The Laboratory Diagnosis of Chlamydia Infections

Abstract

Bacteria of the genus Chlamydia belong to the order Chlamydiales, within the family Chlamydiaceae. These intracellular parasites have a different biphasic reproductive cycle than other bacteria. The important Chlamydiaceae are Chlamydia trachomatis, Chlamydophila pneumoniae and Chlamydophila psittaci. Chlamydia trachomatis and Clamydophila pneumoniae are primary human pathogens. Chlamydia trachomatis is transmitted by sexual contact. It is the causative agent of LGV (lymphogranuloma venoreum) and ocular trachoma in humans. Chlamydophila pneumoniae causes bronchitis, atypical pneumonia, sinusitis, pharyngitis, and inflammatory atherosclerosis. Chlamydia psittaci is the causative agent of psittacosis (pneumonia). It primarily causes infection in birds and domestic animals, and sometimes in humans. Chlamydia trachomatis laboratory diagnosis is based on cytological examination (Giemsa), antigen detection (with enzyme-linked immunosorbent assay and direct immunofluorescence staining), nucleic acid-based tests (nucleic acid probe tests and nucleic acid amplification tests—NAAT), cell culture (in vivo and in vitro), and detection of antibodies (especially microimmunofluorescence—MIF and enzyme immunoassay—EIA, for the diagnosis of LGV). The most specific test in diagnosis is cell culture, and the most sensitive is nucleic acid-based test. NAAT and MIF tests are successful in the diagnosis of C. pneumoniae infections. The diagnosis of psittacosis is usually made by serological testing, and species-specific MIF testing should be performed to confirm.

1. Introduction

Chlamydiae are nonmotile, gram-negative, cocci [1]. They are obligate intracellular microorganisms with a biphasic life cycle. They can only grow in live cell cultures [2]. Chlamydias belong to the Chlamydiaceae family, from the order Chlamydiales. Species that cause disease in humans are Chlamydia trachomatis, Chlamydophila psittaci, and Clamydophila pneumoniae [3, 4]. Among them, C. trachomatis is among the most common sexually transmitted diseases (STIs) in the world today [5]. According to the World Health Organization (WHO) data, around 90 million new cases of chlamydial infections occur worldwide every year [6]. Chlamydia trachomatis has been included in the group D notifiable diseases as a sexually transmitted disease agent in our country since 2005 [7].

There are many diagnostic tests used in the diagnosis of sexually transmitted diseases. Laboratory tests are critical because most of the patients are asymptomatic [8]. Developing countries need cost-effective, fast, reliable, sensitive, and specific laboratory services. The high sensitivity of the tests is important in preventing transmission and complications, directing treatment, and controlling infection [9]. Chlamydia testing is indicated for patients with STIs who have urogenital, anorectal, and ocular symptoms, people who have had sexual contact with patients with STIs, and people who are screened for chlamydia [10].

2. Structure, life cycle

Chlamydia cell wall is rich in lipids like gram-negative bacteria. It does not contain typical bacterial peptidoglycan. It contains N-acetylmuramica acid. They exist in two forms in their life cycle. Elementary body (EC) is the form that is metabolically inactive, resistant to environmental conditions and infective. Reticular Body (RC) is the metabolically active, intracellular proliferative and non-infective form. Elementary bodies show a high affinity for host epithelial cells. Bacterial major outer membrane proteins (major outer membran protein-MOMP, OmcB, PmpD), on the surface of Chlamydia trachomatis interact with host cell receptors (heparan sulfate, proteoglycans, mannase-6-phosphate receptors, and growth factor such as receptors) and enter the cell rapidly. They are then taken up into the cell by receptor-mediated endocytosis or pinocytosis. Meanwhile, they are protected from the environment by inhibiting lysosome fusion. In the cell, the EC transforms into a reticular body. The reticular bodies are larger in size and multiply by dividing each into two, forming an intracytoplasmic inclusion. They leave the cell to infect new cells. This cycle takes 24–48 h [10].

3. Staining properties

Chlamydia has different staining characteristics. Elementary bodies are stained purple on blue-stained host cell cytoplasm with Giemsa stain. Reticular bodies are stained blue. Chlamydia stains with gram-negative or variable staining. Therefore, the gram stain is useless in identification. Chlamydia particles and inclusions (with group-specific, species-specific, or serovar-specific antibodies) are stained bright yellow-green by the immunofluorescence dye method. In culture, McCoy cells are stained with iodide when growth occurs. C. trachomatis appears with brown-stained inclusions on a yellow background [11].

4. History

Trachoma has been described in BC China and Egypt. The role of chlamydia in genital infections was understood at the beginning of the twentieth century. Inclusion bodies were first demonstrated in 1909 in the conjunctival cells of infants with non-gonococcal ophthalmia neonatorum, in the cervical epithelial cells of their mothers, and in the urethral epithelial cells of male patients. In the samples, they showed intracytoplasmic inclusion bodies with the Giemsa method. Thinking they were protozoa, they named them Chlamydiazoae. In Greek, “chlamys” means a curtain that covers the environment and describes the inclusions that surround the cell nucleus [12]. Lymphogranuloma venereum was first described by scientists in 1913. An epidemic was studied in Switzerland in 1879 and was named pneumotyphus. Later, in a study on parrots in 1892, the term psittacosis was used. The term psittacosis comes from the Greek “parrot.” The psittacosis agent, C. psittaci, was first produced in fertilized chicken eggs in 1935 and in cell culture in 1941 [4, 12].

Chlamydia pneumoniae was first isolated from a conjunctival swab of a child participating in the trachoma vaccine trial in Taiwan. It was named TW-183. The agent AR-39, isolated from the throat swab of a student with pharyngitis at Seattle University in 1983, was named. The name TWAR (TW + AR) was formed from the first conjunctival strain and the respiratory strain. C. pneumoniae strain was defined by morphological examinations and DNA sequence analysis performed in 1989 [4, 12].

5. Classification

The genus Chlamydia has been described in the Chlamydiaceae family of the order Chlamydiales. Chlamydiae are classified according to their antigenic structures, sulfonamide resistance, host differences, and the diseases they cause. Characteristics of three species infecting humans have been identified:

Chlamydia trachomatis has intracytoplasmic inclusion bodies containing glycogen. They are inhibited by sulfonamides. They cause eye and genital infections in adults and conjunctivitis and pneumonia in infants.

Chlamidiae (Chlamydophila) pneumoniae have intracytoplasmic inclusion bodies that lack glycogen. It is generally resistant to sulfonamides. It causes respiratory tract infections in humans.

Chlamidiae (Chlamydophila) psittaki, contain dense intracytoplasmic inclusions devoid of glycogen. It is generally resistant to sulfonamides. It causes psittacosis in humans and ornithosis in birds [11].

6. Chlamydia trachomatis

Clinic: Chlamydia trachomatis causes trachoma, adult inclusion conjunctivitis, neonatal conjunctivitis, infant pneumonia, LGV, and urogenital infections. Infections in newborns are directly related to sexually transmitted infections [4].

It is the most common cause of nongonococcal urethritis (NGU) in the sexually active and young population [13]. Although its distribution varies according to region, its prevalence is 20–50% [14]. Chlamydia trachomatis is an exclusively sexually transmitted pathogen and has been more isolated in developing countries. Clinically, it usually causes cervicitis in women, and epididymitis and infertility in men. It should be noted that it can be asymptomatic in both men and women. Azithromycin should be considered in the treatment [15].

7. Diseases by serotype

Chlamydia trachomatis, has 15 serotypes due to antigenic variations in MOMP encoded by ompA [16].

1. A, B, Ba, C serotypes: It is the cause of endemic trachoma. Hand-eye contact is transmitted by flies.

2. D, E, F, G, H, I, J, K serotypes: Causing inclusion conjunctivitis, nongonococcal urethritis, cervicitis, salpingitis, proctitis, epididymitis, neonatal pneumonia, and conjunctivitis. Hand-eye contact, sexually and perinatally transmitted.

3. L1, L2, L3 serotypes: Sexually transmitted L1, L2, L3 serotypes cause LGV [7].

8. Trachoma

Clinic: The incubation period of trachoma is 3–10 days. It is a chronic disease that starts in early childhood and progresses insidiously. Chlamydia trachomatis serovars A, B, Ba, and C are associated with trachoma. It often accompanies a bacterial conjunctivitis and composes the clinical picture together. Trachoma is chronic keratoconjunctivitis involving the conjunctiva and cornea. At the onset of the disease, lacrimation, mucopurulent discharge, and conjunctival redness are the first findings [11]. It is characterized by advanced ocular signs of trachoma, lymphocyte infiltration in the corneal epithelium, granulations in the conjunctiva, eyelid deformities, and complications that progress to blindness [2].

9. Laboratory diagnosis

Culture: Conjunctival scrapings are taken from the upper tarsal conjunctiva, where inclusion bodies are most common. Conjunctival scrapings taken are inoculated into McCoy cell cultures. Usually, McCoy cell cultures were treated with cycloheximide. The adequate number of viable infectious particles and the application of the centrifugation method increase reproduction. Diagnosis can be made by detecting inclusion bodies by one of the staining methods with fluorescently labeled antibodies against Giemsa, iodine, or chlamydia antigens (LPS or MOMP) after 48–72 hours of incubation of the first passage. Detection by staining with MOMP-specific antibodies is highly specific. The disadvantages of the culture method are the need for a long time, the high workload, and the difficulties in standardization. However, some reference laboratories require a culture method to monitor antibiotic susceptibility or when a highly specific test, such as suspected sexual assault, is required [10, 11]. If cultures are to be cultivated from the samples within 24 hours, they can be kept at +4°C. If it is to be kept for a longer period of time, it should be stored frozen at −70°C. The specificity of the culture is 100% and the sensitivity is below 100% under appropriate conditions. In legal cases, the infectious agent should be determined by culture [17].

Morphological detection of inclusions: In the presence of a large number of chlamydial inclusion bodies, a preliminary diagnosis can be made if the preparations are stained with the Giemsa or Gimenez methods. Inclusions are found in the cytoplasm of epithelial cells, usually in the perinuclear space. The incidence of inclusions in preparations prepared from specimens is the highest in neonatal conjunctivitis, lower in inclusion conjunctivitis and trachoma in adults, and rare in urethritis and cervicitis. Detection of inclusions is a rarely used method in diagnosis. Difficulty in the application, low sensitivity, and specificity are the reasons for not using it [18].

Serology: Among the diagnostic methods other than culture, immunofluorescence and enzyme-based immunological methods (enzyme immunoassay, EIA) are the main ones [18]. Both group antibodies and serovar-specific antibodies are frequently found in the serum and eye secretions of infected patients. Antibodies formed against the cell wall structure of the organism are detected. The microimmunofluorescence method is a sensitive method for measuring antichlamydial antibodies [11].

Molecular methods: Polymerase chain reaction (PCR) and other molecular diagnostic methods are not widely used in the diagnosis of trachoma. In developing countries where trachoma is endemic, there are insufficient resources to perform PCR or other molecular tests. In developed countries, which have the opportunity to apply the tests, trachoma is rare and tests are not needed. These methods are generally used in research studies on trachoma [11].

Treatment and prevention: The primary source in endemic areas is children with ocular infections. Transmission occurs between infected children and their caregivers by hand-eye contact and by the feet of black flies. Hygiene rules such as facial cleaning and reducing flying insects are important in protection [11].

Azithromycin is used in the mass treatment of endemic trachoma. After 6–12 months of treatment, clinical manifestations are greatly reduced. It has replaced erythromycin and doxycycline. Topical therapy is of little value [19].

The World Health Organization initiated the S-A-F-E program to eliminate or reduce trachoma (Surgery-Azithromycin-Face-Environmental). It is a program that covers the treatment of trachoma with surgery and azithromycin, facial cleaning, and environmental cleaning by reducing black flies [11].

10. Chlamydia trachomatis genital infections and inclusion conjunctivitis

Clinic: Chlamydia trachomatis D-K serovars cause sexually transmitted diseases and eye infections (conjunctivitis with inclusion). Inclusion conjunctivitis in adults is follicular conjunctivitis involving the lower eyelid. In the first two weeks, there are purulent-mucoid discharge and signs of hyperemia in the eye. It is accompanied by keratitis, indistinguishable from ocular trachoma. It usually heals spontaneously. However, if left untreated, it may become chronic and deformities may develop [20].

In newborns, 20–60% are infected as they pass through the birth canal. Conjunctivitis with inclusions (15–20%) and respiratory system infections (10–40%) may develop in infected infants. Neonatal inclusion conjunctivitis begins 7–12 days after birth as mucopurulent conjunctivitis. It tends to improve within weeks to months, either spontaneously or with erythromycin or tetracycline therapy, rarely can become chronic [11].

Most chlamydial endocervical infections are asymptomatic or have mild symptoms such as dysuria, mild abdominal pain, bleeding, and vaginal discharge. Mucopurulent discharge is seen in cervicitis. In women, it causes urethritis, cervicitis, endometritis, pelvic inflammatory disease, and its complications. Ectopic pregnancy, spontaneous abortion, tubal infertility, and chronic pelvic pain are the main complications. Pelvic inflammatory disease can progress to a widespread disease that progresses to perihepatitis and ascites [21].

In men, it causes nongonococcal urethritis and epididymitis. It is the most common cause of nongonococcal urethritis, up to 50% in men and women [22]. The incubation period is 7–14 days. It has clinical signs such as dysuria, non-purulent discharge, and frequent urination [23]. Infected adults infect their conjunctiva by autoinoculation and inclusion conjunctivitis resembling acute trachoma may develop. The most common cause of epididymitis in men over 35 years of age is Escherichiae coli, and those under 35 years of age are C. tachomatis and Neisseria gonorrhoeae. Azoospermia can be seen in the acute phase in the chlamydial epididymitis. It is not known whether it causes infertility or not. It is the most common cause of proctitis and proctocolitis in both men and women, more often in gay men [24]. Patients with proctocolitis have fever, tenesmus, and rectal pain. Asymptomatic rectal carriage can occur in neonates and adults [11, 12]. However, since infections are often asymptomatic, diagnosis cannot be made in most cases [6]. Chlamydial infections trigger reactive arthritis and some of them develop Reiter’s Syndrome. Reiter’s Syndrome is a disease with urethritis, conjunctivitis, arthritis, mucocutaneous lesions [5, 6]. In addition, chlamydial infections can cause cancerous lesions such as cervical dysplasia [4, 25].

11. Laboratory diagnosis

Taking samples: When taking samples, dacron, rayon (artificial silk), cotton, or calcium alginate swabs with plastic handles should be used. Wooden-handled swabs are toxic to chlamydiae. Because chlamydiae are obligate intracellular bacteria, samples should contain both extracellular material and infected human cells. While collecting samples of the vagina, urethra, endocervix, and conjunctiva, firstly the discharge and secretions are cleaned and then the epithelial cells are scraped with the help of a swab. Samples taken for culture should be placed in chlamydia transport media. It should be kept in the refrigerator until it goes to the laboratory. Urine can be tested to detect the presence of chlamydia. The first 20 ml urine sample can be used for the detection of chlamydia nucleic acid. It may not be detected as it may be diluted in subsequent urine.

12. Molecular methods

Probe tests: There are commercially available hybridization-based tests to detect Chlamydia trachomatis directly from clinical specimens. In these tests, a single-stranded DNA probe labeled with a chemiluminescent substance that is complementary to the ribosomal 16sRNA of the target organism can be used. The sensitivity and specificity of this test are good [11].

Nucleic acid amplification test: These tests use molecular methods such as polymerase chain reaction (PCR), chain removal, and transcription-based amplification. NAATs have very high specificity like culture, but differently, it is not dependent on the viability of the causative microorganism and so sample transfer is easier. Today, the results have been accelerated by the use of fluorescent labeled probes and automatic nucleic acid extraction. The use of two target gene regions in NAATs enabled the detection of new variants. Bead-based extraction systems further increased the specificity and sensitivity of the test. Therefore, NAATs have become the gold standard in the diagnosis of C. trachomatis infections [10]. For these tests, first urine, vagina, cervix, and urethra swab samples are suitable. Approval studies are ongoing for non-genital (conjunctiva, oropharynx, and rectum) specimens [11]. There are also multiplex real-time PCR tests where other sexually transmitted infectious agents (Mycoplasma genitalium, Tricomonas vaginalis, Neisseria Gonorrhoeae vb.) can be detected [24].

Drekt fluorescent antibody (DFA) tests: These tests are used in newborns, conjunctival samples. They are stained with labeled monoclonal antibodies developed against species-specific antigens on chlamydia major outer membrane protein (MOMP). Stained preparations are examined by fluorescence microscopy and samples containing fluorescent smooth-sided round or oval elementary bodies are considered positive [26].

Enzyme immunoassay (EIA): The enzyme immunoassay detects genus-specific antigens in elementary bodies and is less sensitive than NAAT and is not widely used [26].

Culture: Compared to NAATs, culture is a difficult, costly, delayed, and less sensitive method. Swabs from the endocervix, anal canal, urethra, and conjunctiva are suitable specimens for culture. Swab samples are inoculated into McCoy, HeLa229, and Buffalo Green Monkey Kidney cell lines [10]. Samples are incubated at 35–37°C and 48–72 hours. A second inoculation is performed to increase sensitivity. Intra-cytoplasmic inclusions are examined by direct immunofluorescence [11].

Serology: An increase in serum antibody titers is observed in acute genital chlamydia infections. Antibodies are raised against the infecting immunotype. Serum antibodies are higher titer than trachoma. In societies with a high prevalence of genital chlamydia infection, there is a high background of antichlamydial antibodies. Therefore, serology is not useful and common in diagnosis [26].

Treatment: In chlamydia infections, simultaneous treatment of sexual partners to prevent reinfection is the basic principle. Tetracyclines (such as doxycycline) are widely used in nongonococcal urethritis and in non-pregnant infected women. Azithromycin is effective and can be used in infected pregnant women. Topical treatment is not helpful in eye infections due to chlamydia so systemic treatment is preferred. Treatment with doxycycline and erythromycin for 2–3 weeks is recommended [16].

13. Chlamydia trachomatis and newborn pneumonia

Chlamidia trachomatis can be passed from an infected mother to her baby during birth. In pregnant women, the incidence of chlamydial infection is between 2 and 24% [7]. Infants born to infected mothers may develop inclusion conjunctivitis and pneumonia in the first few weeks after birth. Infants with inclusion conjunctivitis have mucopurulent discharge and conjunctival edema. It can also be asymptomatic [5, 20]. Neonatal pneumonia begins at 3–11 weeks with nasal congestion, tachypnea, and cough. Interstitial infiltrates may be seen in the lungs. If left untreated, respiratory failure may develop. Obstructive lung diseases may be more common in these infants later in life [20].

The first choice in diagnostic methods is the isolation of C. trachomatis in respiratory secretion cultures with McCoy or other cell lines. Detection of anti-chlamydia IgM antibodies at higher levels or at titers higher than 1:32 is diagnostic.

Treatment is the same for neonatal inclusion conjunctivitis and pneumonia. Oral erythromycin is effective for 14 days. It also eliminates the carrier [7].

14. Lymphogranuloma venereum

Clinic: Lymphogranuloma venereum is a sexually transmitted disease with suppurative inguinal lymphadenitis and is common in tropical climates. The first sign of LGV, which is a systemic disease, is a painless papule that can ulcerate at the inoculation site. Later, painful and unilateral regional lymphadenopathy develops and may fistulize. In men, lymph nodes above and below the Poupart ligament are frequently involved. Involved lymph nodes become suppurative, purple, and painful over time. Adhesions may occur in the genital area. Perirectal lymph nodes are mainly involved in homosexual men and women. The disease leads to proctitis and bloody mucopurulent anal discharge. Systemic symptoms such as fever, nausea, vomiting, conjunctivitis, skin rashes, arthralgia, and muscle pains are also accompanied [6].

15. Laboratory diagnosis

Culture: Infected lymph node samples are inoculated into McCoy cell cultures. It can be treated with aminoglycoside to prevent bacterial contamination. The agent is then identified by morphology or serological tests [11].

Serology: Sex-specific antibodies are demonstrated by the complement coupling (CF) test. The test becomes positive 2–4 weeks after the onset of the disease. An increased antibody titer or a single titer greater than 1:64 in a patient with clinical findings is a strong indication of active infection. A decrease in CF titer is observed in patients receiving treatment [11].

Treatment: Treatment with tetracyclines and sulfonamides, especially in the early period, yielded successful results. A significant reduction in complement-binding antibodies was observed in most drug-treated patients. This may indicate that the infectious agent has been eradicated from the body. Surgical treatments may be needed in the late stages [27].

16. Chlamydia pneumoniae and respiratory infections

Clinic: Chlamydia pneumoniae is known as the TWAR agent. A serotype has been identified. It is pathogenic in humans. It is transmitted by respiratory secretions. They are causative agents of upper and lower respiratory tract diseases, most of which are asymptomatic. They cause atypical pneumonia and inflammatory atherosclerosis and cardiovascular diseases [28]. The seropositivity rate is quite low under the age of five. The incidence begins to increase in school-age children. The incidence in adults and the elderly is around 50–75% [29]. The sex-related seropositivity rate in children is equal. However, it is significantly more common in males than adults. Today, studies are still ongoing to explain this difference [30].

Laboratory diagnosis: Many laboratory tests have been developed for the diagnosis of Chlamydia pneumoniae. However, serological tests are frequently used.

Culture: For Chlamydia pneumoniae, oropharyngeal swab samples should be transported with a chlamydia transport medium (sucrose phosphate glutamic acid buffer solution). Sputum samples are not suitable. It should be kept in a refrigerator at +4°C before being sent to the laboratory. They die quickly at room temperature. C. pneumoniae grows better in HL and Hep-2 cells. They are incubated for three days at 35°C. They are usually detected by fluorescent antibody staining with monoclonal antibodies specific for C. pneumoniae [11, 31].

Serology: Microimmunofluorescence (MIF), a serological test method, is the most sensitive method. It is species-specific. IgM increases 2–3 weeks after primary infection and IgG increases 6–8 weeks later. In reinfections, there may be no increase in IgM and after 1–2 weeks there may be an increase in IgG [3].

Nucleic acid amplification methods: The PCR method is used to determine the agents in the pharyngeal swab, bronchoalveolar lavage, and sputum samples. This method is a sensitive and rapid diagnostic test [32].

Treatment: Chlamydia pneumoniae are sensitive to macrolides, fluoroquinolones, and tetracyclines. Treatment success is good in patients treated with doxycycline, azithromycin, or clarithromycin. However, after some routine treatments, symptoms of the disease continue or reinfections are observed. Therefore, drugs should be used for 10–14 days [11].

17. Chlamydia psittaci and psittacose

Clinic: Chlamydia psittaci is the causative agent of psittacosis. It is transmitted by contact with birds and by breathing. Human-to-human transmission is rare. It can survive for months at room temperature. Therefore, environmental cleanliness is important [33]. It can be asymptomatic and pulmonary involvement is seen in humans. It can cause sepsis with severe pneumonia and high mortality. It is accompanied by systemic symptoms such as fever, nausea, vomiting, and muscle aches [34]. In birds, liver, kidney, and pericardium involvement is typical [33].

Laboratory diagnosis: For the isolation of Chlamydia psittaci in culture, blood, sputum, and lung tissue samples are suitable. Clinical specimens can be produced by inoculating tissue culture cells, embryonated eggs, and mice. They can be identified by examining them under a microscope. Culture can be dangerous. Molecular and serological tests may be preferred. Antigen detection by DFA or immunoassay (MIF) methods is common and PCR tests are used [11, 34].

Treatment: Since the laboratory diagnosis of Chlamydia psittaci infection is difficult, it is usually treated with clinical findings. According to several clinical studies, azithromycin, clarithromycin, and erythromycin (doxycycline in adults) treat most C. psittaci infections. Clinical improvement is achieved [11, 33].