Malaria parasites of long-tailed macaques in Sarawak, Malaysian Borneo: a novel species and demographic and evolutionary histories.

BACKGROUND: Non-human primates have long been identified to harbour different species of Plasmodium. Long-tailed macaques (Macaca fascicularis), in particular, are reservoirs for P. knowlesi, P. inui, P. cynomolgi, P. coatneyi and P. fieldi. A previous study conducted in Sarawak, Malaysian Borneo, however revealed that long-tailed macaques could potentially harbour novel species of Plasmodium based on sequences of small subunit ribosomal RNA and circumsporozoite genes. To further validate this finding, the mitochondrial genome and the apicoplast caseinolytic protease M genes of Plasmodium spp. were sequenced from 43 long-tailed macaque blood samples. RESULTS: Apart from several named species of malaria parasites, long-tailed macaques were found to be potentially infected with novel species of Plasmodium, namely one we refer to as "P. inui-like." This group of parasites bifurcated into two monophyletic clades indicating the presence of two distinct sub-populations. Further analyses, which relied on the assumption of strict co-phylogeny between hosts and parasites, estimated a population expansion event of between 150,000 to 250,000 years before present of one of these sub-populations that preceded that of the expansion of P. knowlesi. Furthermore, both sub-populations were found to have diverged from a common ancestor of P. inui approximately 1.5 million years ago. In addition, the phylogenetic analyses also demonstrated that long-tailed macaques are new hosts for P. simiovale. CONCLUSIONS: Malaria infections of long-tailed macaques of Sarawak, Malaysian Borneo are complex and include a novel species of Plasmodium that is phylogenetically distinct from P. inui. These macaques are new natural hosts of P. simiovale, a species previously described only in toque monkeys (Macaca sinica) in Sri Lanka. The results suggest that ecological factors could affect the evolution of malaria parasites.

Advanced Genetic Methodologies in Tracking Evolution and Spread of Chikungunya Virus.

Recent advances in genetic methodologies have substantially expanded our ability to track evolution and spatio-temporal distribution of rapidly evolving pathogens. The information gathered from such analyses can be used to decipher host adaptations that shape disease epidemiology. In this chapter, we demonstrate the utilization of freely available resources to track the evolution and spread of Chikungunya virus.

Intra-epidemic evolutionary dynamics of a Dengue virus type 1 population reveal mutant spectra that correlate with disease transmission.

Dengue virus (DENV) is currently the most prevalent mosquito-borne viral pathogen. DENVs naturally exist as highly heterogeneous populations. Even though the descriptions on DENV diversity are plentiful, only a few studies have narrated the dynamics of intra-epidemic virus diversity at a fine scale. Such accounts are important to decipher the reciprocal relationship between viral evolutionary dynamics and disease transmission that shape dengue epidemiology. In the current study, we present a micro-scale genetic analysis of a monophyletic lineage of DENV-1 genotype III (epidemic lineage) detected from November 2012 to May 2014. The lineage was involved in an unprecedented dengue epidemic in Singapore during 2013-2014. Our findings showed that the epidemic lineage was an ensemble of mutants (variants) originated from an initial mixed viral population. The composition of mutant spectrum was dynamic and positively correlated with case load. The close interaction between viral evolution and transmission intensity indicated that tracking genetic diversity through time is potentially a useful tool to infer DENV transmission dynamics and thereby, to assess the epidemic risk in a disease control perspective. Moreover, such information is salient to understand the viral basis of clinical outcome and immune response variations that is imperative to effective vaccine design.

Three-Month Real-Time Dengue Forecast Models: An Early Warning System for Outbreak Alerts and Policy Decision Support in Singapore.

BACKGROUND: With its tropical rainforest climate, rapid urbanization, and changing demography and ecology, Singapore experiences endemic dengue; the last large outbreak in 2013 culminated in 22,170 cases. In the absence of a vaccine on the market, vector control is the key approach for prevention. OBJECTIVES: We sought to forecast the evolution of dengue epidemics in Singapore to provide early warning of outbreaks and to facilitate the public health response to moderate an impending outbreak. METHODS: We developed a set of statistical models using least absolute shrinkage and selection operator (LASSO) methods to forecast the weekly incidence of dengue notifications over a 3-month time horizon. This forecasting tool used a variety of data streams and was updated weekly, including recent case data, meteorological data, vector surveillance data, and population-based national statistics. The forecasting methodology was compared with alternative approaches that have been proposed to model dengue case data (seasonal autoregressive integrated moving average and step-down linear regression) by fielding them on the 2013 dengue epidemic, the largest on record in Singapore. RESULTS: Operationally useful forecasts were obtained at a 3-month lag using the LASSO-derived models. Based on the mean average percentage error, the LASSO approach provided more accurate forecasts than the other methods we assessed. We demonstrate its utility in Singapore's dengue control program by providing a forecast of the 2013 outbreak for advance preparation of outbreak response. CONCLUSIONS: Statistical models built using machine learning methods such as LASSO have the potential to markedly improve forecasting techniques for recurrent infectious disease outbreaks such as dengue. CITATION: Shi Y, Liu X, Kok SY, Rajarethinam J, Liang S, Yap G, Chong CS, Lee KS, Tan SS, Chin CK, Lo A, Kong W, Ng LC, Cook AR. 2016. Three-month real-time dengue forecast models: an early warning system for outbreak alerts and policy decision support in Singapore. Environ Health Perspect 124:1369-1375; http://dx.doi.org/10.1289/ehp.1509981.

Development and Evaluation of a SYBR Green-Based Real-Time Multiplex RT-PCR Assay for Simultaneous Detection and Serotyping of Dengue and Chikungunya Viruses.

Chikungunya virus (CHIKV) and dengue virus (DENV) have emerged as the two most important arbovirus diseases of global health significance. Similar clinical manifestations, transmission vectors, geographical distribution, and seasonal correlation often result in misdiagnosis of chikungunya infections as dengue cases and vice versa. In this study, we developed a rapid and accurate laboratory confirmative method to simultaneously detect, quantify, and differentiate DENV serotypes 1, 2, 3, and 4 and CHIKV. This SYBR Green I-based one-step multiplex real-time RT-PCR assay is highly sensitive and specific for CHIKV and DENV. Melting temperature analysis of PCR amplicons was used to serotype DENV and to differentiate from CHIKV. The detection limit of the assay was 20, 10, 50, 5, and 10 RNA copies/reaction for DENV-1, DENV-2, DENV-3, DENV-4, and CHIKV, respectively. Our assay did not cross-react with a panel of viruses that included other flaviviruses, alphaviruses, influenza viruses, human enteroviruses, and human coronaviruses. The feasibility of using this assay for clinical diagnosis was evaluated in DENV- and CHIKV-positive patient sera. Accordingly, the assay sensitivity for DENV-1, DENV-2, DENV-3, DENV-4, and CHIKV was 89.66%, 96.67%, 96.67%, 94.12%, and 95.74%, respectively, with 100% specificity. These findings confirmed the potential of our assay to be used as a rapid test for simultaneous detection and serotyping of DENV and CHIKV in clinical samples.

2013 dengue outbreaks in Singapore and Malaysia caused by different viral strains.

Characterization of 14,079 circulating dengue viruses in a cross-border surveillance program, UNITEDengue, revealed that the 2013 outbreaks in Singapore and Malaysia were associated with replacement of predominant serotype. While the predominant virus in Singapore switched from DENV2 to DENV1, DENV2 became predominant in neighboring Malaysia. Dominance of DENV2 was most evident on the southern states where higher fatality rates were observed.

Dengue serotype-specific differences in clinical manifestation, laboratory parameters and risk of severe disease in adults, singapore.

Studies on serotype-specific features of dengue and disease severity on adults are limited. We prospectively recruited adult febrile patients without alternate diagnosis to dengue from April 2005 to December 2011. Outcomes were defined using both the World Health Organization (WHO) 1997 and 2009 criteria; Dengue hemorrhagic fever (DHF) and severe dengue (SD). Infecting serotype was identified in 469 dengue-confirmed patients comprising 22.0% dengue virus serotype 1 (DENV-1), 57.1% DENV-2, 17.1% DENV-3, and 3.8% DENV-4. Cases infected with DENV-1 were more likely to present with red eyes whereas presence of joint pain and lower platelet count was associated with DENV-2 cases. After adjusting for potential confounders, DENV-1 was associated with both DHF (adjusted Relative Risk [aRR] = 1.74) and SD (aRR = 2.1) whereas DENV-2 had a lower risk of DHF (aRR = 0.5). DENV-1 genotype 1 and DENV-2 cosmopolitan were the predominant genotypes identified. Infecting dengue serotype and possibly genotype may play an important role in disease severity among adult dengue patients in Singapore.

Clinical outcome and genetic differences within a monophyletic Dengue virus type 2 population.

The exact mechanisms of interplay between host and viral factors leading to severe dengue are yet to be fully understood. Even though previous studies have implicated specific genetic differences of Dengue virus (DENV) in clinical severity and virus attenuation, similar studies with large-scale, whole genome screening of monophyletic virus populations are limited. Therefore, in the present study, we compared 89 whole genomes of DENV-2 cosmopolitan clade III isolates obtained from patients diagnosed with dengue fever (DF, n = 58), dengue hemorrhagic fever (DHF, n = 30) and dengue shock syndrome (DSS, n = 1) in Singapore between July 2010 and January 2013, in order to determine the correlation of observed viral genetic differences with clinical outcomes. Our findings showed no significant difference between the number of primary and secondary infections that progressed to DHF and DSS (p>0.05) in our study cohort. Despite being highly homogenous, study isolates possessed 39 amino acid substitutions of which 10 substitutions were fixed in three main groups of virus isolates. None of those substitutions were specifically associated with DHF and DSS. Notably, two evolutionarily unique virus groups possessing C-P43T+NS1-S103T+NS2A-V83I+NS3-R337K+ NS3-I600T+ NS5-P136S and NS2A-T119N mutations were exclusively found in patients with DF, the benign form of DENV infections. Those mutants were significantly associated with mild disease outcome. These observations indicated that disease progression into DHF and DSS within our patient population was more likely to be due to host than virus factors. We hypothesize that selection for potentially less virulent groups of DENV-2 in our study cohort may be an evolutionary adaptation of viral strains to extend their survival in the human-mosquito transmission cycle.

DNA barcoding: complementing morphological identification of mosquito species in Singapore.

BACKGROUND: Taxonomy that utilizes morphological characteristics has been the gold standard method to identify mosquito species. However, morphological identification is challenging when the expertise is limited and external characters are damaged because of improper specimen handling. Therefore, we explored the applicability of mitochondrial cytochrome C oxidase subunit 1 (COI) gene-based DNA barcoding as an alternative tool to identify mosquito species. In the present study, we compared the morphological identification of mosquito specimens with their differentiation based on COI barcode, in order to establish a more reliable identification system for mosquito species found in Singapore. METHODS: We analysed 128 adult mosquito specimens, belonging to 45 species of 13 genera. Phylogenetic trees were constructed for Aedes, Anopheles, Culex and other genera of mosquitoes and the distinctive clustering of different species was compared with their taxonomic identity. RESULTS: The COI-based DNA barcoding achieved a 100% success rate in identifying the mosquito species. We also report COI barcode sequences of 16 mosquito species which were not available previously in sequence databases. CONCLUSIONS: Our study utilised for the first time DNA barcoding to identify mosquito species in Singapore. COI-based DNA barcoding is a useful tool to complement taxonomy-based identification of mosquito species.

Feeding host range of Aedes albopictus (Diptera: Culicidae) demonstrates its opportunistic host-seeking behavior in rural Singapore.

Aedes albopictus (Skuse) is a competent vector of arboviruses of public health importance, including dengue virus (DENV) and chikungunya virus viruses. Ae. albopictus is the primary vector of chikungunya virus in Singapore. However, despite being ubiquitous, it plays a secondary role in DENV transmission. The vectorial capacity of Ae. albopictus for DENV in field settings appears to be weak because dengue primarily occurs in Aedes aegypti (L.)-dominated, urban settings of the country. As host-seeking behavior is one of the determinants of vectorial capacity, we screened 6,762 female Ae. albopictus from rural, semiurban, and urban locations in Singapore for avian and nonavian bloodmeals using two polymerase chain reaction-sequencing assays developed in-house. The majority (83.2%, n = 79) of bloodmeals from rural and semiurban areas were from humans. However, Ae. albopictus was also found to feed on shrews, swine, dogs, cats, turtles, and multiple hosts in rural settings. In urban areas, all positive bloodmeals were from humans. There were no avian bloodmeals. Our findings testify that Ae. albopictus is highly anthropophagic even in rural settings, but become opportunistic in extremely low human abundance. This opportunistic feeding behavior warrants further investigations into the vectorial capacity of Ae. albopictus to assess its role in arbovirus transmission in endemic habitats.

Evolution and heterogeneity of multiple serotypes of Dengue virus in Pakistan, 2006-2011.

BACKGROUND: Even though dengue has been recognized as one of the major public health threats in Pakistan, the understanding of its molecular epidemiology is still limited. The genotypic diversity of Dengue virus (DENV) serotypes involved in dengue outbreaks since 2005 in Pakistan is not well studied. Here, we investigated the origin, diversity, genetic relationships and geographic distribution of DENV to understand virus evolution during the recent expansion of dengue in Pakistan. METHODS: The study included 200 sera obtained from dengue-suspected patients from 2006 to 2011. DENV infection was confirmed in 94 (47%) sera by a polymerase chain reaction assay. These included 36 (38.3%) DENV-2, 57 DENV-3 (60.6%) and 1 DENV-4 (1.1%) cases. Sequences of 13 whole genomes (6 DENV-2, 6 DENV-3 and 1 DENV-4) and 49 envelope genes (26 DENV-2, 22 DENV-3 and 1 DENV-4) were analysed to determine the origin, phylogeny, diversity and selection pressure during virus evolution. RESULTS: DENV-2, DENV-3 and DENV-4 in Pakistan from 2006 to 2011 shared 98.5-99.6% nucleotide and 99.3-99.9% amino acid similarity with those circulated in the Indian subcontinent during the last decade. Nevertheless, Pakistan DENV-2 and DENV-3 strains formed distinct clades characterized by amino acid signatures of NS2A-I116T + NS5-K861R and NS3-K590R + NS5-S895L respectively. Each clade consisted of a heterogenous virus population that circulated in Southern (2006-2009) and Northern Pakistan (2011). CONCLUSIONS: DENV-2, DENV-3 and DENV-4 that circulated during 2006-2011 are likely to have first introduced via the southern route of Pakistan. Both DENV-2 and DENV-3 have undergone in-situ evolution to generate heterogenous populations, possibly driven by sustained local DENV transmission during 2006-2011 periods. While both DENV-2 and DENV-3 continued to circulate in Southern Pakistan until 2009, DENV-2 has spread in a Northern direction to establish in Punjab Province, which experienced a massive dengue outbreak in 2011.

Clinico-genetic characterisation of an encephalitic Dengue virus 4 associated with multi-organ involvement.

Neurological manifestations due to Dengue virus (DENV) infection are atypical and uncommon. Genomic information of clinically characterised, neurotrophic DENV in humans is extremely limited albeit their importance in deciphering the pathogenicity is substantial. Here, we report a rare case of fatal DENV-4 infection complicated with encephalitis and multi-organ failure. The clinical presentation was unusual due to its rapid onset of encephalitis despite a very low virus titre. Full genomes of serum and CSF-derived viruses shared 99.99% similarity, indicating the virus dissemination across blood-brain barrier. Even though virus genomes did not reveal any of the neurotrophic substitutions of DENV documented so far, case isolates possessed a combination of 8 novel amino acid alterations, predominantly distributed in non-structural genes of DENV-4.

Dengue virus surveillance in Singapore reveals high viral diversity through multiple introductions and in situ evolution.

Dengue fever, a vector-borne disease, has caused tremendous burden to countries in the tropics and sub tropics. Over the past 20 years, dengue epidemics have become more widespread, severe and frequent. This study aims to understand the dynamics of dengue viruses in cosmopolitan Singapore. Envelope protein gene sequences of all four dengue serotypes (DENV-1-DENV-4) obtained from human sera in Singapore (2008-2010) revealed that constant viral introductions and in situ evolution contribute to viral diversity in Singapore and play important roles in shaping the epidemiology of dengue in the island state. The diversity of dengue viruses reported here could be a reflection of the on-going dengue situation in the region given Singapore's location in a dengue hyperendemic region and its role as the regional hub for travels and trade. Though cosmopolitan genotype of DENV-2 has remained as the predominant strain circulating in Singapore, we uncovered evidence of in situ evolution which could possibly result in viruses with improved fitness. While we have previously shown that a switch in the predominant dengue serotype could serve as a warning for an impending outbreak, our current data shows that a replacement of a predominant viral clade, even in the absence of a switch in predominant serotype, could signal a possible increase in dengue transmission. The circulating dengue viruses in Singapore are highly diverse, a situation which could offer ample opportunities for selection of strains of higher fitness, thus increasing the risk of outbreaks despite a low Aedes population.

Plasmodium knowlesi: reservoir hosts and tracking the emergence in humans and macaques.

Plasmodium knowlesi, a malaria parasite originally thought to be restricted to macaques in Southeast Asia, has recently been recognized as a significant cause of human malaria. Unlike the benign and morphologically similar P. malariae, these parasites can lead to fatal infections. Malaria parasites, including P. knowlesi, have not yet been detected in macaques of the Kapit Division of Malaysian Borneo, where the majority of human knowlesi malaria cases have been reported. In order to extend our understanding of the epidemiology and evolutionary history of P. knowlesi, we examined 108 wild macaques for malaria parasites and sequenced the circumsporozoite protein (csp) gene and mitochondrial (mt) DNA of P. knowlesi isolates derived from macaques and humans. We detected five species of Plasmodium (P. knowlesi, P. inui, P. cynomolgi, P. fieldi and P. coatneyi) in the long-tailed and pig-tailed macaques, and an extremely high prevalence of P. inui and P. knowlesi. Macaques had a higher number of P. knowlesi genotypes per infection than humans, and some diverse alleles of the P. knowlesi csp gene and certain mtDNA haplotypes were shared between both hosts. Analyses of DNA sequence data indicate that there are no mtDNA lineages associated exclusively with either host. Furthermore, our analyses of the mtDNA data reveal that P. knowlesi is derived from an ancestral parasite population that existed prior to human settlement in Southeast Asia, and underwent significant population expansion approximately 30,000-40,000 years ago. Our results indicate that human infections with P. knowlesi are not newly emergent in Southeast Asia and that knowlesi malaria is primarily a zoonosis with wild macaques as the reservoir hosts. However, ongoing ecological changes resulting from deforestation, with an associated increase in the human population, could enable this pathogenic species of Plasmodium to switch to humans as the preferred host.

Entomologic and molecular investigation into Plasmodium vivax transmission in Singapore, 2009.

BACKGROUND: Singapore has been certified malaria free since November 1982 by the World Health Organization and despite occasional local transmission, the country has maintained the standing. In 2009, three clusters of malaria cases were reported in Singapore. METHODS: Epidemiological, entomological and molecular studies were carried out to investigate the three clusters, namely Mandai-Sungei Kadut, Jurong Island and Sembawang. RESULTS: A total of 29 malaria patients, with no recent travel history, were reported in the three clusters. Molecular analysis based on the msp3α and msp1 genes showed two independent local transmissions: one in Mandai-Sungei Kadut and another in Sembawang. Almost all cases within each cluster were epidemiologically linked. In Jurong Island cluster, epidemiological link remains uncertain, as almost all cases had a unique genetic profile. Only two cases shared a common profile and were found to be linked to the Mandai-Sungei Kadut cluster. Entomological investigation found Anopheles sinensis to be the predominant Anopheline in the two areas where local transmission of P. vivax was confirmed. Anopheles sinensis was found to be attracted to human bait and bites as early as 19:45 hrs. However, all Anopheles mosquitoes caught were negative for sporozoites and oocysts by dissection. CONCLUSION: Investigation of P. vivax cases from the three cluster areas confirmed the occurrence of local transmission in two areas. Although An. sinensis was the predominant Anopheline found in areas with confirmed transmission, the vector/s responsible for the outbreaks still remains cryptic.

Dengue virus surveillance for early warning, Singapore.

In Singapore, after a major outbreak of dengue in 2005, another outbreak occurred in 2007. Laboratory-based surveillance detected a switch from dengue virus serotype 1 (DENV-1) to DENV-2. Phylogenetic analysis showed a clade replacement within DENV-2 cosmopolitan genotype, which accompanied the predominant serotype switch, and cocirculation of multiple genotypes of DENV-3.

Re-emergence of Chikungunya virus in South-east Asia: virological evidence from Sri Lanka and Singapore.

Chikungunya fever swept across many South and South-east Asian countries, following extensive outbreaks in the Indian Ocean Islands in 2005. However, molecular epidemiological data to explain the recent spread and evolution of Chikungunya virus (CHIKV) in the Asian region are still limited. This study describes the genetic Characteristics and evolutionary relationships of CHIKV strains that emerged in Sri Lanka and Singapore during 2006-2008. The viruses isolated in Singapore also included those imported from the Maldives (n=1), India (n=2) and Malaysia (n=31). All analysed strains belonged to the East, Central and South African (ECSA) lineage and were evolutionarily more related to Indian than to Indian Ocean Islands strains. Unique genetic characteristics revealed five genetically distinct subpopulations of CHIKV in Sri Lanka and Singapore, which were likely to have emerged through multiple, independent introductions. The evolutionary network based on E1 gene sequences indicated the acquisition of an alanine to valine 226 substitution (E1-A226V) by virus strains of the Indian sublineage as a key evolutionary event that contributed to the transmission and spatial distribution of CHIKV in the region. The E1-A226V substitution was found in 95.7 % (133/139) of analysed isolates in 2008, highlighting the widespread establishment of mutated CHIKV strains in Sri Lanka, Singapore and Malaysia. As the E1-A226V substitution is known to enhance the transmissibility of CHIKV by Aedes albopictus mosquitoes, this observation has important implications for the design of vector control strategies to fight the virus in regions at risk of chikungunya fever.

Plasmodium knowlesi from archival blood films: further evidence that human infections are widely distributed and not newly emergent in Malaysian Borneo.

Human infections with Plasmodium knowlesi have been misdiagnosed by microscopy as Plasmodium malariae due to their morphological similarities. Although microscopy-identified P. malariae cases have been reported in the state of Sarawak (Malaysian Borneo) as early as 1952, recent epidemiological studies suggest the absence of indigenous P. malariae infections. The present study aimed to determine the past incidence and distribution of P. knowlesi infections in the state of Sarawak based on archival blood films from patients diagnosed by microscopy as having P. malariae infections. Nested PCR assays were used to identify Plasmodium species in DNA extracted from 47 thick blood films collected in 1996 from patients in seven different divisions throughout the state of Sarawak. Plasmodium knowlesi DNA was detected in 35 (97.2%) of 36 blood films that were positive for Plasmodium DNA, with patients originating from all seven divisions. Only one sample was positive for P. malariae DNA. This study provides further evidence of the widespread distribution of human infections with P. knowlesi in Sarawak and its past occurrence. Taken together with data from previous studies, our findings suggest that P. knowlesi malaria is not a newly emergent disease in humans.

Morphological features and differential counts of Plasmodium knowlesi parasites in naturally acquired human infections.

BACKGROUND: Human infections with Plasmodium knowlesi, a simian malaria parasite, are more common than previously thought. They have been detected by molecular detection methods in various countries in Southeast Asia, where they were initially diagnosed by microscopy mainly as Plasmodium malariae and at times, as Plasmodium falciparum. There is a paucity of information on the morphology of P. knowlesi parasites and proportion of each erythrocytic stage in naturally acquired human infections. Therefore, detailed descriptions of the morphological characteristics and differential counts of the erythrocytic stages of P. knowlesi parasites in human infections were made, photographs were taken, and morphological features were compared with those of P. malariae and P. falciparum. METHODS: Thick and thin blood films were made prior to administration of anti-malarial treatment in patients who were subsequently confirmed as having single species knowlesi infections by PCR assays. Giemsa-stained blood films, prepared from 10 randomly selected patients with a parasitaemia ranging from 610 to 236,000 parasites per microl blood, were examined. RESULTS: The P. knowlesi infection was highly synchronous in only one patient, where 97% of the parasites were at the late trophozoite stage. Early, late and mature trophozoites and schizonts were observed in films from all patients except three; where schizonts and early trophozoites were absent in two and one patient, respectively. Gametocytes were observed in four patients, comprising only between 1.2 to 2.8% of infected erythrocytes. The early trophozoites of P. knowlesi morphologically resemble those of P. falciparum. The late and mature trophozoites, schizonts and gametocytes appear very similar to those of P. malariae. Careful examinations revealed that some minor morphological differences existed between P. knowlesi and P. malariae. These include trophozoites of knowlesi with double chromatin dots and at times with two or three parasites per erythrocyte and mature schizonts of P. knowlesi having 16 merozoites, compared with 12 for P. malariae. CONCLUSION: Plasmodium knowlesi infections in humans are not highly synchronous. The morphological resemblance of early trophozoites of P. knowlesi to P. falciparum and later erythrocytic stages to P. malariae makes it extremely difficult to identify P. knowlesi infections by microscopy alone.

Human Infections with Plasmodium knowlesi, the Philippines.

Five human cases of infection with the simian malaria parasite Plasmodium knowlesi from Palawan, the Philippines, were confirmed by nested PCR. This study suggests that this zoonotic infection is found across a relatively wide area in Palawan and documents autochthonous cases in the country.