Comparative detection of Plasmodium vivax and Plasmodium falciparum DNA in saliva and urine samples from symptomatic malaria patients in a low endemic area

<p>Abstract</p> <p>Background</p> <p>Definite diagnosis of malaria relies on microscopy detection of blood stages of parasites in peripheral blood and requires blood sample collection. The nested PCR method has shown to be more sensitive and superior to microscopy in detecting co-infections of <it>Plasmodium </it>species in circulation while <it>Plasmodium falciparum </it>DNA can be identified in urine and saliva specimens of patients, albeit at a lower sensitivity.</p> <p>Methods</p> <p>Matched blood, saliva and urine samples were collected from 100 microscopy-positive and 20 microscopy-negative febrile patients who attended a malaria clinic in Tak Province, northwestern Thailand for nested PCR analysis targeting the small subunit ribosomal RNA gene of human malaria. Both <it>P. falciparum </it>and <it>Plasmodium vivax </it>have been known to circulate at a comparable rate in the study area.</p> <p>Results</p> <p>Comparing with microscopy results, nested PCR of saliva samples had a sensitivity of 74.1% for <it>P. falciparum </it>detection and 84% for <it>P. vivax </it>detection while 44.4% and 34.0% of the corresponding values were observed for urine samples. Both nested PCR results of saliva and urine samples had a specificity of 100% for identification of <it>P. falciparum </it>and <it>P. vivax </it>when compared with nested PCR results from blood. Co-infections of both species were found in four, 26 and 8 patients by microscopy and nested PCR of blood and saliva samples, respectively. Although the positive rates of nested PCR of saliva samples for <it>P. falciparum </it>increased with parasite density, no tendency occurred in results from nested PCR of saliva samples for <it>P. vivax </it>as well as those of urine samples.</p> <p>Conclusions</p> <p>Saliva and urine samples could be alternative noninvasive sources of DNA for molecular detection of both <it>P. falciparum </it>and <it>P. vivax</it>. Further improvement of the detection method will offer an opportunity to use these samples for diagnosis of malaria.</p

Draft Genome Sequences of Chlamydiales Bacterium STE3 and Neochlamydia sp. Strain AcF84, Endosymbionts of Acanthamoeba spp.

Chlamydiales bacterium STE3 and Neochlamydia sp. strain AcF84 are obligate intracellular symbionts of Acanthamoeba spp. isolated from the biofilm of a littoral cave wall and gills from striped tiger leaf fish, respectively. We report the draft genome sequences of these two environmental chlamydiae affiliated with the family Parachlamydiaceae

Clinical expression and antigenic profiles of a Plasmodium vivax vaccine candidate: Merozoite Surface Protein 7 (PvMSP-7)

Background: Vivax malaria is the predominant form of malaria outside Africa, affecting about 14 million people worldwide, with about 2.5 billion people exposed. Development of a Plasmodium vivax vaccine is a priority, and merozoite surface protein 7 (MSP-7) has been proposed as a plausible candidate. The P. vivax genome contains 12 MSP-7 genes, which contribute to erythrocyte invasion during blood-stage infection. Previous analysis of MSP-7 sequence diversity suggested that not all paralogs are functionally equivalent. To explore MSP-7 functional diversity, and to identify the best vaccine candidate within the family, MSP-7 expression and antigenicity during bloodstream infections were examined directly from clinical isolates. Methods: Merozoite surface protein 7 gene expression was profiled using RNA-seq data from blood samples isolated from ten human patients with vivax malaria. Differential expression analysis and co-expression cluster analysis were used to relate PvMSP-7 expression to genetic markers of life cycle stage. Plasma from vivax malaria patients was also assayed using a custom peptide microarray to measure antibody responses against the coding regions of 12 MSP-7 paralogs. Results: Ten patients presented diverse transcriptional profiles that comprised four patient groups. Two MSP-7 paralogs, 7A and 7F, were expressed abundantly in all patients, while other MSP-7 genes were uniformly rare (e.g. 7J). MSP-7H and 7I were significantly more abundant in patient group 4 only, (two patients having experienced longer patency), and were co-expressed with a schizont-stage marker, while negatively associated with liver-stage and gametocyte-stage markers. Screening infections with a PvMSP-7 peptide array identified 13 linear B-cell epitopes in five MSP-7 paralogs that were recognized by plasma from all patients. Conclusions: These results show that MSP-7 family members vary in expression profile during blood infections; MSP-7A and 7F are expressed throughout the intraerythrocytic development cycle, while expression of other paralogs is focused on the schizont. This may reflect developmental regulation, and potentially functional differentiation, within the gene family. The frequency of B-cell epitopes among paralogs also varies, with MSP-7A and 7L consistently the most immunogenic. Thus, MSP-7 paralogs cannot be assumed to have equal potential as vaccines. This analysis of clinical infections indicates that the most abundant and immunogenic paralog is MSP-7A

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

Coexistence of GP195 Alleles of Plasmodium Falciparum in a Small Endemic Area

Dimorphic variations in the genotype of the precursor to Plasmodium falciparum major merozoite surface antigens or gp195, among wild isolates in a small malaria parasite population were examined using Southern blot hybridization techniques. Hybridization, with DNA fragment probes and oligonucleotide probes derived from variable blocks of known gp195 alleles against 18 wild isolates from Mae Sod district in Thailand, revealed the existence of seven gp195 alleles, two of which were newly identified in this study. In four out of 17 patients, two different alleles coexisted in the circulation. It was furthermore noted that the seven alleles did not occur at the same frequency, but rather several alleles predominated in the population of P. falciparum in this small malaria field

Allelic Variation in the Circumsporozoite Protein of Plasmodium falciparum from Thai Field Isolates

Allelic variation in the Plasmodium falciparum circumsporozoite (CS) protein gene has been examined by sequencing the entire gene in 15 isolates from an endemic area of Thailand. The isolates contain a total of six new allelic forms of the tetrapeptide repeats and eight variants of the T cell epitope (TCE) region of the CS gene. All nucleotide substitutions in the TCE are nonsynonymous. There is no apparent association between the sequence patterns in the repeats and in the TCE. Comparison of the TCE with published sequences has shown that most variants of our isolates are not identical to those found in different geographic areas, suggesting geographic variation in genetic diversity of the CS protein. In a phylogenetic tree, the new Thai alleles did not cluster together, suggesting a considerable heterogeneity within some geographic areas. Furthermore, analyses of tetrapeptide repeats from a number of isolates and strains showed evidence of three genetic mechanisms for the generation of variation in the repeats of the CS gene: point mutation, duplication of one or more repeat units, and intragenic recombination

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

Disseminated Infection Caused by Novel Species of Microsporidium, Thailand

We describe a case of microsporidial myositis in a healthy man from Thailand. The small subunit rRNA sequence of this microsporidium is novel and has a close phylogenetic relationship with Endoreticulatus, a genus of lepidopteran microsporidia. Myositis could be caused by more genera of microsporidia than previously known

Plasmodium knowlesi Malaria in Humans and Macaques, Thailand

This parasite may be transmitted from macaques to humans

Genetic Diversity and Lack of Artemisinin Selection Signature on the Plasmodium falciparum ATP6 in the Greater Mekong Subregion

The recent detection of clinical Artemisinin (ART) resistance manifested as delayed parasite clearance in the Cambodia-Thailand border area raises a serious concern. The mechanism of ART resistance is not clear; but the P. falciparum sarco/endoplasmic reticulum Ca2+-ATPase (PfSERCA or PfATP6) has been speculated to be the target of ARTs and thus a potential marker for ART resistance. Here we amplified and sequenced pfatp6 gene (~3.6 Kb) in 213 samples collected after 2005 from the Greater Mekong Subregion, where ART drugs have been used extensively in the past. A total of 24 single nucleotide polymorphisms (SNPs), including 8 newly found in this study and 13 nonsynonymous, were identified. However, these mutations were either uncommon or also present in other geographical regions with limited ART use. None of the mutations were suggestive of directional selection by ARTs. We further analyzed pfatp6 from a worldwide collection of 862 P. falciparum isolates in 19 populations from Asia, Africa, South America and Oceania, which include samples from regions prior to and after deployments ART drugs. A total of 71 SNPs were identified, resulting in 106 nucleotide haplotypes. Similarly, many of the mutations were continent-specific and present at frequencies below 5%. The most predominant and perhaps the ancestral haplotype occurred in 441 samples and was present in 16 populations from Asia, Africa, and Oceania. The 3D7 haplotype found in 54 samples was the second most common haplotype and present in nine populations from all four continents. Assessment of the selection strength on pfatp6 in the 19 parasite populations found that pfatp6 in most of these populations was under purifying selection with an average dN/dS ratio of 0.333. Molecular evolution analyses did not detect significant departures from neutrality in pfatp6 for most populations, challenging the suitability of this gene as a marker for monitoring ART resistance