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

<p>Abstract</p> <p>Background</p> <p>Rosetting and cytoadherence of <it>Plasmodium falciparum-</it>infected red blood cells have been associated with severity of malaria. ICAM-1 and CD36 are the main host cell receptors, while PfEMP1-DBLα is a major parasite ligand, which can contribute to rosette formation. This study is aimed at demonstrating whether the highly polymorphic PfEMP1-DBLα 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.</p> <p>Methods</p> <p>Two hundred and ninety five PfEMP1-DBLα 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-DBLα sequences were also analysed by network analysis. The binding abilities of parasitized red blood cells (PRBCs) to CD36, wild type ICAM-1, ICAM-1^Kilifiand ICAM-1^S22/Aunder static condition were included.</p> <p>Results</p> <p>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-DBLα 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 (<it>p </it>= 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 DBLα 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 (<it>p </it>= 0.014, <it>p </it>= 0.007, respectively).</p> <p>Conclusion</p> <p>This is the first report of PfEMP1-DBLα analysis in clinical Thai isolates using semi-conserved features (cys/PoLV and PSPBs). The cys/PoLV group 5 gave the highest rosetting rate. PfEMP1-DBLα domains in Thai isolates are highly diverse, however, clinical isolates from severe and uncomplicated malaria shared common sequences.</p

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

<p>Abstract</p> <p>Background</p> <p>The <it>IL4</it>-590 gene polymorphism has been shown to be associated with elevated levels of anti-<it>Plasmodium falciparum </it>IgG antibodies and parasite intensity in the malaria protected Fulani of West Africa. This study aimed to investigate the possible impact of <it>IL4</it>-590C/T polymorphism on anti-<it>P. falciparum </it>IgG subclasses and IgE antibodies levels and the alteration of malaria severity in complicated and uncomplicated malaria patients with or without previous malaria experiences.</p> <p>Methods</p> <p>Anti-<it>P.falciparum </it>IgG subclasses and IgE antibodies in plasma of complicated and uncomplicated malaria patients with or without previous malaria experiences were analysed using ELISA. <it>IL4</it>-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.</p> <p>Results</p> <p>The <it>IL4</it>-590T allele was significantly associated with anti-<it>P. falciparum </it>IgG3 antibody levels in patients with complicated (<it>P </it>= 0.031), but not with uncomplicated malaria (<it>P </it>= 0.622). Complicated malaria patients with previous malaria experiences carrying <it>IL4</it>-590TT genotype had significantly lower levels of anti-<it>P. falciparum </it>IgG3 (<it>P </it>= 0.0156), while uncomplicated malaria patients with previous malaria experiences carrying the same genotype had significantly higher levels <it>(P </it>= 0.0206) compared to their <it>IL4</it>-590 counterparts. The different anti-<it>P. falciparum </it>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 (<it>P </it>= 0.075). In contrast, complicated malaria patients without previous malaria experiences carrying CC genotype had significantly higher anti-<it>P. falciparum </it>IgG1 than those carrying either CT or TT genotypes (<it>P </it>= 0.004, <it>P </it>= 0.002, respectively).</p> <p>Conclusion</p> <p>The results suggest that <it>IL4</it>-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.</p

Accuracy of Loop-Mediated Isothermal Amplification for Diagnosis of Human Leptospirosis in Thailand

There is a lack of diagnostic tests for leptospirosis in technology-restricted settings. We developed loop-mediated isothermal amplification (LAMP) specific for the 16S ribosomal RNA gene (rrs) of pathogenic and intermediate group Leptospira species. The lower limit of detection was 10 genomic equivalents/reaction, and analytical specificity was high; we observed positive reactions for pathogenic/intermediate groups and negative reactions for non-pathogenic Leptospira species and other bacterial species. We evaluated this assay in Thailand by using a case–control study of 133 patients with laboratory-proven leptospirosis and 133 patients with other febrile illnesses. Using admission blood, we found that the rrs LAMP showed positive results in 58 of 133 cases (diagnostic sensitivity = 43.6, 95% confidence interval [CI] = 35.0–52.5) and in 22 of 133 controls (diagnostic specificity = 83.5, 95% CI = 76.0–89.3). Sensitivity was high for 39 patients who were culture positive for Leptospira spp. (84.6, 95% CI = 69.5–94.1). The rrs LAMP can provide an admission diagnosis in approximately half of patients with leptospirosis, but its clinical utility is reduced by a lower specificity

Antibiotic Resistance Profile and association with Integron Type I among Salmonella Enterica Isolates in Thailand

Salmonella infection is the second most common cause of diarrhea in Thailand; however, the data on antimicrobial resistance is limited. There were137 Salmonella strains, isolated from patients and 126 strains isolated from chicken meat, collected from Nonthaburi, Thailand during 2002. The top five serotypes of patients isolates were Enteritidis (22%), Typhimurium (11%), Weltevreden (8.8%), Rissen (8%), and Choleraesuis (6.6%) while the top five serotypes of chicken meat isolates were found as follows: Schwarzengrund (11.91%), Hadar (11.11%), Rissen (8.73%), Amsterdam (7.94%), and Anatum (7.94%). Salmonella strains were most resistance to the class of antibiotics that act as inhibitor to nucleic acid synthesis such as antifolates group (Trimethoprim;SXT) and fluoroquinolones (Nalidixic acid; NA, Ciprofloxacin; CIP),while the β lactam antibiotic was more effective, i.e. the 3rd gen cephalosporin (Ceftazidime; CAZ, Cefotaxime ; CTX), Monobactam (Aztreonam; ATM) and carbapenams group (Imipenem; IMP, Meropenem; MEM). The role of class I integron element in transmission of the resistance gene was revealed by detection the gene cassette associated with a class 1 integron in plasmid preparation among 80% of the isolated strains. The gene cassettes containing resistant genes of dhfrA12 (resistant to trimethoprim) and aadA2 (resistant to streptomycin and spectinomycin), were detected more frequently in the resistant strains. These gene cassettes were likely to be transmitted via plasmid, as it could not be detected in genomic DNA

ELISA based on a recombinant Paragonimus heterotremus protein for serodiagnosis of human paragonimiasis in Thailand

Abstract Background Paragonimus heterotremus is the main causative agent of paragonimiasis in Thailand. In Western blot diagnostic assays for paragonimiasis, the 35 kDa band present in crude P. heterotremus somatic extracts represents one of the known diagnostic bands. This study aimed to use a P. heterotremus cDNA library to create a recombinant version of this antigen for use in immunodiagnosis of paragonimiasis. Methods To accomplish this aim a cDNA expression library was constructed from adult worm mRNA and immuno-screened using antibodies from mice that had been immunized with the 35 kDa antigen. Screening resulted in the identification of an immunoreactive protein encoded by clone CE3, which contained an inserted sequence composed of 1292 base pairs. This clone was selected for use in the construction of a recombinant P. heterotremus protein because of its similarity to proactivator polypeptide. For recombinant protein expression, the CE3 gene sequence was inserted into the plasmid vector pRset and the resulting product had the expected molecular weight of 35 kDa. An IgG-ELISA based on the CE3 recombinant protein was evaluated by using sera from healthy individuals, from patients with paragonimiasis and other parasitic infections. This ELISA was performed by using human sera diluted at 1:2000, an optimized antigen concentration of 1 μg/ml, and anti-human IgG diluted at 1:4000. Results The cut-off optical density value was set as the mean + 2 standard deviations (0.54), which resulted in the test having a sensitivity of 88.89% and a specificity of 95.51%. The recombinant antigen could react with antibodies from P. heterotremus, P. pseudoheterotremus and P. westermani infections. Cross-reactivity occurred with a few cases of Blastocystis hominis infection (2/3), Bancroftian filariasis (1/10), opisthorchiasis (3/10), strongyloidiasis (4/10) and neurocysticercosis (1/11). Conclusions Given the high test sensitivity and specificity, reflected in the low level of heterologous infection cross-reactivity (11/215 serum samples), observed in the IgG-ELISA, this 35 kDa antigen may be useful for the detection of paragonimiasis