported as early as 1980s, its exact global burden is still unknown. Compared to falciparum malaria, drug resistance studies of vivax which focus on epidemiology, drug efficacy and drug resistance mechanism are rare. Drug resistance malaria can be detected by in vivo TES studies, molecular maker analysis, in vitro drug susceptibility testing and drug concentration measurement.
Lack of a standardized culture system limits the usefulness of in vitro susceptibility tests for detection of drug resistance. Although in vivo TES were accepted as a standard method for drug resistance detection, recurrent parasitaemia cases needed to be distinguished between re-infection, recrudescence or relapse. Relapse patterns of vivax malaria also widely differ across geographical regions and no standardized method to exclude relapse or re-infection in TF cases leads to difficulties in interpretation of findings of TES. Currently, clinical surveillance with chloroquine drug level measurement has been accepted to confirm chloroquine-resistant vivax malaria. At least one TF case that showed whole blood    concentration of chloroquine plus desethylchloroquine more than 100 ng/mL was observed in ten countries: Brazil, Ethiopia, Indonesia, Malaysia (Borneo), Myanmar, Papua New Guinea, Peru, the Solomon Islands, and Thailand [6].
Although there are no validated molecular markers for drug-resistant vivax malaria, potential candidates were reported. Most of these candidate markers [7][8][9] were homologues of falciparum drug resistance makers, such as pvmdr1, pvdhp and pvdhfr. K10 insertion (' AAG' insert) in first exon at tenth position of pvcrt-O was also suggested as a chloroquine resistance marker [7,10]; it was observed in Thailand (56-89%) [7,10] and Myanmar (46%) [7]. In this study, the highest rate of K10 inset alleles was observed in Shwegyin (central Myanmar) (72.7%) followed by Kawthaung (southern Myanmar) (66.7%) and Buthidaung (western Myanmar) (48.3%) indicating high chloroquine resistance in southern Myanmar, which is similar to the artemisinin resistance status [11]. As the chloroquine is the first line treatment for vivax malaria in Myanmar, K10 insert of pvcrt-O gene was widely distributed in all three study sites.
Similarly, pvdhps and pvdhfr showed a significant role in antifolate drug-resistant vivax malaria [19]. F57I/L, S58R, T61M, and S117T/N of pvdhfr were found to be associated with pyrimethamine resistance [20] and S382A, S383G, A553G of pvdhps were associated with sulfadoxine resistance [21]. Plasmodium vivax was supposed to have a certain degree of innate resistance to sulfadoxine and S383G and A553G could be responsible inducers for resistance [22]. In Myanmar, relatively high rates of these mutations were noted in southern and central Myanmar.
Within 2009-2016, treatment regimen for vivax malaria was not changed and trend on clinical efficacy of chloroquine is similar except some fluctuations in Kawthaung study site. It is difficult to rule-out the drug resistance factor resulting in treatment failure in this study sites without molecular analysis in previous years. As the drug resistance alone is not responsible for treatment failure [4], clinical response and prevalence of molecular markers is not similar in this study.

Conclusions
This study is the first multisite, clinical and molecular surveillance of drug-resistant vivax malaria in Myanmar, exploring the neglected niche of the infection. According to current anti-malarial treatment guidelines in Myanmar, chloroquine is the first-line treatment for vivax malaria. Most of the study sites showed 100% ACPR except in the southern and western Myanmar sites. Wide distribution of chloroquine and antifolate resistance molecular markers revealed the spread of the drug-resistant parasite population in Myanmar. High mutant rates of most of the vivax drug resistance molecular markers in southern and central Myanmar were similar to that of artemisinin resistance falciparum malaria, indicating higher anti-malarial resistance burden of falciparum and vivax in southern Myanmar. An appropriate strategy and action plan to contain or eliminate drug-resistant vivax malaria in Myanmar is recommended.