Real-Time Loop-Mediated Isothermal Amplification (RealAmp) for the Species-Specific Identification of Plasmodium vivax

Plasmodium vivax infections remain a major source of malaria-related morbidity and mortality. Early and accurate diagnosis is an integral component of effective malaria control programs. Conventional molecular diagnostic methods provide accurate results but are often resource-intensive, expensive, have a long turnaround time and are beyond the capacity of most malaria-endemic countries. Our laboratory has recently developed a new platform called RealAmp, which combines loop-mediated isothermal amplification (LAMP) with a portable tube scanner real-time isothermal instrument for the rapid detection of malaria parasites. Here we describe new primers for the detection of P. vivax using the RealAmp method. Three pairs of amplification primers required for this method were derived from a conserved DNA sequence unique to the P. vivax genome. The amplification was carried out at 64°C using SYBR Green or SYTO-9 intercalating dyes for 90 minutes with the tube scanner set to collect fluorescence signals at 1-minute intervals. Clinical samples of P. vivax and other human-infecting malaria parasite species were used to determine the sensitivity and specificity of the primers by comparing with an 18S ribosomal RNA-based nested PCR as the gold standard. The new set of primers consistently detected laboratory-maintained isolates of P. vivax from different parts of the world. The primers detected P. vivax in the clinical samples with 94.59% sensitivity (95% CI: 87.48–98.26%) and 100% specificity (95% CI: 90.40–100%) compared to the gold standard nested-PCR method. The new primers also proved to be more sensitive than the published species-specific primers specifically developed for the LAMP method in detecting P. vivax.     

Beriberi (Thiamine Deficiency) and High Infant Mortality in Northern Laos

BackgroundInfantile beriberi (thiamine deficiency) occurs mainly in infants breastfed by mothers with inadequate intake of thiamine, typically among vulnerable populations. We describe possible and probable cases of infantile thiamine deficiency in northern Laos.ConclusionThiamine deficiency may be a major cause of infant mortality among ethnic groups in northern Laos. Mothers’ and children’s symptoms are compatible with thiamine deficiency. The severity of this nutritional situation requires urgent attention in Laos.     

Insect Consumption to Address Undernutrition,a National Survey on the Prevalence of Insect Consumption among Adults and Vendors in Laos

Background

Insect consumption (entomophagy) is a potentially high nutritious and healthy source of food with high fat, protein, vitamin, fiber and micronutrient content. At least 2 billion people globally eat insects (over 1900 edible species) though this habit is regarded negatively by others. There is a limited amount of data on the perception and consumption of insects. We conducted a national cross-sectional survey in the Lao People’s Democratic Republic (Laos) to assess the prevalence and characteristics of insect consumption among adult lay people and insect vendors.

Methods

We conducted a multi stage randomized national survey in 1303 households in 96 villages in 16 Lao provinces. Three insect vendors or collectors per village were also included. A standardized pretested questionnaire addressed the following issues: socioeconomic characteristics, type of insects consumed and frequency of consumption, reasons and trends in consumption as well as reports on side effects, over the last 10 years.

Results

A total of 1059 adults (Sex ratio F/M: 1.2, 30 ethnic groups), and 256 vendors were enrolled. A total of 1025 (96.8%) lay people were currently insect consumers, 135 (13.0%) daily or weekly consumers, and 322 (31.1%) consumed several times per month. For the majority (575, 55.6%) the consumption was infrequent (less than a few times per year) and only 22 (2%) had never eaten insects. Consumption started in childhood. Insect availability was seasonal (670, 63.2%) and respondents would have eaten more insects, if they had been more available (919, 86.7%). Hmong and Leu ethnic groups had significantly lower consumption levels than the general population. Eggs of weaver ants, short-tailed crickets, crickets, grasshoppers, and cicadas were the top 5 insects consumed. Consumption had decreased in the last decade, mostly due to less availability (869; 84.0%) and change of life (29; 5.5%). Of 1059, 80 (7.5%) reported allergy problems and 106 (10.0%) reported some use in traditional medicine. A total of 874 (82.6%) were regular collectors.Insect vendors (Sex ratio F/M: 5.3) were also collectors (185; 72.2%). They dedicated a mean time of 4.7 hours during the last harvesting period. The majority sold insects at markets (141, 55.0%). They had earned, on average, USD 6.0 the day before. Five insects (weaver ant eggs; bamboo worms; short-tailed crickets; crickets; wasps) represented 85% of the market.

Conclusion

Entomophagy is general in Laos, and well accepted despite a decreasing trend in consumption over the last decade. Its contribution to the Lao diet is limited to a minority of frequent consumers. Income through insect sales benefits mostly women. Consumption varies according to ethnicity, residence and season. Development of insect farming is still at an early stage. It could however increase availability of insects and contribute to the generation of income.     

A new single-step PCR assay for the detection of the zoonotic malaria parasite Plasmodium knowlesi

BACKGROUND: Recent studies in Southeast Asia have demonstrated substantial zoonotic transmission of Plasmodium knowlesi to humans. Microscopically, P. knowlesi exhibits several stage-dependent morphological similarities to P. malariae and P. falciparum. These similarities often lead to misdiagnosis of P. knowlesi as either P. malariae or P. falciparum and PCR-based molecular diagnostic tests are required to accurately detect P. knowlesi in humans. The most commonly used PCR test has been found to give false positive results, especially with a proportion of P. vivax isolates. To address the need for more sensitive and specific diagnostic tests for the accurate diagnosis of P. knowlesi, we report development of a new single-step PCR assay that uses novel genomic targets to accurately detect this infection. METHODOLOGY AND SIGNIFICANT FINDINGS: We have developed a bioinformatics approach to search the available malaria parasite genome database for the identification of suitable DNA sequences relevant for molecular diagnostic tests. Using this approach, we have identified multi-copy DNA sequences distributed in the P. knowlesi genome. We designed and tested several novel primers specific to new target sequences in a single-tube, non-nested PCR assay and identified one set of primers that accurately detects P. knowlesi. We show that this primer set has 100% specificity for the detection of P. knowlesi using three different strains (Nuri, H, and Hackeri), and one human case of malaria caused by P. knowlesi. This test did not show cross reactivity with any of the four human malaria parasite species including 11 different strains of P. vivax as well as 5 additional species of simian malaria parasites. CONCLUSIONS: The new PCR assay based on novel P. knowlesi genomic sequence targets was able to accurately detect P. knowlesi. Additional laboratory and field-based testing of this assay will be necessary to further validate its utility for clinical diagnosis of P. knowlesi.