CHLAMYDIA INFECTIONS F. O. EKO Professor Microbiology, Biochem & Immunology

Chlamydia Obligate intracellular GN bacteria Major species in human disease: – C. trachomatis (CT) - oculo-genital infections – C. pneumoniae (Cp)– pneumonia – C. psittaci – psittacosis – C. abortus – pneumonia & preterm abortion WHO - genital CT infection is most common bacterial cause of STIs worldwide 2012: Global prevalence of STIs -273 million/yr – Prevalence of CT is about 128m (~47%) – 357m new STI cases; CT accounts 131m (~37%)

Chlamydia High # of chlamydial infections are asymptomatic – 70% of endocervical infections in women and – 50% of urethral infections in men Prevalence in adults (15-49yr) varies globally – Highest in women in the Americas & Western Pacific and men in Western Pacific – Prevalence of trichomoniasis is highest in African region

Estimated prevalence of chlamydia, gonorrhoea, trichomoniasis, & syphilis in women and men aged 15–49 years by WHO region, based on 2005–2012 data Newman, L. et al., PLos One, Dec 8, 2015

Epidemiology: Disease in the U.S.

Incidence and Cost Estimated 3 million new cases in U.S. annually Most frequently reported disease in U.S. Direct and indirect annual costs total approximately $3 billion

Chlamydia — Reported rates by state: United States and outlying areas, 2006 Note: The total rate of chlamydia for the United States and outlying areas (Guam, Puerto Rico and Virgin Islands) was per 100,000 population.

Chlamydia — Reported rates: Total and by sex: United States, 1987–2006 Note: As of January 2000, all 50 states and the District of Columbia had regulations requiring the reporting of chlamydia cases.

Chlamydia Prevalence Among Women and Men Aged Years by Race/Ethnicity (NHANES)*, *National Health and Nutrition Examination Survey

Risk Factors Age, race and gender - CCR5delta32 gene deletion New or multiple sex partners History of STI infection Presence of another STI Oral contraceptive use Lack of barrier contraception

Transmission Transmission is sexual or vertical Highly transmissible Incubation period 7-21 days Infected asymptomatic people significant reservoir Re-infection is common Perinatal transmission results in neonatal conjunctivitis in 30%-50% of exposed babies

Microbiology C. trachomatis is a strict human pathogen Harbor a “cryptic” plasmid Require host cell energy Infect columnar epithelial cells – eye/genital tract Survive by replication that results in the death of the cell Takes on two forms in its life cycle: – Elementary body (EB) – Reticulate body (RB)

Chlamydia: Two morphologic forms 1. Elementary body: a. Metabolically inactive b. Membrane proteins are highly cross-linked: enables it to survive in the extracellular environment c. Infectious form 2. Reticulate body: a. Larger, metabolically active form b. Only found intracellularly c. Divides by binary fission d. Non-infectious form

Chlamydia Developmental cycle

TEM of EB and RB Chlamydia Inclusion body

Virulence factors Clinical effects of C. trachomatis infection –cell destruction and the host’s inflammatory response Adhesins – attach to receptors on surface of epithelial cells Epithelial cell-surface antigens of the EB prevents lysosomal fusion A number of candidate virulence factors have been identified, including –Polymorphic outer membrane (autotransporter proteins) –Cytotoxin - indistinguishable from clostridial toxin B –Type III secretion effectors - promote delivery of pathogen effector proteins to host cell IncA - mediates inclusion vesicle fusion

Virulence factors –Stress response protein - GroEL and GroES homologues –Proteins produced by “cryptic” plasmid –Infection with plasmid free strains results in: –Reduced inflammation –Abrogation of infertility Capacity to sequester iron through the tryptophan biosynthetic pathway

Host response Innate immune system recognizes PAMPs –TLRs are one group of pattern recognition receptors that are expressed on macrophages, dendritic cells (DCs), neutrophils, NK cells, and epithelial cells –Chlamydial LPS is not the component responsible for induction of the proinflammatory cytokines –TLR2 plays a role - significant reduction in oviduct pathology was noted in TLR2-knockout mice –Infection of DC/NK cells induces innate immune responses Expression of co-stimulatory molecules & TLRs Secretion of proinflammatory cytokines

Host response Cell Mediated Immunity (CMI) –predominant component of protective immune responses –major histocompatibility complex class II (MHC II)-restricted CD4+ T cells –Protective CMI strongly associated with the production of IFN-  either by CD4+ or by CD8+ T cells –Immunity induced by natural chlamydial infection does not provide long-lasting protection and may contribute to pathology Humoral immunity –IgG- and IgA: Dominant immunoglobulin isotype in the genital tract is IgG rather than sIgA –Role of Abs in chlamydial immunity not completely clear –Significant role during reinfection & enhancing Th1-mediated responses

Animal models Animal models of genital chlamydial disease – mouse, rat, guinea pig, pigs, nonhuman primate models Reflect aspects of human disease Useful in defining correlates of protection & pathogenesis Mouse most commonly used model Manifests pathologic features of disease in women – shedding, tubal pathologies & infertility Macaca mulatta Guinea pigRat Mouse

Mouse model Intravaginal infection of mice twice or treatment with Depo-Provera to ensure productive infection Vaginal swabs validation of infection/shedding Mice are assessed for development of pathology after 14 or 28 days – UGT evaluated for tubal inflammation, hydrosalpinx Infected females mated with males to evaluate fertility – assessed by enumerating recovered pups from each pregnant mouse

Molecular basis of CT-induced infertility Currently, the mechanisms by CT induce disease are not completely understood We showed mice infected with plasmid free (PF) CT failed to develop oviduct damage and remained fertile indicating that plasmid related factors are implicated in pathogenesis

Chlamydia-induced infertility in female mice (a) Chlamydia-infected ( WT-CT ) – Infertile (b) Non-infected or PF CT-infected - Fertile ** Results: indicate CT-induced infertility requires the presence of the Chlamydia plasmid

Pathogenesis Air-dried chlamydiae may remain infective because of the cross- linked membrane proteins of the elementary body Ascending route of genital infection in women: – Lower genital tract (cervicitis) – Upper genital tract-pelvic inflammatory disease (PID) – Tubal factor infertility

Pathogenesis Infection characterized by chronic, persistent infection over many years: Little protective immunity –Host inflammatory response to chlamydial antigens –Tissue scarring and damage: Cornea in trachoma Fallopian tubes in genital infections (partial obstruction: increased risk of ectopic pregnancy; complete obstruction: tubal infertility) Uterine horn dilation Glandular duct dilation Normal: Uninfected

Clinical manifestations Humans are natural host Trachoma caused by serovars A-C Genital infections caused by serovars D-K Lymphogranuloma venereum caused by serovars L1, L2, L3 –More severe sexually transmitted disease characterized by a transient genital lesion, swollen, painfully inflamed, inguinal lymph nodes and systemic symptoms such as fever Reactive arthritis may occur - probably immunologically mediated

Types of Chlamydia infections C. trachomatis – 2 biovars Non-LGV: –STD and trachoma in all ages –conjunctivitis in adults –conjunctivitis and pneumonia in infants LGV: lymphogranuloma venerum (LGV) C. pneumoniae - causes pharyngitis, bronchitis, and pneumonia C. psittaci (Parrot fever) - causes pneumonia (Psittacosis) C. abortus – pneumonia & preterm abortion

Clinical Syndromes Caused by C. trachomatis Local InfectionComplicationSequelae Conjunctivitis Urethritis Proctitis Epididymitis Reiter’s syndrome (rare) Infertility (rare) Chronic arthritis (rare) Conjunctivitis Urethritis Cervicitis Proctitis Cervical cancer Endometritis Salpingitis Reiter’s syndrome (rare) Infertility Ectopic pregnancy Chronic pelvic pain Chronic arthritis (rare) Conjunctivitis Pneumonitis Pharyngitis Rhinitis Chronic lung disease? Rare, if any Men WomenInfants

Chlamydia trachomatis: Clinical Genital infections 1. Most common cause of sexually transmitted disease in U.S. 2. Dysuria and urinary frequency,nonpurulent urethral discharge vs. purulent discharge for gonorrhea 3. Men: nongonococcal urethritis White-gray urethral discharge, which may be slight A 46 yr old male with chlamydial urethritis

C. trachomatis Complications in Men Epididymitis - Swollen or tender testicles Reiter’s Syndrome –a type of reactive arthritis - joint swelling and pain, often in knees, ankles, and feet, along with inflammation of the eyes and urinary tract

C. trachomatis Infections in Women Cervicitis –Majority are asymptomatic –Local signs of infection, when present, include: Mucopurulent endocervical discharge Edematous cervical ectopy with erythema and friability Urethritis –Usually asymptomatic –Signs/symptoms, when present, include dysuria, frequency, pyuria Mucopurulent cervicitis

C. trachomatis Complications in Women Pelvic Inflammatory Disease (PID) ( Can have PID even if there are no symptoms) –Salpingitis –Endometritis –Ectopic pregnancy and tubal infertility Perihepatitis (Fitz-Hugh-Curtis Syndrome) –Inflammation of the serous or peritoneal coating of the liver often resulting from PID Reiter’s Syndrome –a type of reactive arthritis

Chlamydia trachomatis: Clinical Genital infections: Neonates 1. Spread from infected mother during passage through birth canal 2. Inclusion conjunctivitis and pneumonia Holmes K.K. et. al. Sexually Transmitted Diseases. 3rd edition, 1999, plate 10

DIAGNOSIS Specimen: conjunctival scraping, urethral or cervical swab: need adequate specimen that will contain chlamydiae-infected cells 3 main types of tests: 1.Fluorescent antibody test- Detects intact bacteria with a fluorescent antibody Chlamydiae stained green Holmes K.K. et. al. Sexually Transmitted Diseases. 3rd edition, 1999, plate 93

2(a). Giemsa stain: stains purple Hundreds of organisms inside a cytoplasmic vacuole DIAGNOSIS Chlamydial vacuole (= chlamydial inclusion) Giemsa stain Holmes K.K. et. al. Sexually Transmitted Diseases. 3rd edition, 1999, plate 94

DIAGNOSIS

2(b). Gram stain: Weakly Gram negative; not identifiable on Gram stain = No cell wall (envelope without peptidoglycan layer) DIAGNOSIS

2. Nucleic acid amplification: e.g. PCR – very sensitive (can be performed on non-invasive specimen like urine, which will detect infected cells that have been shed) 3. Culture –Historically the “gold standard” –Variable sensitivity (50%-80%) –High specificity

PREVENTION

Why Screen for Chlamydia? Most infections are asymptomatic Screening can reduce the incidence of PID by more than 50% Screening decreases the prevalence of infection in the population and reduces disease transmission

Prevention Counseling Nature of the infection –Chlamydia is commonly asymptomatic in men and women –In women, there is an increased risk of upper reproductive tract damage with re-infection Transmission issues –Effective treatment of chlamydia may reduce HIV transmission and acquisition Abstinence and other behavioral changes

Chlamydia trachomatis: Clinical Trachoma 1. Chronic infection of the conjunctiva 2. A leading cause of preventable blindness 3. Mainly in the developing world – North Africa, Southeast Asia and the Middle East 4. Global estimate of 150 million 5. 6 million are severely visually impaired or irreversibly blinded Tom Lietman, UCSF

Chlamydia trachomatis: Clinical Trachoma EarlyMidLate Peters, W. & Gilles, H.M. A Colour Atlas of Tropical Medicine & Parasitology. 3rd edition, 1989, p Treatment: azithromycin Reinfection occurs frequently 6. Main intervention is through improving sanitary conditions by increasing socioeconomic level