PCR Molecular Method Research Articles

Molecular Identification of Free-living Amoebae Isolated from Hospital Water Systems in Southwest Iran.

Free-living amoebae are opportunistic amoebae that usually live freely in various environmental conditions, including warm water and even in water supply network pipes and reservoirs connected to water. In addition to living freely, these protozoa are able to attack the host's body when they enter a human or animal body. Therefore, it is necessary to identify their presence in water resources. So, this study aimed to identify free-living amoebae isolated from water reservoirs of hospitals in southwest Iran. A total of 80 water samples were isolated from the hospitals of Ahvaz city, southwest Iran, and their physical and chemical parameters were measured. The samples were then put into non-food agar culture medium and stained using the Wright-Giemsa staining. Finally, the samples were identified by the PCR molecular method. The mean pH and turbidity values were 7.57 ± 0.03 and 3.31 ± 0.26 nephelometric turbidity unit (NTU), respectively. The mean residual chlorine and electrical conductivity were 0.91 ± 0.02 and 1122.39 ± 24.31, respectively. In addition, 9 (11.25%) and 3 (3.75%) samples were contaminated with Acanthamoeba spp. and Naegleria spp., respectively. However, no positive cases of Balamuthia spp. infection were observed. Moreover, two samples were co-infected with Acanthamoeba spp. and Naegleria spp. Due to the existence of free-living amoebae in water storage tanks at hospitals, it is necessary to prevent possible contamination with these amoebae and infectious agents by using new methods of disinfection and purification of water resources.

Research Article MQS-PCR: a simple and quick multiplex PCR molecular method for human chromosomal sex determination in DNA samples

Research Article MQS-PCR: a simple and quick multiplex PCR molecular method for human chromosomal sex determination in DNA samples

Use of BRAF immunohistochemistry as a screening test in detecting BRAFV600E mutation in melanomas.

BRAF mutation is detected in 50-70% of melanomas. The molecular methods used to detect BRAF mutations are 80-90% sensitive, specific, and expensive methods. Immunohistochemistry is a relatively common, rapid, relatively inexpensive method in pathology practice compared to molecular techniques. We aimed to compare immunohistochemical and molecular methods in our case of malign melanoma in which we investigated BRAF mutation with "real time PCR" method and to investigate the compatibility of molecular test results of BRAF immunohistochemistry results as a preliminary test. Selected blocks of 30 patients with metastatic melanoma who came to our department for BRAF mutation detection were subjected to real time PCR molecular method and immunohistochemical study was performed with BRAF primer antibody. BRAF mutation was detected by molecular method in 7 of 30 cases (23.33%). In all of these 7 cases, positive immunohistochemical staining was identified (100%). In conclusion, the use of BRAF immunohistochemistry as a screening test in the detection of mutant disease will allow the cost-effective use of molecular testing.

Prevalence of Malaria with Anaemia and HIV Status in Women of Reproductive Age in Onitsha, Nigeria

The major health problems affecting pregnant women in sub-Saharan African are anaemia, malaria and HIV. A case-control study aimed at determining the prevalence of malaria, anaemia and HIV status among women of reproductive age between 20-49 years attending General Hospital, Onitsha was conducted. Blood samples of three hundred and sixty-two apparently healthy gravid women attending antenatal and 181 non-gravid apparently healthy women in Onitsha were recruited using random sampling method. A structured questionnaire was used for data collection. Haemoglobin was estimated using automated method, Malaria, diagnosed microscopically using gold standard staining method and HIV screened using qualitative immunochromatographic method, confirmed molecularly. Statistical analysis was done using SPSS version 21. Results revealed that out of the 362 pregnant test groups,125 (34.5%) had malaria parasite (mp) in their blood and were anaemic 53(37.6%) though fewer in control groups ;23,14 (12.7%; 16.1%) (p = 0.195; p =0.055). Out of 347 HIV sero-negative pregnant women, 110 (31.7%) tested HIV positive when confirmed by PCR molecular method while only 1(0.6%) sero-negative controls were confirmed positive with molecular method with statistical significance observed in the test and control groups (p = 0.000; p = 0.000). However, as pregnancy affects HIV serology testing, it is necessary to use molecular method to increase its sensitivity and more enlightment programmes on the importance of balanced diet, compliance to Malaria control and routine gynecological drugs intake in pregnant women.

Study of toxoplasmosis and toxocariasis in patients suffering from ophthalmic disorders using serological and molecular methods

IntroductionToxoplasma gondii is an intracellular protozoan parasite that can cause ocular toxoplasmosis with most complications such as retinal detachment. Toxocara parasite, round worm, found in dogs and cats appears as larva migrans in humans can cause serious ocular complications such as debilitating vision loss.In Khuzestan province, southwest of Iran, T. gondii infection has been reported to be significant but toxocariasis was rare. However, the frequency of ocular toxoplasmosis and toxocariasis has not been studied in this area. The aim of this study was to evaluate the ocular toxoplasmosis and ocular toxocariasis using serological and molecular methods.MethodIn this case control study, 310 patients were identified by ophthalmologist as ocular toxoplasmosis and then 5 cc of venous blood samples were taken from each of them. Serum samples and buffy coat were prepared and ELISA was used to detect IgG and IgM anti-Toxoplasma antibodies and the molecular PCR was used to detect Toxoplasma DNA parasite in buffy coats. ELISA test was used to detect of IgG anti-Toxocara antibodies.ResultsTotally, for ocular toxoplasmosis, 130 (41.93%) of 310 patients were positive by ELISA, of them 121 (39%) IgG positive and nine (2.9%) IgM positive were diagnosed. Of 121 cases with IgG+, 119 (98.35%) were diagnosed with high IgG avidity indicating chronic phase of the infection. For ocular toxocariasis evaluation, antibodies against Toxocara were not detected in any of the samples. By PCR molecular method, 11 out of 310 patients (3.54%) had T. gondii DNA in the blood. In control, in total, 21 cases were detected positive by serology method, which showed a significant difference with the results of the case group(P < 0.05).By PCR method, only three cases showed positive which also indicated significant difference with result of case group (3 vs 9) (P < 0.05). In the control group, also no anti-toxocara antibodies were found.ConclusionIt can be concluded that T. gondii in Khuzestan province as the etiologic agent of ocular toxoplasmosis and physicians should consider diagnostic methods for identifying the infection when they visit the patients.

Frequency Assessment of OXA-10 and PER β-Lactamase Genes and Determination of Minimum Inhibitory Concentration in Klebsiella Strains Isolated from Urinary Tract Infections

Background: Klebsiella is one of the Enterobacteriaceae family that causes infections such as pneumonia, urinary tract infections (UTI), and meningitis. Klebsiella strains are capable of producing enzymes that can degrade the third-generation of cephalosporins known as broad-spectrum beta-lactamase enzymes. The resistance of Klebsiella strains to β-lactam antibiotics is related to the presence of β-lactamase genes. Methods: In this study, 90isolates of Klebsiella were isolated from two inpatient andoutpatient groups, each of themwas 45 isolates, which were collected from patients with urinary tract infection in educational hospitals of Shahrekord. The isolates were identified using phenotypic agar diffusion, disc phenotypic confirmation tests, and E-test of extended-spectrum beta-lactamase (ESBL). The PCR molecular method was used to diagnose and determine the strains containing broad-spectrum β-lactamases. Results: Thirty (66%) inpatients and 8 (17.8%) outpatients had broad-spectrum β-lactamase enzymes. The frequency of β-lactamase OXA-10 genes and PER in inpatients were 90% and 33%, respectively and also in outpatients were 50% and 12.5%, respectively. Conclusions: This study showed that the prevalence of isolated Klebsiella producing broad-spectrum β-lactamases is higher in inpatients in comparison to outpatients. Therefore, the rapid and accurate identification of bacteria and their resistance genes in clinical microbiology labs are highly recommended

Staphylococcal Enterotoxin A Detection from Rheumatoid Arthritis Patients' Blood and Synovial Fluid.

IntroductionDirect detection of microbial super antigens in synovial fluid of patients with rheumatoid arthritis may be able to guide to the design of cost-effective therapies. The purpose of this study was to assess the existence of Staphylococcal enterotoxin A (superantigen A) in the synovial fluid of patients with RA by the PCR and ELISA methods.MethodsThis experimental study was conducted on the synovial fluid of 103 RA patients from Baqiyatallah University of Medical Sciences’ Rheumatology Clinic in Tehran, Iran in 2011–2014. Bacterial cultures, polymerase chain reaction with specific primer pairs and enzyme-linked immunosorbent assay (ELISA) methods were used. The PCR products were subjected to sequence as a confirmatory molecular method results. The data were descriptively analyzed by SPSS Version 19.ResultsThe bacteriological study result indicated that, in four cases (3.8%) of the patients, bacterial strains were isolated. The result of PCR molecular method for staphylococcal enterotoxin A gene showed that, 42 of the patients (40.7%) tested positive for the ent A gene. The results of ELISA were positive for staphylococcal enterotoxin A (superantigen A) in 51 cases (49.51%) of the patients’ synovial fluids. The results indicated that the possibility of detecting superantigen A in the SF of RA patients, but the origin of the enterotoxin A gene remained unknown.ConclusionsThe findings of this study may be able to alter the actual theory on the pathogenesis, diagnosis, and treatment of RA patients. In addition, the results have shown the probability of an endogenous origin for the involved superantigen A in RA patients’ synovial fluids.

First report of Klebsiella pneumonia carbapenemase-producing Pseudomonas aeruginosa isolated from burn patients in Iran: phenotypic and genotypic methods.

Wound infection associated with carbapenem-resistant Pseudomonas aeruginosa in burn patients is a growing problem. One of the main mechanisms of resistance to carbapenem antibiotics is the ability of P. aeruginosa to produce carbapenemase enzymes. Klebsiella pneumonia carbapemenase (KPC) is an important type of carbapenemase which can hydrolyze carbapenem antibiotics. The Modified Hodge Test (MHT) and boronic acid as a KPC inhibitor are two phenotypic methods used for detection of carbapenemase. The sensitivity and specificity of these two phenotypic tests for the identification of KPC can be measured by PCR.In this study, 241 P. aeruginosa strains were isolated from wounds of hospitalized burn patients. Carbapenem-resistant P. aeruginosa isolates were determined by the disk diffusion method. KPC-producing carbapenem-resistant strains were examined using the Modified Hodge Test, followed by boronic acid. Further, strains with positive responses to MHT and boronic acid tests were analyzed with the PCR molecular method. One hundred eighty-six of 241 isolates were resistant to carbapenems and 75 were positive in the MHT. Three exhibited an at least 5-mm diameter difference when meropenem was combined with boronic acid vs meropenem alone in the boronic acid test. Two strains had a specific band with primer No.1 after gel electrophoresis. This study showed that MHT, despite excellent sensitivity, has variable specificity independent of bacterial species. Further, the use of KPC inhibitors such as boronic acid did not yield favorable sensitivity and specificity among the specimens from Iranian patients. Thus, it seems that sequencing after PCR should be considered the gold standard for the detection of KPC-producing P. aeruginosa.

Comparison between phenotypic and PCR for detection of OXA-23 type and metallo-beta-lactamases producer Acinetobacter spp.

Background: Resistance to carbapenems is developing around the world and can cause many problems for treatment of patients. Production of metallo-beta-lactamase (MBL) is one of the main mechanism for this type of resistance. So, detection of MBL-producer microorganisms can prevent the spread of this type of resistance.Materials and methods: In this study 94 Acinetobacter spp. were investigated. Resistance to imipenem was conducted after purification and identification. Combination disc (CD) and Double Disc Synergy Test (DDST) were performed for phenotypic detection of MBL and the molecular PCR method was done for vim-1, vim-2, imp-1 and OXA-23 genes.Results: According to TSI, SIM and oxidation-fermentation (OF) test and PCR assay 93 Acinetobacter baumannii and one strain Acinetobacter lwoffii were identified. 85% of them were resistant to imipenem. 34% of them have a positive combination disc test (CD) while Double Disc Synergy Test (DDST) was negative for all of them. The vim-1, vim-2 and imp-1 genes were not detected in PCR molecular method, however in 74% of strains with positive results in combination disc, were positive for the OXA-23 gene after PCR test. This study shows that the blaOXA-23 resistance determinant may become an emerging therapeutic problem.Discussion: According to the results, it seems that combination disc does not have enough specificity for detection of MBL-producer Acinetobacter and using Double Disc Synergy Test (DDST) can be more convenient.

Real-time PCR assay in differentiating Entamoeba histolytica, Entamoeba dispar, and Entamoeba moshkovskii infections in Orang Asli settlements in Malaysia

BackgroundAmebiasis caused by Entamoeba histolytica is the third leading cause of death worldwide. This pathogenic amoeba is morphologically indistinguishable from E. dispar and E. moshkovskii, the non-pathogenic species. Polymerase chain reaction is the current method of choice approved by World Health Organization. Real-time PCR is another attractive molecular method for diagnosis of infectious diseases as post-PCR analyses are eliminated and turnaround times are shorter. The present work aimed to compare the results of Entamoeba species identification using the real-time assay against the established nested PCR method.MethodsIn this study, a total of 334 human faecal samples were collected from different Orang Asli settlements. Faecal samples were processed by direct wet smear and formalin ethyl acetate concentration methods followed by iodine staining and was microscopically examined for Entamoeba species and other intestinal parasites. Microscopically positive samples were then subject to nested PCR and real-time PCR.ResultsThe overall prevalence of Entamoeba infection was 19.5% (65/334). SK Posh Piah recorded highest Entamoeba prevalence (63.3%) while Kampung Kemensah had the lowest prevalence (3.7%) of Entamoeba. Microscopically positive samples were then tested by real-time PCR and nested PCR for the presence of Entamoeba histolytica, Entamoeba dispar, and Entamoeba moshkovskii infection. Real-time PCR showed higher Entamoeba detection (86.2%) compared to nested PCR (80%), although the McNemar test value showed no significant difference between the two methods (p = 0.221).ConclusionsThis study is the first in Malaysia to report the use of real-time PCR in identifying and differentiating the three Entamoeba infections. It is also proven to be more effective compared to the conventional nested PCR molecular method.

Clostridium perfringens isolate typing by multiplex PCR

Clostridium perfringens is an important pathogen that provokes numerous different diseases. This bacterium is classified into five different types, each of which capable of causing a different disease. There are various methods for the bacterial identification, many are labor-intensive, time-consuming, expensive and also present low sensitivity and specificity. The aim of this research was to identify the different types of C. perfringens using PCR molecular method. In this study, 130 sheep-dung samples were randomly collected from areas around the city of Kerman, southeastern Iran. After processing and culturing of samples, the produced colonies were morphologically studied, gram stain test was also carried out and the genera of these bacteria were identified through biochemical tests. DNA extracted from isolated bacteria for genotyping was tested by multiplex PCR with specific primers. Based on length of synthesized fragments by PCR, toxin types and bacterial strains were detected. C. perfringens isolated types were divided as follows: 17.39% type A, 21.74% type B, 34.78% type C and 26.09% type D. It should be emphasized that, up to the present moment, C. perfringens type A has not been reported in Iran.

Inquilinus limosusand Cystic Fibrosis

<i>Inquilinus limosus</i>and Cystic Fibrosis

A novel Spiroplasma pathogen causing systemic infection in the crayfish Procambarus clarkii (Crustacea: Decapod), in China

A novel disease of crayfish Procambarus clarkii appeared in the summer of 2004 in freshwater aquaculture in Jiangsu province of China. Light and transmission electron microscopy (TEM), molecular biological methods and in vitro culture were used to identify the pathogen. The agent was unique in having a helical morphology and rotary motility as observed by phase-contrast light microscopy and was found in haemolymph, muscles, nerves and connective tissues by smear method and TEM. Ultra-thin sections under TEM revealed the wall-free membrane of the microbe. The agent could pass through membrane filters with pores 220 nm in diameter and was cultivated in vitro in M1D medium. 16S rDNA of the crayfish pathogen was amplified by PCR using primers specific for Spiroplasma-specific 16S rDNA. The resultant 271 bp PCR product showed 99% identity with Spiroplasma mirum 16S rDNA, having a close relationship with the spiroplasma from the Chinese mitten crab Eriocheir sinensis. This is the second time a spiroplasma has been found in a freshwater crustacean. The 271 bp PCR product was also amplified from the bottom mud in the ponds associated with the disease. The PCR molecular method is an effective way to detect spiroplasma in freshwater environment. The results from this study are significant in expanding the host range of spiroplasma and freshwater ecology.