comparative analysis of routine techniques: Reverse transcriptase polymerase chain reaction [RT-PCR] and five cell lines for detection of enteroviruses in stool specimens

Each year, Enteroviruses infect millions of people and cause different diseases. The agents are usually detected using cell culture. RD [Rhabdomyosarcoma] and L20B [L cells] are among the recommended cells by the World Health Organisation [WHO] for this purpose. Even though cell culture is the most common method used in diagnosing Enteroviruses in stool specimens, this particular method poses some problems, which include false positive or negative results, lack of a unique cell line for diagnosing all Entero virus types in addition to being time consuming. For these reasons, an attempt was made to find better techniques of Entero virus detection. RT-PCR [Reverse Transcriptase Polymerase Chain Reaction] is a technique used in place of the cell culture method. In this study, the cell culture method was compared with RT-PCR for detection of Enteroviruses in stool specimens. First, the chloroform treated stool samples were inoculated onto five cell lines, including RD, L20B, Hep-2 [Human Epidermoid carcinoma cell line], Vero [Verde Reno] and GMK [Green Monkey Kidney]. The results were then compared with data from Entero virus detection using the RT-PCR technique. The difference between RT-PCR and cell culture results was significant. Enteroviruses were detected in 24% of specimens using RT-PCR while cell lines could isolate Enteroviruses in just 14.4% of the samples

[Prevalence of anti-HCV antibody and related risk factors among bleeding disorder patients in Yazd province of Iran]

Inherited bleeding disorder is a disease due to deficiency in clotting factors or platelets. Replacement therapy of unscreened blood and blood products or unviricidal concentrated clotting factors would expose these patients to risk of acquired hepatitis C. The aim of this study was to determine the seroprevalence of hepatitis C virus and related risk factors among bleeding disorder patients in Yazd province. This cross-sectional study was performed in Yazd province in summer 2006 using census method. Following data collection through questionnaires, blood samples were taken from 77 [82.8%] patients. Plasma samples were tested for the presence of anti-HCV antibody via a third-generation ELISA kit. To exclude false positive cases, ELISA positive samples were re-tested by the confirmatory third generation RIBA test. The assessment of risk factors was done from the information analysis of both the questionnaires and test results by Chi-square test and multivariate logistic regression analysis, using SPSS 11.5 statistic software. The frequency of anti-HCV antibody was found in 38 [49.4%] patients. Chi-square test showed a statistically significant relationship between the severe form of the disease [in terms of blood products needed] [P<0.001], treatment duration of more than 121 months [P<0.001] and family history to HCV [P<0.05] antibody positivity. Successful execution of the screening of the blood and blood products as well as more scrutiny in preparing concentrated factors have been led to a remarkable decrease in the new infections among the recipients of these products in Yazd province

Comparison of Integrated Cell Culture -RT-PCR and Cell Culture Methods for detection of Enteroviruses

Generally, sewage exposed water could be potentially contaminated with enteroviruses. For this reason, enterovirus isolation from sewage specimens is one of the most sensitive indicators for virus circulation in the population. We evaluated the ICC-RT-PCR and cell culture methods for detection of enteroviruses in Tehran sewage system. This research utilized 63 specimens provided through Grab sample method to concentrate by two-phase method and cultured in RD and HEp-2 cells, respectively. All specimens then were inoculated using sensitive cell cultures of RD and HEp-2. After 24 hours incubation at 36[degree sign] by means of Pan E.V primers and afterwards Pan P.V Primers along with specific sabin primers, RT-PCR was carried out on the cell culture specimens. Data were analyzed using SPSS Software [SPSS for and ANOVA test as well as Chi-square test. Out of 63 collected specimens, enteroviruses were isolated from 33 specimens [52.38%] and 41[65.01%] specimens which utilized cell-culture and ICC-RT-PCR methods respectively. Polioviruses were also isolated from 6 specimens. Statistical analysis indicated that there was a significant relationship [0.05 level] between cell culture and ICC-RT-PCR methods to isolate enteroviruses. Further the sensitivity of ICC-RT-PCR method to detect enteroviruses less than 0.01 TCID 50 was evaluated, which indicated that this method is acceptable and sensitive enough to detect enteroviruses in sewage

Distinguishing between primary infection and reinfection with rubella vaccine virus by IgG avidity assay in pregnant women

During the mass measles/rubella vaccination campaign in 2003 in Iran, many pregnant women were vaccinated mistakenly or became pregnant within 1 month of vaccination. To distinguish pregnant women who were affected by rubella vaccine as primary infection from those who had rubella reinfection from the vaccine, serum samples were collected 1-3 months after the campaign from 812 pregnant women. IgG avidity assay showed that 0.3% of the women had no rubella-specific IgG response; 14.4% had low-avidity anti-rubella IgG and were therefore not immune to rubella before vaccination; 85.3% had high-avidity antirubella IgG and were regarded as cases of reinfection

Environmental surveillance of Polio and Non-Polio Enteroviruses in Sistan and Balouchestan Province

Enteroviruses can easily circulate in the population through sewage and they are suitable indicators for environmental surveillance. On the other hand, in some countries there are evidences of silent circulation of viruses in sewage specimens despite no virus isolation from clinical specimens. Therefore, WHO has suggested environmental surveillance using surface water and sewage specimens for final confirmation of Poliovirus eradication. In this research, according to wild Poliovirus circulation in Afghanistan and Pakistan and probability of virus entrance to Iran, and also to assure wild Poliovirus eradication, the environmental surveillance was performed in Sistan and Balouchestan Province of Iran. From March 2004 to February 2005, 86 specimens from 2 sewage disposal systems, 5 hospitals and surface water from several villages were collected by Grab Sample method and tested for Enteroviruses directly and using 2 concentration methods: Pellet and Two-phase. Then Poliovirus and Non-Polio Enteroviruses [NPEV] were serotyped by microneutralization method and Polioviruses were intratypically differentiated using ELISA and Probe Hybridization techniques. From a total of 86 specimens, Enteroviruses and Non-Polio Enteroviruses were isolated from 49[56.98%] and 46[53.49%] of specimens respectively. Polioviruses were isolated from 18[20.93%] specimens and none of them was wild Poliovirus fortunately. 13[17.81%], 39[53.42%] and 57[78.08%] of enteroviruses were isolated using Direct, Pellet and Two-phase methods, respectively. The results of this research confirm the validity of environmental surveillance and Polio eradication in Sistan and Balouchestan Province

Distinguishing between primary measles infection and vaccine failure reinfection by IgG avidity assay

In this study in the Islamic Republic of Iran 365 measles cases were evaluated to distinguish between primary infection with measles and reinfection due to secondary vaccine failure. All cases previously confirmed by detection of specific IgM were tested for IgG avidity. A secondary immune response was seen in 18.4% of patients. All unvaccinated patients [16.7%] showed a primary immune response. Of 244 patients with documented vaccination, 75.8% showed a primary immune response and 24.2% showed a secondary immune response, thereby indicating a secondary vaccine failure. Almost all measles reinfections [99%] were seen in patients >10 years old, indicating that vaccination for 10- year- old children is recommended

[Assessment of different methods to remove organic inhibitors in order to create a sensitive method for direct surveillance of enteroviral infectious diseases in sewage specimens]

Enteroviruses in sewage are considered among the most sensitive indicators for virus circulation in society. These are mainly detected by sensitive cell cultures, however, since it is time consuming, molecular direct methods have also been considered as sensitive techniques. This study accomplished to assess different methods of removing organic inhibitors of sewage in order to detect Enteroviruses with RT-PCR. For this cross sectional study, 63 sewage specimens of Tehran were prepared with Grab sample method and concentrated with Pellet and Two-Phase methods and cultured in RD and Hep-2 cells. Then, with 12 different methods, removing organic inhibitors in sewage with RT-PCR method was assessed. Of investigated methods, we succeeded to isolate all of expected viruses only with the use of ICC-RT-PCR method. With respect to 0.01 TCID50 sensitivity of ICC-RT-PCR method, further studies are strongly recommended in order to confirm the utility of this method as one of the most sensitive methods of detecting Enteroviruses

Poliovirus environmental surveillance in sistan-balouchestan province, isolation and identification of viruses by cell culture and microneutralization, and intratypic differentiation by ELISA and probe hybridization

In some countries, wild polioviruses have been isolated from environment despite the absence of viruses being recovered from clinical cases, therefore to confirm of final Polio eradication, WHO has recommended environmental surveillance using sewage specimens and surface water. During the present study, in order to assure the polio eradication in Iran, Sistan-Balouchestan province was chosen as the target area. During a 12-month period, 86 specimens from 2 sewage disposal systems and 5 hospitals, as well as surface water from several rural areas were collected by Grab Sampling and tested for polioviruses using direct and concentrated specimens with 2 concentration methods: Pellet and Two-phase. Then the isolated viruses were serotyped by microneutralization method and differentiated intratypically by ELISA and probe hybridization techniques. Of all studied specimens, 18 [20.9%] were identified as poliovirus, none of which were wild virus, fortunately. Among these, 2 [2.3%], 8 [9.3%] and 13 [15.1%] were isolated from direct specimens, Pellet and Two-phase concentrated specimens, respectively. The most frequent viruses were Polio 2 [72.2%] and Polio 3 [27.8%]. Results have revealed the efficacy of immunization coverage in Iran. Meanwhile, sufficient surveillance programs have been observed during the recent years

Influenza surveillance in the Islamic Republic of Iran from 1991 to 2001

To better underst and the annual distribution of influenza virus in our country, we isolated and typed 45 viruses from 1043 patients with acute respiratory illnesses in a 10-year study conducted by the National Influenza Centre of the Islamic Republic of Iran. The seasonal distribution of influenza typically ran from November to April. Type A influenza was most common during the winters of 1991-92, 1997-98 and 2000-01 and type B influenza was most common during 1992-5 and 1996-97. Both type A and type B viruses circulated in 1995-96 and 1998-2000. A serological survey based on haemagglutination inhibition test confirmed our findings. The annual pattern of strains isolated was similar to the worldwide pattern during the same interval

Prevalence of human papillomavirus in Mazandaran Province, Islamic Republic of Iran

We report the prevalence of human papillomavirus [HPV] types in 100 cervical biopsy specimens in Mazandaran province. HPV DNA was detected in 78.6% of cervical carcinoma cases, 64.3% of dys/ metaplasia and 9% of normal cases. Significant correlation was found between the presence of HPV DNA and development of cervical carcinoma. HPV types 16 and 18 were detected in 60.6% of HPV-positive cervical carcinoma cases, whereas HPV31 and 33 were found in 21.2%, and HPV6 and 11 in 18.2%. Among HPV-positive dys/metaplasia cases, 55.6% were positive for HPV16 and 18, 22.3% for HPV6 and 11, and 11.1% for HPV31 and 33. Only HPV6 and 11 were detected in 4 [100%] normal biopsy specimens