Loop-mediated isothermal amplification (LAMP), a novel nucleic acid amplification method, was developed for the clinical detection of four species of human malaria parasites: Plasmodium falciparum, P. vivax, P. malariae, and P. ovale. We evaluated the sensitivity and specificity of LAMP in comparison with the results of microscopic examination and nested PCR. LAMP showed a detection limit (analytical sensitivity) of 10 copies of the target 18S rRNA genes for P. malariae and P. ovale and 100 copies for the genus Plasmodium, P. falciparum, and P. vivax. LAMP detected malaria parasites in 67 of 68 microscopically positive blood samples (sensitivity, 98.5%) and 3 of 53 microscopically negative samples (specificity, 94.3%), in good agreement with the results of nested PCR. The LAMP reactions yielded results within about 26 min, on average, for detection of the genus Plasmodium, 32 min for P. falciparum, 31 min for P. vivax, 35 min for P. malariae, and 36 min for P. ovale. Accordingly, in comparison to the results obtained by microscopy, LAMP had a similar sensitivity and a greater specificity and LAMP yielded results similar to those of nested PCR in a shorter turnaround time. Because it can be performed with a simple technology, i.e., with heat-treated blood as the template, reaction in a water bath, and inspection of the results by the naked eye because of the use of a fluorescent dye, LAMP may provide a simple and reliable test for routine screening for malaria parasites in both clinical laboratories and malaria clinics in areas where malaria is endemic.

The Greater Mekong Subregion (GMS), comprised of six countries including Cambodia, China's Yunnan Province, Lao PDR, Myanmar (Burma), Thailand and Vietnam, is one of the most threatening foci of malaria. Since the initiation of the WHO's Mekong Malaria Program a decade ago, malaria situation in the GMS has greatly improved, reflected in the continuous decline in annual malaria incidence and deaths. However, as many nations are moving towards malaria elimination, the GMS nations still face great challenges. Malaria epidemiology in this region exhibits enormous geographical heterogeneity with Myanmar and Cambodia remaining high-burden countries. Within each country, malaria distribution is also patchy, exemplified by ‘border malaria’ and ‘forest malaria’ with high transmission occurring along international borders and in forests or forest fringes, respectively. ‘Border malaria’ is extremely difficult to monitor, and frequent malaria introductions by migratory human populations constitute a major threat to neighboring, malaria-eliminating countries. Therefore, coordination between neighboring countries is essential for malaria elimination from the entire region. In addition to these operational difficulties, malaria control in the GMS also encounters several technological challenges. Contemporary malaria control measures rely heavily on effective chemotherapy and insecticide control of vector mosquitoes. However, the spread of multidrug resistance and potential emergence of artemisinin resistance in Plasmodium falciparum make resistance management a high priority in the GMS. This situation is further worsened by the circulation of counterfeit and substandard artemisinin-related drugs. In most endemic areas of the GMS, P. falciparum and P. vivax coexist, and in recent malaria control history, P. vivax has demonstrated remarkable resilience to control measures. Deployment of the only registered drug (primaquine) for the radical cure of vivax malaria is severely undermined due to high prevalence of glucose-6-phosphate dehydrogenase deficiency in target human populations. In the GMS, the dramatically different ecologies, diverse vector systems, and insecticide resistance render traditional mosquito control less efficient. Here we attempt to review the changing malaria epidemiology in the GMS, analyze the vector systems and patterns of malaria transmission, and identify the major challenges the malaria control community faces on its way to malaria elimination.

This parasite may be transmitted from macaques to humans.

Giemsa-stained blood smears from each of 2,190 patients from Thai government-operated clinics on the Thailand-Myanmar border were independently examined by the on-duty microscopists at the clinics and by 2-3 research microscopists, each blinded to the clinics' and each other's reports. Using a strictly defined protocol, a consensus reference-standard blood smear interpretation for each sample was produced by the research microscopists. This result was compared with the clinic's diagnostic interpretation for the corresponding sample with respect to detection of parasitemia and diagnosis of infecting species. Reference-standard results reported parasitemia in 13.2% of the samples reported negative by the clinic. Reference-standard results were negative in 24.3% of the samples reported parasite-positive by the clinic. For samples in which both the reference-standard result and the clinic result reported parasitemia, species identification differed for 13.7% of the samples. The likelihood of parasite detection and correct diagnosis at the clinic varied in accordance with the referencestandard estimates of parasite density.

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Immunity induced by Plasmodium vivax infections leads to memory T-cell recruitment and activation during subsequent infections. Here, we investigated the role of regulatory T cells (Treg) in coordination with the host immune response during P. vivax infection. Our results showed a significant increase in the percentage of FOXP31 Treg, IL-10-secreting Type I Treg (Tr1) and IL-10 levels in patients with acute P. vivax infection as compared with those found in either naïve or immune controls. The concurrent increase in the Treg population could also be reproduced in vitro using peripheral blood mononuclear cells from naïve controls stimulated with crude antigens extracted from P. vivax-infected red blood cells. Acute P. vivax infections were associated with a significant decrease in the numbers of DC, indicating a general immunosuppression during P. vivax infections. However, unlike P. falciparum infections, we found that the ratio of myeloid DC (MDC) to plasmacytoid DC (PDC) was significantly lower in acute P. vivax patients than that of naïve and immune controls. Moreover, the reduction in PDC may be partly responsible for the poor antibody responses during P. vivax infections. Taken together, these results suggest that P. vivax parasites interact with DC, which alters the MDC/PDC ratio that potentially leads to Treg activation and IL-10 release.Key words: Dendritic cell . IL-10 . Malaria . Plasmodium vivax . Regulatory T cell Eur. J. Immunol. 2008. 38: 2697-2705 DOI 10.1002 Immunity to infection 2697 IntroductionMalaria is a common tropical disease causing deaths among Plasmodium falciparum-infected children mainly in Sub-Saharan Africa [1]. P. falciparum causes malignant tertian malaria that accounts for most malaria-associated deaths, whereas P. vivax causes relapsing fever and the infection rarely becomes fatal. Although a better understanding of immunity is needed for the design of effective vaccines, immune regulation in the host during malaria infection is not fully understood, and few studies have been conducted in patients with P. vivax infections. Our recent study has shown that anti-P. vivax antibody levels were very low in immune individuals living in endemic area and in patients with acute P. vivax malaria. For the cell-mediated arm, an acute P. vivax infection was associated with the activation of memory T cells belonging to either a cytotoxic or helper phenotype [2]. Additionally, previous evidence [3,4] shows that immunization with pre-erythrocytic antigens can induce IFN-g release. This suggests that P. vivax can activate the immune system via the Th1 pathway. However, a possible suppressing mechanism arises from the activation of regulatory T cells (Treg) as has been shown in a murine malaria study [5]. Treg constitutively express CD25, which is the IL-2/a chain receptor [6]. Co-presentation of CD25 with forkhead box protein P3 (FOXP3) dictates the immune-suppressive role of Treg via the release of . Treg have been shown to alter the balance between myeloid dendritic cells (MDC) and plasmacytoi...

Malaria incidence data at the district level from 1997 to 2002 and total malaria case data from 1965 to 2002 in Thailand were analyzed to determine the spatial and temporal dynamics of Plasmodium falciparum and P. vivax malaria incidence. Over the 37-year period, there was a 35-fold reduction in the incidence rates of P. falciparum malaria (11.86% in 1965 versus 0.34% in 2002) and a 7-fold reduction in P. vivax malaria (2.89% in 1965 versus 0.40% in 2002). The incidence ratio of P. falciparum to P. vivax malaria was reduced from 4.1 to 0.8 during this period. Malaria incidence rate exhibited the most rapid reduction between 1975 and 1985, coinciding with the introduction of a combination of antifolate drugs (sulfadoxine-pyrimethamine). The distribution maps of P. falciparum and P. vivax malaria incidence rates indicated a high spatial heterogeneity. The Thailand-Myanmar and Thailand-Cambodia border areas, where migration of foreign workers was pronounced, had the highest incidence rates for P. falciparum, P. vivax, and mixed-species infections. Transition probability analysis based on the malaria incidence rate among Thai residents indicated that there was an overall trend of decrease in the number of malaria cases and the number of high incidence districts between 1997 and 2002. High spatial variation in malaria incidence and local human migration patterns suggest that malaria control measures need to be adjusted according to local environmental and demographic settings.

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