Chlamydia

A Journey From Embryonated Egg To LCR
AUTHOR: Dr. Jordan Dimitrakov
HIGHER MEDICAL INSTITUTE
PLOVDIV, BULGARIA
e-mail: jdim@medscape.com
A Short Historical Note
"Currently, what might be called the fifth epidemic of interest in the chlamydiae is occcurring," noted Professor Julius Schachter, Head of Chlamydiology Laboratory at UCLA. Ludwig Waelsch, a famous German microbiologist was the first to describe the so-called "non-gonococcal urethritis" and noted that "it was difficult to cure".
But what set the cycle of the first epidemic of interest in Chlamydiae as a group was the famous Java expedition of Neisser who discovered a bug that was already, and still is, named after him ( Neisseria gonorrhoeae) . In 1903, interested in the experimental infection by Treponema pallidum (the pathogen responsible for syphilis) Neisser undertook a journey to the island of Java together with two other famous scientistists — Ludwig Halberstaedter and Stanislaus von Prowazek. As it often happens, the latter, against the will of Neisser, inoculated the eyes of orangutans with trachoma material (obtained from eye scrapings from patients with trachoma) and found that the monkeys developed conjunctival inclusions. Similar inclusions were found in the conjunctiva of infants, the cervix of their mothers, and the urethral scrapings from male patients with non-gonococcal urethritis. But it was not until 1930 that Philip Thygeson and his colleagues were the first ones to prove the causal baby-mother-NGU connection.
What is Chlamydia?
Chlamydiae are obligate intracellular prokaryotic parasites of eukaryotic cells which means that these organisms can survive only by establishing residence within human or animal (eukaryotic) cells unlike the remaining Gram-negative organisms ( E.coli, Pseudomonas, Proteus etc), which are able to develop extracellullarly. The reason for this intracellular parasitism is the fact that Chlamydiae are energy parasites, i.e. they require an energy source for their replication and survival. All human cells have specific systems that they use to form a special substance called ATP (adenosine triphosphate) whih is a major source of energy. Chlamydiae have a cell membrane transport system that steals an ATP from host cells .
(click on medium to view medium-sized image, large to view large-sized image. Use your browser's "back" button to return to this page.)	Scanning electron microscopy of chlamydia trachomatis attacking epithelial cells. A transverse section through an epithelial cell.
Why are Chlamydiae considered bacteria?
Chlamydiae used to be considered viruses since they cannot be cultured on routine artificial (nonliving) media used in the clinical microbiology lab since, as it was already pointed out, they require living cells so that they can replicate and use them as a source of energy. Unlike viruses, Chlamydiae are sensitive to antibiotics (tetracyclines and macrolides) and therefore are considered bacteria. They are very small (about 350 nm) so they cannot be visualized (seen) using the Gram stain and the usual magnification of the light microscope found in the microbiology lab. Using greater magnification, Chlamydiae appear red on Gram staining (that is, they are Gram-negative). Unlike other Gram-negatives, however, they do not have a peptidoglycan layer and muramic acid (those are the structures attacked by penicillins and their derivative antibiotics) and therefore Chlamydial infections do not respond to treatment with penicillins.
A comparison of Chlamydiae with the Enterobacteriaceae (Gram-negative rods) and Viruses
Criteria Enterobacteriaceae Chlamydiae Viruses Size (nm) 30-3000 350 15-350 Obligatory intracellular parasites No YES YES Nucleic acids RNA and DNA RNA and DNA RNA OR DNA Reproduction Fission Complex cycle with fission Synthesis and assembly Antibiotic sensitivity YES YES NO Ribosomes YES YES NO Energy production YES NO NO
Because of their unique developmental cycle these microorganisms have been classified into a separate order, Chlamydiales , with one family, Chlamydiaceae, containing one genus, Chlamydia. There are three species, C. trachomatis, C. psittaci and C. pneumoniae. Our review is based mainly on C. trachomatis.
Species Acute Diseases Sequelae/Chronic Diseases C. trachomatis Serovars A-C Conjunctivitis Trachoma Serovars D-K (Men) Urethritis Chronic urethritis     Prostatitis     Epididymitis     Infertility     Reiter’s syndrome     Proctitis Serovars D-K (Women) Acute urethral syndrome     Bartholinitis   Cervicitis Infertility, ectopic pregnancy and pelvic inflammatory disease   Endometritis   Salpingitis   Periappendicitis Chronic abdominal pain   Perihepatitis LGV-serovars Lymphogranuloma venereum
Compiled from:
Guidelines for the prevention of genital chlamydial infections. Published on behalf of the World Health Organization, regional Office for Europe. Uppsala, Sweden, 1989, page 13
Peeling RW, Brunham RC. Chlamydiae as pathogens: new species and new issues. Emerging Infectious Diseases 1996;2(4):307-19.
Stamm WE, Holmes KK. Chlamydial infections. In: Isselbacher KJ, Braunwald E, Wilson JD, Martin JB, Fauci AS, Kasper DL, editors. Harrison’s principles of internal medicine. McGraw Hill:International Edition;1994:760.
Chlamydia developmental cycle
Chlamydiae exist in two forms:
Elementary body (EB): a small, infectious, metabolically inactive form with a rigid outer membrane adapted for surviving outside the cell; Reticulate body (RB): larger intracellular form, non-infectious, metabolically active, replicating form that is unstable out of the cell.	(click on medium to view medium-sized image, large to view large-sized image. Use your browser's "back" button to return to this page.) Scanning electron microscopy appearance of Chlamydia Trachomatis attacking epithelial cells. Arrows show reticulate bodies.
	The elementary body is adapted for extracellular survival and for establishing infection but does not replicate. It is relatively resistant to the usual methods of desinfection (sonication) and trypsin treatment and is relatively impermeable. In contrast, the reticulate body is relatively sensitive to sonication and trypsin and is more permeable.
Chlamydiae have a special preference for columnar epithelial cells. After attachment the EBs are endocytosed(engulfed) by the host cell and once inside the cell, the EB loses its infectivity and undergoes a number of changes associated with its transition to RB. Continued development occurs with a the formation of a cytoplasmic vacuole bounded by the host cell membrane, which continues to increase in size though protein synthesis in the cell is inhibited(similar to the invasion of cells by viruses). Therefore, Chlamydiae are secluded into their own "shell" within the cell. Endocytosis and growth within the vacuole do not incite phagolysosomal fusion which normally forms the basis for the subsequent killing of any foreign agent directed against the cell integrity. Therefore, Chlamydiae escape the normal cellular defense mechanisms. The RB divides by binary fission(meaning that it splits into two) resulting in 4-26 RBs within the vacuole in 8-12 hours. After 20 hours, if examined under an electron microscope, a central condensation is noted within some of the particles and these are typical EBs resulting from the RBs. The inclusions within the cell increase in number and begin to displace the nucleus. After 48-60 hours approximately 100 or more EBs rupture extracellularly to release the particles.
Epidemiology of chlamydial infections
C. trachomatis infection is the most common bacterial sexually transmitted disease. Although the actual incidence of chlamydial infections is not yet known due to lack of reporting data from the CDC based on data from states , reported cases in 1996, sentinel surveillance, surveys, and models based on proxies of infection show that:
About 4 million new Chlamydial infections occur annually in the United States alone ( Centers for Disease Control and Prevention. 1993 Recommendations for the Prevention and management of Chlamydial trachomatis infections. MMWR 42(no. RR-12):1-39; Black CM. Current Methods of Laboratory Diagnosis of Chlamydia trachomatis Infections. Clinical Microbiology Reviews 1997;10(1):161.) at an estimated cost exceeding 2.4 billion dollars.
Worldwide, it is estimated that there are 50-70 million new cases of C. trachomatis infection annually (Paavonen J and Lehtinen M. Chlamydial pelvic inflammatory disease. Human Reproduction Update 1996;2(6):515)
As many as 70 to 80 % of women and more than 50 % of men who are infected do not experience any symptoms — i.e. MOST CHLAMYDIAL INFECTIONS ARE ASYMPTOMATIC (SCHACHTER J, STONER E, MONCADA J. Screening for Chlamydial infection in women attending family planning clinics. West J Med 1983;138:375-9.; STAMM WE, COLE B. Asymptomatic Chlamydia trachomatis urethritis in men. Sex Transm Dis 1986;13:163-5; Zellin JM, Robinson AJ, Ridgway GL, Allason-Jones E, Williams P. Chlamydial urethritis in heterosexual men attending a genito-urinary medicine clinic: prevalence, symptoms, condom usage and partner change. Int J STD AIDS 1995;6:27-30.) This results in a large reservoir of unrecognized, infected individuals capable of transmitting the infection to their sexual partners. Besides, immunity following Chlamydial infections is type-specific (i.e. it protects only against the individual infecting Chlamydial organism) and only partially protective which results in frequent recurrent infections (Pearlman MD, McNeeley SG. A review of the microbiology, immunology, and clinical implications of Chlamydia trachomatis infections. Obstet Gynecol Surv 1992;47:448-61).
PATHOGENESIS OF CHLAMYDIAL INFECTIONS
C. trachomatis is spread by contact, with the RB binding only to receptor-bearing, nonciliated epithelial cells of mucous membranes. Target cells (cells primarily attacked) are those of the: Urethra (including the epithelial cells of the prostatic part of the urethra — so called "pars prostatica urethrae"); Vagina; Fallopian tubes (ovarian tubes in women); Anorectal tract; Respiratory tract; Conjunctiva; Synovium (joints); Hepatic (Glisson’s) capsule in women. Symptoms of infections result from: destruction of target cells bacterial replication and severe host inflammatory reactions.
Chlamydia and Prostatitis
Does Chlamydia trachomatis cause prostatitis? The answer to this question has plagued physicians and urologists for more than 20 years now.
Several confounding facts account for this.
First, as it has already been stated Chlamydiae are a special kind of bacteria that do not grow or multiple on routine culture media and has been "obscured" for years. They have been considered major agents leading to the so-called "Persistent non-gonococcal urethritis" in patients who presented to STD clinics for evaluation and treatment. Routine evaluation of such patients still requires a Gram-stain (a special staining technique that will be explained in detail in another article of this series) where, if the Gram stain reveals the presence of more than 5 white blood cells (WBC) per high power field and Gram-negative intracellular diplococci (gonococci) the diagnosis of gonorrhea (or gonococcal urethritis) is made and the patient should receive treatment for gonorrhea and his partner(s) should also be treated. (Burnstein GR and Zenilman JM. Nongonococcal urethritis — a new paradigm. Clin Inf Dis 1999 March; 28(Suppl 1):S66-S73).
However, it has been known that a certain proportion of such patients in the early years of STD treatment (during the 1950-1960) even if they had been adequately treated for gonorrhea, if the strain was susceptible to the prescribed antibiotic and if the patient was compliant with the treatment and there was no reinfection, most such patients developed persistent urethral discharge and urethral burning and the control Gram-exams and cultures failed to identify the pathogens involved. The patients were termed to have persistent (idiopathic, without any known reason) NGU and were told that there was no treatment available for the condition. Occasionally, some of these cases could be treated with tetracyclines without a plausible explanation.
It was not until 1964 that Eric Dunlop and Barrie Jones were the first to isolate Chlamydia trachomatis from men with NGU and publish their findings in the British Journal of Venereal Diseases. As in other developed countries, in the US in the 1970s and the 1980s the cases of NGU increased dramatically , outstripping the cases of gonorrhea, until a peak was reached in 1986-1987, followed by a decline.
Table 1. Detection of Chlamydia trachomatis in Men with Acute Nongonococcal Urethritis: Data from Published Studies Using Tests Other Than Nucleic Acid Amplification Tests
Year Published Method of Detection Number of Men Studied % Men Chlamydia positive Reference Number 1976 Culture 81 31 1 1979 Culture 217 43 2 1979 Culture 263 36 3 1981 Culture 221 35 4 1981 Culture 62 31 5 1984 DFA* 100 35 6 1985 Culture 22 45 7 1988 DFA 146 45 8 1990 DFA 281 40 9 1991 DFA 112 43 10 1991 DFA 37 49 11 1992 DFA 112 43 12 1992 DFA 108 33 13 1993 DFA 98 41 14 1995 DFA 67 40 15
*DFA - direct immunofluorescent test
Prentice MJ,
DIAGNOSIS OF CHLAMYDIAL INFECTIONS
Before we consider the reliability of current methods of Chlamydia diagnosis several terms need to be defined (definitions found in Segen JC. The Dictionary of Modern Medicine, 1992):
SENSITIVITY — the degree to which a test or clinical assay is capable of confirming or at least supporting the diagnosis of a disease X. Represents the proportion of subjects with a disease which a test is capable of detecting (ideal sensitivity 100 %) SPECIFICITY - Represents the proportion of subjects without a condition which a test will designate as negative (ideal specificity 100 %) POSITIVE PREDICTIVE VALUE (PPV) — the proportion of subjects with a positive test who actually have the disease (EFFICIENCY) NEGATIVE PREDICTIVE VALUE (NPV) — the proportion of subjects with a negative test who actually do not have the disease INCIDENCE — the number of new cases of a disease that occur in a population divided by a unit of time, usually a year PREVALENCE - the cases of a disease divided by the total number of subjects in the population.
Thus, it is very important to know if the test is done in a high-risk (high prevalence) population or in a low-risk (low-prevalence) population since even with nonculture tests with a specificity of 98 % and a sensitivity of 80 % the PPV decreases from 87.6 % to 45.1 % as the prevalence decreases from 15 % to 2%. In other words, if Chlamydiae are not frequently encountered in a certain area and population due to public health efforts and the frequency of infection is low , then a positive test for C. trachomatis should be interpreted very carefully and best repeated since it will be a true-positive result in 45.1 % of the cases if the prevalence is 2 % as in the example above. On the other hand, if it is a high-risk population, and the prevalence of the infection is at least 15 % the positive test will be reliable in 87.6 % of the cases and need not be repeated but instead should receive prompt treatment.
CULTURE ON MCCOY OR HELA CELLS
Until recently, culture was considered the gold standard in the diagnosis of Chlamydial infections since it is nearly 100 % specific (only Chlamydiae multiple and can be visualized).
PRINCIPLE:
Specimens from patients are inoculated (plated) onto cell culture monolayers. Usually, McCoy or HeLa cell lines are employed and monolayers allow visualization. If sufficient numbers of viable chlamydial elementary bodies are present, they infect the cells and grow to form intracytoplasmic inclusions, thus we have to imitate in the cell culture system the natural history of Chlamydial infections as described above. The inclusions (these are the formations that result from Chlamydial infections of the cells are visualized following 48 to 72 hours of incubation (depending on the inoculum, i.e. the severity of infection)by staining with fluorescently-labeled antibodies (these are special substances aimed at the membrane of Chlamydiae). Antibodies form complexes with the membrane structures of Chlamydia, called antigens, and when examined under a special kind of microscope, called immunofluorescent, they emit light, or "fluoresce". Antibodies are aimed at two types of antigen — the chlamydial lipopolysaccharide antigen (LPS) which is common to the three chlamydial species (and therefore, this results in cross reaction thus introducing false-positive results because other chlamydiae could also give a positive signal) and the so-called MOMP (major outer membrane protein) which is specific for C. trachomatis and therefore desirable, resulting in a nearly 100 % specificity (i.e. the structure visualized is a Chlamydia trachomatis with nearly 100 % reliability).
WHY CAN’T CELL CULTURE BE USED IN THE DIAGNOSIS OF CHRONIC PROSTATITIS ?
Semen and expressed prostatic secretions (EPS) are highly toxic to the cells in the cell culture line (Schachter 1985) thus preventing the incorporating of Chlamydia into the cell cultures and resulting in false-negative results (i.e. the patient may have Chlamydia in the prostate and semen but he will be given a negative result; Cell culture has a sensitivity of 75% - 85 % in expert laboratories on urethral swabs, but the advent of the newer nucleic acid amplification tests (NAAT) has shown even this to be an overestimate (Jaschek G, Gaydos CA, Welsh LE, Quinn TC. Direct detection of Chlamydia trachomatis in urine specimens from symptomatic and asymptomatic men by using a rapid polymerase chain reaction. J Clin Microbiol 1993;31:1209-1212).The sensitivity (the number of reliable positive test results for C. trachomatis from EPS and semen is even lower; The performance and accuracy of cell culture depends on many factors as detailed by Schachter (Chlamydia trachomatis: The More You Look, the More You Find: How Much Is There? Sex Trans Dis 1998 May; 25(5):229-231): The quality of the specimen (inhibition is a major problem with EPS and semen — my commentary) The type of swab used for collecting the specimen ( some types of swabs have been shown to be toxic to chlamydia thus resulting in false-negative results) Transportation system used ( stringent cold chain) Type of cells used ( McCoy or HeLa resulting in difference in sensitivity and the recovery of the organisms, thus influencing sensitivity or the number of reliable positive test results) Length of time in storage of the specimen Type of microscopy used to demonstrate the inclusions Limited availability to clinicians due to the expense, high level of technical expertise required and time required to obtain results (3 to 7 days)(Black CM. Current Methods of Laboratory Diagnosis of Chlamydia trachomatis Infections. Clin Microbiol Rev 1997 Jan;10(1):165).
OTHER METHODS FOR THE DIAGNOSIS OF CHLAMYDIAL INFECTIONS
DFA (DIRECT FLUORESCENT ASSAY)
(click on medium to view medium-sized image, large to view large-sized image. Use your browser's "back" button to return to this page.) DFA of Chlamydia Trachomatis (Microtrak, Syva, Palo Alto, USA) Chlamydiae appear as green dots and the remaining large areas are epithelial cells.
PRINCIPLE: A technique in which the molecule of interest (an antigen in the membrane of Chlamydia trachomatis, usually MOMP) is detected directly by an antibody labelled or tagged with a fluorochrome. The specimen for examination is obtained using an urethral swab or sediment (pellet) from centrifuged first-catch or post-prostatic massage urine samples. The specimen is examined under ultraviolet light. DFA SENSITIVITY: 80 — 90 % relative to culture DFA SPECIFICITY: 98-99 % relative to culture (Chernesky M, et al. Detection of Chlamydia trachomatis antigens by enzyme immunoassay and immunofluorescence in genital specimens from symptomatic and asymptomatic men and women. J Infect Dis 1986;154:141-148) DFA ADVANTAGES: Relatively high specificity for urethral specimens; Permits direct assessment of specimen adequacy (i.e. whether a sufficient amount of epithelial cells have been collected to allow the detection of Chlamydial EBs; if the number of cells is too low, the specimen is considered indeterminate and another specimen is requested bu the lab because otherwise a false-negative result may be reported); Relatively rapid (results available in 30 minutes); Easy transportation of specimens (no refrigeration required) DISADVANTAGES OF DFA: Invasive (requires the insertion of urethral swabs); Requires highly trained personnel; Performance varies with the specimen fixation technique; Performance varies with the number of EBs present; Performance varies with the serotype; Performance varies with the antibody type used.
EIA (ENZYME IMMUNOASSAY) PRINCIPLE:In the direct EIA, enzyme-labeled antibodies that recognize all species of Chlamydia bind to LPS extracted from elementary bodies in the specimen (therefore, the false-positive results are often encountered because of non-specificity). In the indirect EIA, a primary LPS-antibody is used as the detector reagent, followed by a secondary enzyme-linked antibody which is directed aginst the first antibody (the first antibody here serves as an antigen) That is why this model has been called "the sandwich model" because we presumptively have an antigen (Chlamydia) in the patient specimen (the outer part of the sandwich). Then we have the first antibody directed against the antigen (detector reagent or middle piece of the sandwich) and then we have the outermost part of thee sandwich (the seconadry enzyme-linked antibody which is directed against the first antibody). The enzyme component of the specifically-bound antibody converts a colourless substrate (substance) to a colored product detected by an equipment which measures the length of waves and is called spectrophotometer. A MAJOR DRAWBACK of the older EIA is the fact that the antibodies directed at the LPS may cross-react with the LPS of other Gram-negative organisms (E.coli, Proteus, Klebsiella, Enterobacter)and therefore produce false-positive results (Centers for Disease Control and Prevention. False-positive results with the use of chlamydial tests in the evaluation of suspected sexual abuse. Morbidity and Mortality Weekly Report 1991;39:932-5). Therefore, some manufacturers have added blocking reagents to overcome this problem. EIA has a lower sensitivity as compared with culture — 73 % and good specificity — 98 % (data for the best studied to date EIA Chlamydiazyme (Abbott Diagnostics, North chicago, Ill) (Ehret JM et al. Evaluation of chlamydiazyme enzyme immunoassay for detection of Chlamydia trachomatis in urine specimens from men. J Clin Microbiol 1993;31:2702-5).
NUCLEIC ACID AMPLIFICATION TESTS
"There has been a revolution in diagnostic methodology in recent years with the introduction of nucleic acid amplification tests (NAAT). We became aware that the estimates of prevalence of infection determined in the past by either tissue culture or early nonculture tests (antigen detection methods or the direct nucleic acid probes) were gross underestimates."Julius Schachter, "Chlamydia trachomatis: The More You Look, the More You Find: How Much Is There?" Sex Trans Dis 1998 May; 25(5):229-231).	(click on medium to view medium-sized image, large to view large-sized image. Use your browser's "back" button to return to this page.) Polymerase Chain Reaction - Principle.
"NAAT has been the most important advance in the field of chlamydial diagnosis since in vitro cell culture techniques replaced the yolk sac for culture and isolation of the organism from clinical specimens." Black CM. Current Methods of Laboratory Diagnosis of Chlamydia trachomatis Infections. Clin Microbiol Rev 1997 Jan;10(1):165). POLYMERASE CHAIN REACTION(PCR)(AMPLICOR, ROCHE DIAGNOSTIC SYSTEMS, BRANCHBURG, NEW JERSEY. FDA APPROVED 1993) PRINCIPLE: Every organism on earth possesses unique DNA sequences which consist of nitrogenous bases, sugars and phosphate and are termed "nucleotides". The sequence of bases along a polynucleotide chain carries the genetic information and can be visualized as "bricks". In the PCR, the reaction is carried out in a special instrument, called a thermocycler. First the two strands of Chlamydial DNA, if present, should be separated which is accomplished by heating at 95 degrees. Then, two synthetic oligonucleotide primers (special synthetic DNA pieces of nucleotides) which initiate the reaction bind to specific regions of Chlamydia DNA (if it is present in the patient sample) a process, known as "primer annealing" at a lower temperature . The target DNA sequence is copied from free nucleotides ("bricks")in the mixture in the presence of a special substance (enzyme) called thermostable (active at high temperature) DNA polymerase where the process gets its name. The product is visualized, if present, in the final step of the reaction by a change in the color (positive samples get yellow). ADVANTAGES OF PCR: Highest sensitivity: 90 % Highest specificity: 99 to 100 % (Table)
AUTHOR/YEAR POPULATION STUDIED No. OF SUBJECTS LOCATION TEST MEN WOMEN Bianchi A et al/1994 STD clinic 756 France PCR 14.4% 4.5 % Bauwens JE et al/1993 STD clinic 949 Seattle PCR No data 8 % Jaschek G et al /1993 STD clinic 530 Baltimore PCR 9.8 % No data Cohen DA et al/1997 High school 1933 New Orleans LCR 4 % 9.7 % Oh MK et al /1997 Youth detention center 263 Birmingham LCR 8.8 % 28.3 % Rietmeijer CA et al /1997 High-risk urban youth 820 Denver PCR 7.6 % 7.3 % Burstein GR et al/1997 Family planning, STD, school 18,617 Baltimore PCR No data 10.6 % Brodine S et al/1998 Marines — shipboard 618 US military LCR 3.4 % No data Marines- Okinawa 406 US military LCR 5.2 % No data Marrazzo JM et al /1997 Teens/young adults 10,118 Seattle LCR 5.4 % 8.6 % Mertz KJ et al/1998 Convenience samples from NHANES Not stated Not stated LCR 2.8 % 4.5 %
PCR is the only method available for studying EPS and semen samples for the presence of Chlamydia trachomatis.
PCR is not dependent on the viability of the organism but it detects even 10 copies of Chlamydial DNA, if present, in the sample.
DISADVANTAGES OF PCR:
Inhibition due to some inhibitory substances — a problem which has been resolved in the commercial PCR test of Roche Molecular Systems — the AMPLICOR test.
False-positive results — resulting from the so-called "carry-over" contamination in the lab which is easily overcome once stringent conditions are implemented in the lab. LIGASE CHAIN REACTION (LCR) (ABBOTT LABORATORIES, FDA APPROVED IN LATE 1995) PRINCIPLE: In an effort to reduce false-positive results and improve specificity a second primer pair is introduced in the reaction. SENSITIVITY: 94 % SPECIFICITY: 99-100 %
GUIDELINES FOR DIAGNOSIS OF INFECTIONS WITH C. TRACHOMATIS ( After Black CM. Current Methods of Laboratory Diagnosis of Chlamydia trachomatis Infections. Clin Microbiol Rev 1997 Jan;10(1):165). Based on: "Centers for Disease Control and Prevention. 1998 Sexually Transmitted Diseases Clinical Practice Guidelines." Centers for Disease Control and Prevention, Atlanta, Ga. DEFINITIVE DIAGNOSIS (requires 1 or 2) 1. Isolation and confirmed identification of C. trachomatis in tissue culture from cervical, rectal, or urethral exudate and identification of characteristic intracellular inclusions. 2. Identification of C. trachomatis by one of the following methods and confirmation by a second culture or nonculture test method
Identification of the organism by DFA test of exudate Detection of antigen by EIA of exudate Detection of nucleic acid from exudate by DNA probe or DNA amplification technique a
PRESUMPTIVE DIAGNOSIS (requires 1 and 2) 1. Presence or absence of clinical symptoms(mucopurulent cervicitis, urethritis, epididymitis, pelvic inflammatory disease) 2. Detection of C. trachomatis by a nonculture test SUGGESTIVE DIAGNOSIS (requires 1 and either 2 or 3) b 1. Clinical symptoms (e.g. mucopurulent cervicitis, urethritis, epididymitis, pelvic inflammatory disease) 2. Exclusion of other causes of discharge or exudate (e.g. gonorrhoea) 3. Sexual exposure to a person infected with C. trachomatis or recently diagnosed with non-gonococcal urethritis, mucopurulent cervicitis, pelvic inflammatory disease.
a At present, there are no standard procedures for confirmation of DNA amplification methods.
b Testing is recommended to determine a specific diagnosis and to expedite management of sexual partners.
A BRIEF REVIEW OF THE STUDIES EXAMINING THE ROLE OF CHLAMYDIA TRACHOMATIS IN CHRONIC PROSTATITIS Chlamydia trachomatis, together with Ureaplasma urealyticum, is the most frequent cause of non-and post-gonococcal urethritis. So, the assumption appears plausible that, by way of intracanalicular ascension, Chlamydia might subsequently infect the prostate (Weidner W et al, 1988). However, its role in chronic abacterial prostatitis is still unclear, since, in most studies to date contamination of prostatic specimens by urethral microorganisms cannot be excluded. The role of Chlamydia in chronic prostatitis is still a mystery largely due to methodologic problems. Besides, most of the studies have yielded conflicting results due to different selection criteria and heterogeneity of the groups. As evidenced from the above, most of the earlier studies examined the role of C. trachomatis using cell-culture, which, as already pointed out, is unsuitable for diagnosing prostatitis since EPS and semen contain inhibitory substances. On the other hand, it is almost impossible to perform DFA from EPS (due to the small amount of material obtained and the concurrent presence of leukocytes, which may obscure the interpretation of slides) and semen. A short outline with a discussion of some of the most important studies examining the role of C. trachomatis in chronic prostatitis follows.
AUTHOR/YEAR OF STUDY NUMBER OF PATIENTS POSITIVE CHLAMYDIAL FINDINGS IN PROSTATIC PART OF URETHRA POSITIVE CHLAMYDIAL FINDINGS IN PROSTATE METHOD FOR OBTAINING MATERIAL DIAGNOSTIC METHOD Poletti et al (1985) 30 ND* 10 TRANSRECTAL BIOPSY CELL CULTURE Pust et al (1986) 32 6 1 URETHRAL ANR PERINEAL BIOPSY DFA** Shurbaji et al (1988) 16 ND 5 TURP***, OPEN OPERATION IMMUNOHISTOCHEMISTRY Doble et al (1989) 50 ND None PERINEAL ULTRASONOGRAPHICALLY GUIDED BIOPSY CELL CULTURE, DFA Abdelatif et al (1991) 23 ND 7 TURP IN SITU HYBRIDISATION Weidner et al (1991) 22 4 None PERINEAL, ULTRASONOGRAPHICALLY GUIDED BIOPSY CELL CULTURE Dan et al (1991) 100 ND 3 TURP CELL CULTURE * ND — not done ** DFA — direct immunofluorescence assay ***TURP — transurethral resection of the prostate The paper by Poletti and his colleagues published in the Journal of Urology in 1985 was the first one to contribute to the highly controversial topic of elucidating the role of C. trachomatis in chronic prostatitis. Until then, most studies relied on and quoted data published by Mardh and colleagues in 1972 (Mardh PA, Colleen S, and Holmquist B. Chlamydia in chronic prostatitis. British Medical Journal 1972; volume 4; page 361.) who examined 79 men with "non-acute prostatitis" and 72 blood donors as controls using a complement fixing test. 33 % of the men with "non-acute prostatitis" and 3 % of the controls had a positive test. A major shortcoming of this study was the lack of a good definition of prostatitis and the fact that the method used - complement fixation - is known to be quite non-specific (the antibodies found in those patients can actually reflect an infection with other pathogens). Therefore, the findings were unreliable and the same authors 6 years later reported contradicting results: Chlamydia trachomatis was isolated from urethral secretions of only 1 among 53 patients with chronic prostatitis, whereas Chlamydiae could not be cultured from the EPS (due to inhibiton, as it became obvious several years later). Those findings were published in the British Journal of Venereal Diseases 1978, volume 54, page 330-334 (Mardh PA et al. Role of Chlamydia trachomatis in non-acute prostatitis). So, the study by Poletti and colleagues contributed by examining epithelial cells obtained by transrectal aspiration biopsy and found that cultures were positive in 10 of 30 patients (33 %). In a review on chronic prostatitis Dr. Andrew Doble from Cambridge in UK categorized this study in his editorial in the British Journal of Urology as "poorly controlled" since in his opinion the Chlamydiae were actually "isolated from their urethra. Not only would it appear that these patients did not have chronic prostatitis, but their prostates must have been awash with organisms to yield such a high positive rate." (Doble A. Chronic prostatitis. British Journal of Urology 1994;74:537-541). Since a major critique of the Poletti study was that the material for examination of Chlamydia was obtained using TRANSRECTAL biopsy (and so the study did not control or take into account the possibility of a co-exiting asymptomatic Chlamydia proctitis (inflammation of the rectal mucosa as reported by Quinn TC et al. (Chlamydia trachomatis proctitis. New England Journal of Medicine 1981;305:195-200) and Munday PE et al. (Chlamydia trachomatis proctitis. New England Journal of Medicine 1981;305: 1158-9) a later study was performed by Pust and colleagues in the Federal Republic of Germany using transperineal biopsy (the perineum is the part situated between the testes and the anus) and published in 1986 in a book entitled "Therapy of Prostatitis"(Weidner W, Brunner H, Krause W, Rothauge CF (eds) Therapy of prostatitis. Klinische und experimentelle Urologie 11. Zuckschwerdt, Munich, pp. 102-9). Pust and colleagues examined 32 patients with a diagnosis of "urethritis posterior" and found Chlamydiae in the urethras of 6 and in the prostate of 1 patient. A landmark study in the field was published in 1988 by Shurbaji and associates (Shurbaji MS et al. Immunohistochemical demonstration of Chlamydial antigens in association with prostatitis. Mod Pathol 1988;1:348-51). The authors evaluated formalin-fixed paraffin-embedded section from 16 patients with histologically proven prostatitis and found Chlamydial antigens in 5 cases (31 %) whereas Chlamydial antigens were not found in any of the specimens from patients with benign prostatic hyperplasia although Chlamydial antigens were found in the urothelium of the prostatic urethra of one of the controls. So, the authors concluded that the presence of Chlamydia trachomatis in the prostate of patients is almost always correlated with leukocytes and it could be a possible etiologic agent of prostatitis. Doble and his colleagues performed an elegant study using ultrasound-guided biopsy (Doble A, et al. The role of Chlamydia trachomatis in chronic abacterial prostatitis: a study using ultrasound guided biopsy. J Urol. 1989 Feb;141(2):332-3)The authors found Chlamydia trachomatis using transrectal ultrasound with a subsequent transperineal biopsy of the abnormal areas only in 1 patient (2 %) and they concluded that "there is no evidence that chlamydiae are directly implicated in the disease, although the possibility of an earlier active role cannot be excluded." Abdelatif and colleagues (1991) (Abdelatif OM et al. Chlamydia trachomatis in chronic abacterial prostatitis: demonstration by colorimetric in situ hybridization. Hum Pathol. 1991 Jan;22(1):41-4) performed a study on 23 transurethral resected specimens and evaluated them for the presence of Chlamydia trachomatis by a sophisticated technique called "colorimetric in situ hybridization" which detects Chlamydia genetic material in the prostate tissue and found Chlamydial EBs in 7 out of 23 specimens (30.4 %). It is important to note that Chlamydial inclusion bodies were present both within epithelial cells and intraluminal histiocytes (those are a specialkind of leukocytes, transformed into "defense cells") in 5 of those 7 cases. In two of 7 cases extracellular (RBs) Chlamydial bodies were identified in conjunction with intraepithelial inclusions and in only one case they observed extraductal, extraacinar, intrahistiocytic inclusion bodies. Weidner et al (1991) performed a number of studies examining the role of Chlamydia and Mycoplasma in abacterial prostatitis. In one of their studies (1988) and a later one (1991) they critically correlated the results of culture, cytological examination of leukocytes in urine after prostatic massage (VB3) and microimmunofluorescence tests and found that positive chlamydial cultures from EPS correlate with high leukocyte counts in VB3. (Weidner W et al. Role of Chlamydia trachomatis and mycoplasmas in chronic prostatitis. A review. Urol Int. 1988;43(3):167-73 and Weidner W et al. Chronic prostatitis: a thorough search for etiologically involved microorganisms in 1,461 patients. Infection. 1991;19 Suppl 3:S119-25). Dan et al (1991) studied 100 prostate biopsy samples obtained by transurethral resection and found C. trachomatis DNA in 3 patients (Dan M et al. Isolation of Chlamydia trachomatis from prostatic tissue of patients undergoing transurethral prostatectomy. Infection 1991 May-Jun;19(3):162-3). A REVIEW OF THE PROSTATITIS STUDIES EXAMINING THE ROLE OF CHLAMYDIA TRACHOMATIS Major caveats: Most earlier studies used methods that could not reliably detect C. trachomatis even if it were present in the EPS. A major flaw for many studies was the lack of a clear definition of what constitutes "chronic prostatitis" Many studies lacked a standardized design or examination technique which makes it difficult to compare and interpret the results. It is very important to examine the local immune response to Chlamydia in the prostate. So, an elevated IgA (immunoglobulin A) antibody level against C. trachomatis in the EPS (not in the serum samples or blood from the patient) would be evidence for a prostate infection with C. trachomatis. An interesting study published in Japanese was performed by Koroku M and colleagues. The analyzed anti-Chlamydia trachomatis specific IgA levels in EPS using Western blot ( a sensitive and sophisticated technique) and, in addition, they used the presence of anti-heat-shock protein (HSP: 60 kDa) IgA in EPS as a marker of infection. Heat-shock proteins have numerous functions mainly in the folding and unfolding or translocation of proteins and they are called "molecular chaperones". They are involved in antigen recognition by promoting the assembly of immunoglobulins, T-cell lymphocytes and gene products of the major histocompatibility complex (MHC). Several lines of evidence indicate that HSP synthesis is increased in inflammation. IgA was measured in the EPS of 192 subjects, including 92 patients with chronic idiopathic prostatitis. Anti-C. trachomatis IgA in the EPS was found in 44 (26 %) of 169 patients and in 29 % of those with more than 10 leukocytes/hpf (20 out of 69 patients). Besides, of those patients with more than 10 leukocytes/hpf, 38.5 % (5 out of 13 subjects)had anti-HSP IgA; In contrast, none of the atients with 5-9 leukocytes/hpf had anti-HSP IgA. Immunologic data of the 69 subjects with more than 10 leukocytes/hpf suggested that 29 % (20 of these patients) had chronic "idiopathic" prostatitis which was caused by C. trachomatis. Ludwig et al in a landmark study reported the prevalence of chlamydial antibodies using a specific immunofluorescence test in 101 men. The results were correlated with the clinical diagnosis , cell culture of urethral swabs, demonstration of chlamydial DNA in the ejaculate and signs of inflammation by counting peroxidase-positive leukocytes and elastase levels in semen. Serum specific IgG and IgA antibodies were found in 26 and 15 % of men, respectively, whereas seminal IgG and IgA antibodies were present in 6 and 7 % of men, respectively. Serum-specific antibodies showed no correlation with the infection, isolation rate of C. trachomatis in culture, PCR-findings and leukocyte count or elastase levels, thus proving to be an unreliable and useless marker for C. trachomatis genital infection. A significant correlation was found between seminal antibody levels and PCR-positive results in the ejaculate. Therefore, seminal antibodies against C. trachomatis can be used for diagnosing asymptomatic or ascending infection but their absence does not preclude an infection. CHLAMYDIA AND THE PROSTATE: FOOD FOR THOUGHT OR THE TWILIGHT ZONE In our opinion, based in the results from our lab and our PCR findings of PCR Chlamydia trachomatis is the etiologic agent in about 30 % of cases with non-bacterial prostatitis. Perhaps, the varying rate between different study groups can be explained by the fact that in the early stages Chlamydial invasion of the prostate is usually asymptomatic and in the later stages can be cryptic. The role of leukocytes, so widely used in the basis of prostatitis classification, and more specifically the role of neutrophils is complex. The complexities, as outlined in a recent article " the complexities span beneficial effects of neutrophils in the early stages of chlamydial infection to detrimental effects in later stages" (Wyrick PB et al. Persistent Chlamydial Envelope antigens in antibiotic-infected cells trigger neutrophil chemotaxis. J Inf Dis 1999;179:954-66). Chlamydiae, like the remaining Gram-negative microorganisms, possess a structure called lipopolysaccharide (LPS) and it has been experimentally shown that it is this LPS that can induce the production of the inflammatory cytokines like TNF-alpha (tumor necrosis factor alpha) although this induction is about 100 times less potent than the one induced by Neisseria gonorrhoeae LPS (Ingalls RR et al. Inflammatory cytokine response to Chlamydia trachomatis is endotoxin-mediated. Infect Immun 1995;63:125-30). On the other hand, the accumulation of chlamydial LPS in the cellular membrane has been shown by a very sophisticated technique (spin-probe electron-spin resonance spectroscopy) to decrease plasma membrane fluidity, which compromises the recognition of Chlamydia by the immune system and their destruction. Ingalls and his colleagues proposed that a chlamydial LPS-induced a less vigorous inflammatory response which correlates with the predominantly asymptomatic nature of chlamydial infections. Our data support the findings that the rise in the levels of pro-inflammatory cytokines precedes the appearance of leukocytes in the EPS. Most of the studies have studied different patient groups with different dynamics of Chlamydial infection which can explain the difference in the isolation rate excluding the difference due to technique sensitivity and specificity. The dynamics of Chlamydial infections in the prostate has not been fully elucidated. The role of chlamydial outer membrane protein in the recruitment of leukocytes to the prostate is not clear. One possible explanation is that the MOMP can be acquired by surveillant dendritic cells (local cells of the immune system), processed and presented to T-cells which explains the early and predominant antibody response to MOMP in infected individuals. The differences in isolation rate can be associated with the polymorphism of the MOMP gene similarly to the differences found in a study of 33 women in the San Francisco Bay Area where serovar E MOMP genotypes were associated with asymptomatic infection, nonvariant F genotypes correlated with lower genital tract infection and omp1 F variants were associated with pelvic inflammatory disease. Probably only omp1 F variants are discovered in symptomatic men with Chlamydial prostatitis but this question has not been fully studied. Besides, it is well known that LPS and MOMP antigens can escape and get to the surface of infected cells, thereby remaining undiscovered by traditional methods with the exception of PCR and LCR triggering an autoimmune response. It is interesting to note that numerous such vacuoles containing chlamydial antigens have been observed during a stage in the antibiotic destruction of a metabolically active chlamydial infection by azithromycin. It could be speculated that inadequate antibiotic therapy could contribute to the chronicity of infection. We have designed a scheme for the role of Chlamydia trachomatis in prostatitis that will be presented in a later installment or at the November Meeting on Chronic Prostatitis. AUTHOR: Dr. Jordan Dimitrakov HIGHER MEDICAL INSTITUTE PLOVDIV, BULGARIA e-mail: jdim@medscape.com

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