Nguitragool et al. Parasites & Vectors (2017) 10:512 DOI 10.1186/s13071-017-2407-y RESEARCH Open Access Very high carriage of gametocytes in asymptomatic low-density Plasmodium falciparum and P. vivax infections in western Thailand Wang Nguitragool1†, Ivo Mueller2,3,4†, Chalermpon Kumpitak5, Teerawat Saeseu5, Sirasate Bantuchai5, Ritthideach Yorsaeng5, Surapon Yimsamran6, Wanchai Maneeboonyang6, Patiwat Sa-angchai6, Wutthichai Chaimungkun6, Prasert Rukmanee6, Supalarp Puangsa-art6, Nipon Thanyavanich6, Cristian Koepfli3,4, Ingrid Felger7, Jetsumon Sattabongkot5 and Pratap Singhasivanon6* Abstract Background: Low-density asymptomatic infections of Plasmodium spp. are common in low endemicity areas worldwide, but outside Africa, their contribution to malaria transmission is poorly understood. Community-based studies with highly sensitive molecular diagnostics are needed to quantify the asymptomatic reservoir of Plasmodium falciparum and P. vivax infections in Thai communities. Methods: A cross-sectional survey of 4309 participants was conducted in three endemic areas in Kanchanaburi and Ratchaburi provinces of Thailand in 2012. The presence of P. falciparum and P. vivax parasites was determined using 18S rRNA qPCR. Gametocytes were also detected by pfs25 / pvs25 qRT-PCRs. Results: A total of 133 individuals were found infected with P. vivax (3.09%), 37 with P. falciparum (0.86%), and 11 with mixed P. vivax/ P. falciparum (0.26%). The clear majority of both P. vivax (91.7%) and P. falciparum (89.8%) infections were not accompanied by any febrile symptoms. Infections with either species were most common in adolescent and adult males. Recent travel to Myanmar was highly associated with P. falciparum (OR = 9.0, P = 0.001) but not P. vivax infections (P = 0.13). A large number of P. vivax (71.5%) and P. falciparum (72.0%) infections were gametocyte positive by pvs25/pfs25 qRT-PCR. Detection of gametocyte-specific pvs25 and pfs25 transcripts was strongly dependent on parasite density. pvs25 transcript numbers, a measure of gametocyte density, were also highly correlated with parasite density (r2 = 0.82, P < 0.001). Conclusions: Asymptomatic infections with Plasmodium spp. were common in western Thai communities in 2012. The high prevalence of gametocytes indicates that these infections may contribute substantially to the maintenance of local malaria transmission. Keywords: Malaria, Asymptomatic infection, Gametocytes, Transmission, Thailand * Correspondence: [email protected] †Equal contributors 6Department of Tropical Hygiene, Faculty of Tropical Medicine, Mahidol University, Bangkok, Thailand Full list of author information is available at the end of the article © The Author(s). 2017 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Nguitragool et al. Parasites & Vectors (2017) 10:512 Page 2 of 9 Background was strongly linked to Anopheles dirus infectivity; only a The last 30 years have seen a great reduction in the bur- few patient blood samples without LM-detectable game- den of malaria in South East Asia in general, and par- tocytaemia were found to infect mosquitoes [24, 25]. In ticularly in Thailand [1]. As a consequence of sustained contrast, P. vivax appears to be more infectious, and a control and economic development in many rural areas, gametocyte density under LM limit of detection can the incidence of malaria has dropped from a peak of readily lead to mosquito infection [26]. 10.7 cases/1000 in early 1981 to currently less than 1/ Molecular techniques are much more sensitive at de- 1000. Malaria has been eliminated from most areas of tecting gametocytes than LM [22]. In a study from Thailand except the border regions with Myanmar in Thailand that compared LM with molecular methods to the west and Cambodia in the east [2, 3]. The reductions detect gametocytes in patients from Tak Province, the have been more pronounced for P. falciparum than for proportion of gametocyte positive infections increased P. vivax, which now accounts for most local cases in from 8.9 to 89.5% for P. falciparum and from 31.1 to Thailand [1]. The advent of artemisinin resistance on 91.1% for P. vivax [27]. To date, there are no data on the Thai-Cambodian border [4] and more recently in the prevalence of gametocytes by PCR in community western Thailand [5, 6], has emphasized the need to surveys in Thailand. move from control to elimination of malaria in these We, therefore, conducted a cross-sectional survey of areas [7, 8]. Despite this renewed focus, there is still a 4309 people living in three malaria endemic communi- substantial lack of understanding on how transmission ties in Kanchanaburi and Ratchaburi provinces. We in the remaining endemic areas is sustained. While combined full population sampling with the sensitive Thailand has an excellent system that detects, treats and molecular diagnosis of both asexual blood-stage para- monitors clinical malaria cases even in relatively remote sites and gametocytes. rural areas [9], there is no routine monitoring of the in- fectious reservoir. Methods Low density, asymptomatic infections with Plasmo- Description of study sites dium spp. are common in low endemicity areas world- Three sites on a 250 km central span of the Thai- wide [10–14]. Studies in migrant populations in Burmese border were selected for our cross-sectional Thailand have also detected high rates of asymptomatic survey: (i) Suan Phueng district of Ratchaburi Province; infections [15, 16]. However, community-based studies (ii) Ban Kong Mong Tha, Sangkhla Buri district of are scarce [17–19] and, except for one recent study [20], Kanchanaburi Province; and (iii) Ban Bong Ti, Sai Yok were small and assayed relatively small volumes of blood district of Kanchanaburi Province. All areas had stable from dried filter paper blood spots. More studies with populations (main ethnic groups Thai and Karen) with higher detection sensitivity are thus urgently needed to less than 2% reporting to have travelled in the previous quantify the asymptomatic reservoir of P. falciparum month. Agriculture, farming and forest foraging were and P. vivax infections in Thai communities. main occupations. The annual peak malaria season of Little is known about the role of asymptomatic infec- these areas was April-July [28]. tions in sustaining local malaria transmission in The southernmost study site, Suan Phueng district, is Thailand. In a single study conducted in Mae Hong Son, located 163 km west of Bangkok and encompasses the it was found that blood from 15.4% and 60% of study mountainous regions of the Tanaosri Mountain range. participants with afebrile P. falciparum and P. vivax in- This study site was composed of four connected small vil- fections could infect mosquitos [21]. In this study 46% lages: Wangko, Huai Krawan, Pong Hang and Huai Phak. of all microscopy-positive individuals were afebrile, and The combined population size was 2188. Most houses the number of submicroscopic infections is unknown. were clustered along a stream. Anopheles minimus and Given the prevalence of such afebrile infections and the Anopheles maculatus, exophagic and nighttime feeders, low incidence rate of clinical malaria, it was concluded were the primary malaria vectors in this area. A large- that asymptomatic infection constituted the main reser- scale cross-sectional survey conducted in 2003–2004 re- voir of transmission in this population [21]. ported monthly prevalence rates of 0.1–1.5% for P. falcip- Light microscopy (LM) is relatively insensitive for de- arum and 0.2–0.6% for P. vivax by microscopy [28]. tecting gametocytes (and thus potentially infectious The central site, Bong Ti, was composed of three parasite carriers), particularly in asymptomatic individ- villages: Ban Bong Ti Bon, Ban Bong Ti Lang and Ban uals with low-density infections [22]. In Thailand, game- Thai Muang. The combined population size was 3176. tocytes were more commonly observed in P. vivax The site was situated 65 km west of Kanchanaburi in compared to P. falciparum patients [23]. In membrane hilly terrain. To date, no detailed malaria epidemio- feeding studies using south-east Asian mosquito Anoph- logical and entomological study has been conducted in eles dirus, P. falciparum gametocyte observation by LM this area. Most houses were accessible by paved roads. Nguitragool et al. Parasites & Vectors (2017) 10:512 Page 3 of 9 The northernmost site Kong Mong Tha was a single vil- kit, on-column treatment with RNase-Free DNase set lage located in a remote valley surrounded by moun- (Qiagen) was used on all samples to further remove trace tains. The population size was 959. Once accessible only genomic DNA contaminant. by boats or foot for 10 months of the year, the village had become accessible by trucks throughout the year at the time of this study. Previous studies done in 2000 Detection of parasite DNA and gametocyte-specific estimated malaria prevalence to be 5.2% for P. falciparum transcripts by qPCR and 5.9% for P. vivax by LM and identified the predomin- All qPCR and qRT-PCR measurements were performed ant anophelines as A. minimus, A. sawadwongporni, A. on a CFX96 Realtime PCR Detection System and ana- maculatus, A. campestris and A. barbirostris [29]. lysed with CFX Manager Software Version 3.0 (Bio-Rad Laboratories, California, USA). All qPCRs used iTaq Survey methods Universal Probes Supermix (Bio-Rad Laboratories), and Participation in the study was household based. Conveni- all qRT-PCRs used Superscript III One-Step Quantitative ence sampling of households was used, and all household RT-PCR System (Invitrogen, Massachusetts, USA).