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      Use of Malaria Rapid Diagnostic Test to Identify Plasmodium knowlesi Infection


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          Reports of human infection with Plasmodium knowlesi, a monkey malaria, suggest that it and other nonhuman malaria species may be an emerging health problem. We report the use of a rapid test to supplement microscopic analysis in distinguishing the 5 malaria species that infect humans.

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          Naturally Acquired Plasmodium knowlesi Malaria in Human, Thailand

          We describe a case of naturally acquired infection with Plasmodium knowlesi in Thailand. Diagnosis was confirmed by the small subunit ribosomal RNA and the mitochondrial cytochrome b sequences. The occurrence of simian malaria in human has signified the roles of wild primate populations in disease transmission in some malaria-endemic areas.
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            Human Infections with Plasmodium knowlesi, the Philippines

            Human malaria is commonly caused by Plasmodium falciparum, P. vivax, P. malariae, and P. ovale. However, a large focus of human infections with the simian malaria parasite, P. knowlesi ( 1 ), has recently been reported in Malaysian Borneo ( 2 ), and single case reports of infections acquired in Thailand ( 3 ) and Myanmar ( 4 ) have been documented. The diagnosis of P. knowlesi in humans may be missed by microscopy since the early blood stages of P. knowlesi morphologically resemble P. falciparum; the mature blood stages and gametocytes are similar to those of P. malariae ( 2 ). The Study Palawan is an island province lying southwest of the main islands of the Philippines. One of its smaller islands, Balabac, located off the southern tip, is separated from Borneo by the Balabac Strait (Figure). Malaria transmission occurs in all 19 municipalities of the province throughout the year. The Anopheles flavirostris mosquito is the reported primary vector in the area ( 5 ). Based on national control program data in 2005, a total of 16,339 malaria cases were reported from Palawan, accounting for 35% of the country’s total. Of these, 11,580 (≈71%) were P. falciparum, 4,194 (26%) were P. vivax, 430 (3%) were P. malariae, and the remainder (135, 40 years of age) who lived in the villages of Tagbarungis and Bacungan near Puerto Princesa City, Palawan (Figure). The patients were interviewed in July 2006 after their blood films were found to contain P. knowlesi. Both were subsistence farmers who had engaged in livelihood activities at night (charcoal making) in forested areas within a 50-km radius from their homes; neither had traveled out of Palawan within the past year. Before seeking medical treatment at the Palawan provincial malaria laboratory, they experienced chills, minor headaches, and daily low-grade fever, consistent with the reported quotidian fever pattern of this infection ( 1 ). P. malariae infection was diagnosed in both men by the local microscopist; each claimed to have responded well to chloroquine and primaquine drug therapy. The usual treatment regimen for P. falciparum/P. malariae was followed: 4 tablets of chloroquine (150 mg base/tablet) on days 1 and 2, and 2 tablets on day 3; 3 tablets of primaquine (15 mg/tablet) on day 4). The farmers reportedly stayed overnight before onset of their illness in forested foothills that contained many breeding sites ideal for A. flavirostris mosquitoes. Long-tailed macaques (Macaca fascicularis), the natural hosts for P. knowlesi, were observed to be roaming freely in the area. An additional 9 samples (D,E, G, H, I, J, K, O, and P), consisting of 5 blood films and 4 blood spots on filter paper, were obtained from patients at Bataraza and Roxas municipalities (also in Palawan) and P. malariae infection was diagnosed by the local microscopists. These samples were subsequently examined by nested PCR assays at UNIMAS after DNA extraction. Three were identified as P. knowlesi, 4 as P. malariae, and the remaining 2 as mixed species infections (Table). The findings of autochthonous P. malariae infections further compounded the problem of accurate diagnosis of P. knowlesi by microscopy. The P. knowlesi patients came from 3 different villages in Roxas, 80–100 km north of where the original 2 P. knowlesi case-patients resided, near Puerto Princesa (Figure). This suggests that human P. knowlesi infections are found across a relatively wide area in Palawan. PCR examination of more blood samples in other areas where P. malariae infections have been diagnosed by microscopy are necessary to determine the geographic distribution and public health importance of human knowlesi infections in the Philippines. Table Microscopy and PCR results of blood samples from Palawan, the Philippines Patient Age, y/sex Location Plasmodium species Microscopy PCR A 50/M Bacungan, Puerto Princesa P. falciparum (gametocytes), 
P. malariae P. falciparum, P. malariae, 
P. knowlesi B 49/M Inagawan, Tagbarungis, Puerto Princesa P. falciparum, P. malariae P. knowlesi D 55/F Caibulo, Iraan, Roxas P.malariae P. knowlesi E 3/M Balogo, San Miguel, Roxas P.malariae P. knowlesi G 6/M Maninguin, Iraan, Roxas P. malariae P. malariae H 25/M Minara, Roxas P. malariae P. malariae I 10/F Taradungan, Roxas P. malariae P. knowlesi J 5/M Bono-Bono, Bataraza P. vivax, P. malariae P. falciparum, P. vivax, P. malariae K 14/F Bono-Bono, Bataraza P. malariae P. malariae O 9/M Inogbong,Bataraza P. malariae P. malariae P 5/F Inogbong, Bataraza P. falciparum, P. malariae P. falciparum, P. vivax Conclusions This report extends the geographic range of human P. knowlesi infections from Thailand ( 3 ), Myanmar ( 4 ), peninsular Malaysia ( 8 ), and Malaysian Borneo ( 2 ) to Palawan Island in the Philippines. Although the parasite has been isolated from local macaques in the Philippines in 1961 ( 6 ) and 1978 ( 7 ), this report documents autochthonous human cases in the country. Major progress in malaria control has been achieved in many malarious areas in the Philippines ( 9 ). However, P. knowlesi forms a previously unrecognized pool of infections that may be maintained in forested areas through its presence in a simian reservoir, despite control efforts in the human population. Current data suggest that human knowlesi malaria is strictly a zoonotic disease. To confirm this theory, further knowledge of the dynamics of human infection is needed.
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              Naturally Acquired Human Plasmodium knowlesi Infection, Singapore

              Plasmodium knowlesi is one of the simian malarias that causes human infection ( 1 , 2 ). All 6 published reports of naturally acquired P. knowlesi infection were in rural settings with the largest case series being reported from East Malaysia ( 3 – 8 ). P. knowlesi is commonly misidentified as P. malariae since the blood stages are morphologically similar on microscopy, and molecular methods of detection are necessary for accurate diagnosis ( 5 , 8 ). Singapore is an urban city-state, which was declared free of human malaria by the World Health Organization in 1982 ( 9 ). However, we report a case of locally acquired P. knowlesi malaria, which indicates that this emerging zoonotic parasite should be considered as an etiologic agent of acute febrile illness acquired in Singapore, the southern-most locale reported thus far. The Case A previously healthy 20-year-old soldier in the Singapore Army sought treatment on April 28, 2007. He had had a fever for 4 days, along with myalgia, anorexia, nausea, and occasional vomiting. For a year leading up to his illness, he had trained in a forested area inhabited by the long-tailed macaque (Macaca fascicularis) in Lim Chu Kang, northwestern Singapore. His only travel out of Singapore was a 3-week training visit to a non–malaria-endemic foreign country in September 2006 and another visit to Bukit Batok Nature Reserve in western Singapore, an area with monkeys (M. fascicularis) 1 month before onset of symptoms. On initial examination, his temperature was 39.5°C with a pulse rate of 106 beats/min. He was lethargic with tender hepatomegaly. Laboratory investigations showed thrombocytopenia (platelet count 66 × 109/L), hyperbilirubinemia (bilirubin 33 mmol/L [reference level 7–31 mmol/L]), and mild transaminitis (alanine transaminase 64 U/L [reference level 17–63 mmol/L] and aspartate transaminase 67 U/L [reference level 15–41 mmol/L]). Initial diagnosis was dengue fever, which is endemic in Singapore. The patient experienced daily fever spikes from 39.5°C to 40.4°C (Figure 1). When fever persisted (40.4°C on day 6 of his illness, hospital day 3), the clinical picture was atypical for dengue fever. Blood films for malaria parasites were ordered, because introduced cases of malaria have been reported in Singapore ( 10 ). Microscopy showed Plasmodium parasitemia of 0.2% (equivalent to 7,700 parasites/mmol/L blood) with morphologic features consistent with P. malariae. Results of dengue reverse transcription–PCR (RT-PCR) on serum, 2 sets of blood cultures, and Rickettsia typhi serologic testing were negative. Results of a chest radiograph and ultrasound of the abdomen were normal. Figure 1 Patient’s temperature chart showing fever spikes 24 h apart at approximately 7 PM daily (red arrow). The black arrow denotes 38°C, and each blue arrow denotes a difference of 1°C from the neighboring arrow. Oral chloroquine was started with an initial dose of 600 mg base, followed by 300 mg base 6 h later and another 2 doses over the next 2 days. He defervesced rapidly; blood smears were negative 3 days after chloroquine therapy. At 2 weeks follow-up, he was clinically well. Because P. malariae infection was not consistent with the clinical findings of the initial examination, we investigated further to determine the etiology of this case. End-point nested Plasmodium genus- and species-specific nested PCR carried out on DNA extracted from whole blood samples were positive for Plasmodium sp. but negative for the 4 species that cause human malaria (Table) ( 11 ). Similarly, the sample was negative on real-time PCR for the 4 human parasites ( 12 ). P. knowlesi species-specific PCR resulted in a 153-bp fragment indicative of P. knowlesi ( 5 ). This 153-bp PCR product was directly sequenced and verified in the BLAST database (www.ncbi.nlm.nih.gov/blast/Blast.cgi) to match only P. knowlesi small subunit ribosomal RNA (SSU rRNA). Table Primers used for the PCR investigation of the clinical sample from Singapore* Primers Forward Sequence (5′ → 3’) Reverse Sequence (5′ → 3’) Results Nest 1, genus specific primers Genus specific ( 11 ) rPLU 1 TCA AAG AAT AAG CCA TGC AAG TGA rPLU 2 TAC CCT GTT GTT GCC TTA AAC TCC + Nest 2, genus- and species-specific primers Genus specific ( 11 ) rPLU 3 TTT TTA TAA GGA TAA CTA CGG AAA AGC TGT rPLU 4 TAC CCG TCA TAG CCA TGT TAG GCC AAT ACC + Plasmodium knowlesi 
 specific ( 5 ) Pmk8 GTT AGC GAG AGC CAC AAA AAA GCG AAT Pmkr9 ACT CAA AGT AAC AAA ATC TTC CGT A + P. vivax specific ( 11 ) rVIV1 CGC TTC TAG CTT AAT CCA CAT AAC TGA TAC rVIV2 ACT TCC AAG CCG AAG CAA AGA AAG TCC TTA – P. falciparum specific ( 11 ) rFAL1 TTA AAC TGG TTT GGG AAA ACC AAA TAT ATT rFAL2 ACA CAA TGA ACT CAA TCA TGA CTA CCC GTC – P. malariae specific ( 11 ) rMAL1 ATA ACA TAG TTG TAC GTT AAG AAT AAC CGC rMAL2 AAA ATT CCC ATG CAT AAA AAA TTA TAC AAA – P. ovale specific ( 11 ) rOVA1 ATC TCT TTT GCT ATT TTT TAG TAT TGG AGA rOVA2 GGA AAA GGA CAC ATT AAT TGT ATC CTA GTG – *PCR was carried out at the Environmental Health Institute and cycling conditions used were as described in the references shown in parentheses. 
+, positive; –,negative. We confirmed the pathogen by using previously described approaches to compare the sequences of the 5′ and 3′ ends of the circumsporozoite protein (csp) gene ( 13 ), as well as the gene encoding of the sSSU rRNA ( 5 ) in our case sample, to other Plasmodium parasites. Sequences were obtained by direct sequencing of PCR products and aligned by using the ClustalW method (EMBL-EBI, Hixton, Cambridge, UK); we constructed phylogenetic trees by using the MegAlign software (DNASTAR Inc, Madison, WI, USA). The case sample (denoted as SingPk1) clustered with other P. knowlesi isolates and is clearly distinct from other Plasmodium species (Figure 2). Figure 2 Phylogenetic trees comparing our case sample (denoted as SingPk1) with other Plasmodium species, based on SSU rRNA (A) and csp (B) sequences. Species and sequences used were selected to match those previously reported ( 5 ). Figures on the branches are bootstrap percentages based on 1,000 replicates, and only those above 80% are shown. GenBank accession numbers are in parentheses. Conclusions We describe an unequivocal case of P. knowlesi infection supported by clinical findings and laboratory diagnostics classic for this pathogen. Similar to our patient, the classic scenario that raises the suspicion of P. knowlesi infection is a blood smear consistent with P. malariae but with parasitemia exceeding 5,000 per mmol/L blood, daily fever spikes, and pronounced symptoms, features atypical for P. malariae infection ( 1 , 5 ). The daily fever spike is due to the P. knowlesi 24-hour asexual life cycle, the shortest of all primate malarias ( 8 ). P. malariae has a 72-hour asexual life cycle and manifests as chronic, asymptomatic infection with low level parasitemia ( 5 ). As in this case, P. knowlesi is commonly mistaken for P. malariae by microscopy due to similarity of the blood stages ( 5 ). P. knowlesi can be misidentified as P. falciparum if only ring forms are identified ( 5 ). The P. knowlesi–specific primers used by both independent laboratories have previously been shown not to detect any of the 4 Plasmodium species that cause human infection or the 3 agents that cause simian malaria: P. cynomolgi, P. fieldi, and P. fragile ( 5 ). PCR detection using P. knowlesi–specific primers, followed by sequencing and phylogenetic analyses of the csp and SSU rRNA genes confirmed P. knowlesi infection in our patient. This report extends the range of natural P. knowlesi human infection from East Malaysia, peninsular Malaysia, Thailand, and Myanmar to Singapore, an industrialized country that had been declared malaria-free by WHO ( 3 – 8 ). Our patient likely acquired the infection in the forested area in Lim Chu Kang where he had been training for the entire year before his illness. Experimental P. knowlesi studies show a prepatent period of 9–12 days in humans ( 14 ). P. knowlesi has no liver hypnozoite stage and does not cause relapse ( 1 ). The patient’s pevious overseas travel 7 months before and his visit to Bukit Batok Nature Reserve a month before onset of illness are beyond the incubation period. P. knowlesi’s natural hosts are the macaques, M. fascicularis and Macaca nemestrina ( 1 ). Notably, the first studies on P. knowlesi were on a parasite isolated from a macaque imported into India from Singapore ( 2 ). M. fascicularis and Presbytis femoralis are the 2 native monkeys in Singapore, with M. fascicularis being the only species in Lim Chu Kang and Bukit Batok Nature Reserve ( 15 ). Mosquitoes of the Anopheles leucophyrus group have been identified as vectors of P. knowlesi and are present in surrounding countries in southeast Asia ( 1 , 8 ). Studies are ongoing to determine potential mosquito vectors and whether macaques are hosts of P. knowlesi in Singapore. Our patient’s condition was diagnosed within 6 days of illness, and the infection responded rapidly to oral chloroquine. Although most patients’ infections respond well to antimalarial agents, 4 fatal cases of P. knowlesi infection were reported recently in patients ages 39 to 69 years, whose conditions were all diagnosed within 7 days of symptom onset ( 8 ). Common clinical features included fever, abdominal pain, thrombocytopenia (platelet count <30 × 109/ μL), renal impairment, and jaundice. All of the patients received a misdiagnosis of P. malariae infection. P. knowlesi infection should be considered as an etiologic agent of malaria acquired in Singapore, particularly in cases with daily fever spikes and blood smears suggestive of P. malariae. Epidemiologic studies into the parasite’s reservoir and mosquito vector will be important in the prevention of this emerging zoonotic disease.

                Author and article information

                Emerg Infect Dis
                Emerging Infectious Diseases
                Centers for Disease Control and Prevention
                November 2008
                : 14
                : 11
                : 1750-1752
                [1]National Institute of Allergy and Infectious Diseases, Bethesda, Maryland, USA (T.F. McCutchan)
                [2]University of Iowa, Iowa City, Iowa, USA (R.C. Piper)
                [3]Flow Incorporated, Portland, Oregon, USA (M.T. Makler)
                Author notes
                Address for correspondence: Thomas F. McCutchan, Laboratory of Malaria and Vector Research, National Institute of Allergy and Infectious Diseases, National Institutes of Health, 12735 Twinbrook Pkwy, Rockville, MD 20892, USA; email: tmccutchan@ 123456niaid.nih.gov

                Infectious disease & Microbiology
                borneo,dispatch,plasmodium knowlesi,plasmodium cynomolgi,mimetopes,lactate dehydrogenase,monkey malaria,malaria diagnostic


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