Application of multiplex PCR for detection and differentiation of entamoeba histolytica, entamoeba dispar and entamoeba moshkovskii

Entamoeba moshkovskii and E. dispar are impossible to differentiate microscopically from the pathogenic species E. histolytica. Multiplex polymerase chain reaction [Multiplex PCR] is a widespread molecular biology technique for amplification of multiple targets in a single PCR experiment. For detection and differentiation of the three-microscopy indistinguishable Entamoeba species in human, multiplex PCR assay using different DNA extraction methods was studied. A conserved forward primer was derived from the middle of the small-subunit rRNA gene, and reverse primers were designed from signature sequences specific to each of these three Entamoeba species. A 166-bp PCR product with E. histolytica DNA, a 580-bp product with E. moshkovskii DNA and a 752-bp product with E. dispar DNA were generated in a single-round and multiplex PCR reaction. We recommend this PCR assay as an accurate, rapid, and effective diagnostic method for the detection and discrimination of these three Entamoeba species in both routine diagnosis of amoebiasis and epidemiological surveys

Development and assessment of loop-mediated isothermal amplification [LAMP] assay for the diagnosis of human visceral leishmaniasis in Iran

Parasitological methods for the diagnosis of Visceral leishmaniasis [VL] require invasive procedures, so serological and molecular approaches have been developed but are not generally applicable in the field. We evaluated a loop mediated isothermal amplification [LAMP] assay using blood from VL patients and compared it to nested PCR. Forty-seven subjects with clinical features [fever, hepatosplenomegaly and anemia] were confirmed positive for VL by the direct agglutination test [DAT] at titers >3200. Forty DAT negative individuals from non-endemic areas with no clinical signs or symptoms of VL served as controls. A LAMP assay was performed using a set of six primers targeting leishmania infantum kinetoplast DNA [kDNA] minicircle gene under isothermal [64 degree C] conditions. For nested PCR we used primers targeting the kDNA minicircle gene. The LAMP assay provided a detection limit of 1 parasite in 1 ml of j peripheral blood and detected L. infantum DNA in 44 of 47 DAT-confirmed VL cases, with diagnostic sensitivity of 93.6% [95% CI]. No L. infantum DNA was amplified in controls, indicating a specificity of 100%. The nested PCR yielded sensitivity of 96% [95% CI] and a specificity of 100% [95% CI]. The LAMP assay gave results similar to those of nested PCR but in a shorter time. The LAMP method is simple; requires no sophisticated equipment; has a short reaction time; and results, indicated by turbidity of the reaction mixture, are observable with the naked eye

[Serological evaluation of anti-toxoplasma IgM and IgG antibodies in renal transplant recipient's before and after transplant by ELFA, ELISA and ISAGA methods]

Toxoplasmosis is a shared human-animal disease with worldwide distribution caused by Toxoplasma gondii. More than half of the world's population is seropositive for toxoplasmosis. The possibility of reactivation of the old infection or acquisition of infection from donor's tissue increases in the transplant recipient patients who receive immunosuppressive therapy. In this study, IgM and IgG antitoxoplasma immunoglobulins seroconversion in renal transplant recipients was evaluated before and after transplantation. This was a prospective cohort study on a total of 102 recipients. Two serum samples were obtained from each patient. The first sample was taken before administration of any immunosuppressive drugs and second sample was taken 3 months after transplantation. The IgM and IgG anti-toxoplasma antibodies were assayed by ELFA and ELISA techniques. IgM/ISAGA method was also used. ELFA identified 65 [63.7%] pre-transplantation samples as IgG+ and did not detect any positive IgM samples. However, IgM was detected in 3 [2.9%] post-transplantation samples by this method. Forty nine [48%] pre-transplantation samples were reported IgG+ by ELISA and no IgM positive sample was identified by this method. ELISA method detected 2 [1.9%] IgM positive reactions in post-transplantation samples. By IgM/ISAGA method, we detected no IgM positive reactions in pre-transplantation samples whereas 3 months later [second sampling] IgM antibody was detected in 3 [2.9%] cases. Secondary toxoplasmosis infection was observed in 30 cases per 1000 recipients, which indicates that screening for toxoplasmosis infection should be performed in developed countries for these patients. On the other hand, as the risk of re-active toxoplasmosis infection exists in developing nations, they should consider the necessary preventive measures to control this condition. In addition, we suggest that ELFA is the best method because of the most valuable results

Miltefosine-Induced Apoptotic Cell Death on Leishmania major and L. tropica Strains

The aim of this study was to assess the cytotoxic effects of various concentrations of miltefosine on Leishmania major (MRHO/IR/75/ER) and L. tropica (MHOM/IR/02/Mash10) promastigotes and to observe the programmed cell death features. The colorimetric MTT assay was used to find L. major and L. tropica viability and the obtained results were expressed as 50% inhibitory concentration (IC50). Also, 50% effective doses (ED50) for L. major and L. tropica amastigotes were also determined. Annexin-V FLUOS staining was performed to study the cell death properties of miltefosine using FACS analysis. Qualitative analysis of the total genomic DNA fragmentation was performed by agarose gel electrophoresis. Furthermore, to observe changes in cell morphology, promastigotes were examined using light microscopy. In both strains of L. major and L. tropica, miltefosine induced dose-dependent death with features of apoptosis, including cell shrinkage, DNA laddering, and externalization of phosphatidylserine. The IC50 was achieved at 22 microM and 11 microM for L. major and L. tropica after 48 hr of incubation, respectively. ED50 of L. major and L. tropica amastigotes were 5.7 microM and 4.2 microM, respectively. Our results indicate that miltefosine induces apoptosis of the causative agent of cutaneous leishmaniasis in a dose-dependent manner. Interestingly, L. major did not display any apoptotic changes when it was exposed to miltefosine in concentrations sufficient to kill L. tropica.