Knowledge, Behavior, and Free-Living Amoebae Contamination of Cosmetic Contact Lens Among University Wearers in Thailand: A Cross-Sectional Study.

OBJECTIVE: To assess the general knowledge, behavior, and presence of potentially pathogenic amoebae in cosmetic contact lens (CCL) wearers. METHODS: One hundred CCL asymptomatic wearers were randomly selected. A questionnaire regarding their lens use, and a pair of their CCL was obtained. Identification of free-living amoeba (FLA) strains was based on morphological diagnosis, enflagellation tests (for non-Acanthamoeba strains), and sequencing of the small-subunit rRNA gene fragments. RESULTS: Most (92%) of the participants surveyed were women, and the average age of the participants was 21.5±0.2 years. The CCL wearers generally showed a moderate (47%) or good (35%) level of knowledge, and good (51%) or excellent (40%) use of CCL. Two CCL samples were positive for Acanthamoeba genotype T3 or Vahlkampfia. The Acanthamoeba-contaminated CCL was from a wearer who used saline for treating lenses, and the Vahlkampfia-contaminated CCL was from a wearer who used CCL while swimming. CONCLUSIONS: This is the first report of the presence of potentially pathogenic FLA in used CCL from asymptomatic wearers in Thailand. Although there was satisfactory knowledge and practice of lens care use, the public should be aware of CCL contaminated with potentially pathogenic FLA that can directly or indirectly cause keratitis.

Assessment of an Immuno-Diagnostic Method for Hookworm-Related Cutaneous Larva Migrans Using Crude Extracts of Ancylostoma caninum.

People can become infected with cutaneous larva migrans (CLM) through skin penetration by the infective zoonotic larvae of hookworms. Few studies have investigated CLM's immunodiagnosis, and the existing studies were limited to crude somatic or excretory/secretory antigens (Ags) from adult worms. Here, we aimed to develop an indirect enzyme-linked immunosorbent assay (ELISA) to differentiate and diagnose hwCLM by detecting immunoglobulin (Ig)E, IgG, and IgG subclasses 1-4 (IgG1-4) against the somatic Ag of adult Ancylostoma caninum checkerboard titrations of adult A. caninum worm extract. Pooled serum controls were immunocharacterized using an indirect ELISA. The IgG1-4 and IgE results were unsatisfactory; however, the use of total IgG achieved results comparable to those of immunoblotting. Thus, we continued to analyze the IgG-ELISA using serum samples from patients with hwCLM and heterologous infections as well as from healthy controls. The sensitivity and excellent specificity of the total IgG-ELISA were 93.75% and 98.37%, respectively, and its positive and negative predictive values were 75% and 99.67%, respectively. Antibodies from five cases of angiostrongyliasis, gnathostomiasis, and dirofilariasis cross-reacted with the somatic Ag of adult A. caninum. This new assay can adequately serodiagnose hwCLM when combined with clinical features and/or histological examination.

Cloning of a trypsin-like serine protease and expression patterns during Plasmodium falciparum invasion in the mosquito, Anopheles dirus (Peyton and Harrison).

Understanding specific gene regulation during responses to malaria infection is key to dissecting the mosquito defense mechanisms and host/parasite interactions. A full-length serine protease cDNA was isolated from the fat body of female Anopheles dirus, a major malaria vector in Thailand. The predicted amino acid sequence of SERF4 identifies it as a member of the serine protease family containing a single trypsin-like protease domain. Digestive trypsins function in the female mosquito midgut and are inducible in two phases in this tissue upon blood intake. However, the gene was highly upregulated in the midgut at day 3 postinfection by Plasmodium falciparum. In situ hybridization confirmed that SERF4 transcripts were located in the midgut epithelial cells rather than hemocytes or other tissues associated with the midgut. SERF4 was also strongly downregulated in the whole insects at day 16 after infection in comparison with the blood-fed control. Changes in the expression of the SERF4 gene in response to infection with this human malaria parasite suggest a role in parasite-specific innate immunity.

Comparison of physiological, cytopathogenic and immunological properties between two environmental isolates of Acanthamoeba spp.

The aim of this study was to determine whether pathogenic and less-pathogenic isolates of environmental Acanthamoeba exhibit differences in adhesion to human erythrocytes. Based on physiological properties of temperature, tolerance, and rapid growth, Acanthamoeba were divided into pathogenic and less-pathogenic isolates. Acanthamoeba were tested for their ability to produce cytopathic effects (CPE) using two human cell lines, HEp-2 and KB cells. Both ameba isolates caused CPE to both cell lines with the same pattern without significant difference. Human erythrocytes from 20 healthy volunteers were used to study the erythrocyte reactivity of Acanthamoeba by co-incubation with trophozoites. The pathogenic Acanthamoeba exhibited significantly higher erythrocyte adhesion as compared to the less-pathogens (p<0.05). Erythrocyte activity occurred in the presence of plasma in all blood samples, suggesting the role of plasmatic components and contact-dependent mechanisms to produce host cell cytotoxicity. The present results showed correlation between the physiological properties and erythrocyte reactivity of Acanthamoeba.

Development of a rapid, simple method for detecting Naegleria fowleri visually in water samples by loop-mediated isothermal amplification (LAMP).

Naegleria fowleri is the causative agent of the fatal disease primary amebic meningoencephalitis. Detection of N. fowleri using conventional culture and biochemical-based assays is time-consuming and laborious, while molecular techniques, such as PCR, require laboratory skills and expensive equipment. We developed and evaluated a novel loop-mediated isothermal amplification (LAMP) assay targeting the virulence-related gene for N. fowleri. Time to results is about 90 min and amplification products were easily detected visually using hydroxy naphthol blue. The LAMP was highly specific after testing against related microorganisms and able to detect one trophozoite, as determined with spiked water and cerebrospinal fluid samples. The assay was then evaluated with a set of 80 water samples collected during the flooding crisis in Thailand in 2011, and 30 natural water samples from border areas of northern, eastern, western, and southern Thailand. N. fowleri was detected in 13 and 10 samples using LAMP and PCR, respectively, with a Kappa coefficient of 0.855. To the best of our knowledge, this is the first report of a LAMP assay for N. fowleri. Due to its simplicity, speed, and high sensitivity, the LAMP method described here might be useful for quickly detecting and diagnosing N. fowleri in water and clinical samples, particularly in resource-poor settings.

In vitro effect of artesunate against Acanthamoeba spp.

The in vitro effects of artesunate, the antimalarial agent, and metronidazole against Acanthamoeba spp were studied. Acanthamoeba Group II and Acanthamoeba polyphaga-like were isolated from natural water courses in Buri Ram Province, northeastern Thailand. The trophozoites were axenically cultured in PPYG medium and treated with artesunate in a concentration of 5-700 microg/ml. Artesunate showed its ability to inhibit the growth of acanthamoeba trophozoites: 54% at 50 mg/ml (after six days of exposure) and 93.2% at 100 microg/ml (after two days). The 500-700 microg/ml concentration caused inhibition on the first day of more than 93.2%; excystation did not occur in drug-treated medium. The present study shows that artesunate is amebastatic rather than amebicidal in an axenic culture of trophozoites at the highest concentration of 100 microg/ml. Metronidazole, in concentrations of 5-1,000 microg/ml, had no effects on either trophozoites or cysts.

Factors affecting the hatching of human pinworm ova.

Parasite life-history traits reflect past environmental and host selective pressure that act to produce strategies that maximize successful transmission. Pooled human pinworm eggs were pretreated with 0.9% NaCl, acid digestive enzyme, and alkaline solutions (pH 9.0) and then incubated in 0.9% NaCl at room temperature and 37 degrees C both with and without 5% CO2. Eggs pretreated with both acid and base had the same hatching pattern, which was markedly different to that of the untreated eggs. At room temperature (RT), hatching of the pretreated eggs occurred on the first day and reached its peak rate (>90%) on day 3; at 37 degrees C hatching occurred on the second day and was more than 80% by day 5. Hatching of the untreated eggs was evident on day 2 at RT and between days 3-5 at 37 degrees C although in smaller numbers (<20%). The CO2 did not affect the hatching of larvae. The larvae could survive after hatching in 0.9% NaCl for 2 and 4 days at 37 degrees C and 25 degrees C, respectively. The present investigation gives a different information that human pinworm ova can hatch into larvae with or without exposure to acid digestive enzyme or alkaline solutions.

Linkage disequilibrium structure of the 5q31-33 region in a Thai population.

A number of loci related to the immune response are located on human chromosomal region 5q31-33, and polymorphisms in this region have been reported to be associated with autoimmune and infectious diseases. In Southeast Asian populations, no systematic survey with dense SNP markers has been performed for the 5q31-33 region. In this study, the LD and haplotype structures for a 472-kb region on 5q31 were investigated in a Thai population to provide useful information for association studies. In addition, the LD structure in Thais was compared with that of the CHB and JPT HapMap populations (CHB + JPT) to evaluate the transferability of tagging SNPs from CHB + JPT for Thais. We show that the minor allele frequency, pattern of LD block, and genetic structure in the 5q31-33 region were highly concordant between Thais and CHB + JPT. A high transferability of tagging SNPs from CHB + JPT for Thais was observed. Our results suggest that tagging SNPs from CHB + JPT (Northeast Asians) can efficiently capture common variants in Southeast Asians, and that the HapMap data are useful for association studies in Southeast Asian populations.