Clonal diversity of the glutamate dehydrogenase gene in Giardia duodenalis from Thai isolates: evidence of genetic exchange or mixed infections?

BACKGROUND: The glutamate dehydrogenase gene (gdh) is one of the most popular and useful genetic markers for the genotypic analysis of Giardia duodenalis (syn. G. lamblia, G. intestinalis), the protozoan that widely causes enteric disease in humans. To determine the distribution of genotypes of G. duodenalis in Thai populations and to investigate the extent of sequence variation at this locus, 42 fecal samples were collected from 3 regions of Thailand i.e., Central, Northern, and Eastern regions. All specimens were analyzed using PCR-based genotyping and recombinant subcloning methods. RESULTS: The results showed that the prevalence of assemblages A and B among these populations was approximately equal, 20 (47.6%) and 22 (52.4%), respectively. Sequence analysis revealed that the nucleotide diversity of assemblage B was significantly greater than that in assemblage A. Among all assemblage B positive specimens, the allelic sequence divergence within isolates was detected. Nine isolates showed mixed alleles, ranged from three to nine distinct alleles per isolate. Statistical analysis demonstrated the occurrence of genetic recombination within subassemblages BIII and BIV was likely. CONCLUSION: This study supports increasing evidence that G. duodenalis has the potential for genetic exchange.

Determination of discriminatory power of genetic markers used for genotyping Giardia duodenalis.

Small subunit ribosomal DNA (SSU-rDNA), glutamate dehydrogenase (gdh), beta-giardin, triosephosphate isomerase (tpi), and elongation factor 1-alpha (ef1-alpha) genes are useful genetic markers for genotypic analysis of the intestinal protozoan, Giardia duodenalis (syn. G. lamblia, G. intestinalis), the cause of enteric disease in humans. To quantitatively compare the discriminatory power of these loci, 43 fecal samples were collected from central, northern and eastern Thailand and G. duodenalis specimens were analyzed using PCR-based genotyping and subcloning methods. Approximately equal prevalence of assemblage A (21) and B (22) were present among these populations. Analysis of Simpson's index and Wallace coefficient values from assemblage B isolates together with the data obtained from GenBank showed that the combination of two loci provides a higher discrimination power for subgenotyping G. duodenalis than using any single locus.

Improved sensitivity of PCR amplification of glutamate dehydrogenase gene for detection and genotyping of Giardia duodenalis in stool specimen.

A modified set of primers was developed to increase the sensitivity of nested PCR amplification of glutamate dehydrogenase (gdh) gene to detect and genotype Giardia duodenalis cysts in stool specimens. This modified set of primers had a significantly higher sensitivity (82%) than that of a previously published PCR primer set (53%).

In vitro growth characteristics and morphological differentiation of Leishmania martiniquensis promastigotes in different culture media.

The protozoan hemoflagellate, Leishmania martiniquensis, is the causative agent of cutaneous and visceral leishmaniasis among humans. This parasite was first isolated from an autochthonous case of cutaneous leishmaniasis in Martinique Island (French West Indies) in 1995 and its taxonomical position was later established in 2002. At present, the emergence of this globally infectious disease caused by L.martiniquensis raises serious concerns and has gained attention from the national public health policy. Epidemiological studies indicated that Thailand is one of the endemic areas of L.martiniquensis with hundreds of cases, both symptomatic and asymptomatic, have been reported among patients positive for HIV/AIDS. Information on its basic biology including suitable conditions for parasite propagation is limited. To assess this, we used four established media, that is, Medium 199 (M199), RPMI 1640 medium (RPMI), Grace's insect medium (GIM), and Schneider's insect medium (SIM) to investigate the promastigote growth by evaluating the growth characteristics, viability, and kinetics of stage differentiation in each medium. The findings from this study showed that parasites growing in different media exhibited different biological characteristics, which would be suitable for very specific research purposes, i.e., RPMI; for long term parasite maintenance, M199; for mass culture of parasites, M199 and GIM; for initial isolation of the parasites in clinical specimens, and SIM; for metacyclogenesis study.

Prevalence of giardiasis and genotypic characterization of Giardia duodenalis in hilltribe children, Northern Thailand.

A cross-sectional study was performed to determine the prevalence of giardiasis in hilltribe children of 2 different remote districts (Mae-chaem and Hod), Chiang Mai, Northern Thailand from November 2006-April 2007. The overall prevalence of giardiasis was 5.2%. Genetic characterization of Giardia duodenalis isolated from these children was performed using PCR methods specific for small subunit ribosomal rRNA (SSU-rRNA) and glutamate dehydrogenase (gdh) gene. This study shows that the distribution of Giardia assemblages varied in these 2 populations. Assemblage BIV was found predominantly in children from Hod District while assemblage AII was more common in children from Mae-Chaem District. Our result showed that assemblage A was significantly associated with loose/watery stool (p = 0.001). In addition, children harbouring assemblage B had shed a significantly higher number of cysts (p = 0.019) in stools than those infected with assemblage A. Further study on the epidemiology of giardiasis especially risk factors associated with genotyping would help to understand the nature of this disease in each population.