*7.1.1 Tissue culture*

For conjunctival specimens, any purulent exudate should be removed before collecting epithelial cells by rubbing a dry swab over the everted palpebral conjunctiva. The conjunctivae are often friable and may bleed after swabbing so great attention is needed. For suspected pneumonia, material should be obtained from nasopharyngeal aspiration or deep suctioning of the trachea.

Culture of the bacterium is considered as the gold standard for diagnosing neonatal conjunctivitis and pneumonia. Proficiency in specimen collection and transport is paramount to accuracy in diagnostic testing for Chlamydiae [5, 42]. Both the sensitivity and the specificity of culture is nearly 100% but is directly related to the adequacy of the specimen. For optimal isolation of the bacteria, specimens should be refrigerated immediately after collection at 2 to 8 °C and kept at this temperature during transport to the laboratory. The intervening period between collection and laboratory processing of specimens should ideally not exceed the 48 hours [42].

### *7.1.2 Nucleic acid amplification test (NAAT)*

This method amplifies the nucleic acid sequences of chlamydiae. Non-viable bacteria can be detected contrary to cell culture. All relevant clinical materials can be analyzed by NAATs, including urethral, cervical, vulvo-vaginal, anorectal and ocular swabs, first void urine, sperm or tissues [43]. The test is recommended for routine use in adults and older children but data relating to infants are insufficient. The NAATs have not been approved by the US Food and Drug Administration (FDA) for testing of conjunctival specimens from infants with suspected C. trachomatis conjunctivitis or for testing of nasopharyngeal swab, tracheal aspirate or lung biopsy specimens from infants with suspected C. trachomatis pneumonia [33, 35].

#### *7.1.3 Antigen detection tests*

Antigen tests based on the detection of either chlamydial lipopolysaccharides (enzyme immunoassay, EIA) or direct fluorescent antibodies (DFA) have also been found to perform relatively well when used with conjunctival specimens. Sensitivity of nasopharyngeal samples is poor. Antigen tests are no longer recommended for chlamydia testing in infants due to insufficient diagnostic accuracy [43].

## *7.1.4 Serum anti-chlamydial antibody concentration*

Chlamydial IgG antibodies detected from infants during the first months of life reflect maternally transferred antibodies and correlate with the levels of maternal serum antibodies. Maternal IgG antibodies do not protect infants from developing chlamydial infection and infants born to antibody positive mothers usually lose their maternally transferred IgG-antibodies by nine months of age [24]. In infants with chlamydial pneumonia a microimmunofluorescence serum titer of ≥1:32 is considered diagnostic for infection [31, 33]. The IgM antibodies have been observed to develop as early as five days after infection and to persist for three months. Their determination is difficult and availability limited [24].

#### *7.1.5 Other tests*

Obtaining lung biopsies is not a routine practise for confirming chlamydial pneumonia, since the course of the disease is rarely fatal, but when a biopsy is obtained the material should be examined for chlamydiae [44]. Additionally in pneumonia, eosinophilia in the peripheral blood is usual with white blood cell count being normal. Blood gas measurements reveal mild to moderate hypoxemia [33, 35, 36].
