Antimicrobial Susceptibility of Neisseria gonorrhoeae Isolates From Peru, 2018 and 2019

Abstract

Background 

Currently, in Latin America, including Peru, the treatment of gonorrhea is still empiric and information regarding antimicrobial resistance is scarce in some countries because of the limited resources, which can contribute to the rising rates of reported multidrug-resistant gonococcal strains. In that context, it is mandatory to continuously monitor and report antimicrobial resistance in N. gonorrhoeae to update treatment recommendations.

Methods 

This descriptive study analyzed genital and anal samples from symptomatic patients who attended 15 sexually transmitted infections health facilities from 8 different regions in Peru during the years 2018 to 2019 within the framework of Sentinel Surveillance. After establishing the presumptive diagnosis, the isolates were sent to the Laboratory of Sexually Transmitted Bacteria of the National Institute of Health of Peru in Lima where the species were confirmed (N = 165) and susceptibility profiles were determined.

Results 

Among the 165 isolates, 95.2% corresponded to male patients, between 18 and 22 years of age (40.6%), half reported having a sexual partner and being heterosexual. Clinically, 89.7% manifested the presence of urethral exudate. Microbiology showed 95.2% of the isolates resistant to ciprofloxacin and 9.1% non-susceptible to azithromycin. Reduced susceptibility to ceftriaxone and cefixime was observed in 1.2% and 3.6% of the isolates respectively. All strains tested were susceptible to spectinomycin.

Conclusions 

This study demonstrated that in Peru, fluoroquinolones should not be recommended or used in N. gonorrhoeae infections due to the high percentage of resistant strains. In addition, nationwide access to gonococcal resistance testing, molecular diagnostics and antimicrobial stewardship should be implemented to control the spread of gonococcal antimicrobial resistance.

In Brief

In this Peruvian descriptive study, among the 165 Neisseria gonorrhoeae isolates collected between 2018 and 2019, 95.2% were resistant to fluoroquinolones; in addition, 1.2% and 3.6% of the isolates showed reduced susceptibility to ceftriaxone and cefixime, respectively.

Gonorrhea is the second most frequently reported sexually transmitted infection (STI) with the highest antimicrobial resistance (AMR) rate worldwide and is caused by the gram-negative diplococcus N. gonorrhoeae (NG).¹ This STI causes mostly cervicitis in women and urethritis in men, but it is often asymptomatic in women, and when untreated, it may lead to complications, such as infertility, pelvic inflammatory disease, and ectopic pregnancy,² whereas neonatal conjunctivitis and blindness are also possible.³ In addition, gonorrhea facilitates both the acquisition and transmission of the human immunodeficiency virus (HIV).

Nevertheless, there is a lack of gonococcal AMR reports, surveillance, and diagnosis in many countries, making it difficult to establish the true global burden of gonorrhea.

In recent years, an increased resistance to all therapeutic options for this STI has been reported, including the extended-spectrum cephalosporins (ESCs) ceftriaxone and cefixime, which are currently the last option of single-dose empirical treatment available.⁴ According to the Latin American Surveillance Network of Antimicrobial Resistance, gonococcal resistance to ciprofloxacin has increased from 35% in 2009 to 62% in 2015.¹ Furthermore, reduced susceptibility and resistance to macrolides have also been reported in Latin America and the Caribbean (LAC).^1,5–7

The World Health Organization (WHO) Global Gonococcal Antimicrobial Surveillance Programme, which has been monitoring gonococcal AMR worldwide since 1990, has reported confirmed therapeutic failures with ceftriaxone monotherapy or combined with doxycycline or azithromycin in Europe, Asia, and Australia.⁸ Also, resistance or decreased susceptibility to ESCs has been reported in LAC.^9,10 In Peru, there has been also a rising trend of gonococcal AMR; in 1998 to 1999, 11.1% of 130 strains showed reduced susceptibility to azithromycin, whereas 100% were susceptible to ciprofloxacin, spectinomycin, and ceftriaxone.¹¹ Later, in 2016, the NG Sentinel Surveillance Group, reported 82.3% resistance to ciprofloxacin, 54.2% to tetracycline, 51% to penicillin, 15.6% to azithromycin, and 4.2% none susceptibility to ceftriaxone in 96 strains.¹⁰ According to WHO, countries should update their national gonorrhea treatment guidelines based on recent local AMR surveillance data, and where this information is not available, recommends dual empiric therapy (eg, ceftriaxone plus azithromycin) over monotherapy for asymptomatic and symptomatic gonorrhea.¹²

The aim of this article is to describe the AMR profiles and some clinical features of NG isolates collected between 2018 and 2019, identified from the Sentinel Surveillance Group.

MATERIALS AND METHODS

This is a descriptive cross-sectional study on 165 NG isolates (1 per patient) recovered from 970 samples collected in 15 STI health facilities (sentinel centers) located in 8 regions of Peru: Lima, Madre de Dios, Ayacucho, Ancash, Arequipa, Lambayeque, Loreto, and Puno.

From these samples, 316 suspicious specimens were sent to the Laboratory of Sexually Transmitted Bacteria of the National Institute of Health of Peru (INS) in Lima, during the period of 2018 to 2019, as part of the NG Sentinel Surveillance in Peru. The genital (urethral, endocervical, vaginal) and extragenital (anal) samples were obtained from symptomatic patients and sexual contacts of NG cases at the sentinel centers for Gram stain and culture. Once the suspicious isolates were obtained, these were sent at room temperature on Thayer Martin agar or Amies transport medium to the Specialized Reference Centers at the INS, within 48 hours.

Subsequently, all samples were cultured on Thayer Martin and chocolate agar at the INS, and incubated for 18 to 24 hours at 36°C in an enriched atmosphere of 5% CO₂ and 70% to 80% humidity. Presumptive identification of NG was performed analyzing colony features, Gram stain, superoxol, and oxidase test; for the confirmatory identification of NG a commercial kit (API NH system, BioMérieux, France) and conventional methods by carbohydrate degradation were used. For the ß-lactamase production, commercial discs were used (Nitrocefin Remel). For determining antimicrobial susceptibility, NG isolates were tested using the agar dilution method for the determination of the minimum inhibitory concentration (MIC) using GC agar enriched with 1% Isovitalex or Vitox, according to the recommendations of the Clinical and Laboratory Standards Institute (CLSI-M100)¹³ to the following antibiotics: penicillin, tetracycline, ciprofloxacin, ceftriaxone, cefixime, azithromycin, and spectinomycin (Sigma-Aldrich Brand). For gentamicin, 140 of 165 isolates were evaluated and analyzed using the breakpoints provided by the Latin-American Reference Laboratory for Sexually Transmitted Diseases, National Institute of Infectious Diseases “Dr. Carlos G. Malbrán,” Argentina.¹⁴

NG ATCC 49226 (American Type Culture Collection) and WHO K, L, M, N and O strains were used as controls, according to the Technical Verification Report of Antimicrobial Susceptibility method by MIC and the INS internal quality control.¹⁵

The phenotypes were categorized using the nomenclature of resistance phenotypes in NG, also provided by the Latin-Americans Reference Laboratory (Table 2).^16,17

TABLE 2 - Resistance Phenotypes of N. gonorrhoeae Isolates (N = 165)

Number	Resistance Phenotypes	%
1	PPNG/CMTR/QRNG/AZNS	0.61
2	PP-TRNG/QRNG	1.82
3	CMRNG/QRNG/AZNS	1.21
4	PPNG/CMRT/QRNG	5.45
5	CMTR/QRNG/AZNS	2.42
6	CMPR/TRNG/QRNG	1.21
7	CMPR/QRNG	8.48
8	CMPR/TRNG	0.61
9	CMRNG/QRNG	4.24
10	CMTR/QRNG	8.48
11	CMTR/AZNS	0.61
12	QRNG/AZNS	4.24
13	TRNG/QRNG	5.45
14	PPNG/QRNG	15.15
15	PP-TRNG	0.61
16	PPNG	1.21
17	QRNG	36.36
18	SENSIBLE	1.82
Total		100.00

Interpretation:

• PPNG: penicillinase-producing N. gonorrhoeae. Strains with plasmid resistance to penicillin: β-lactamase positive, MIC ≥2 μg/mL, usually inhibition halo ≤19 mm.

• CMPR: strain of N. gonorrhoeae with chromosomal resistance to penicillin: β-lactamase negative, MIC ≥2 μg/mL, inhibition halo ≤26 mm.

• TRNG: N. gonorrhoeae with plasmid resistance to tetracycline: MIC ≥16 μg/mL, inhibition zone ≤19 mm.

• CMTR: N. gonorrhoeae with chromosomal resistance to tetracycline: MIC ≥2 μg/mL, inhibition halo ≤30 mm and >19 mm.

• PP-TRNG: N. gonorrhoeae with plasmid resistance to penicillin and tetracycline.

• CMRNG: N. gonorrhoeae with chromosomal resistance to penicillin and tetracycline.

• QRNG: N. gonorrhoeae with chromosomal resistance to quinolones.

• AZNS: N. gonorrhoeae not susceptible to azithromycin (MIC ≥2 μg/mL).

RESULTS

Most of the isolates included in this study were recovered from patients that resided in Lima city (63%), in Madre de Dios region located in the Amazon basin (13.9%), and in the Ayacucho region in the south-central Andes (6.7%). Most isolates (95.2%, 157 of 165) were recovered from male patients, 49% (77 of 157) of which were classified as men who had sex with men (MSM) and 3.82% (6 of 157) were bisexual men. Most of the isolates were obtained from urethral samples (89.7%, 148 of 165), whereas the rest were recovered from cervix, vagina and anus. From the 165 people sampled, patients' age ranged from 18 to 52 years (median age, 24 years). There were 59.4% (98 of 165) who had high school and 32.1% (53 of 165) had university as their highest education level. Most of the patients (73.9%, 122 of 165) lived in an urban area, and 26.7% (44 of 165) stated as being students, whereas 3.6% (6 of 165) were sex workers.

Regarding clinical features, as all collected samples were obtained from patients with suggestive symptoms of gonorrhea, 89.7% (148 of 165) of them had urethral discharge and 72.1% (119 of 165) presented dysuria, among other signs and symptoms as pruritus and lower abdominal pain.

Antimicrobial susceptibility testing of the 165 isolates was performed against 8 antibiotics as shown in Table 1. It was observed a high percentage of ciprofloxacin resistance (95.2%, 157 of 165) among all sampled patients, and also in MSM (94.3%, 66 of 70) and in men who had sex with women (96.3%, 78 of 81). Regarding macrolides, only 9.1% (15 of 165) of the isolates were not susceptible to azithromycin with the highest MIC of 8 μg/mL.

TABLE 1 - Antimicrobial Susceptibility and MIC of N. gonorrhoeae Isolates (N = 165)

Antibiotic	Susceptible		Intermediate		Resistant		NS*		RS†
	n	%	n	%	n	%	n	%	n	%	MIC Range (μg/mL)	MIC90	MIC50
Ciprofloxacin	5	3.0	3	1.8	157	95.2					0.002–32	16	4
Tetracycline	9	5.5	102	61.8	54	32.7					0.125–64	16	1
Penicillin	1	0.6	97	58.8	67	40.6					0.06–128	64	1
Azithromycin	150	90.9					15	9.1			0.025–8	1	0.25
Ceftriaxone	163	98.8							2	1.2	0.002–0.06	0.03	0.008
Cefixime	159	96.4							6	3.6	0.004–0.25	0.06	0.016
Gentamicin‡	33	23.6	107	76.4	0	0					4–16	16	8
Spectinomycin	165	100	0	0	0	0					8–32	32	32

*Nonsusceptibility.

^†Reduced susceptibility.

^‡25 unprocessed isolates.

Reduced susceptibility to ceftriaxone (MIC, 0.06–0.25 μg/mL) and cefixime (0.125–0.25 μg/mL) was observed in 1.2% (2 of 165) and 3.6% (6 of 165) of the isolates, respectively. There were 76.4% (107 of 140) that was categorized as intermediate susceptibility (MIC = 8–16 μg/mL) and isolates with resistance to gentamicin were not detected. All isolates were susceptible to spectinomycin. The MIC distributions and ranges are shown in Figure 1. In addition, several resistance phenotypes were determined (Table 2).

Figure 1: MIC distributions for N. gonorrhoeae isolates (N = 165).

Of note, as part of the syndromic management, among the 165 patients whose samples were analyzed, we had information from 46.7% (77 of 165) patients, and among those, 64.9% (50 of 77) received intravenous ceftriaxone monotherapy or as a combination therapy, 32.5% (25 of 77) received oral ciprofloxacin monotherapy or as a combination therapy and 7.8% (6 of 77) received other antimicrobials during the study period.

DISCUSSION

This article describes the AMR patterns from the largest sample of NG isolates in Peru to date, as part of the National Sentinel Surveillance Program. Because the national reports on gonococcal susceptibility profiles are still scarce in Peru, monitoring emerging resistance and its trends is essential for updating NG treatment guidelines.

Our results showed that 95.2% of the isolates were resistant to ciprofloxacin. This situation was already reported in 2016 to 2017 in Peru (82.3%)¹⁰ and has been described in multiple countries around the world since the mid-2000s.¹⁸ In fact, ciprofloxacin resistance in LAC has shown a rising trend through the years, staying below 5% until 2004, increasing over 15% in 2006 and reaching levels over 40% in 2010.¹⁴ In Peru, an increased consumption and an inappropriate use of ciprofloxacin have been reported during the years 2016 to 2017 in addition with the local STI guidelines, which recommended ciprofloxacin as first line therapy for gonorrhea, this would explain the rising trend of resistance to this antimicrobial.^19,20 These results indicate that ciprofloxacin should not be used as a first-line antibiotic, and its use should be limited to those isolates in which sensitivity to quinolones is proven. The MIC values found in our study for tetracycline and gentamicin had the same antimicrobial wild type distributions showed by the European Committee on Antimicrobial Susceptibility Testing.²¹

In addition, despite there was no high-level of resistance to azithromycin among these strains, the finding of azithromycin non-susceptible isolates (15 isolates with a MIC ≥2 μg/mL) increase the possibilities of selecting higher MIC levels if no measures in the use of this antibiotic are taken.²² As shown in a previous publication,¹⁰ we also found isolates with reduced susceptibility to ESCs. Our findings indicate that these isolates are still rare (less than 5% of the population), confirming that these antibiotics are still useful for gonorrhea treatment in Peru. This is particularly important for ceftriaxone, the ESC recommended for dual empiric therapy, with a very low prevalence of non-susceptibility (less than 2%).^10,23 Gonococcal resistance to ceftriaxone has been reported in many countries around the world, including treatment failures with monotherapy and dual therapy with ceftriaxone plus doxycycline or azithromycin.^8,24 In the Latin American region, isolates resistant to ceftriaxone have been described since 2007 in Argentina, Chile, Uruguay, and Brazil.^4,9,18 This regional emergency calls for continued surveillance of gonococcal AMR in Peru for decreased susceptibility to ESCs, the current last option in empiric gonorrhea therapy. This aspect is crucial because the WHO recommends suspending the use of an empirical antimicrobial treatment when the rates of resistance or therapeutic failure reach a level of 5%.³ Unfortunately, despite being a global major public health concern, gonococcal AMR surveillance is still very limited in many settings worldwide including Latin America.

Regarding therapy, until a gonococcal vaccine is developed and available, an appropriate, opportune and affordable syndromic treatment is necessary, and this depends on the recent and local gonococcal AMR data. In Peru, the current National Guidelines for STIs Management (including N. gonorrhoeae) are being updated; the following syndromic treatment scheme is now being indicated: ceftriaxone 500 mg intramuscularly single dose plus azithromycin 1 g orally single dose and as an alternative treatment gentamicin 240 mg intramuscularly in a single-dose plus azithromycin 2 g orally in a single dose, taking into consideration the PAHO/WHO guidelines for the treatment of NG and the national reports.^12,25,26

It is important to mention that because of the unstoppable AMR, new treatment regimens will be essential in the upcoming years, and for this purpose, 3 approaches might be considered: first, the use of novel antibiotics; second, new combinations of already existing antibiotics; and third, the use of alternative drugs.²⁷

The limitations of this study should be noted. First, the data presented has been obtained from populations with high-risk sexual behaviors, which attended STI health facilities, mostly men and symptomatic patients. Second, although this is the largest sample of NG isolates described in Peru, there are concerns regarding the representativeness of data (WHO recommends testing approximately 100 isolates per year); however, efforts are being done to surpass this goal in the upcoming years. Third, genomic analysis of NG isolates was not performed; and lastly, we had no information regarding possible NG treatment failures in these 165 patients.

In conclusion, to control and mitigate the rising gonococcal AMR in our country, it is mandatory to use the right antibiotic. First, the use of fluoroquinolones is no longer recommended because of the high level of gonococcal resistance; likewise, combined antimicrobial therapy should be used to treat Chlamydia trachomatis coinfection and preferably with single doses to improve medication adherence. Also, increased detection and timely treatment of gonorrhea based on the local AMR prevalence are also critical; and, to achieve this goal, it is important to increase nationwide access to gonococcal resistance testing and molecular diagnostics to implement antimicrobial stewardship.

Finally, an improved surveillance system collecting data regarding key factors, such as age, occupation, or same-sex partnership, might identify specific risk factors of gonococcal AMR to prevent and mitigate the spread of ESC-resistant strains in our country.