Abstract
We examined the factors influencing gonorrhea detection at the pharynx. One hundred men infected with Neisseria gonorrhoeae were swabbed from the tonsils and posterior oropharynx. N. gonorrhoeae was reisolated from the tonsils and posterior oropharynx in 62% and 52%, respectively (P = 0.041). Culture positivity was greater with higher gonococcal DNA loads at the tonsils (P = 0.001) and oropharynx (P < 0.001). N. gonorrhoeae can be cultured from the tonsils and posterior oropharynx with greater isolation rates where gonococcal loads are higher.
TEXT
Rising gonorrhea rates, growing antimicrobial resistance, and increasingly limited antimicrobial options globally have made gonorrhea an urgent antibiotic-resistant threat (1). Pharyngeal gonorrhea is believed to be an important factor in the development of Neisseria gonorrhoeae antimicrobial resistance by facilitating the transfer of genetic elements of antibiotic resistance from commensal Neisseriae, prevalent in the pharynx, to N. gonorrhoeae (2).
Regular screening for pharyngeal gonorrhea is recommended for men who have sex with men (MSM). There are few published data, however, that indicate where gonococci reside within the pharynx and whether swabs should be taken from the tonsils, posterior oropharynx, or both. The primary aim of this study was to establish the specific anatomical sites from which pharyngeal gonorrhea can be detected, comparing culture and nucleic acid amplification testing (NAAT). The secondary aim was to determine the factors that influence detection using these methods, including sampling and load of N. gonorrhoeae.
This was a secondary study embedded within a larger study that was undertaken at the Melbourne Sexual Health Centre, Australia, as previously detailed elsewhere (3). Briefly, MSM were screened for pharyngeal gonorrhea using culture. Men who were culture positive for pharyngeal N. gonorrhoeae and who had not already been treated were recalled 7 days later, reswabbed from the pharynx, and retested for gonorrhea using culture and NAAT prior to treatment. First, one swab was taken from the posterior oropharynx (henceforth referred to as oropharynx), which involved sweeping the tip of a cotton swab across the posterior oropharynx without making contact with the tonsils. Then, a second swab was taken from both palatine tonsils (henceforth referred to as tonsils) where the tip of a cotton swab was swept across each tonsil, avoiding contact with the oropharynx (4, 5). All study swabs obtained from the tonsils and oropharynx were collected carefully by three sexual health clinicians (M. Bissessor, A. F. Snow, and D. M. Lee) trained in a standardized method to ensure consistent sampling.
The oropharyngeal and tonsillar swabs were plated onto separate plates for gonorrhea culture. Culture was undertaken using modified Thayer Martin medium. After inoculating culture plates, swabs were placed into 10-ml tubes containing phosphate-buffered solution for NAAT detection (6).
Detection of N. gonorrhoeae DNA in samples was performed using individual real-time PCR methods targeting the gonococcal porA pseudogene and multicopy opa genes (porA monoplex and opa monoplex) as previously described (6). An NAAT result was reported as positive when the two assays were positive. The cycle threshold (CT) values obtained using the porA monoplex and opa monoplex real-time PCR were used as semiquantitative measures of gonococcal DNA. CT values are inversely proportional to the gonococcal load; that is, the lower the CT value, the higher the gonococcal load. A difference of 3.3 cycles is generally considered to represent a 10-fold difference in DNA load (7).
To assess sampling adequacy, DNA extracts were tested using a real-time PCR assay targeting human endogenous retrovirus 3 (ERV3) as previously described (8). ERV3 real-time PCR values were used as semiquantitative measures of human DNA.
The calculation of the sample size has been previously described for the larger study (5). The chi-square test was used to compare categorical data, and McNemar's test was used to compare paired proportions. The Mann-Whitney U test was used to compare organism loads between groups. Ethical approval for this study was granted by the Alfred Hospital Research Ethics Committee (no. 453/11).
One hundred men with pharyngeal gonorrhea were recruited into the study. The median time between the diagnosis of pharyngeal gonorrhea and retesting and treatment was 7 days (range, 6 to 9 days). The demographic and behavioral characteristics of men and pharyngeal isolate MICs have been detailed elsewhere (3).
N. gonorrhoeae was cultured from the tonsils as well as the oropharynx (62% versus 52%, respectively; P = 0.041). In 47 men, the culture was positive at the tonsils and the oropharynx. There were 15 men whose culture was positive at the tonsils but negative at the oropharynx, and there were 5 men whose culture was positive at the oropharynx but negative at the tonsils (Fig. 1A). A further 33 men were culture negative at the two sites.
FIG 1.
Number of positive gonorrhea swabs among 100 men with culture-positive pharyngeal gonorrhea according to site of sampling.
N. gonorrhoeae was detected using NAAT in 84% of tonsillar and 81% of oropharyngeal samples. These results are summarized Table 1. NAAT for N. gonorrhoeae isolates from the tonsils and oropharynx was significantly more sensitive than culture at the tonsils (84% versus 62%; P < 0.001) and the oropharynx (81% versus 52%; P < 0.001). There was no significant difference in detection between the tonsils and oropharynx using NAAT (P = 0.61). In 75 men, NAAT was positive at the tonsils and oropharynx. In 9 men, NAAT was positive at the tonsils but negative at the oropharynx, and in 6 men, NAAT was positive at the oropharynx but negative at the tonsils (Fig. 1B). A further 10 men were NAAT negative at the two sites.
TABLE 1.
Frequency of results for differing combinations of N. gonorrhoeae culture, real-time PCR, and ERV3 real-time PCR
| No. of men | Tonsilsa |
Oropharynxa |
||||
|---|---|---|---|---|---|---|
| Culture | N. gonorrhoeae PCR (CT [range; mean]) | ERV3 PCR (CT [range; mean]) | Culture | N. gonorrhoeae PCR (CT [range; mean]) | ERV3 PCR (CT [range; mean]) | |
| 45 | + | + (23–36; 29) | + (22–29; 26) | + | + (22–35; 28) | + (23–30; 26) |
| 2 | + | − | + (26–26; 26) | + | + (33–37; 36) | + (28–28; 28) |
| 12 | + | + (26–31; 28) | + (24–28; 26) | − | + (25–35; 30) | + (24–30; 27) |
| 2 | + | + (35–37; 36) | + (27–29; 28) | − | − | + (29–29; 29) |
| 1 | + | − | + (29) | − | + (35) | + (29) |
| 4 | − | + (31–32; 31) | + (24–27; 26) | + | + (29–30; 29) | + (25–29; 26) |
| 1 | − | − | + (29) | + | + (33) | + (27) |
| 14 | − | + (24–36; 31) | + (24–31; 27) | − | + (24–37; 32) | + (24–29; 27) |
| 7 | − | + (32–38; 35) | + (23–31; 27) | − | − | + (26–32; 28) |
| 2 | − | − | + (27–29; 28) | − | + (35–38; 36) | + (28–28; 28) |
| 10 | − | − | + (24–30; 27) | − | − | + (25–30; 27) |
+, positive; −, negative.
Analysis of ERV3 CT values indicated that detection of gonorrhea was influenced by the adequacy of sampling (Table 1). At the oropharynx, the median ERV3 CT value was significantly lower (indicating higher human DNA load) in culture-positive men than that in culture-negative men (26.8 cycles versus 27.8 cycles; P = 0.013). At the tonsil, the median ERV3 CT value was not significantly lower in culture-positive men than that in culture-negative men (26.3 cycles versus 27.0 cycles; P = 0.182). Sampling also influenced gonorrhea detection by NAAT. At the tonsils, the median ERV3 CT value was significantly lower in NAAT-positive men than that in NAAT-negative men (26.2 cycles versus 27.3; P = 0.040). Similarly, at the oropharynx, the median ERV3 CT value was significantly lower in NAAT-positive men than that in NAAT-negative men (26.9 cycles versus 27.5; P = 0.045). However, in all of these observations, the difference in ERV3 CT values corresponded to a 10-fold difference in DNA load.
At the tonsils, the median opa monoplex real-time PCR CT values were significantly lower (indicating higher gonococcal DNA load) in culture-positive specimens than those in culture-negative specimens (28.0 cycles and 31.4 cycles; P = 0.001). Similarly, at the oropharynx, the median CT values of opa monoplex real-time PCR were significantly lower in culture-positive specimens than those in culture-negative specimens (28.5 cycles and 33 cycles; P < 0.001). In the above observations, the difference in CT values corresponded to a >10-fold difference in DNA load.
In this study, we determined the sites within the pharynx from which N. gonorrhoeae can be detected using two different laboratory methods. Using culture, N. gonorrhoeae was isolated from the tonsils and oropharynx separately. NAAT also detected N. gonorrhoeae separately from the tonsils and oropharynx but with significantly greater sensitivity. The isolation rate from culture was significantly higher with higher gonococcal load and better sampling. Detection using NAAT was also improved with better sampling.
We included culture in this study because isolation of N. gonorrhoeae requires a live organism and is, therefore, indicative of productive infection. While pharyngeal screening for gonorrhea using NAAT has increased in clinical practice, culture remains important for the determination of antimicrobial susceptibility and test of cure following treatment for gonorrhea (3).
Adequate sampling is important for optimal detection of pharyngeal gonorrhea using culture (4, 5). We have also shown that sampling adequacy is important for the detection of N. gonorrhoeae at the tonsils and the oropharynx using NAAT even though NAAT is a more sensitive method for detecting N. gonorrhoeae. In addition, even when NAAT testing is used, it is important to sample the oropharynx and tonsils.
Previously, we showed that lower-load gonococcal infections in the pharynx were more likely to be missed using culture (9). In this study, we found that the failure of culture to detect lower-load gonococcal infections applied to the tonsils and the posterior oropharynx independently.
Although the positivity from culture taken from the tonsils was significantly higher than that from the oropharynx, both were low considering that all men in the study were culture-positive from screening that was undertaken a median of 7 days prior. Previous studies using culture suggest that pharyngeal gonorrhea is generally transient (10, 11) and self-limiting over a period of weeks or months. It would, therefore, seem unlikely that spontaneous clearance of infection would account for the poor yield when each site was sampled independently within 7 days. Rather, it may reflect the fact that optimal yield using culture requires swabbing from the two sites to obtain sufficient bacterial load to support culture. Ostensibly, this would also aid in detection of pharyngeal N. gonorrhoeae by NAAT.
There are strengths and limitations to this study. Culture for gonorrhea requires stringent transport and laboratory conditions. The on-site location of our laboratory with the ability to culture at the point of collection, as well as the rigorous laboratory methods, ensured optimal conditions for N. gonorrhoeae growth. A limitation is that although there was a high level of emphasis placed on careful sampling from the tonsils and oropharynx, there was still some variability in sampling as indicated by the analyses of ERV3.
Previous studies have investigated the sensitivity of oral throat rinses for the detection of pharyngeal gonorrhea. These are potentially more acceptable, as swabs may induce a gag reflex making collection uncomfortable for patients and impeding proper sampling. However, in a study of 25 cases of pharyngeal gonorrhea, the sensitivity of NAAT on oral-rinse specimens was only 72% (12).
The asymptomatic nature of pharyngeal gonorrhea, its propensity for selection of antimicrobial-resistant clones, and treatment failures underscore the importance of optimal detection of pharyngeal gonorrhea and verification of cure.
This study has established that N. gonorrhoeae can be isolated from the tonsils and the oropharynx. Clinicians screening for pharyngeal gonorrhea using culture or NAAT should be aware of the higher sensitivity of NAAT over that of culture and should be trained to ensure that one swab is collected carefully and thoroughly from each of these sites for optimal detection of N. gonorrhoeae, regardless of the laboratory method used.
ACKNOWLEDGMENTS
We thank Jun Kit Sze and Afrizal for data extraction and the laboratory staff at the University of Melbourne Microbiology Diagnostic Unit for handling specimens and performing cultures and MIC determination. We also wish to thank staff at the Melbourne Sexual Health Centre that supported the study, including Htun Lin Aung who assisted with data analysis. Melanie Bissessor was supported by a National Health and Medical Research Council postgraduate scholarship (no. 1038734).
We declare that we have no conflicts of interest.
REFERENCES
- 1.Centers for Disease Control and Prevention. 2013. Antibiotic resistance threats in the United States, 2013. Centers for Disease Control and Prevention, Atlanta, GA: http://www.cdc.gov/drugresistance/threat-report-2013/pdf/ar-threats-2013-508.pdf Accessed 2 February 2015. [Google Scholar]
- 2.Unemo M, Nicholas RA. 2012. Emergence of multidrug-resistant, extensively drug-resistant and untreatable gonorrhea. Future Microbiol 7:1401–1422. doi: 10.2217/fmb.12.117. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 3.Bissessor M, Whiley DM, Fairley CK, Bradshaw CS, Lee DM, Snow AS, Lahra MM, Hocking JS, Chen MY. 2015. Persistence of Neisseria gonorrhoeae DNA following treatment for pharyngeal and rectal gonorrhea is influenced by antibiotic susceptibility and reinfection. Clin Infect Dis 60:557–563. doi: 10.1093/cid/ciu873. [DOI] [PubMed] [Google Scholar]
- 4.Razali MF, Fairley CK, Hocking J, Bradshaw CS, Chen MY. 2010. Sampling technique and detection rates for pharyngeal gonorrhea using culture. Sex Transm Dis 37:522–524. [DOI] [PubMed] [Google Scholar]
- 5.Mitchell M, Rane V, Fairley CK, Whiley DM, Bradshaw CS, Bissessor M, Chen MY. 2013. Sampling technique is important for optimal isolation of pharyngeal gonorrhoea. Sex Transm Infect 89:557–560. doi: 10.1136/sextrans-2013-051077. [DOI] [PubMed] [Google Scholar]
- 6.Goire N, Nissen MD, LeCornec GM, Sloots TP, Whiley DM. 2008. A duplex Neisseria gonorrhoeae real-time polymerase chain reaction assay targeting the gonococcal porA pseudogene and multicopy opa genes. Diagn Microbiol Infect Dis 61:6–12. doi: 10.1016/j.diagmicrobio.2007.12.007. [DOI] [PubMed] [Google Scholar]
- 7.Whiley DM, Goire N, Ray ES, Limnios A, Lambert SB, Nissen MD, Sloots TP, Tapsall JW. 2010. Neisseria gonorrhoeae multi-antigen sequence typing using non-cultured clinical specimens. Sex Transm Infect 86:51–55. doi: 10.1136/sti.2009.037689. [DOI] [PubMed] [Google Scholar]
- 8.Alsaleh AN, Whiley DM, Bialasiewicz S, Lambert SB, Ware RS, Nissen MD, Sloots TP, Grimwood K. 2014. Nasal swab samples and real-time polymerase chain reaction assays in community-based, longitudinal studies of respiratory viruses: the importance of sample integrity and quality control. BMC Infect Dis 14:15. doi: 10.1186/1471-2334-14-15. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 9.Bissessor M, Tabrizi SN, Fairley CK, Danielewski J, Whitton B, Bird S, Garland S, Chen MY. 2011. Differing Neisseria gonorrhoeae bacterial loads in the pharynx and rectum in men who have sex with men: implications for gonococcal detection, transmission, and control. J Clin Microbiol 49:4304–4306. doi: 10.1128/JCM.05341-11. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 10.Templeton DJ, Jin F, McNally LP, Imrie JC, Prestage GP, Donovan B, Cunningham PH, Kaldor JM, Kippax S, Grulich AE. 2010. Prevalence, incidence and risk factors for pharyngeal gonorrhoea in a community-based HIV-negative cohort of homosexual men in Sydney, Australia. Sex Transm Infect 86:90–96. doi: 10.1136/sti.2009.036814. [DOI] [PubMed] [Google Scholar]
- 11.Wallin J, Siegel MS. 1979. Pharyngeal Neisseria gonorrhoeae: coloniser or pathogen? Br Med J 1:1462–1463. doi: 10.1136/bmj.1.6176.1462. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 12.Papp JR, Ahrens K, Phillips C, Kent CK, Philip S, Klausner JD. 2007. The use and performance of oral-throat rinses to detect pharyngeal Neisseria gonorrhoeae and Chlamydia trachomatis infections. Diagn Microbiol Infect Dis 59:259–264. doi: 10.1016/j.diagmicrobio.2007.05.010. [DOI] [PubMed] [Google Scholar]