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

Roya Torabizadeh^1,2*, Gita Eslami², Mohammad Hossein Dehghantarzejani³, Zahra Zahirnia² and Malihe Habibi²

¹Departement of Medical Microbiology, Alborz university of Medical Sciences, Karaj, Iran.
²Departement of Medical Microbiology, School of Medicine, Shahid Beheshti University of Medical Sciences, Tehran, Iran.
³Departement of Medical Biochemistry, Alborz University of Medical Sciences, Karaj, Iran.

ABSTRACT

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, wherase 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 this study which collected from women with genital problems Dacron swab samples were cultured in chocolate agar in 37°C with 5-10% CO2 for 24-48hours. Gram staining, oxidase and carbohydrate utilization tests were used to identify the isolated species. Extraction of DNA of All isolates recognize as Neisseria gonoroheae were done, PCR for amplification of 390 bp fragment of CPPB plasmid for confirmation of isolates were performed. From 300 specimens, 7 isolates recognize as 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 differrent sequelle in young age or pregnant women so it is very important that with culture or PCR methods, screening of bacteria should be done.

Keywords:  Phenotyping and Genotyping methods, STD.

Introduction

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.in women 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 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.in order 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,s Hospitals,Tehran,Iran.the  specimens  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  stwart medium , the other for gram staining.All  smear of gram stain  were seen for extracellular or intracellular G- diplococcic.   specimens  in stwart medium were transport to chocolate agar  and  were placed in 37^οC 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)by Nano  plus plasmid Extraction kit BioNEER  were done,  PCR were performed with following format:

Primer Forward:5′ GCT ACG CAT ACCCGC GTT GC3′
Primer Reverse:5′ CGA AGA CCT TCG AGC AGA CA3′
PCRprotocol :Primer forward=1µl,primer revers1µl,DNAtemplate3
µl,Mastermix12 µl, ,8µlH20 →   Totalvolume =25µl
(94 ⁰c 30sec,55⁰c 1min,74⁰c 30sec)94⁰_{C 5min}
Lenght of product was 390bp by using 2% agaroze gel.

So the isolates which, with culture methods were Neisseria gonoroheae ,with molecular method were confirmed  as Neisseria gonoroheae too.

Results

300 specimens were  enrolled in this study consist of 150 cervicitis,100 D&C,50 spontanous abortion ,53 specimens were Oxidase and  Catalase  positive.The result of utilization of sugares according to table 2 revealed  that 7 isolates were Glucose positive,Lactose ,Maltose and Sucrose negative.which confirm 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.

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 1995to 2002.In London ,wales and Northern Ireland gonorroheae diagnosed  more than doubled between 1996and 2001,in a study in Austria there was 414cases of  N .gonorroheae increase to 995cases in 2002.with increasing 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  370endocervical 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 method(14,18) these studies which has done in recent years have similar and near result, specially the result of afrasiabi,(kashan ) and akya  (Kermanshah ) confirm our study  ,but in a study which performed by Batool  Hossein Rashid et al ,in Tehran,they examined  209 infertile women as cases and 170 pregnant women as controlle by using PCR for detection bacteria in urine ,they found no gonorroheae infection among the society which were selected(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 .

Table 1. Result of Oxidaze test in3 groups

Oxidaze pos	Oxidaze Neg	G – diplococci	Number	Specimen
23	29	52	150	Vaginal discharge
14	8	22	100	D&C
16	3	19	50	Spontaneous abortion

 

Table 2. Result of utilization of sugars in Neisseria gonoroheae

	Sucrose	Maltose		Lactose		Glucose		Oxidase pos specimen
20+	2-	8-	16+	2 –	20+		23+	Group 1=22
12+	2-	4-	10+	2 –	12+		14+	Group 2=14
14+	2-	2-	14+	2 –	14+		16+	Group 3=16

group 1=vaginal discharge
group 2=D&C
group3=spontaneous abortion

Table 3. Frequency of Neisseria gonoroheae among 3 groups

Frequency=%	Positive for Neisseria gonoroheae	Total number	specimen
2%	N=3	150	group1= vaginal discharge
2%	N=2	100	group 2= D&C
4%	N=2	50	group3= spontaneous abortion

 

390bp band of Neisseria gonoroheae

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