Prevalence of antimicrobial resistance of neisseria gonorrhoeae in national hospital of dermatology and venereology in 2017 – Le Van Hung

JMR 116 E3 (7) - 2018 125 JOURNAL OF MEDICAL RESEARCH PREVALENCE OF ANTIMICROBIAL RESISTANCE OF NEISSERIA GONORRHOEAE IN NATIONAL HOSPITAL OF DERMATOLOGY AND VENEREOLOGY IN 2017 Le Van Hung, Nguyen Phuong Thuy, Le Ha Long Hai Hanoi Medical University The antimicrobial resistance of Neisseria gonorrhoeae is a complicated problem that causes many diffi- culties for diagnosis and treatment. Thus, more research into all aspects of antimicrobial resistance of N. gonorrhoeae is necessary. Objectives: 1. To determine the prevalence of N. gonorrhoeae infections in pa- tients with urethral and/or vaginal discharge syndrome, who attended National Hospital of Dermatology and Venereology in 2017. 2. To describe the antimicrobial resistant levels of isolated N. gonorrhoeae. Methods: This was a cross sectional study. N. gonorrhoeae was isolated from pus or discharge of 1582 patients with urethral and/or vaginal discharge syndromes. Isolates were determined by using Gram staining, culture, and identification test, followed by antimicrobial susceptibility method. Minimum inhibitory concentration (MIC) was performed to differentiate the between resistant and susceptible isolates. Results: The prevalence of N. gonorrhoeae in patients with urethral and/or vaginal discharge syndrome was 12.45%; the isolates of N. gonorrhoeae showed resistance to ciprofloxacin (97.97%), nalidixic acid (85.79%), penicillin (45.69%) and tetracycline (85.28%). N. gonorrhoeae was susceptible to spectinomycin (100%), azithromycin (99.49%), cefotaxime (99.49%), cefixime (99.49%) and ceftriaxone (98.98%). As for MIC results: 7/12 isolates showed its resistance to ciprofloxacin (MIC ≥ 1 àg/ml), 7/10 to tetracycline (MIC ≥ 2 àg/ml), 5/10 to penicillin (MIC ≥ 2 àg/ml), 2/17 to ceftriaxone (MIC ≤ 0,25 àg/ml) và 100% of isolates were susceptible to spectinomycin (MIC ≤ 32 àg/ml). Conclusion: Ceftriaxone, spectinomycin, azithromycin, cefixime and cefotaxime were recom- mended for N. gonorrhoeae treatments while ciprofloxacin, cefuroxime, tetracycline and penicillin were not. Keywords: Neisseria gonorrhoeae, antimicrobial resistance Corresponding author: Le Van Hung, Hanoi Medical University Email: levanhungvdl@yahoo.com Received: 9/7/2018 Accepted: 22/11/2018 I. INTRODUCTION Neisseria gonorrhoeae infection is a com- mon sexually transmitted disease (STD). Ac- cording to The World Health Organization re- port, it was estimated there were 106 million new cases of gonorrhea in 2012 [1]. Each year at the National Hospital of Dermatology and Venereology, more than 20% of all STD cases are diagnosed as gonorrhea. Gonorrhea may spread within a community and cause many different genitourinary tract complications, even infertility, if it is not correctly diagnosed and treated in time. The antimicrobial resis- tance of Neisseria gonorrhoeae is a major topic of interest due to the appearance of the multiple drug resistant species that can resist current antibiotics used for treatment, which remains a major public health concern world- wide [2]. In Vietnam, using antibiotics without prescription or antibiotics abuse in community and health stations also may increase the an- timicrobial resistance of N. gonorrhoeae, which cause the difficulties for diagnosis and treatment. Thus, we performed this study to investigate the prevalence of N. gonorrhoeae infections in patients with urethral and/or vagi- nal discharge syndrome who attended Na- 126 JMR 116 E3 (7) - 2018 JOURNAL OF MEDICAL RESEARCH tional hospital of Dermatology and Venereol- ogy in 2017 and to describe the antimicrobial resistant levels of isolated N. gonorrhoeae. II. METHODS 1. Subjects This study included all patients with ure- thral and/or vaginal discharge syndrome who visited the National Hospital of Dermatology and Venereology, Hanoi, Vietnam in 2017. Patients with AIDS or using antibiotics within 7 days were excluded from this study. A total of 1582 male and female patients agreed to par- ticipate in this study. 2. Materials Thayer-Martin agar (Oxoid) was used for Neisseria gonorrhoeae isolation. This agar medium is a selective medium used for the isolation of Neisseria gonorrhoeae due high nutritional content suitable for N. gonorrhoeae growth. This medium includes the supplemen- tary (Iso Vitalex) and the inhibitors (VCN- vancomycin, colistin, nystatin) which can sup- press other gram-negative diplococci, gram- negative bacilli, gram-positive organisms, and yeast. Remel BactiCard Neisseria test kit (Thermo Scientific) were used for N. gonorrhoeae iden- tification. This test uses enzyme technology for identification of pathogenic Neisseria spp. (including N. gonorrhoeae, N. meningitis and N. lactamica) and Moraxella catarrhalis iso- lated on selective media. - Antibiotic disks (Oxoid) were used for determination of antimicrobial susceptibility. - E-test (Abbiodisk, Sweden), a quantitative technique, was used to determine the mini- mum inhibitory concentration (MIC). 3. Methods: A cross-sectional study Sample collection: (patients must be in- formed and explained prior sample collection). In women, samples were collected from urethra, cervix, 2 sides of Bartholin’s Gland, and 2 sides of Skene’s Gland. For cervical samples, distilled water was used to pre-wet the vaginoscope. Next, the vaginoscope was inserted into the vagina to open the cervix, then samples were obtained by inserting a sterilized swab approximately 2 - 3 cm in the cervix and keeping for 5 - 10 seconds to ab- sorb cervical discharge. The swab was pulled out without touching vaginal wall, followed by making smear for gram staining, and culturing in Thayer-Martin agar with VCN inhibitors. Male patients were required to block urination 2 - 3 hours before sample collection. Sodium Chloride 0.9% was used to pre-wet a sterilized swab. Sample was collected by inserting the swab approximately 1.5 - 2 cm in urethral meatus, rubbing against the inner wall and keeping for 5 - 10 second to absorb discharge. The swab was pulled out, followed by making smear for gram staining and culturing in Thayer-Martin agar with VCN inhibitors. Cultured plates were incubated at 36 - 37 0 C in a humid atmosphere (70% humidity) containing 3 - 10% carbon dioxide (CO2) for 18 - 24 hours. As for N. gonorrehoeae identification, cultured colony could be visualized in an S form, 1mm in diameter, mucus, gray and twinkle. Next, the cultured colony underwent gram staining and showed negative gram results, with a coffee bean shape, in and outside of neutrophils in the microscope field. Finally, a cultured colony was confirmed by JMR 116 E3 (7) - 2018 127 JOURNAL OF MEDICAL RESEARCH using oxidase test and Remel BactiCard Neisseria test kit [3]. For antibiotic sensitivity testing, N. gonor- rhoeae colony was cultured in Thayer-Martin agar without inhibitors, then incubated at 36 - 37 0 C in > 70% humidity containing 3 - 10% CO2 for 18 - 24 hours. Colony purification was rechecked by Gram staining. A suspension of 0.5 McFarland turbidity was made by mixing colonies with Sodium Chloride 0.9%, then cul- tured as monolayer in Thayer-Martin agar without inhibitors. The agar surface was kept dry up in room temperature. Next, 5 antibiotic disks were put on each 9cm diameter plate with a distance of 2 cm from plate wall and kept at room temperature for 10 minutes for the antibiotic diffusion. Cultured plates were incubated at 36 - 37 o C in > 70% humidity con- taining 3 - 10% CO2 for 18 - 24 hours. The zone of inhibition was measured by using mm ladder and interpreted according to The Clini- cal & Laboratory Standards Institute (CLSI) guideline in 2015 [4]. Minimum inhibitory concentrations (MICs) was performed by using E-test (Abbiodisk, Sweden). MIC is the lowest concentration of an antibiotic, which prevents visible growth of bacterium. In our study, MIC was performed for only N. gonorrhoeae with the zone of inhi- bition close to sensitive or resistant level. For example, Ceftriaxone ≥ 35mm was sensitive result, thus, those strains with the inhibition zone of 34 - 35 mm were examined for MICs test. III. RESULTS 1. The prevalence of N. gonorrhoeae infections in patients with urethral and/or vaginal discharge syndrome (n = 1582) Table 1. The prevalence of N. gonorrhoeae infections in patients with urethral and/or vaginal discharge syndrome Test results Gram staining (%) Culture (%) p value Positive 190 (12.01) 197 (12.45) 0.71 Negative 1392 (87.99) 1385 (87.55) Of 1582 patients with urethral and/or vaginal discharge syndrome, 197 (12.45%) were positive for N. gonorrhoeae. As compared to gram staining method, the detection rate of N. gonorrhoeae using culture method was higher (12.45% vs 12.01%) thought not significantly significant (p = 0.71). 2. Describe the antimicrobial resistant level of isolated N. Gonorrhoeae Of 197 N. gonorrhoeae isolates, 97.97% were resistant to ciprofloxacin, 85.79% to nalidixic acid, 85.28% to tetracycline, 45.69% to Penicillin and 1.02% to Ceftriaxone. No resistance to Spectinomycin was detected among isolates (Table 2). Of 197 N. gonorrhoeae isolates, 99.49% were sensitive to Azithromycin, Cefotaxim and Cefixim (Table 3). 128 JMR 116 E3 (7) - 2018 JOURNAL OF MEDICAL RESEARCH Antibiotics Abbreviation Sensitive Intermediate Resistant n % n % n % Penicillin PG 0 0 107 54.31 90 45.69 Spectinomycin SPT 197 100 0 0 0 0 Ciprofloxacin CIP 0 0 4 2.03 193 97.97 Ceftriaxon CRO 195 98,98 0 0 2 1.02 Nalidixic acid NAL 0 0 28 14.21 169 85.79 Tetracyclin TE 1 0.51 28 14.21 168 85.28 Tetracycline TE TRNG: 97(49.24%) Table 2. The resistant levels against different antibiotics from main group (n = 197) TRNG: tetracycline resistant Neisseria gonorrhoeae is the term used to describe isolates with inhibition zone of Tetracycline being less than or equal to 20 mm. Table 3. The resistant levels against antibiotics from additional groups Antibiotics Abbreviation Sensitive Intermediate Resistant n % n % n % Azithromycin AZM 196 99.49 0 0 1 0.51 Cefotaxim CTX 196 99.49 0 0 1 0.51 Cefixim CFM 196 99.49 0 0 1 51 Table 4. MIC value within several antibiotics to N. gonorrhoeae Antibiotics Strains MIC (àg/ml) Sensitive Intermediate Resistant Penicillin 10 ≤ 0.06 0.12 - 1 ≥ 2 5 5 Spectinomycin 11 ≤ 32 64 ≥ 128 11 Ciprofloxacin 12 ≤ 0.06 0.12 - 0.5 ≥ 1 5 7 Ceftriaxon 17 ≤ 0.25 - - 12 2 Tetracyclin 10 ≤ 0.25 0.5 - 1 ≥ 2 3 7 JMR 116 E3 (7) - 2018 129 JOURNAL OF MEDICAL RESEARCH The MIC value showed that 5/10 strains resisted to penicillin (MIC ≥ 2 àg/ml), 7/12 strains to ciprofloxacin (MIC ≥ 1 àg/ml), 7/10 to tetracyclin (MIC ≥ 2 àg/ml) and 2/17 to ceftriaxone (MIC > 0,25 àg/ml). No strain showed its resistance to spectinomycin. IV. DISCUSSION Since 2017, the World Health Organization has supported five hospitals in Vietnam, in- cluding the National hospital of Dermatology and Venereology, to perform the project of surveillance of antimicrobial resistance in Neisseria gonorrhoeae. Therefore, specimens from patients with urethral and/or vaginal dis- charge syndrome in the hospital were proc- essed in a clinical laboratory for bacterial cul- ture, identification and antimicrobial suscepti- bility test. Of 1582 patients with urethral and/or vaginal discharge syndrome who attended National hospital of Dermatology and Venereology in 2017, 197 (12.45%) were posi- tive for N. gonorrhoeae. This finding was higher than previous studies. Nguyen Huu Sau reported that of 20,260 patients who attend National hospital of Dermatology and Venereology for STDs from 2006 to 2010, 1946 (9.6%) were positive for N.gonorrhoeae [5]. From November 2006 to April 2007 at Na- tional hospital of Dermatology and Venereol- ogy, Nguyen Dinh Ha reported that the preva- lence of N. gonorrhoeae in STDs patients was 11.08%. Nguyen Hong Hinh reported that prevalence of N. gonorrhoeae was 10.16% among STDs patients in their study from 1/2001 to 12/2003. However, the real number of gonorrhea patients in the community may be higher because patients usually decide to be examined in private clinics or even to treat themselves without a prescription from a health care professional. On the other hand, the prevalence of N. gonorrhoeae also de- pends on several factors, such as the experi- ence of technicians and quality of laboratory tests. The antimicrobial resistance of N. gonor- rhoeae becoming more widespread across different classes of antibiotics, including peni- cillin, tetracyclin, fluoroquinolone, erythromy- cin, and azithromycin. Eventually, some N. gonorrhoeae strains have shown the resis- tance to broad-spectrum cephalosporin in vitro which is recommended for treatment in many countries [6]. In this study, we showed that 97.97% of N. gonorrhoeae isolates were resis- tant to ciprofloxacin. According to the results of Le Hong Hinh, there was an upward trend of the resistant rate of N. gonorrhoeae to ciprofloxacin with 42.26% in 2001; 46.01% in 2002 and 50% in 2003. Remarkably, in 2014, the resistant rate of 96.33% was shown in Le Van Hung’s study [7]. WHO has reported that prevalence of antimicrobial resistance of N. gonorrhoeae was 85.2% in China, 79.9% in Hong Kong and 40% in Japan [8]. This preva- lence was 4.89% in England 4.89%, 7.8% in Scotland 7.8% and 0.9% in Papua New Guinea [9]. The antimicrobial resistance of N. gonorrhoeae has increased rapidly due to the usage of quinolone not only for gonorrhea treatment but also for some other infections like urinary tract infections and injuries. Thus, quinolone has not been recommended for gonorrhea treatment. As for tetracycline, our study showed that the resistant rate to this antibiotic was 85.28%. Of these tetracycline resistant isolates, 130 JMR 116 E3 (7) - 2018 JOURNAL OF MEDICAL RESEARCH 49.24% was TRNG with inhibition zone being less than and equal to 20mm. . The study con- ducted at The National Hospital of Dermatol- ogy and Venereology in 2000 showed that the resistant rate of N. gonorrhoeae to tetracycline was 42.7% [7]. The results from study of Le Hong Hinh showed that this resistant rate had a downward trend as follows: 40.47% in 2001; 26.29% in 2002 and 27.9% in 2003. In Asia Pacific, this rate ranged from 25% to 70% [9]. Thus, this resistant rate in Vietnam showed an decreasing trend. This may be explained by the multiple-dose regimens of tetracycline – an old antibiotic, for the treatment of urethritis and cervicitis due to N. gonorrhoeae and C. tra- chomatis infections. As for penicillin, the first class of antibiotics to be discovered, the resistance rate to this drug was 45.69% in our study. In 2014, Le Van Hung et al reported this rate was 47.7%. According to WHO report, in Asia Pacific countries, the resistant rate was more than 90% in Korea and the Philippines; 80% in China and 60% in Singapore [8]. Currently, WHO recommended penicillin to not be used for gonorrhea treatment among countries where this resistant rate was high. As for ceftriaxone, our study showed that N. gonorrhoeae was sensitive (98.98%), how- ever, we detected two resisted strains with MIC > 0.25 àg/ml. This finding was consistent with previous studies of Le Van Hung and other authors in Vietnam and Asia Pacific countries [7; 10]. However, a study reported that 5% of N. gonorrhoeae strains showed the resistance to ceftriaxone in Vietnam in 2011 [11]. Regarding spectinomycin, 100% of isolates were sensitive to this drug, which was also reported from other research groups in Viet- nam. Therefore, this antibiotic is still recom- mended for treatment. However, it was re- ported that spectinomycin-resistant strains have appeared with 0.47% in other country. The additional antibiotic group which is se- lected by the particular condition of each na- tion showed the high sensitive result (99.49%) in our study, including azithromycin, cefotaxim and cefixim. These results were consistent with study of Ana Paula Ramalho da Costa- Lourenỗo et al in 2017 [12]. E-test was used for the identification of MIC of N. gonorrhoeae. However, due to the high costs, MIC was performed only for strains which showed the difficult interpretation with antibiotic sensitivity results closed to sensitive, intermediate or resistance with five antibiotics of main group. MIC results showed 5/10 strains resistant to penicillin (MIC ≥ 2 àg/ml), 7/12 strains resis- tant to ciprofloxacin (MIC ≥ 1 àg/ml), 7/10 strains resistant to tetracycline (MIC ≥ 2 àg/ ml), 2/17 strains resistant to ceftriaxone (MIC > 0.25 àg/ml). No strain showed the resistance to spectinomycin. These finding were consis- tent with other reports. According to Le Van Hung et al in 2014, N. gonorrhoeae resisted to penicillin, ciprofloxacin, tetracycline at 47.7%; 96.3% and 83.4%, respectively. Coinciden- tally, N. gonorrhoeae showed its high sensitiv- ity to ceftriaxone and spectinomycin [6]. In 2011 at National Hospital of Dermatology and Venereology, Pham Thi Lan reported that the sensitivity and resistance of N. gonorrhoeae to ceftriaxone with 95% and 5%, respectively, and 100% of N. gonorrhoeae strains were sensitive to spectinomycin [10]. JMR 116 E3 (7) - 2018 131 JOURNAL OF MEDICAL RESEARCH V. CONLUSION Ciprofloxacin, tetracycline and penicillin should not be used for N. gonorrhoeae treat- ment due to the high resistance of N. gonor- rhoeae to these antibiotics. Spectinomycin, ceftriaxon, azithromycin, cefotaxim and cefixim should be used for the treatment of N. gonorrhoeae infection. ACKNOWLEDGEMENTS We acknowledge the support of Prof. Tran Hau Khang, Assoc.Prof. Nguyen Van Thuong, Bsc. Ninh Thi Dan and Bsc. 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