1. Chlamydia trachomatis: WRONG Target for Sexually Transmitted Diseases (STD) in Hong Kong! Clement Leung-kwok CHAN MBBS (HK), ECFMG (USA), AMC (Aust), MD (S’pore), FRCOG (UK), FRANZCOG (Aust/NZ), FHKCOG (HK), FHKAM (OG), FAMS (S’pore), FICS (USA), FACS (USA) 1. Director, Women’s Health And Reproductive Medicine (WHARM) Centre, Hong Kong 2. Hon. Clinical Assoc. Professor, Dept. of O & G, Hong Kong University, Hong Kong 3. Hon. Clinical Assoc. Professor, Dept. of O & G, Chinese University of Hong Kong Hong Kong 4. Adjunct Assoc. Professor, School of Biomedical Sciences, Chinese University of Hong Kong Hong Kong 5. Visiting Professor, Cebu Institute of Medicine, Philippines 6. Asian Federation Secretary & Executive Board Member, International College of Surgeons, USA 7. Ex-Chairman & Professor, University of Tasmania, Australia

2. Chlamydia trachomatis: WRONG Target for Sexually Transmitted Diseases (STD) in Hong Kong! Clement Leung-kwok CHAN MBBS (HK), MD (S’pore), FRCOG (UK), FRANZCOG (Aust/NZ), FHKCOG (HK), FHKAM (OG) 1. Consultant, Women’s Health And Reproductive Medicine (WHARM) Centre, Hong Kong 2. Adjunct Assoc. Professor, School of Biomedical Sciences, Chinese University of Hong Kong, Hong Kong Wai Yee CHAN BSc (CUHK), PhD (Florida) Director & Professor, School of Biomedical Sciences, Chinese University of Hong Kong, Hong Kong Chrssitopher CHENG Bsc (CUHK), PhD (Lond) Professor, School of Biomedical Sciences, Chinese University of Hong Kong, Hong Kong

3. Chlamydia urethritis

4. Chlamydia trachomatis: WRONG Target for sexually transmitted disease in Hong Kong? -Chlamydia trachomatis- obligate intracellular human pathogen, is 1 of 3 species in genus Chlamydia. - Gram (-) bacteria, therefore cell wall components retain the counter-stain safranin & appear pink under light microscope - appear as either coccoid or rod shape - inclusion bodies of C trachomatis 1 st described 1942 - 1st cultured in yolk sacs of eggs by Prof.Feifan Tang et al in 1957 - 3 human biovars: a) Serovars Ab, B, Ba, or C  trachoma (infection of eyes  blindness, prevalent in Africa) b) serovars D-K  urethritis, pelvic inflammatory disease, ectopic pregnancy, neonatal pneumonia & neonatal conjunctivitis c) serovars L1, L2 & L3  lymphogranuloma venereum(LGV) - Many, but not all strains have an extrachromosomal plasmid

5. Chlamydia trachomatis: WRONG Target for sexually transmitted disease in Hong Kong? Chlamydia trachomatis: 1) Both sexes - urethritis, proctitis (rectal disease and bleeding), trachoma & infertility. 2) Men- prostatitis & epididymitis 3) Women- cervicitis, pelvic inflammatory disease (PID), ectopic pregnancy (EP), & acute or chronic pelvic pain 4) Neonate- trachoma & pulmonary Cx -single most important infectious agent associated with blindness; - ≈ 600 M worldwide - C. trachomatis eye infections  20 M blindness

6. Chlamydia trachomatis: WRONG Target for sexually transmitted disease in Hong Kong? Tests for Chlamydia trachomatis: 1) Nucleic acid amplification tests (NAAT)  chlamydia DNA; e.g polymerase chain reaction (PCR) -can use urine - most sensitive, very accurate & very unlikely false-negative 2) Nucleic acid hybridization tests (DNA probe test)  Also chlamydia DNA- very accurate but not as sensitive 3) Enzyme-linked immunosorbent assay (ELISA, EIA) - quick test finds chlamydia antigens 4) Direct fluorescent antibody test (DFA). - quick test also finds chlamydia antigens. 5) Chlamydia cell culture. -suspected chlamydia sample grown in a vial of cells in laboratory. The pathogen infects the cells and after a set incubation time (48 hours) the vials are stained and viewed on a fluorescent light microscope. - more expensive, longer time (2 days)

7. Chlamydia trachomatis: WRONG Target for sexually transmitted disease in Hong Kong? -Chlamydial genital infection - most common bacterial STD in USA (Cates W. Sex Transm Dis 1999; 26:S2–7) - prevalence highest in persons </ =25 yrs of age (Centers for Disease Control & Prevention. STD surveillance 2002 suppl. Chlamydia Prevalence Monitoring. Project Ann Report 2002. Atlanta, GA: US Dept of Health & Human Services, Centers for Disease Control & Prevention, Oct 2003) - also commonest curable STD in England &Wales (Taylor-Robinson D. BMJ 1994; 308: 150-151) (Sexually transmitted diseases quarterly report. Communicable Dis Rep 1995; 5: 122-123) - Asymptomatic (up to 70%); common among both sex; is one of main causes of pelvic inflammatory disease (PID) in developed world (Cates W, Wasserheit JN. AJOG 1991; 164: 1771-81) - PID  infertility, ectopic pregnancy (EP) & miscarriage (US Dept of Health & Human Services. Morbidity MortalityWeekly Rep 1993; 42: 1-38) (Oakeshott P, Hay P. Br J Gen Pract 1995; 45: 615-20)

8. Chlamydia trachomatis: WRONG Target for sexually transmitted disease in Hong Kong? -detection often relies on screening - Centers for Disease Control & Prevention (CDC) & US Preventive Services Task Force each recommend annual chlamydial screening for a) all sexually active women </=25 yrs of age & b) older women with risk factors (e.g. new or multiple sex partners) (CDC & Prevention. STD treatment guidelines 2002. MMWR Recomm Rep 2002; 51(RR-6):1–78) (US Preventive Services Task Force. Screening for chlamydial infection: recommendations & rationale. Am J Prev Med 2001; 20(Suppl 3): 90– 4) - estimated cost of Dx & Mx genital chlamydia & its Cx in UK  > £50 M /yr (Taylor-Robinson D. BMJ 1994; 308: 150-151) (Sexually transmitted diseases quarterly report. Communicable Dis Rep 1995; 5: 122-123)

9. Chlamydia trachomatis: WRONG Target for sexually transmitted disease in Hong Kong? Screening programs in women  a)  prevalence of chlamydia (Mertz KJ etal. Sex Transm Dis 1997; 24:169–75) (Centers for Disease Control and Prevention. Chlamydia trachomatis genital infections United States, 1995. MMWR Morb Mortal Wkly Rep 97; 46:193–8) & b)  rates of PID (Scholes D et al. NEJM 96; 334:1362–6) (Kamwendo F et al. STD 96; 23:384–91)

10. Chlamydia trachomatis: WRONG Target for sexually transmitted disease in Hong Kong? Mx of uncomplicated genital chlamydial infection in adults 1)Px of patients   Cx & prevent transmission to partners 2)Px of sex partners  prevent reinfection of index patient & infection of other partners 3)risk-reduction counselling, & 4)repeat chlamydial testing few months after Px (to detect failure/recurrent / persistent infections).

15. Ureaplasma Mycoplasma

16. Chlamydia trachomatis: WRONG Target for sexually transmitted disease in Hong Kong! Ureaplasma & Mycoplasma (Bryan Larsen,Joseph Hwang. Infect Dis Obstet Gynecol. 2010;2010. pii: 521921) - Mycoplasma hominis, Ureaplasma urealyticum for several decades - among experts in genital tract infectious disease with indications that former can be part of normal flora of sexually experienced women & both may  chorioamnionitis, salpingitis, bacterial vaginosis, & postpartum endometritis. - availability of molecular methods substantially  valid information about the pathogenic potential of these bacteria which lack rigid cell walls (Molicutes, specifically the family Mycoplasmataceae) - Mycoplasma hominis & Ureaplasma urealyticum have historically been the subject of attention, but Mycoplasma genitalis  male urethritis & colonizing female genital tract - Ureaplasma - two species, urealyticum & parvum

17. Chlamydia trachomatis: WRONG Target for sexually transmitted disease in Hong Kong! Viscardi RM. Ureaplasma species: role in neonatal morbidities and outcomes. Arch Dis Child Fetal Neonatal Ed. 2014;99:F87-92. -In our institution, ≈ 50% preterm infants < 32 wk Ureaplasma (+) in >/= 1 compartment (respiratory, blood &/or CSF)  most common pathogens affecting population. -compelling epidemiological & experimental evidence linking perinatal Ureaplasma species exposure to important morbidities of prematurity, e.g. bronchopulmonary dysplasia, intraventricular haemorrhage & necrotising enterocolitis

18. Chlamydia trachomatis: WRONG Target for sexually transmitted disease in Hong Kong! Capoccia R et al. Ureaplasma urealyticum, Mycoplasma hominis and adverse pregnancy outcomes. Curr Opin Infect Dis. 2013;26:231-40. -review recent data on epidemiology & clinical role in pregnancy  many observational studies  assoc. with preterm labour, but proof of causality remains limited. -PCR- excellent (cf culture) for Dx, but culture  CST. -?? Px effectively  adverse pregnancy outcomes  more Ix SUMMARY: 1)Mycoplasma & U. urealyticum  adverse pregnancy outcomes is increasingly accepted. 2)However, sole presence of these in vaginal flora might be insufficient to cause pathological issues, but + other factors (e.g. bacterial vaginosis or cervical incompetence) may be additionally needed  preterm birth

19. Chlamydia trachomatis: WRONG Target for Sexually Transmitted Diseases (STD) in Hong Kong! Aims of study 1)Is Chlamydis trachomatis the most prevalent organism in HK? 2)What are the relative prevalence of the various STD organisms? 3)For male, which is the more sensitive method of collection of specimen, semen or urine?

20. Chlamydia trachomatis: WRONG Target for Sexually Transmitted Diseases (STD) in Hong Kong! Methods and Materials - from 1 July 2005 to 30 June 2013  8 yr solo private practice - Patient selection: 1)Subfertility or discharge - initial screen - C trachomatis & U urealyticum DNA for females (Real Time PCR using Applied Biosystem 7500- based on 36% prevalence of infection, sensitivity, specificity, PPV & NPV = 87, 96, 94, & 93% respectively); - only if anyone is (+), then counsel COUPLE, explain to couples to do STD full check package ( DNA for C. trachomatis, U urealyticum, M hominis, N gonorrhoea; VDRL or RPR for syphilis; Herpes I & I IgG; HIV only on request)

21. Chlamydia trachomatis: WRONG for Sexually Transmitted Diseases (STD) in Hong Kong! Methods and Materials 2) Pelvic (or genital) infection, or pain- counsel Patient, explain & suggest: do STD full check package - RPR: ASI RPR Card Test for Syphilis, which is a qualitative and semi-quantitative non-treponemal flocculation test for the detection of regain antibodies in human serum and plasma as a screening test in syphilis serology. - HSV II Antibody IgG: Euroimmun Anti-HSV-2 (gG2) ELISA (IgG) kit. The kit is claimed to have 95.5% specificity and 100% sensitivity HSV I Antibody IgG: Euroimmun Anti-HSV-1 (gC1) ELISA (IgG) kit. The kit is claimed to have 100% sensitivity and specificity

22. Overall Percentage of C. trachomatis DNA & U. urealyticum DNA (including patients who have either the SCREENING or FULL STD CHECK) Chlamydia trachomatis Ureaplasma urealyticum P-value FEMALE48/862 (5.57%) 334/901 (37.07%) < 0.001 MALE10/177 (5.65%) 40/289 (13.84%) 0.006 P-value0.966 < 0.001 TOTAL:1,039 cases1,190 cases

23. STD in Relation to Patient’s Indication Chlamydia trachomatis Neisseria gonorrhoea Mycoplasma hominis Ureaplasma urealyticum VDRL/ RPR HSV II IgG HSV I IgG INFECTION* 10/81 (12.35%) 0/81 (0.00%) 10/81 (12.35%) 9/81 (11.11%) 0/81 (0.00%) 10/81 (12.35%) 58/81 (71.60%) PELVIC DISCOMFORT 10/92 (10.87%) 1/92 (1.09%) 13/92 (14.13%) 36/92 (39.13%) 1/92 (1.09%) 21/92 (22.83%) 78/92 (84.78%) SUBFERTILITY 19/343 (5.54%) 4/343 (1.17%) 46/343 (13.41%) 139/343 (40.52%) 3/343 (0.87%) 67/343 (19.53%) 247/343 (72.01%) VAGINAL DISCHARGE 4/63 (6.35%) 0/63 (0.00%) 10/63 (15.87%) 26/63 (41.27%) 0/63 (0.00%) 10/63 (15.87%) 53/63 (84.13%) UNCLASSIFIED 1/41 (2.44%) 0/41 (0.00%) 6/41 (14.63%) 10/41 (24.39%) 0/41 (0.00%) 10/41 (24.39%) 26/41 (63.41%) TOTAL 44/620 (7.1%) 5/620 (0.8%) 85/620 (13.7%) 220/620 (35.5%) 4/620 (0.64%) 118/620 (19%) 462/620 (74.5%) P-value 0.2000.7080.979 < 0.001 0.7790.350 0.015

24. % of Frequency C. trachomatis22/620 (3.55%) N. gonorrhoea1/620 (0.16%) U. urealyticum173/620 (27.90%) M. hominis47/620 (7.58%) C. trachomatis + U. urealyticum13/620 (2.10%) C. trachomatis + M. hominis6/620 (0.97%) N. gonorrhoea + U. urealyticum4/620 (0.65%) U. urealyticum + M. hominis27/620 (4.35%) C. trachomatis + U. urealyticum + M. hominis 4/620 (0.65%) All negative323/620 (52.10%)

25. PAIRED COUPLE WITH COMPLETE STD PROFILE Chlamydia trachomatis Neisseria gonorrhoea Mycoplasma hominis Ureaplasma urealyticum RPR/ Syphilis HSV II IgG HSV I IgG FEMALE 14/127 (11.03%) 3/127 (2.36%) 23/127 (18.11%) 83/127 (65.35%) 1/127 (0.79%) 29/127 (22.83%) 100/127 (78.74%) MALE 4/127 (3.15%) 0/127 (0%) 16/127 (12.60%) 31/127 (24.41%) 1/127 (0.79%) 18/127 (14.17%) 86/127 (67.71%) p-value 0.014 0.2470.223 < 0.001 1.00.076 0.047 Comment : this slide should be paired analysis. P –values are based on unpaired

26. Semen vs urine Chlamydia trachomatis Neisseria gonorrhoea Mycoplasma hominis Ureaplasma urealyticum RPR/ Syphilis HSV II IgG HSV I IgG semen 10/166 (6.02%) 0/166 (0.00%) 21/166 (12.65%) 31/166 (18.67%) 1/166 (0.60%) 24/166 (14.46%) 111/166 (66.87%) urine 1/21 (4.76%) 0/21 (0.00%) 2/21 (9.52%) 5/21 (23.81%) 0/21 (0.00%) 4/21 (19.05%) 14/21 (66.67%) P-value0.512-0.9810.6741.00.6700.640 other doctors (urine sample) 0/11 (0.00%) 2/11 (18.19%) 0/11 (0.00%) 2/11 (18.19%) 7/11 (63.64%) Total: 199 cases

27. Chlamydia trachomatis: WRONG for Sexually Transmitted Diseases (STD) in Hong Kong! SUMMARY: Chlamydia trachomatis 7.4% Neissera gonorrhoea 0.8% Mycoplasma hominus 14% Ureaplasma urealyticum 35.3% Syphilis 0.5% HSV I IgG 74.2% HSV II IgG 18.7%

28. Chlamydia trachomatis: WRONG for Sexually Transmitted Diseases (STD) in Hong Kong! CONCLUSION: 1 ) Chlamydia trachomatis incidence is much lower than U urealyticum, M hominus 2) This is more obvious in those presenting with pelvic pain cf subfertility 3) Guideline in testing C trachomatis should be changed to U urealyticum, M hominus (with/ without C trachomatis & N gonorhoea) 4) Apparently, there is no significant difference using urine or semen specimen

29. Chlamydia trachomatis: WRONG for Sexually Transmitted Diseases (STD) in Hong Kong! Problems to solve: 1) How to diagnose Upper Genital Tract infection? 2) Role of U parvum? 3) More sensitive & specific tests? 4) Efficacy of different treatment regimes?

30. Chlamydia trachomatis: WRONG for Sexually Transmitted Diseases (STD) in Hong Kong! THANK YOU