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| RESEARCH ARTICLE |
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| Year : 2021 | Volume
: 58
| Issue : 3 | Page : 257-264 |
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Pretreatment gametocyte carriage in symptomatic patients with Plasmodium falciparum and Plasmodium vivax infections on the Thai-Myanmar border
Pongsakorn Martviset1, Sirima Kitvatanachai2, Mayuri Tarasuk3, Phunuch Muhamad4, Kesara Na-Bangchang5
1 Division of Parasitology, Department of Preclinical Sciences, Faculty of Medicine; Center of Excellence in Molecular Biology and Pharmacology of Malaria and Cholangiocarcinoma, Thammasat University, Pathumthani, Thailand
2 Faculty of Medical Technology, Rangsit University, Pathumthani, Thailand
3 Center of Excellence in Molecular Biology and Pharmacology of Malaria and Cholangiocarcinoma; Graduate Studies, Chulabhorn International College of Medicine, Thammasat University, Pathumthani, Thailand
4 Center of Excellence in Molecular Biology and Pharmacology of Malaria and Cholangiocarcinoma; Drug Discovery and Development Center, Office of Advanced Science and Technology, Thammasat University, Pathumthani, Thailand
5 Center of Excellence in Molecular Biology and Pharmacology of Malaria and Cholangiocarcinoma; Graduate Studies, Chulabhorn International College of Medicine, Pathumthani, Thailand; Drug Discovery and Development Center, Office of Advanced Science and Technology, Thammasat University, Thailand
| Date of Submission | 12-Feb-2020 |
| Date of Acceptance | 02-Oct-2020 |
| Date of Web Publication | 15-Feb-2022 |
Correspondence Address:
Kesara Na-Bangchang
Chulabhorn International College of Medicine, Thammasat University, Pathumthani
Thailand
 Source of Support: None, Conflict of Interest: None  | Check |
DOI: 10.4103/0972-9062.316274
Background&objectives: Changes in parasite biology, particularly the gametocytogenesis process, could be one of the important contributing factors for worldwide malaria resurgence. The present study investigated the prevalence rates of pretreatment gametocyte carriage and density in Plasmodium falciparum and P. vivax infections in the low malaria-endemic area on the Thai-Myanmar border.
Methods: One hundred and twenty-six blood samples were collected from patients with signs and symptoms of malaria who attended malaria clinics. Malaria positive cases detected by microscopic examination were confirmed by species-specific nested-PCR in 97 (29 and 68 samples for P. falciparum and P. vivax, respectively).
Results: The proportion of P. vivax and P. falciparum-infected samples was 70.1: 29.9%. The density in P. falciparum positive samples [median (95%CI): 10,340 (5280-19,200) μ/l] was significantly higher than P. vivax positive samples [4508 (3240-6120) μ/l]. Sixteen out of twenty-nine (55.2%) and 36 out of 68 (52.9%) P. falciparum- and P. vivax-infected samples, respectively, were gametocyte-positive. Gametocyte density in the P. falciparum-infected[124 (69-253) /μl] was significantly higher than that of the P. vivax-infected [54 (45-70)/μl] samples. A significant correlation between gametocyte density and pretreatment parasitemia was only detected in P. falciparum-infected, but not P. vivax-infected samples.
Interpretation & conclusion: The observed high prevalence rates of pretreatment gametocyte carriage of both malaria species, which serves as a large malaria reservoir, particularly in P. falciparum infection, could have a significant impact on malaria control in the endemic populations. Keywords: Plasmodium falciparum; Plasmodium vivax; gametocyte; symptomatic; Thai-Myanmar border
How to cite this article:
Martviset P, Kitvatanachai S, Tarasuk M, Muhamad P, Na-Bangchang K. Pretreatment gametocyte carriage in symptomatic patients with Plasmodium falciparum and Plasmodium vivax infections on the Thai-Myanmar border. J Vector Borne Dis 2021;58:257-64 |
How to cite this URL:
Martviset P, Kitvatanachai S, Tarasuk M, Muhamad P, Na-Bangchang K. Pretreatment gametocyte carriage in symptomatic patients with Plasmodium falciparum and Plasmodium vivax infections on the Thai-Myanmar border. J Vector Borne Dis [serial online] 2021 [cited 2023 Mar 29];58:257-64. Available from: http://www.jvbd.org//text.asp?2021/58/3/257/316274 |
| Introduction | |  |
Malaria remains one of the most important parasitic infections worldwide despite tremendous efforts on human resources, infrastructure, and financial inputs through the control programs at both national and international levels[1]. This vector-borne disease is caused by Apicomplexan unicellular parasite in the genus Plasmodium and transmitted to humans by Anopheles mosquitoes. The number of malaria cases has been declining globally, particularly in Southeast Asia, from 17 cases per 1000 population in 2010 to 7 cases per 1000 population in 2017[1]. The estimated number of cases worldwide in 2017 was 219 million with 435000 deaths, 92% of which occurred in Africa[1]. The malaria burden in Thailand has been decreasing over the past five years. Nevertheless, the change in the epidemiology and distribution of malaria species is remarkable.
Plasmodium falciparum and P. vivax are the predominant species. Since 2000, the incidence ratio has been reversed from 70:30% to 50:50%, and recently 12:82% P. falciparum: P. vivax[2]. Several factors account for this reversion, including malaria parasite factors (biology and resistant development), vector factors (mosquito’s species and behaviors), drug factors (efficacy of antimalarial drugs and pattern of drug resistance), and environmental factors (weather, humidity, and water pollution)[3]. Concerning malaria biology, the gametogenesis process is one of the crucial factors accountable for this change. Gametocyte, the sexual stage of malaria life-cycle, is the only developmental stage that is responsible for malarial transmissionfrom an infected person to Anopheles mosquitoes,which finally develops into an infective stage-- sporozoite[4]. Despite intensive research efforts, the biology of gametogenesis and its impact on malaria control and elimination remain unclear[5],[6]. In almost all cases, monitoring of gametocyte carriage and density have been performed following treatment with antimalarial drugs. Investigation of the patterns of pretreatment gametocyte carriage and density in both symptomatic and asymptomatic malaria patients in different populations would provide important information for malaria control and elimination.
The present study aimed to investigate the prevalence of pretreatment gametocyte carriage and density in P. falciparum and P. vivax infections in the low malaria-endemic area in Kanchanaburi Province, on the Thai-Myanmar border. Also, factors associated with gametocyte carriage and density were also determined.
| Material & Methods | | |
Study site and population
The study was conducted during June 2016 and February 2018 at the malaria clinics in Kanchanaburi province, the area with low malaria transmission (entomological inoculation rate <1 per species) on the Thai-Myanmar border. This province is well documented as the top ten highest malaria cases in the countrywith multidrug-resistant P. falciparum[7],[8]. The intervention strategies as well as surveillance activities remained unchanged during the study period. The annual peak malaria season is during April and July[9]. Anopheles maculatus,the exophagic and nighttime feeder, is the primary malaria vector in this area. A large-scale cross-sectional survey conducted in 2003–2004 reported monthly prevalence rates of 0.1–1.5% for P. falciparum and 0.2–0.6% for P. vivax by microscopy[9].
Approval of the study protocol was obtained from the Ethics Committee of the Ministry of Public Health of Thailand (No. IHRP 2017012) and Rangsit University (No. RSEC 52/2559). Informed consent for study participation was obtained from each patient with confirmed Plasmodium infection. Demographic information (sex, age, ethnic lineage, occupation, and household location) of all patients was recorded in the case recorded form.
Microscopic examination
Finger-prick blood samples were collected before treatment from all symptomatic malaria patients for microscopic examination of peripheral blood smears for identification and determination of malaria species and density,as well as gametocytes. Parasite density (perμL) was estimated from thick blood smear and calculated as the number of parasites/500 WBCs × WBC count perμL, using average WBC count in the Thai population of 8,000 cells/μL. The presence of asexual stage gametocyte was examined and reported as a positive or negative result.
Molecular analysis of malaria species
Blood samples (200–500 μL) from all P. falciparum and P. vivax-infected cases were collected onto the dried blood spot (3MM Whatman filter paper, Clifton, NJ, USA) for confirmation of malaria infection as well as the infected species. The samples were air-dried and stored in the zippered plastic bags containing desiccator. Malaria parasite DNA was extracted from dried blood spots using QIAamp® Blood Kit (Qiagen Inc.,Valencia, CA,USA) following the manufacturer’s protocol and the concentration quantified using Nanodrop™ ND-1000 UV-Vis spectrophotometer (ThermoFisher Scientific,Wilmington, DE, USA). Malaria species was identified using species-specific nested-PCR[10]. The first round of amplification was performed using Plasmodium 18s ribosomal DNA specific primers (Forward: 5′-TCAAAGATTAAGCCATGCAAGTGA-3′; and Reverse: 5′-CCTGTTGTTGCCTTAAACTCC-3′). The amplification steps consisted of predenaturation at 94°C for 4 min, 30 cycles of denaturation at 94°C for 30 sec, annealing at 55°C for 1 min, extension at 72°C for 1 min, and a final extension at 72°C for 10 min. The amplicons from the first round were used as the template for species-specific amplification using P. falciparum (Pf) and P. vivax (Pv) 18s ribosomal DNA specific primers (Pf Forward: 5′-TTAAACTGGTTTGGGAAAACCAAATATATT-3′; Pf Reverse: 5′-ACACAATGAACTCAATCAATCATGACTACCCGTC-3′; PvForward: 5′-CGCTTCTAGCTTAATCCACATAACTGATAC-3′; and PvReverse: 5′-ACTTCCAAGCCGAAGCAAAGAAAGTCCTTA-3′). The amplification steps consisted of predenaturation at 94°C for 4 min, 35 cycles of denaturation at 94°C for 30 sec, annealing at 58°C for 1 min, extension at 72°C for 1 min, and a final extension at 72°C for 10 min. The laboratory cultured P. falciparum and P. vivax were used as positive control samples for PCR amplification. The PCR products were separated on 2% agarose gel electrophoresis.
Molecular analysis of gametocytes
Reverse transcriptase-polymerase chain reaction (RT-PCR) was used to detect the gametocyte stage of Plasmodiumin all blood samples (positive and negative malaria cases)[11],[12],using specific primers for 18s rRNA--pfs25 and pvs25. Total RNA (50 ng each) was used as a template. The primers used were pfs 25forward (5′-GAAATCCCGTTTCATACGCTTG-3′), pfs25 reverse (5′-AGTTTTAACAGGATT GCTTGTATCTAA-3′), pvs25 forward (5′-ACACTTGTGTGCTTGATGTATGTC-3′), and pvs25 reverse (5′-ACTTTGCCAATAGCACATGAGCAA-3′). Human GAPDH was used as an internal control of the samples mixed with human and malaria genetic materials. The amplification steps consisted of predenaturation at 94°C for 4 min, 35 cycles of denaturation at 94°C for 30 secs, annealing at 60°C for 1 min, extension at 72°C for 1 min, anda final extension at 72°C for 10 min. The laboratory cultured P. falciparum gametocyte and confirmed clinical P. vivax gametocyte-positive samples were used as positive controls for PCR amplification. The PCR products were confirmed for correct sequences by DNA sequencing.
The density of gametocytes in the gametocyte-positive samples was determined by quantitative real-time PCR (qRT-PCR). The pfs25 and pvs25 PCR amplicons were used for standardization of SYBR green, using specific primers as previously described with modification[13]. Briefly, the PCR amplicons were diluted in sterile water to 1000, 500, 200, 100, 50, and 10 copy/μL. The RT-PCR was performed using iTaq™ Universal SYBR® Green Supermix (BIORAD, Hercules, CA, USA)with the same condition as described above (internal controls: Pfβ-actin and PvTubulin-1). The copy number of gametocytemia was determined from the calibration curve[13],[14].
Statistical analyses
The statistical analyses were performed using SPSS version 17 (IBM Inc., Armonk, NY, USA). Qualitative data are presented as number and percentage (%), and the association between variables was determined using the chi-square test. Quantitative data are presented as median (95% confidence interval: 95% CI), and a significant difference between groups was analyzed by the Mann- Whitney U test (unpaired data) or Wilcoxon Signed Rank test (paired data). Correlation between two quantitative variables was determined using the Spearman Correlation test. The statistical significance level was set at p = 0.05 for all tests.
| Results | | |
A total of 126 blood samples were collected from patients with signs and symptoms of malaria who attended malaria clinics in Kanchanaburi (66 males, 37 females, and 23 unspecified sex, aged 1 to 68 years). These included 49, 8, 38, 6 and 1 samples for Thai, Burmese, Karen, Mon and Tawai, respectively (24 unidentified ethnics). Malaria positive cases detected by microscopic examination were confirmed by species-specific nested-PCR in 97 (29 and 68 samples for P. falciparum and P. vivax, respectively) out of 126 samples. Twenty-six microscopic-negative and three PCR-negative samples were excluded from further analysis. One sample was identified as Plasmodium-negative by microscopic examination but was confirmed P. falciparum-positiveby molecular analysis. The distribution of P. vivax and P. falciparum-infected samples during the two periods, i.e., June 2016 to December 2016 and January 2017 to February 2018 was comparable (10 vs. 20 samples and 19 vs. 48 samples, respectively). The proportion of P. vivax and P. falciparum-infected samples was 70.1: 29.9%. The parasite density P. falciparum positive samples [median (95%CI): 10,340 (5,280-19,200) parasite/μL] was significantly higher than P. vivax positive samples [median (95%CI): 4,508 (3,240-6,120) parasite/μL] (p = 0.005).
Sixteen out of twenty-nine (55.2%) and 36 out of 68 (52.9%) P. falciparum- and P. vivax-infected samples, respectively, were gametocyte-positive. Parasite densities of the gametocyte-positive and gametocyte-negative samples were comparable [Table 1]. However, the gametocyte density in the P. falciparum-infected [124 (69-253)/μL was significantly (p<0.001) higher than that of the P. vivax-infected [54 (45-70)/μL] samples. There was no association between gametocyte carriagein both malaria species and demographic variables (sex, ethnics lineage, occupation, and home location) as well as pretreatment parasitemia when classified into two levels (< or > 10,000 μL) [Table 1]. Significant correlation between pretreatment parasitemia and gametocyte density was however, detectedin P. falciparum-infected (p = 0.017, r = +0.585), but not P. vivax-infected (p = 0.167, r = +0.331) samples. | Table 1: Demographics and parasitological parameters (pretreatment parasitemia and gametocytemia) of blood samples collected from patients with P. vivax and P. falciparum infections in Kanchanaburi province during June 2016 and February 2018. Data are presented as number (percentage) or median (95% confidence interval), where appropriate.
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| Discussion | | |
Gametocyte is the infective malaria parasite stage which is responsible for malaria transmission via the Anopheles mosquitoes[5],[6]. It is developed from sexually committed merozoites or schizont in the circulation and inside the hepatocellular tissue before releasing to the circulation[15],[16],[17]. The generation of gametocyte is initiated through the activation of AP2-G transcription factor in the committed progeny, which triggers the expression of an early gametocyte gene series including the surface antigens such as pfs16, pfs25, pfs27 in P. falciparum and pvs25 in P. vivax[15],[18],[19],[20]. The gametocytes often circulate in blood at very low density particularly in asymptomatic cases, with the densityunderestimated by routine microscopic examination (detection limit = 10-1-10° gametocytes/μL blood)[21],[22],[23]. The more sensitive molecular detection tools (detection limit 0.02-0.1 gametocyte/μL blood) such as reverse transcriptase-polymerase chain reaction (RT-PCR)[24],[25], quantitative nucleic acid sequence-based amplification (QT-NASBA),[25],[26] and RT loop-mediated isothermal amplification (RT-LAMP)[27], are commonly used for quantitative determination of gametocyte density in patients, blood[25],[26],[27],[28],[29].
The proportion of P. vivax and P. falciparum cases in the present study was 70.1:29.9%. The high parasite density in P. falciparum compared with P. vivax-infected samples (10340 vs. 4508 /μL) is explained by the biology of P. falciparum which produces a higher number of merozoites from one erythrocytic schizont. Furthermore, the number of merozoites produced per one hepatocyte cell is twice as many as in P. vivax[30],[31]. The proportions of gametocyte-positive samples in both P. vivax- and P. falciparum-infected samples were comparable (52.9% and 55.2%, respectively), but the gametocyte density in P. falciparum-infected samples (124/μL) was significantly higher than P. vivax-infected samples (54 /μL). This high prevalence rate of gametocyte carriage and level of density may potentially impact the transmissibility and thus, malaria control, particularly P. falciparum infection. In previous studies, gametocyte carriage was reported to be more common in P. vivax than P. falciparum infection due to the higher rate of gametocyte production, short longevity, and effective transmissibility of P. vivax[30],[31]. The observed high prevalence of pretreatment gametocyte carriage before treatment in symptomatic patientsis consistent with that previously reported from Africa and Asia[32],[33],[34],[35]. In a recent study conducted in the same area of Thai-Myanmar border with low malaria transmission level, 71.5% and 72.0% of P. vivax and P. falciparum infections, respectively, were gametocyte positive by pvs25/ pfs25 RT-PCR[36]. It was noted for a considerable variation of prevalence rates of gametocyte carriages ranging from 3.5% to 77% in various reports as most were the analysis from post-treatment blood samples[30],[37],[38],[39]. With routine microscopy, the gametocyte-positive rate was lower than 50% of clinical[40],[41],[42],[43],[44],[45],[46] and asymptomatic[47],[48],[49]P. falciparum-infected cases. Low density, asymptomatic infections with Plasmodium spp. are common in low malaria endemicity areas,including Thailand[34],[50],[51],[52]. Studies in Thailand showed high rates of asymptomatic infections of 15.4% to 60%,[36],[53],[54] suggesting a substantial contribution to the transmission reservoir. In the present study, all samples were obtained from only symptomatic patients. This group of patients presents a significant challenge to the malaria control program which is based exclusively on case management and vector control. Gametocyte carriage has also been shown to be a very sensitive measure of deteriorating and the first warning sign of antimalarial drug resistance. Two examples that best illustrate this claim are sulfadoxine-pyrimethamine resistance in South Africa[40] and chloroquine resistance in Sri Lanka[55]. Monitoring the effects of antimalarial chemotherapy on gametocyte carriage and identifying drugs that promote gametocytocidal activity are therefore priorities in efforts to eliminate malaria[56].
Several factors have been reported to be associated with gametocyte carriage. These include patients’ demographics (age) and genetic factors (e.g., HbAS, HbAC, and HbCC genotypes), malaria disease severity (pretreatment parasite density, fever, and duration of malaria symptoms, hemoglobin level, and platelet count), co-infections with the virus (HIV), and treatment with antimalarial drugs[57],[58],[59],[60],[61],[62],[63]. Parasite density before treatment was reported to be associated with the gametogenesis of P. vivax and P. falciparum in Thailand[36]. In our study, only the level of pretreatment parasitemia was found to be associated with gametocyte density. No association was found between gametocyte carriage/density in both malaria species and patients’ demographics (sex, age, ethics lineage, occupation, and home location). A significant positive correlation between gametocyte density and pretreatment parasitemia was found in the P. falciparum gametocyte-positive samples. This finding corresponded to the observation of a 2-fold higher parasite density in P. falciparum compared with P. vivax infection. P. falciparum acquires higher parasitemia than P. vivax for starting the development of gametocytes and generates sexual stages immediately in the blood-stage infection[64],[65]. A 10-fold increase in parasite density was found to increase the probability of detecting peripheral blood gametocytemia by 3.14-fold and 1.83-fold in P. vivax and P. falciparum infections, respectively, in this low transmission area[36]. Gametocyte carriage was reported to be most prevalent in younger age groups who also have the highest prevalence and density of asexual parasite in malaria high transmission areas[35],[66],[67],[68],[69],[70]. Certain antimalarial drugs, particularly artemisinins, exhibit promising gametocytocidal activity and thus, provide significant interruption of malaria transmission. The 3-day artesunate-mefloquine and dihydroartemisinin-piperaquine combinations are the artemisinin-based combination therapies (ACTs) used as first-line treatment for acute uncomplicated P. falciparum together with the gametocytocidal drug primaquine (for individuals living in malaria-endemic areas) in the current study area. The uses of ACTs have been associated with lower gametocyte carriage and post-treatment malaria transmission in most areas[28],[41],[61],[62],[71],[72]. For P. vivax infection, 3-day chloroquine in combination with the anti-hypnozoite primaquine is the first-line treatment for P. vivax infection in Thailand for more than 60 years. Unfortunately, data on post-treatment gametocytemia was not available in the current study to assess the impact of antimalarial drugs on gametocyte clearance in this populationas most patients did not come back for follow-up after recovery from acute malaria symptoms. Furthermore, it was unclear whether the included blood samples were obtained from patients who had previous episodes of malaria infection and received treatment with antimalarial drugs.
In conclusion, results from the present study provided information on the patterns of pretreatment gametocyte carriage and density in symptomatic P. falciparum and P. vivax infections in the population residing on the Thai- Myanmar border which would contribute to treatment and control policy for both infections. The observed high prevalence rate of pretreatment gametocyte carriage of both malaria species, which serves as a large malaria reservoir, particularly in P. falciparum infection, could have a significant impact on malaria control in the population in the endemic areas.
Conflict of interest: None
| Acknowledgements | | |
This study was financially supported by Thammasat University through the Center of Excellence in Pharmacology and Molecular Biology of Malaria and Cholangiocarcinoma, the Ministry of Higher Education, Science, Research and Innovation of Thailand through the National Research University Project, the National Research Council of Thailand, and Rangsit University research grant (Grant No. 2/2560).
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