Cielo Pasay

Plasmodium vivax synonymous substitution frequencies, evolution and population structure deduced from diversity in AMA 1 and MSP 1 genes.

Polymorphic regions of the genes encoding Plasmodium vivax apical membrane antigen 1 (PvAMA1) and P. vivax merozoite surface protein 1 (PvMSP1) were sequenced to examine population diversity both within and between geographical areas. Sequences were obtained for 219 isolates for PvAMA1 and for 175 isolates for PvMSP1 from Africa, China, India, Indonesia, Philippines, Papua New Guinea, Solomon Islands and Thailand. Over half of the isolates were obtained from different regions within the Philippines, and this was used to look at the diversity within a country. Sixty nine haplotypes and 22 polymorphic sites in a 414-bp region of PvAMA1 and 41 haplotypes and 34 polymorphic sites in a 249-bp fragment of PvMSP1 were detected. For both PvAMA1 and PvMSP1, four previously unreported polymorphic nucleotide positions were identified. Population analysis indicated that there were significant differences in allele frequencies between different regions but these differences were small compared to the diversity within populations (Fixation index, F(ST), of 0.126 and 0.078 for PvAMA1 and PvMSP1, respectively). PvAMA1 and PvMSP1 had similar nonsynonymous substitution frequencies but surprisingly, the synonymous substitution frequency for PvMSP1 was eight times the frequency for PvAMA1 suggesting that synonymous substitutions in at least PvAMA1 are not neutral.

pfcrt Allelic Types with Two Novel Amino Acid Mutations in Chloroquine-Resistant Plasmodium falciparum Isolates from the Philippines

ABSTRACT Mutations in the pfcrt and pfmdr1 genes have been associated with chloroquine resistance in Plasmodium falciparum. Ten and five mutations, respectively, have been identified in these genes from chloroquine-resistant parasites worldwide. Mutation patterns in pfcrt revealed that chloroquine resistance evolved independently in southeast Asia, South America, and Papua New Guinea. However, the evolution of chloroquine resistance in the rest of the Pacific region is unclear. In this study, we examined sequence polymorphisms in these genes in isolates from Morong, Philippines, and compared them to known chloroquine resistance sequences. Two novel mutations, A144T and L160Y, were identified outside of the 10 known mutations in pfcrt in Morong isolates. These novel mutations were identified only in parasites with K76T and N326D but without the common A220S mutation found in most chloroquine-resistant isolates. This represents a unique chloroquine resistance allelic type (K76T/A144T/L160Y/N326D) not previously found elsewhere in the world. One Morong isolate also had an additional C72S mutation, whereas only one isolate possessed an allelic type typical of chloroquine resistance in Asia. Parasites with the novel pfcrt allelic types were resistant to chloroquine in vitro and were unresponsive to verapamil (0.9 μM) chemosensitization, similar to chloroquine-resistant parasites from South America and Papua New Guinea. These results suggest that chloroquine resistance evolved independently in the Philippines and represents a second chloroquine resistance founder event in the South Pacific.

Acaricidal Activity of Eugenol Based Compounds against Scabies Mites

Backgound Human scabies is a debilitating skin disease caused by the “itch mite” Sarcoptes scabiei. Ordinary scabies is commonly treated with topical creams such as permethrin, while crusted scabies is treated with topical creams in combination with oral ivermectin. Recent reports of acaricide tolerance in scabies endemic communities in Northern Australia have prompted efforts to better understand resistance mechanisms and to identify potential new acaricides. In this study, we screened three essential oils and four pure compounds based on eugenol for acaricidal properties. Methodology/Principal Findings Contact bioassays were performed using live permethrin-sensitive S. scabiei var suis mites harvested from pigs and permethrin-resistant S. scabiei var canis mites harvested from rabbits. Results of bioassays showed that clove oil was highly toxic against scabies mites. Nutmeg oil had moderate toxicity and ylang ylang oil was the least toxic. Eugenol, a major component of clove oil and its analogues –acetyleugenol and isoeugenol, demonstrated levels of toxicity comparable to benzyl benzoate, the positive control acaricide, killing mites within an hour of contact. Conclusions The acaricidal properties demonstrated by eugenol and its analogues show promise as leads for future development of alternative topical acaricides to treat scabies.

A Tractable Experimental Model for Study of Human and Animal Scabies

Background Scabies is a parasitic skin infestation caused by the burrowing mite Sarcoptes scabiei. It is common worldwide and spreads rapidly under crowded conditions, such as those found in socially disadvantaged communities of Indigenous populations and in developing countries. Pruritic scabies lesions facilitate opportunistic bacterial infections, particularly Group A streptococci. Streptococcal infections cause significant sequelae and the increased community streptococcal burden has led to extreme levels of acute rheumatic fever and rheumatic heart disease in Australias Indigenous communities. In addition, emerging resistance to currently available therapeutics emphasizes the need to identify potential targets for novel chemotherapeutic and/or immunological intervention. Scabies research has been severely limited by the availability of parasites, and scabies remains a truly neglected infectious disease. We report development of a tractable model for scabies in the pig, Sus domestica. Methodology/Principal Findings Over five years and involving ten independent cohorts, we have developed a protocol for continuous passage of Sarcoptes scabiei var. suis. To increase intensity and duration of infestation without generating animal welfare issues we have optimised an immunosuppression regimen utilising daily oral treatment with 0.2mg/kg dexamethasone. Only mild, controlled side effects are observed, and mange infection can be maintained indefinitely providing large mite numbers (>6000 mites/g skin) for molecular-based research on scabies. In pilot experiments we explore whether any adaptation of the mite population is reflected in genetic changes. Phylogenetic analysis was performed comparing sets of genetic data obtained from pig mites collected from naturally infected pigs with data from pig mites collected from the most recent cohort. Conclusions/Significance A reliable pig/scabies animal model will facilitate in vivo studies on host immune responses to scabies including the relations to the associated bacterial pathogenesis and more detailed studies of molecular evolution and host adaption. It is a most needed tool for the further investigation of this important and widespread parasitic disease.

High-resolution melt analysis for the detection of a mutation associated with permethrin resistance in a population of scabies mites

Abstract Permethrin as a topical acaricide cream is widely used to treat scabies. The neuronal voltage‐sensitive sodium channel (Vssc), necessary for the generation of action potentials in excitable cells, is the target of pyrethroid acaricides such as permethrin. Pyrethroid resistance has been linked to specific mutations in the Vssc gene. Following the partial sequencing of the Vssc gene in the scabies mite Sarcoptes scabiei (L.) (Astigmata: Sarcoptidae), we compared Vssc gene sequences from permethrin‐sensitive and ‐tolerant S. scabiei var. canis Gerlach mites, and identified a G to A single nucleotide polymorphism (SNP) in permethrin‐tolerant mites resulting in an amino acid change from glycine to aspartic acid in domain III S6. The mutation is in a region of the gene where mutations have been identified in a range of pyrethroid‐resistant arthropods. Results of in vitro permethrin exposure assays showed that survival rates for mites bearing the mutation were similar to those previously reported for mites from human subjects where clinical tolerance to permethrin had been observed. A real‐time polymerase chain reaction−high‐resolution melt (PCR‐HRM) assay was developed to detect this SNP. This assay provides a useful methodology for screening for this and other mutations associated with permethrin resistance in scabies mite populations and thus facilitates surveillance for acaricide resistance.

The effect of insecticide synergists on the response of scabies mites to pyrethroid acaricides.

Background Permethrin is the active component of topical creams widely used to treat human scabies. Recent evidence has demonstrated that scabies mites are becoming increasingly tolerant to topical permethrin and oral ivermectin. An effective approach to manage pesticide resistance is the addition of synergists to counteract metabolic resistance. Synergists are also useful for laboratory investigation of resistance mechanisms through their ability to inhibit specific metabolic pathways. Methodology/Principal Findings To determine the role of metabolic degradation as a mechanism for acaricide resistance in scabies mites, PBO (piperonyl butoxide), DEF (S,S,S-tributyl phosphorotrithioate) and DEM (diethyl maleate) were first tested for synergistic activity with permethrin in a bioassay of mite killing. Then, to investigate the relative role of specific metabolic pathways inhibited by these synergists, enzyme assays were developed to measure esterase, glutathione S-transferase (GST) and cytochrome P450 monooxygenase (cytochrome P450) activity in mite extracts. A statistically significant difference in median survival time of permethrin-resistant Sarcoptes scabiei variety canis was noted when any of the three synergists were used in combination with permethrin compared to median survival time of mites exposed to permethrin alone (p<0.0001). Incubation of mite homogenates with DEF showed inhibition of esterase activity (37%); inhibition of GST activity (73%) with DEM and inhibition of cytochrome P450 monooxygenase activity (81%) with PBO. A 7-fold increase in esterase activity, a 4-fold increase in GST activity and a 2-fold increase in cytochrome P450 monooxygenase activity were observed in resistant mites compared to sensitive mites. Conclusions These findings indicate the potential utility of synergists in reversing resistance to pyrethroid-based acaricides and suggest a significant role of metabolic mechanisms in mediating pyrethroid resistance in scabies mites.

Increased transcription of Glutathione S-transferases in acaricide exposed scabies mites

BackgroundRecent evidence suggests that Sarcoptes scabiei var. hominis mites collected from scabies endemic communities in northern Australia show increasing tolerance to 5% permethrin and oral ivermectin. Previous findings have implicated detoxification pathways in developing resistance to these acaricides. We investigated the contribution of Glutathione S-transferase (GST) enzymes to permethrin and ivermectin tolerance in scabies mites using biochemical and molecular approaches.ResultsIncreased in vitro survival following permethrin exposure was observed in S. scabiei var. hominis compared to acaricide naïve mites (p < 0.0001). The addition of the GST inhibitor diethyl maleate restored in vitro permethrin susceptibility, confirming GST involvement in permethrin detoxification. Assay of GST enzymatic activity in mites demonstrated that S. scabiei var. hominis mites showed a two-fold increase in activity compared to naïve mites (p < 0.0001). Increased transcription of three different GST molecules was observed in permethrin resistant S. scabiei var. canis- mu 1 (p < 0.0001), delta 1 (p < 0.001), and delta 3 (p < 0.0001). mRNA levels of GST mu 1, delta 3 and P-glycoprotein also significantly increased in S. scabiei var. hominis mites collected from a recurrent crusted scabies patient over the course of ivermectin treatment.ConclusionsThese findings provide further support for the hypothesis that increased drug metabolism and efflux mediate permethrin and ivermectin resistance in scabies mites and highlight the threat of emerging acaricide resistance to the treatment of scabies worldwide. This is one of the first attempts to define specific genes involved in GST mediated acaricide resistance at the transcriptional level, and the first application of such studies to S. scabiei, a historically challenging ectoparasite.

Origin and Dissemination of Chloroquine-Resistant Plasmodium falciparum with Mutant pfcrt Alleles in the Philippines

ABSTRACT The pfcrt allelic type and adjacent microsatellite marker type were determined for 82 Plasmodium falciparum isolates from the Philippines. Mutant pfcrt allelic types P1a and P2a/P2b were dominant in different locations. Microsatellite analysis revealed that P2a/P2b evolved independently in the Philippines, while P1a shared common ancestry with Papua New Guinea chloroquine-resistant parasites.

Sensitive Detection of Plasmodium vivax Using a High-Throughput, Colourimetric Loop Mediated Isothermal Amplification (HtLAMP) Platform: A Potential Novel Tool for Malaria Elimination

Introduction Plasmodium vivax malaria has a wide geographic distribution and poses challenges to malaria elimination that are likely to be greater than those of P. falciparum. Diagnostic tools for P. vivax infection in non-reference laboratory settings are limited to microscopy and rapid diagnostic tests but these are unreliable at low parasitemia. The development and validation of a high-throughput and sensitive assay for P. vivax is a priority. Methods A high-throughput LAMP assay targeting a P. vivax mitochondrial gene and deploying colorimetric detection in a 96-well plate format was developed and evaluated in the laboratory. Diagnostic accuracy was compared against microscopy, antigen detection tests and PCR and validated in samples from malaria patients and community controls in a district hospital setting in Sabah, Malaysia. Results The high throughput LAMP-P. vivax assay (HtLAMP-Pv) performed with an estimated limit of detection of 1.4 parasites/ μL. Assay primers demonstrated cross-reactivity with P. knowlesi but not with other Plasmodium spp. Field testing of HtLAMP-Pv was conducted using 149 samples from symptomatic malaria patients (64 P. vivax, 17 P. falciparum, 56 P. knowlesi, 7 P. malariae, 1 mixed P. knowlesi/P. vivax, with 4 excluded). When compared against multiplex PCR, HtLAMP-Pv demonstrated a sensitivity for P. vivax of 95% (95% CI 87–99%); 61/64), and specificity of 100% (95% CI 86–100%); 25/25) when P. knowlesi samples were excluded. HtLAMP-Pv testing of 112 samples from asymptomatic community controls, 7 of which had submicroscopic P. vivax infections by PCR, showed a sensitivity of 71% (95% CI 29–96%; 5/7) and specificity of 93% (95% CI87-97%; 98/105). Conclusion This novel HtLAMP-P. vivax assay has the potential to be a useful field applicable molecular diagnostic test for P. vivax infection in elimination settings.

Field epidemiological studies on malaria in a low endemic area in the Philippines.

Field epidemiological studies were conducted to examine factors affecting endemicity in an area with a low prevalence of malaria. Two annual cross sectional surveys were done to estimate parasite prevalence rates at two periods in time, to determine the distribution of the parasitemic population and to describe the serological status of the population. A longitudinal study of a sample of infected people was used to measure reinfection rates and antibody dynamics. A 2 year passive case detection was done to estimate the number and distribution of people with symptomatic infections. Malaria was found in all age groups, with marked clustering of cases. Active and passive case detection and serological surveys all gave a similar pattern of malaria distribution: generally low prevalence with small foci of relatively high endemicity. The infection frequencies were generally similar in all age groups, measured by both active and passive case detection. There was a high frequency of P. falciparum gametocytemic infections in the asymptomatic cases found through active case detection. Twenty to 39 year old males had the highest frequency of infection by active case detection, and 10-19 year old males by passive case detection. These two groups were also more likely to be gametocyte positive than their female counterparts, suggesting that in this community, this portion of the population acts as the main reservoir of infection.