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

BACKGROUND AND OBJECTIVES: 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 Enterovirus types in addition to being time consuming. For these reasons, an attempt was made to find better techniques of Enterovirus 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. MATERIAL AND METHOD: 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 Enterovirus detection using the RT-PCR technique. RESULTS AND CONCLUSION: 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.

Sequence and phylogenetic analyses of neuraminidase gene of Iranian seasonal influenza H1N1 viruses from 2005-2009 and corresponding vaccine strains.

Prophylaxis of influenza A virus infections is based on the vaccines inducing antibodies to the major viral antigens, hemagglutinin (HA) and neuraminidase (NA). Since these antigens continuously change during virus replication in various hosts, only the currently circulating strains should be used in the vaccines. Besides, monitoring of the naturally occurring changes in HA, NA, and respective genes, especially those associated with resistance to the NA inhibitors is necessary. The NA genes of 30 Iranian isolates of influenza H1N1 virus from the seasons 2005-2009 were sequenced and subjected to the sequence and phylogenetic analyses. The seasonal isolates turned out to be closely related to the corresponding vaccine strains, except for the 2007-2008 isolates, which also displayed a higher nucleotide variation. A resistance to the NA inhibitors was found in the 2008-2009 isolates only. The average nucleotide identities of the isolates with corresponding vaccine strains for the years 2005-2009 were 98.83%, 98.55%, 98.7%, 97.55%, and 98.76%, respectively.

Amantadine-resistant influenza A (H3N2) viruses in Iran.

Adamantanes have been used for the prophylaxis and treatment of Influenza A virus (IAV) infections worldwide. However, they have limited use because of increasing number of resistant viruses during recent years. In investigating the frequency of amantadine-resistant IAVs (H3N2) circulating in Iran in 2005-2008, we found that M2 sequences of recently circulating viruses that were amantadine-resistant contained a Ser31Asn mutation. Thus, adamantanes should not be used for treatment or prophylaxis of recent IAVs (H3N2) infections. In future, their potential use will depend on the resistance of circulating viruses.

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 anti-rubella IgG and were regarded as cases of reinfection.

Direct identification of non-polio enteroviruses in residual paralysis cases by analysis of VP1 sequences.

BACKGROUND: The 66 serotypes of human enteroviruses (EVs) are classified into four species A-D, based on phylogenetic relationships in multiple genome regions. Partial VP(1) amplification and sequence analysis are reliable methods for identifying non-polio enterovirus serotypes, especially in negative cell culture specimens from patients with residual paralysis. OBJECTIVES: In Iran during the years 2000-2002, there were 29 residual paralysis cases with negative cell (RD, HEp(2) and L(20)B) culture results. STUDY DESIGN: The genomic RNA was extracted from stool specimens from cases of residual paralysis and detected by amplification of the 5'-nontranslated region using RT-PCR with Pan-EV primers. Partial VP(1) amplification by semi-nested RT-PCR (snRT-PCR) and sequence analysis were done. RESULTS: Specimens from the 29 culture-negative cases contained echoviruses of six different serotypes. CONCLUSIONS: The global eradication of wild polioviruses is near and study of non-polio enteroviruses, which can cause poliomyelitis, is increasingly important to understand their pathogenesis. The VP(1) sequences, derived from the snRT-PCR products, allowed rapid molecular analysis of these non-polio strains.

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

Characterization of mutations in the VP(1) region of Sabin strain type 1 polioviruses isolated from vaccine-associated paralytic poliomyelitis cases in Iran.

BACKGROUND: The live-attenuated oral polio vaccine used to interrupt poliovirus transmission is genetically unstable. Reversion of some attenuating mutations, which normally occurs during vaccine strain replication in some recipients, and can rarely cause vaccine-associated paralytic poliomyelitis (VAPP). The poliovirus eradication program designed by the World Health Organization (WHO) includes immunization with OPV in addition to careful surveillance of all acute-flaccid paralysis (AFP) cases. OBJECTIVES: In Iran we last isolated imported wild poliovirus in 2000 and the immunization coverage was 100% in 2002. During 2001, there were three AFP cases with residual paralysis from which Sabin-like type 1 polioviruses were isolated in our national polio laboratory. STUDY DESIGN: The complete VP(1) region of the three isolates was sequenced and amino acid substitutions associated with these neurovirulent isolates were recorded. RESULTS: These isolates had either 4, 2 or 1 nucleotide substitution(s) in the VP(1) region, corresponding to amino acid change in the VP(1) of isolate 1 of either (H-[149]->Y), (T-[106]->A) or (I-[90]->L), respectively. CONCLUSIONS: Surveillance of the VAPP cases in countries where endemic transmission has recently ceased increases our understanding of the important neurovirulent mutations in vaccine-strain isolates and assists in planning the next step in the eradication program in these countries.

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.

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

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

To better understand 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.

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.

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