Case Study Pathogenic Bacteriology 2009 Case #3 Mamadou Diallo Anne Roberts

Case Summary Patient is 16 year old female Complaints of ongoing abdominal pain for days and vaginal bleeding No nausea or vomiting and denies urinary tract infection symptoms, chills or fever Abdominal pain increased 24 hours before being admitted to the emergency room Upon examination, exquisite tenderness in the upper right and lower left quadrants of the abdomen, and had a fever of 38.3°C (101 °F) Cervical motion tenderness present upon pelvic examination as well as right and left adnexal (ovary) tenderness

Key Information Pointing to Diagnosis PHYSICAL EXAMINATION Fever of 38.5°C or greater Abnormal vaginal discharge Lower abdominal tenderness when felt Tenderness when cervix is moved Tenderness in female organs Pelvic Inflammatory Disease (PID) believed to be the cause of the symptoms. 2 organisms known to cause PID: Neisseria gonorrhoeae and Chlamydia trachomatis LABORATORY EXAMINATION Endocervical swab obtained from patient used to inoculate McCoy cells. After incubation, cells were stained and showed the presence of inclusions.

The Diagnosis for Case #3 After results from the physical examination and a laboratory examination that determined the presence of elementary and reticulate body inclusions in inoculated cells it was established that Chlamydia trachomatis was the infectious agent.

Classification,Gram Stain Results, and Microscopic Appearance of Chlamydia trachomatis Chlamydia trachomatis Gram indeterminate – cannot be stained with Gram stain (no peptidoglycan), structurally organism is Gram negative (outer / inner membrane & LPS) Family: Chlamydiaceae Genus: Chlamydia Species: C. trachomatis

Diseases and Pathogenesis of Disease – C. trachomatis Chlamydia is a STD. It is an OBLIGATE intracellular bacteria. Alternate between two development stages with different functions: 1. Elementary Bodies (EB) – Infectious form Spore-like to permit survival outside of host cell Metabolically inert, Dense, compacted chromosomes Infect non-ciliated, epithelial cells by phagocytosis 2. Reticulate Bodies (RB) – Non-infectious form Active metabolism, and divides rapidly To complete the cycle, the RB must develop into EB before exiting the host. Cell will lyse and release the EB DISEASES CAUSED BY Chlamydia Eye infections - Infant conjunctivitis and Trachoma (Chronic eye infection) Lymphogranuloma Venereum (STD where organism disseminate to lymph nodes and result in buboes) Urethritis, epididymitis, prostatitis, cervicitis

Methods to Isolate/Identification/Detect C.trachomatis MOST SENSITIVE / SPECIFIC Culture organism in tissue/cells where Giemsa stain is used Considered the “gold standard” for specificity / sensitivity OTHER METHODS DIRECT FLUORESCENT ANTIBODY Mainly used for Trachoma / Conjunctivitis Highly sensitive/specific but technically demanding ENZYME IMMUNOASSAY Detects outer membrane LPS chlamydial antigen Significantly less sensitive than culture, but high in specificity ANTIBODY DETECTION Least sensitive and specific – Individuals have antibodies from previous infection Used as a complementary diagnostic tool in certain scenarios NUCLEIC ACID AMPLIFICATION TESTS (NAATS) Detect organism specific DNA/RNA sequences Used more today due to higher sensitivity than the other non culture methods

Therapy, Prevention and Prognosis of Patient Infected with C.trachomatis LONG TERM THERAPY NECESSARY! Doxycycline, Azithromycin, Erythromycin and Tetracycline used Beta Lactam drugs are… NOT effective for treatment No peptidoglycan in the organism!!! Beta Lactam drugs can be used in addition to the antibiotics above to treat patient for concomitant infection with Neisseria gonorrhoeae

Primary Research Article – C. trachomatis Kevin Hybiske and Richard Stephens. Mechanisms of Host Cell Exit by the Intracellular Bacterium Chlamydia. PNAS. Vol. 104: EXPERIMENTAL HeLa cells generated to express cytosolic GFP gene. HeLa cells infected with C.trachmatis. Visualization of real-time inclusion dynamics by fluorescence microscopy took place to determine mechanism of host cell exit. WHAT WAS FOUND 2 mutually exclusive pathways of release take place in the inclusion: Lysis – rupture of membrane, destructive mode of release mediated by Ca +2 activated proteases. Extrusion – packaged elementary bodies released, no conclusive results as to how mediated

Conclusion Rate of inclusion release was reduced when cells treated with protease inhibitors Rate of lysis duration was increased when host cells placed in Ca 2+ free conditions Deciphering the cellular exit mechanisms of C. trachomatis will be of fundamental importance to understanding Chlamydia pathogenesis. This is because cellular release directly affects its ability to infect new cells and transmit to new hosts

Take Home Message PID often involves infection with Chlamydia trachomatis… Typical symptoms fever, severe abdominal tenderness, cervical/ovary tenderness, vaginal bleeding/discharge Pathogen is an OBLIGATE INTRACELLULAR ORGANISM with a complex life cycle Diagnostics include culture in cells, enzyme immunoassays, direct fluorescent antibody test, NAATs Therapy is based on long term antibiotic treatment Prognosis is based on patient symptoms and inclusions of organism within host cells Prevention of organism is through use of condoms, talking openly with sexual partners, etc. Transmission is via sexual contact or through infected mothers to their babies Threats associated with this disease include ectopic pregnancy, infertility, blindness

References CDC – Chlamydia Factsheet. Department of Health and Human Services. Dec 20, March 1, Hybiske, Kevin and Richard Stephens. Mechanisms of Host Cell Exit by the Intracellular Bacterium Chlamydia. PNAS. Vol. 104: Mahon, Connie, Donald Lehman, George Manuselis. Textbook of Diagnostic Microbiology. 3 rd Edition. Saunders-Elsevier, St. Louis, Missouri, University of South Carolina School of Medicine – Microbiology / Immunology Online. The Board of Trustees of the University of South Carolina. April 11, March 8, 2009.