Shirzad Fallahi

Background: There is an increasing disease trend for SARS-COV-2, so need a quick and affordable diagnostic method. It should be highly accurate and save costs compared to other methods. The purpose of this research is to achieve these goals. Methods: This study analyzed 342 samples using TaqMan One-Step RT-qPCR and fast One-Step RT-LAMP (Reverse Transcriptase Loop-Mediated Isothermal Amplification). The One-Step LAMP assay was conducted to assess the sensitivity and specificity. Results: The research reported positive samples using two different methods. In the RT-LAMP method, saliva had 92 positive samples (26.9%) and 250 negative samples (73.09%) and nasopharynx had 94 positive samples (27.4%) and 248 negative samples (72.51%). In the RT-qPCR method, saliva had 86 positive samples (25.1%) and 256 negative samples (74.8%) and nasopharynx had 93 positive samples (27.1%) and 249 negative samples (72.8%). The agreement between the two tests in saliva and nasopharynx samples was 93% and 94% respectively, based on Cohen’s kappa coefficient (κ) (P < 0.001). The rate of sensitivity in this technique was reported at a dilution of 1 × 10¹ and 100% specificity. Conclusions: Based on the results of the study the One-Step LAMP assay has multiple advantages. These include simplicity, cost-effectiveness, high sensitivity, and specificity. The One-Step LAMP assay shows promise as a diagnostic tool. It can help manage disease outbreaks, ensure prompt treatment, and safeguard public health by providing rapid, easy-to-use testing.

In the absence of effective antiviral drugs or vaccines, early and accurate detection of SARS-CoV-2 infection is essential to the COVID-19 pandemic. This study developed and evaluated a novel rapid One-Step LAMP assay to directly detect the SARS-CoV-2 RNA from nasopharyngeal (NP) swab samples of patients with suspected SARS-CoV-2 infection living in deprived areas in comparison to One-Step Real-time PCR. Two hundred fifty-four NP swab samples from patients suspected of COVID-19 infection living in deprived western areas of Iran were tested by TaqMan One-Step RT-qPCR and fast One-Step LAMP assays. Tenfold serial dilutions of SARS-CoV-2 RNA standard strain where the viral copy number in each dilution was previously determined using the qPCR and various templates were used to investigate the analytical sensitivity and specificity of the One-Step LAMP assay in triplicate. Also, the efficacy and reliability of the method compared to TaqMan One-Step RT-qPCR were evaluated using SARS-CoV-2 positive and negative clinical samples. The results of the One-Step RT-qPCR and One-Step LAMP tests were positive in 131 (51.6%) and 127 (50%) participants, respectively. Based on Cohen’s kappa coefficient (κ), the agreement between the two tests was 97%, which was statistically significant (P < 0.001). The detection limit for the One-Step LAMP assay was 1 × 10¹ copies of standard SARS-CoV-2 RNA per reaction in less than an hour in triplicates. Negative results in all samples with non-SARS-CoV-2 templates represent 100% specificity. The results showed that the One-Step LAMP assay is an efficient consistent technique for detecting SARS-CoV-2 among suspected individuals due to its simplicity, speed, low cost, sensitivity, and specificity. Therefore, it has great potential as a useful diagnostic tool for disease epidemic control, timely treatment, and public health protection, especially in poor and underdeveloped countries.

Background: Cryptosporidium spp. are opportunistic intestinal protozoans with global distribution and are of great importance as zoonotic protozoans are common to humans and domestic animals, including cattle and calves. Identification and detection of parasite species using precise methods including molecular methods can be an effective step in treating and controlling parasites.Objectives: This study aimed to investigate the prevalence of Cryptosporidium among breeding calves of Khorramabad city, Lorestan province, Western Iran, using PCR.Methods: The faecal samples were taken from 181 healthy and diarrhoeal calves and after the Ziehl Neelsen Acid‐fast staining and microscopic evaluation, the genomic DNA was extracted for molecular evaluations. To detect Cryptosporidium species, specific primers targeting the SAM‐1 gene of Cryptosporidium and a commercial master mix were used for PCR.Results: Out of 181 faecal samples of breeding calves in Khorramabad city, 9 samples (5%) were positive for Cryptosporidium spp. using the PCR method. Statistical analysis of the data showed that there was no significant statistical relationship between Cryptosporidium infection of the calves and variables of age, breed, type of water consumption, clinical signs of diarrhoea, and sampling location, while parasite infection had a significant relationship with calf gender so that all Cryptosporidium positive samples were from male calves (p ≤ 0.05).Conclusions: Considering the presence of Cryptosporidium infection, the region's traditional grazing system, and the close relationship between livestock and humans, there is a possibility of human infection in the region. So preventive measures such as periodic animal testing with sensitive and accurate diagnostic techniques including PCR, pharmacological treatment of livestock, water hygiene and the use of industrial grazing instead of traditional grazing to improve the hygiene of food consumed by livestock are recommended.

Background Blastocystis spp. is one of the most prevalent intestinal parasites with worldwide distribution. Various diagnostic methods with different sensitivities and specificities have been used to detect Blastocystis in clinical samples. The present study aims to develop and evaluate a LAMP assay to detect Blastocystis spp. in AIDS patients for the first time. Methods In this cross-sectional study, 98 AIDS patients with an average CD4 + T lymphocyte count lower than 150 cells/mm³ participated in the study. The presence of Blastocystis spp. in the stool samples of AIDS patients was examined by parasitology (direct wet mount and concentration assays) and molecular (PCR and LAMP) methods. The 18 SSU rRNA genomic target was used to design the specific primers for the PCR and LAMP assays. The specificity of designed primers for the LAMP assay was evaluated using the sequencing of a conventional PCR product by the external LAMP primers. The data were analyzed using the SPSS software and chi-square test and Fischer’s exact tests were used and Cohen’s Kappa calculates the agreement of the molecular tests. Associations were tested using odd ratios (OR) and 95% confidence intervals (CI) after adjustments. Results Out of 98 stool samples from patients with AIDS, 9 (9.18%), 13 (13.26%), and 15 (15.30%) samples were detected positive for Blastocystis spp. by parasitology, PCR, and LAMP techniques, respectively. PCR amplification and subsequent sequencing of the product sequences revealed that the obtained partial sequences were identical to the corresponding 18 SSU rRNA sequences reported in GenBank. The higher positivity rate for Blastocystis spp. among studied AIDS patients by LAMP technique compared to other diagnostic methods showed the higher potential and effectiveness of this relatively new described molecular assay for the detection of Blastocystis spp. in AIDS patients. Conclusion The accurate and rapid detection of emerging intestinal protozoa such as Blastocystis is of clinical importance for better prevention and timely treatment of the disease, especially in immunocompromised patients. The results obtained for the first time showed that the sensitivity and accuracy of the LAMP technique in the diagnosis of Blastocystis spp. in AIDS patients is very high.

Fascioliasis is a parasitic infection caused by Fasciola spp. in humans and animals. Despite significant advances in vaccination and new therapeutic agents, little attention has been paid to validate methods for the diagnosis of fascioliasis in animals. This study aimed to compare the loop‐mediated isothermal amplification (LAMP) technique with PCR assay for the diagnosis of F. hepatica in sheep. In this cross‐sectional study, 195 stool samples were collected from sheep for 3 months in Lorestan province, West of Iran. Specimens’ parasitological examination was performed by using the direct wet mount and formalin‐ether concentration method. After DNA extraction from the samples, molecular analysis was done using PCR and LAMP techniques based on the Fasciola ribosomal intergenic spacer (IGS) sequence. Of 195 specimens of sheep, 11 specimens were identified as F. hepatica‐positive infection by using microscopic, PCR and LAMP assays. Kappa agreement test results showed that there was a significant agreement between the results of microscopic examination diagnostic tests, PCR and LAMP (Kappa = 0.51–0.72 and p < .001). According to the results of chi‐square comparisons between parasite prevalence applying different techniques and variables of age, sex breed, and type of drinking water, there was no significant relationship (p ≥ .05). However, most of the infected sheep with Fasciola were 3‐ to 4‐year‐old females, of the Lori breed and consumed tap water. In many endemic areas, successful prevention and treatment of fascioliasis in animals depend on rapid and accurate diagnosis. Based on the results of the Kappa agreement, the significant agreement among the results of the microscopic examination, PCR and LAMP indicates the accuracy and reliability of these tests in the diagnosis of F. hepatica in sheep. However, molecular methods, especially the LAMP technique, are suggested because of their higher sensitivity and reliability for the diagnosis of F. hepatica even under field conditions.