**6. Diagnosis**

#### **6.1. Clinical**

A presumptive diagnosis of trachoma can be made based on clinical features, especially in an area where trachoma is considered to be present. The following signs are impor‐ tant indicators for trachoma, and at least two must be present in person diagnosed: folli‐ cles in the upper tarsal conjunctiva, limbal follicles or their sequelae, Herbert's pits, typical conjunctival scarring, and pannus. These signs along with conjunctival detection of C. trachomatis in the laboratory confirm the presence of endemic or hyperendemic trachoma in the respective area.

#### **6.2. Laboratory**

Serotyping has been the most widely accepted technique for classifying C. trachomatis or‐ ganisms. However, within the last decade, a new technique has been developed based on sequencing of ompA and is referred to as ompA genotyping. (ompA was previously called omp1, but the nomenclature has changed to be consistent with that of other bacteria.) This latter technique has been and continues to be invaluable for evaluating the molecular epi‐ demiology, disease pathogenesis, and transmission dynamics of chlamydiae for STD and

Chlamydia trachomatis are obligate intracellular parasites that are unable to synthesize their own energy (ATP) and are completely dependent on their host for energy. It has a unique biphasic developmental cycle not found in any other bacteria. There is the ele‐ mentary body (EB) is the infectious form (spore-like particle) that posses a rigid outer membrane that bind to receptors on host cells and initiate infection. and the reticulate body (RB), which is the metabolically active form. Once the EB comes in contact with susceptible epithelial cells, it attaches by divalent cations and polycations, using heparin sulfate as a bridge between receptors on the EB and the cell surface. The EB is taken up into a phagosome by receptor-mediated endocytosis. There is ineffectual lysosomal fu‐ sion with the endophagosome because of their rigid outer membrane, and hence intracel‐

A vacuole encloses the elementary body and the bacterium is now a reticulate body. Reticu‐ late bodies obtain their energy by sending forth "straw-like" structures into the host cell cy‐ toplasm. It can then replicate itself through binary fission. After division, the reticulate body becomes the elementary body. Anywhere from 100 to 1000 EBs can be produced per infected cell. In many cases, the cell ruptures and dies releasing the infectious progeny, but the cell can also extrude the inclusion body by a process of exocytosis and is released trough reverse

A presumptive diagnosis of trachoma can be made based on clinical features, especially in an area where trachoma is considered to be present. The following signs are impor‐ tant indicators for trachoma, and at least two must be present in person diagnosed: folli‐ cles in the upper tarsal conjunctiva, limbal follicles or their sequelae, Herbert's pits, typical conjunctival scarring, and pannus. These signs along with conjunctival detection of C. trachomatis in the laboratory confirm the presence of endemic or hyperendemic

trachoma populations.

236 Common Eye Infections

lular survival is insured.

endocytosis

**6. Diagnosis**

trachoma in the respective area.

**6.1. Clinical**

**5.2. Chlamydia trachomatis development cycle**

#### *6.2.1. Cytology*

The conjunctiva is swabbed with a Dacron or cotton swab, and the smear is made by rolling the swab over a clean glass slide. Alternatively, the swab can be placed in a special transport media for the respective diagnostic test. Epithelial cells that are clearly separated and the presence of PMNs, lymphocytes, plasma cells, or Leber cells (giant macrophages that con‐ tain phagocytosed material) can denote an adequate sampling of the conjunctiva. The de‐ gree of inflammation and bacterial superinfection can also be appreciated from these smears. Thus, although not diagnostic for chlamydiae, these findings are suggestive of tra‐ choma. Giemsa stain is inexpensive, and the test is easy to perform, which makes it attrac‐ tive for developing countries where trachoma is endemic or hyperendemic. However, the sensitivity is only about 60% and, thus, should not be used in areas of low endemicity. With this stain, the inclusion body is visualized as a basophilic, stippled inclusion in contrast to the dark blue to purple color of the cell. However, other entities can also stain similarly: These include goblet cells, bacteria, keratin, nuclear extrusions, and eosinophilic granules. Lugol's iodine stains the glycogen-containing inclusion of C. trachomatis. It imparts a dark yellow-brown color to the inclusion but is infrequently used, because it is insensitive. Com‐ mercially available fluorescent [fluorescein isothiocyanate (FITC)] conjugated monoclonal antibodies against the MOMP, which is species-specific for C. trachomatis, or the LPS, which is genus-specific for Chlamydia, are used in this test. The EBs are stained an apple green col‐ or and are visualized as extracellular round dots. The sensitivity for this test is approximate‐ ly 80% to 90%.

#### *6.2.2. Tissue culture*

Although the intracellular inclusions of trachoma were first identified by Halberstaedter and von Prowazek4 in 1907, the actual organisms were finally cultured in 1957 by using chick embryos. Today, tissue culture has supplanted the use of eggs, which has made isola‐ tion of chlamydiae more widely available, although it is still only performed in specialized reference laboratories. Tissue culture remains the gold standard for C. trachomatis identifi‐ cation but is not 100% sensitive, probably because of the difficulty in maintaining a cold chain (4°C for no longer than 24 hours and then -70°C) from the field site to a specialized reference lab where the culture will actually be performed. Also, because some viability is lost on freezing and some of the trachoma serovars are more difficult to propagate, culture requires technical expertise, can take 3 to 6 days for results, and is very expensive. Many dif‐ ferent cell lines are now available for culture, including HeLa and McCoy cells. Additional passages in tissue culture can increase the positive rate but have other drawbacks, including a delay in the reporting time of the results. Fluorescein-conjugated antibodies are used to detect the inclusions in cell culture, which are visualized as intracytoplasmic ovoid, round, or irregularly shaped inclusions. This stain imparts a fluorescing, apple green color to the inclusion body that stands out against the dark red cells that have been counterstained with Evans blue. Peroxidase-conjugated monoclonal antibodies are also available for the detec‐ tion of chlamydial inclusion bodies.

#### *6.2.3. Antigen detection*

ELISA or enzyme immunoassay (EIA) commercial assays are available to detect chlamydiae, but the sensitivity is only 70% to 85%. However, these assays can be cost effective compared with other commercially available tests such as the DNA detection assays. These tests detect the EB via polyclonal or monoclonal antibodies directed against the genus-specific chlamy‐ dial LPS. The antibodies are conjugated with an enzyme that reacts with a substrate to pro‐ duce a change in color that can be detected by a specific wavelength in a spectrophotometer. One advantage is that a 96-well format can be used to process multiple samples at one time. Less technical expertise is required than for the above-mentioned tests. Another advantage is that the kits contain a confirmatory test.

#### *6.2.4. DNA detection*

The commercial LCR and PCR tests are the most recent assays to be developed for detecting Chlamydia. Primers that are specific for the organism anneal to the complementary strand of DNA after denaturation. This target DNA is usually the plasmid, which is only present in C. trachomatis and C. psittaci species. LCR amplifies a signal that occurs when the primers hybridize with the plasmid DNA. In PCR, the actual DNA is amplified after hybridization. Both tests can be used in a 96-well format in which 92 to 94 samples can be assayed at one time. Both products are detected by spectrophotometers that are set at specific wavelengths for the particular assay. An advantage to the commercial PCR test is that an internal control plate can be run in parallel with the chlamydial detection plate to identify which samples have inhibitors. Those samples that contain inhibitors can then be run by in-house PCR as‐ says that employ a DNA purification protocol that removes the inhibitors.

Chlamydial DNA can also be detected by commercially available hybridization probes. These also hybridize with complementary plasmid or ompA DNA. The sample is usually a swab of the conjunctiva that has been applied to a special filter paper immediately after the sample has been obtained from the patient. Occasionally, DNA is extracted from a swab that has been placed in a special collection media and then is applied to a filter. In both cases, the filter is what is probed. The advantage of this technique is that the filter paper that contains the samples can be stored at room temperature under field conditions and transported back to the lab at a convenient time, without the necessity of a cold chain. The sensitivity of the probes is 70% to 90%.

#### *6.2.5. Serology*

There are two serologic tests for Chlamydia: the microimmunofluorescent (MIF) test and the complement fixation (CF) test. However, neither is specific for the organism because patient sera can cross react with different serovars and species and may represent current or previ‐ ous sexually transmitted infection as opposed to conjunctival infection. The highest antibod‐ ies detected in the assay, however, are usually found against the initial infecting serovar, even on subsequent infection. This concept is referred to as original antigenic sin. Further‐ more, ocular chlamydial infections tend to be chronic and endemic. Thus, these assays can‐ not be used to diagnose active infection, although occasionally MIF has been used for epidemiologic studies. They are also only available in reference laboratories.

The CF test is the older of the two and detects group-reactive antigen on C. psittaci and C. trachomatis. This test can be used for diagnosing ocular infections resulting from LGV or C. psittaci. The MIF test employs EBs representing C. trachomatis serovars and usually one or two strains of C. pneumoniae and C. psittaci. Sera, tears, and other bodily fluids can be used in this assay. The fluids are serially diluted and reacted against the EBs that have been ap‐ plied and fixed to a slide in groups of dots. A FITC conjugated antihuman IgM, IgG, IgA or secretory IgA antibody is used as the secondary antibody to detect antigen-antibody bind‐ ing. The slides are screened under fluorescent microscopy for fluorescing EBs that repre‐ sents the respective serovar or species. Serum IgM and IgG antibodies appear around 2- to 3-weeks postinfection and persist for 4 to 8 weeks, although the IgG antibodies persist for much longer. Occasionally, IgM titers can rise again with reinfection or relapse of infection. Approximately 80% of children in trachoma endemic areas and 90% of adults with inclusion conjunctivitis will have detectable MIF antibodies. About half of the population in trachoma endemic areas will have both serum and tear antibodies, and the titers are directly propor‐ tional to the severity of disease and to the presence of chlamydial organisms in the conjunc‐ tiva. In one study in Tunisia, 80% of children with severe disease, 31% with moderate disease, and 17% with mild disease had tear antibodies to chlamydiae. Although the highest titers are usually against the infecting serovar in ocular infections, the MIF test can be used only for a diagnosis of active infection in neonates not in older children or adults. Neonates acquire IgG antibodies from their mothers, and when they develop conjunctivitis, these tit‐ ers usually do not change during the course of the ocular infection. Most infected neonates do develop a small rise in serum IgM antibodies, usually less than 1:32, which persists for a few weeks.
