Prevalence and Antimicrobial Susceptibility Pattern of Neisseria Gonorrhoeae from Selected Public Health Centers, Addis Ababa, Ethiopia


 Background: Neisseria gonorrhea (NG) is gram negative diplococcic bacteria that cause most important sexually transmitted infections (STIs) worldwide. In most developing countries including Ethiopia, NG infections are diagnosed syndromically, and its antibiotic susceptibility is rarely tested. Thus, the objective of the present study was to determine the prevalence and antimicrobial susceptibility patterns of N. gonorrhoeae among syndrome diagnosed and treated cases attending selected public health centers, in Addis Ababa, Ethiopia.Methods: a cross-sectional study was conducted from April 2019 to March 2020, at selected health centers STIs clinics in Addis Ababa, Ethiopia. Urethral discharge or vaginal discharge specimens were cultured on Modified Thayer Martín media and suspected gonococcal colonies were confirmed using Oxidase, Superoxol tests. Antimicrobial susceptibility testing was performed by Minimum Inhibitory Concentration (MIC) using concentration gradient strips (E-test) of the same antimicrobial agents of ciprofloxacin, penicillin, Ceftriaxone, Azytromicin, Cefoxitin, and SpectinomicinResults: Among the total 325 NG suspects 163 (50 %) were culture positive. Among the culture positive cases 126 were male and 37 females. Around 4% of isolates are non-susceptible to Ceftriaxone which is the current antimicrobial agent used for urethral discharge/ vaginal discharge syndrome treatment.Conclusion: In the current study, the proportion of N. gonorrhoeae culture positivity rate among male was found to be high. The antimicrobial agent ceftriaxone used for the treatment of gonococccal isolates found to become non susceptible. Antimicrobial agents outside of ciprofloxacin and natural penicillin antimicrobial agents are eligible for the national STI syndromic management.


Introduction
Neisseria gonorrhea (NG) is gram-negative intracellular diplococci, non-capsulated & Oxidase positive organisms. N. gonorrhoeae is one of the most common sexually transmitted diseases in developing countries (1). According to World Health Organization (WHO) estimate in 2016, around 87 million new gonorrhea infections recorded worldwide (2). The bacteria can reside at different anatomical sites such as the urethra, endocervix, pharynx, or rectum and transmitted from infected host to the other during unsafe sexual intercourse (3,4).
Syndromic management of sexually transmitted infections are becoming increasingly challenging due to antimicrobial resistance (AMR) to various classes of available antibiotic therapy (5). In the syndromic management of NG, ceftriaxone is the last remaining option for empirical first-line antimicrobial monotherapy, in most part of the world. (6). However, NG developing resistance to ceftriaxone at different regions worldwide with the proportion of varying extensively, from 1.3% to 55.8% (7). Therefore regular assessment and updates on prevalence of NG, as well as antimicrobial sensitivity pattern of the bacteria so important, mainly for those developing courtiers including Ethiopia, which practiced only sydromic diagnosis and treatment of the diseases. Besides, in order to control and prevent occurrence of NG prevalence and antimicrobial worldwide, such kind of studies are so essential.

Study Area
This research was carried out in three health centers; Janmeda health center woreda 06, Arada health center woreda 10, and Gerji health center woreda 13, Addis Ababa, Ethiopia. The selected health centers are administered under the Addis Ababa Health Bureau in Addis Ababa, Ethiopia.

Study Design and Period
A cross-sectional study was conducted to compare the Syndromic approach versus etiologic based approach on STI at a selected Health center in Addis Ababa, Ethiopia, from April 2019-March 2020.

Source Population
All clients who visited in selected health center outpatient department, OPD for medical service with a compliant of sign and symptoms of urethral /vaginal discharge.

Study Population
The study populations were patients in the selected health facilities of Addis Ababa who were participated in the study. All Out Patient Department (OPD) clients for STI according to WHO standard Syndromic approach STI management guideline between April 2019-March 2020 were included in this study.

Inclusion Criteria
Clients who visited the study site OPD and have a history of urethral /vaginal discharge Neisseria gonorrhoeae and willing to participate in the study.

Exclusion Criteria
Clients who have no signs and symptoms of Neisseria gonorrhoeae and patients having a history of taking antibiotics for the past 10 days are to be excluded.

Sample Size Calculation and Sampling Method
In this study sample size was calculated by using single population proportion formula with a con dence interval of 95%and5%marginal error by taking prevalence of the case(74.1%)from a study conducted at Gondar town hospital and health centers, Ethiopia (8). And by adding 10% of calculated sample sizes for contingency, nally, the sample size was determined to be three hundred twenty-ve. And convenience sampling method is used (9, 10).

Sample collection
Appropriate ureteral and vaginal discharge and blood (serum) samples was collected by a trained clinical nurse. The ureteral and vaginal discharges were transported by Amies with charcoal transport media.

Laboratory Analysis
A sterile cotton-tipped swab was used to obtain swab specimens. Then sterile Dacron swabs tipped applicator were used to collect urethral secretions. The swabs were inoculated on in-house prepared Modi ed Thayer Martin Agar plates made of Gonococcal agar base supplemented with isovitalex (vitox); vancomycin, colistin, nystatin, and trimethoprim (VCNT); and synthetic hemoglobin (Oxoid and BBL).The inoculated plates were incubated on the site using candle jar and transported to the Ethiopian Public Health Institute (EPHI), Clinical Bacteriology and Mycology Reference Laboratory within the same day of collection. Swab was rolled onto a microscopy slide, labeled, heat xed, placed in a slide box and sent to EPHI for Gram-stain analysis. Gram stain was performed to look for polymorph nuclear leucocytes with Gram-negative intracellular diplococci microscopically. Then the isolates were identi ed as Neisseria gonorrhoeae by presumptive diagnosis method based on colony morphology, Gram staining, catalase test, Oxidase test, and superoxide test.

Culture and identi cation:
In the clinical bacteriology laboratory, inoculated plates were incubated at 35 o C in carbon dioxide enriched environment (5-8 % CO2) for 72 hours inspecting every day for the growth of small, translucent and non-pigmented colonies. Gram negative diplococcic convex, glistening, elevated, mucoid colony characteristics and oxidase, catalase and supercool (30% H2O2) positive were considered as probable N. gonorrhoeae. Antimicrobial susceptibility testing was performed by on enriched GC agar (Oxoid Ltd) plus 1% BBL Isovitalex Enrichment. Minimum Inhibitory Concentration (MIC) was done using concentration gradient strips (E-test) of the same antibiotics. The range of inhibition zones and MIC for each type of antibiotic disk were interpreted according to Clinical Laboratory Standard Institute (CLSI) guideline (11). N. gonorrhoeae reference strain ATCC 49226 was used a positive control.

Data Analysis and Interpretation
For data entry and analysis, SPSS (the Statistical Package for Social Sciences for Windows) version 23 statistical software was used. Bivariate analysis was used to determine Socio-demographic At the enrollment visit, all participants with urethral discharge or vaginal discharge were given written consent diagnosed according to the syndromic treatment guidelines currently approved in Ethiopia. Those who are eligible (> 18 years of age) and willing to participate in the study were asked using structured questionnaire for their demographic and behavioral data. All data were kept con dential anonymously. Participants were informed of their rights to resign from the study at any level of the study without any restriction if they were feeling uncomfortable in the study. Their results of all positive individuals were reported to the respective health facilities for better management of the patients.

Socio-demographic characteristics
A total of 325 STI symptomatically diagnosed and treated patients were enrolled in the study, and of them 167 (51.4%) were males. The majority of the study participants were between the ages of 26 and 35. (54.5 %).According to the marital status analysis, the majority of people were single 178(54.8%); whereas only 19 (5.8%) were illiterate. Out of 325 participants, one hundred seventy eight (54.8%) were singles in their marital status. Table1.
Of all the specimens analyzed, only 163 (50 %) gonococcal isolates were identi ed. Among these gonococcal isolates 126 were male and 37 females. Table 2.
The present study has revealed that N. gonorrhoeae isolates recovered from this study have shown high level of resistance to natural penicillin and uoroquinolone class of antibiotics but best susceptible for cephalosporin, macrolides, and spectinomycin in Ethiopia. Figure1.

Discussion
The present study showed that among sydromic diagnosed cases, more than half of participants had laboratory-con rmed gonococcal infection. A similar community based Syndromic versus Laboratory Diagnosis of Sexually Transmitted Infections study conducted in Northern Tanzania, indicated that of the total participated 1,520 women, gonorrhea was con rmed in 0.2 % cases (12). Another study conducted in India indicated that out of 1120 Syndromic STI cases only 409 (36.5%) cases were con rmed in the laboratory for pathogens (13). Another study conducted in Jimma (western Ethiopia) private clinics indicated that of the total l315 samples examined, 31(9.8%) were confirmed to have gonococcal infection (14). The ndings from the present study compared with others has relatively high culture positivity rate of gonococcal etiologies. The difference might be due to, the study setting, study participates (more male participants), laboratory diagnostic approaches, and disease prevalence of the study areas. In this study the culture positivity rate compared with other studies it has high prevalent. The difference might be nature of participants in this study. In this study more participants were male and the positivity rate from urethral discharge higher than vaginal discharge (7).
The present study has revealed that N. gonorrhoeae isolates recovered from this study have shown high level of resistance to commonly prescribed uoroquinolone class of antibiotics in Ethiopia. This nding is in agreement with other study conducted previously in Ethiopia which reported a resistance level of 67% (15). The Non-susceptibility to ceftriaxone which is used for the national N. gonorrhoeae treatment was detected in 5% all of the isolates tested during the study period. This nding is in agreement with other studies conducted in different part of Ethiopia (16, 17, and 18). This indicated that the resistance is increasing through time compared with the susceptibility pattern of this antimicrobial agent six years before (. Antimicrobial agents tested in this study except cipro oxacin and natural penicillin have susceptibility pattern of greater than 95% which could be used for the treatment of syndromic management.

Conclusion
In the current study, the proportion of N. gonorrhoeae culture positivity rate among male was found to be high. The antimicrobial agent ceftriaxone used for the treatment of gonococccal isolates found to become non susceptible. Still the antimicrobial agent is competent agent for the syndromic management of urethral / vaginal discharge syndrome. Antimicrobial agents outside of cipro oxacin and natural penicillin antimicrobial agents are eligible for the national STI syndromic management. The data found in this study could be input for the national syndromic management.