Prevalence of antimicrobial resistance of neisseria gonorrhoeae in national hospital of dermatology and venereology in 2017

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.

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 difficulties for diagnosis and treatment Thus, more research into all aspects of antimicrobial resistance of N gonorrhoeae is necessary Objectives: To determine the prevalence of N gonorrhoeae infections in patients with urethral and/or vaginal discharge syndrome, who attended National Hospital of Dermatology and Venereology in 2017 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 ≥ µg/ml), 7/10 to tetracycline (MIC ≥ µg/ml), 5/10 to penicillin (MIC ≥ µg/ml), 2/17 to ceftriaxone (MIC ≤ 0,25 µg/ml) 100% of isolates were susceptible to spectinomycin (MIC ≤ 32 µg/ml) Conclusion: Ceftriaxone, spectinomycin, azithromycin, cefixime and cefotaxime were recommended for N gonorrhoeae treatments while ciprofloxacin, cefuroxime, tetracycline and penicillin were not Keywords: Neisseria gonorrhoeae, antimicrobial resistance I INTRODUCTION even infertility, if it is not correctly diagnosed Neisseria gonorrhoeae infection is a com- and treated in time The antimicrobial resis- mon sexually transmitted disease (STD) Ac- tance of Neisseria gonorrhoeae is a major cording to The World Health Organization re- topic of interest due to the appearance of the port, it was estimated there were 106 million multiple drug resistant species that can resist new cases of gonorrhea in 2012 [1] Each year current antibiotics used for treatment, which at the National Hospital of Dermatology and remains a major public health concern world- Venereology, more than 20% of all STD cases wide [2] In Vietnam, using antibiotics without are diagnosed as gonorrhea Gonorrhea may prescription or antibiotics abuse in community spread within a community and cause many and health stations also may increase the an- different timicrobial resistance of N gonorrhoeae, genitourinary tract complications, which cause the difficulties for diagnosis and Corresponding author: Le Van Hung, Hanoi Medical University Email: levanhungvdl@yahoo.com Received: 9/7/2018 Accepted: 22/11/2018 JMR 116 E3 (7) - 2018 treatment Thus, we performed this study to investigate the prevalence of N gonorrhoeae infections in patients with urethral and/or vaginal discharge syndrome who attended Na- 125 JOURNAL OF MEDICAL RESEARCH tional hospital of Dermatology and Venereology in 2017 and to describe the antimicrobial resistant levels of isolated N gonorrhoeae II METHODS Methods: A cross-sectional study Sample collection: (patients must be informed and explained prior sample collection) In women, samples were collected from urethra, cervix, sides of Bartholin’s Gland, Subjects and sides of Skene’s Gland For cervical This study included all patients with ure- samples, distilled water was used to pre-wet thral and/or vaginal discharge syndrome who the vaginoscope Next, the vaginoscope was visited the National Hospital of Dermatology inserted into the vagina to open the cervix, and Venereology, Hanoi, Vietnam in 2017 then samples were obtained by inserting a Patients with AIDS or using antibiotics within sterilized swab approximately - cm in the days were excluded from this study A total of cervix and keeping for - 10 seconds to ab- 1582 male and female patients agreed to par- sorb cervical discharge The swab was pulled ticipate in this study out without touching vaginal wall, followed by 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 supplementary (Iso Vitalex) and the inhibitors (VCNvancomycin, colistin, nystatin) which can suppress other gram-negative diplococci, gramnegative bacilli, gram-positive organisms, and yeast Remel BactiCard Neisseria test kit (Thermo making smear for gram staining, and culturing in Thayer-Martin agar with VCN inhibitors Male patients were required to block urination - 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 - cm in urethral meatus, rubbing against the inner wall and keeping for - 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 - Scientific) were used for N gonorrhoeae iden- 37 C in a humid atmosphere (70% humidity) tification This test uses enzyme technology containing - 10% carbon dioxide (CO2) for for identification of pathogenic Neisseria spp 18 - 24 hours (including N gonorrhoeae, N meningitis and N lactamica) and Moraxella catarrhalis isolated on selective media - Antibiotic disks (Oxoid) were used for determination of antimicrobial susceptibility 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 - E-test (Abbiodisk, Sweden), a quantitative results, with a coffee bean shape, in and technique, was used to determine the mini- outside of neutrophils in the microscope field mum inhibitory concentration (MIC) Finally, a cultured colony was confirmed by 126 JMR 116 E3 (7) - 2018 JOURNAL OF MEDICAL RESEARCH using oxidase test and Remel BactiCard incubated at 36 - 37oC in > 70% humidity con- Neisseria test kit [3] taining - 10% CO2 for 18 - 24 hours The For antibiotic sensitivity testing, N gonor- zone of inhibition was measured by using mm rhoeae colony was cultured in Thayer-Martin ladder and interpreted according to The Clini- agar without inhibitors, then incubated at 36 - cal & Laboratory Standards Institute (CLSI) 370C in > 70% humidity containing - 10% guideline in 2015 [4] CO2 for 18 - 24 hours Colony purification was Minimum inhibitory concentrations (MICs) rechecked by Gram staining A suspension of was performed by using E-test (Abbiodisk, 0.5 McFarland turbidity was made by mixing Sweden) MIC is the lowest concentration of colonies with Sodium Chloride 0.9%, then cul- an antibiotic, which prevents visible growth of tured as monolayer in Thayer-Martin agar bacterium In our study, MIC was performed without inhibitors The agar surface was kept for only N gonorrhoeae with the zone of inhi- dry up in room temperature Next, antibiotic bition close to sensitive or resistant level For disks were put on each 9cm diameter plate example, Ceftriaxone ≥ 35mm was sensitive with a distance of cm from plate wall and result, thus, those strains with the inhibition kept at room temperature for 10 minutes for zone of 34 - 35 mm were examined for MICs the antibiotic diffusion Cultured plates were test III RESULTS The prevalence of N gonorrhoeae infections in patients with urethral and/or vaginal discharge syndrome (n = 1582) Table The prevalence of N gonorrhoeae infections in patients with urethral and/or vaginal discharge syndrome Test results Gram staining (%) Culture (%) Positive 190 (12.01) 197 (12.45) Negative 1392 (87.99) 1385 (87.55) p value 0.71 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) 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) JMR 116 E3 (7) - 2018 127 JOURNAL OF MEDICAL RESEARCH Table The resistant levels against different antibiotics from main group (n = 197) Antibiotics Sensitive Intermediate Resistant n % n % n % Abbreviation Penicillin PG 0 107 54.31 90 45.69 Spectinomycin SPT 197 100 0 0 Ciprofloxacin CIP 0 2.03 193 97.97 Ceftriaxon CRO 195 98,98 0 1.02 Nalidixic acid NAL 0 28 14.21 169 85.79 Tetracyclin TE 0.51 28 14.21 168 85.28 Tetracycline TE TRNG: 97(49.24%) 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 The resistant levels against antibiotics from additional groups Antibiotics Abbreviation Sensitive Intermediate Resistant n % n % n % Azithromycin AZM 196 99.49 0 0.51 Cefotaxim CTX 196 99.49 0 0.51 Cefixim CFM 196 99.49 0 51 Table MIC value within several antibiotics to N gonorrhoeae Antibiotics Strains Penicillin 10 Spectinomycin 11 Ciprofloxacin 12 Ceftriaxon 17 Tetracyclin 10 128 MIC (µg/ml) Sensitive Intermediate Resistant ≤ 0.06 0.12 - ≥2 5 64 ≥ 128 0.12 - 0.5 ≥1 - - ≤ 32 11 ≤ 0.06 ≤ 0.25 12 ≤ 0.25 0.5 - ≥2 JMR 116 E3 (7) - 2018 JOURNAL OF MEDICAL RESEARCH The MIC value showed that 5/10 strains resisted to penicillin (MIC ≥ µg/ml), 7/12 strains to ciprofloxacin (MIC ≥ µg/ml), 7/10 to tetracyclin (MIC ≥ µg/ml) and 2/17 to ceftriaxone (MIC > 0,25 µg/ml) No strain showed its resistance to spectinomycin IV DISCUSSION pends on several factors, such as the experi- Since 2017, the World Health Organization has supported five hospitals in Vietnam, in- ence of technicians and quality of laboratory tests cluding the National hospital of Dermatology The antimicrobial resistance of N gonor- and Venereology, to perform the project of rhoeae becoming more widespread across surveillance of antimicrobial resistance in different classes of antibiotics, including peni- Neisseria gonorrhoeae Therefore, specimens cillin, tetracyclin, fluoroquinolone, erythromy- from patients with urethral and/or vaginal dis- cin, and azithromycin Eventually, some N charge syndrome in the hospital were proc- gonorrhoeae strains have shown the resis- essed in a clinical laboratory for bacterial cul- tance to broad-spectrum cephalosporin in vitro ture, identification and antimicrobial suscepti- which is recommended for treatment in many bility test Of 1582 patients with urethral and/or countries [6] In this study, we showed that vaginal discharge syndrome who attended 97.97% of N gonorrhoeae isolates were resis- National and tant to ciprofloxacin According to the results Venereology in 2017, 197 (12.45%) were posi- of Le Hong Hinh, there was an upward trend tive for N gonorrhoeae This finding was of the resistant rate of N gonorrhoeae to higher than previous studies Nguyen Huu Sau ciprofloxacin with 42.26% in 2001; 46.01% in reported that of 20,260 patients who attend 2002 and 50% in 2003 Remarkably, in 2014, National and the resistant rate of 96.33% was shown in Le Venereology for STDs from 2006 to 2010, Van Hung’s study [7] WHO has reported that 1946 (9.6%) were positive for N.gonorrhoeae prevalence of antimicrobial resistance of N [5] From November 2006 to April 2007 at Na- gonorrhoeae was 85.2% in China, 79.9% in tional hospital of Dermatology and Venereol- Hong Kong and 40% in Japan [8] This preva- ogy, Nguyen Dinh Ha reported that the preva- lence was 4.89% in England 4.89%, 7.8% in lence of N gonorrhoeae in STDs patients was Scotland 7.8% and 0.9% in Papua New 11.08% Nguyen Hong Hinh reported that Guinea [9] The antimicrobial resistance of N prevalence of N gonorrhoeae was 10.16% gonorrhoeae has increased rapidly due to the among STDs patients in their study from usage of quinolone not only for gonorrhea 1/2001 to 12/2003 However, the real number treatment but also for some other infections of gonorrhea patients in the community may like urinary tract infections and injuries Thus, be higher because patients usually decide to quinolone has not been recommended for be examined in private clinics or even to treat gonorrhea treatment hospital hospital of of Dermatology Dermatology themselves without a prescription from a As for tetracycline, our study showed that health care professional On the other hand, the resistant rate to this antibiotic was 85.28% the prevalence of N gonorrhoeae also de- Of JMR 116 E3 (7) - 2018 these tetracycline resistant isolates, 129 JOURNAL OF MEDICAL RESEARCH 49.24% was TRNG with inhibition zone being reported from other research groups in Viet- less than and equal to 20mm The study con- nam Therefore, this antibiotic is still recom- ducted at The National Hospital of Dermatol- mended for treatment However, it was re- ogy and Venereology in 2000 showed that the ported that spectinomycin-resistant strains resistant rate of N gonorrhoeae to tetracycline have appeared with 0.47% in other country 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 The additional antibiotic group which is selected by the particular condition of each nation 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 CostaLourenỗo et al in 2017 [12] old antibiotic, for the treatment of urethritis and E-test was used for the identification of cervicitis due to N gonorrhoeae and C tra- MIC of N gonorrhoeae However, due to the chomatis infections high costs, MIC was performed only for strains 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% which showed the difficult interpretation with antibiotic sensitivity results closed to sensitive, intermediate or resistance with five antibiotics of main group According to WHO report, in Asia Pacific MIC results showed 5/10 strains resistant countries, the resistant rate was more than to penicillin (MIC ≥ µg/ml), 7/12 strains resis- 90% in Korea and the Philippines; 80% in tant to ciprofloxacin (MIC ≥ µg/ml), 7/10 China and 60% in Singapore [8] Currently, strains resistant to tetracycline (MIC ≥ µg/ WHO recommended penicillin to not be used ml), 2/17 strains resistant to ceftriaxone (MIC for gonorrhea treatment among countries > 0.25 µg/ml) No strain showed the resistance where this resistant rate was high to spectinomycin These finding were consis- As for ceftriaxone, our study showed that tent with other reports According to Le Van N gonorrhoeae was sensitive (98.98%), how- Hung et al in 2014, N gonorrhoeae resisted to ever, we detected two resisted strains with penicillin, ciprofloxacin, tetracycline at 47.7%; MIC > 0.25 µg/ml This finding was consistent 96.3% and 83.4%, respectively Coinciden- with previous studies of Le Van Hung and tally, N gonorrhoeae showed its high sensitiv- other authors in Vietnam and Asia Pacific ity to ceftriaxone and spectinomycin [6] In countries [7; 10] However, a study reported 2011 at National Hospital of Dermatology and that 5% of N gonorrhoeae strains showed the Venereology, Pham Thi Lan reported that the resistance to ceftriaxone in Vietnam in 2011 sensitivity and resistance of N gonorrhoeae to [11] ceftriaxone with 95% and 5%, respectively, Regarding spectinomycin, 100% of isolates were sensitive to this drug, which was also 130 and 100% of N gonorrhoeae strains were sensitive to spectinomycin [10] JMR 116 E3 (7) - 2018 JOURNAL OF MEDICAL RESEARCH V CONLUSION The National hospital of Dermatology and Venereology Journal of Vietnam Medical Re- Ciprofloxacin, tetracycline and penicillin search, 1, 32 - 35 should not be used for N gonorrhoeae treatment due to the high resistance of N gonorrhoeae to these antibiotics Spectinomycin, ceftriaxon, azithromycin, Unemo M and Nicolas RA (2012) Emergence of mutidrug resistance, extensively drug-resesistant and untreatable gonorrhoeae Future Microbiol, 7, 1401 - 1422 cefotaxim and cefixim should be used for the treatment of N gonorrhoeae infection ACKNOWLEDGEMENTS We acknowledge the support of Prof Tran Le Van Hung., Tran Kim Thuy., Nguyen Huu Sau (2014) Survey on antibiotic resistance of gonorrhoeae at The National hospital of Dermatology and Venereology in 2014 Journal of Vietnam Dermatology, 21, 28 - 36 Hau Khang, Assoc.Prof Nguyen Van Thuong, WHO (2001) Surveillance of Antibiotic Bsc Ninh Thi Dan and Bsc Le Phuong Thao resistance in Neisseria gonorrhoeae in the for this study WHO/WPRO 2000 CDI, 15(4) Creightion S (2003) Co-infection with REFERENCES Newman, L., J Rowley (2015) Global gonorrhoeae and Chlamydia how much is there and what does it mean Int J STD-AIDS, 14, 109 - 113 Estimates of the Prevalence and Incidence of 10 WHO (2000) Antibiotic susceptibility of Four Curable Sexually Transmitted Infections Neisseria gonorrhoeae in the WHO/WPRO in 2012 Based on Systematic Review and Report of surveillance of GASP 1999, 16, 11 - Global Reporting PLoS One, 10(12), e0143304 13 Magnus Unemo and William M Shafer (2014) Antimicrobial Resistance in Neisseria gonorrhoeae in the 21st Century: Past, Evolution and Future, 27(3), 587 - 613 Nguyen Vu Trung 2014 (2014) Textbook of Clinical Microbiology and Parasitolgy Medical Publishing House, 1, 173 - 177 CLSI (2017) Clinical and Laboratory Standards Instituts M100, 27th ed, 72 - 74 Nguyen Huu Sau (2012) Study the situation, characteristics of gonorrhoeae at JMR 116 E3 (7) - 2018 11 Birgitta Plsen (2013) Antimicrobial susceptibility and generic characteristics of Neisseria gonorrhoeae from Vietnam BMC Infectious Diseases, 13, 40 12 Ana Paula Ramalho da CostaLourenỗo, Kộsia Thaớs Barros dos Santos, et al (2017) Antimicrobial resistance in Neisseria gonorrhoeae: history, molecular mechanisms and epidemiological aspects of an emerging global threat, 48(4), 617 - 628 131 ... ence of technicians and quality of laboratory tests cluding the National hospital of Dermatology The antimicrobial resistance of N gonor- and Venereology, to perform the project of rhoeae becoming... Hospital of Dermatology inserted into the vagina to open the cervix, and Venereology, Hanoi, Vietnam in 2017 then samples were obtained by inserting a Patients with AIDS or using antibiotics within... positive for N .gonorrhoeae prevalence of antimicrobial resistance of N [5] From November 2006 to April 2007 at Na- gonorrhoeae was 85.2% in China, 79.9% in tional hospital of Dermatology and Venereol-