Abstract
Background:
Pharyngeal Neisseria gonorrhoeae (phNG) infections are difficult to treat and contribute to antimicrobial resistance (AMR) in NG, but studies on phNG and differences with urogenital NG (uNG) are limited.
Methods:
Participants with urethral discharge syndrome (UDS) were recruited at health clinics in Kampala, Uganda, between July 2020 and August 2023. Penile-meatal and pharyngeal swabs were cultured for NG and tested by nucleic acid amplification test (NAAT) for NG, Chlamydia trachomatis, and Mycoplasma genitalium. Paired uNG and phNG isolates were subjected to susceptibility testing and whole genome sequencing (WGS). Demographic and behavioral data were analyzed for correlates of uNG and phNG.
Results:
The participants’ (n=411) median [IQR] age was 25 [22, 32] years; 16.7% (67/401) were living with HIV. By NAAT and/or culture, 273 (66.4%) participants were positive for uNG and 29 (7.1%) for phNG. Of those with uNG, 10.6% (29/273) were NAAT-positive for phNG; 69.0% (20/29) were culture-positive for phNG. All (n=40) NG isolates were susceptible to cefixime and ceftriaxone and displayed intermediate resistance/resistance to ciprofloxacin, penicillin, and tetracycline; eight displayed high-level azithromycin resistance. WGS showed that 52.6% (10/19) of participants had different strains at urethral and pharyngeal sites. Having different strains at two anatomical sites was more common in men self-reporting transactional sex (p=0.019).
Conclusions:
phNG is common in Ugandan men with uNG, both displaying high rates of AMR. Different strains at the two anatomical sites were common, supporting the need for pharyngeal testing and phNG susceptibility testing as the pharynx is a critical site for AMR emergence.
Keywords: Neisseria gonorrhoeae, pharyngeal gonorrhea, antimicrobial resistance, sexually transmitted infections (STI), extragenital STI
Introduction
As the second most common bacterial sexually transmitted infection (STI) with an estimated 82 million cases in 2020 [1], gonorrhea is a major public health threat with no vaccine and limited treatment options [2]. Neisseria gonorrhoeae (NG) has progressively developed antimicrobial resistance (AMR) to all first-line treatments, including ceftriaxone (CRO), the currently recommended treatment in the United States and many other high-income countries [3, 4]. NG with decreased susceptibility/resistance to CRO and associated with treatment failures are increasing globally [5] especially in the Asia-Pacific region [6] and England [7] and threaten the future successful treatment of gonorrhea.
Pharyngeal NG (phNG) infections are more difficult to treat than urogenital NG (uNG) infections [8, 9]. A review of reported cases of NG treatment failures found that the site of infection most associated with confirmed treatment failures was the pharynx with 75% (6/8) of cases; in the case of probable treatment failures – defined as probable because the isolated strain was found to be ceftriaxone susceptible – the pharynx was the common site with 60% (15/25) of cases [5]. PHNG infections also play an important role in the emergence of AMR in NG [8, 10]. Previous studies have shown that CRO resistance in NG emerged from recombination events with commensal Neisseria species, which are common in the oropharynx [10]. Furthermore, as phNG infections are primarily asymptomatic, they serve as a transmission reservoir of undiagnosed infections [11], which provides an optimal setting for selection of AMR [12]. Despite the clinical importance of pharyngeal gonorrhea, surveillance and epidemiological data on AMR from phNG isolates are lacking. A reason for the lack of data from this anatomical site is that pharyngeal gonococcal cultures, required for antimicrobial susceptibility testing (AST), lack sensitivity for isolation of NG due to low culture recovery rates [8, 13, 14]. Additionally, the increased use of nucleic acid amplification tests (NAATs) for the diagnosis of NG has resulted in a significant decrease in culture isolates for AST and surveillance [15].
As surveillance data informs treatment guidelines and public health responses, it should include a representative sample of NG isolates from all anatomical sites to provide an accurate portrayal of AMR trends. However, the collection of pharyngeal samples for NG AMR surveillance is not routinely performed globally [16]. Therefore, it is critical to evaluate phNG isolates to ascertain potential differences in the antimicrobial susceptibility profiles of uNG and phNG. A study compared uNG and phNG isolates from two patients and reported that one patient was infected with the same strain at both sites, while the second patient was infected with different strains [17]. A larger study compared the antimicrobial susceptibility profiles of 55 paired uNG and phNG isolates from men who have sex with men (MSM) collected by the CDC Gonococcal Isolate Surveillance Program (GISP) between 2011 and 2013 [18]. The study reported that >90% of urethral-pharyngeal NG pairs from the same participant had concordant minimum inhibitory concentrations (MICs) within 1 dilution of each other [18]. A study of Korean men with urethritis reported that 30% of men tested positive for NG at both the urethra and pharynx and that 87.5% (14/16) of men had strains with the same sequence type at both anatomical sites [19]. However, the study did not compare antimicrobial susceptibility profiles between uNG and phNG. Another study reported that 18.2% (2/11) of patients with paired uNG and phNG had isolates with discordant MICs [20]. However, in addition to performing the analysis with non-contemporary NG isolates (collected between 2014–2015), genomic analysis was limited to paired isolates with discordant MICs, preventing detection of strain differences between isolates with identical or similar antimicrobial susceptibility profiles. Despite these studies, there are knowledge gaps on phNG in low-resource settings, especially on potential differences in antimicrobial susceptibility profiles between uNG and phNG.
Our group has previously reported high prevalence of urogenital gonorrhea and AMR in Ugandan men with urethral discharge syndrome (UDS) attending government health clinics in Kampala, Uganda [21]. In this study, we report the results of a multi-year, cross-sectional study to determine the prevalence of phNG infections, concurrent uNG and phNG infections among men with UDS. Secondary aims were to determine the microbiological (including AMR profile), clinical, and sociodemographic differences between men with only uNG and those with concurrent phNG. Finally, we assessed genomic differences between uNG and phNG.
Methods
Male participants with UDS were recruited at six government health clinics in Kampala, Uganda between July 2020 and August 2023. Following written consent, participants underwent onsite point-of-care testing for HIV and syphilis as previously described [21] and self-collected three penile-meatal swabs and urine. Self-collected penile-meatal swabs and urine are comparable for single or multiple STI detection in Ugandan men with UDS [22]. Additionally, two oropharyngeal swabs were collected by a study nurse. All samples were transported to the Translational Research Laboratory at the Infectious Diseases Institute (IDI) in Kampala for processing, testing, and storage. Clinical care was not a component of this study; participants received treatment based on the Ugandan national guidelines on syndromic case management [23]. Demographic and behavioral data, including self-reported condom use, number of sexual partners, engagement in transactional sex, and alcohol use, were collected by a trained research nurse using a structured interview format. A transport reimbursement of Ugandan shillings 20,000 (equivalent to approximately $5–6) was provided to each participant.
At the IDI lab, a penile-meatal and pharyngeal swab from each participant were cultured on selective media for NG. Following overnight incubation, a discrete translucent colony was isolated and sub-cultured overnight on non-selective media (enriched GC agar). Biochemical tests; oxidase reaction, superoxol reaction, and Gram stain were performed; gram-negative diplococci, superoxol-positive, and oxidase-positive bacterial isolates were classified as NG, and stored frozen at −80°C. The second penile-meatal and pharyngeal swab was eluted in phosphate-buffered saline (PBS), frozen at −80°C, and together with the NG isolates transported to the Johns Hopkins University School of Medicine for additional testing. At Johns Hopkins, the penile-meatal and/or urine samples were tested using the Aptima NAAT (Hologic Inc, Marlborough, MA, USA) for NG, Chlamydia trachomatis (CT), and Mycoplasma genitalium (MG); pharyngeal samples were tested for NG and CT.
At Johns Hopkins, all NG isolates recovered from participants with paired uNG and phNG underwent AST for azithromycin, cefixime, ceftriaxone, ciprofloxacin, penicillin, and tetracycline using the agar dilution method. If a culture appeared to be mixed (based on colony morphology) with more than one NG strain, each colony type was sub-cultured, tested by NAAT to confirm that it was NG, and subjected to AST. The WHO reference strains L, V, W, X, and the ATCC49226 reference strains were included as quality control to ensure the accuracy of agar dilution results. Breakpoints for resistance classification were selected in accordance with Clinical and Laboratory Standards Institute (CLSI) guidelines [24]. Isolates with azithromycin MIC ≥ 16 μg/mL were classified as resistant and underwent AST using E-test to determine if they displayed high-level resistance (HLR) to azithromycin (MIC ≥ 256 μg/mL). Paired uNG and phNG isolates were classified as having discordant MICs if there was a difference ≥ 2 logs for at least one of the antimicrobials. Paired isolates with discordant MICs and isolates displaying HLR to azithromycin were subjected to repeat AST to confirm MICs.
Genomic DNA from the NG isolates was extracted using the QIAamp DNA Micro Kit (Qiagen) and used for library construction for whole genome shotgun deep sequencing on the Illumina NovaSeq S4 platform, yielding a minimum of 10M 150 bp paired-end reads per genome. Genome assembly with SPAdes v4.0.0 [25] resulted in draft genomes assembled into 89 to 257 contigs of size ≥ 500 bp. Assemblies were annotated with Prokka v1.13 [26].
Variants in known resistance markers (Supplemental Table 1) were identified by sequencing read mapping with breseq V0.38.1 [27]. Raw sequencing reads were used to perform molecular epidemiologic analysis using the multilocus sequence typing (MLST) method [28]. MLST defines sequence types (STs) based on a combination of alleles at seven housekeeping genes [28]. A matrix of pairwise distances between whole genome sequences was generated using Mash v2.3 [29] and a neighbor-joining phylogenetic tree constructed from this matrix using quicktree v2.5 [30] with default parameters. Trees were viewed in Dendroscope v3.8.10 [31]. Sequencing data for the 40 NG isolates have been deposited at NCBI under BioProject accession number PRJNA1460746 (accession numbers JBYDFB000000000, JBYDFA000000000, JBYDEZ000000000, JBYDEY000000000, JBYDEX000000000, JBYDEW000000000, JBYDEV000000000, JBYDEU000000000, JBYDET000000000, JBYDES000000000, JBYDER000000000, JBYDEQ000000000, JBYDEP000000000, JBYDEO000000000, JBYDEN000000000, JBYDEM000000000, JBYDEL000000000, JBYDEK000000000, JBYDEJ000000000, JBYDEI000000000, JBYDEH000000000, JBYDEG000000000, JBYDEF000000000, JBYDEE000000000, JBYDED000000000, JBYDEC000000000, JBYDEB000000000, JBYDEA000000000, JBYDDZ000000000, JBYDDY000000000, JBYDDX000000000, JBYDDW000000000, JBYDDV000000000, JBYDDU000000000, JBYDDT000000000, JBYDDS000000000, JBYDDR000000000, JBYDDQ000000000, JBYDDP000000000, JBYDDO000000000.
Descriptive statistical analysis was performed to provide the prevalence of uNG as well as concurrent phNG and summarize the distribution of key demographics, clinical, behavioral characteristics of men with UDS by urogenital and pharyngeal positivity. Chi-square test (or Fisher’s exact test) and non-parametric Mann-Whitney U test were performed for comparing categorical data and continuous data between two groups, respectively. SAS version 9.4 software (SAS Institute, Cary, North Carolina) was used for all analyses and a 2-sided p-value < .05 was considered statistically significant.
The studies were approved in Uganda by the Joint Clinical Research Center at Makerere University (JC0919, JC1920), and the Ugandan National Council for Science and Technology (HS455ES, HS1338ES) approved the study. Approval was also obtained from the Johns Hopkins Institutional Review Board (IRB00215298, IRB00273945). Written informed consent was obtained in Luganda or English per participant preference.
Results
Of the 416 participants enrolled in the study, 411 (98.8%) were included in the analysis (Figure 1). The remaining five participants were excluded because of pharyngeal NAAT equivocal NG results (n=4) or missing pharyngeal sample (n=1). The median [IQR] age of participants was 25 [22, 32] years, 16.7% (67/401) were living with HIV, and 6.8% (27/399) tested positive for syphilis antibodies.
Figure 1. Participants and recovery of urogenital NG and pharyngeal NG isolates.
NAAT – nucleic acid amplification test. uNG – urogenital NG. phNG – pharyngeal NG,
By culture (NG only) and/or NAAT, 80.0% (329/411) of participants had at least one urogenital STI (gonorrhea, chlamydia, and/or Mycoplasma genitalium) (Table 1). Of those, 22.8% (75/329) had ≥ 2 urogenital STIs. Of the 329 participants positive for an urogenital STI, 244 (74.2%) participants were positive for uNG and negative for phNG, including 184 (55.9%) with NG mono-infection, 39 (11.9%) with NG and CT, 16 (4.9%) with NG and MG, and 5 (1.5%) with NG, CT, and MG. Co-infection with CT and MG were detected in 1.5% (n=5) of participants; mono-infection with CT or MG was detected in 8.5% (28/329) and 5.4% (18/329) of participants, respectively.
Table 1.
Demographics, clinical, behavioral characteristics of men with urethral discharge syndrome by urogenital and pharyngeal positivity.
| Characteristics | Any STI | Urogenital NG only | Urogenital & pharyngeal NG | Non-NG STI urogenital | P |
|---|---|---|---|---|---|
| N=329 n (%) | N=244 n (%) | N=29 n (%) | N=56 n (%) | ||
| Age (years) | 24 [22, 31] | 24 [22, 31] | 25 [23, 32] | 25.5 [21.5, 31] | 0.637 |
| 16–19 | 15 (4.6) | 11 (4.5) | 3 (10.3) | 1 (1.8) | |
| 20–24 | 150 (45.6) | 113 (46.3) | 11 (37.9) | 26 (46.4) | |
| 25–29 | 66 (20.1) | 47 (19.3) | 6 (20.7) | 13 (23.2) | |
| 30–34 | 46 (14.0) | 34 (13.9) | 4 (13.8) | 8 (14.3) | |
| ≥ 35 | 52 (15.8) | 39 (16.0) | 5 (17.2) | 8 (14.3) | |
| HIV | 0.780 | ||||
| Negative | 273 (83.0) | 199 (84.3) | 26 (89.7) | 48 (85.7) | |
| Indeterminate | 1 (0.3) | 1 (0.4) | 0 (0.0) | 0 (0.0) | |
| Positive | 47 (14.3) | 37 (15.2) | 3 (10.3) | 7 (12.5) | |
| Unknown | 8 (2.4) | 7 (2.9) | 0 (0.0) | 1 (1.8) | |
| Syphilis | 0.704 | ||||
| Negative | 295 (89.7) | 217 (88.9) | 28 (96.6) | 50 (89.3) | |
| Indeterminate | 1 (0.3) | 1 (0.4) | 0 (0.0) | 0 (0.0) | |
| Positive | 24 (7.3) | 18 (7.4) | 1 (3.4) | 5 (8.9) | |
| Unknown | 9 (2.7) | 8 (3.3) | 0 (0.0) | 1 (1.8) | |
| # sex partners (2 months) | 0.833 | ||||
| 0 | 7 (2.1) | 5 (2.1) | 0 (0.0) | 2 (3.6) | |
| 1 | 142 (43.2) | 103 (42.2) | 14 (48.3) | 25 (44.6) | |
| 2 | 110 (33.4) | 86 (35.3) | 9 (31.0) | 15 (26.8) | |
| 3 | 32 (9.7) | 20 (8.2) | 1 (3.5) | 11 (19.6) | |
| 4 | 13 (4.0) | 10 (4.1) | 2 (6.9) | 1 (1.8) | |
| ≥5 | 25 (7.6) | 20 (8.2) | 3 (10.3) | 2 (3.6) | |
| Transactional Sex | 0.554 | ||||
| Given | 72 (21.9) | 58 (23.8) | 2 (6.9) | 12 (21.4) | |
| Received | 24 (7.3) | 14 (5.7) | 4 (13.8) | 6 (10.7) | |
| Both given and received | 33 (10.0) | 26 (10.7) | 4 (13.8) | 3 (5.4) | |
| No | 200 (60.8) | 146 (59.8) | 19 (65.5) | 35 (62.5) | |
| Condom use (6 months) | 0.321 | ||||
| Always | 8 (2.4) | 6 (2.5) | 2 (6.9) | 0 (0.0) | |
| Sometimes | 137 (41.6) | 105 (43.0) | 10 (34.5) | 22 (39.3) | |
| Never | 150 (56.0) | 133 (54.5) | 17 (58.6) | 34 (60.7) | |
| # of episodes of UDS (6 months) | 0.181 | ||||
| 0 | 24 (7.3) | 19 (7.8) | 1 (3.4) | 4 (7.1) | |
| 1 | 209 (63.7) | 167 (68.4) | 19 (65.5) | 23 (41.1) | |
| 2 | 74 (22.6) | 43 (17.6) | 7 (24.1) | 24 (42.9) | |
| ≥3 | 21 (6.4) | 15 (6.1) | 1 (3.4) | 5 (8.9) | |
| Unknown | 1 (0.3) | 0 (0.0) | 1 (3.4) | 0 (0.0) |
P values compared participants with urogenital NG only to participants with urogenital NG and pharyngeal NG.
By culture (NG only) and/or NAAT, phNG and pharyngeal CT were detected in 7.1% (29/411) and 0.5% (2/411) of participants, respectively. Of those with uNG, 10.6% (29/273) were positive for phNG. All participants with phNG were also positive for uNG, most (90.0%, n=26) were HIV-negative, the majority (69.0%, [20/29]) were < 30 of age, and were only positive for uNG and no other urogenital STIs (Tables 1 and 2). Of the 29 participants with urogenital and pharyngeal gonorrhea, 27 (93.1%) and 20 (69.0%) were culture-positive for uNG and phNG, respectively (Table 2). The uNG and phNG isolates from one participant were excluded from the analysis because the pharyngeal isolate was grossly contaminated, and a pure pharyngeal isolate could not be recovered; uNG and phNG isolates from 19 participants were included in the final analysis.
Table 2.
Distribution of urogenital STIs in Ugandan men with pharyngeal NG.
| Pattern number | Number of participants | HIV status | Urogenital | Pharyngeal | |||
|---|---|---|---|---|---|---|---|
| CT | NG | MG | CT | NG | |||
| A. Participants with culture-positive pharyngeal NG (n=20) & | |||||||
| 1 | 12 | − | − | + | − | − | + |
| 2 | 5 | − | + | + | − | − | + |
| 3 | 2 | + | − | + | − | − | + |
| 4 | 1 | − | − | + | − | − | + |
| B. Participants with culture-negative/NAAT-positive pharyngeal NG (n=9) | |||||||
| 5 | 3 | − | − | + | − | − | + |
| 6 | 2 | − | + | + | − | − | + |
| 7 | 2 | − | − | + * | − | − | + |
| 8 | 1 | − | − | + | + | − | + |
| 9 | 1 | + | − | + | + | − | + |
CT – Chlamydia trachomatis; NG – Neisseria gonorrhoeae; MG – Mycoplasma genitalium; NAAT – Nucleic Acid Amplification Test.
Of the 20 participants with culture-positive urogenital and pharyngeal NG, the NG isolates from 19 participants (n=40) were included in the analysis.
These two participants were only positive for uNG by NAAT.
Table 3 describes the characteristics of participants and antimicrobial susceptibility profiles of the uNG and phNG isolates recovered from those participants. A total of 40 uNG and phNG isolates were included in the analysis; participant MN_220 had two unique urogenital (penile) isolates, while participant SA_285 had two unique pharyngeal isolates. Genomic analysis revealed that 47.4% (9/19) of participants with uNG and phNG had the same strain type at both anatomical sites (Figure 2); most (88.9%; 8/9) of these paired isolates had identical antimicrobial susceptibility profiles with the exception of the isolates from participant SA_281 in which the penile and pharyngeal isolate displayed intermediate resistance and resistance to penicillin, respectively (Table 3A). Concurrent uNG and phNG infections caused by different strains were identified in 52.6% (10/19) of participants (Table 3B). In seven (SA_057, SA_166, SA_285, MN_102, MN_220, MN_221, and MN_228) of the 10 participants, the paired urogenital and pharyngeal isolates had different antimicrobial susceptibility profiles to at least one antimicrobial (Table 3B). Self-reported transactional sex was more common in men with different strain types between the urethra and pharynx than in men with the same strain (70% (7/10) vs 11.1% (1/9), p=0.019).
Table 3.
Participants’ characteristics and antimicrobial susceptibility profiles of paired uNG and pNG.
| ID | Anatomical site (isolate #) | Age | Transactional sex | MLST ST | AZM | CIP | PEN | TET |
|---|---|---|---|---|---|---|---|---|
| Antimicrobial susceptibility (Minimum Inhibitory concentration (MIC) (μg/mL) All isolates were susceptible to CEF and CRO | ||||||||
| A. Participants with the same NG strain at both anatomical sites | ||||||||
| SA_005 | P & Ph | 38 | No | 17833 | S | R | R | R |
| SA_014 | P & Ph | 32 | No | 11368 | S | R | I | R |
| SA_030 | P & Ph | 33 | No | 1587 | S | R | R | R |
| SA_041 | P & Ph | 35 | No | 11365 | S | R | R | I |
| SA_102 | P & Ph | 18 | No | 1919 | S | R | R | R |
| SA_120 | P & Ph | 25 | Yes | 11994 | HLR | R | I | R |
| SA_121 | P & Ph | 24 | No | 11365 | S | R | R | I |
| SA_171 | P & Ph | 24 | No | 1587 | S | R | R | R |
| SA_281 | P | 28 | No | 1588 | S | R | I (0.25) | R |
| Ph | R (8) | |||||||
| Median age | 28 & | |||||||
| B. Participants with different NG strains | ||||||||
| SA_057 | P | 23 | No | 13778 | S | R | R (64) | R |
| Ph | 13781 | I (0.25) | ||||||
| SA_110 | P | 29 | No | 11365 | S | R | I | R |
| Ph | 13781 | |||||||
| SA_166 | P | 42 | No | 13778 | S | R (2) | I (0.125) | R |
| Ph | 13765 | R (8) | R (>32) | |||||
| SA_241 | P | 37 | Yes | 1588* | S | R | R (2) | R |
| Ph | 1588* | R (8) | ||||||
| SA_285 | P | 17 | Yes | 1588^ | S (0.125) | R | I | R |
| Ph (1) | 11994 | HLR | ||||||
| Ph (2) | 1588^ | S (0.125) | ||||||
| SA_290 | P | 20 | Yes | 1901 | S | R | R (8) | R |
| Ph | 1588 | R (32 | ||||||
| MN_102 | P | 22 | Yes | 18034 | S | R | R | R (32) |
| Ph | 11367 | I (0.5) | ||||||
| MN_220 | P (1) | 32 | Yes | 11994 | HLR | R (16) | I (0.25) | R (>32) |
| P (2) | 18034 | S (0.125) | R (4) | R (48) | R (32) | |||
| Ph | 14451 | S (0.03) | R (4) | I (0.25) | R (>32) | |||
| MN_221 | P | 24 | Yes | 11994 | HLR | R (16) | I (0.25) | R |
| Ph | 1597 | S (0.25) | R (4) | R (>32) | R | |||
| MN_228 | P | 23 | Yes | 11994 | HLR | R (16) | I | R |
| Ph | 11368 | S (0.25) | R (4) | |||||
| Median age | 24 & | |||||||
MLST ST – Multilocus Sequence Type, Sequence Type; AZM – azithromycin; CEF – cefixime; CRO – ceftriaxone, CIP – ciprofloxacin; PEN – penicillin; TET - Tetracycline; P – penile; Ph – pharyngeal; S – susceptible, I – intermediate resistance; R – resistant. High-level resistance (HLR) – isolates with AZM MIC ≥ 256 μg/mL. The penile culture of participant MN_220 and the pharyngeal culture of participant SA_285 had two different NG strains each.
Both strains belonged to MLST ST1588 but were distinct strains (Figure 2).
Both strains belonged to MLST ST1588 but were two different strains (Figure 2). For simplicity, the MIC is not shown if the MIC was identical for both the penile and pharyngeal isolate.
The difference in age between participants with the same or different strain types at both anatomical was not statistically significant (p=0.347).
Figure 2. Maximum-likelihood phylogenetic tree of 40 Neisseria gonorrhoeae (NG) isolates from participants with urogenital (penile - P) and pharyngeal (Ph) NG.
★ - Participants with the same strain at both anatomical sites. & - Participant SA_241 had two different strains belonging to MLST ST1588. $ - Participant SA_285 was infected with three different strains. # - Participant MN_220 was infected with three
All NG isolates displayed intermediate resistance or resistance to ciprofloxacin, penicillin, and tetracycline; 15.0% (6/40) of isolates displayed intermediate resistance, resistance, or high-level resistance to four antimicrobials (azithromycin, ciprofloxacin, penicillin, and tetracycline); all isolates were susceptible to cefixime and ceftriaxone (Table 3). Of the 19 participants with uNG and phNG, 10 (52.6%) had paired uNG and phNG isolates with discordant (≥ 2 logs) MICs to a least one antimicrobial; in five pairs, the MIC was higher in the pharyngeal isolate (SA_166, SA_241, SA_281, SA_285, SA_290) and higher in the urogenital isolate in three pairs (SA_057, MN_102, MN_228). In the remaining two pairs (MN_220, MN_221), the MIC to one antimicrobial was higher in the penile isolate, but higher in the pharyngeal isolate to a different antimicrobial. Phenotypic antimicrobial susceptibility profiles correlated well with known AMR markers (Supp Table 1).
Discussion
In this cross-sectional study, we assessed prevalence of pharyngeal gonorrhea, AMR, and phenotypic and genomic differences between uNG and phNG in men seeking treatment for UDS at Ugandan government health centers. Our study showed that phNG infections are common (~11%) in Ugandan men with urogenital gonorrhea, which is consistent with previous findings that extragenital infections can be common in key target populations in high-income settings [32, 33]. Notably, the majority of participants with pharyngeal gonorrhea in our study were infected with different strains at the urethra and pharynx, which often displayed distinct antimicrobial susceptibility profiles.
In our study, the overall rate of pharyngeal gonorrhea among all participants was 7.1% which increased to 10.6% among those with urogenital gonorrhea suggesting that Ugandan men with UDS could be a key target population for pharyngeal testing to help enhance surveillance of AMR in NG in Uganda. Our findings are similar to those from a study of MSM from South Africa which reported that 18% of oropharyngeal samples from men with UDS were positive for NG [34]. Assessment of antimicrobial susceptibility in phNG provides an opportunity to detect emerging and increasing AMR as the pharynx is an important niche for the emergence of AMR in NG; the pharynx is a reservoir of AMR markers which could be transferred from commensal Neisseria species to NG through horizontal gene transfer [10, 35].
Although cultures are less sensitive than NAAT for detection of phNG [8, 13, 14], phNG isolates were recovered from 69% of study participants with NAAT-confirmed pharyngeal gonorrhea, allowing for assessment of differences in uNG and phNG from most of the participants. Our study found that 52.6% of the participants with culture-positive uNG and phNG had different strains at the two anatomical sites, higher than reported by a previous study of Korean men, which compared isolates from multisite infection and found that 9.6% (4/41) of patients had different strains [19]. Another study assessing differences in heterosexual and homosexual networks among patients attending STI clinics in Amsterdam reported that 40% of patients with multi-site infections were infected with different NG strains [36]. Our study found that self-reported transactional sex was associated with having different NG strains between the urethra and pharynx. This finding and that of another study which reported high rates of non-traceable partners as an indicator of high-risk behavior [37], suggest that participants infected with multiple strains may have acquired different strains from different sexual partners. Our study did not systematically assess for the presence of mixed-strain infections but found that two participants were infected with two different strains at one anatomical site (one participant in the urethra and another participant in the pharynx). Other studies have reported that mixed gonococcal infections at a single anatomical site may be relatively infrequent [37, 38]. Goire et al. reported that, despite intensively sampling of isolates from NG-positive samples, only 3.2% (2/63) of NG-positive samples provided evidence of mixed-strain infections [38].
All of the uNG and phNG isolates in our study displayed intermediate resistance or resistance to ciprofloxacin, tetracycline, and penicillin which is consistent with results from our previous study on urogenital gonorrhea in Ugandan men with UDS [21]. However, this study identified isolates with HLR to azithromycin, which have not been previously reported in Uganda, reflecting the evolving nature of AMR in this setting with high rates of gonorrhea. Although most of the isolates with HLR to azithromycin were recovered from urogenital samples, one participant had an azithromycin-susceptible uNG infection while the phNG isolate displayed HLR to azithromycin, highlighting the importance of extragenital testing and performing antimicrobial susceptibility testing on phNG isolates.
Our study has several limitations. First, while we enrolled and tested a large number of participants, the number of participants with concurrent uNG and phNG was relatively small when compared to those with only uNG, limiting our ability to detect robust statistical differences between those with single-site and multi-site NG infections. Due to Ugandan laws criminalizing some sexual behaviors [39], we did not collect information on sexual orientation, sexual behavior, and anatomical sites of exposures, which prevented us from assessing relevant predictors of pharyngeal gonorrhea. We did not collect data on HIV pre-exposure prophylaxis (PrEP) use or pharyngeal symptoms. However, previous studies have shown that pharyngeal NG infections are primarily asymptomatic [35]. Our study included only men recruited from government health centers, limiting the generalizability of our results to the general population. Additional studies involving women and sexual partners of those diagnosed with NG are warranted. Genomic analysis was limited to cultured NG isolates, which prevented the assessment of differences between uNG and phNG from all participants with NAAT-confirmed gonorrhea. Performing WGS on samples (urogenital and pharyngeal) from participants without NG isolates may help to identify potential differences in uNG and phNG from those participants.
In the largest, to our knowledge, prospective study to assess pharyngeal gonorrhea in men with UDS, our study showed that pharyngeal gonorrhea is common in Ugandan men with urogenital gonorrhea. As such, pharyngeal gonorrhea may contribute to the ongoing gonorrhea epidemic in Uganda given that phNG infections are primarily asymptomatic, and that cefixime – which has decreased efficacy for the treatment of phNG – is used for the syndromic management of gonorrhea in Uganda. These data have public health implications beyond surveillance planning; eight in ten men had urogenital STIs, consistent with our previous study in which 84% of Ugandan men with UDS attending government clinics were positive for at least one urogenital STI (gonorrhea, chlamydia, Mycoplasma genitalium or trichomoniasis) [21] suggesting suboptimal STI control measures. Consistent condom use was rare and HIV prevalence was high in Ugandan men with UDS; prioritizing men with UDS for curable STI and HIV prevention strategies may reduce the population burden [40]. Future surveillance programs of NG AMR in Uganda, which currently only collect urogenital isolates, should include pharyngeal isolates as our study demonstrated that multi-site infections caused by strains with different antimicrobial susceptibility profiles are common. Furthermore, assessment of antimicrobial susceptibility in phNG could help to identify emerging AMR as the pharynx is a critical site for emergence of AMR.
Supplementary Material
Article’s main point:
Pharyngeal gonorrhea is common in Ugandan men with urogenital gonorrhea. The majority of these multi-site infections were caused by different Neisseria gonorrhoeae strains highlighting the importance of susceptibility testing of pharyngeal isolates to more effectively monitor emerging gonococcal antimicrobial resistance.
Financial support.
This work was funded by the National Institutes of Health (NIH): K01AI153546 to J. H. M. and U54EB007958 to Y. C. M.).
Footnotes
Potential conflict of interest. The authors have no conflict of interest.
Data availability statement:
The sequencing data is available via the National Center for Biotechnology Information (NCBI) under BioProject accession number PRJNA1460746. The data underlying this article will be shared on reasonable request to the corresponding author.
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Associated Data
This section collects any data citations, data availability statements, or supplementary materials included in this article.
Supplementary Materials
Data Availability Statement
The sequencing data is available via the National Center for Biotechnology Information (NCBI) under BioProject accession number PRJNA1460746. The data underlying this article will be shared on reasonable request to the corresponding author.