Lower genital tract infection in women is the most common disease in gynecological clinics.1 The majority of these infections are caused by mixed pathogens. Treatment is frequently neglected or delayed,2 especially for sexually transmitted infections (STIs), resulting in subsequent fulminant infection (pelvic inflammatory diseases).3 STIs are also some of the most common serious diseases in the emergency department. Even though intensive treatment is used, some STIs are ultimately complicated with severe sequelae, resultant socioeconomic problems, infertility, ectopic pregnancy, preterm labor, and chronic pelvic pain.4 Therefore, it is important to identify these highly invasive pathogens accurately and rapidly, since prompt and appropriate antibiotic treatment can lead to patients avoiding the majority of these complications.
To make an early diagnosis of STIs, familiarity with the diseases, a high degree of suspicion and easy-to-use diagnostic methods are important. Chlamydia infection may be one of the most well-known examples, because of the development of artificial reproductive techniques and the well-recognized adverse effects of chlamydia infection on fertility. The polymerase chain reaction makes the diagnosis of chlamydia infection easier and quicker. By contrast, the diagnosis of Neisseria gonorrhoeae is often overlooked. The causes include the lack of a formal and organized curriculum in medical schools or house officer training,5 the lower incidence in the general population, nonspecific symptoms or signs of infection, and rare discomfort of infected women. Of course, lack of simple, accurate and easy-to-use diagnostic methods further hinders our attention to this detrimental infection in women.
According to the estimate of the World Health Organization,6 62 million cases of gonorrhea occur annually worldwide, and the sequelae of gonorrhea infection in women, such as the facilitation of human immunodeficiency virus transmission, ocular infections of the newborn, disseminated infection and infertility, are severe and profound.7 Nearly blind use of antibiotics for the N. gonorrhoeae infection has enabled resistant strains to spread widely and rapidly.8 All of these factors contribute to the necessity of revisiting N. gonorrhoeae infection.
In the general population, the estimated prevalence of gonorrhea infection is around 0.7% to 7%,9,10 and it increases to 15–35% in specific high-risk groups,11,12 including sex workers or groups with different risk factors, such as young age, black ethnicity, frequent sexual contacts,9 and cases with some specific symptoms, such as mucopurulent cervical discharge and lower abdominal pain.10
There are several tools that are used currently to establish diagnosis of gonorrhea infection.2,7,8,10 This review attempts to describe and compare these methods, and hopefully help physicians better understand this common but overlooked sexually transmitted disease.
2. Syndromic approach
This is a subjective and non-consistent method for the diagnosis of a specific cervical infection. There should be various symptoms in a cervical infection caused by different and multiple pathogens in different persons with various physical conditions. It is nearly impossible to identify the pathogen simply from symptoms. Moreover, so many asymptomatic women are not aware of the risk of gonorrhea infection.
It is believed that gonorrhea infection is most likely confirmed if mucopurulent discharge is present during vaginal speculum examination.6 Other factors include history of being a sex worker and tenderness induced by bimanual examination. However, mucopurulent discharge (non-clear, yellowish discharge from the endocervix), friability (easy bleeding) when the cervix is touched with a swab, or a positive swab test (yellow discoloration of the swab when inserted in the endocervix) predict only a 50% chance of gonorrhoeae infection.13
In different populations, such as patients from a family practice, in a prenatal clinic, or in a setting especially for sex workers, the sensitivity, specificity and positive predictive value of various methods differ due to fluctuating incidence.10 The average sensitivity and specificity for an approach by syndrome are <50%.6
Combined with a scoring system using risk factors,14 such as age, marital status, dyspareunia, and discolored vaginal discharge, higher sensitivity (60–80%) and specificity (∼60%) have been sporadically reported in certain groups of patients, using a syndromic approach.10 Despite the low sensitivity and specificity, it is still an easy, rapid and economic tool for diagnosis. The syndromic approach remains the main diagnostic choice in most developing countries with limited laboratory facilities.15
3. Gram’s stain
Spreading of smear samples from the cervix on a glass slide and staining with dyes seems an inexpensive and convenient screening method for diagnosis of infection and malignancy. Identification of intracellular Gram-negative diplococci under the microscope after Gram staining is considered an instant and standard finding, with specificity around 95%.16,17 However, it is not recommended by the World Health Organization for diagnosis of cervical infection,6 because of its low detection rate of near 30%,18 even when well-trained technicians are available.6 Some have tried to raise the detection ability of Gram’s stain by using the criteria of finding ≥10 polymorphonuclear neutrophils per high-power field. However, as an aid to diagnosis, a result with sensitivity <50% is disappointing.16 This might also indicate the minimal role of Pap smear in identifying cervical infection.
Microbial culture is still accepted by some investigators as the gold standard for the diagnosis of gonorrhea infection,19,20 and is used as a standard to be compared with the results of nucleic acid amplification tests (NAATs)19 or other tools.20 With a high specificity (95%) but a questionable detection rate (50%), this tool no longer satisfies the needs of modern medicine.
Optional conditions such as a CO2-enriched (5–7%) and body-temperature (37°C) environment, and an enriched medium such as modified Thayer–Martin medium or chocolatized medium will achieve a better culture rate.21 These complicated conditions might explain the questionable sensitivity of this tool. To increase the detection rate, the high suspicion of an alert clinician is essential for choosing the right culture medium and conditions, and a significant improvement in culture rate could also be achieved by urgent transportation to and processing of the specimens in the laboratory.22
Compared to the advanced NAATs, the culture method seems not sensitive enough to be the gold standard of diagnosis of gonorrheal infection. However, sometimes, the culture method might provide more accurate information when a NAAT is targeted on a changing gene.23 Moreover, this is still the only method that provides information about antimicrobial agent susceptibility. Although resistant strains become increasingly prevalent, high-quality culture methods will need to be maintained to ensure a representative sample for susceptibility testing.24 Moreover, the culture method can be used as a rescue approach when symptoms persist even after optimal treatment.
Different immunoassay kits are used as a rapid method to confirm the genus in the Neisseria family. The products available on the market include GonoGen, GonoGen II, the Syva Micro Trak Direct Fluorescent-Antibody test, the Phadebact Monoclonal GC OMNI test, and the Gonobio-Test.25 These tools are useful in telling us if the culture is N. gonorrhoeae or not. Some use this tool to detect possible infection from original cervical swabs directly. The sensitivity rates of currently available rapid tests are between 50% and 70%. The change of target antigen after routine use may decrease sensitivity after extinguishing the related subtypes, but improve the specificity up to 90–100%.11,17
In spite of their lower sensitivity, immunoassays, as rapid point-of-care tests, might outperform other standard tests in populations with high sexual activity, such as sexual workers and/or in those with low return rates in developing countries.26 If treatment can be started at the initial visit, the possibility of onward transmission of N. gonorrhoeae can be minimized7,18 Immunoassays are an easy-to-use method with rapid diagnosis, low cost and no requested microscopy, therefore, they seem more acceptable than other tools in developing countries.11,17,18,20
To date, NAATs have provided high specificity of 95–100%, and the best sensitivity of all diagnostic methods, at around 95%.19,20,22 There are also consistent data among different NAATs, such as the strand displacement assay (BD ProbeTec C trachomatis/N gonorrhoeae Amplified DNA Assay) APTIMA Combo 2 and APTIMA GC assays,19 Ligase Chain Reaction (Abbott Laboratories, Abbott Park, IL, USA),20 and Cobas Amplicor (Roche).20 With their extremely high sensitivity and specificity, they are considered the gold standard for diagnosis27 and good for different samples, such as cervical swabs from intrusive speculum examination or urine from the noninvasive route.28
However, there are still some pitfalls in using these tools. They can cross-react with other Neisseria species and can be affected by specimen transport conditions. False-negative reports, compared with culture data, indicate the variation in the target sequence. Therefore, confirmation with another NAAT method, which may double the cost, has been recommended.23 In addition, the widespread use of NAATs might result in decreased isolation of N. gonorrhoeae and ignorance of possible resistant organisms.
Due to their high cost, NAATs are not recommended as the only tool for screening. The use of a molecular testing strategy may be cost-effective when it is supplemented with microscopy and culture, to provide prompt treatment and further microbial susceptibility testing.29 In fact, NAATs are not only expensive and complicated, but also dependent on highly trained staff and delicate equipment. These factors all hinder the widespread use of these techniques in developing countries.11 Therefore, they might not be suitable as a diagnostic method in a point-of-care setting.11,18
7. Prospecting method
Advances in high throughput assays such as protein microarray chip fabrication30 have made it possible to perform multiple immunoassays with one minimal vaginal sample. Up to several thousand proteins can be printed and evaluated on one slide simultaneously.31 In addition, liposomal nanovesicles might be used to build up a signal amplifying system and increase the sensitivity rate of immunoassays.32,33 This strategy has been used successfully for the detection of an infectious Escherichia coli strain, with a detection limit 100CFU/mL,32 and similar immunoassay techniques have been applied to detect several food-borne pathogens simultaneously, including E. coli O157:H7, Salmonella enterica serovar typhimurium, and Listeria monocytogenes, with detection limits between 100 and 15,000CFU/mL.33
The liposomal nanovesicles are composed of outer-surface bilayer and inner vesicles; there are several hundred thousand fluorescent dyes contained within the vesicle (Fig. 1), which amplify the fluorescent signals. These are excellent and easy-to-use signal carriers in biosense analysis.34 In addition, antibody microarray is an excellent tool for multiplex detection of analytes (Fig. 2). The illustrated procedures comprise several steps. Capture antibodies are first printed on the slide. After interaction with pathogens, detection antibodies conjugated with biotin are added. Finally, the amplifying liposome system is used to label the biotin.
Another advantage is the multiplexed nature of a microarray assay. STIs involve not only the conventionally named female reproductive organs, but also the anus or oral cavity; therefore, there are plenty of microorganisms and pathogens found, including different kinds of bacteria and viruses from the cervix. In this situation, it is reasonable and convenient to use a single approach with a multiplexed nature to detect all the common microorganisms in one test either by culture35 or targeting of nucleic acid.36 For an inflammatory disease caused by various and multiple pathogens, detection of them all in one multiplexed tool is the best policy. Moreover, a thorough and detailed therapeutic plan can be useful in eradicating the infection.35,36
Besides the benefits of high throughput and being multiplexed, the amount of sample in this immunoassay can be lowered to tens of microliters.30 These advantages make the method more convenient and easier to sue. The best part is that the estimated cost for each chip is US$3, if 200 chips are fabricated at the same time. Now, this technique is also being tested in our laboratory for the detection of gonorrhea infection. We hope to report the results in the near future.
In conclusion, a brief summary of available methods for the diagnosis of N. gonorrhoeae infection is listed in Table 1. Although the traditional methods have higher scores, the sensitivity of these tools is too low and is not acceptable for a screening method. In contrast, the NAATs show the best sensitivity and specificity; however, the application of NAATs to the clinical practice might not be cost-effective since the score, as shown in Table 1 is not high. This main defect limits their popularity, and they are not widely used in point-of-care settings. When considering a screening tool in a developing country, the tools reviewed above do not fit the routine clinical practice, which means that we are not still satisfied with these methods. To achieve the goals of accurate diagnosis and effective treatment of infectious diseases, such as gonococcal infection, advances in techniques to aid the detection of pathogens should be encouraged and supported.24
Factors determining the choice of screening tests for gonorrhea infection in women might include test sensitivity, specificity, available antibiotic susceptibility tests, ease of specimen collection, time, cost, degree of technique difficulty, and laboratory facilities.21 NAATs supplemented with microscopy and culture remain the best choice in an ideal setting of a developed country.24,29 However, in settings where laboratory facilities are not fully available, especially in a developing country or in high-risk populations where return rates are low, rapid tests using immunoassay may be the most effective way of diagnosing gonorrhea infection.11,18 The optimal use in these settings requires the development of rapid tests that are simpler and cheaper.7 An easy, fast, inexpensive, high-throughput, and nano-scale-sensitive multiplexed detection system might be the answer. A clinician might be able to detect the different pathogens causing cervical and vaginal infections, including gonorrhoea or chlamydiasis,37 immediately and accurately using a microarray-based immunoassay. With a sensitive test for the point-of-care setting, a correct prescription of antibiotics will be made rapidly and confidently and over- or under-treatment can be avoided.
This work was supported in part by grants from the Veterans General Hospitals University System of the Taiwan Joint Research Program (VGHUST99-G4) and Taipei Veterans General Hospital (V99-C1-085 and V99F-014), Taiwan, ROC.
1. Donders G. Diagnosis and management of bacterial vaginosis and other types of abnormal vaginal bacterial flora: a review. Obstet Gynecol Surv
2. Field E, Heel K, Palmer C, Vally H, Beard F, McCall B. Evaluation of clinical management of gonorrhoea using enhanced surveillance in South East Queensland. Sex Health
3. Shattock RM, Patrizio C, Simmonds P, Sutherland S. Detection of Chlamydia trachomatis
in genital swabs: comparison of commercial and in-house amplification methods with culture. Sex Transm Infect
4. Heinonen PK, Leinonen M. Fecundity and morbidity following acute pelvic inflammatory disease treated with doxycycline and metronidazole. Arch Gynecol Obstet
5. Sweet RL, Gibbs RS, editors. Infectious diseases of the female genital tract 5th ed. Philadelphia: Wolters Kluwer/Lippincott Williams & Williams; 2009.
6. World Health Organization. Sexually transmitted and other reproductive tract infections. A guide to essential practice. 2005. 40. 49. http://www.who.int/reproductivehealth/publications/rtis/9241592656/en/index.html
7. Vickerman P, Peeling RW, Watts C, Mabey D. Detection of gonococcal infection: pros and cons of a rapid test. Mol Diagn
8. Unemo M, Palmer HM, Blackmore T, Herrera G, Fredlund H, Limnios A, et al. Global transmission of prolyliminopeptidase-negative Neisseria gonorrhoeae
strains: implications for changes in diagnostic strategies. Sex Transm Infect
9. Rao GG, Bacon L, Evans J, Dejahang Y, Michalczyk P, Donaldson N. Prevalence of Neisseria gonorrhoeae
infection in young subjects attending community clinics in South London. Sex Transm Infect
10. Ryan CA, Zidouh A, Manhart LE, Selka R, Xia M, Moloney-Kitts M, et al. Reproductive tract infections in primary healthcare, family planning, and dermatovenereology clinics: evaluation of syndromic management in Morocco. Sex Transm Infect
11. Benzaken AS, Galban EG, Antunes W, Dutra JC, Peeling RW, Mabey D, et al. Diagnosis of gonococcal infection in high risk women using a rapid test. Sex Transm Infect. 2006;82(suppl):v26-v28.
12. Diallo MO, Ghys PD, Vuylsteke B, Ettiegne-Traore V, Gnaore E, Soroh D, et al. Evaluation of simple diagnostic algorithms for Neisseria gonorrhoeae
and Chlamydia trachomatis
cervical infections in female sex workers in Abidjan, Côte d’Ivoire. Sex Transm Infect
13. Wi T, Mesola V, Manalastas R, Tuazon C, Mugrditchian DS, Perine P, et al. Syndromic approach to detection of gonococcal and chlamydial infections among female sex workers in two Philippine cities. Sex Transm Infect
14. Monteiro EF, Lacey CJ, Merrick D. The interrelation of demographic and geospatial risk factors between four common sexually transmitted diseases. Sex Transm Infect
15. Romoren M, Sundby J, Velauthapillai M, Rahman M, Klouman E, Hjortdahl P. Chlamydia and gonorrhoea in pregnant Batswana women: time to discard the syndromic approach? BMC Infect Dis
16. Myziuk L, Romanowski B, Brown M. Endocervical Gram stain smears and their usefulness in the diagnosis of Chlamydia trachomatis
. Sex Transm Infect
17. Schachter J, McCormack WM, Smith RF, Parks RM, Bailey R, Ohlin AC. Enzyme immunoassay for diagnosis of gonorrhea. J Clin Microbiol
18. Alary M, Gbenafa-Agossa C, Aina G, Ndour M, Labbe AC, Fortin D, et al. Evaluation of a rapid point-of-care test for the detection of gonococcal infection among female sex workers in Benin. Sex Transm Infect. 2006;82(suppl):v29-v32.
19. Hardwick R, Gopal Rao G, Mallinson H. Confirmation of BD ProbeTec Neisseria gonorrhoeae
reactive samples by Gen-Probe APTIMA assays and culture. Sex Transm Infect
20. Van Dyck E, Ieven M, Pattyn S, Van Damme L, Laga M. Detection of Chlamydia trachomatis
and Neisseria gonorrhoeae
by enzyme immunoassay, culture, and three nucleic acid amplification tests. J Clin Microbiol
21. Bignell C, Ison CA, Jungmann E. Gonorrhoea. Sex Transm Infect
22. Gopal Rao G, Bacon L, Evans J, Dejahang Y, Hardwick R, Michalczyk P, et al. Can culture confirmation of gonococcal infection be improved in female subjects found to be positive by nucleic acid amplification tests in community clinics? Sex Transm Infect
23. Lum G, Freeman K, Nguyen NL, Limnios EA, Tabrizi SN, Carter I, et al. A cluster of culture positive gonococcal infections but with false negative cppB gene based PCR. Sex Transm Infect
24. Ison C. GC NAATs: is the time right? Sex Transm Infect
25. Kellogg JA, Orwig LK. Comparison of GonoGen, GonoGen II, and MicroTrak direct fluorescent-antibody test with carbohydrate fermentation for confirmation of culture isolates of Neisseria gonorrhoeae
. J Clin Microbiol
26. Vickerman P, Watts C, Alary M, Mabey D, Peeling RW. Sensitivity requirements for the point of care diagnosis of Chlamydia trachomatis
and Neisseria gonorrhoeae
in women. Sex Transm Infect
27. CDC MMWR. 2010 treatment guidelines for sexually transmitted diseases. Morb Mortal Wkly Rep
28. Suzuki K, Matsumoto T, Murakami H, Tateda K, Ishii N, Yamaguchi K. Evaluation of a rapid antigen detection test for Neisseria gonorrhoeae
in urine sediment for diagnosis of gonococcal urethritis in males. J Infect Chemother
29. Ho MK, Lo JY, Lo AC, Cheng FK, Chan FK. Evaluation of replacing the existing diagnostic strategy for Neisseria gonorrhoeae
and Chlamydia trachomatis
infections with sole molecular testing of urine specimens in a sexually transmitted infection clinic setting. Sex Transm Infect
30. Chen CS, Zhu H. Protein microarrays. Biotechniques
31. Chen CS, Korobkova E, Chen H, Zhu J, Jian X, Tao SC, et al. A proteome chip approach reveals new DNA damage recognition activities in Escherichia coli
. Nat Methods
32. Chen CS, Baeumner AJ, Durst RA. Protein G-liposomal nanovesicles as universal reagents for immunoassays. Talanta
33. Chen CS, Durst RA. Simultaneous detection of Escherichia coli
spp. and Listeria monocytogenes
with an array-based immunosorbent assay using universal protein G-liposomal nanovesicles. Talanta
34. Ho JA, Durst RA. Detection of fumonisin B1: comparison of flow-injection liposome immunoanalysis with high-performance liquid chromatography. Anal Biochem
35. Verhelst R, Verstraelen H, Claeys G, Verschraegen G, Van Simaey L, De Ganck C, et al. Comparison between Gram stain and culture for the characterization of vaginal microflora: definition of a distinct grade that resembles grade I microflora and revised categorization of grade I microflora. BMC Microbiol
36. De Backer E, Verhelst R, Verstraelen H, Alqumber MA, Burton JP, Tagg JR, et al. Quantitative determination by real-time PCR of four vaginal Lactobacillus
species, Gardnerella vaginalis
and Atopobium vaginae
indicates an inverse relationship between L. gasseri
and L. iners
. BMC Microbiol
37. Su WH, Tsou TS, Chen CS, Ho TY, Lee WL, Yu YY, et al. Diagnosis of Chlamydia infection in females. Taiwan J Obstet Gynecol