Chlamydia

A concise presentation highlighting the clinical, microbiological and diagnostic aspects of Chlamydial infections

1. CHLAMYDIA Dr. Suprakash Das M.D (Microbiology)

2. Introduction Chlamydiae are obligate intracellular bacteria that replicate within eukaryotic cells. C. trachomatis , like all other Chlamydia spp., has one of the smallest bacterial genomes . Its 1.04 Mb genome encodes approximately 900 genes, which is less than a fifth of the genes in Escherichia coli. The large majority of these genes (668) are shared among all Chlamydia, representing a core set of genes necessary for the intracellular chlamydial lifestyle. The small genome size is due to reductive evolution in which C. trachomatis has lost enzymes and metabolic pathways for nutrients that are readily available from the host cell.

3. Taxonomy The Chlamydia spp. are members of the order Chlamydiales and the family Chlamydiaceae . Members of the family Chlamydiaceae had been regrouped by Everett et al. (1999) from one genus, Chlamydia, into two genera, Chlamydia and Chlamydophila , based on differences in phenotype, 16S ribosomal ribonucleic acid ( rRNA ), and 23S rRNA . This classification was not universally accepted by the Chlamydia scientific community, and recently it was agreed that the family Chlamydiaceae contains a single genus , Chlamydia.

4. Important Species The genus Chlamydia contains nine recognized species: Chlamydia trachomatis , C. psittaci (agent of psittacosis; many species of birds), C. pneumoniae , C. pecorum (ruminants and koalas), C. muridarum (formerly the agent of mouse pneumonitis ), C. suis (an important pathogen of swine) , C. abortus (causes abortion in cattle and sheep; rarely causes abortion in humans), C. caviae (formerly C. psittaci , the guinea pig inclusion conjunctivitis strain), and C. felis (causes epidemic keratoconjunctivitis in cats). C. trachomatis and C. pneumoniae are the most significant human pathogens, and C. psittaci is an important zoonosis .

5. History Chlamydia-like disease affecting the eyes of people was first described in ancient Chinese and Egyptian manuscripts. A modern description of Chlamydia-like organisms was provided by Halberstaedter and von Prowazek in 1907. Chlamydial isolates cultured in the yolk sacs of embryonating eggs were obtained from a human pneumonitis outbreak in the late 1920s and early 1930s, and by the mid-20th Century isolates had been obtained from dozens of vertebrate species. The term Chlamydia (a cloak) appeared in the literature in 1945, although other names continued to be used, including Bedsonia , Miyagawanella , ornithosis -, TRIC ( Trachoma and Inclusion Conjunctivitis) , and PLT ( Psittacosis, Lymphogranuloma Venereum and Trachoma) agents.

6. Stanislaus von Prowazek Ludwig halberstaedter

7. Morphology and Characteristics Members of the order Chlamydiales are obligate intracellular bacteria that were once regarded as viruses because, like viruses, the chlamydiae require biochemical resources of eukaryotic host cells to fuel their metabolism for growth and replication by providing high-energy compounds such as adenosine triphosphate . Chlamydia spp. are similar to gram-negative bacilli in that they have lipopolysaccharide (LPS) as a component of the cell wall. The chlamydial LPS, however, has little endotoxic activity. The chlamydiae have a major outer membrane protein (MOMP) that is very diverse. The variation in MOMP in Chlamydia trachomatis is used to separate the species into 18 distinct serovars .

8. Morphology and Characteristics Chlamydia aren’t viruses- The chlamydiae were once considered viruses because they are small enough to pass through 0.45 μm filters and are obligate intracellular parasites; however, the organisms have the following properties of bacteria: 1. Possess inner and outer membranes similar to those of gram negative bacteria. 2. Contain both deoxyribonucleic acid (DNA) and ribonucleic acid (RNA). 3. Possess prokayotic ribosomes . 4. Synthesize their own proteins, nucleic acids, and lipids. 5. Suceptible to numerous antibacterial antibiotics.

9. C. trachomatis - Biovars & Serotypes Strains of C. trachomatis are divided into three biovars and are further subtyped by serovar . The trachoma biovar ( serovars A–C) is the leading cause of non-congenital blindness in developing nations, whereas T he genital tract biovar ( serovars D–K) is the most prevalent sexually transmitted bacterium. In women, 70–80% of genital tract infections with C. trachomatis are asymptomatic, but 15–40% ascend to the upper genital tract, which can lead to serious sequelae , including pelvic inflammatory disease, infertility and ectopic pregnancy. The lympho granuloma venereum (LGV) biovar ( serovars L1–L3) causes invasive urogenital or anorectal infection.

10. Life cycle Chlamydiae have a unique developmental life cycle reminiscent of parasites, with an intracellular, replicative form, the reticulate body (RB), and an extracellular, Metabolically inert, infective form, the elementary body (EB) (200-400 nm in diameter). The EB cannot survive outside of a host cell for an extended period. After infection of a host cell, the EB differentiates into an RB. The RB divides by binary fission within vacuoles. As the numbers of RB increase, the vacuole expands, forming an intracytoplasmic inclusion.

11. Life cycle The RB then revert to EB, and 48 to 72 hours postinfection , the EB are released from the host cell. In addition to the replicative cycle associated with acute chlamydial infections, there is evidence that Chlamydia can persist in an aberrant form in vitro, depending on the amount of interferon-gamma (IFN-g) and tryptophan in the host cell as well as function of the tryptophan synthase encoded by the organism. Removal of IFN-g or increase in tryptophan will result in differentiation of chlamydiae into an active EB infection.

12. Immune Response Immune response- Chlamydia survives and multiplies by- Controlling host cell survival and death Modulating the host cell cycle Modulation of innate immune response. Virulence factors- Chlamydial polymorphic outer membrane proteins Chlamydial type iii secretion systems Chlamydial stress response proteins Chlamydial toxin Chlamydial lipopolysaccharide ( lps ) and other glycolipids

13. Diseases caused by- C. trachomatis - 1. Trachoma Trachoma is manifested by a chronic inflammation of the conjunctiva and remains a major cause of preventable blindness worldwide. The organism is acquired as a result of contact with infected secretions on towels or fingers or by flies. Early symptoms of infection include mild irritation and itching of the eyes and eyelids. There may also be some discharge from the infected eye. The infection progresses slowly with increasing eye pain, blurred vision, and photophobia. Repeated infections result in scarring of the inner eyelid that may then turn the eyelid in toward the eye ( entropion ). As the inner eyelid continues to turn in, the eyelashes follow ( trichiasis ), resulting in rubbing and scratching of the cornea. The combined effects of the mechanical damage to the cornea and inflammation result in ulceration, scarring, and loss of vision.

14. Diseases caused by- C. trachomatis - 2. LGV Lymphogranuloma venereum (LGV) is a sexually transmitted disease rarely identified in North America but relatively frequent in Africa, Asia, and South America. It is reemerging in Europe, especially in homosexual males. C. trachomatis serovars L1, L2, L2b, and L3 are invasive, causing LGV, which leave the mucosa to spread to the regional lymph nodes. The disease is characterized by a brief appearance of a primary genital lesion at the initial infection site . This lesion is often small and may be unrecognized, especially by female patients. The second stage, acute lymphadenitis , often involves the inguinal lymph nodes, causing them to enlarge and become matted together, forming a large area of groin swelling, or bubo. During this stage, infection may become systemic and cause fever or may spread locally, causing granulomatous proctitis . In a few patients (more females than males), the disease progresses to a chronic third stage, causing the development of genital hyperplasia, rectal fistulas, rectal stricture, draining sinuses , and other manifestations.

15. Diseases caused by- C. trachomatis - 3. Nongonococcal and Postgonococcal Urethritis Nongonococcal urethritis (NGU) is a diagnosis of exclusion that is applied to men with symptoms and/or signs of urethritis who do not have gonorrhea. Postgonococcal urethritis (PGU) refers to NGU developing in men 2–3 weeks after treatment of gonococcal urethritis with single doses of agents such as penicillins or cephalosporins that lack antimicrobial activity against chlamydiae . Since current treatment regimens for gonorrhea include tetracycline, doxycycline , or azithromycin for possible concomitant chlamydial infection, both the incidence of PGU and the causative role of chlamydiae in this syndrome have declined. C. trachomatis causes 20–40% of cases of NGU in heterosexual men but is less commonly isolated from homosexual men with this syndrome. The cause of most of the remaining cases of NGU is uncertain; considerable evidence suggests that Ureaplasma urealyticum and Mycoplasma genitalium cause many cases of NGU, whereas Trichomonas vaginalis and herpes simplex virus (HSV) cause some cases.

16. Diseases caused by- C. trachomatis - 3. Nongonococcal and Postgonococcal Urethritis NGU is diagnosed by documentation of a leukocytic urethral exudate and by exclusion of gonorrhea by Gram’s staining or culture. C. trachomatis urethritis is generally less severe than gonococcal urethritis , although in an individual patient, these two forms of urethritis cannotbe reliably differentiated solely on clinical grounds. Symptoms include urethral discharge (often whitish and mucoid rather than frankly purulent), dysuria , and urethral itching. Physical examination may reveal meatal erythema and tenderness and a urethral exudate that is often demonstrable only by stripping of the urethra.

17. Diseases caused by- C. trachomatis - 3. Nongonococcal and Postgonococcal Urethritis Such patients generally have first-glass pyuria (≥15 leukocytes per 400× microscopic field in the sediment of first-void urine), a positive leukocyte esterase test, or an increased number of leukocytes on a Gram stained smear prepared from a urogenital swab inserted 1–2 cm into the anterior urethra. For the enumeration of leukocytes, the smear is first scanned at low power to identify areas of the slide containing the highest concentration of leukocytes. These areas are then examined under oil immersion (1000×). An average of four or more leukocytes in at least three of five 1000× oil-immersion fields is indicative of urethritis and correlates with the recovery of C. trachomatis .

18. Diseases caused by- C. trachomatis - 4. Epididymitis C. trachomatis is the foremost cause of epididymitis in sexually active heterosexual men <35 years of age, accounting for ∼70% of cases. N. gonorrhoeae causes most of the remaining cases, and some men have simultaneous infections with both pathogens, usually accompanied by asymptomatic urethritis . Men with chlamydial epididymitis typically present with unilateral scrotal pain, fever, and epididymal tenderness or swelling on examination. The illness may be mild enough to treat with oral antibiotics on an outpatient basis or severe enough to require hospitalization and parenteral therapy.

19. Diseases caused by- C. trachomatis - 5. Reiter’s Syndrome Reiter’s syndrome consists of- conjunctivitis, urethritis (or, in female patients, cervicitis ), arthritis, and characteristic mucocutaneous lesions. C. trachomatis has been recovered from the urethra of up to 70% of men with untreated nondiarrheal Reiter’s syndrome and associated urethritis . In the absence of overt urethritis , it is important to exclude subclinical urethritis in the men in whom this diagnosis is suspected. More than 80% of affected patients have the HLA-B27 phenotype.

20. Diseases caused by- C. trachomatis - 5. Reiter’s Syndrome Chlamydial infection is thought to initiate an aberrant and hyperactive immune response that produces inflammation at the involved target organs in these genetically predisposed individuals. Evidence of exaggerated cell-mediated and humoral immune responses to chlamydial antigens in Reiter’s syndrome supports this hypothesis. The presumptive demonstration of chlamydial EBs and chlamydial DNA in the joint fluid and synovial tissue of patients with Reiter’s syndrome suggests that chlamydiae may actually spread from genital to joint tissues in these patients, perhaps in macrophages.

21. Diseases caused by- C. trachomatis - 6. Proctitis C. trachomatis strains of either the genital immunotypes D through K or the LGV immunotypes cause proctitis in homosexual men who practice receptive anorectal intercourse. These infections may develop in heterosexual women as well. Patients present with mild rectal pain,mucous discharge, tenesmus , and (occasionally) bleeding. Nearly all have neutrophils in Gram-stained rectal samples. Anoscopy in these non-LGV cases of chlamydial proctitis reveals mild, patchy mucosal friability and mucopurulent discharge, and the disease process is limited to the distal rectum. LGV strains produce more severe ulcerative proctitis or proctocolitis that can be confused clinically with HSV proctitis (severe rectal pain, rectal bleeding, discharge, and tenesmus ) and that histologically resembles Crohn’s disease in that giant cell formation and granulomas can be seen. In the United States, these cases of LGV proctitis occur almost exclusively in homosexual men, many of whom are positive for HIV.

22. Diseases caused by- C. trachomatis - 7. Mucopurulent Cervicitis Although many women with C. trachomatis infection of the cervix have no symptoms or signs, a careful speculum examination reveals evidence of MPC in 30–50% of cases. MPC is associated with yellow mucopurulent endocervical discharge and with ≥20 neutrophils per 1000× microscopic field within strands of cervical mucus on a thinly smeared, Gram-stained preparation of endocervical exudate . Other characteristic findings include edema of the zone of cervical ectopy and a propensity of the mucosa to bleed on minor trauma —e.g., when specimens are collected with a swab. A Pap smear shows increased numbers of neutrophils as well as a characteristic pattern of mononuclear inflammatory cells, including plasma cells, transformed lymphocytes, and histiocytes . Cervical biopsy shows a predominantly mononuclear cell infiltrate of the sub-epithelial stroma , often with follicular cervicitis .

23. Diseases caused by- C. trachomatis - 8. Pelvic Inflammatory Disease In the United States, C . trachomatis has been identified in the fallopian tubes or endometrium of up to 50% of women with pelvic inflammatory disease (PID), and its role as an important etiologic agent in this syndrome is well accepted. PID occurs via ascending intraluminal spread of C. trachomatis from the lower genital tract. MPC is thus followed by endometritis , endosalpingitis , and finally pelvic peritonitis. Chlamydial salpingitis produces milder symptoms than does gonococcal salpingitis and may be associated with less marked adnexal tenderness. Thus mild adnexal or uterine tenderness in sexually active women with cervicitis suggests PID. Infertility associated with fallopian-tube scarring has been strongly linked to antecedent C. trachomatis infection in serologic studies. Since many infertile women with tubal scarring and antichlamydial antibody have no history of PID, it appears that subclinical tubal infection (“silent salpingitis ”) may produce scarring.

24. Diseases caused by- C. trachomatis - 9. Urethral Syndrome in Women In the absence of infection with uropathogens such as coliforms or Staphylococcus saprophyticus , C. trachomatis is the pathogen most commonly isolated from college women with dysuria , frequency, and pyuria . This organism can also be isolated from the urethra of women without symptoms of urethritis , and up to 25% of female STD clinic patients with chlamydial urogenital infection have cultures positive for C. trachomatis from the urethra only.

25. Diseases caused by- C. trachomatis - 10. Infection in Pregnancy and the Neonatal Period Studies in the United States have demonstrated that 5–25% of pregnant women have C. trachomatis infections of the cervix . approximately one-half to two thirds of children exposed during birth have acquired C . trachomatis infection . Roughly half of the infected infants (25% of the group exposed) have developed clinical evidence of inclusion conjunctivitis . Pneumonia develops in 10% of children infected perinatally , and otitis media may in some cases result from perinatally acquired chlamydial infection . Neonatal chlamydial conjunctivitis has an acute onset 5–14 days after birth and often produces a profuse mucopurulent discharge .

26. Neonatal chlamydial conjunctivitis

27. Diseases caused by- C. psittaci - Psittacosis is primarily an infectious disease of birds and mammals that is caused by C. psittaci . Transmission of infection from birds to humans results in a febrile illness characterized by pneumonitis and systemic manifestations. Inapparent infections or mild influenza-like illnesses may also occur. The term ornithosis is sometimes applied to infections contracted from birds other than parrots or parakeets, but psittacosis is the preferred generic term for all forms of the disease. C. psittaci is an endemic pathogen of all bird species. Psittacine birds (e.g., parrots, parakeets) are a major reservoir for human disease, but outbreaks have occurred among turkey processing workers and pigeon aficionados.

28. Diseases caused by- C. psittaci - The birds may show diarrheal illness or may be asymptomatic. Humans acquire the disease by inhalation of aerosols. The organisms are deposited in the alveoli; some are ingested by alveolar macrophages and then carried to regional lymph nodes. From there they are disseminated systemically, growing within cells of the reticuloendothelial system. Human-to human transmission is rare, thus obviating the need for isolating patients if admitted to the hospital.

29. Psittacosis- Clinical Manifestations The clinical manifestations and course of psittacosis are extremely variable. After an incubation period of 7–14 days or longer, the disease may start abruptly with shaking chills and fever, with temperatures ranging as high as 40.5°C (105°F); however, the onset is often gradual, with fever increasing over 3–4 days. Headache is almost always prominent, is usually diffuse and excruciating, and is often the chief complaint. Many patients present with a dry hacking cough that is usually nonproductive, but small amounts of mucoid or bloody sputum may be raised as the disease progresses.

30. Psittacosis- Clinical Manifestations Cough may begin early in the course of the disease or as late as 5 days after the onset of fever. Chest pain, pleurisy with effusion, or a friction rub may all occur but are rare. Pericarditis and myocarditis have been reported. Most patients have a normal or slightly increased respiratory rate; marked dyspnea with cyanosis occurs only in severe psittacosis with extensive pulmonary involvement. In psittacosis, as in mycoplasmal pneumonias, the physical signs of pneumonitis tend to be less prominent than symptoms and x-ray findings would suggest.

31. Psittacosis- Clinical Manifestations Patients often report generalized myalgia , and spasm and stiffness of the muscles of the back and neck may lead to an erroneous diagnosis of meningitis. Lethargy, mental depression, agitation, insomnia, and disorientation have been prominent features of the illness in some epidemics but not in others; delirium and stupor develop near the end of the first week in severe cases. Occasional patients are comatose when first seen; the diagnosis of psittacosis may be elusive in these cases.

32. Psittacosis- Clinical Manifestations Gastrointestinal manifestations such as abdominal pain, nausea, vomiting, or diarrhea are noted in some cases; constipation and abdominal distention sometimes occur as late complications. Icterus , the result of severe hepatic involvement, is a rare and ominous finding. A faint macular rash ( Horder’s spots) resembling the rose spots of typhoid fever has been described.

33. Diseases caused by- C . pneumoniae - The clinical spectrum of C. pneumoniae infection includes acute pharyngitis , sinusitis, bronchitis, and pneumonitis , primarily in young adults. The pneumonitis resembles that of Mycoplasma pneumonia in that leukocytosis is frequently lacking and patients often have prominent antecedent upper respiratory tract symptoms, fever, nonproductive cough, mild to moderate illness, minimal findings on chest auscultation, and small segmental infiltrates on chest x-ray. In elderly patients, pneumonia due to C. pneumoniae can be especially severe and may necessitate hospitalization and respiratory support. It causes 5% to 10% of cases of community-acquired pneumonia.

34. Laboratory Diagnosis- C.trachomatis Specimen Collection and Transport- The organism can be recovered from or detected in infected cells of the urethra, cervix, conjunctiva, nasopharynx , rectum, and material aspirated from the fallopian tubes and epididymis . The endocervix is the preferred anatomic site to collect screening specimens from female patients. The specimen for C. trachomatis culture should be obtained after collection of all other specimens (e.g., those for Gramstained smear, Neisseria gonorrhoeae culture, or Papanicolaou [Pap] smear). A large swab should first be used to remove all secretions from the cervix. The appropriate swab (for nonculture tests, use the swab supplied or specified by the manufacturer) or endocervical brush is inserted 1 to 2 cm into the endocervical canal, rotated against the wall for 10 to 30 seconds, withdrawn without touching any vaginal surfaces, and then placed in the appropriate transport medium or applied to a slide prepared for direct fluorescent antibody (DFA) testing.

35. Laboratory Diagnosis- C.trachomatis Urethral specimens should not be collected until 2 hours after the patient has voided. A urogenital swab (or one provided or specified by the manufacturer) is gently inserted into the urethra (females, 1-2 cm; males, 2-4 cm), rotated at least once for 5 seconds, and then withdrawn. Again, swabs should be placed into the appropriate transport medium or onto a slide prepared for DFA testing. Screening of rectal or pharyngeal specimens for C. trachomatis by nucleic acid tests has proven useful in homosexual male patients. Because chlamydiae are relatively labile, viability can be maintained by keeping specimens cold and minimizing transport time to the laboratory. For successful culture, specimens should be submitted in a chlamydial transport medium such as 2SP (0.2 M sucrose-phosphate transport medium with antibiotics. Specimens should be refrigerated upon receipt, and if they cannot be processed for culture within 24 hours, they should be frozen at –70°C.

36. Laboratory Diagnosis- C.trachomatis Culture- Several different cell lines have been used to isolate C. trachomatis in cell culture, including McCoy, HeLa , and monkey kidney cells; cycloheximide -treated McCoy cells are commonly used. After shaking the clinical specimens with 5-mm glass beads, centrifugation of the specimen onto the cell monolayer (usually growing on a coverslip in the bottom of a vial, commonly called a “shell vial”) facilitates adherence of elementary bodies. After 48 to 72 hours of incubation, monolayers are stained with a fluorescein labeled monoclonal antibody that is either species specific, targeting the MOMP of C. trachomatis , or genus specific, targeting the LPS. The monolayers are examined microscopically for inclusion.

37. Laboratory Diagnosis- C.trachomatis Although its specificity approaches 100%, the sensitivity of culture has been estimated at between 70% and 90% in experienced laboratories. Limitations of Chlamydia culture contributing to the lack of sensitivity include prerequisites to maintain viability of patient specimens by either rapid or frozen transport and to ensure the quality of the specimen submitted for testing (i.e., endocervical specimens devoid of mucus and containing endocervical epithelial or metaplastic cells or urethral epithelial cells). In addition, successful culture requires a sensitive cell culture system and a minimum of at least 2 days turnaround time between specimen receipt and the availability of results. Despite these limitations, culture is still recommended as the test of choice in some situations

38. C. trachomatis DFA

39. Laboratory Diagnosis- C.trachomatis Direct Detection Methods- Cytological Examination Cytologic examination of cell scrapings from the conjunctiva of newborns or persons with ocular trachoma can be used to detect C. trachomatis inclusions, usually after Giemsa staining. Cytology has also been used to evaluate endocervical and urethral scrapings, including those obtained for Pap smears. However, this method is insensitive compared with culture or other methods

40. Laboratory Diagnosis- C.trachomatis Antigen Detection and Nucleic Acid Hybridization- The sensitivity and specificity of DFA are similar to those of culture. Chlamydial antigen can also be detected by enzyme immunoassays (EIA). Numerous U.S. Food and Drug Administration (FDA)–approved kits are commercially available. These assays use polyclonal or monoclonal antibodies that detect chlamydial LPS. These tests are not species-specific for C. trachomatis and may cross react with LPS of other bacterial species present in the vagina or urinary tract and thereby produce a false-positive result.

41. Epithelial cells of conjunctiva containing intra- cytoplasmic inclusions "draped" around nucleus

42. Laboratory Diagnosis- C.trachomatis Nucleic acid hybridization tests for Chlamydia were first available for the clinical microbiology laboratory in the late 1980s. The Digene Hybrid Capture II assay uses a ribonucleic acid (RNA) probe to detect chlamydial deoxyribonucleic acid (DNA) in a sample. The DNA/RNA hybrids are captured using monoclonal antibodies imbedded on the side of the well that recognize the unique structure produced by the DNA/RNA hybrid. A second enzyme labeled anti-DNA/ RNA hybrid antibody binds to captured hybrids, and enzyme activity is measured by chemiluminescence . The assay is species specific for the detection of C. trachomatis .

43. Laboratory Diagnosis- C.trachomatis FDA-approved nucleic acid amplification tests (NAATs) for the laboratory diagnosis of C. trachomatis infection use three different formats: polymerase chain reaction (PCR), strand displacement amplification (SDA), and transcription-mediated amplification (TMA). The first two assay formats amplify DNA sequences present in the cryptic plasmid that is present in 7 to 10 copies in the chlamydial EB,

44. Laboratory Diagnosis- C.trachomatis whereas the last format amplifies 23S ribosomal RNA (rRNA) sequences. Studies clearly indicate that NAATs are more sensitive than culture and other non–nucleic acid amplification assays. Because of the increased sensitivity of detection, first-voided urine specimens from symptomatic and asymptomatic males and females are acceptable specimens to detect C. trachomatis , thereby affording a noninvasive means of chlamydia testing. NAATs are the preferred methodology for detecting C. trachomatis in most clinical stuations .

45. Psittacosis- Laboratory Diagnosis Diagnosis of psittacosis is almost always by serologic means. Because of hazards associated with working with the agent, only laboratories with Biosafety Level 3 biohazard containment facilities can culture C. psittaci safely. State health departments take an active role in consulting with clinicians about possible cases. Complement fixation and indirect micro- immunofluorescence have been used to detect anti– C. psittaci antibodies in patients with suspected psittacosis infections. Either a fourfold rise in titer between acute and convalescent serum samples or a single IgM titer of 1:32 or greater in a patient with an appropriate illness is considered diagnostic of an infection. Finally, amplification of rRNA sequences using a PCR assay followed by restriction fragment length polymorphism (RFLP) analysis was able to identify and distinguish all nine chlamydial species, including C. psittaci .

46. C. pneumoniae - Laboratory Diagnosis In the laboratory, C. pneumoniae infections are diagnosed by cell culture, serology, or NAATs. Direct Detection Methods To date, assays to directly detect C. pneumoniae antigens have poor sensitivity. A variety of NAAT, including conventional and real-time PCR assays, have been developed to detect C. pneumoniae nucleic acid sequences in clinical specimens. Several of these amplification assays are commercially available. Using these methods, the organism has been detected in throat swabs and other specimens, such as nasopharyngeal, bronchoalveolar lavage fluids, and sputum.

47. C. pneumoniae - Laboratory Diagnosis Cultivation Specimens for isolation are usually swabs of the oropharynx ; techniques for isolation of the organism from sputum are unsatisfactory. Swabs should be placed into chlamydial transport media, transported on ice, and stored at 4°C; organisms are rapidly inactivated at room temperature or by rapid freezing or thawing. A cell culture procedure similar to that used for C. trachomatis but using the more sensitive HL or Hep-2 cell lines must be substituted for McCoy cells.

48. Treatment- C. trachomatis A 2-week course is recommended for complicated chlamydial infections (e.g ., PID, epididymitis ) and at least a 3-week course of doxycycline (100 mg orally bid) or erythromycin base (500 mg orally qid) for LGV . C . trachomatis is eradicated from the urethra in nearly all cases by treatment with tetracycline hydrochloride (500 mg qid for 7 days) or doxycycline (100 mg by mouth bid for 7 days ). Azithromycin (a single oral 1-g dose) is the regimen of choice for pregnant women with C . trachomatis infection. However , amoxicillin (500 mg tid for 7 days) can also be given to pregnant women . Tetracycline hydrochloride (500 mg qid ) or doxycycline (100 mg bid) for 14 days produces clinical and microbiologic cure of epididymitis and PID associated with C . trachomatis infection, but in this situation a tetracycline should always be used together with a drug that is highly effective against gonorrhea . Inclusion Conjuctivitis-azithromycin -(a 1-g single oral dose) or doxycycline (100 mg bid for 7 days).

49. Treatment- C. psittaci The tetracyclines are consistently effective in the treatment of psittacosis. Defervescence and alleviation of symptoms usually take place within 24–48 h after the institution of therapy with 2 g daily in four divided doses. To avoid relapse, treatment should probably be continued for at least 7–14 days after defervescence . Doxycycline (100 mg by mouth bid) can also be used.

50. Treatment- C. pneumoniae Recommended therapy consists of 2 g/d of either tetracycline or erythromycin for 10–14 days . Other macrolides (e.g., azithromycin ) and some fluoroquinolones (e.g., levofloxacin and gatifloxacin ) also appear to be effective.

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