Phylogenetic relationship of Plasmodium falciparum populations in the Philippines.

Malaria is one of the major infectious diseases in the Philippines. It is being targeted for control through sustained early diagnosis, treatment and mosquito control. It is in this light that understanding the genetic background of the parasite population is important not only for basic biology of the organism but also for epidemiology and control of the disease. In the present study, molecular phylogenetic relationships of the 3 Plasmodium falciparum populations in the Philippines with the other populations in the world were inferred based on polymorphisms of 9 highly polymorphic microsatellite DNA loci in the parasite genome. A total of 92 P. falciparum isolates collected from 3 provinces (Kalinga, Palawan and Davao del Norte) in the Philippines, and 8 from other populations (3 African, 2 South American, 2 Papua New Guinean, and 1 Thai) that were previously reported, were used for the analysis. The phylogenetic tree showed that the 3 Philippine populations were genetically divergent from each other as compared to the other populations. The branching pattern of the tree suggests that the 3 Philippine populations were relatively close to the Thai population, rather than the Papua New Guinean populations, indicating that the ancestor of the 3 Philippines populations were introduced from Indochina peninsula, and not from countries located south of the Philippines such as Papua New Guinea or Indonesia.

Efficacy of Artequick versus artesunate-mefloquine in the treatment of acute uncomplicated falciparum malaria in Thailand.

To determine the efficacy, safety and tolerability of an alternative short-course, artemisinin-based combination therapy for acute uncomplicated Plasmodium falciparum malaria, we compared Artequick--a fixed-dosed combination of artemisinin (80 mg), piperaquine (400 mg), and primaquine (4 mg), per tablet--with a standard regimen of artesunate-mefloquine. A total of 130 patients were randomly assigned to treatment with an orally administered, once-daily, 3-day regimen of either Artequick (Group A: 3.2 mg/Kg/day of artemisinin, 16 mg/Kg/day of piperaquine, and 0.16 mg/Kg/day of primaquine) or artesunate-mefloquine (Group B: artesunate, 4 mg/Kg/day, with mefloquine, 8 mg/Kg/day). Patients receiving each regimen had a rapid clinical and parasitological response. All treatments were well tolerated, and no serious adverse effects occurred. No significant differences were found in fever- and parasite-clearance times between the two study groups. The 28-day cure rates were similarly high, at 98.5% and 100%, in groups A and B, respectively. We conclude that Artequick was as effective and well tolerated as artesunate-mefloquine and could be used as an alternative treatment for multidrug-resistant Plasmodium falciparum malaria in Southeast Asia.

Chemoprophylaxis according to the guidelines on malaria prevention for Japanese overseas travelers.

Mefloquine has been licensed and registered in Japan for chemoprophylaxis against malaria since 2001. Guidelines for the prevention of malaria for Japanese overseas travelers were published by a group of malaria specialists under the auspices of the Japanese Society of Tropical Medicine and the Ministry of Health, Labor and Welfare, but not until March 2005. We implemented these guidelines in our clinic at the International Medical Center of Japan in Tokyo and, to better understand whether these guidelines are optimally useful, we conducted a study of Japanese travelers who visited our clinic seeking pertinent information and prophylaxis against malaria. The study group comprised 52 individuals who visited our clinic during the period October 2004 through June 2005 prior to travel abroad. On the basis of the above-mentioned guidelines, mefloquine was given to 27 of these individuals, 22 of whom were judged to need regular chemoprophylaxis. Mefloquine was not recommended to the other 25 individuals because their stays abroad would have been too long to avoid possible side effects or too short for symptoms to appear. In fact, some were traveling to malaria-free areas. Of the 27 individuals given mefloquine, 7 (26%) reported side effects, such as headache, vertigo, and nausea, 17 (63%) reported no side-effects, and the other 3 (11%) were unable to be followed. The diversity of destinations and accompanying malaria risks makes it very difficult for us to administer chemoprophylaxis to overseas travelers appropriately. The guidelines proved to be somewhat useful, but further experience in malaria chemoprophylaxis is needed for physicians to provide reliable pre-travel consultation.

In vitro susceptibility and genetic variations for chloroquine and mefloquine in Plasmodium falciparum isolates from Thai-Myanmar border.

In vitro drug susceptibility to chloroquine (CQ) and mefloquine (MF) were assessed in 39 P. falciparum isolates from the Thai-Myanmar border area. To further characterize CQ- and MF-resistance profiles in this area, we also analyzed pfcrt K76T mutation that is critical for CQ resistance, and pfmdr1 polymorphism that has an association with MF resistance. Eighteen isolates were successfully examined by in vitro tests for CQ, and 17 of them had resistance to the drug. Geometric mean concentration of CQ that inhibited the growth of parasites at 50% (IC50) was 371 +/- 227 nM (105-971 nM). Sixteen isolates were successfully examined by in vitro tests for MF, and 8 of them were resistant to the drug. Geometric mean of IC50 for MF was 41 +/- 31 nM (4-125 nM). Genotypes of drug resistance, such as pfcrt and pfmdr1 mutations, were also analyzed. All the 39 isolates had the same haplotype (CVIET) for PfCRT at its 72-76th amino acids. A pfmdr1 Y86 mutation was found in 95% of isolates. A pfmdr1 D1042 mutation was also present in 7 isolates, while no pfmdr1 Y1246 mutation was observed. These results indicated a correlation between CQ resistance and the pfcrt T76 and pfmdr1 Y86 mutations.

Pyrimethamine-sulfadoxine treatment of uncomplicated Plasmodium falciparum malaria in Lao PDR.

A 28-day in vivo treatment trial to evaluate the efficacy of pyrimethamine/sulfadoxine (Fansidar, PS) was conducted in 21 Lao patients with uncomplicated Plasmodium falciparum malaria. Sixteen patients (76%) were completely cured with PS without any reappearance of asexual stage parasitemia during the follow-up examination. On the other hand, 5 patients (24%) failed to respond to this trial medication, resulting in recrudescence of asexual stage P. falciparum malaria. PS resistance resulted in higher prevalence of post-treatment gametocytemia, 25% gametocyte carriers among PS sensitive cases versus 75% of the resistant cases. These findings suggest that although the level of PS resistance is still valid for treatment of malaria in the study area of Lao PDR, post-treatment induction of gametocytemia among resistant cases may result an increase in transmission rate of PS resistant falciparum malaria.

The use of flow cytometry as a diagnostic test for malaria parasites.

A total of 453 clinical blood samples were determined for malaria parasites by flow cytometric assay (FCM) and reagents from Sysmex Corporation, Japan. In this study, the FCM greatly simplified and accelerated parasite detection, with sensitivity of 91.26%, specificity 86.28% and accuracy 87.42%. Overall, the parasite counts by flow cytometric measurement correlated well with the parasitemia measured by microscopic assay (regression coefficient = 0.9409). The detection limit was 0.05-0.1% parasitemia. No evidence of malaria parasites in either blood donor volunteers or other disease patients groups was determined by FCM. However, 48 samples who had been treated with antimalarial drugs and whose parasite microscopic counts were negative, showed false-positive results. When the data of these 48 samples were analyzed, they were found to have high levels of reticulocytes, ranging from 2.0-18.9%. This finding suggested that a high reticulocyte concentration in the blood may interfere with the performance of the FCM. Further improvement, by eliminating this interference, will make the FCM one of the most promising tests for malaria diagnosis.

Evaluation of the KAT-Quick Malaria Rapid Test for rapid diagnosis of falciparum malaria in Thailand.

In recent years, several rapid diagnostic tests for falciparum malaria have been developed. KAT test results were compared with microscopy on 90 consecutive patients hospitalized at the Hospital for Tropical Diseases, Bangkok, Thailand. Fifty-one patients had P. falciparum infections while 49 had malaria due to other plasmodium species. For a geometric mean +/-SD (Min;Max;range) parasitemia of 11,481 +/- 5.0 (88;713,838;713,750), the sensitivity of the KAT test was 96% (95% CI = 86-99.5), the specificity was 92% (95% CI = 80-99), the accuracy was 94% and the reliability was 85%. These findings suggest that the KAT test is of potential interest in the diagnosis of falciparum malaria in Thailand.

Application of real-time polymerase chain reaction (PCR) analysis for detection and discrimination of malaria parasite species in Thai patients.

A TaqMan real-time PCR system was used to detect and discriminate the 4 species of human malaria parasites in clinical blood samples. A 150-base pair (bp) region of the small subunit ribosomal RNA (SSU rRNA) gene of each malaria parasite, including species-specific sequences to be detected by TaqMan probe, was used as a target for PCR analysis. The PCR method used universal primers and species-specific TaqMan probes for Plasmodium falciparum, P. vivax, P. ovale, and P. malariae. The detection threshold for the method, as determined with serial dilution of cultured P. falciparum-infected erythrocytes, was 5 parasite-infected erythrocytes per reaction. Fifty blood samples of falciparum malaria and a second set of 50 samples of vivax malaria, diagnosed by microscopic examination at the Hospital for Tropical Diseases, Mahidol University, Thailand, were analyzed by real-time PCR. In the 50 samples of microscopically-diagnosed falciparum malaria, 40 were regarded as P. falciparum single infection, 7 were P. falciparum and P. vivax mixed infections, and 3 were P. vivax single infection by real-time PCR. In the second set of 50 samples of microscopically diagnosed vivax malaria, all were considered P. vivax single infection by PCR. Neither P. ovale nor P. malariae infection was identified in the 100 blood samples. Real-time PCR analysis was shown to be more sensitive and accurate than routine diagnostic methods. Application and extension of the PCR method reported here will provide a powerful tool for further studies of malaria.

In vitro susceptibility of Plasmodium falciparum isolates to chloroquine and mefloquine in southeastern Mindanao Island, the Philippines.

Although the presence of multi-drug-resistant falciparum malaria has been reported in the Philippines, the distribution of drug-resistant malaria parasites has not yet been determined in Mindanao Island. In vitro susceptibility of P. falciparum to both chloroquine and mefloquine was assessed to forecast the spread of drug-resistant parasites in various foci in southeastern Mindanao Island. Of the 33 isolates of P. falciparum successfully tested, 10 (30%) were susceptible, 12 (36%) showed decreased susceptibility (80 nM < or = IC50 < 114 nM), and 11 (33%) were resistant (IC50 > or = 114 nM) to chloroquine. Ten (91%) of the resistant isolates and 9 (75%) of those with decreased susceptibility were from northern and northwestern Davao del Norte Province. Chloroquine-susceptible isolates were found among patients in the eastern parts of Davao del Norte and Davao Oriental provinces. Seven isolates from several foci in the study area were all mefloquine- susceptible (IC50 < 10 nM). This is the first report indicating the potential emergence of chloroquine-resistant P. falciparum on Mindanao Island, which is presently regarded as a drug-susceptible area.

PCR-amplification, sequencing, and comparison of the var/PfEMP-1 gene from the blood of patients with falciparum malaria in the Philippines.

The adhesion of Plasmodium falciparum-infected erythrocytes to vascular endothelium and to uninfected red blood cells (RBCs) plays a key role in the pathology of severe malaria. Adhesion is known to be mediated in part by the antigenically-variant erythrocyte membrane protein-1 (PfEMP-1), which is encoded by the var-gene family of P. falciparum. It has recently been reported that in vitro a single parasite simultaneously transcribes multiple var-genes but that, through a developmentally regulated process, the parasite selects only one PfEMP-1 that will to reach the surface of the host RBC. Were this to be true in vivo, one would expect a correlation between the type of var/PfEMP-1 that is expressed on the parasite-infected RBC and the severity of clinical disease. In order to test this assumption, we determined the sequence of the var-gene that was expressed by the parasites in patients' blood samples. Seven blood samples were collected from patients with or without severe clinical symptoms (cerebral malaria): two samples were from patients diagnosed as having imported falciparum malaria at the International Medical Center of Japan (IMCJ); the five others were from patients of the Davao Regional Hospital in Davao, the Philippines. The parasites (ring stage) in the blood samples were cultured for 24 hours; the matured trophozoites, in which the var-gene selection had taken place, served as material for mRNA isolation. The cDNA corresponding to the Duffy-binding-like (DBL)-1 domain of the var-gene was amplified by RT-PCR, using a region-specific primer set. The amplified cDNAs were cloned into the plasmid vector; the resultant clones (32) were sequenced on both strands. The results indicated that there was considerable diversity in the sequence of the DBL-1 domain among the clones, even among those from a single patient. In conclusion, it was difficult to demonstrate the correlation between the type of var-gene transcripts found in the RBCs of malaria patients and the severity of their symptoms.

A study of chloroquine resistance of plasmodium falciparum using the in-vitro sensitivity test and polymerase chain reaction (PCR)

The widespread and increasing resistance of Plasmodium falciparum to antimalarial drugs is one of several factors that contributed to the persistence and even worsening of the malaria problem. Resistance to Chloroquine is of utmost concern, considering that it had been the most reliable antimalarial until the emergence and spread of Chloroquineresistant P. falciparum. Until recently, Chloroquine and Sulfadoxine-Pyrimethamine were the first and second line antimalarials in use in the Philippines. However, this has been changed to a combination of Chloroquine and Sulfadoxine-Pyrimethamine, because of the high percentage of treatment failures in therapeutic efficacy studies done in northern and southern Philippines. The objective of this study is to apply PCR in determining the occurence of Chloroquine resistance in southeastern Mindanao using in-vitro test and polymerase chain reaction (PCR)In the first study, the in-vitro susceptibility of P. falciparum to Chloroquine was tested in 33 isolates using the World Health Organization (WHO) Semi-Microtest Method. These isolates were collected from patients who consulted or were admitted at the regional hospital of Davao del Norte. The results showed that 10 (30.3 percent) were susceptible with IC50 80 nM, 12 (36.4 percent) isolates had decreased sensitivity with 80 nM /- IC50 114 nM, and 11 (33.3 percent) were resistant with IC 114 nM. Ten of these 11 (91 percent) were from Davao del Norte. A closer look at the municipalities of this province showed that the geometric mean (SD) of IC50 of Asuncion was 133 (41) nM and was significantly higher (p=0.025) than nearby Kapalong at 82 (25) nMThe PCR, Apo1 restriction revealed that 30 (90.9 percent) of the isolates manifested the PfCRT (K76T) mutation. These findings are indicative of the presence of Chloroquine resistance among the isolates. Comparison with the results of the invitro test (33.3 percent resistance) showed that the frequency of the PfCRT gene (90.9 percent) was very high. This finding suggests that the mere presence of the PfCRT gene does not mean the expression of Chloroquine resistance. It is possible that other genes such as the Pfmdr and cg2 are also involved in the expression of Chloroquine resistance. The study also shows that PCR and Apo1 restriction may be limited in generating results that can be used to correlate with those of the in-vitro or even in-vivo tests