Application of Multiplex PCR for Detection and Differentiation of Entamoeba histolytica,Entamoeba dispar and Entamoeba moshkovskii

Background

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.

Methods

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.

Results

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.

Conclusion

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.     

Seventy-kilodalton protein density in Porites spp.: possible useful proxy for cold stress in coral reefs

During the last two decades, significant deterioration of tropical coral reef communities has been observed, and based on current theories, environmental stressors are among the main causes of this. The density of 70-kDa heat shock proteins (Hsps) may present as a potential tool for the determination of cold stress on Porites spp. Therefore, density of Hsp70 proteins, concentration of total protein and seawater temperature was measured on a seasonal basis. Sampling was conducted, from the dominant genus of Porites spp. at four separate time points over 1?year (2003–2004) and at a depth of 3?m. Our results showed no significant correlations between total protein and seawater temperature. Our results also indicated that Porites spp. from the Persian Gulf is sensitive to low range of temperature (15–27°C). Maximum Hsp70 density and minimum average number of colonies in Porites spp. were obtained in winter. Also, a significant negative correlation was observed between Hsp70 density and water temperatures (p?>?0.05). Therefore, density of Hsp70 may be used as indicator of cold stress in Porites spp.     

Epidemiological Study of Gastrointestinal Helminthes of Canids in Chaharmahal and Bakhtiari Province of Iran

Background

The present study was carried out to describe the epidemiological aspects of gastrointestinal helminthic infections of canids in Chaharmahal and Bakhtiari Province, the central western part of Iran.

Methods

Forty nine canid species including, dogs, jackals, foxes and wolves were included in this study. The contents of their alimentary canal were inspected in order to isolate and identify the parasitic helminthes of this system. To identify the worms, the Soulsbey and Anderson identification key and light microscopy were used.

Results

Based on necropsy findings, 35 (71.4%) of examined animals were infected with at least one helminth. The prevalence of identified worms was as follows: Mesocestoides lineatus (55.1%), Joyeuxiella echinorinchoides (26.5%), Taenia hydatigena (12.2%), T. multiceps (8.2%), T. ovis (2%), Dipylidium caninum (2%) and Spirura spp. (2%). No significant difference was noticed between the sampling areas, age and helminth infection. Only a significant difference was observed for prevalence of T. multiceps in wolf (25%), dog (21.4%), jackal and fox (0%), respectively (P < 0.05).

Conclusion

The canids in Chaharmahal and Bakhtiari harbor several parasites that some kind of them have zoonotic importance and may pose a threat to community health specially in rural areas.     

Early detection and impaired quality of life in COPD GOLD stage 0: a pilot study

This pilot study aimed to identify early stages of chronic obstructive pulmonary disease (COPD) in an urban population of smokers and ex-smokers using the Global Initiative for Chronic Obstructive Lung Disease (GOLD 2001, 2003) classification guidelines and to assess the impact of early disease on quality of life. Smokers and ex-smokers of >or= 10 pack years and age >or= 50 years were recruited. After an initial telephone interview, eligible subjects completed a clinical assessment, spirometry tests, and the St. George's Respiratory Questionnaire (SGRQ). A total of 244 subjects completed the study; 91 subjects (37%) were normal, 153 subjects (63%) met the criteria for GOLD stages 0 to III: 65 stage 0 (27%), 43 stage I (18%), 38 stage II (16%), 7 stage III (3%) and 0 in stage IV. The stage 0 patients were younger than any other COPD groups (p<0.0005), including normal subjects (55.5+/-5.4 years vs. 59.6+/-7.2 years; p=0.0005). The frequency of current smoking in stage 0 patients was greater than those in the normal category (80% vs. 33%; p<0.0001). There were significant impairments in quality of life measures between normal subjects and all GOLD stages (SGRQ total scores; p<0.0001) except for stage I (SGRQ total scores; p=0.1409). Subjects with COPD at GOLD stage 0 were markedly under-diagnosed. These subjects had a significant impairment in their health-related quality of life measures, were younger than other categories, and were mostly current smokers. Thus, detection of COPD at GOLD stage 0 may provide a unique opportunity for early intervention and smoking cessation and the removal of GOLD stage 0 from the 2006 update should be re-assessed.     

Comparison of loop-mediated isothermal amplification (LAMP) and nested-PCR assay targeting the RE and B1 gene for detection of Toxoplasma gondii in blood samples of children with leukaemia

Toxoplasmosis diagnosis constitutes an important measure for disease prevention and control. In this paper, a newly described DNA amplification technique, loop-mediated isothermal amplification (LAMP), and nested-PCR targeting the repeated element (RE) and B1 gene, were compared to each other for the detection of Toxoplasma gondii DNA in blood samples of children with leukaemia. One hundred ten blood samples from these patients were analyzed by LAMP and nested-PCR. Out of 50 seropositive samples (IgM+, IgG+), positive results were obtained with 92% and 86% on RE, B1-LAMP and 82% and 68% on RE, B1-nested PCR analyses, respectively. Of the 50 seronegative samples, three, two and one samples were detected positive by RE-LAMP, B1-LAMP and RE-nested PCR assays, respectively, while none were detected positive by B1-nested PCR. None of the 10 IgM−, IgG+ samples was detected positive after testing LAMP and nested-PCR assays in duplicate. This is the first report of a study in which the LAMP method was applied with high sensitivity and efficacy for the diagnosis of T. gonii in blood samples of children with leukaemia.