Epidemiology of rotavirus-norovirus co-infection and determination of norovirus genogrouping among children with acute gastroenteritis in Tehran, Iran

Background: Enteric viruses, particularly human rotavirus and norovirus, have been shown to replace bacteria and parasites, as the most common pathogens responsible for acute diarrhea. However, there are still few epidemiological data on the simultaneous occurrence of these viruses in Iran. In this regard, the aim of this study was to assess the useful epidemiological data on the gastroenteritis associated with rotavirus-norovirus mixed infection and to examine the prevalence of norovirus genogrouping among children aged less than five years old in Iran

Methods: A total of 170 stool samples were collected from children under five years of age with the clinical signs and symptoms of acute gastroenteritis, from May 2013 to May 2014. For the detection of both rotavirus and norovirus, total RNA was extracted from all samples, followed by reverse transcription polymerase chain reaction [RT-PCR]. For both detected rotaviruses and noroviruses, genogrouping was performed

Results: Of 170 samples, 49 [28.8%] and 15 [8.8%] samples were found to be positive for rotavirus and norovirus infections by RT-PCR. Interestingly, 6 [3.5%] patients were positive for both infections. Among the 15 norovirus-positive patients, 13 [86.6%] and 2 [13.3%] belonged to genogroups GII and GI

Conclusions: The norovirus genogroup GII and rotavirus lead to the serious infections in children with acute gastroenteritis. However, more well-designed studies are needed to further elucidate the role of other enteric viruses in acute gastroenteritis

[Bioinformatic evaluations for locating the microRNA suppressing PI3K/AKT pathway and analysis in prostate cancer cell lines]

Objective: Prostate cancer is the fifth most common cancer. In 2012, it was the second leading cause of cancer death for men worldwide. The PI3K/AKT pathway plays an essential role in pathogenesis of prostate cancer; the key role of this pathway in cancer progression makes it an attractive target for prostate cancer therapy. MicroRNAs [miRNAs] that regulate gene expression have a special ability to simultaneously control multiple genes and pathways which make them candidates for therapeutics. This study aims to determine miRNAs which target the PI3K/AKT pathway and evaluate them in prostate cancer cell lines

Methods: In order to determine an effective miRNA for the PI3K/AKT pathway, we assessed six genes from this pathway which have been proposed as drug targets in ten different prediction algorithms. Next, the candidate miRNAs were analyzed in expression profile and pathway analysis databases. Expression of candidate miRNAs in control and prostate cancer cell lines were subsequently evaluated

Results: According to bioinformatics, the miR-29 family could target the most genes from this list. Other bioinformatic estimates confirmed these results. The miR-29 family showed significant downregulation in prostate cancer cell lines LNCAP, PC3 and DU-145 compared to control samples

Conclusion: These results propose the possibility of using the miR-29 family to inhibit the PI3K/AKT pathway in prostate cancer

Establishment of national laboratory standards in public and private hospital laboratories

In September 2007 national standard manual was finalized and officially announced as the minimal quality requirements for all medical laboratories in the country. Apart from auditing laboratories, Reference Health Laboratory has performed benchmarking auditing of medical laboratory network [surveys] in provinces. 12[th] benchmarks performed in Tehran and Alborz provinces, Iran in 2010 in three stages. We tried to compare different processes, their quality and accordance with national standard measures between public and private hospital laboratories. The assessment tool was a standardized checklist consists of 164 questions. Analyzing process show although in most cases implementing the standard requirements are more prominent in private laboratories, there is still a long way to complete fulfillment of requirements, and it takes a lot of effort. Differences between laboratories in public and private sectors especially in laboratory personnel and management process are significant. Probably lack of motivation, plays a key role in obtaining less desirable results in laboratories in public sectors

simple and rapid method for the detection of HIV-1/HCV in co-infected patients

Due to some limitations of serological methods in diagnosis of patients infected with HIV-1 [human immunodeficiency virus] and HCV [hepatitis C virus], it is profoundly important to use molecular methods for the detecting of these infectious agents. However, the most significant problems are the exorbitant cost of these methods and the need of a thermocycler which is an expensive instrument. The current research recruits a multiplex nucleic acid sequence base amplification [NASBA] in order to simultaneously detect HIV-1 and HCV genomes in patients' plasma samples. Sensitivity and specificity of this method have been evaluated using clinical samples. A multiplex NASBA assay for simultaneous detection of HCV and HIV-1 by the use of specific primers were designed and validated. A well-conserved region in the HIV-1 pol gene and 5'-NCR of HCV genome were used. A total of 40 samples of HIV-1 [20 samples] and HCV [20 samples] were used in the NASBA assay. The specificity and sensitivity of the assay were evaluated. Our results have demonstrated that the primers used in the assay had no interrelation with each other and other possible interfering agents in the assay. The analytical sensitivity of the assay for both HIV-1 and HCV was determined to be 1000 copies/mL and the clinical sensitivity and specificity were 93.3% and 100%, respectively. By exploiting this multiplex NASBA assay, it is possible to detect HIV-1 and HCV infection/co-infection in patients' plasma with a suitable sensitivity and specificity. Furthermore, due to its simplicity and multiplexing feature, it could be used in limited access laboratories in a cost-effective manner.

Comparison of qualitative PCR and pp65 antigenemia for the diagnosis of CMV infection in hematopoietic stem cell transplanted patients

Human cytomegalovirus [CMV] is a major life-threatening pathogen for hematopoietic stem cell transplant recipients. Specific tests are used for the diagnosis and monitoring of CMV infection in transplant patients. This study evaluates the performance of pp65 antigenemia and qualitative PCR assays for monitoring CMV in such patients. We analyzed 179 clinical samples from 41 patients by using a validated home-brewed qualitative PCR and a commercial antigenemia assay. The obtained results were evaluated using quantitative real-time PCR as the gold standard. CMV was observed in 26.8% of samples analyzed by the antigenemia assay and in 42.6% of the samples by qualitative PCR. Among 179 clinical samples, 50.8% were negative and 21.2% were positive by both assays. On the other hand, 26.3% were only positive by qualitative PCR whereas 1.7% were positive by the antigenemia assay. A comparison of the results with real-time PCR showed that qualitative PCR has a higher sensitivity than the antigenemia assay [98.7% vs. 45.7%]. The specificity of both assays was equal [96.8%]. Quantitative results of the antigenemia assay showed good correlation with real-time PCR [r=0.715; p<0.001] Both the qualitative PCR and antigenemia assays have special deficiencies for efficient diagnosis of CMV infection. Therefore, effective management of CMV infection in transplant patients requires the use of other sensitive quantitative methods such as qPCR

Rapid low-cost detection of hepatitis C virus RNA in HCV-infected patients by real-time RT-PCR using SYBR green I

We intend to design and validate a low-cost assay for the detection of hepatitis C virus [HCV] RNA using rapid-cycle RT-PCR. The procedure is performed in a closed system with little risk of contamination allowing PCR and product identification to be performed within one or two hours. A SYBR Green-based real-time RT-PCR for rapid detection of HCV. Amplicon synthesis was monitored continuously by SYBR Green I, which binds to double stranded DMA during PCR. The PCR products were identified by melting curve analysis. Standard sera with known concentrations of HCV RNA and 150 clinical samples were used to validate our assay. The minimum detection level of our assay was less than 50 ID/mL. The results on 100 plasma samples were comparable with commercial assays. This method is useful for rapid qualitative detection of HCV infection and particularly suitable for routine diagnostic applications

[ study on single nucleotide polymorphisms in Plasmodium fakiparum chloroquine resistance during two years in Chabahar]

Plasmodium falciparum chloroquine resistance is a major problem in malaria endemic areas. Single nucleotide polymorphisms in pfcrt and pfmdr1 genes are known to be associated with chloroquine resistance in some parts of the world. The major goal of the present study was to detect the five single nucleotide polymorphisms in pfmdr1 gene and one single nucleotide polymorphisms in pfcrt gene. Total of 26 blood samples were collected from falciparum malaria infectious person with chloroquine failure in Chabahar, a harbor located in Sistan baluchestan during 2 years. Detection of single nucleotide polymorphisms were carried out by Real-Time PCR using Light CyclerTM hybridization probe assay. Our data showed that the pfmdr1 N86Y mutation was detected in 6[23%] samples. Although this mutation was not observed in the first year but in the second year it was substancial. In addition the pfcrt K76T mutation was detected in 11 samples [42.3%] of CVMNT haplotype, 7 samples [26.9%] of CVIET haplotype, 5 samples [19.2%] of SVMNT haplotype and 2 samples [7.6%] of SVIET haplotype. The mutations considerably have increased during 2 years. Our results showed single nucleotide polymorphisms in pfmdr1 and pfcrt genes. This could be considered as chloroquine resistance markers for malaria control in Chabahar

use of a sensitive RT-Nested PCR method for detection of Hepatitis C virus

Hepatitis C virus is the major cause of viral hepatitis and its diagnosis in suspected specimens is of great importance. The risk of transfusion- transmitted virus infection is primarily the result of failure in serological screening tests to detect recently infected donors in the pre-seroconversion window period of infection. Therefore, sensitive and accurate diagnosis of HCV prior to antibody production to reduce window period is necessary. In the present study, a sensitive and specific RT-Nested PCR method for detection of a conserved HCV 5 UTR sequence was developed. Two pairs of primers for amplification of the target sequence in two rounds of PCR were selected. The developed RT- Nested PCR assay was performed on HCV-antibody confirmed positive samples as well as negative controls and standard samples. In order to compare the results, One Step RT-PCR kit was used in this study. 25 HCV-positive plasma samples whose positivity were confirmed by ELISA and Western Blot tests, also as well as 10 fold dilutions of a high viral load plasma sample obtained from a HCV-positive patient as standard samples and 25 negative control plasmas from healthy blood donors were collected and tested by this assay. In all of positive samples a 175bp band was observed on agarose gel electrophoresis, but no band could be detected in negative control plasma. Results from developed RT-PCR assay and One Step RT-PCR kit showed a good correlation. According to the results of this study, the developed RT-Nested PCR assay has a good sensitivity and specificity for diagnosis of HCV infection. It has the advantage of viral genome detection prior to seroconversion and can be used to detect HCV infection during window period