General Characteristics

C. trachomatis infects humans almost exclusively and is responsible for various clinical syndromes. Based on MOMP antigenic differences, C. trachomatis is divided into 18 different serovars that are associated with different primary clinical syndromes (Table 44-2).

Epidemiology and Pathogenesis

C. trachomatis causes significant infection and disease worldwide. In the United States, C. trachomatis is the most common sexually transmitted bacterial pathogen and a major cause of pelvic inflammatory disease (PID), ectopic pregnancy, and infertility (see Chapter 74 for more information on PID). An estimated 3 million cases of C. trachomatis infection occur annually in the United States. In 2010, more than 1.3 million cases of C. trachomatis infection were reported to the Centers for Disease Control and Prevention (CDC), corresponding to a rate of infection of 426 per 100,000 population and a 5.1% increase over the cases reported in 2009. In fact, of all the organisms causing sexually transmitted disease reported to the CDC, only C. trachomatis cases have increased every year. Genital tract infections caused by C. trachomatis were identified most frequently in women between the ages of 15 and 24 years. It is important to note, however, that data reported to the CDC, especially with regards to Chlamydia, come as a result of screening programs that primarily target women between the ages of 15 and 24 years.

Ocular trachoma, on the other hand, is a much more prevalent disease, affecting 84 million individuals worldwide, with 7 to 9 million infections resulting in blindness. Remote rural areas of Africa, Asia, Central and South America, Australia, and the Middle East are hyperendemic for trachoma, where the prevalence rate of C. trachomatis is 60% to 90% in preschool children. Trachoma is the cause for 3% of the cases of blindness in individuals around the world, with adult women more likely to be affected as a result of their exposure to children who serve as the major reservoir of the organism.

C. trachomatis infections are primarily transmitted from human to human by direct contact with infected secretions. Some infections, such as neonatal pneumonia or inclusion conjunctivitis, are transmitted from mother to infant during birth. The various routes of transmission for C. trachomatis infection are summarized in Table 44-2.

The natural habitat of C. trachomatis is humans. The mechanisms by which C. trachomatis cause inflammation and tissue destruction are not completely understood. The chlamydiae can infect a variety of different cells, including epithelial cells of the mucosa as well as blood vessels, smooth muscle cells, and monocytes. The chlamydial EB is phagocytosed into a host cell and resides in a vacuole that fails to fuse with a lysosome, leading to the intracellular persistence of the organism and escape from the host immune response. Chlamydiae are able to either turn on or turn off apoptosis (programmed cell death pathways) in infected host cells. By inducing host cell death, the organism facilitates its transmission to neighboring host cells and down-regulating inflammation in the acute disease process, whereas, by inhibiting apoptosis, the organism keeps the host cell alive, allowing for sustained survival in chronic infections.

The host’s immune response accounts for the majority of the tissue destruction following infection with C. trachomatis. Infected epithelial cells secrete pro-inflammatory cytokines including Interleukin-1α (IL-1α), tumor necrosis factor (TNF) and IL-6. Quickly upon infection, neutrophils and monocytes migrate to the mucosa and eliminate exposed EB. Later CD4 T helper cells migrate to the site of infection. Responding neutrophils and T helper cells release cytokines, resulting in the influx of additional immune cells. The importance of multiple, recurrent infection with C. trachomatis is associated with the development of ocular trachoma. Immunity provides little protection from reinfection and appears to be short lived following infection with C. trachomatis.

Spectrum of Disease

As previously mentioned, infection with different C. trachomatis serovars can lead to several clinical syndromes. These infections are summarized in Table 44-2.

Perinatal Infections

Approximately one fourth to half of infants born to women infected with C. trachomatis develop inclusion conjunctivitis. Usually, the incubation period is 5 to 12 days from birth, but it may be as long as 6 weeks. Although most develop inclusion conjunctivitis, about 10% to 20% of infants develop pneumonia. Perinatal acquired C. trachomatis infection may persist in the nasopharynx, urogenital tract, or rectum for more than 2 years.

Laboratory Diagnosis

C. trachomatis can be diagnosed by cytology, culture, direct detection of antigen or nucleic acid, and serologic testing.

Direct Detection Methods

Cytologic 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 discussed in the following sections.

Antigen Detection and Nucleic Acid Hybridization.

To circumvent the shortcomings of cell culture, antigen detection methods are commercially available.

Direct fluorescent antibody (DFA) staining methods use fluorescein-isothiocyanate conjugated monoclonal antibodies to either MOMP or LPS of C. trachomatis to detect elementary bodies in smears of clinical material (Figure 44-2). 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.

Nucleic acid hybridization tests for Chlamydia were first available for the clinical microbiology laboratory in the late 1980s. Two hybridization tests are currently available, Gen-Probe PACE 2C (Hologic-Gen-Probe, San Diego, California) and Digene Hybrid Capture II assay (Digene, Silver Spring, Maryland). The Gen-Probe PACE 2C assay uses a chemiluminescent-labeled DNA probe complementary to a sequence of ribosomal RNA (rRNA) in the chlamydial genome. If chlamydial rRNA is present in the sample, the labeled DNA probe will bind. In a unique hybridization protection assay, only bound label is detected by measuring chemiluminescence in a luminometer. The Digene Hybrid Capture II assay uses an RNA probe to detect chlamydial 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. Both assays are species specific for C. trachomatis.

Based on numerous studies, these nonculture tests are more reliable for the detection of infection in patients who are symptomatic and shedding large numbers of organisms than in those who are asymptomatic and most likely shedding fewer organisms. For the most part, these assays have sensitivities of greater than 70% and specificities of 97% to 99% in populations with a prevalence of C. trachomatis infection of 5% or more. In a low-prevalence population—that is, less than 5%—a significant proportion of positive tests will be falsely positive. Therefore, a positive result in a low-prevalence population should be handled with care, and a positive result should be verified. Positive results can be validated by the following methods:

• Culture

• Performing a second nonculture test that identifies a C. trachomatis antigen or nucleic acid sequence that is different from that used in the screening test

• Using a blocking antibody or competitive probe that verifies a positive test result by preventing attachment of a labeled antibody or probe used in the standard assay

Nucleic Acid Amplification Tests.

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, whereas the last format amplifies 23S ribosomal RNA 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 men and women 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 situations because of increased sensitivity, ease of specimen collection, and the availability of automated high volume methods. Table 44-3 summarizes the possible uses of the different methodologies available for the detection of C. trachomatis; however, NAATs are used almost exclusively for the laboratory detection of C. trachomatis.

Antibiotic Susceptibility Testing and Therapy

Because C. trachomatis is an obligate intracellular bacterium, susceptibility testing is not practical in the routine clinical microbiology laboratory setting and is performed in only a few laboratories. In addition no standardized in vitro assay or an understanding of the relationship between in vitro test results and clinical outcome following treatment exist. Antibiotics typically used in infections with C. trachomatis include erythromycin and other macrolide antibiotics, tetracyclines, and fluoroquinolones.

Prevention

Because no effective vaccines are available, strategies to prevent chlamydial urogenital infections focus on trying to manifest behavioral changes. By identifying and treating persons with genital chlamydia before infection is transmitted to sexual partners or from pregnant women to babies, the risk of acquiring or transmitting infection may be significantly decreased.

General Characteristics

C. psittaci differs from C. trachomatis in that it is sulfonamide resistant and in the morphology of its EB and inclusion bodies (see Table 44-1).

Epidemiology and Pathogenesis

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. 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.

Spectrum of Disease

Disease usually begins after an incubation period of 5 to 15 days. Onset may be insidious or abrupt. Clinical findings associated with this infection are diverse and include pneumonia, severe headache, mental status changes, and hepatosplenomegaly. The severity of infection ranges from unapparent or mild disease to a life-threatening systemic illness with significant respiratory problems.

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 microimmunofluorescence 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 rDNA 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.

Antibiotic Susceptibility Testing and Therapy

Because C. psittaci is an obligate intracellular pathogen and its incidence of infection is rare, susceptibility testing is not practical in the routine clinical microbiology laboratory. Tetracycline is the drug of choice for psittacosis. If left untreated, the fatality rate is about 20%.

Prevention

Disease is prevented by treating infected birds or by quarantining imported birds for a month.

General Characteristics

C. pneumoniae is considered more homogeneous than either C. trachomatis or C. psittaci, because all isolates tested are immunologically similar because of the homogeneity of the MOMP. One significant difference between C. pneumoniae and the other chlamydiae is the pear-shaped appearance of its EB (Figure 44-3).

Epidemiology and Pathogenesis

C. pneumoniae appears to solely be a human pathogen; no bird or animal reservoirs have been identified. It is transmitted from person to person by aerosolized droplets via the respiratory route. The spread of infection is low. Antibody prevalence to C. pneumoniae starts to rise in school-aged children and reaches 30% to 45% in adolescents; more than half of adults in the United States and in other countries have C. pneumoniae antibody. Of interest, C. pneumoniae infections are both endemic and epidemic. Unfortunately, little is known about the pathogenesis of C. pneumoniae infections, but it is similar to C. trachomatis in inducing inflammation that contributes to tissue damage.

Spectrum of Disease

C. pneumoniae has been associated with pneumonia, bronchitis, pharyngitis, sinusitis, and a flulike illness. It causes 5% to 10% of cases of community-acquired pneumonia. Infection in young adults is usually mild to moderate; the microbiologic differential diagnosis primarily includes Mycoplasma pneumoniae. Severe pneumonia may occur in older or respiratory-compromised patients. Of note, asymptomatic infection or unrecognized, mildly symptomatic illnesses caused by C. pneumoniae are common. In addition, an association exists between C. pneumoniae infection and the development of asthmatic symptoms. Finally, an association between coronary artery disease and other atherosclerotic syndromes and C. pneumoniae infection has been suggested by seroepidemiologic studies and the demonstration of the organism in atheromatous plaques (yellow deposits within arteries containing cholesterol and other lipid material). Such an etiologic role by this organism is still under intense scrutiny. An excellent and comprehensive review of the literature related to whether C. pneumoniae is a cause of atherosclerosis was published in 2008 by Watson and Alp. Their research indicated that “it is difficult to attribute causality to a common infectious agent in a highly prevalent multifactorial disease.” They also stated that “C. pneumoniae is neither alone sufficient nor is it necessary to cause atherosclerosis or its clinical consequences in humans,” but they allowed for the possibility that treatment of C. pneumoniae may reduce the risk of atherosclerosis development.

Laboratory Diagnosis

In the laboratory, C. pneumoniae infections are diagnosed by cell culture, serology, or NAATs.

Antibiotic Susceptibility Testing and Therapy

Methods for susceptibility testing of C. pneumoniae have been largely adapted from those used for C. trachomatis. Similar to C. trachomatis, susceptibility testing is not practical for the clinical microbiology laboratory and, because methods are not yet standardized, the results can be influenced by several variables. Treatment with tetracycline, doxycycline, macrolides, fluroquinolones, and erythromycin has been successful.

Prevention

Little is known regarding effective ways to prevent C. pneumoniae infections beyond avoiding aerosolized droplets from infected people.

Direct Detection Methods

Immunohistology and conventional and real-time PCR have been used to diagnose rickettsial and ehrlichial infections. Biopsy of skin tissue from the rash caused by the spotted fever group rickettsiae is the preferred specimen. Organisms are identified using polyclonal antibodies and are detected with the use of fluorescein-labeled antibodies or enzyme-labeled indirect procedures. The sensitivity of these techniques is about 70% and depends on correct tissue sampling, examination of multiple tissue levels, and biopsy before or during the first 24 hours of therapy (see Table 44-4).

Direct detection of Ehrlichia and Anaplasma from peripheral blood or cerebrospinal fluid (CSF) includes PCR amplification, direct microscopic examination of Giemsa-stained or Wright’s stained specimens, or immunocytologic or immunohistologic stains with E. chaffeensis or Anaplasma species antibodies. Direct microscopic examination of Giemsa-stained or Diff-Quik–stained peripheral blood buffy-coat smears can detect morulae (cytoplasmic vacuoles containing enriched organisms) during the febrile stage of infection in ehrlichiosis; morulae-like structures also can be observed in CSF cells and tissues. Finally, recent reports have described the development of rapid, species-specific real-time PCR assays to detect single or co-infections with Anaplasma species or Ehrlichia species in peripheral blood specimens.

Cultivation

Although the rickettsiae can be cultured in embryonated eggs and in tissue culture, the risk of laboratory-acquired infection is extremely high, limiting the availability of culture to a few specialized laboratories. Blood should be collected as early as possible in the course of disease in a sterile, heparin-containing vial. Similarly, punch biopsies of skin or eschars (slough or dead skin) are also acceptable but must be collected early in the course of disease. These same specimens are also acceptable for PCR.

To date, culture of Ehrlichia and Anaplasma is limited and culture conditions are still being optimized. Currently, the preferred specimen for culture is peripheral blood obtained in a sterile, EDTA- or acid-citrate-dextrose-anti-coagulated blood tube; if specimens must be moved, they should be transported overnight at approximately 4° C.

Serodiagnosis

Although it is not fast, the diagnosis of rickettsial disease and ehrlichiosis is primarily accomplished serologically. Serologic assays for the diagnosis of rickettsial infections include the indirect immunofluorescence assay (IFA), enzyme immunoassay (EIA), Proteus vulgaris OX-19 and OX-2 and Proteus mirabilis OX-K strain agglutination (the Weil-Felix reaction), line blot, and Western immunoblotting. The Weil-Felix reaction (see Procedure 44-1 on the Evolve site), the fortuitous agglutination of certain strains of P. vulgaris by serum from patients with rickettsial disease, may still be performed in developing countries, but because false-positive and false-negative tests are a continuing problem, these tests have been replaced by more accurate serologic methods such as IFA.

Except for latex agglutination, IFA, and DFA testing for diagnosing Rocky Mountain spotted fever, none of the serologic tests is useful for diagnosing disease in time to influence therapy. This lack of utility for serology is because antibodies to rickettsiae other than R. rickettsii cannot be reliably detected until at least 2 weeks after the patient has become ill. With newer immunologic recombinant reagents under development, the potential exists for new tests for all the rickettsial diseases.

To date, the sensitivity and specificity of serologic assays for ehrlichiosis is unknown but is presumed to be relatively high; indirect immunofluorescent antibody testing is available for E. chaffeensis or A. phagocytophilum. A fourfold or greater rise in antibody titer during the course of disease is considered significant.