Reliable interpretation and long-term stability using SYBRTM safe fluorescent assay for loop-mediated isothermal amplification (LAMP) detection of Leishmania spp.

Leishmaniasis, a vector-borne disease caused by Leishmania, is the second leading mortality after malaria. Continuously increasing cases of cutaneous and visceral leishmaniasis (CL/VL) have been documented in Thailand. Recently, loop-mediated isothermal amplification (LAMP) based on malachite green (MG) colorimetric assay that detects Leishmania DNA was developed to facilitate epidemiological studies of leishmaniasis in affected areas. However, ambiguous reading interpretation sometimes occurred using the MG-LAMP assay. In this study, the efficiency and effectiveness of the SYBRTM Safe fluorescent assay for LAMP detection of Leishmania siamensis (MON-324) and Leishmania martiniquensis (MON-229) were compared under two different light sources, i.e., blue light and ultraviolet light transilluminators. Regarding the SYBRTM-LAMP assay, the detection limit of DNA of both L. siamensis and L. martiniquensis was 103 parasites/mL. The assay exhibited consistency and reproducibility without requiring any post-reaction preparations. The dye is generally available, affordable and safe while reliable interpretation can be easily visualized under both blue light and ultraviolet light transilluminators. Using buffy coat of VL patients, the SYBRTM-LAMP offers an alternative method for screening samples with high sensitivity and specificity. This cost effective SYBRTM Safe fluorescent assay is simple to use without ambiguous evaluation which could provide another suitable choice of a standard LAMP assay in molecular laboratories as well as further development in field studies.

Reliable interpretation and long-term stability using SYBRTM safe fluorescent assay for loop-mediated isothermal amplification (LAMP) detection of Leishmania spp

@#Leishmaniasis, a vector-borne disease caused by Leishmania, is the second leading mortality after malaria. Continuously increasing cases of cutaneous and visceral leishmaniasis (CL/VL) have been documented in Thailand. Recently, loop-mediated isothermal amplification (LAMP) based on malachite green (MG) colorimetric assay that detects Leishmania DNA was developed to facilitate epidemiological studies of leishmaniasis in affected areas. However, ambiguous reading interpretation sometimes occurred using the MG-LAMP assay. In this study, the efficiency and effectiveness of the SYBRTM Safe fluorescent assay for LAMP detection of Leishmania siamensis (MON-324) and Leishmania martiniquensis (MON-229) were compared under two different light sources, i.e., blue light and ultraviolet light transilluminators. Regarding the SYBRTM-LAMP assay, the detection limit of DNA of both L. siamensis and L. martiniquensis was 103 parasites/mL. The assay exhibited consistency and reproducibility without requiring any post-reaction preparations. The dye is generally available, affordable and safe while reliable interpretation can be easily visualized under both blue light and ultraviolet light transilluminators. Using buffy coat of VL patients, the SYBRTM-LAMP offers an alternative method for screening samples with high sensitivity and specificity. This cost effective SYBRTM Safe fluorescent assay is simple to use without ambiguous evaluation which could provide another suitable choice of a standard LAMP assay in molecular laboratories as well as further development in field studies.

Incidence and risk factors of Giardia duodenalis infection in an orphanage, Thailand.

A cohort study was performed to evaluate the incidence and risk factors of Giardia duodenalis infection in an orphanage in suburban area outside Bangkok, central Thailand. Stool specimens were examined for the presence of G. duodenalis in January 2007, May 2007 and January 2008. Of 892 stool specimens from 481 individuals, simple wet preparation, PBS ethyl-acetate sedimentation and PCR amplification of the SSU-rRNA gene were performed to detect G. duodenalis. Using PCR of the glutamate dehydrogenase gene and sequence analysis, G. duodenalis assemblages were identified. Associated risk factors were analysed using Fisher's exact test which revealed significant infection of G. duodenalis in boys and specific rooms where orphans aged 25-48 months old lived. Genotypic characterization of G. duodenalis revealed that assemblage A subtype AII was the most predominant found in orphans living in the specific rooms, thus the transmission was likely to occur via person-to-person. Other modes of transmission were less likely to occur. This study showed that the incidence rate of Giardia infection gradually decreased significantly after the implementation of health education and appropriate treatment of infected orphans.

Epidemiology of giardiasis and genotypic characterization of Giardia duodenalis in preschool children of a rural community, central Thailand.

A cross-sectional study was conducted to determine the prevalence and risk factors of Giardia duodenalis infection in 189 preschool children at Sanamchaiket District, Chachoengsao Province, central Thailand in February 2007. Stool specimens were examined for the presence of Giardia using simple wet preparation and PBS-ethyl acetate concentration technique. The prevalence of G. duodenalis in preschool children was 5.8%. Using PCR-RFLP analysis of the glutamate dehydrogenase gene (gdh), genotypes of G. duodenalis revealed assemblage AII (3, 30.0%), BIII (1, 10.0%), and BIV (6, 60.0%). Using multivariate analysis, children who kept cat(s) at home were at 5.1 times (95% CI; 1.3- 20.3) greater risk of acquiring giardiasis. This study possibly represents the information supporting the potential zoonotic transmission of G. duodenalis between cats and preschool children. Unfortunately, in this study, we did not determine G. duodenalis infection in cats, so further studies in cats should be performed to confirm this postulation.

Intestinal parasitic infections: prevalences in HIV/AIDS patients in a Thai AIDS-care centre.

During 2005, the intestinal parasitic infections in the adult HIV/AIDS patients in a combined AIDS-care centre and hospice in Thailand were identified in a cross-sectional study. Overall, 41 (45.6%) of the 90 stool samples investigated, by microscopy and/or PCR, were found parasite-positive. Cryptosporidium was the genus most commonly encountered, with 27 (30.0%) of the patients positive for Cr. hominis and four (4.4%) positive for Cr. meleagridis. Enterocytozoon bieneusi, always of genotype D, was detected in five (5.6%) of the patients while two patients (2.2%) were found infected with Blastocystis, just one patient (1.1%) was found infected with Cyclospora cayetanensis and one more (1.1%) was found positive for Isospora belli. The only helminth infection detected was opisthorchiosis and that was only seen in two (2.2%) of the subjects. Patients with stools of unusual consistency (categorized as mucous, loose-watery or watery) were more likely to be found infected with one or more species of protozoan parasite than the patients with stools of normal consistency (P=0.022). In a multivariate analysis, compared with the other subjects, the patients with stools of unusual consistency were 4.9-fold more likely to have (opportunistic) intestinal infection with parasitic protozoa (with a 95% confidence interval for the adjusted odds ratio of 1.370-17.350; P=0.014). Although there was also a trend for stools of unusual colour (red-brown, red-orange, green, black or grey) to be positively associated with intestinal parasitic infection, this was not statistically significant (P=0.658). The observation of the consistency of stools of HIV/AIDS patients could help in the presumptive diagnosis of opportunistic protozoan infections and allow parasitological investigation to be targeted at the cases most likely to be found positive.

Direct characterization of Blastocystis from faeces by PCR and evidence of zoonotic potential.

In vitro propagation followed by PCR, and a PCR-based method capable of the direct detection of Blastocystis in faeces were utilized to detect Blastocystis from various hosts in Australia, including primates and their handlers from the Perth Zoo. In addition, Blastocystis isolates from dogs and humans living in a localized endemic community in Thailand were also characterized genetically. PCR-based detection directly from faeces was shown to be more sensitive compared with in vitro culture for the detection of Blastocystis. Moreover, phylogenetic analysis of Blastocystis isolates amplified utilizing in vitro techniques prior to PCR revealed that this method favoured the preferential amplification of Blastocystis subtype 5 over subtype 1. This study is the first to provide molecular-based evidence supporting the zoonotic potential of Blastocystis in dogs, possums and primates in a natural setting.

Molecular epidemiology of drug resistance markers of Plasmodium falciparum malaria in Thailand.

To determine differences in the distribution of drug resistance mutations in the Plasmodium falciparum chloroquine resistance transporter (pfcrt) and P. falciparum multi-drug resistance 1 (pfmdr1) genes of P. falciparum isolates in Thailand, a study was conducted using polymerase chain reaction-restriction fragment length polymorphism to detect mutations in P. falciparum isolates obtained from three areas with different levels of in vivo mefloquine (MQ) resistance. All isolates carried mutant allele T76 of the pfcrt gene and wild-type allele D1246 of the pfmdr1 gene except for one isolate, which showed the wild-type K76 allele. This isolate was obtained from Chanthaburi Province, an area with high MQ resistance. Relatively low rates of the mutant alleles D1042 and Y86 of the pfmdr1 gene were found among Thai isolates of P. falciparum. However, a statistically significant difference in the distribution was noted. Most of the mutant isolates were found among isolates from areas with moderate or low MQ resistance. Only one isolate with mixed mutant and wild-type N1042 and D1042 and two mutants of Y86 were found among the isolates from areas with high MQ resistance. The findings provide limited support for the hypothesis that mutant alleles of pfmdr1 may be associated with increased sensitivity to MQ.

In-vitro cultivation: a sensitive method for detecting Blastocystis hominis.

Currently, the detection of human infection with Blastocystis hominis is usually based on the examination under a light microscope of faecal samples, either directly, as 'simple smears', or after some form of concentration. Whether short-term, in-vitro cultivation would increase the sensitivity of such detection remains a matter of controversy. Over 900 fresh stool specimens, from soldiers in the Royal Thai Army, were each checked for the parasite using three methods: simple smears; formalin-ethyl-acetate concentration; and cultivation in Jones' medium. Although 334 of the samples were found to be culture-positive, the parasites were only detected in 142 of the simple smears, and faecal concentration led to an even lower sensitivity (64 positive samples). In-vitro cultivation does seem worthwhile in the detection of B. hominis carriage in field studies.

Intestinal microsporidiosis in HIV-infected children with acute and chronic diarrhea.

A prospective study of intestinal microsporidiosis in HIV-positive children was conducted at the Queen Sirikit National Institute of Child Health and Phramongkutklao Hospital, Bangkok, Thailand. Hospitalized HIV-positive children with and without diarrhea were enrolled in this study. Microsporidial spores identified by calcofluor fluorescent and gram-chromotrope stain were confirmed by electron microscopy. As well as Cryptosporidium parvum, Microsporidia was the most common protozoa found in the present study, each was 7.1%. Microsporidia was significantly more common in those who had diarrhea. Intestinal microsporidiosis was found in HIV-positive children with both acute and chronic diarrhea. This study emphasizes the importance of Microsporidia in HIV-infected children. Early detection of microsporidia could be of benefit for the patients, since the infection is treatable.

Transmission of intestinal blastocystosis related to the quality of drinking water.

A cross-sectional study was performed to evaluate the risk factors of Blastocystis hominis infection in the Thai army population of the 11th Infantry Division, Chachoengsao Province, Thailand. 201 army personnel and their family members were enrolled in this study. Intestinal parasitic infections in this population were assessed by stool examination using simple smear, formalin/ether technique and Kato-thick smear. Approximately one third of the specimens were positive for one or more intestinal parasites. With the prevalence of 21.9%, B. hominis was the most common intestinal parasite found in this population. Our data indicated that blastocystosis in this army population was significantly linked to the quality of drinking water. After being adjusted for potential confounders, consuming neither filtered nor boiled water was independently associated with blastocystosis.

Phenotypic and genotypic characteristics of recently adapted isolates of Plasmodium falciparum from Thailand.

The drug sensitivity characteristics and Plasmodium falciparum pfmdr1 status of five isolates of P. falciparum recently isolated from patients presenting for treatment from the Thailand/Myanmar border have been investigated. The aim of the study was to avoid the criticisms of some earlier studies by focusing on newly collected isolates from a specific geographic location. Three of the isolates studied exhibited clear resistance to chloroquine similar to that observed in the K1 Thai standard isolate obtained in the 1970s, and the other two isolates were of intermediate sensitivity to chloroquine with concentrations of drug that inhibit parasite growth by 50% of 50 and 43 nmol. The sensitivity of all isolates was enhanced by verapamil but we found no clear association between chloroquine sensitivity and gene copy number or intra-allelic variation of pfmdr1. In contrast, clear cross-resistance was seen between mefloquine and halofantrine, with the most sensitive isolates carrying the K1 mutation in pfmdr1.

Cellular uptake of chloroquine is dependent on binding to ferriprotoporphyrin IX and is independent of NHE activity in Plasmodium falciparum.

Here we provide definitive evidence that chloroquine (CQ) uptake in Plasmodium falciparum is determined by binding to ferriprotoporphyrin IX (FPIX). Specific proteinase inhibitors that block the degradation of hemoglobin and stop the generation of FPIX also inhibit CQ uptake. Food vacuole enzymes can generate cell-free binding, using human hemoglobin as a substrate. This binding accounts for CQ uptake into intact cells and is subject to identical inhibitor specificity. Inhibition of CQ uptake by amiloride derivatives occurs because of inhibition of CQ-FPIX binding rather than inhibition of the Na+/H+ exchanger (NHE). Inhibition of parasite NHE using a sodium-free medium does not inhibit CQ uptake nor does it alter the ability of amilorides to inhibit uptake. CQ resistance is characterized by a reduced affinity of CQ-FPIX binding that is reversible by verapamil. Diverse compounds that are known to disrupt lysosomal pH can mimic the verapamil effect. These effects are seen in sodium-free medium and are not due to stimulation of the NHE. We propose that these compounds increase CQ accumulation and overcome CQ resistance by increasing the pH of lysosomes and endosomes, thereby causing an increased affinity of binding of CQ to FPIX.

Central role of hemoglobin degradation in mechanisms of action of 4-aminoquinolines, quinoline methanols, and phenanthrene methanols.

We have used a specific inhibitor of the malarial aspartic proteinase plasmepsin I and a nonspecific cysteine proteinase inhibitor to investigate the importance of hemoglobin degradation in the mechanism of action of chloroquine, amodiaquine, quinine, mefloquine (MQ), halofantrine, and primaquine. Both proteinase inhibitors antagonized the antiparasitic activity of all drugs tested with the exception of primaquine. An inhibitor of plasmepsin I, Ro40-4388, reduced the incorporation of radiolabelled chloroquine and quinine into malarial pigment by 95%, while causing a 70% reduction in the incorporation of radiolabelled MQ. Cysteine proteinase inhibitor E64 reduced the incorporation of chloroquine and quinine into malarial pigment by 60 and 40%, respectively. This study provides definitive support for the central role of hemoglobin degradation in the mechanism of action of the 4-aminoquinolines and the quinoline and phenanthrene methanol antimalarials.

Access to hematin: the basis of chloroquine resistance.

The saturable uptake of chloroquine by parasites of Plasmodium falciparum has been attributed to specific carrier-mediated transport of chloroquine. It is suggested that chloroquine is transported in exchange for protons by the parasite membrane Na+/H+ exchanger [J Biol Chem 272:2652-2658 (1997)]. Once inside the parasite, it is proposed that chloroquine inhibits the polymerization of hematin, allowing this toxic hemoglobin metabolite to accumulate and kill the cell [Pharmacol Ther 57:203-235 (1993)]. To date, the contribution of these proposed mechanisms to the uptake and antimalarial activity of chloroquine has not been assessed. Using sodium-free medium, we demonstrate that chloroquine is not directly exchanged for protons by the plasmodial Na+/H+ exchanger. Furthermore, we show that saturable chloroquine uptake at equilibrium is due solely to the binding of chloroquine to hematin rather than active uptake: using Ro 40-4388, a potent and specific inhibitor of hemoglobin digestion and, by implication, hematin release, we demonstrate a concentration-dependent reduction in the number of chloroquine binding sites. An equal number of chloroquine binding sites are found in both resistant and susceptible clones, but the apparent affinity of chloroquine binding is found to correlate with drug activity (r2 = 0.93, p < 0.0001). This completely accounts for both the reduced drug accumulation and activity observed in resistant clones and the "reversal" of resistance produced by verapamil. The data presented here reconcile most of the available biochemical data from studies of the mode of action of chloroquine and the mechanism of chloroquine resistance. We show that the activity of chloroquine and amodiaquine is directly dependent on the saturable binding of the drugs to hematin and that the inhibition of hematin polymerization may be secondary to this binding. The chloroquine-resistance mechanism regulates the access of chloroquine to hematin. Our model is consistent with a resistance mechanism that acts specifically at the food vacuole to alter the binding of chloroquine to hematin rather than changing the active transport of chloroquine across the parasite plasma membrane.

Relationship between antimalarial drug activity, accumulation, and inhibition of heme polymerization in Plasmodium falciparum in vitro.

We have investigated the contribution of drug accumulation and inhibition of heme polymerization to the in vitro activities of a series of antimalarial drugs. Only those compounds exhibiting structural relatedness to the quinolines inhibited heme polymerization. We could find no direct correlation between in vitro activity against chloroquine-susceptible or chloroquine-resistant isolates and either inhibition of heme polymerization or cellular drug accumulation for the drugs studied. However, in vitro activity against a chloroquine-susceptible isolate but not a chloroquine-resistant isolate showed a significant correlation with inhibition of heme polymerization when the activity was normalized for the extent of drug accumulation. The importance of these observations to the rational design of new quinoline-type drugs and the level of agreement of these conclusions with current views on quinoline drug action and resistance are discussed.

Physicochemical properties correlated with drug resistance and the reversal of drug resistance in Plasmodium falciparum.

At high molar excess, verapamil can selectively increase the accumulation and cytotoxicity of structurally dissimilar natural product drugs in many multidrug-resistant tumor cell lines. Such concentrations of verapamil are also capable of increasing the accumulation and activity of chloroquine in chloroquine-resistant strains of the human malaria parasite Plasmodium falciparum. Despite such similarities, it is not clear why chloroquine-resistant P. falciparum is often susceptible to closely related compounds such as amodiaquine, whereas cancer cells are cross-resistant to many structurally unrelated drugs. For 13 aminoquinoline and aminoacridine compounds, relative drug resistance was negatively correlated with lipid solubility at physiological pH (r2 = 0.90, p < 0.0001). The ability of verapamil (5 microM) to reverse drug resistance was also negatively correlated with lipid solubility (r2 = 0.88, p < 0.0001). Furthermore, molar refractivity was weakly correlated with relative drug resistance (r2 = 0.46, p < 0.05) and reversal of drug resistance (r2 = 0.52, p < 0.005). Verapamil increases chloroquine accumulation by resistant parasites, a mechanism suggested to account for its selective chemosensitization effect. We show that the initial rate of chloroquine accumulation by resistant parasites is increased by verapamil. This effect of verapamil is abolished when deoxy-glucose is substituted for glucose. Therefore, verapamil produces an energy-dependent increase in the permeability of resistant parasites to chloroquine. For a panel of four chloroquine-resistant and two chloroquine-susceptible isolates, the effect of verapamil on the accumulation of chloroquine and monodesethyl amodiaquine was found to be correlated (r2 = 0.96, p < 0.001). Verapamil chemosensitization was also correlated for the two drugs (r2 = 0.92, p < 0.005), suggesting a common mechanism. In summary, the degree of drug resistance and the extent of verapamil chemosensitization for a particular drug seem to be dependent on general physical features such as lipid solubility and molar refractivity rather than on closely defined structural parameters. These studies provide insight into this important resistance mechanism of malaria parasites and may provide direction for the development of new drugs that are effective against resistant parasites.

In vitro selection of halofantrine resistance in Plasmodium falciparum is not associated with increased expression of Pgh1.

Recent investigations into quinoline and phenanthrene methanol resistance in Plasmodium falciparum have described a linkage between amplification of the mdr homologue pfmdr1 and decreased susceptibility to mefloquine (MQ) and halofantrine (HF). We have examined the current theories on cross-resistance patterns and pfmdr1 gene expression by comparing the chloroquine (CQ) resistant isolate K1 with K1Hf, developed from the K1 isolate by intermittent exposure to halofantrine. Reduced halofantrine susceptibility in K1Hf was accompanied by reduced sensitivity to mefloquine and increased sensitivity to chloroquine. These sensitivity changes were reflected by changes in parasite drug accumulation. The loss of high level chloroquine resistance in K1Hf was associated with an inability of verapamil to enhance chloroquine sensitivity or accumulation. In contrast verapamil retained the chemosensitising potential against quinine in this isolate. The changes in phenotype were achieved without any amplification or increased expression of pfmdr1 or reversion of the Tyr86 mutation in the gene. Our data indicates that acquisition of halofantrine and mefloquine resistance and the loss of high level chloroquine resistance can be achieved without enhanced expression of the pfmdr1 gene product.

Current views on the mechanisms of resistance to quinoline-containing drugs in Plasmodium falciparum.

The issue of chloroquine resistance in Plasmodium falciparum and cross-resistance patterns with other related chemotherapeutic agents has been the subject of intense interest for many years. Despite this level of investigation, the picture remains very unclear. Although it is accepted that chloroquine resistance is, at least in part, a function of reduced drug accumulation, the question of reduced drug uptake versus enhanced efflux is yet to be resolved at both the molecular and biochemical levels. Further, the absolute cross-resistance patterns of chloroquine-resistant isolates to closely related analogues is a matter for debate, although there appears to be a reciprocal arrangement between resistance to chloroquine and resistance to mefloquine, halofantrine and possibly quinine. Evidence is presented for the coexistence of two or more chloroquine-resistance mechanisms in isolates of P. falciparum, only one of which is verapamil sensitive. In addition, an analysis of cross-resistance patterns, as measured by the inoculum effect, is presented.