In Vitro and In Silico Studies of Kinase Inhibitor of MAPK3 Protein to Determine Leishmania martiniquensis Treatment.

PURPOSE: Leishmaniasis is a parasitic disease transmitted by the bite of the phlebotomine female sand fly. Currently, no reported effective vaccines are available for the treatment of leishmaniasis; consequently, restricting this disease completely depends on controlling its transmission. Mitogen-activated protein (MAP) kinases have been reported to be involved in the regulation of the flagellum length and hence play an important role in disease transmission, especially the MAPK3 protein. Therefore, the current work focused on identifying approved drugs that can inhibit the MAPK3 protein. METHODS: First, the recombinant plasmid (pET28b( +) MAPK3) was cloned into E. coli strain BL21 using the heatshock method. Afterward, E. coli was induced using IPTG, and cells were harvested for protein purification in the next step. After that, the MAPK3 protein was purified using Ni-NTA column. Then, the inhibition kinase activity of the purified MAPK3 protein was performed using an ADP-Glo™ Kinase Assay kit. Furthermore, the cytotoxicity of Leishmania cells were detected by alamarBlue™ Cell Viability Reagent. Finally, the binding affinity within the binding site of MAPK3 protein was performed by computational methods. RESULTS: Purification of the MAPK3 protein was done using an Ni-NTA column and a protein band was identified at the expected 44 kDa molecular weight. Afterward, the ability of commercial drugs (afatinib and lapatinib) to inhibit the purified MAPK3 kinase activity was performed using an ADP-Glo™ Kinase Assay kit. The half-maximal inhibitory concentrations (IC50) of two drugs inhibited the MAPK3 protein within the same range of IC50 values (3.27 and 2.22 µM for afatinib and lapatinib, respectively). Furthermore, the cytotoxicity assay of compounds toward the extracellular promastigote and intracellular amastigote stages was investigated using alamarBlue™ Cell Viability Reagent. The results showed that both drugs were more efficient against extracellular promastigotes and intracellular amastigotes of both Leishmania donovani and Leishmania martiniquensis. Finally, the molecular dynamics simulation (MD) was performed to study the intermolecular interactions of both drugs with MAPK3 protein. From 100 ns molecular dynamics simulation, the structural stability of both drugs in a complex with MAPK3 was quite stable. CONCLUSION: This work was suggesting that afatinib and lapatinib act as MAPK3 inhibitors and might be developed for leishmaniasis treatment.

Development of loop-mediated isothermal amplification (LAMP) assay using SYBR safe and gold-nanoparticle probe for detection of Leishmania in HIV patients.

Asymptomatic leishmaniasis cases have continuously increased, especially among patients with HIV who are at risk to develop further symptoms of cutaneous and visceral leishmaniasis. Thus, early diagnosis using a simple, sensitive and reliable diagnostic assay is important because populations at risk mostly reside in rural communities where laboratory equipment is limited. In this study, the highly sensitive and selective determination of Leishmania infection in asymptomatic HIV patients was achieved using dual indicators (SYBR safe and gold-nanoparticle probe; AuNP-probe) in one-step LAMP method based on basic instruments. The assay can be simply evaluated under the naked eye due to clear interpretation of fluorescent emission of LAMP-SYBR safe dye-complex and colorimetric precipitate of specific AuNP-probes. The sensitivities and specificities of fluorescent SYBR safe dye and AuNP-probe indicators were equal, which were as high as 94.1 and 97.1%, respectively. Additionally, detection limits were 102 parasites/mL (0.0147 ng/µL), ten times more sensitivity than other related studies. To empower leishmaniasis surveillance, this inexpensive one-step SYBR safe and AuNP-LAMP assay is reliably fast and simple for field diagnostics to point-of-care settings, which can be set up in all levels of health care facilities including resource limited areas, especially in low to middle income countries.

An Epidemiological Survey of Opisthorchis viverrini Infection in a Lightly Infected Community, Eastern Thailand.

Human liver fluke infection caused by Opisthorchis viverrini increases the risk of cholangiocarcinoma (CCA) reported along the Mekong basin including Thailand, Lao People's Democratic Republic (PDR), Cambodia, and Vietnam. The highest incidence of CCA has been reported in northeastern Thailand where liver fluke infection is prevalent. This study aimed to investigate the prevalence of O. viverrini infection in a northeastern-descendent community in rural Sa Kaeo Province, eastern Thailand, using stool examination and molecular technique. The Kato-Katz method was performed to determine eggs per gram (EPG) for infection intensity. Phosphate-buffered saline-ethyl acetate concentration was used to prepare specimens for polymerase chain reaction (PCR) and restriction fragment length polymorphism of the internal transcribed spacer 2 (ITS2) region of the ribosomal RNA. From 1,245 specimens, 105 (8.4%) samples were identified as Opisthorchis-like eggs from stool examination, and all positive specimens indicated light infection (< 1,000 EPG). From positive Opisthorchis-like egg samples, 55.2% (58/105) were identified as O. viverrini eggs from ITS2-PCR assay for which low infection intensity might result in a negative PCR result (44.8%). Using multiple logistic regression analysis, males were at 3.1 times higher risk of acquiring O. viverrini infection than females. From phylogenetic analysis, in eastern Thailand, nucleotide sequences of O. viverrini were grouped as a monoclade as those isolated from Greater Mekong, Vietnam, Myanmar, and west Siberia. The results revealed that the surveyed community is a low-grade endemic area of O. viverrini infection. Thus, data from this study can be used to improve health-promoting programs and activities to control the infection and its subsequent CCA.

Comparison of PCR methods for detection of Leishmania siamensis infection.

BACKGROUND: Leishmania siamensis, a newly identified species, has been reported as a causative agent of leishmaniasis in Thailand. This organism has been identified and genetically characterized using PCR techniques based on several target genes. However, the sensitivities and specificities of these methods for the diagnosis of L. siamensis infection have never been evaluated. METHODS: To evaluate the sensitivities and specificities of PCR methods to detect L. siamensis infection, PCR for different genetic markers, i.e., the small subunit ribosomal RNA region (SSU-rRNA), the internal transcribed spacer 1 region (ITS1), cysteine protease B (cpb), cytochrome b (cyt b), heat shock protein 70 (hsp70), the spliced leader mini-exon, and the triose-phosphate isomerase (tim) genes were compared. RESULTS: Both the ITS1-PCR and the SSU rRNA-PCR could detect promastigote of L. siamensis at concentrations as low as 0.05 parasites/µl or the DNA concentration at 2.3 pg/µl. Though the ITS1-PCR method only recognized 8 samples as positive, all of these could be assessed as true positive according to microscopic diagnosis and/or amplifying the results of the PCR and their sequencing, while other methods also produced false positive results. Compared with the ITS1-PCR method, the PCR amplified SSU-rRNA and cpb gene showed 100% sensitivity for the detection of L. siamensis in clinical specimens. The PCR amplified mini-exon and hsp70 gene also gave a high sensitivity of 87.5%. In contrast, the PCR methods for cyt b and tim gene showed low sensitivity. The PCR methods for cyt b, mini-exon and tim gene showed 100% specificity compared with the ITS1-PCR. CONCLUSION: As a result, the ITS1-PCR method is a suitable target for PCR-based detection of L. siamensis infection in clinical specimens due to its high sensitivity and specificity. The results of this study can be used for molecular diagnosis as well as in epidemiological studies of L. siamensis in affected areas.

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.

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%).