Antimicrobial Peptide Modifications against Clinically Isolated Antibiotic-Resistant Salmonella.

Antimicrobial peptides are promising molecules to address the global antibiotic resistance problem, however, optimization to achieve favorable potency and safety is required. Here, a peptide-template modification approach was employed to design physicochemical variants based on net charge, hydrophobicity, enantiomer, and terminal group. All variants of the scorpion venom peptide BmKn-2 with amphipathic α-helical cationic structure exhibited an increased antibacterial potency when evaluated against multidrug-resistant Salmonella isolates at a MIC range of 4-8 µM. They revealed antibiofilm activity in a dose-dependent manner. Sheep red blood cells were used to evaluate hemolytic and cell selectivity properties. Peptide Kn2-5R-NH2, dKn2-5R-NH2, and 2F-Kn2-5R-NH2 (variants with +6 charges carrying amidated C-terminus) showed stronger antibacterial activity than Kn2-5R (a variant with +5 charges bearing free-carboxyl group at C-terminus). Peptide dKn2-5R-NH2 (d-enantiomer) exhibited slightly weaker antibacterial activity with much less hemolytic activity (higher hemolytic concentration 50) than Kn2-5R-NH2 (l-enantiomer). Furthermore, peptide Kn2-5R with the least hydrophobicity had the lowest hemolytic activity and showed the highest specificity to Salmonella (the highest selectivity index). This study also explained the relationship of peptide physicochemical properties and bioactivities that would fulfill and accelerate progress in peptide antibiotic research and development.

Genetic association study of interferon lambda 3, CD27, and human leukocyte antigen-DPB1 with dengue severity in Thailand.

BACKGROUND: Dengue patients develop different disease severity ranging from mild (dengue fever [DF]) to severe forms (dengue hemorrhagic fever [DHF] and the fatal dengue shock syndrome [DSS]). Host genetics are considered to be one factor responsible for the severity of dengue outcomes. To identify genes associated with dengue severity that have not been studied yet, we performed genetic association analyses of interferon lambda 3 (IFNL3), CD27, and human leukocyte antigen-DPB1 (HLA-DPB1) genes in Thai dengue patients. METHODS: A case-control association study was performed in 877 children (age ≤ 15 years) with dengue infection (DF, n = 386; DHF, n = 416; DSS, n = 75). A candidate single nucleotide polymorphism of each of IFNL3, CD27, and HLA-DPB1 was selected to be analyzed. Genotyping was performed by TaqMan real-time PCR assay, and the association with dengue severity was examined. RESULTS: The rs9277534 variant of HLA-DPB1 was weakly associated with DHF. The genotype GG and G allele conferred protection against DHF (p = 0.04, odds ratio 0.74 for GG genotype, p = 0.03, odds ratio 0.79 for G allele). The association became borderline significant after adjusting for confounders (p = 0.05, odds ratio 0.82). No association was detected for IFNL3 or CD27. CONCLUSIONS: The present study demonstrated the weak association of the rs9277534 variant of HLA-DPB1 with protection against DHF. This variant is in the 3' untranslated region and affects HLA-DPB1 surface protein expression. Our finding suggests that HLA-DPB1 may be involved in DHF pathogenesis.

MULTIPLE-LOCUS VARIABLE-NUMBER TANDEM REPEAT ANALYSIS OF BRUCELLA ISOLATES FROM THAILAND.

Brucellosis-induced abortion can result in significant economic loss to farm animals. Brucellosis can be transmitted to humans during slaughter of infected animals or via consumption of contaminated food products. Strain identification of Brucella isolates can reveal the route of transmission. Brucella strains were isolated from vaginal swabs of farm animal, cow milk and from human blood cultures. Multiplex PCR was used to identify Brucella species, and owing to high DNA homology among Brucella isolates, multiple-locus variable-number tandem repeat analysis (MLVA) based on the number of tandem repeats at 16 different genomic loci was used for strain identification. Multiplex PCR categorized the isolates into B. abortus (n = 7), B. melitensis (n = 37), B. suis (n = 3), and 5 of unknown Brucella spp. MLVA-16 clustering analysis differentiated the strains into various genotypes, with Brucella isolates from the same geographic region being closely related, and revealed that the Thai isolates were phylogenetically distinct from those in other countries, including within the Southeast Asian region. Thus, MLVA-16 typing has utility in epidemiological studies.

Cloning and expression of a 16-kDa recombinant protein from Angiostrongylus cantonensis for use in immunoblot diagnosis of human angiostrongyliasis.

Angistrongylus cantonensis is a zoonotic nematode parasite and causative agent of human angiostrongyliasis, which clinically presents as eosinophilic meningitis or meningoencephalitis. Diagnosis of the disease is problematic since parasitologic findings are infrequent, and infection determinations must be based on the clinical symptoms and serological tests with limited specificities and sensitivities. The aim of the present study was to identify and generate a novel recombinant protein from A. cantonensis and evaluate its efficacy in the diagnosis of human angiostrongyliasis when incorporated into a Western blot serodiagnostic system. A cDNA protein expression library from adult A. cantonensis was constructed, followed by immunoscreening with serum from confirmed infected patients to identify and isolate immunoreactive clones. One clone, designated fAC40, possessed a partial sequence encoding a LisH protein domain with a predicted molecular weight of 16 kDa and containing four predicted antigenic peptides. By incorporating recombinant fAC40 in Western immunoblot tests using a serum panel consisting of confirmed and clinically diagnosed cases of human angiostrongyliasis and other helminthic infections, fAC40 exhibited a sensitivity and specificity of 91.8 and 100 %, respectively, and a positive and negative predictive value of 100 and 97.19 %, respectively, in the diagnosis of angiostrongyliasis. Importantly, it was not reactive with antibodies from serum of patients infected with Gnathostoma spinigerum and Cysticercus cellulosae, infections that clinically present neurological symptoms similar to angiostrongyliasis. These data demonstrate that the 16-kDa recombinant protein from A. cantonensis possesses high potential as a candidate antigen for a more sensitive and specific serodiagnosis of human angiostrongyliasis.

Molecular genotyping of human papillomavirus l1 gene in low-risk and high-risk populations in Bangkok.

BACKGROUND: Human papillomavirus (HPV) infections in Thailand are a public health concern, but information on HPV infection in sex workers and men who have sex with men (MSM) is limited. The aim of this study was to measure the prevalence and genotype distribution of HPV among low- and high-risk, HIV-negative populations. METHODS: A total of 300 participants were categorized as general women, female sex workers, MSM, and MSM sex workers. Human papillomavirus infections were identified by the Papanicolaou test and nested polymerase chain reaction. A phylogenetic analysis of partial HPV L1 genes was performed. RESULTS: Abnormal cytology was found in 5% of general women, 10% of female sex workers, 24% of MSM, and 28% of MSM sex workers. Human papillomavirus was detected in 9% of general women, 13% of female sex workers, and 30% in both MSM and the MSM sex workers. The prevalence of HPV high-risk genotypes was significantly higher in female sex workers and MSM, whereas low-risk genotypes and genital warts were significantly higher in MSM sex workers. Significantly more patients with genital warts and cervical intraepithelial neoplasia I/anal intraepithelial neoplasia I harbored low-risk genotypes, whereas those with cervical intraepithelial neoplasia II/anal intraepithelial neoplasia II harbored high-risk genotypes. CONCLUSIONS: High- and low-risk HPV genotypes persist in high-risk groups in Bangkok. Some genotypes infecting at-risk populations are not vaccine preventable. These findings may help to elucidate the prevalence of HPV infections in Thailand and serve as the basis for additional investigations into risk factors for these populations.

ELISA for brucellosis detection based on three Brucella recombinant proteins.

Control of brucellosis among farm animals, wildlife and humans require reliable diagnosis. Rose Bengal serological test (RBT) is based on lipopolysaccharide antigen of Brucella, which may cross react with other gram-negative bacteria and produce false positive result. Immunoreactive proteins, such as outer-membrane protein BP26, ribosome recycling factor protein CP24 and Brucella lumazine synthase (BLS), previously reported to be recognized by infected sheep sera, were selected for production of recombinant proteins for use in an ELISA in order to investigate immune response among goats and cows, in comparison with commercial RBT. Cut-off value for ELISA was based on the immune response of in vitro fertilized goats and cows. Goats positive for Brucella culture or by RBT were ELISA positive for either IgG or IgM against at least one recombinant protein. For animals with negative RBT, animals with positive ELISA could be detected, and 61.6% possessed ELISA values as high as in infected animals. Thus, this ELISA procedure is proposed as an alternative to RBT for screening of brucellosis in farm animals.

Cloning and application of recombinant dengue virus prM-M protein for serodiagnosis of dengue virus infection.

We studied the use of the precursor to the M structural protein (prM) found only on the surface of mature dengue virus as a target protein to detect dengue virus infection. Recombinant D2-16681 prM-M protein was constructed and tested for immunogenicity with dengue and Japanese encephalitis patient sera by Western blot analysis and indirect ELISA. The sensitivity and specificity of indirect ELISA were 48.1 and 85.5%, respectively, and Western blot assay were 23.1 and 98.7%, respectively, for detection of dengue virus. Although the sensitivity of the indirect ELISA is low, the indirect ELISA using recombinant D2-16681 prM-M proteins as antigen may be used for early detection of dengue virus infection.

A Use of 56-kDa Recombinant Protein of Orientia tsutsugamushi Karp Serotype in Serodiagnosis of Scrub Typhus by Enzyme-Linked Immunosorbent Assay in Thais.

Scrub typhus is a mite-borne disease caused by a Gram-negative obligately intracellular bacillus, Orientia tsutsugamushi. The disease is endemic in the Asia-Australia-Pacific region, including Thailand. Scrub typhus generally manifests as acute undifferentiated febrile fever along with myalgia, rash, and lymphadenopathy. An eschar can be a valuable diagnostic clue, but this skin lesion may be missed in some patients. The disease symptoms resemble those of other febrile illnesses such as leptospirosis, typhoid, murine typhus, malaria, and dengue fever, making a laboratory diagnosis necessary for the definitive diagnosis. In this study, we expressed a recombinant protein derived from 56-kDa type-specific antigen of O. tsutsugamushi Karp serotype and tested its ability to detect and differentiate scrub typhus infection. IgM and IgG antibodies were determined in sera from scrub typhus (n = 92) and other febrile illness patients (murine typhus (n = 25), melioidosis (n = 36), leptospirosis (n = 42), and dengue (n = 35)) from Thailand. Sensitivities of 87.0% and 59.8% with a specified assay cut-off were obtained for IgM and IgG indirect ELISAs, respectively, with a specificity of 100% in both tests. The sensitivity was increased to 95.7% when a combination of IgM and IgG ELISAs results was considered. Our study suggested a potential of the 56-kDa recombinant protein for further development and evaluation for use in scrub typhus serodiagnosis.

Roles of partially purified antigens from Gnathostoma spinigerum larvae on antibody production by human B cell culture.

A 24 kDa protein from advanced third stage Gnathostoma spinigerum larvae (GsAL3) is used for gnathostomiasis serodiagnosis. This study investigated whether partially purified protein antigen (Ag) from GsAL3 (Gnath Ag), prepared by simple gel filtration chromatography, could be used for serodiagnosis. Using DNA microarray analysis, significant gene expression related to immunoreactivity was examined in peripheral blood mononuclear cells (PBMC) cocultured with Gnath Ag. Antigenicity was then determined by its capacity to induce antibody production among purified naive B cells stimulated with Gnath Ag and anti-CD40. Seven and 14 days post-exposure, immunoglobulin levels (Igs) in culture supernatants were determined by enzyme-linked immunosorbent assay. The Gnath Ag stimulated PBMC had a significant increase in gene expression related to an innate immune response and decreased cell mediated immunity, but the expression of gene related antibody production was not markedly increased. The Gnath Ag stimulated naive B cells or lipopolysaccharide primed B cells to produce low levels of specific antibody. Our findings support the assertion that partially purified Gnath Ag possess low antigenicity for Ig induction. Further studies are needed to improve G. spinigerum larva Ag for serodiagnosis.

Evaluation of a genus-specific rGroEL1-524 IgM-ELISA and commercial ELISA kits during the course of leptospirosis in Thailand.

In the present study, we developed a genus-specific rGroEL1-524 IgM-ELISA assay for use in screening diagnosis of suspected leptospirosis among acute undifferentiated febrile illness patients during acute fever. The diagnostic accuracies of the rGroEL1-524 IgM-ELISA, commercial Panbio IgM-ELISA, and Virion-Serion Classic IgG-ELISA were evaluated using 133 Thai leptospirosis sera and 210 controls. Sensitivities were 91.7%, 59.6%, and 17.7% for acute infection, and the specificities were 92.6%, 90.2%, and 88.3% for the non-leptospirosis control, respectively. The rGroEL1-524 IgM-ELISA had high sensitivity, at 92.3% and 91.7%, among culture-positive and MAT-negative cases at 1-3 days post-onset of symptoms (DPO1-3), respectively. Impaired specificity on scrub typhus was found, possibly from antibody cross-reaction to ortholog GroEL. Commercial Panbio IgM-ELISA had sensitivities at DPO1-3 of 30.8% and 41.7% for culture-positive and MAT-negative cases whereas Virion-Serion IgG-ELISA showed sensitivities of 5.9% and 13.3%, respectively. The rGroEL1-524 IgM-ELISA could be useful as a screening test for early diagnosis. The performance of the commercial ELISA suggests the applicability of IgM-ELISA for diagnosis, while IgG-ELISA is useful for seroprevalence surveys. However, confirmation by reference tests is recommended.

Effects of CYP2B6 G516T polymorphisms on plasma efavirenz and nevirapine levels when co-administered with rifampicin in HIV/TB co-infected Thai adults.

BACKGROUND: Cytochrome P450 2B6 (CYP2B6) metabolizes efavirenz and nevirapine, the major core antiretroviral drugs for HIV in Thailand. Rifampicin, a critical component of tuberculosis (TB) therapy is a potent inducer of CYP enzyme activity. Polymorphisms of CYP2B6 and CYP3A4 are associated with altered activity of hepatic enzyme in the liver and pharmacokinetics resulting in treatment efficacy. This study aimed to investigate whether CYP2B6 or CYP3A4 polymorphisms had effects on plasma efavirenz and nevirapine concentrations when co-administered with rifampicin in HIV/TB co-infected Thai adults. RESULTS: We studied 124 rifampicin recipients with concurrent HIV-1/TB coinfection, receiving efavirenz (600 mg/day) (n = 65) or nevirapine (400 mg/day) (n = 59) based antiretroviral therapy (ART). The frequencies of GG, GT and TT genotypes of CYP2B6-G516T were 38.46%, 47.69% and 13.85% in efavirenz group and 44.07%, 52.54% and 3.39% in nevirapine group, respectively. The mean 12-hour post-dose plasma efavirenz concentration in patients with TT genotype at weeks 6 and 12 of ART and 1 month after rifampicin discontinuation (10.97 +/- 2.32, 13.62 +/- 4.21 and 8.48 +/- 1.30 mg/L, respectively) were significantly higher than those with GT (3.43 +/- 0.29, 3.35 +/- 0.27 and 3.21 +/- 0.22 mg/L, respectively) (p < 0.0001) or GG genotypes (2.88 +/- 0.33, 2.45 +/- 0.26 and 2.08 +/- 0.16 mg/L, respectively) (p < 0.0001). Likewise, the mean 12-hour post-dose plasma nevirapine concentration in patients carrying TT genotype at weeks 6 and 12 of ART and 1 month after rifampicin discontinuation (14.09 +/- 9.49, 7.94 +/- 2.76 and 9.44 +/- 0.17 mg/L, respectively) tended to be higher than those carrying GT (5.65 +/- 0.54, 5.58 +/- 0.48 and 7.03 +/- 0.64 mg/L, respectively) or GG genotypes (5.42 +/- 0.48, 5.34 +/- 0.50 and 6.43 +/- 0.64 mg/L, respectively) (p = 0.003, p = 0.409 and p = 0.448, respectively). Compared with the effects of CYP2B6-516TT genotype, we could observe only small effects of rifampicin on plasma efavirenz and nevirapine levels. After 12 weeks of both drug regimens, there was a trend towards higher percentage of patients with CYP2B6-TT genotype who achieved HIV-1 RNA levels <50 copies/mL compared to those with GT or GG genotypes. This is the first report to demonstrate the effects of CYP2B6 G516T polymorphisms on plasma efavirenz and nevirapine concentrations when co-administered with rifampicin in HIV/TB co-infected Thai adults. CONCLUSIONS: CYP2B6-TT genotype had impact on plasma efavirenz and nevirapine concentrations, while rifampicin co-administration had only small effects.

Iso-electricfocusing of Bithynia snail antigens for IgG- and IgG(1-4)-ELISA detection of human opisthorchiasis.

Diagnosis of opisthorchiasis is confirmed by the presence of characteristic eggs and worms. However, misdiagnosis may occur in light infections, and also due to the morphological similarity of opisthorchid eggs to other species. A finding of specific immune mediators can help confirm infection. This study used indirect ELISA to detect total IgG and IgG(1-4) with selected antigens of Bithynia siamensis goniomphalos extract, which were derived by liquid-phase iso-electricfocusing (IFE). Antigens (Iso-AgF) from 20 IEF fractionated fractions were selected based on a high ELISA-OD ratio between pooled-positive and pooled-negative sera. Iso-AgF 7, 7, 6, 2, and 10 resulted in high OD-ratio to total IgG, IgG1, 2, 3, and 4, respectively. A full-scale ELISA was conducted with sera from 50 opisthorchiasis cases, 196 from other parasitic-disease cases, and 35 healthy controls. Iso-AgF7 to IgG1 showed the best result, with sensitivity, specificity, positive and negative predictive value of 100, 96, 86, and 100%, respectively, at a cut-off 0.221. Low cross-reactivity to IgG1 was found in one case each of gnathostomiasis, trichinellosis, toxocariasis, angiostrongyliasis, bancroftian filariasis, enterobiasis, neurocysticercosis, and taeniasis. Thus, Iso-AgF7 to IgG1 was a good candidate antigen to be developed for detection of antibodies against Opisthorchis viverrini.

Application of recombinant SMR-domain containing protein of angiostrongylus cantonensis in immunoblot diagnosis of human angiostrongyliasis.

The aim of this study was to find novel proteins expressed from an Angiostrongylus cantonensis adult female worm cDNA library for serodiagnosis of angiostrongyliasis. An immuno-dominant clone, fAC22, was identified by immunoscreening with pooled positive sera from proven angiostrongyliasis patients. The clone contained an open reading frame of 2,136 bp encoding a 80.5 kDa protein with a predicted isoelectric point of 5.8. The deduced amino acid sequence (712 amino acids) contained the conserved domain of Small mutS related (Smr) superfamily protein, with similarity with the Smr domain protein of Brugia malayi. The fusion His-tagged 81 kDa recombinant protein expressed as inclusion body in Escherichia coli was solubilized and purified by Ni-affinity chromatography for use in immunoblot analysis. Its sensitivity, specificity, positive and negative predictive values in immunodiagnostic test was 93.5, 91.5, 79.0 and 97.5%, respectively. Although some cross-reactivity of the antigen was observed among gnathostomiasis, bancroftian filariasis, ascariasis, echinococcosis, paragonimiasis and opisthorchiasis, sera from 14 other infections were all negative. These data indicate its possible application in immunodiagnosis of clinically suspected angiostrongyliasis. Key words: Angiostrongylus cantonensis,eosinophilic meningitis, recombinant fusion protein, immunodiagnosis

Copro-DNA diagnosis of Opisthorchis viverrini and Haplorchis taichui infection in an endemic area of Lao PDR.

The utility of differential copro-DNA diagnosis using modified sample preparation steps of small liver and minute intestinal fluke infections was tested. Fecal samples containing parasite eggs were washed extensively with diluted detergent solution. Parasite eggs were concentrated by sedimentation and broken by microwaving before DNA extraction. PCR targeting ITS1 and ITS2 regions were performed using primer specific for Opisthorchis viverrini, Haplorchis taichui and other related species. Of 125 fecal samples, 94 were positive for small trematode eggs by a modified cellophane thick smear method. By ITS1-PCR, 52 samples were positive for O. viverrini, 12 H. taichui and 7 mixed infection. By ITS2-PCR, 63 were positive for O. viverini, 17 H. taichui, and 19 mixed infection. The ITS-PCR assay identified a higher number of opisthorchiasis cases than those with O. viverrini expelled after treatment, but for H. taichui, ITS-PCR identified less than half of the worm expelled cases. These results showed that copro-DNA diagnosis was useful for the differential diagnosis of O. viverrini and H. taichui infection, which could not be discriminated by microscopy.

Interferon lambda 1 is associated with dengue severity in Thailand.

OBJECTIVES: Patients with dengue exhibit a range of symptoms from an acute febrile illness (dengue fever, DF), to dengue hemorrhagic fever (DHF), and to the most severe outcome, dengue shock syndrome (DSS). This study was performed to determine the host genetic factors responsible for dengue severity. Two single nucleotide polymorphisms (SNPs) of the interferon lambda 1 (IFNL1) gene (rs30461 and rs7247086) were analyzed for their association with dengue severity in a Thai population. METHODS: This was a case-control association study involving 877 patients under the age of 15 years (DF, n = 386; DHF, n = 416; DSS, n = 75). Genotyping was performed by TaqMan real-time PCR assay. RESULTS: It was found that the rs7247086 variant of IFNL1 was associated with DHF, but not DSS. Genotypes CT and TT and the T allele were protective against DHF (p = 0.03, odds ratio 0.62 for CT, odds ratio 0.13 for TT; and p = 0.01, odds ratio 0.54 for the T allele). The other SNP tested was not associated with DHF or DSS. CONCLUSIONS: The rs7247086 variant of IFNL1 (the T allele) was found to be protective against DHF, suggesting that IFNL1 may play a role in the pathogenesis of DHF.

Immunodominance of LipL3293-272 peptides revealed by leptospirosis sera and therapeutic monoclonal antibodies.

BACKGROUND/PURPOSE: Leptospirosis is a neglected zoonosis, imposing significant human and veterinary public health burdens. In this study, recombinant LipL3293-147 and LipL32148-184 middle domain of LipL3293-184, and LipL32171-214, and LipL32215-272 of c-terminal LipL32171-272 truncations were defined for immunodominance of the molecule during Leptospira infections revealed by leptospirosis sera. RESULTS: IgM-dominant was directed to highly surface accessible LipL32148-184 and Lipl32171-214. IgG dominance of LipL32148-184 revealed by rabbit anti-Leptospira sera and convalescent leptospirosis paired sera were mapped to highly accessible surface of middle LipL32148-184 truncation whereas two LipL32148-184 and LipL32215-272 truncations were IgG-dominant when revealed by single leptospirosis sera. The IgM-dominant of LipL32148-214 and IgG-dominant LipL32148-184 peptides have highly conserved amino acids of 70% identity among pathogenic and intermediate Leptospira species and were mapped to the highly surface accessible area of LipL32 molecule that mediated interaction of host components. IgG dominance of two therapeutic epitopes located at LipL32243-253 and LipL32122-130 of mAbLPF1 and mAbLPF2, respectively has been shown less IgG-dominant (<30%), located outside IgG-dominant regions characterized by leptospirosis paired sera. CONCLUSION: The IgM- and IgG-dominant LipL32 could be further perspectives for immunodominant LipL32-based serodiagnosis and LipL32 epitope-based vaccine.

Sequence variation of PfEMP1-DBLalpha in association with rosette formation in Plasmodium falciparum isolates causing severe and uncomplicated malaria.

BACKGROUND: Rosetting and cytoadherence of Plasmodium falciparum-infected red blood cells have been associated with severity of malaria. ICAM-1 and CD36 are the main host cell receptors, while PfEMP1-DBLalpha is a major parasite ligand, which can contribute to rosette formation. This study is aimed at demonstrating whether the highly polymorphic PfEMP1-DBLalpha sequences occurring among Thai isolates causing severe and uncomplicated malaria are associated with their ability to form rosettes and reflected the clinical outcome of the patients. METHODS: Two hundred and ninety five PfEMP1-DBLalpha sequences from Thai clinical isolates causing severe and uncomplicated malaria were evaluated by sequencing and direct comparison using the specific text string analysis functions in Microsoft Excel and Perl. The relationships between the PfEMP1-DBLalpha sequences were also analysed by network analysis. The binding abilities of parasitized red blood cells (PRBCs) to CD36, wild type ICAM-1, ICAM-1Kilifi and ICAM-1S22/A under static condition were included. RESULTS: Two hundred and eighty one non-identical amino acid sequences were identified (< 95% sequence identity). When the distributions of semi-conserved features (PoLV1-4 and sequence group) within the rosetting domain PfEMP1-DBLalpha were observed, close similarity was found between isolates from the two disease groups. The sequence group 1 representing uncomplicated malaria was significantly different from the sequence group 3 representing the majority of severe malaria (p = 0.027). By using a simple non-phylogenetic approach to visualize the sharing of polymorphic blocks (position specific polymorphic block, PSPB) and cys/PoLV among DBLalpha sequences, the sequence group 1 was split from the other five sequence groups. The isolates belonging to sequence group 5 gave the highest mean rosetting rate (21.31%). However, within sequence group 2 and group 6, the isolates causing severe malaria had significantly higher rosetting rate than those causing uncomplicated malaria (p = 0.014, p = 0.007, respectively). CONCLUSION: This is the first report of PfEMP1-DBLalpha analysis in clinical Thai isolates using semi-conserved features (cys/PoLV and PSPBs). The cys/PoLV group 5 gave the highest rosetting rate. PfEMP1-DBLalpha domains in Thai isolates are highly diverse, however, clinical isolates from severe and uncomplicated malaria shared common sequences.

IL4 gene polymorphism and previous malaria experiences manipulate anti-Plasmodium falciparum antibody isotype profiles in complicated and uncomplicated malaria.

BACKGROUND: The IL4-590 gene polymorphism has been shown to be associated with elevated levels of anti-Plasmodium falciparum IgG antibodies and parasite intensity in the malaria protected Fulani of West Africa. This study aimed to investigate the possible impact of IL4-590C/T polymorphism on anti-P. falciparum IgG subclasses and IgE antibodies levels and the alteration of malaria severity in complicated and uncomplicated malaria patients with or without previous malaria experiences. METHODS: Anti-P.falciparum IgG subclasses and IgE antibodies in plasma of complicated and uncomplicated malaria patients with or without previous malaria experiences were analysed using ELISA. IL4-590 polymorphisms were genotyped using RFLP-PCR. Statistical analyses of the IgG subclass levels were done by Oneway ANOVA. Genotype differences were tested by Chi-squared test. RESULTS: The IL4-590T allele was significantly associated with anti-P. falciparum IgG3 antibody levels in patients with complicated (P = 0.031), but not with uncomplicated malaria (P = 0.622). Complicated malaria patients with previous malaria experiences carrying IL4-590TT genotype had significantly lower levels of anti-P. falciparum IgG3 (P = 0.0156), while uncomplicated malaria patients with previous malaria experiences carrying the same genotype had significantly higher levels (P = 0.0206) compared to their IL4-590 counterparts. The different anti-P. falciparum IgG1 and IgG3 levels among IL4 genotypes were observed. Complicated malaria patients with previous malaria experiences tended to have lower IgG3 levels in individuals carrying TT when compared to CT genotypes (P = 0.075). In contrast, complicated malaria patients without previous malaria experiences carrying CC genotype had significantly higher anti-P. falciparum IgG1 than those carrying either CT or TT genotypes (P = 0.004, P = 0.002, respectively). CONCLUSION: The results suggest that IL4-590C or T alleles participated differently in the regulation of anti-malarial antibody isotype profiles in primary and secondary malaria infection and, therefore, could play an important role in alteration of malaria severity.

Evaluation of an electronic air filter for filtrating bacteria and viruses from indoor air.

This study compared the filtrating efficiency (FE) of a commercial electronic air filter for filtering bacteria and viruses from contaminated air with a high efficiency particulate air (HEPA) filter. An enclosed chamber was constructed, in the middle of which an air filter was placed for testing. MTB H37Ra and T7 virus at concentrations of 5x10(8) each were sprayed into one side of the chamber using a nebulizer and the sprayed samples were collected by an impinger air-sampler on the other side. MTB and T7 viruses were detected by PCR and culture. The PCR could detect samples down to 10 fg for MTB H37Ra and 1 pg for T7 virus. Most MTB H37Ra sprayed failed to culture. S. aureus at a concentration of 10(5) cfu and E. coli at a concentration of 10(4) cfu along with T7 virus were filtered out with a FE of more than 99%. T7 virus has a particle size of 0.04 microm, S. aureus has a particle size of 1 microm and E. coli has a particle size of 2 microm.

Identification of Entamoeba histolytica and Entamoeba dispar by PCR assay of fecal specimens obtained from Thai/Myanmar border region.

Due to the indistinguishable morphology between Entamoeba histolytica (pathogenic) and Entamoeba dispar (non pathogenic), PCR-based assays were conducted. Based on microscopy, suspected Entamoeba cells were detected in 30 out of 455 fecal samples obtained from individuals residing at Thai/Myanmar border region. The target genes for PCR amplification included genes encoding small subunit rRNA (SSU-rRNA), chitinase and serine rich Entamoeba protein. PCR primers derived from SSU-rRNA gene amplified both E. histolytica and E. dispar genes producing an amplicon of 1,080 bp, and detected 3 out of 30 samples. PCR primers derived from chitinase gene of E. histolytica generating amplicons of 500 and 1,260 bp, samples were positive in 12 out of 30 samples. Due the large difference of gene encoding serine rich protein between E. histolytica and E. dispar, two specific sets of primers were designed. SREH-primer set, specific for E. histolytica, generated amplicons of 550 and 700 bp and detected 22 out of 30 samples. SED-primer set, specific to E. dispar, produced an amplicon of 550 bp, and together with a nested primer pair generating an amplicon of 477 bp, detected 16 out of 30 samples. Thus, detection of single and mixed infections of the two Entamoeba species could be effectively achieved directly from DNA extracted from feces without the need to culture the parasites.