Introduction

Schistosomiasis is a disease caused by blood flukes of the genus Schistosoma which are transmitted by freshwater snails [1,2]. There are three identified species that cause human infections in Asia. Schistosoma japonicum has the widest distribution in China, the Philippines, and small pockets of Indonesia. S. malayensis is restricted to Malaysia, and S. mekongi is present in small areas of Cambodia and Lao PDR, and previously Thailand [1]. All three are zoonotic [3–7]. Morphologically and genetically these species are very similar; it is difficult to differentiate species based on morphology alone (Fig 1). The main diagnostic method for schistosomiasis is the microscopic Kato-Katz (KK) technique, which has poor sensitivity at low intensity infections and relies solely on morphology to diagnose parasite infection.

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Fig 1. A) Alignment of 28S ribosomal primer binding region [8] for the three Asian schistosome species (Schistosoma mekongi, S. japonicum, and S. malayensis) showing high genetic similarity.

B) S. mekongi eggs (50 – 80 µm long by 40 – 65 µm wide) and C) S. malayensis eggs (52 – 90 µm long x 33 – 62 µm wide) are generally smaller and rounder [9] than D) S. japonicum eggs (70 – 100 µm long by 55 – 64 µm wide), while E) S. incognitum eggs (85 µm long x 50 µm wide) have a boxy shape with a large terminal spine. Panel B is from the CDC website, panel C is an artistic representation of ‌‌S. malayensis, panel D was provided by author CAG, panel E is reproduced from Ajjampur, 2024 [10].

https://doi.org/10.1371/journal.pntd.0014384.g001

Myanmar has been considered non-endemic for schistosomiasis, although there have been unconfirmed historical reports of both S. japonicum and S. mekongi, with diagnosis based primarily on morphology. Meanwhile there have been several studies over the last 13 years indicating that schistosomiasis is either emerging or re-emerging in the country (Fig 2). The first of these studies showed a serological prevalence of 23.8% for schistosomiasis around Lake Inle (Inlay) between 2012 and 2013 [11]. The serological method used, enzyme-linked immunosorbent assay (ELISA) IgG (AccuDiag Schistosoma IgG) does not differentiate between S. japonicum and S. mekongi. According to the manufacturer’s website the test has a sensitivity of 100% and specificity of 85%, which indicates there may be some cross-reactivity [12]. In 2018 there was an outbreak of schistosomiasis in Rakhine State in Mrauk-U and Sittwe townships with more than 400 cases confirmed and another 800 cases suspected [13]. Diagnosis in that outbreak was via KK. A prevalence of 2.9% (6/205 participants) was identified in 2016 in Shwegyin (Shwe Kyin) Township, Bago region by KK, while 8 samples were positive by conventional Polymerase Chain Reaction (PCR) [14] with bands of 300 bp, associated with S. mekongi, identified [15]. A year later in 2017 a Schistosoma spp. prevalence of 3% was identified across four villages in Shwegyin across inland and riverside villages (n = 698) by KK (n = 20) and wet mount microscopy (n = 7) [16]. A case study associated with the 2018 outbreak in Rakhine reported schistosomiasis in a patient using an S. mansoni ELISA [17]. As S. mansoni is restricted to Africa and South America, and not found in Asia, the result almost certainly reflects cross-reactivity with the local Schistosoma species responsible for infection. Thus, there remains some uncertainty regarding which Schistosoma species are responsible for infections in Myanmar. Historical movements of Japanese troops during World War II and the country’s proximity to endemic areas in Thailand raise the possibility of multiple species infecting humans. However, the detection of a ~ 300 bp amplicon consistent with S. mekongi by conventional PCR suggests that S. mekongi is the most likely species involved.

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Fig 2. Showing locations of previous schistosomiasis outbreaks in Rakhine State (2018) and Lake Inlay (Inle) in Shan State (2012 and 2013), Shwegyin in Bago Region (2016, 2017), the location of Phyu Township, also in Bago Region, where the current study was performed (2016), and Yezin Dam, site of a snail survey in 2019 – 2020 [17].

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Schistosoma parasites have a high degree of specificity for their molluscan hosts. Even within the same species complex of Schistosoma, certain geographical strains and their associated snail hosts are not compatible and cannot establish infection [18,19]. In Cambodia, Lao PDR, and Thailand Neotricula aperta snails are intermediate hosts for S. mekongi while Oncomelania hupensis snails are hosts of S. japonicum in China (O. h. hupensis), the Philippines (O. h. quadrasi), and Indonesia (O. h. lindoensis); in Malaysia Robertsiella kaporenisis is the molluscan host. There is limited information on snail intermediate hosts or definitive hosts for human schistosomiasis in Myanmar. The ruminant infecting S. spindale has been identified in Indoplanorbis exustus snails from Yezin Dam, a man-made reservoir located in Zayarthiri township in Nay Pyi Taw City [17] (Fig 2). I. exustus snails were also identified from Lake Inlay in the same survey. Notably, I. exustus is also an intermediate host for S. incognitum (Fig 1) which has previously been identified in animals from Thailand and Indonesia [20–22], and more recently excreted by two humans from India [10]; this may not be representative of true infection and merely eggs passing through the gastrointestinal tract. Bithynia siamensis, the snail host for Opisthorchis sinensis, was identified from Lake Inlay and Yezin Dam, along with cercariae of O. sinensis [17,23].

Definitive hosts between the three species potentially causing schistosomiasis in Myanmar differ significantly, with both S. mekongi and S. incognitum identified from dogs, pigs, and rats, as well as sheep and goats for S. incognitum, while S. japonicum has 46 known mammalian hosts with water buffalo a notable major reservoir host. Experimental infections of primates with S. incognitum have been successful, indicating human infection may be possible [10,24,25]. With the high number of potential hosts for S. japonicum it is important to confirm which species is occurring in Myanmar and identify any possible animal hosts which would need to be considered as part of a One Health approach to schistosomiasis control.

Methods

Ethics statement

Ethical approval was provided by the Protocol and Ethics Review Committee, the Department of Medical Research, Ministry of Health and Sports, Myanmar, (Approval Number: Ethics/DMR/2016/099), the Science and Medical Delegated Ethics Review Committee, The Australian National University, Australia (Protocol: 2016/406), and QIMR Berghofer Human ethics committee (P1271). A waiver of consent for reanalysis of these samples for other parasites was granted by the QIMRB Human ethics committee in 2025 (P1271). In each of the selected schools, the study team conducted a meeting with parents or guardians of the fifth graders to explain the study and obtain written consent from parents or guardians for students participating in the study.

In August 2016 we conducted a stool survey in schoolchildren from Phyu township, Bago Region, for soil-transmitted helminths (STH) [26]. We initially did not consider schistosomiasis due to the focus of the grant on the STH. Notably, strongyloidiasis which has been called the most neglected of the neglected tropical diseases was only formally added to the STH grouping by the WHO in 2024 and was overlooked. The development of a sensitive qPCR assay for A. ceylanicum in 2017 has further helped identification of this important zoonotic helminth [27].

Stool samples were collected from 274 schoolchildren and examined by KK for any helminth infection; 264 samples were also tested by real-time PCR (qPCR) for STH (Ascaris lumbricoides, Ancylostoma spp., Necator americanus, Trichuris trichiura). Prevalence of STH was high with 78.8% of schoolchildren infected by at least one STH by qPCR, and 33.3% by KK [26] (Fig 3). A single sample was identified by KK to be positive for Schistosoma spp. and another for Strongyloides spp., but was not reported at the time. Other helminths also identified by KK but not previously reported are Diphyllobothrium latum (n = 3), Clonorchis sinensis (n = 1), and Hymenolepsis nana (n = 3) (Fig 3).

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Fig 3. A) Flow diagram showing timing of sample collection, Kato-Katz, initial qPCR assay and more recent follow up assay and attempted sequencing in 2025 B) Total number of helminth detections by KK and qPCR assays.

https://doi.org/10.1371/journal.pntd.0014384.g003

Here, we detail the results of new qPCR assays for further helminth detection. Stool samples had previously undergone DNA extraction and qPCR for STH in 2017 [26] and it was these DNA extracts that we then revisited to test additionally for schistosomiasis using two different assays capable of detecting both S. japonicum and S. mekongi [8,28], and additionally ran qPCR assays to detect Strongyloides spp. [29] and S. stercoralis [30], Opisthorchis viverrini [31], and a duplex qPCR for A. duodenale and A. ceylanicum [27] (Fig 3), PCRs were performed as per the original paper methods, with some changes. GoTaq Probe qPCR mastermix (Promega) was used for qPCR, and all probes were ordered as FAM (Integrated DNA Technologies). We also re-ran the two Schistosoma spp. samples for T. trichiura and A. lumbricoides as per the previous paper to test sample degradation, noting that both samples had previously been positive for these parasites. Internal controls were not used in the recent assays.

Results

Of the 264 samples tested by qPCR, two were positive for Schistosoma using one of the assays, including the KK positive sample; twelve samples were positive for Strongyloides, which were not the same as the positive sample detected by KK, and eleven samples were positive for A. ceylanicum. No samples were positive for O. viverrini or A. duodenale. Full results can be found in S1 File. We attempted Big Dye sequencing of the Schistosoma amplicons, however our attempts failed (no visible bands on gel), highlighting limitations in template quality or primer-target mismatch. The average cycle threshold (ct) scores for both positive samples were 36.02 and 30.56. We also attempted to use generic trematode primers that we know amplify Schistosoma spp. DNA but were also unsuccessful [29], making low template quality the most likely cause of failure to sequence. These two Schistosoma spp. positive samples were previously positive for T. trichiura and A. lumbricoides and were re-tested to test the hypothesis that low template quality was causing issues with sequencing [26]. Neither sample was positive upon re-test in 2026 for T. trichiura, and only one was positive for A. lumbricoides with a ct of 32.42, 10 cycles higher than it had originally tested at, further confirming degradation of the DNA.

Discussion

These molecular findings, though limited in number, reinforce evidence that schistosomiasis may be more widespread in Myanmar than previously acknowledged. Phyu and Shwegyin townships are roughly 100 km from each other, making local transmission in Bago Region likely. Inle Lake is farther away, about 400 km from Phyu, while the Rakhine mountain range and poor road conditions present a notable barrier between Phyu and the capital city of Rakhine State, which is approximately 800 km away. The inability to sequence the amplicon prevents species identification; clarifying whether infections are due to S. japonicum, S. mekongi, or other species is essential for guiding control strategies. We also cannot rule out multiple strains occurring, or that a specific species or strain is occurring in Myanmar as occurs in Malaysia (S. malayensis). The age of the DNA samples, although stored in -20°C since the original study, means that the DNA may have degraded over time leading to lower detection. Samples had undergone a minimum of three freeze-thaw cycles, and there were several occasions of the freezer malfunctioning over the intervening years which may have led to additional full or partial thaws. Additionally, just over 31% of samples had less than 200mg of stool for DNA extraction which also decreases the sensitivity of qPCR (S1 File). The two primer assays used for schistosomiasis were originally designed for S. japonicum detection but also show evidence of amplifying S. mekongi when testing specificity [8,28]. The first assay [8] which detected zero positive cases is designed on the ribosomal 28 large subunit (28LSU) (Fig 1), while the second assay [28] that detected the two positives is designed on the a repetitive tandem sequence of S. japonicum. The latter was previously shown a higher sensitivity than other published S. japonicum assays, indicating a higher copy number of the tandem repeat than targets of these other assays including mitochondrial gene NADH1, ribosomal 16S rRNA, and a putative DNA phot-lyase gene [28,32]. These sequences are also shorter, which can improve detection in cases where DNA may be more fragmented due to low template quality. The SjTR1 assay has an amplicon length of 80 bp, including primers, and a reported limit of detection (LOD) of 200 attograms for S. japonicum DNA [28]. The Sme assay, which did not identify any S. mekongi in this study, has an amplicon length of 264 bp which is quite long for a qPCR assay and could also result in the lower assay sensitivity observed here. The Sme assay does not have a stated LOD. To address this we ran a ten-fold serial dilution of S. mekongi DNA to 1x10^-10 for both assays (S2 File). The SjTR1 assay was able to detect S. mekongi DNA to 1x10^-8, corresponding to 1.73 femtograms (fg) of DNA, compared to 1x10^-6, corresponding to 173 fg of DNA for the Sme assay. The SjTR1 also had higher Cts across all dilutions compared to the Sme assay, reinforcing that the SjTR1 assay has higher sensitivity.

Two assays were run for Strongyloides spp. detection. The first assay targeting SSU rRNA [33] only detected one positive sample, while the second assay which identifies a repeat sequence [30], detected 12 samples as positive for S. stercoralis. The repeat assay is highly sensitive and highly specific for S. stercoralis [34], while the SSU rRNA assay can detect other Strongyloides spp. including the zoonotic S. fuelleborni [35,36]. Much like the Sme assay above, the amplicon for the SSY rRNA assay is quite long at 471 bp than that of the repeat assay which has a bp of 138. Neither assay was positive for the single sample identified as positive by KK. It should also be noted that KK is not an ideal diagnostic for strongyloidiasis and this may be a mis-diagnosis [37]. This result indicates additional hidden burdens that warrant integrated surveillance, and is line with a previous survey which identified a Strongyloides spp. prevalence of 5.7% (n = 703) in three villages in Yangon and Ayeyarwady regions by agar plate culture [38]. Displaced populations and deteriorating water and sanitation infrastructure may be facilitating the transmission and persistence of parasitic infections, including schistosomiasis and strongyloidiasis.

The duplex for Ancylostoma speciation did not identify as many positives as the first Ancylostoma spp. qPCR assay performed in the initial study [26], and none were positive for A. duodenale specifically. This may again be due to decline in DNA template quality over time. The identification of the zoonotic A. ceylanicum in Myanmar is in line with detection in other countries within Asia [39–43], cementing that this species is a major parasite of humans in Asia. Dogs are considered the major reservoir host of this species worldwide although it has also been identified in cats and wild canid species, and is the only animal hookworm species where patent infections have been identified [43,44]. A. ceylanicum infections have previously been identified in Myanmar with positive samples from Bago region and Mon state, which is just South of Bago region [45]. With the high prevalence of dogs, cats, and other canids in Asia A. ceylanicum transmission risk is high and it is crucial that animal health and treatment as well as environmental contamination with parasite eggs is taken into consideration in traditional STH control programs as part of a One Health approach. As previously mentioned Asian schistosome species are also zoonotic, with a range of definitive mammalian hosts and reliance on a molluscan intermediate host making One Health control for schistosomes an absolute must.

Snail survey and environmental DNA (eDNA) detection to identify the intermediate host would be invaluable in determining which species is causing infection as well as providing parasite samples (from infected snails) for sequencing. The intermediate snail hosts for S. japonicum (O. hupensis) and S. malayensis (Robertsiella spp.), and S. mekongi (N. aperta) have very different habitats with the O. hupensis and Robertsiella spp. snails present in rice paddies, streams, and other slow-moving water sources with vegetation along the sides while N. aperta live specifically in the Mekong river, a fast-moving river, on the underside of rocks. The snail intermediate host in Myanmar for S. japonicum or S. mekongi is currently unknown. Identifying the Schistosoma spp. causing human infection would help narrow down potential snail hosts. Knowing the snail intermediate host will be crucial for control efforts.

Alongside snail surveys and eDNA detection, human and animal host sampling should also be performed to help fully elucidate the epidemiology of schistosomiasis in Myanmar. eDNA could be used for initial mapping of sites, allowing then for more targeted approaches to areas where parasite DNA has been identified. Although the case numbers are small, the findings are epidemiologically significant—suggesting cryptic transmission with potential for focused regional spread. We also recommend to those performing similar studies in endemic regions to look beyond the focus of the project, whether that be on STH or schistosomiasis or other specific species, and where time and funds allow, to look at identification of all possible parasites in a sample. Doing this early allows for limited DNA degradation and gives the complete picture of helminth ‌‌epidemiology in endemic regions which so often exhibit high polyparasitism.

Supporting information