The Prevalence of Neisseria gonoroheae among Iranian Women by Phenotyping and Genotyping Methods

Sexually transmitted disease (STD) consist of Neisseria gonoroheae, Chlamydia trachomatis, Syphilis and Trichomonyasis are a major public health concern. The prevalence of these organisms are increasing level because of homosexuality, migration and resistance to antibacterial agents and also usage of high technique for diagnosis of infection, according to WHO information 88 million people from 448 million which infected with STD, is Neisseria gonoroheae, whereas, Iran as a developing country have not complete prevalence information about Neisseria gonoroheae. In this study it has been tried to estimate the prevalence of Neisseria among Iranian women with phenotypin and genotyping methods. 300 specimens were enrolled in the present investigation which collected from women with genital problems. Dacron swab samples were cultured in chocolate agar in 37°C with 5-10% CO2 for 24-48 hours. Gram staining, oxidase and carbohydrate utilization tests were used to identify the isolated species. Extraction of DNA of all isolates recognize as Neisseria gonoroheae. PCR for amplification of 390 bp fragment of CPPB plasmid for confirmation of isolates were performed. From 300 specimens, 7 isolates recognize as Neisseria gonoroheae with culture and PCR methods. It means N. gonorrhoeae was detected in 2.6% cases which enrolled in this study. The study revealed that although primary report found no prevalence of gonorrohea in Iran but other study revealed different prevalence of bacteria in Iran, even in the recent studies, the results are very near to this result with consideration that gonoccocal infection have different sequelle in young age or pregnant women so it is very important that with culture or PCR methods, screening of bacteria should be done.

The Neisseriae are gram-negative cocci that usually occur in pairs (diplococci).Neisseria gonorrhoeae (gonococci) is pathogenic for humans and typically are found associated with or inside polymorphonuclear cells.Gonococci attack mucous membranes of the genitourinarytract, eye, rectum, and throat, producing acute suppuration that may lead to tissue invasion, this is followed by chronic inflammation and fibrosis.inwomen cuses genitourinary tract infection with sequel of pelvic inflamatory disease,ectopic pregnancy 1,2 .
The prevalence of organism has increasing level because of homosexuality, migration and resistance to antibacterial agents, according to WHO information ,88 million people from 448 million which infected with STD is Neisseria gonoroheae, from 1990s th prevalence TORABIZADEH et al.: PREVALENCE OF N. gonoroheae AMONG IRANIAN WOMEN grow up in most countries of the world , but differs from one country to another (3,4),information of iran is spars and spread , so in this study it has beeb tried to investigate the perevalenc of gonoroheae in 3 groups of women,group 1 with cervicitis,group 2 reffer for (D&C) and group 3 women with spontaneous abortion.inorder to have more and correct information about the prevalence of Neisseria gonorrhoeae with using of phenotyping (culture )and genotyping( PCR) methods.

MATERIALS AND METHODS
During a 20 months study from September 2013 to March 2014, specimens were collected from 300 patients with genital problems who reffered to university of shahid beheshtie, Hospitals, Tehran,Iran.thespecimens consist of 3 groups of women Group 1: women with vaginal discharge ,abdominal pain ,bleeding (Cervicitis) .Group 2: women who reffered to hospital for (D&C).Group 3: pregnant women who reffered for spontaneous abortion .The mean age of group 1: was : 36year , group 2: 39year , group 3:27year.
Specimens were collected with 2 Dacron swab for each patients, one for stuart medium , the other for gram staining.All smear of gram stain were seen for extracellular or intracellular Gdiplococcic.Specimens in stwart medium were transport to chocolate agar and were placed in 37°C incubator, CO2 5% for 24-48 hours.Then oxidase test were done , for suspicious colonies utilaztion of sugars(glucose,lactose,maltose and sucrose) were used.(Table1,2).
For confirmation of isolates which recognize as Neisseria gonoroheae, extraction of 4.2 kb CPPB plasmid (5,6)

DISCUSSION AND CONCLUSION
This study sought to understand the prevalence of Neisseria gonoroheae among Iranian women who have genital problems by using of phenotyping(culture) and genotyping( PCR) methods.The result revealed that in 3 groups of women the prevalence of Neisseria gonoroheae was: group 1: 2%, group 2 :2%,and group 3:4%.The mean of 3 groups was 2.6%.according to WHO information.The prevalence of Neisseria gonorroheae has an increasing level from 1990s and differs from one country to another,also these informations indicate that the prevalence of gonoroheae between 2005 to 2008 grow up to 21% (4).In USA, overall rate of gonorroheae was 111.6 per 100000 population between 1989-2008.(5,7).In Africa there are a reviw article which consist of studies about prevalence of STD infection among African men and women ,they reports the prevalence of Neisseria gonoroheae in women 5%, and in high risk groups like sexworkers and women attending STIs clinics was between (10-31%).(6,8).
In Europe, a study has done with conributation of 12 European countries, an increasing rate in prevalence of N.gonorroheae observed from 1995 to 2002.In London ,wales and Northern Ireland gonorroheae diagnosed more than doubled between 1996 and 2001, in a study in Austria there was 414cases of N. gonorroheae increase to 995cases in 2002.withincreasing rate both in men and women (7,9).
In neighborhood countries,like Turkey the Fahriye et al by using PCR found that the prevalence of gonorohae was3.7% among 370 endocervical samples (7,10), other study which has done in Palestine by using PCR and 213 endocervical specimens, revealed that the prevalence of gonoroheae was 1.4% (8,12), the other study in Bangladesh done by rahman et al ,found that prevalence of gonorroheae in women with vaginal discharge was 3.8% , (9,13).
Studies which have done in recent years In Iran, cleared that in north west of Iran Zanjanprovince the prevalence of gonoroheae was o.9% by using culture methods among pregnant and impregnant women.(10,14), in Babol north of Iran the prevalence was 0.2% by culture method (11,15), in Sabzevar the prevalence was o.9% by using triplex PCR (12,16), in Kermanshah west of Iran,the overall incidence of gonoroheae was 2.4%among 255 married women BY doing PCR.( 13,17),the other study which has done in Kashan found the prevalence of gonorrohae 2.38 % among 294 married women and using culture

Table 1 .
Result of Oxidaze test in3 groups

Table 2 .
Result of utilization of sugars in Neisseria gonoroheae according to table 2 revealed that 7 isolates were Glucose positive,Lactose ,Maltose and Sucrose negative.whichconfirm that there were 7 isolates of Neisseria gonoroheae with phenotyping methods.Also with genotyping and molecular method and amplification of CPPB plasmid there were 7 isolates with 390bp band in PCR ,which confirm of 7 isolates as Neisseria gonoroheae.So in our study with phenotyping and genotyping methos there were 7 isolates of Neisseria gonoroheae.

Table 3 .
(15,uency of Neisseria gonoroheae among 3 groups(15,19),their result is oppose to other study which has done in iran , different result could because of the source of specimen which they selected, it is likely that vaginal and genital discharge are better source for searching bacteria in women than urine.with consideration that young women and female adolescents are more susceptible to STIs compared to the male due to their anatomy , so infection in women could have different sequle like ectopic pregnancy or pelvic inflammatory disease (PID),it is better to culture of vaginal discharge become a routin test for women especially in young age for diagnosis and treatment of gonococcal infection .