Antimicrobial susceptibility of Neisseria gonorrhoeae isolates from patients attending a public referral center for sexually transmitted diseases in Belo Horizonte , State of Minas Gerais , Brazil

Introduction: The aim of this study was to determine the antimicrobial susceptibility of Neisseria gonorrhoeae isolates obtained from patients attending a public referral center for sexually transmitted diseases and specialized care services (STD/SCS) in Belo Horizonte, Brazil. Methods: Between March 2011 and February 2012, 201 specimens of Neisseria gonorrhoeae were consecutively obtained from men with symptoms of urethritis and women with symptons of cervicitis or were obtained during their initial consultation. The strains were tested using the disk diffusion method, and the minimum inhibitory concentrations of azithromycin, cefi xime, ceftriaxone, ciprofl oxacin, chloramphenicol, penicillin, tetracycline and spectinomycin were determined using the E-test. Results: The specimens were 100% sensitive to cefi xime, ceftriaxone and spectinomycin and exhibited resistances of 4.5% (9/201), 21.4% (43/201), 11.9% (24/201), 22.4% (45/201) and 32.3% (65/201) to azithromycin, ciprofl oxacin, chloramphenicol, penicillin and tetracycline, respectively. Intermediate sensitivities of 17.9% (36/201), 4% (8/201), 16.9% (34/201), 71.1% (143/201) and 22.9% (46/201) were observed for azithromycin, ciprofl oxacin, chloramphenicol, penicillin and tetracycline, respectively. The specimens had plasmid-mediated resistance to penicillin PPNG 14.5% (29/201) and tetracycline TRNG 11.5% (23/201). Conclusions: The high percentage of detected resistance to penicillin, tetracycline, chloramphenicol and ciprofl oxacin indicates that these antibiotics are not appropriate for gonorrhea treatment at the Health Clinic and possibly in Belo Horizonte. The resistance and intermediate sensitivity of these isolates indicates that caution is recommended in the use of azithromycin and emphasizes the need to establish mechanisms for the surveillance of antimicrobial resistance for the effective control of gonorrhea.

Gonorrhea remains a serious public health problem and is the second most prevalent bacterial infection and sexually transmitted disease worldwide, with 106 million new cases annually 1,2 .Its incidence is higher in underdeveloped countries and developing countries but remains unacceptably high in developed countries, particularly among those associated with vulnerable populations, marginalized ethnicities or sexu al orientations 3,4 .Gonorrhea can cause serious complications such as epididymitis, pelvic infl ammatory disease, ectopic pregnancy and infertility and can also enhance the acquisition and transmission of human immunodefi ciency virus (HIV) 2,5 .
Neisseria gonorrhoeae has a high ability to develop resistance to antimicrobials, resulting in the progressive loss of cheap and effective treatments and the need to use more expensive drugs that are inaccessible in many countries 4 .An issue of current signifi cance is the appearance of strains of N. gonorrhoeae that are resistant to multiple antimicrobials, including penicillin, tetracycline, macrolides, fl uoroquinolone and spectinomycin.The emergence of resistance occurs, in general, by rapid diffusion and is highly prevalent 6,7 .Over the past three years, gonococcal resistance to third-generation cephalosporins, which are recommended as the fi rst choice for treatment, has emerged rapidly in Japan and other countries.This resistance constitutes an epidemiological situation that prevents healing and increases the likelihood of the occurrence of serious consequences associated with gonorrhea 8 .

RESULTS
The control of gonorrhea is a vital issue to public health and requires an integrated approach to provide effective treatment that breaks the chain of transmission and reduces complications.Knowledge of local susceptibility patterns of N. gonorrhoeae is essential to establish the best treatment 9,10 .Control of gonococcal infection requires the continuous and high-quality surveillance of resistance 2,6 ; however, few studies on this subject have been conducted in Brazil.The current study provides insight into the resistance of Neisseria gonorrhoeae in Belo Horizonte, Minas Gerais.This study also contributes to the collection of epidemiological data to support the formulation of health policies for disease prevention and control and will facilitate the standardization of the most appropriate and effective treatment.
This study is a descriptive and cross-sectional observational study that was conducted at the Department of STD/SCS, Secondary Reference Unit Centre-South (SRU-CS), the Secretaria Municipal de Saúde of Belo Horizonte and the Microbiology Laboratory of Hospital João XXIII, Fundação Hospitalar do Estado de Minas Gerais.
The isolates of Neisseria gonorrhoeae were consecutively obtained from men with symptoms of urethritis and women with symptoms of cervicitis or were obtained during their initial consultation at the STD/SCS Department during the period March 2011 to February 2012.A total of 232 isolates of Neisseria gonorrhoeae were obtained, of which 201 were viable for use in the proposed analysis.
Eligible patients underwent the collection of endocervical or urethral material according to the appropriate technique 11 .Strains were cultivated and identifi ed according to standard procedures 12,13 .The collected material was inoculated directly onto modifi ed Thayer Martin (Polyvitex/VCAT3-Biomérieux®) and incubated immediately at 35.5ºC to 36.5°C in an atmosphere of 5% CO 2 for 72h.Morphologically suggestive colonies were screened by Gram staining and by their positive responses to the oxidase and catalase tests.Positive colonies were subcultivated on chocolate supplement agar (Chocolate Polyvitex/Biomérieux®), and their identifi cation was confi rmed using the automatic system VITEK II® (NH -Biomérieux).The production of ß-lactamase was tested by the chromogenic cephalosporin method (Cefi nase Paper Disc-BBL®).
The susceptibility of the Neisseria gonorrhoeae isolates to azithromycin, cefixime, ceftriaxone, ciprofloxacin, chloramphenicol, penicillin, tetracycline and spectinomycin was tested by the disk diffusion 14,15 and E-test 16 (Biomérieux®) methods, and minimum inhibitory concentrations (MICs) were determined.The tests were performed on GC agar supplemented with 1% growth factors (GC Agar with 1% Vitox/PlastLabor®) according to the methodology of the Clinical Laboratory Standards Institute (CLSI) and the Centers for Disease Control (CDC) 14,15,17 .Inocula were prepared using colonies that had been grown for 24h by direct suspension in sterile saline to a turbidity equivalent to the 0.5 McFarland standard; inocula were adjusted using a turbidimeter.Within 15min, the suspension was inoculated on the surface of a 150-mm diameter GC agar plate.After the absorption of the inoculum, antimicrobial discs (OXOID®) and E-test strips (Biomerieux®) were applied.The plates were incubated at 35.5°C to 36.5°C in an atmosphere of 5% CO 2 for 20 to 24h.
The susceptibility was interpreted according to the criteria defi ned by CLSI 15 .The CDC criteria were used for azithromycin, whereas the method of Dick Van and collaborators was used for chloramphenicol 12,17 .The isolates were classifi ed as having a presumed plasmid-mediated resistance to penicillin (PPNG) when they were ß-lactamase positive and a presumed resistance to tetracycline (TRNG) when the MIC was ≥16µg/mL 15 .Chromosomal resistance to penicillin was defi ned as an MIC≥2µg/mL in ß-lactamase-negative specimens, and tetracycline resistance was defi ned for specimens with MICs between 2 and < 16µg/mL.
Quality control was performed using reference strains of Neisseria gonorrhoeae ATCC 49226 (American Type Culture Collection), which were kindly provided by Professor Sergio Eduardo Longo Fracalanzza, Universidade Federal of Rio de Janeiro.
Information was obtained on the socio-demographic and behavioral characteristics of patients using a questionnaire and medical records.
Statistical analysis was performed using SPSS software version 17.0.For the age variable, an estimated median was performed, as with the measures of variability.For the qualitative variables, proportions were performed.

Ethical considerations
This study was authorized by the Research Ethics Committee of the the Secretaria Municipal de Saúde of Belo Horizonte and Universidade Federal de Minas Gerais, and specimens were collected after the subjects agreed with and completed the Free and Informed Consent Terms.

Evaluation of the susceptibility profi le
The susceptibility of 201 isolates of Neisseria gonorrhoeae to azithromycin, cefixime, ceftriaxone, ciprofloxacin, chloramphenicol, penicillin, tetracycline and spectinomycin was assessed as shown in Table 1.

Frequency of gonorrhea in the Department of STD/SCS of the SRU-CS/SMSA in Belo Horizonte
During the period March 2011 to February 2012, 7,646 patients were cared for in the Department of STD/SCS of the SRU-CS/SMSA in Belo Horizonte, including 5,851 (76.5%) males and 1,795 (23.5%) females.Of these, 676 men and 583 women underwent endocervical or urethral sample collection.There were 287 cases of gonorrhea, with a relative frequency of 40.5% (274/676) for men and 2.2% (13/583) for women.

Studied population at the Department of STD/SCS of the SRU-CS
The 201 analyzed specimens of Neisseria gonorrhoeae were obtained from 200 patients of both sexes, including two specimens from one patient who returned to the clinic with a new infection.Of the patients studied, 95% (190/200) were male, and 5% (10/200) were female, with ages ranging from 14 to 61 years (median 26 years) and 17 to 40 years (median 22 years), respectively.Among the men, 21% (40/200) declared that they were men who have sex with men (MSM).
The degree of formal schooling at the elementary, secondary and university level was 64% (128/200), 32 Of the patients who returned with symptoms, 1 patient had non-gonococcal urethritis, and 4 had gonococcal urethritis, as confi rmed by laboratory testing.Among those with gonorrhea, two returned within 7 days with no improvement and were infected with strains of N. gonorrhoeae that were resistant to the treatment used (thiamphenicol), but there was no confi rmation of treatment failure.

DISCUSSION
The latest data on the antimicrobial susceptibility of N. gonorrhoeae reported in Belo Horizonte were reported in 1984 18 ; in that study, tetracycline resistance was observed in 5.6%, chloramphenicol in 13.9% and intermediate sensitivity to penicillin in 15.3% of the studied patients.
The data reported here reveal a high level of resistance to tetracycline, penicillin, chloramphenicol and ciprofl oxacin, as well as intermediate sensitivities to azithromycin, chloramphenicol, penicillin and tetracycline.The occurrence of resistance, except in the case of azithromycin, exceeded the limit of 5%, which does not permit the use of these antimicrobials in the treatment of gonorrhea according to the World Health Organization and the CDC 6,9 .
The tetracycline resistance observed (32.3%) in this study is similar to that observed in Rio de Janeiro 21 (33.9%) and São Paulo 20 (30.8%) and much lower than that in Manaus 19 (80%).These fi ndings are consistent with observations in countries such as Chile (33%) and Uruguay (32%) and lower than that obtained in Bolivia (54%), Peru (90%) and Venezuela 23 (100%).The frequency of TRNG strains (11.5%) is similar to that observed in Manaus (12.7%) but lower than that detected in Rio de Janeiro (20%).The frequency reported in this study is also compatible with that observed in Peru (10%) and higher than that reported in Argentina (5%) and Uruguay (4%).
Specimens with resistance to penicillin and tetracycline have been reported in Brazil since 1984 18,26 .Gonorrhea has also become resistant to azithromycin (4.5%), consistent with observations in Manaus 19 (6.4%), but our reported rate is higher than the 20% intermediate sensitivity observed in Rio de Janeiro 21 .Azithromycin resistance has been reported in several countries such as Uruguay (9%), Chile (46%), Peru 23 (75%), the United States of America 3 (0.5%) and Germany 27 (7.7%).Although it is not recommended for treatment for gonorrhea in Brazil 28 , azithromycin could be used as a therapeutic option, albeit with caution.In this study, less than 5% of the specimens were resistant to azithromycin, but we also observed large numbers of specimens with reduced susceptibility.
Ciprofl oxacin is recommended as a fi rst-line treatment for gonococcal infections in Brazil 28 .Ciprofl oxacin resistance is greatly increased (21.4%) compared to other national studies.These studies initially observed decreased susceptibility 19,21 followed by drug resistance in Manaus 23 (2%), São Paulo 29 (3.7%) and Rio de Janeiro 30 (7.7%).These fi ndings are consistent with a recent study in Rio de Janeiro 30 that reported 16.5% resistance in the period 2005 to 2010 and 33.3% in 2010.
According to the GASPLAC surveillance program 31 , resistance increased in Latin America and the Caribbean from 2% in 2000 to 30% in 2009, reaching 60% in some countries such as Peru 23 .Many countries, such as the U.S.A (12.5%) 3 and Germany 27 (47.7%),have detected high levels of resistance to ciprofl oxacin, and thus its use for the treatment of gonorrhea has been discontinued in these locations 4,32,33 .In addition, specimens with a high level of resistance to ciprofl oxacin (MIC>32µg/ml) that also exhibited resistance to chloramphenicol and tetracycline were identifi ed.These specimens are phenotypically similar to those detected in Manaus 34 , although this similarity has not been confi rmed by molecular analysis.

Costa LMB et al -Antimicrobial susceptibility of Neisseria gonorrhoeae isolates
The 11.9% resistance to chloramphenicol observed in this study is higher than that obtained in Rio de Janeiro 21 (3.5%) and Manaus (5%) 23 but lower than that observed in Belo Horizonte in 1984 18 (13.8%)and in Indonesia (36.8%) 25 and African countries (57.3%) 24 .
In the Department of STD/SCS of the SRU-CS, thiamphenicol is commonly used to treat gonorrhea.Ciprofl oxacin is also often used in combination with doxycycline or azithromycin for infections concomitant with chlamydia.Azithromycin and ciprofl oxacin are also widely used, both for other STDs and for various other community-acquired or nosocomial infections.Although thiamphenicol is no longer recommended by the Ministry of Health 28 , it is still currently used because of its reduced cost, ease of administration and clinical effi cacy.The level of resistance to chloramphenicol (an analog of thiamphenicol) is concerning, although it is lower now than in 1984 18 , presumably due to reduced use.Nonetheless, the level of resistance still exceeds the percentage of resistance allowed for empirical use.The patients are presumably considered healed, but the return rate is too low (32%) to ensure the effectiveness of the treatment.Spectinomycin exhibits excellent sensitivity, but it is only effective for urethral and rectal infections 33 .However, Pharyngeal (P) infections are asymptomatic, and although increasing in importance 6,7 , particularly in the MSM population, this site is rarely screened.In addition to the high cost and intramuscular administration of spectinomycin, high-level resistance has emerged quickly when spectinomycin has been used in the past due to a single mutation 33 .
Although no resistance to cephalosporin was observed in this study, all antimicrobials used extensively in the hospital should be monitored.Cefi xime, despite its advantage of oral administration, is not available in Brazil.
Factors such as self-medication, possibly due to insuffi cient control of the sale of antimicrobials, and the lack of medical care for treatment may contribute to the development of resistance.
This study was limited by the impossibility of molecular tests for the detection of resistance mechanisms and the typing of strains.The data originate from an STD health service referral center for healthcare in Belo Horizonte and the metropolitan area, which may have a higher prevalence of resistance than the general population.Further studies are needed to verify the effectiveness of treatment with thiamphenicol.
This study supports the use of third-generation cephalosporins and spectinomycin as a fi rst-choice treatment for gonorrhea in Belo Horizonte.The results of our study confirm the recommendations for the non-use of penicillin and tetracycline and advise against the use of ciprofl oxacin.Based on our in vitro results, the rate of resistance to chloramphenicol, which is structurally related to thiamphenicol, discourages its empirical use for the treatment of gonorrhea.
The effective treatment of gonorrhea is important to reduce its transmission and to reduce the transmission of HIV.The continuous monitoring of treatment efficacy and regional systematic surveillance of antimicrobial susceptibility, are critical components of disease control and prevention.Further studies are urgently needed to identify new alternative treatment regimens for gonorrhea.
Patients in the Department of STD/SCS are predominantly young and have multiple partners, a low level of education and income and a history of recurrent STDs.More effective programs to promote health must be developed that include considerations of sexual practices and encourage the practice of safe sex.
The study of the susceptibility of N. gonorrhoeae to antimicrobials represents a step toward more effi cient therapies.This type of strategy must become a routine part of monitoring reference services of microbial resistance.

TABLE 1 -
Susceptibility and MIC, as determined by E-test, of 201 strains of Neisseria gonorrhoeae sampled in the Department of Sexually Transmitted Diseases/Specialized Care Services of the SRU-CS/SMSA in Belo Horizonte, State of Minas Gerais, Brazil.

TABLE 2 -
Plasmid-mediated and chromosomal-mediated resistance of 201 Neisseria gonorrhoeae isolates to penicillin and tetracycline.