Lab d of clamydia trachomatis
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Chlamydia trachomatis inclusion bodies laboratory diagnosis
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Laboratory Diagnosis of Chlamydia trachomatis
Biology Relevant to Lab Diagnosis
C. trachomatis is an obligate intracellular bacterium with a biphasic life cycle:
- Elementary body (EB): the infectious, extracellular, metabolically inert form; ~0.3 µm
- Reticulate body (RB): the intracellular, replicating form; ~1 µm
Because it cannot replicate outside living cells, culture requires cell lines. The organism has a cell wall similar to gram-negative bacteria (contains LPS and MOMP - major outer membrane protein), both of which are exploited in antigen detection tests. It targets columnar epithelial cells preferentially.
Methods of Laboratory Diagnosis
1. Nucleic Acid Amplification Tests (NAATs) - Gold Standard
NAATs are the method of choice for all genital C. trachomatis infections and are recommended by the CDC.
Targets used:
- Cryptic plasmid (present in ~7-10 copies per cell - enhances sensitivity)
- 23S rRNA gene
- MOMP gene (ompA)
Amplification technologies:
| Technology | Example Assay |
|---|---|
| PCR | Abbott RealTime CT/NG |
| Transcription-mediated amplification (TMA) | Hologic Aptima Combo 2 |
| Strand displacement amplification (SDA) | BD ProbeTec |
| Real-time PCR | Roche cobas CT/NG; GeneXpert (Cepheid) |
Specimen types accepted:
- First-void urine (males and females) - non-invasive, preferred for screening
- Vaginal swab (self-collected or clinician-collected)
- Endocervical swab
- Urethral swab
- Extragenital: rectal and oropharyngeal swabs (FDA cleared for some platforms as of 2019 - Hologic Aptima Combo 2 and GeneXpert)
Performance:
- Sensitivity: ~90-95% (significantly higher than culture)
- Specificity: ~99%
- Can simultaneously detect N. gonorrhoeae (dual-target assays)
Limitation: A 23S rRNA C1515T mutation in Finnish strains caused false-negative results with some assays - an example of target failure.
- Henry's Clinical Diagnosis and Management by Laboratory Methods, p. 1381-1383
2. Cell Culture - Historical Gold Standard
Although replaced by NAATs in clinical practice, culture retains a role in medico-legal cases (sexual assault/abuse) because it offers 100% specificity and strain viability.
Cell lines used:
- McCoy cells (most widely used for C. trachomatis)
- HeLa 229
- HEp-2 cells
Method (Shell Vial Technique):
- Cells grown as monolayers on coverslips in shell vials
- Cells pre-treated with cycloheximide to inhibit host cell metabolism and enhance chlamydial replication
- Clinical specimen is centrifuged onto the monolayer (centrifugation enhances uptake)
- Incubated at 35-37°C for 48-72 hours
- Monolayer stained and examined for intracytoplasmic inclusions
Detection of inclusions in culture:
| Stain | Notes |
|---|---|
| Direct immunofluorescence (DIF) | Most sensitive; uses species-specific monoclonal antibodies against MOMP |
| Giemsa stain | More sensitive than iodine; inclusions stain dark blue/purple |
| Iodine stain | Detects glycogen in C. trachomatis inclusions (NOT C. pneumoniae or C. psittaci - iodine positivity distinguishes C. trachomatis) |
Performance:
- Sensitivity: ~80% (varies with method)
- Specificity: 100%
- A second passage (blind passage after sonication) can improve sensitivity
- Jawetz Melnick & Adelberg's Medical Microbiology 28E, p. 377; Henry's
3. Direct Fluorescent Antibody (DFA) Test
- Uses fluorescein-labeled monoclonal antibodies directed against the species-specific antigen on the chlamydial MOMP (major outer membrane protein)
- Detects EBs directly in smears from clinical specimens
- Useful for: conjunctival swabs in neonates, extragenital sites where NAATs may not be validated
- Requires trained microscopist; labour-intensive
- Lower sensitivity than NAATs; still used in some reference labs
- Jawetz Melnick & Adelberg's Medical Microbiology 28E; Henry's
4. Enzyme Immunoassay (EIA) for Antigen Detection
- Detects genus-specific LPS antigen extracted from EBs
- Commercially available but largely being phased out due to much lower sensitivity compared to NAATs
- No longer recommended for screening genital tract specimens
- Positive results should be confirmed by NAAT or culture
5. Cytology / Direct Microscopy
- Giemsa staining of conjunctival/cervical scrapings (not swabs) can reveal intracytoplasmic inclusion bodies within epithelial cells
- Inclusions appear as dense, dark-staining intracytoplasmic masses
- Sensitivity is very low (~10-30%) - not recommended for routine diagnosis
- Historically used for trachoma diagnosis (Halberstaedter-Prowazek bodies)
- Iodine stain: inclusions of C. trachomatis stain brown (glycogen-containing inclusions), unlike C. pneumoniae and C. psittaci
6. Serology
Serology is generally not useful for diagnosing genital tract infections due to high background antibody prevalence in sexually active populations. It is used in specific situations:
| Test | Use |
|---|---|
| Microimmunofluorescence (MIF) | Most sensitive and specific; detects type/species-specific IgG and IgM; gold standard for serology |
| Complement fixation (CF) | Used for LGV and psittacosis; detects genus-specific (LPS) antibodies |
| ELISA | Genus-specific antibodies; less specific than MIF |
When serology IS useful:
- LGV (serovars L1-L3): MIF titer ≥1:64 (or fourfold rise in paired sera) supports diagnosis
- Neonatal chlamydial pneumonia: IgM titer ≥1:32 by MIF is diagnostic (maternal IgG crosses placenta, so IgM is more meaningful)
- Pelvic inflammatory disease / salpingitis: high IgG titers correlate with tubal damage
- C. trachomatis pneumonia in infants: IgM detection is particularly useful
MIF interpretation:
- IgM: appears 2-3 weeks after onset; declines over months
- IgG: detected 6-8 weeks post-infection; persists lifelong; rises 1-2 weeks after reinfection
- A fourfold rise in paired (acute + convalescent) IgG titers is diagnostic
- Jawetz Melnick & Adelberg's Medical Microbiology 28E, p. 782-783
Specimen Collection by Disease Syndrome
| Disease | Specimen |
|---|---|
| Mucopurulent cervicitis | Endocervical swab |
| Urethritis (male) | Urethral swab or first-void urine |
| PID / Endometritis | Endometrial biopsy or endocervical swab |
| Neonatal conjunctivitis | Conjunctival scraping/swab |
| Neonatal pneumonia | Nasopharyngeal aspirate/swab |
| LGV | Bubo aspirate; rectal swab; urine |
| Trachoma | Conjunctival scraping |
| Proctitis | Rectal swab |
Important collection notes:
- Use Dacron or rayon-tipped swabs with plastic/wire shafts
- Avoid wooden-shaft swabs (toxic to organism), calcium alginate swabs (toxic to chlamydiae and cell lines), and cotton swabs (occasionally toxic)
- Purulent discharge should be removed before sampling - it contains predominantly dead PMNs, not infected epithelial cells
- For urine: first-void urine (first 20-30 mL) is preferred
- Henry's Clinical Diagnosis and Management by Laboratory Methods, p. 1374-1376
Comparison of Methods
| Method | Sensitivity | Specificity | Clinical Role |
|---|---|---|---|
| NAAT | 90-95% | ~99% | First-line for all genital infections |
| Cell culture | ~80% | 100% | Medico-legal; susceptibility testing |
| DFA | 70-80% | ~99% | Conjunctival specimens; extragenital |
| EIA | 60-75% | 97% | Largely phased out |
| Cytology (Giemsa) | <40% | Variable | Trachoma; historical |
| MIF serology | Moderate | Moderate | LGV; neonatal pneumonia; epidemiology |
Special Situations
- LGV (L1-L3 serovars): NAAT is the test of choice on bubo aspirate or rectal swab; serology (MIF titer ≥1:64) is supportive. Genotype confirmation requires sequencing or LGV-specific PCR.
- Neonates: Culture or NAAT from conjunctival swab; DFA acceptable if NAATs not validated for conjunctival use; IgM serology for pneumonia diagnosis.
- Medico-legal (sexual assault): Culture preferred (100% specificity); NAAT acceptable if culture unavailable, but positive NAAT should be confirmed by a second NAAT targeting a different gene.
- Trachoma (A, B, Ba, C): Conjunctival scraping for Giemsa/DFA; NAAT increasingly used in field studies.
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