Fighting drug-resistant Plasmodium falciparum: the challenge of artemisinin resistance.

Following a decade-long scale up of malaria control through vector control interventions, the introduction of rapid diagnostic tests and highly efficacious Artemisinin-based Combination Therapy (ACT) along with other measures, global malaria incidence declined significantly. The recent development of artemisinin resistance on the Cambodia-Thailand border, however, is of great concern. This review encompasses the background of artemisinin resistance in Plasmodium falciparum, its situation, especially in the Greater Mekong Sub-region (GMS), and the responses taken to overcome this resistance. The difficulties in defining resistance are presented, particularly the necessity of measuring the clinical response to artemisinins using the slow parasite-clearance phenotype. Efforts to understand the molecular basis of artemisinin resistance and the search for molecular markers are reviewed. The markers, once identified, can be applied as an efficient tool for resistance surveillance. Despite the limitation of current surveillance methods, it is important to continue vigilance for artemisinin resistance. The therapeutic efficacy "in vivo study" network for monitoring antimalarial resistance in the GMS has been strengthened. GMS countries are working together in response to artemisinin resistance and aim to eliminate all P. falciparum parasites. These efforts are crucial since a resurgence of malaria due to drug and/or insecticide resistance, program cuts, lack of political support and donor fatigue could set back malaria control success in the sub-region and threaten malaria control and elimination if resistance spreads to other regions.

Identification of Rickettsia spp. and Bartonella spp. in ffrom the Thai-Myanmar border.

During a survey for possible rickettsial vectors in villages of the central part of the Thai-Myanmar border from September 2001 to February 2002, four species of fleas were collected from common peridomestic animals. All fleas were tested by PCR to detect DNA of bacteria of the genera Rickettsia (gltA and ompB genes) and Bartonella (ITS and ftsZ genes). Sequencing of PCR-amplified products was done using gltA fragments for Rickettsia and ftsZ fragments for BARTONELLA: Two genotypes related to Rickettsia felis were identified in three Ctenocephalides canis and one C. felis specimen. Further, the following Bartonella spp. were detected: Bartonella henselae in two C. felis specimens; Bartonella clarridgeiae in three C. felis specimens; and a new Bartonella genotype in one Nosopsylla fasciatus specimen. Rickettsia and Bartonella may be frequently detected in fleas infesting peridomestic animals from the western border of Thailand.

Drug-resistant malaria in Bangladesh: an in vitro assessment.

Forty-four Plasmodium falciparum isolates from Bangladesh and 22 from western Thailand were successfully tested for their drug susceptibility. High degrees of resistance were observed against chloroquine with geometric mean IC50s of 114.25 and 120.5 nM, respectively, for Bangladesh and western Thailand. Most isolates from both sites were sensitive to quinine, and all were sensitive to artesunate. Many isolates were considered in vitro resistant to mefloquine, but the geometric mean IC50 for the Thai isolates (98.79 nM) was 1.6 times (P = 0.002) higher than that of isolates from Bangladesh (60.3 nM). The high prevalence of in vitro mefloquine resistance in Bangladesh suggests that close surveillance is necessary to delay widespread multidrug resistant problems in the area.

In vitro susceptibility of Plasmodium falciparum isolates from Myanmar to antimalarial drugs.

In vitro drug susceptibility profiles were assessed in 75 Plasmodium falciparum isolates from 4 sites in Myanmar. Except at Mawlamyine, the site closest to the Thai border, prevalence and degree of resistance to mefloquine were lower among the Myanmar isolates as compared with those from Thailand. Geometric mean concentration that inhibits 50% (IC50) and 90% (IC90) of Mawlamyine isolates were 51 nM (95% confidence interval [CI], 40-65) and 124 nM (95% CI, 104-149), respectively. At the nearest Thai site, Maesod, known for high-level multidrug resistance, the corresponding values for mefloquine IC50 and IC90 were 92 nM (95% CI, 71-121) and 172 nM (95% CI, 140-211). Mefloquine susceptibility of P. falciparum in Myanmar, except for Mawlamyine, was consistent with clinical-parasitological efficacy in semi-immune people. High sensitivity to artemisinin compounds was observed in this geographical region. The data suggest that highly mefloquine-resistant P. falciparum is concentrated in a part of the Thai-Myanmar border region.

Drug resistant malaria on the Thai-Myanmar and Thai-Cambodian borders.

We describe the changing epidemiology of drug resistant malaria in Thailand over the past decade. Factors determining the characteristic patterns of the development and spread of resistance to anti-malarial drugs on the Thai-Cambodian border and the Thai-Myanmar border are explored, namely, population dynamics, drug usage and malaria control measures. The introduction of artesunate-mefloquine combination in selected areas along the two borders in 1995 is believed to be one of the multiple factors responsible for stabilizing the multidrug resistance problems in Thailand today. Other control measures and inter-governmental co-operation must continue to be strengthened in order to limit the spread of drug resistance malaria in the Southeast Asian region.

Malaria rapid diagnostic devices: performance characteristics of the ParaSight F device determined in a multisite field study.

Microscopic detection of parasites has been the reference standard for malaria diagnosis for decades. However, difficulty in maintaining required technical skills and infrastructure has spurred the development of several nonmicroscopic malaria rapid diagnostic devices based on the detection of malaria parasite antigen in whole blood. The ParaSight F test is one such device. It detects the presence of Plasmodium falciparum-specific histidine-rich protein 2 by using an antigen-capture immunochromatographic strip format. The present study was conducted at outpatient malaria clinics in Iquitos, Peru, and Maesod, Thailand. Duplicate, blinded, expert microscopy was employed as the reference standard for evaluating device performance. Of 2,988 eligible patients, microscopy showed that 547 (18%) had P. falciparum, 658 (22%) had P. vivax, 2 (0.07%) had P. malariae, and 1,750 (59%) were negative for Plasmodium. Mixed infections (P. falciparum and P. vivax) were identified in 31 patients (1%). The overall sensitivity of ParaSight F for P. falciparum was 95%. When stratified by magnitude of parasitemia (no. of asexual parasites per microliter of whole blood), sensitivities were 83% (>0 to 500 parasites/microl), 87% (501 to 1,000/microl), 98% (1,001 to 5,000/microl), and 98% (>5,000/microl). Device specificity was 86%.

Rapid diagnostic techniques for malaria control.

The past decade was a milestone in the development of malaria diagnostic technology. Today, a variety of simplified and rapid malaria diagnostic devices, collectively referred to as 'dipsticks', is available. This paper discusses the potential roles of these devices, and obstacles to their use, in supporting malaria control strategies.

PCR-based ELISA technique for malaria diagnosis of specimens from Thailand.

We performed a field evaluation of polymerase chain reaction (PCR)-based enzyme-linked immuno-sorbent assays (ELISA) for the diagnosis of malaria. A commercially available PCR-ELISA microplate hybridization (MPH) assay was used. Blood specimens were collected from 300 volunteers seeking care at malaria clinics in Thailand. Examination of 200 high power fields by Giemsa-stained thick and thin smear (GTTS) revealed 51 P. falciparum (Pf), 45 P. vivax (Pv), seven mixed Pf-Pv infections. These plus a random sample of 48 GTTS-negative specimens were selected for this study. All 151 specimens were processed for parasite DNA extraction and assayed by PCR-MPH. The target DNA sequence of the 18S small subunit ribosomal RNA (SSUrRNA) gene was amplified by PCR and hybridized with species-specific probes for Pf, Pv, P. malariae (Pm) and P. ovale (Po) immobilized in the wells of the microtiter plate and detected by colorimetric assay. Colour development was assessed at an optical density (OD) of 405 nm. An absorbance reading of > or = 0.1 was used as a positive cut-off. In comparison with GTTS results, PCR-MPH sensitivity was 91.4% (53/58, 95% CI 84.2-98.6) for Pf, 94.2% (49/52, 87.9-100) for Pv and specificity was 95.8% (46/48, 95% CI 90.2-100). There was statistically significant positive correlation between parasite densities < or = 7000/microl blood and absorbance reading, suggestive of PCR-MPH being semiquantitative. PCR-MPH also detected additional Pf and Pv cases as well as Pm and Po.

Antimalarial drug combination policy: a caveat.

PIP: This paper presents facts on malaria epidemiology and historical perspectives of antimalarial drug use in Thailand. It also suggests that the use of an artesunate-mefloquine combination for treating falciparum malaria may be one of the factors responsible for the success of the country's control strategies. It is noted that in Thailand Plasmodium falciparum has evolved resistance to chloroquine, sulfadoxine-pyrimethamine, and mefloquine in succession. In view of this, administration of oral artesunate plus mefloquine became the standard treatment for microscopically confirmed uncomplicated falciparum malaria in 1995. The regimen requires administration of 300 mg/day of artesunate for 2 days plus 750 mg mefloquine on the first day, followed by 500 mg on the second day. Overall, it is too early to assume that the addition of artesunate has halted the progression of mefloquine resistance in Thailand. In terms of applicability of the regimen worldwide, the complexity of the factors involved makes it impossible to predict the useful lifespan of the artesunate-mefloquine combination on the Thai-Myanmar border. Further research is needed into the determination and validation of the most suitable antimalarial regimens for each epidemiologically distinct area and each operationally different circumstance.^ieng

Comparison of a rapid field immunochromatographic test to expert microscopy for the detection of Plasmodium falciparum asexual parasitemia in Thailand.

We assessed a rapid, Plasmodium falciparum histidine rich protein 2 (PfHRP2)-based immunochromatographic test (ICT Malaria Pf Test), for detection of asexual P. falciparum parasitemia in 551 subjects in three groups: (1) symptomatic patients self-referring for diagnosis, (2) villagers in a screening survey, and (3) patients recently treated for P. falciparum malaria. Expert light microscopy was the reference standard. ICT test performance was similar for diagnostic and screening modes. Four findings emerged: (1) test sensitivity correlated directly with parasite density, (2) test band intensity correlated directly with parasite density, (3) persistent test positivity after parasite clearance precludes its use for monitoring early therapeutic responses, and (4) a false negative test at 18,000 parasites/microl is unexplained. We conclude that a strong positive ICT test is highly predictive of falciparum asexual parasitemia for the diagnosis of new cases of falciparum malaria in Thailand, but a negative test result is inadequate to exclude parasitemia < 300/microl, and in some instances, even a higher parasitemia.

Field trials of a rapid test for G6PD deficiency in combination with a rapid diagnosis of malaria.

A rapid single-step screening method for detection of glucose-6-phosphate dehydrogenase (G6 PD) deficiency was evaluated on Halmahera Island, Maluku Province, Indonesia, and in Shan and Mon States, Myanmar, in combination with a rapid diagnosis of malaria by an acridine orange staining method. Severe deficiency was detected by the rapid test in 45 of 1126 volunteers in Indonesia and 54 of 1079 in Myanmar, but it was difficult to distinguish blood samples with mild deficiency from those with normal activity. 89 of 99 severely deficient cases were later confirmed by formazan ring method in the laboratory, but 5 with mild and 5 with no deficiency were misdiagnosed as severe. Of the samples diagnosed as mild and no deficiency on-site, none was found to be severely deficient by the formazan method. Malaria patients were simultaenously++ detected on-site in 273 samples on Halmahera island and 277 samples from Shan and Mon States. In Mon State, primaquine was prescribed safely to G6 PD-normal malaria patients infected with Plasmodium vivax and/or gametocytes of P. falciparum. The new rapid test for G6 PD deficiency may be useful for detecting severe cases under field conditions, and both rapid tests combined are can be useful in malaria-endemic areas, facilitating early diagnosis, prompt and radical treatment of malaria and suppression of malaria transmission.

In vitro sensitivity of Plasmodium falciparum to artesunate in Thailand.

Reported are the in vitro susceptibilities of Plasmodium falciparum to artesunate, mefloquine, quinine and chloroquine of 86 isolates and to dihydroartemisinin of 45 isolates collected from areas of high resistance to mefloquine within Thailand near the borders with Myanmar and Cambodia, and from southern Thailand where P. falciparum is generally still sensitive to mefloquine. All the isolates were highly sensitive to artesunate, but the geometric mean IC50S were higher in isolates from the Thai-Myanmar and Thai-Cambodian borders than in those from southern Thailand. The IC50S for mefloquine and artesunate were strongly correlated (Pearson r = 0.605; n = 86; P < 0.00001). As expected, the in vitro sensitivities to dihydroartemisinin and artesunate were similar and strongly correlated (at IC50, Pearson r = 0.695; n = 45; P < 0.00002). The correlation between the activity of mefloquine and artesunate requires further investigation in order to determine the potential for development of cross-resistance in nature. Our results suggest that combination with mefloquine is not the ideal way of protecting the usefulness of artemisinin and its derivatives. A search for more suitable partner drugs to these compounds and careful regulation of their use are necessary in the interest of ensuring their long therapeutic life span.

High prevalence of Plasmodium malariae and Plasmodium ovale in malaria patients along the Thai-Myanmar border, as revealed by acridine orange staining and PCR-based diagnoses.

The prevalence of the four human malaria parasites was investigated among malaria patients at northern, central and southern towns in Thailand along the border with Myanmar between September 1995 and May 1996. Thin smears obtained from 548 Thai and Burmese patients were reviewed by an acridine orange staining method, and many mixed infections with two to four species, including P. malariae and P. ovale, were detected. These diagnostic results were compared with those by two PCR-based diagnoses, microtitre plate hybridization (MPH) and a nested PCR method, both of which targets the same, species-specific regions in the 18S rRNA genes. In both PCR diagnoses, many P. malariae and P. ovale infections were also detected. Detection sensitivity of P. malariae infection was higher in nested PCR than MPH, and a total prevalence of P. malariae infection estimated by nested PCR reached 24.3% (133/548). In 16 of them, the size of PCR products amplified by the P. malariae-specific primer was about 20-bp shorter than the expected size of 115-bp. Four of 16 possessed two different bands with normal and shorter sizes, suggesting that P. malariae isolates may be separated into two types, and that those with shorter products may be new variant form (s) with a nucleotide deletion in the target region. On the other hand, 21 P. ovale infections (3.8%) were detected by nested PCR, but four of them were MPH-negative because of the sequence variation at the probe region. These results indicated that the prevalence of P. malariae and P. ovale along the Thai-Myanmar border may be substantially higher than previously reported.

SPf66 malaria vaccine is safe and immunogenic in malaria naive adults in Thailand.

In preparation for an efficacy trial of malaria vaccine SPf66 in Thailand, a series of overlapping Phase I trials were conducted of US-manufactured SPf66. Here, two clinical lots were evaluated for safety and immunogenicity in a combined open-label trial. Eleven healthy, malaria naive, 18-44 year-old Thai men and women received three doses by subcutaneous injection in alternate arms at 0, 1 and 6 months. Safety was assessed by monitoring local and systemic reactogenicity and laboratory parameters. Common side effects were mild erythema, induration and tenderness at the site of injection which resolved within 24-48 h. At third immunization, two volunteers developed acute bilateral reactions with induration, erythema and pruritus limited to the sites of the second and third immunizations. Eight of 11 volunteers sero-converted by ELISA, six of whom would be classified as high responders by Colombian standards. Eight of 11 volunteers developed a lymphoproliferative response to the SPf66 antigen. Side effects were more common and antibody and lymphoproliferative responses greatest, among the four female volunteers. This initial study of SPf66 malaria vaccine in Asia constitutes an essential link between the initial Phase I study in the US and subsequent field studies in a semi-immune population in a malaria endemic area of Thailand. This study further establishes comparability of US-manufactured SPf66 with that of Colombian provenance and substantiates the validity of the subsequent negative efficacy results of SPf66 in a field trial in Thailand.

In vitro susceptibility of Plasmodium falciparum isolates in Vietnam to artemisinin derivatives and other antimalarials.

We performed in vitro drug susceptibility assays of ten Plasmodium falciparum isolates collected from Vict Nam in 1995. All isolates were found to be highly sensitive to artesunate, dihydro-artemisinin and artemisinin. They were also sensitive to quinine. All of them were resistant to chloroquine and mefloquine in vitro. This study provides the baseline estimates of in vitro susceptibility levels of the Vietnamese isolates to artermisinin and their derivatives because the data were collected soon after these drugs were introduced countrywide. It also describes some quantitative profiles of the in vitro response of other standard antimalarial drugs in Viet Nam, which is presently limited.