Chlamydia and Gonorrhoea Nucleic Acid Amplification Testing

Chlamydia trachomatis and Neisseria gonorrhoeae are the commonest bacterial sexually transmitted infections. Nucleic acid amplification tests (NAATs) are the reference test for both, as they have higher sensitivity than culture or antigen tests, work on non-invasive samples (first void urine, self-collected vaginal swabs) and can be combined in one assay.

Culture and susceptibility testing of N. gonorrhoeae is still needed to monitor resistance, particularly in treatment failure. C. trachomatis L1 to L3 serovars cause lymphogranuloma venereum, which needs specific testing.

About 129 million new chlamydia infections and 82 million new gonorrhoea infections occur worldwide each year in adults aged 15 to 49 years. Most infections in women (70 to 80% of chlamydia and 50% of gonorrhoea) and many in men are asymptomatic.

Untreated infection causes pelvic inflammatory disease in 10 to 15% of women with chlamydia, with ectopic pregnancy and tubal infertility, and epididymitis, reactive arthritis, neonatal conjunctivitis and pneumonia.

Gonococcal resistance to ciprofloxacin, penicillin, tetracycline and azithromycin is widespread, and ceftriaxone is the remaining reliable first line drug, with emerging resistant strains.

Indications. Urethral or vaginal discharge, dysuria, pelvic pain, dyspareunia, intermenstrual bleeding, epididymo orchitis, proctitis, pharyngitis in the exposed, conjunctivitis in newborns, and arthritis with urethritis.

Screening of sexually active women under 25 years, people with new or multiple partners, men who have sex with men (3-site testing every 3 to 6 months), partners of infected people, pregnant women (first visit and in the third trimester if at risk), and before termination of pregnancy or intrauterine device insertion in some programmes. Sexual assault.

Specimen.

• Women: self-collected or clinician collected vaginal swab (preferred, best sensitivity), endocervical swab, or first void urine.
• Men: first void urine (first 10 to 20 mL, with 1 hour since last voiding) or urethral swab.
• Pharyngeal and rectal swabs in people with exposure at these sites, using validated assays.
• Conjunctival swab in neonates and adults with conjunctivitis.
• Gonococcal culture: urethral, endocervical, rectal or pharyngeal swab in transport medium sent promptly, plated on selective media (modified Thayer-Martin) and incubated in CO2.

NAAT performance.

• Chlamydia: sensitivity 90 to 98% and specificity over 99%, for vaginal swabs and male urine. Endocervical swab and female urine are slightly less sensitive (85 to 90%).
• Gonorrhoea: sensitivity 90 to 98% and specificity 98 to 99.5%. The lower specificity at pharyngeal sites (cross-reaction with commensal Neisseria) means pharyngeal positives are confirmed with a second NAAT targeting a different gene.
• Positive predictive value falls in low prevalence populations (under 1 to 2%), and positive results in a low risk person are confirmed.
• Reporting is qualitative (detected or not detected).

Gonococcal culture and susceptibility.

• Culture sensitivity 70 to 90% from genital sites if transported correctly, and lower from the pharynx and rectum.
• Susceptibility is tested to ceftriaxone, cefixime, azithromycin, ciprofloxacin and spectinomycin, with minimum inhibitory concentrations. Culture is done before treatment in all patients with positive NAAT where possible, in treatment failure and in suspected resistance, and for surveillance.
• Molecular resistance assays for ciprofloxacin (gyrA) permit directed oral therapy in susceptible strains.

Gram stain. Intracellular Gram negative diplococci in urethral discharge from symptomatic men: sensitivity over 95% and specificity over 99% for gonorrhoea. Low sensitivity in women and asymptomatic men.

Lymphogranuloma venereum. C. trachomatis positive on a rectal swab in a man who has sex with men, with proctitis, is tested for L1 to L3 serovars (ompA genotyping or specific PCR), since 3 weeks of doxycycline are needed.

Test of cure.

• Chlamydia: not routine for uncomplicated infection with first line treatment, but done in pregnancy, for rectal infection, with persistent symptoms and when adherence is doubtful. NAAT is repeated not earlier than 4 weeks after treatment (residual nucleic acid remains for up to 3 weeks and gives false positives).
• Gonorrhoea: NAAT test of cure at least 2 weeks after treatment (or culture at 3 to 7 days) in pharyngeal infection, treatment failure and with resistance concern. Retest at 3 months for all infected people, because reinfection is common (10 to 20%).

Critical values and findings needing immediate action.

• Positive result in a pregnant woman: treatment and neonatal prophylaxis plans, and test of cure.
• Neonatal conjunctivitis (within 1 to 14 days of birth): urgent culture and NAAT, and systemic treatment for gonococcus to prevent corneal perforation.
• Disseminated gonococcal infection (fever, tenosynovitis, skin lesions, septic arthritis): blood and joint fluid cultures, admission and intravenous ceftriaxone.
• Pelvic inflammatory disease with tubo ovarian abscess or peritonitis.
• Treatment failure or ceftriaxone resistant gonorrhoea: notify public health, culture and susceptibility, and specialist treatment.
• Positive result in a child: investigation for sexual abuse by a specialist team, with forensic procedures and culture.

Interferences and pitfalls.

• Urine samples taken after recent voiding dilute the organism load. Vaginal swabs are preferred in women.
• Inhibitors in samples (blood, lubricant, spermicide, talc) cause invalid results, and the sample is repeated.
• Repeat NAAT within 3 weeks of treatment detects residual DNA and does not prove failure.
• Cross-reactions between N. gonorrhoeae and commensal Neisseria species, and false positive results with some assays at extragenital sites.
• Antibiotics before sampling reduce culture yield.
• Co-infection is frequent: 20 to 40% of gonorrhoea cases have chlamydia, and treatment covers both unless chlamydia is excluded. Test for HIV, syphilis, hepatitis B and C, and Mycoplasma genitalium where symptoms persist.
• Self-collected samples have equivalent accuracy to clinician collected samples.
• Partner notification and treatment of partners from the preceding 60 days (or last partner) prevent reinfection and sequelae.

Special groups.

• Pregnancy: test at first visit and repeat in the third trimester in under 25 years or at risk. Azithromycin or amoxicillin is used for chlamydia. Doxycycline is avoided.
• Adolescents and young adults: highest rates, with annual screening of sexually active women under 25 years in many programmes.
• Men who have sex with men: 3-site testing (urine, rectal, pharyngeal), high rates of asymptomatic extragenital infection, and Mycoplasma genitalium co-infection.
• Neonates: conjunctivitis and pneumonia from chlamydia at 5 to 14 days and 4 to 12 weeks respectively.
• Regions: culture capability for gonorrhoea susceptibility is limited in low resource settings, where syndromic management is used, with rising resistance.

Follow up tests. HIV test, syphilis serology, hepatitis B surface antigen and hepatitis A and B vaccination status, hepatitis C antibody in men who have sex with men, Mycoplasma genitalium and trichomonas testing where indicated, pregnancy test, repeat NAAT at 3 months, test of cure where indicated, gonococcal susceptibility testing, and pelvic ultrasound if pelvic inflammatory disease complicates.