The malaria surveillance study, ongoing since March 2019 in Yala, Sisaket, Ubon Ratchathani, and Ratchaburi Provinces in Thailand, looks to consent and enroll individuals diagnosed with malaria, defined as evidence of any species of parasites in the blood using malaria rapid diagnostic test (RDT) and or microscopy. First, a single venous blood sample is drawn, with a complete blood count (CBC), glucose 6-phosphate dehydrogenase (G6PD) CareStart™ RDT (Access Bio, Inc., USA) and fluorescent spot testing (R&D Diagnostics Ltd., Greece) performed by local Ministry of Public Health (MoPH) or Royal Thai Army (RTA) staff. The remaining blood sample shipped to US Armed Forces Research Institute of Medical Sciences (AFRIMS) in Bangkok, Thailand for additional testing: repeat blood smears, speciation verification using polymerase chain reaction (PCR), quantitative G6PD testing (Pointe Scientific, USA), PCR for molecular markers of resistance and submicroscopic gametocytemia as well as ex-vivo and in-vitro drug susceptibility assays. At the time of writing, 149 malaria patients have been enrolled: 128 P. vivax cases, 14 P. falciparum and four P. knowlesi cases. A short description of three P. cynomolgi cases and the locations within Thailand (Figure 1) follows.
A 53-year-old woman presented at a malaria clinic in Ban Nang Sata District, Yala Province, in March, 2021 with 38°C fever, headache, and chills for five days. The hematological assessment showed white blood count (WBC) at 4,200/mm3, hemoglobin at 10.9 g/dL, and platelets at 191,000/mm3. Further history revealed she worked a rubber plantation and that her husband had recently been diagnosed and treated for P. vivax infection.
A 55-year-old woman was part of a malaria active case detection investigation by malaria clinic staff from Ka Bang District, Yala Province, in February, 2021. The patient reported a history of headache and fever for eight days, although on the day of examination, the subject’s tympanic temperature was 37°C. Laboratory examination revealed WBC at 4,800/mm3, hemoglobin at 11.7 g/dL, and platelet count at 330,000/mm3. The patient reported to be living and working on a rubber plantation.
In June 2021, a 25-year-old male on active duty in the Royal Thai Army presented at a malaria clinic in Yala District, Yala Province, with a complaint of five days of fever and nighttime chills. His temperature was 37.8°C. Hematology findings showed slight thrombocytopenia at 123,000/mm3, WBC at 6,900/mm3, and hemoglobin at 12.5 g/dL. The patient stated he had been stationed in Yala District for at least 20 months, going out on daily patrols and sleeping overnight in the forest. He reported using mosquito repellent and mosquito coils for personal protection.
Using microscopy, all three subjects were diagnosed with P. vivax; all presented with uncomplicated illness, had normal G6PD activity and reported no prior history of malaria. Each patient was treated by local health care staff with chloroquine and a two-week radical cure course of primaquine following Thai national treatment guidelines. All were found to be clinically well within five days of initiating the antimalarials, with no recurrences at subsequent follow-up visits required by the Thai MoPH scheduled at 14-, 28-, 60- and 90-days post-treatment.
Blood smears were prepared and read by two World Health Organization (WHO)-certified microscopists at the AFRIMS labs in Bangkok, Thailand. In brief, thick and thin smears were prepared on the same glass slide and air-dried and fixed in methanol, stained for 45 minutes (min) in 3% diluted Giemsa stain, and examined at an oil immersion magnification of ×100. Parasite counting was done per 500 white blood cells (WBC) in thick films, and percent parasitemia was calculated based on the actual WBC count. Parasites resembling P. vivax were detected, with densities of 25, 10, and 2,718 parasites/µL blood for Case A, B, and C, respectively. Only Case C had gametocytemia at four gametocytes per 200 WBCs. Malaria parasite morphologies (Figure 2) showed growing trophozoite stages with amoeboid-shaped cytoplasm and prominent Schüffner’s stippling. Yellowish-brown pigments dispersed within the cytoplasm were also found in some infected cells. Trophozoites with either single, double, or triple chromatin dots were detected in the blood films. No ring forms were detected in any slide. Only Case C had rare parasites visible on thin film, although erythrocytes were not clearly enlarged or distorted.
This study conducts 5-species (P. falciparum, P. vivax, P. malariae, P. ovale and P. knowlesi) real-time PCR testing at AFRIMS on all malaria isolates received. Briefly, genomic DNA is extracted from ethylenediaminetetraacetic acid (EDTA) whole blood using EZ1 DNA blood kit with automated EZ1 Advanced XL purification system (QIAGEN, Valencia, CA, USA) and confirmed for Plasmodium speciation by multiplex-real time PCR, using species-specific primers and probes [9, 10]. Two of the study patients (A and B) were found to be negative by multiplex real-time PCR, with P. vivax reported for Case C.
Since asexual parasites had been observed on blood smear for Cases A and B, speciation singleplex real-time PCR testing for P. cynomolgi was then performed on these isolates as well as all other samples collected from Yala (n=60). Primers and probes specific to small subunit rRNA, sexual (sporozoite) stage (Genbank accession number L08242.1) were selected, with sequences as follows: Forward: 5’-ATTGCGGTCGCAAATAATGAAG-3’, Reverse: 5’-GGTATGATAAGCCAGGGAAGTG-3’and Probe: 5’-FAM-TACTCGCTCCTTCTGTTCCCTGGA-BHQ1’). The reaction was carried out in a 25 µl reaction using Rotor-Gene Multiplex PCR kit (QIAGEN, Hilden, Germany) with cycling conditions consisting of an initial activation step at 95°C for 5 min, followed by 45 cycles of denaturation at 95°C for 15 seconds and annealing /extension at 60°C for 15 seconds. Blood from a macaque infected with P. cynomolgi was used as a positive control. Mono-infection with P. cynomolgi was confirmed by PCR in Cases A and B, with Case C having co-infection with P. vivax. All other Yala samples were negative for P. cynomolgi.