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  Table of Contents  
Year : 2016  |  Volume : 34  |  Issue : 2  |  Page : 139-145

Gonorrhoea diagnostics: An update

Department of Microbiology, All India Institute of Medical Sciences, New Delhi, India

Date of Submission22-Nov-2015
Date of Acceptance10-Jan-2016
Date of Web Publication14-Apr-2016

Correspondence Address:
S Sood
Department of Microbiology, All India Institute of Medical Sciences, New Delhi
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/0255-0857.180278

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 ~ Abstract 

Diagnosis of gonorrhoea is an ongoing challenge. The organism is fastidious requiring meticulous collection and transport for successful cultivation. Asymptomatic infections are common which go undetected by conventional methods thereby leading to continued transmission and the risk of complications. The nucleic acid amplification tests, now increasingly used in developed countries, offer improved sensitivity compared to bacterial culture. However, these continue to suffer sequence related problems leading to false positive and false negative results. Further, these cannot be used for generation of data on antibiotic susceptibility because genetic markers of antibiotic resistance to recommended therapies have not been fully characterised. They are unaffordable in a setting like ours where reliance is placed on syndromic approach for sexually transmitted infection (STI) management. The use of syndromic approach has resulted in a considerable decline in the number of Neisseria gonorrhoeae isolates that have been cultured for diagnostic purposes. Many laboratories formerly doing so are no longer performing culture for gonococci, and the basic skills have been lost. There is a need to not only revive this skill but also adopt newer technologies that can aid in accurate diagnosis in a cost-effective manner. There is room for innovation that can facilitate the development of a point-of-care test for this bacterial STI.

Keywords: Biosensor, culture, Neisseria gonorrhoeae, nucleic acid amplification tests, sexually transmitted infection

How to cite this article:
Verma R, Sood S. Gonorrhoea diagnostics: An update. Indian J Med Microbiol 2016;34:139-45

How to cite this URL:
Verma R, Sood S. Gonorrhoea diagnostics: An update. Indian J Med Microbiol [serial online] 2016 [cited 2019 Sep 16];34:139-45. Available from:

 ~ Introduction Top

Gonorrhoea is one of the oldest known bacterial sexually transmitted infections (STIs) that continue to cause a significant morbidity among the sexually active individuals. [1] The principal strategy for the control of gonorrhoea involves the diagnosis and treatment of symptomatic cases together with contact tracing and treatment of sexual partners. [2] The high proportion of asymptomatic infections which is of the order of 50-80% in women and approximately 10% in men, the lack of a sensitive and specific test suitable for mass screening, the requirement of invasive samples for tests and the social stigma attached with the disease are some of the reasons because of which the disease is under-diagnosed and under-reported. [3],[4] Although there are many tests available to detect gonococcal infection, most require laboratory facilities, and so are costly; and the results are not usually available before the patient has left the clinic. [5] Currently, tools for gonorrhoea diagnosis include microscopy, culture, and various nonculture methods. Although laboratory tests are highly sensitive, in practice their high costs and technical requirements make their routine use difficult and compromise their potential impact on STI control and management. [6] Consequently, in most resource-limited settings the World Health Organization recommends the use of the syndromic approach for the management of urethral discharge in men and vaginal discharge and lower abdominal pain in women. [7],[8] Attempts to infer a presumptive etiological diagnosis based on clinical manifestations eliminate the laboratory component and thus eliminates treatment delays associated with aetiological management, but they are often inaccurate or incomplete. [9],[10] While the syndromic approach appears to be satisfactory in men, it has several important limitations in women. The studies evaluating the syndromic approach in women have shown that it generally has a poor sensitivity (30-80%) and specificity (40-80%) for the diagnosis of  Neisseria More Details gonorrhoeae. Such low sensitivities and specificities result in many false diagnosis, massive overtreatment, and many STIs remaining untreated. [5]

In light of the constraints described above, development of a dependable test with potential for point of care adaptation could greatly improve gonorrhoea control.

 ~ Current Diagnostic Tools Top

Gonorrhoea usually produces purulent exudates, but signs and symptoms of the disease may either be absent or indistinguishable from those caused by Chlamydia trachomatis. Therefore, laboratory procedures are needed for diagnosis. Microscopy is a rapid, inexpensive, point-of-care test available that facilitates early treatment. Diagnosis of gonorrhoea by microscopy is based on observation of paired intracellular bean-shaped diplococci within neutrophils. The sensitivity and specificity of Gram-stain for males with urethritis are comparable with those of culture when used for a "presumptive" diagnosis being as high as 95% and 99%, respectively. For women and asymptomatic men, the performance is much poorer with a sensitivity of 37-70% and 40-60% respectively. [11],[12] In fact, Gram stain of endocervical specimens, pharyngeal, or rectal specimens are not recommended. [13]

Culture is still considered to be the "Gold Standard" for the definitive diagnosis of gonorrhoea. The gonococci are very exacting with respect to the composition of the culture media. Highly nutritious media that contain a certain concentration of essential amino acids such as cysteine, purines, and pyrimidines, as well as usable energy source (i.e., glucose, pyruvate, or lactate) are needed to be able to culture all divergent gonococcal strains. [14] Primary isolation of gonococci requires a selective culture media containing antimicrobial agents that inhibit the growth of other bacteria and fungi such as modified Thayer-Martin, Martin-Lewis, New York City or GC-Lect medium in combination with a medium without inhibitors, for example, chocolate agar. In laboratories with good quality control (QC) measures, the sensitivity of culture may range from 85% to 95%. [14],[15] The retention of the organism for other tests, such as antimicrobial susceptibility determination and subtyping, can be accomplished only with the use of culture-based methods. Although the methods of gonococcal culture have been well described, there are certain challenges associated with these methods. These include the fastidious nature of the organism, which requires meticulous specimen collection, transport and use of optimal culture media and laboratory techniques. In addition, the procedure requires at least 48-72 h. Moreover, in our country the skill for gonococcal cultivation with QC procedures is restricted to few laboratories only. Despite these disadvantages, N. gonorrhoeae culture is required as a test of cure to evaluate suspected cases of gonorrhoea treatment failure and assess instances of child sexual assault in boys and extragenital infections in girls. [13]

Direct detection of N. gonorrhoeae cell components in biological samples can also be undertaken. These nonculture methods of diagnosis can have a great public health impact as they allow us to test populations that currently escape surveillance. They hold the promise of making an accurate diagnosis of gonococcal infection more readily available. These methods have the advantage that they can be carried out on dead organisms and, therefore, feasible even when sample transport is suboptimal. Several nonculture tests have been introduced. Among these, the commercially available nucleic acid hybridisation assays include the Gen-Probe PACE 2 and the Digene Hybrid Capture II assays. The reported sensitivity and specificity values for these tests suggest that they are not as sensitive or specific as a culture. [16] Moreover, the Hybrid Capture assay is not widely available and the PACE 2 test was discontinued in December 2012. [13] Besides these, sophisticated DNA technologies, especially nucleic acid amplification tests (NAATs) (polymerase chain reaction [PCR] and ligase chain reaction [LCR]), have been developed and evaluated in industrialised countries over the past few years. The NAATs have gained popularity not only because we are dealing with a delicate organism but also because a low detection limit is of crucial importance for the diagnosis of asymptomatic infections. Commercial as well as in-house assays targeting different genes in N. gonorrhoeae have been developed. NAATs offer greatly expanded sensitivities of detection, usually well above 90%, while maintaining very high specificity, usually ≥99%. [13]

There have been five main commercial N. gonorrhoeae NAAT assays. These include the Roche Cobas Amplicor (Roche Moelcular Sytems, Branchburg, NJ, USA), the Gen-Probe APTIMA Combo 2 (AC2; GenProbe), the Becton Dickinson ProbeTec assay (Becton Dickinson, Sparks, MD, USA), the Abbott Real Time m 2000 (Abbott Molecular Inc., Des Plaines, Illinois, USA) and the Xpert NG assay (Cepheid, Sunnyvale, California, USA). [13] The Abbott LCR (LCx) test (by Abbott Laboratories) that was commercially available has been withdrawn because of issues related to quality assurance. [16] NAATs are recommended for detection of urogenital infections in women and men with and without symptoms. However, data on the use of NAATs for detection of N. gonorrhoeae in children are limited. [13]

The commercial tests differ in their amplification methods and the genes they target. The Roche Amplicor assay targets the CMT gene that encodes a putative DNA methyltransferase. This test lacks the desired specificity as it cross-reacts with commensal species of Neisseria and also with lactobacilli. [17],[18] The ProbeTec strand displacement amplification assay is directed to the pilin gene and also shows cross-reaction with some commensal Neisseria strains. The AC2 assay is the transcription-mediated amplification assay. The problem with this assay is that it shows competitive inhibition in case of co-infections. [16] A high false positivity has been reported in some of the commercial assays. Apart from the already mentioned commercial assays, several laboratories world over are developing in-house NAATs predominantly PCRs using different gene targets. The most frequently used targets include the CMT gene, the pilin gene, cppB gene, 16S rRNA gene, the opa gene, and the porA pseudogene. Varying degrees of sensitivity and specificity of the NAATs has been reported mainly depending on the gene targeted. [19],[20],[21],[22],[23],[24] In addition, there are sequence-related limitations that are unique to N. gonorrhoeae NAATs leading to false positive and false negative results. [16]

Therefore, the Centers of Disease Control and Prevention (CDC-2002), Australian Public Health Laboratory Network, and the UK's Heath Protection Agency guidelines, recommend the use of 2 PCRs targeting different genes known to have a discriminatory capacity for determination of "true positives" in urogenital samples. [25],[26] One PCR is used as a screening assay and is highly sensitive while the second PCR that is used as a supplemental assay should be highly specific along with having sensitivity comparable to that of screening assay. For extra-genital samples, three different PCRs need to be performed. [26],[27] However, studies since 2002 addressing the utility of routine repeat testing of positive specimens demonstrated >90% concurrence with the initial test. Therefore, as per CDC 2014 guidelines, only when NAATs that detect nongonococcal Neisseria sp. are used that consideration should be given to retest the specimens with an alternate target assay if the anatomic site from which the specimen was collected is typically colonised with commensal organisms, for example, oropharyngeal specimens. As with any diagnostic test, if there is a clinical or laboratory reason to question a test result, a repeat test should be considered. [13]

The authors have designed an in-house PCR targeting the opa gene of N. gonorrhoeae and evaluated its performance with two already established PCRs, one targeting the 16S rRNA gene and the other targeting the porA pseudogene. [28] In compliance with the guidelines, the same has been validated for use for Indian patients. In addition, as per recommendations, an ongoing assessment of this gene target for diagnostic purposes is being undertaken. [29]

Overall, there are difficulties in the use of traditional methods and obstacles in the existing NAATs. This allows room for innovation for the development of newer tests to contribute to gonorrhoea control.

 ~ Innovation in Diagnostics-DNA Based Electrochemical Biosensor Top

Rapid advances in molecular biology in the last 20 years have provided a vast array of techniques aimed at detection and identification of microorganisms. Several commercial identification systems based on a variety of biochemical and immunological methods are available for rapid identification of N. gonorrhoeae. Recently, DNA probe technology has created new diagnostic approaches for use in clinical laboratories. Several tests have been developed for the direct detection of target sequences of N. gonorrhoeae either in patient samples [30],[31],[32],[33] as a means of direct noncultural diagnosis or in the cultured organism to provide confirmatory identification. This includes the chemiluminescent Gen-Probe PACE system for identification of N. gonorrhoeae in clinical samples such as urogenital and endocervical swabs and the biotinylated ORTHOProbe and chemiluminescent AccuProbe for confirmatory identification of N. gonorrhoeae. [34],[35],[36],[37] Biosensors are an emerging technology in diagnostics. Electrochemical DNA biosensors are primarily based on the integration of a sequence-specific probe with an electrochemical signal transducer which converts the base pairing event of DNA hybridisation into a useful electric signal. The high sensitivity they offer coupled with their compatibility with modern microfabrication technologies, portability, low cost, and minimal power requirements make them excellent candidates for DNA diagnostics. [38] Researchers are continuously evaluating several new engineered materials for the fabrication of bioelectrode for bacterial pathogen detection. Among them, the thin films of polymers such as polyaniline, nanostructured metal oxides such as ZrO 2 , gold, and carbon nanostructures like carbon nanotubes, etc., have been widely used. [39]

The authors have developed an electrochemical DNA biosensor using DNA-modified gold electrode. A self-designed probe targeting the opa gene was immobilised after modification and methylene blue was utilised as hybridisation monitor agent. Optimisation of working conditions was done using the standard strain of N. gonorrhoeae and synthetic DNA. The authors have further evaluated the performance of the biosensor for detection of N. gonorrhoeae using the amplicons of known positive and negative clinical samples. Based on the variation in the signal intensity produced by the different samples, a cut-off value using the receiver operating characteristic curve was determined to distinguish the infected from the noninfected individuals. The sensitivity, specificity, positive predictive value, and negative predictive value of the biosensor assay was 96.2%, 88.2%, 92.6%, and 93.8%, respectively when compared to culture, which is the "gold standard." The CUI (+) was 0.89 and CUI (−) was 0.83, rating the test as excellent. [40]

Thereafter, the authors have worked on genomic DNA from culture of N. gonorrhoeae (ATCC 49226) along with other Gram-negative and Gram-positive bacteria such as Klebsiella pneumoniae (ATCC 700603), Pseudomonas aeruginosa (ATCC 27853), Staphylococcus aureus (ATCC 25923),  Escherichia More Details coli (ATCC 25922),  Neisseria meningitidis More Details (ATCC 13077 serogroup A), and Neisseria sicca (ATCC 29193). The DNA fragmentation studies were carried out following sonication using Sartoriuos Labsonic for different time intervals 2-40 min at 30 kHz and visualised by gel electrophoresis (1.5% agarose gel) for determination of ideal sonication time required to obtain the desired band length of 150-600 bps. The same sonication time was applied to other bacterial isolates. Sonication for 20 min at 30 kHz was required to fragment the genomic DNA to require a length of 150-600 bps [Figure 1]. Best result was obtained after sonication for 20 min. [Figure 2] depicts graph showing the performance of biosensor with DNA sonicated for different time intervals [Figure 2].
Figure 1: Gel electrophoresis of sonication products obtained at a step-up time interval of 2 min 6, 8, 10, 12 … 36 min with continuous sonication at 30 kHz. Best results obtained at 18-22 min (Lanes 7-8)

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Figure 2: Performance of biosensor with DNA sonicated for different time intervals

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Serial dilutions of sonicated genomic DNA were tested to determine the detection limit, and the specificity was determined against the panel mentioned above. The detection limit of the bioelectrode for genomic DNA was found to be 10−18 M (4.336 × 10−15 g) [Figure 3] and the biosensor was found to be 100% specific with the panel of strains tested showing a decrease in signal intensity only for the gonococcal DNA [Figure 4].
Figure 3: Sensitivity graph for genomic DNA sonicated for 20 min

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Figure 4: Result of specificity studies of the bioelectrode

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This was followed by hybridisation studies directly on clinical samples. DNA was extracted from 10 culture and PCR positive and 10 culture and PCR negative patient samples using the QIAamp DNA extraction kit. The extracted DNA was sonicated, diluted and used for biosensor evaluation. The sensor performed well in known negative samples as none showed a decrease in signal intensity [Figure 5]. On the other hand, the performance lacked reproducibility in the case of positive samples [Figure 6]a and b. Response curves with variable signal intensities were generated for positives that may be due to the combination of the high complexity of DNA and the variable bacterial load in the sample. It is pertinent to mention here that the gold electrode used in this study was fabricated with the short oligonucleotide probe. Problems could arise when dealing with clinical samples as a result of the huge steric hindrance encountered by very large targets (thousand to several hundred thousand base pairs). [41] Although the sample preparation involved sonication, sonication being a random process may have fragmented the DNA at its binding site, decreasing the efficiency of the biosensor. The use of restriction enzymes for targeted excision of strands may prove to be useful. Further, the use of capture and detector probe strategy may give good results as seen for uropathogens in clinical urine samples. [42]
Figure 5: Performance of biosensor with negative clinical samples

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Figure 6: (a and b) Performance of biosensor with positive clinical samples at different dilutions

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 ~ Conclusions Top

Asymptomatic infections are common in gonorrhoea especially in women and are typically associated with a low bacterial load. These tend to be missed by traditional techniques of identification. Nevertheless, these methods continue to hold a place in gonorrhoea diagnostics as they allow antimicrobial susceptibility testing and genetic analysis of the isolate obtained and continue to have a role in certain clinical situations. The widespread use and reliance on NAATs in the developed countries allows enhanced diagnosis, but their use is restricted in our setting due to prohibitive cost. Availability of an inexpensive in-house assay overcomes this limitation and permits usage in a resource-limited setting like ours that currently rely on syndromic case management for treatment of STIs. More and more modern day technologies can be applied to STI diagnostics and the use of biosensors is one such strategy. Biosensor technology has so far been used in preamplified samples to distinguish the infected from uninfected individuals. Studies using sonicated genomic DNA were very encouraging as the biosensor could accurately distinguish gonococcal DNA from that of other bacteria and therefore, may be used as a nucleic-acid based confirmation method for culture. However, further modifications are required by improving the matrix and the preprocessing of samples before the developed biosensor could be taken to the next phase of direct detection of gonococcal DNA in clinical samples.

Presently, there is a need to maintain the skill for gonococcal culture, embrace the already established technologies such as PCR and adopt innovative techniques to improve gonorrhoea diagnostics.


We thank Dr. B. D.Malhotra, Scientist G, BECPRL, National Physical Laboratory, New Delhi, India for his support and guidance for biosensor studies. We also thank Dr G. Sumana, Scientist C and Dr. Renu Singh, Ph.D. student, BECPRL, National Physical Laboratory, New Delhi, India for their contribution. We gratefully acknowledge Dr. V. K. Sharma, Prof. & Head, Department. of Dermatology and Venereology, AIIMS, New Delhi for his valuable inputs.

Financial support and sponsorship

This work was supported by grant No. BT/PR7667/ MED/14/1057/2006 from Department of Biotechnology, Ministry of Science and Technology, Government of India.

Authors would like to thank Council of Scientific and Industrial Research, India for financial assistance.

Conflicts of interest

There are no conflicts of interest.

 ~ References Top

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