Natural Human Infections with Plasmodium cynomolgi, P. inui, and 4 other Simian Malaria Parasites, Malaysia.

We detected the simian malaria parasites Plasmodium knowlesi, P. cynomolgi, P. inui, P. coatneyi, P. inui-like, and P. simiovale among forest fringe-living indigenous communities from various locations in Malaysia. Our findings underscore the importance of using molecular tools to identify newly emergent malaria parasites in humans.

New vectors that are early feeders for Plasmodium knowlesi and other simian malaria parasites in Sarawak, Malaysian Borneo.

Plasmodium knowlesi is the main cause of malaria in Sarawak, where studies on vectors of P. knowlesi have been conducted in only two districts. Anopheles balabacensis and An. donaldi were incriminated as vectors in Lawas and An. latens in Kapit. We studied a third location in Sarawak, Betong, where of 2169 mosquitoes collected over 36 days using human-landing catches, 169 (7.8%) were Anopheles spp. PCR and phylogenetic analyses identified P. knowlesi and/or P. cynomolgi, P. fieldi, P. inui, P. coatneyi and possibly novel Plasmodium spp. in salivary glands of An. latens and An. introlatus from the Leucosphyrus Group and in An. collessi and An. roperi from the Umbrosus Group. Phylogenetic analyses of cytochrome oxidase subunit I sequences indicated three P. knowlesi-positive An. introlatus had been misidentified morphologically as An. latens, while An. collessi and An. roperi could not be delineated using the region sequenced. Almost all vectors from the Leucosphyrus Group were biting after 1800 h but those belonging to the Umbrosus Group were also biting between 0700 and 1100 h. Our study incriminated new vectors of knowlesi malaria in Sarawak and underscores the importance of including entomological studies during the daytime to obtain a comprehensive understanding of the transmission dynamics of malaria.

Malaria parasites in macaques in Thailand: stump-tailed macaques (Macaca arctoides) are new natural hosts for Plasmodium knowlesi, Plasmodium inui, Plasmodium coatneyi and Plasmodium fieldi.

BACKGROUND: Certain species of macaques are natural hosts of Plasmodium knowlesi and Plasmodium cynomolgi, which can both cause malaria in humans, and Plasmodium inui, which can be experimentally transmitted to humans. A significant number of zoonotic malaria cases have been reported in humans throughout Southeast Asia, including Thailand. There have been only two studies undertaken in Thailand to identify malaria parasites in non-human primates in 6 provinces. The objective of this study was to determine the prevalence of P. knowlesi, P. cynomolgi, P. inui, Plasmodium coatneyi and Plasmodium fieldi in non-human primates from 4 new locations in Thailand. METHODS: A total of 93 blood samples from Macaca fascicularis, Macaca leonina and Macaca arctoides were collected from four locations in Thailand: 32 were captive M. fascicularis from Chachoengsao Province (CHA), 4 were wild M. fascicularis from Ranong Province (RAN), 32 were wild M. arctoides from Prachuap Kiri Khan Province (PRA), and 25 were wild M. leonina from Nakornratchasima Province (NAK). DNA was extracted from these samples and analysed by nested PCR assays to detect Plasmodium, and subsequently to detect P. knowlesi, P. coatneyi, P. cynomolgi, P. inui and P. fieldi. RESULTS: Twenty-seven of the 93 (29%) samples were Plasmodium-positive by nested PCR assays. Among wild macaques, all 4 M. fascicularis at RAN were infected with malaria parasites followed by 50% of 32 M. arctoides at PRA and 20% of 25 M. leonina at NAK. Only 2 (6.3%) of the 32 captive M. fascicularis at CHA were malaria-positive. All 5 species of Plasmodium were detected and 16 (59.3%) of the 27 macaques had single infections, 9 had double and 2 had triple infections. The composition of Plasmodium species in macaques at each sampling site was different. Macaca arctoides from PRA were infected with P. knowlesi, P. coatneyi, P. cynomolgi, P. inui and P. fieldi. CONCLUSIONS: The prevalence and species of Plasmodium varied among the wild and captive macaques, and between macaques at 4 sampling sites in Thailand. Macaca arctoides is a new natural host for P. knowlesi, P. inui, P. coatneyi and P. fieldi.

Naturally Acquired Human Plasmodium cynomolgi and P. knowlesi Infections, Malaysian Borneo.

To monitor the incidence of Plasmodium knowlesi infections and determine whether other simian malaria parasites are being transmitted to humans, we examined 1,047 blood samples from patients with malaria at Kapit Hospital in Kapit, Malaysia, during June 24, 2013-December 31, 2017. Using nested PCR assays, we found 845 (80.6%) patients had either P. knowlesi monoinfection (n = 815) or co-infection with other Plasmodium species (n = 30). We noted the annual number of these zoonotic infections increased greatly in 2017 (n = 284). We identified 6 patients, 17-65 years of age, with P. cynomolgi and P. knowlesi co-infections, confirmed by phylogenetic analyses of the Plasmodium cytochrome c oxidase subunit 1 gene sequences. P. knowlesi continues to be a public health concern in the Kapit Division of Sarawak, Malaysian Borneo. In addition, another simian malaria parasite, P. cynomolgi, also is an emerging cause of malaria in humans.

A panel of recombinant proteins from human-infective Plasmodium species for serological surveillance.

BACKGROUND: Malaria remains a global health problem and accurate surveillance of Plasmodium parasites that are responsible for this disease is required to guide the most effective distribution of control measures. Serological surveillance will be particularly important in areas of low or periodic transmission because patient antibody responses can provide a measure of historical exposure. While methods for detecting host antibody responses to Plasmodium falciparum and Plasmodium vivax are well established, development of serological assays for Plasmodium knowlesi, Plasmodium ovale and Plasmodium malariae have been inhibited by a lack of immunodiagnostic candidates due to the limited availability of genomic information. METHODS: Using the recently completed genome sequences from P. malariae, P. ovale and P. knowlesi, a set of 33 candidate cell surface and secreted blood-stage antigens was selected and expressed in a recombinant form using a mammalian expression system. These proteins were added to an existing panel of antigens from P. falciparum and P. vivax and the immunoreactivity of IgG, IgM and IgA immunoglobulins from individuals diagnosed with infections to each of the five different Plasmodium species was evaluated by ELISA. Logistic regression modelling was used to quantify the ability of the responses to determine prior exposure to the different Plasmodium species. RESULTS: Using sera from European travellers with diagnosed Plasmodium infections, antigens showing species-specific immunoreactivity were identified to select a panel of 22 proteins from five Plasmodium species for serological profiling. The immunoreactivity to the antigens in the panel of sera taken from travellers and individuals living in malaria-endemic regions with diagnosed infections showed moderate power to predict infections by each species, including P. ovale, P. malariae and P. knowlesi. Using a larger set of patient samples and logistic regression modelling it was shown that exposure to P. knowlesi could be accurately detected (AUC = 91%) using an antigen panel consisting of the P. knowlesi orthologues of MSP10, P12 and P38. CONCLUSIONS: Using the recent availability of genome sequences to all human-infective Plasmodium spp. parasites and a method of expressing Plasmodium proteins in a secreted functional form, an antigen panel has been compiled that will be useful to determine exposure to these parasites.

Plasmodium knowlesi and other malaria parasites in long-tailed macaques from the Philippines.

BACKGROUND: Macaca fascicularis (long-tailed macaque) is the most widespread species of macaque in Southeast Asia and the only species of monkey found naturally in the Philippines. The species is the natural host for the zoonotic malaria species, Plasmodium knowlesi and Plasmodium cynomolgi and for the potentially zoonotic species, Plasmodium inui. Moreover, other Plasmodium species such as Plasmodium coatneyi and Plasmodium fieldi are also natural parasites of M. fascicularis. The aims of this study were to identify and determine the prevalence of Plasmodium species infecting wild and captive long-tailed macaques from the Philippines. METHODS: A total of 95 blood samples from long-tailed macaques in the Philippines were collected from three locations; 30 were from captive macaques at the National Wildlife Rescue and Rehabilitation Center (NWRRC) in Luzon, 25 were from captive macaques at the Palawan Wildlife Rescue and Conservation Center (PWRCC) in Palawan and 40 were from wild macaques from Puerto Princesa Subterranean River National Park (PPSRNP) in Palawan. The Plasmodium spp. infecting the macaques were identified using nested PCR assays on DNA extracted from these blood samples. RESULTS: All 40 of the wild macaques from PPSRNP in Palawan and 5 of 25 captive macaques from PWRCC in Palawan were Plasmodium-positive; while none of the 30 captive macaques from the NWRRC in Luzon had any malaria parasites. Overall, P. inui was the most prevalent malaria parasite (44.2%), followed by P. fieldi (41.1%), P. cynomolgi (23.2%), P. coatneyi (21.1%), and P. knowlesi (19%). Mixed species infections were also observed in 39 of the 45 Plasmodium-positive macaques. There was a significant difference in the prevalence of P. knowlesi among the troops of wild macaques from PPSRNP. CONCLUSION: Wild long-tailed macaques from the island of Palawan, the Philippines are infected with P. knowlesi, P. inui, P. coatneyi, P. fieldi and P. cynomolgi. The prevalence of these Plasmodium spp. varied among the sites of collection and among troops of wild macaques at one site. The presence of these simian Plasmodium parasites, especially P. knowlesi and P. cynomolgi in the long-tailed macaques in Palawan presents risks for zoonotic transmission in the area.

Spontaneous splenic rupture in Plasmodium knowlesi malaria.

BACKGROUND: Plasmodium knowlesi, a malaria parasite typically found in long-tailed and pig-tailed macaques, is the most common cause of human malaria in Malaysian Borneo. Infections in humans result in a spectrum of disease, including fatal outcomes. Spontaneous splenic rupture is a rare, but severe complication of malaria and has not been reported previously for knowlesi malaria. CASE PRESENTATION: A 46-year-old man presented with fever and acute surgical abdomen with concomitant P. knowlesi malaria infection at Kapit Hospital. He was in compensated shock upon arrival to the hospital. He had generalized abdominal tenderness, maximal at the epigastric region. Bedside focused abdominal ultrasonography revealed free fluid in the abdomen. He underwent emergency exploratory laparotomy in view of haemodynamic instability and worsening peritonism. Intraoperatively, haemoperitoneum and bleeding from the spleen was noted. Splenectomy was performed. Histopathological examination findings were suggestive of splenic rupture and presence of malarial pigment. Analysis of his blood sample by nested PCR assays confirmed P. knowlesi infection. The patient completed a course of anti-malarial treatment and recovered well post-operation. CONCLUSIONS: Spontaneous splenic rupture is a rare complication of malaria. This is the first reported case of splenic rupture in P. knowlesi malaria infection. Detection of such a complication requires high index of clinical suspicion and is extremely challenging in hospitals with limited resources.

Correction to: Absence of Plasmodium inui and Plasmodium cynomolgi, but detection of Plasmodium knowlesi and Plasmodium vivax infections in asymptomatic humans in the Betong division of Sarawak, Malaysian Borneo.

After publication of the article [1], it has been brought to our attention that two of the labels on Figure 4 have transposed. The labels "S-type SSU rRNA" and "A-type SSU rRNA" should be in opposite places.

Absence of Plasmodium inui and Plasmodium cynomolgi, but detection of Plasmodium knowlesi and Plasmodium vivax infections in asymptomatic humans in the Betong division of Sarawak, Malaysian Borneo.

BACKGROUND: Plasmodium knowlesi, a simian malaria parasite, has become the main cause of malaria in Sarawak, Malaysian Borneo. Epidemiological data on malaria for Sarawak has been derived solely from hospitalized patients, and more accurate epidemiological data on malaria is necessary. Therefore, a longitudinal study of communities affected by knowlesi malaria was undertaken. METHODS: A total of 3002 blood samples on filter paper were collected from 555 inhabitants of 8 longhouses with recently reported knowlesi malaria cases in the Betong Division of Sarawak, Malaysian Borneo. Each longhouse was visited bimonthly for a total of 10 times during a 21-month study period (Jan 2014-Oct 2015). DNA extracted from blood spots were examined by a nested PCR assay for Plasmodium and positive samples were then examined by nested PCR assays for Plasmodium falciparum, Plasmodium vivax, Plasmodium malariae, Plasmodium ovale, Plasmodium knowlesi, Plasmodium cynomolgi and Plasmodium inui. Blood films of samples positive by PCR were also examined by microscopy. RESULTS: Genus-specific PCR assay detected Plasmodium DNA in 9 out of 3002 samples. Species-specific PCR identified 7 P. knowlesi and one P. vivax. Malaria parasites were observed in 5 thick blood films of the PCR positive samples. No parasites were observed in blood films from one knowlesi-, one vivax- and the genus-positive samples. Only one of 7 P. knowlesi-infected individual was febrile and had sought medical treatment at Betong Hospital the day after sampling. The 6 knowlesi-, one vivax- and one Plasmodium-infected individuals were afebrile and did not seek any medical treatment. CONCLUSIONS: Asymptomatic human P. knowlesi and P. vivax malaria infections, but not P. cynomolgi and P. inui infections, are occurring within communities affected with malaria.

Distribution and prevalence of malaria parasites among long-tailed macaques (Macaca fascicularis) in regional populations across Southeast Asia.

BACKGROUND: Plasmodium knowlesi and Plasmodium cynomolgi are two malaria parasites naturally transmissible between humans and wild macaque through mosquito vectors, while Plasmodium inui can be experimentally transmitted from macaques to humans. One of their major natural hosts, the long-tailed macaque (Macaca fascicularis), is host to two other species of Plasmodium (Plasmodium fieldi and Plasmodium coatneyi) and is widely distributed in Southeast Asia. This study aims to determine the distribution of wild macaques infected with malarial parasites by examining samples derived from seven populations in five countries across Southeast Asia. METHODS: Plasmodium knowlesi, P. cynomolgi, P. coatneyi, P. inui and P. fieldi, were detected using nested PCR assays in DNA samples from 276 wild-caught long-tailed macaques. These samples had been derived from macaques captured at seven locations, two each in the Philippines (n = 68) and Indonesia (n = 70), and one each in Cambodia (n = 54), Singapore (n = 40) and Laos (n = 44). The results were compared with previous studies of malaria parasites in long-tailed macaques from other locations in Southeast Asia. Fisher exact test and Chi square test were used to examine the geographic bias of the distribution of Plasmodium species in the macaque populations. RESULTS: Out of 276 samples tested, 177 were Plasmodium-positive, with P. cynomolgi being the most common and widely distributed among all long-tailed macaque populations (53.3 %) and occurring in all populations examined, followed by P. coatneyi (20.4 %), P. inui (12.3 %), P. fieldi (3.4 %) and P. knowlesi (0.4 %). One P. knowlesi infection was detected in a macaque from Laos, representing the first documented case of P. knowlesi in wildlife in Laos. Chi square test showed three of the five parasites (P. knowlesi, P. coatneyi, P. cynomolgi) with significant bias in prevalence towards macaques from Malaysian Borneo, Cambodia, and Southern Sumatra, respectively. CONCLUSIONS: The prevalence of malaria parasites, including those that are transmissible to humans, varied among all sampled regional populations of long-tailed macaques in Southeast Asia. The new discovery of P. knowlesi infection in Laos, and the high prevalence of P. cynomolgi infections in wild macaques in general, indicate the strong need of public advocacy in related countries.