Chapter 77
Pathogenesis of Chlamydial Ocular Diseases
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Chlamydial ocular infections include trachoma, adult inclusion conjunctivitis (paratrachoma), and neonatal inclusion conjunctivitis (also referred to as neonatal inclusion blennorrhea or ophthalmia neonatorum). These diseases have been well described since antiquity yet the infectious chlamydial particles responsible for each were not discovered until the first decade of the 20th century AD.1 Trachoma is a chronic ocular infection caused by Chlamydia trachomatis. It was first reported in China during the 27th century BC in relation to a treatment for trichiasis. Trichiasis refers to in-turned eyelash(es) that touch the globe and is a sequela of trachoma that occurs years to decades following infection. The Ebers Papyrus of 1800 BC also made reference to trachomatous disease, but the actual term trachoma did not appear until the first century BC, when it was used as the Greek word to describe “rough swelling” of the conjunctiva. During the Middle Ages, endemic trachoma in the Middle East was spread to Europe by the crusaders. Trachoma subsequently became a significant blinding disease of epidemic proportions among civilian and military populations during the Napoleonic era. Although trachoma has disappeared from most developed countries of the world because of improved hygiene, sanitation, and economic development, more than 600 million inhabitants of Africa and the Eastern Mediterranean, as well as inhabitants of parts of Central and South America, Asia, and Australia,2 are infected with C trachomatis. Of these 600 million, 150 million people, mostly children, have active trachoma. Approximately 6 million persons are already blind from this disease, and 10 million will require surgery for trichiasis to prevent progression of visual deficits and blindness.3

Adult and neonatal inclusion conjunctivitis is caused primarily by the sexually transmitted disease (STD) strains of C. trachomatis. The historic link between these chlamydial strains and STDs, as well as the ocular diseases, was not made until diagnostics were developed for Neisseria gonorrhoeae (GC). Once it became clear that many cases of cervicitis, neonatal conjunctivitis, and nongonococcal urethritis could not be attributed to GC, the involvement of another infectious agent was suspected. In 1907, Halberstaedter and Prowazek4 identified typical chlamydial intracytoplasmic inclusions in conjunctival scrapings from an orangutan infected with a human trachoma strain. Similar inclusions were later seen in scrapings from the genital tracts of the parents of an infant with neonatal conjunctivitis.5 But it was not until 1959 that a strain responsible for chlamydial STDs was actually isolated. Because the natural history of chlamydial STDs is not well understood, including the propensity for latent or persistent infections to reactivate and be transmitted, the true incidence and prevalence of adult and neonatal inclusion conjunctivitis are not known. Adult inclusion conjunctivitis is usually a unilateral, self-limited disease of presumed low prevalence, although chlamydial genital strains are the most common cause of STDs in the developed and developing world today. This disease can also be caused by other species of Chlamydia such as Chlamydia psittaci and Chlamydia pneumoniae.6–8 Neonatal conjunctivitis can be severe if untreated, and approximately 20% of infected neonates will develop pneumonitis within the first 6 months of life along with the long-term sequela of small airways disease in adulthood.

The last two decades have provided scientific advances in almost all areas of chlamydial research. These include clinical, diagnostic, epidemiologic, physiologic, pathologic, microbiologic, genetic, and immunologic characteristics of chlamydial diseases. Furthermore, the advent of a new, simplified trachoma grading scheme9 and a more specific strain typing technique referred to as ompA genotyping10 has enhanced the understanding of the clinical epidemiology, molecular epidemiology, transmission, risk factors, and prevalence of trachoma. However, the pathogenesis of trachomatous disease requires further study, and the best approach to designing an efficacious vaccine remains controversial.

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Chlamydia are prokaryotes. Chlamydial organisms were historically referred to as Bedsonia or Miyagawanella and were initially thought to be protozoa. Because of their small size and the problems encountered with propagation, they were subsequently thought to be viruses. In the 1960s they were classified as bacteria because Chlamydia express proteins (e.g., lipopolysaccharides) that are functionally analogous to other bacteria, divide by binary fission, are inhibited by antibacterial drugs, contain ribosomes, and are structurally and morphologically similar to gram-negative bacteria.1 However, Chlamydia are only distantly related to other eubacterial orders based on phylogenies of ribosomal ribonucleic acid (rRNA) gene sequences.11 Chlamydiae comprise their own order, Chlamydiales; a single family, Chlamydiaceae; and one genus, Chlamydia. The genus Chlamydia is comprised of four known species: C. trachomatis, C. psittaci,12 C. pneumoniae,13 and Chlamydia pecorum.1,14 C. trachomatis is made up of three biologic variants or biovars: trachoma, lymphogranuloma venereum (LGV), and the rodent biovar that includes the mouse pneumonitis (MoPn)15 and hamster strains.16 There is 87% to 99% deoxyribonucleic acid (DNA) homology among the human strains and biovars of C. trachomatis but only 30% homology for the rodent strains.

With the exception of the rodent strains, C. trachomatis is currently known to infect only humans. The infections in humans include the conjunctiva and lower and upper genital tracts, including the rectum and lymphatics that drain the perineum. These infections are caused by the 19 currently recognized serologic variants (serovars) of the trachoma and LGV biovars. Serovars are defined by monoclonal and polyclonal antibodies that react to epitopes on the major outer membrane protein (MOMP) of C. trachomatis. Additional serovars presumably exist based on sequence data of the gene (ompA) that encodes for MOMP, but these strains have not been fully characterized to date.17 Serotyping has distinguished these serovars into different serogroups or classes: B class (serovars B, Ba, D, Da, E, L2, and L2a), intermediate class (serovars F and G), and C class (serovars A, C, H, I, Ia, J, Ja, K, Ka, L1, and L3).1 Serovars A through K and Ba, Da, Ia, Ja, and Ka were previously referred to as trachoma-inclusion conjunctivitis (TRIC) strains.18 Trachoma is primarily caused by serovars A, B, Ba, and C, whereas adult and neonatal inclusion conjunctivitis are caused by serovars B or Ba, D through K, Da, Ia, Ja, Ka, L1, L2, L2a, and L3, which are the sexually transmitted strains of the organism. The LGV serovars tend to cause more severe disease and can invade regional lymphatics, whereas the non-LGV serovars are currently known to infect epithelial cells at ocular, respiratory, rectal, and genital mucosal surfaces.

Serotyping has been the most widely accepted technique for classifying C. trachomatis organisms. However, within the last decade, a new technique has been developed based on sequencing of ompA and is referred to as ompA genotyping.1,19,20 (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 epidemiology, disease pathogenesis, and transmission dynamics of chlamydiae for STD and trachoma populations.

New genus and species designations were proposed for chlamydiae in 1997 based on sequence analysis of small segments of rRNA.21,22 However, this type of analysis provides relatively constant evolutionary relationships that do not necessarily translate into biologically important designations that help to distinguish one strain from another within a species or genus. This has become increasingly apparent from evidence of intragenic and intergenic recombination of the ompA gene for C. trachomatis, C. psittaci, and C. pneumoniae that affect persistence and tissue and host tropism for different stains.17 Although the rate of or time frame for mutations and recombination events is not known for ompA or other genes that encode for surface proteins on Chlamydia, a new taxonomy may not be appropriate because these events do occur. Furthermore, genomic analyses of other organisms have revealed biologic relationships that are not reflected in the phylogenies of rRNA-based sequences.23 This continues to be an area of debate in the field of Chlamydia.

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Chlamydia are obligate intracellular pathogens that require nutrients from the host cell for replication. They have a unique biphasic developmental cycle not found in any other bacteria. Chlamydia have two forms: the elementary body (EB), which is the infectious, spore-like particle of the organism; and the reticulate body (RB), which is the metabolically active form. The outer membrane of the EB has disulfide, cross-linked cysteine residues on and between MOMP as well as 12 and 60 kilodaltons (kDa) cysteine-rich outer membrane proteins. These proteins provide a relatively rigid membrane that facilitates short-term viability of the organism outside the host cell. Once the EB comes in contact with susceptible epithelial cells, it attaches by divalent cations and polycations,24 using heparin sulfate as a bridge between receptors on the EB and the cell surface25 or attaching via an as yet unknown receptor. Heterologous serovars competitively inhibit attachment.26 The EB is taken up into a phagosome by receptor-mediated endocytosis,27 although pinocytosis and phagocytosis have also been described. There is ineffectual lysosomal fusion with the endophagosome, and hence intracellular survival is insured. At this point in the cycle, the endophagosome is called an inclusion body.

The developmental cycle is regulated by transcriptional events.28 Cleavage of the disulfide bonds occurs within 6 to 8 hours of EB uptake and heralds the differentiation into the 800 to 1000 nm RB. The energy for RB replication, which occurs via binary fission starting at about 10 to 15 hours postinfection,29 comes from its own stores of adenosine triphosphate (ATP) and activated ATPase, as well as essential metabolites and phosphate compounds from the host cell that are transported across the inclusion body membrane.30 MOMP contains pore-like (porin) structures during the RB stage that are thought to facilitate this exchange.31 There appears to be a differential requirement for amino acids depending on the serovar: The LGV strains require methionine unlike the other STD strains, whereas the trachoma serovars require tryptophan. Leucine, phenylalanine, and valine, however, are necessary for growth for all known serovars and species of Chlamydia.32 As replication proceeds, the inclusion body expands and displaces the nucleus to the side of the cell. In addition, glycogen accumulates, which can be visualized by staining with iodine but is only present in C. trachomatis.

Within 36 to 72 hours, depending on the serovar, chromatin within the RB compacts down into an electron-dense nucleoid and, along with formation of membrane-bound disulfide bonds, produces an EB. The signal for these latter events is the reduction and oxidation of both intramolecular and intermolecular disulfide bonds.31 Anywhere from 100 to 1000 EBs can be produced per infected cell.33 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, thereby, survive.

The EB is composed of a number of different proteins. MOMP comprises approximately 60% with a mass of 40 kDa34 and is the most immunogenic of the surface proteins. The mass of this protein does differ somewhat according to the serovar. Both the lipopolysaccharide (LPS) and MOMP have genus-specific determinants, whereas MOMP also contains serovar-, serogroup-, and species-specific epitopes. There are four variable segments (VSs) interspersed by five constant regions on MOMP.35 VS1, 2, and 4 are surface exposed and contain the serotyping epitopes of the organism, whereas VS3 has important T-cell determinants.24,36 The genus-specific epitope of chlamydial LPS is a trisaccharide of 2-keto-3-deoxy-octanoic acid (KDO).37 This approximately 7000 kDa glycolipid is similar to the LPS of enteric bacteria such as Salmonella typhimurium.38 Both the LPS and MOMP are expressed throughout the developmental cell cycle, whereas the 12 and 60 kDa proteins discussed previously are expressed only at the time when the RB condenses back into an EB.24

There are some important similarities and differences among the four species of Chlamydia. Although they differ in host and host cell preference, genomic composition, epitopes, drug susceptibility, and metabolism, they are similar in morphology and structure. However, the EB of C. pneumoniae can appear pear-shaped, instead of round, with a large periplasmic space. In addition, when more than one EB enters a cell, the inclusion bodies usually fuse into a single body but can remain distinct for C. pneumoniae, C. psittaci, and C. pecorum. The size of these bodies can be variable, especially for C. psittaci. Recently, nonfusing inclusions of C. trachomatis have been identified that have mutations in the inclusion membrane protein A (IncA) gene and lack IncA expression in the inclusion membrane.39 Chlamydia contain a 7.5 kilobase (kb) plasmid that has been found only in C. trachomatis and C. psittaci strains. The sequence is relatively conserved within a species but varies considerably between the two species. The function of the plasmid is not known, although it is presumed to have a role in replication.40 However, C. trachomatis strains that lack the plasmid can still be propagated in cell culture.41

Other genomic differences have been highlighted by the recent publications of the entire genome of C. trachomatis serovar D42; C. pneumoniae strains CWL029, AR39, and J13843,44; and C. psittaci strain GPIC (Read et al., unpublished data). The genome of C. pecorum has not yet been sequenced. Several open reading frames (ORFs) were found in each species that likely encode outer membrane proteins, including a polymorphic membrane protein (Pmp) gene (pmp) family including 9 genes for C. trachomatis, 21 for C. pneumoniae and 16 for C. psittaci. The pmp family is important because it is unique to the genus Chlamydia, comprises a surprising 10% of the genome, and interspecies amino acid sequence homology is less than 50% compared with 70% to 80% for other surface proteins. Although the function(s) of these proteins have not been determined, they are likely important in host immune response and pathogenesis. This is supported by some preliminary data that revealed differential immune responses to certain C. trachomatis Pmps in patients with chlamydial STDs.45

All species of Chlamydia share the genus-specific epitope located on LPS. Serovar-, subspecies-, and species-specific epitopes on MOMP have been characterized for C. trachomatis and C. psittaci but do not appear to be immunogenic for C. pneumoniae. No data are available yet for C. pecorum. All species of Chlamydia are susceptible to tetracyclines, and all serovars of C. trachomatis are susceptible to sulfonamides. However, few strains of C. psittaci and C. pneumoniae strains respond to treatment with sulfonamides.

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Trachoma is the leading infectious cause of preventable blindness in the world today and occurs as mesoendemic, endemic, or hyperendemic disease.1 The distribution is primarily in tropical developing countries of the world including North and sub-Saharan Africa, the Middle East, and the Northern Indian subcontinent where the highest affliction rates are reported.2 However, disease has also been reported in Southeast Asia and specific regions of Central and South America, Australia, and the South Pacific Islands.3 Of the more than 600 million people afflicted, approximately 150 million have visual deficits,3 and 12 million are predicted to be blind by the year 2020.46 However, the precise prevalence of disease or disease severity is not known because surveys have not been conducted in many countries where trachoma is suspected nor have they been performed in many areas where trachoma is considered endemic or hyperendemic.

C. trachomatis is transmitted via eye to hand to eye contact where the primary reservoir of infection is the conjunctival mucosa. Other reservoirs include the nasopharynx and oropharynx and rectum, but their role in infection and reinfection of the ocular mucosa is not completely understood.47 In one study, nasal secretions were not found to play a role in reinfection in a trachoma community in Tanzania,48 but additional studies are required. The STD strains of C. trachomatis are also present in these communities but are not felt to contribute to trachoma. The incubation period is not known but is thought to range from 5 to 12 days based on nonhuman primate studies.49 Children are considered the primary source of infection because they usually comprise more than 50% of the population and become easily infected from close contacts during frequent play and within small, crowded households. However, female caregivers can also serve as an important reservoir in which the infection is passed to them and then back to their children. Thus, children younger than 10 years are at greatest risk for infection and reinfection. Young women of childbearing age and other female caregivers are reported to have infection rates that range from 5% to 10%.47,48,50–52 These infections likely represent transmission within the household from and to children. Adult women develop more severe disease and sequelae than their male counterparts; repeat infection is considered an important factor in disease progression to trichiasis.

Patterns of trachomatous disease and disease severity are dependent on the degree of endemicity in villages or communities where C. trachomatis ocular infections occur. In hyperendemic areas, high prevalence rates of infection among children are found and reinfection is common. Indeed, most children by the age of 1 year have been infected at least once. Both sexes are equally at risk in childhood, but, because the rates of infection decline with age, adolescent and young adult females tend to have higher rates compared with their male counterparts. These communities have higher rates of conjunctival scarring, occurring as early as 4 years of age. Scarring is common in young adults and older age groups and progresses to trichiasis and entropion (in-turned eyelid) in many cases. These sequelae occur 10 to 40 years following the initial childhood disease53; trichiasis rates range from 8% to 17.5% depending on the geographic locale.54–56 Blindness occurs indirectly through corneal abrasion and bacterial superinfection, both of which may be facilitated by dry eyes, which is a common condition among older individuals. These lesions typically heal by scarring and fibrosis. Trachoma is the primary cause of blindness in hyperendemic areas.

In mesoendemic or endemic communities, trachoma takes two forms. In one, the prevalence is low but there are a number of households in which infection rates are high, which provides a reservoir for repeat infection that can lead to the sequelae mentioned previously. In the other form, infection occurs later, starting at school age, and blindness is rare. However, the community rate of infection may be moderate to high.57,58 Where infection rates are declining, scarring in older individuals may still be present along with progression to trichiasis and blindness.

C. trachomatis cannot always be detected in all cases of active trachoma. In one report from Tanzania in which direct fluorescent antibody (DFA) and tissue culture tests were used, 43% of patients with follicular disease (TF) and 23% with intense inflammatory disease (TI) were negative for C. trachomatis.50 Similar findings have also been reported for newer tests such as the ligase chain reaction (LCR)59 and the polymerase chain reaction (PCR)60 that have a higher sensitivity than DFA or culture. The most likely explanations for these findings are inadequate sampling of the conjunctiva, resolution of infection with residual inflammation, inflammation resulting from infections with other bacteria, or autoimmunity or some combination thereof.1 In one study in The Gambia61 household members were tested for chlamydiae every 2 weeks. Organisms were detected 2 weeks before onset of inflammation and for an additional 2 to 6 weeks after disease development in children. Clinical disease persisted for 30 weeks in children and for only 2 weeks in adults. In other studies, 5% to 24% of villagers without active disease were found to be infected,47,48,50–52 including 24% of children in Tanzania51 who were PCR positive but had no evidence for trachoma. Thus, the time at which the sample is obtained can be critical for detection of the organism.

The degree of inflammatory disease varies considerably from one child to the next but can remain constant at different time points for a particular child despite treatment. Historically, environmental factors were considered to be responsible.62 However, a combination of factors is now thought to contribute to this phenomenon: environmental conditions, infectious load of C. trachomatis,63 duration of infection,64 host immune response,1,65,66 and possibly host genetic susceptibility.67,68 Severe inflammatory disease and the duration of inflammation in childhood is thought to be a risk factor for progression to scarring, and trichiasis and entropion later in life. Thus, age is also an important risk factor for trachomatous disease.69 Additional interacting factors likely contribute. With age, there is continued exposure to the organism and risk of reinfection especially among females of childbearing age and female caregivers of all age groups. There can be changes in biologic susceptibility as in the development of dry eye syndrome that can occur from age or scarring. Also, host immune response to chlamydial antigens such as Hsp60 have been associated with trachomatous scarring, and this response may be potentiated with increasing age.66

Other risk factors for trachoma are low socioeconomic status, poor facial hygiene, more than one child in a bedroom, many children per household, lack of water or lack of use of water, and proximity to cows.50,70–72 Flies have historically been considered vectors for transmission in Africa. However, data from recent studies do not support the theory.73 In one study, fluorescein was used to stain the secretions in the eyes of children.74 Within 15 to 30 minutes, the legs and bodies of the flies were also stained with fluorescein. Eye-seeking flies such as Musca sorbens have been shown to land on the eyes of multiple children as was reported in a study in Africa,75 but it is not clear how many infectious EBs can be carried on the flies, how long they are viable, and whether the inoculum is sufficient to cause infection. In The Gambia,75 of 395 flies captured from the eyes of C. trachomatis-infected children, only two were positive by PCR and could not be confirmed. It is certainly possible that flies carry other bacteria from eye to eye, which might promote inflammatory disease and trachoma. Indeed, in many trachoma endemic countries, there are seasonal outbreaks of conjunctivitis resulting from multiple bacterial species including Haemophilus influenzae, Haemophilus aegyptius, Streptococcus pneumoniae, Neisseria meningitidis, N. gonorrhoeae, and Moraxella spp. These infections may actually precede periods of increased trachoma prevalence rates. In a study in Tunisia,57 moderate to severe trachoma was found significantly more often among children with bacterial coinfections. Furthermore, pathogenic and nonpathogenic bacteria commonly colonize children who reside in trachoma areas. Coinfection of C. trachomatis with these bacteria may be one mechanism that is important for promoting severe inflammation, which results in conjunctival scarring and corneal vascularization years later.76 In the study of trachoma pathogenesis, coinfection will be an important area to examine.


Although chlamydial STDs account for more than 500 million cases worldwide with approximately 4 million annually in the United States,77 the exact prevalence of adult inclusion conjunctivitis remains unknown, partly because infection is usually self-limited and does not always reach medical attention. The spread of chlamydial STD serovars is from hand to genital tract to eye contact or during sexual activity. However, there are reports of transmission occurring among family members from an infected neonate.18 Persons between the ages of 15 and 30 years are at highest risk for adult conjunctivitis.18 The incubation period is considered to be approximately 5 to 19 days, and disease may continue for months without treatment. There is usually complete resolution of disease following a 3-week course of systemic therapy with either tetracycline or erythromycin. Adult inclusion conjunctivitis is characterized by a serosanguineous to mucopurulent discharge with keratoconjunctivitis or a follicular inflammatory disease identical to trachoma. This clinical condition is not considered a precursor to the sequelae of trachoma. Cases that persist or in which reinfection occurs may lead to keratitis, corneal vascularization, and conjunctival scarring.

The LGV strains of C. trachomatis are responsible for a much more severe ocular disease referred to as Parinaud's oculoglandular syndrome.78 This syndrome is comprised of an inflammatory conjunctival response with severe lymphadenopathy involving the preauricular, cervical, and submandibular nodes. The LGV serovars are uncommon in developed countries with few reports in the literature79 but are very common in tropical and subtropical developing countries. Occasionally keratoconjunctivitis resulting from L2 has been reported as a consequence of laboratory accidents.80 The female cervix and rectum of homosexual men81 are important primary reservoirs of these infections.


Approximately 5% of pregnant women have cervical infections with C. trachomatis.49 If left untreated, there is a 50% chance that the infant will develop conjunctivitis. Neonatal inclusion conjunctivitis is about 10 times more common than conjunctivitis resulting from N. gonorrhoeae. The incubation period for chlamydial conjunctivitis is 1 to 3 weeks, unlike GC in which the incubation period is less than 7 days. Earlier infection can occur if there is evidence for rupture of membranes.82 If left untreated, the conjunctivitis can persist for 3 to 12 months. The outcome for late or no treatment is conjunctival scarring and corneal vascularization in 100% of these cases.83 Infants born to an infected mother also carry a 20% to 30% chance of pneumonia. C. trachomatis is the leading cause of pneumonitis within the first 6 months of life. Rectal shedding of C. trachomatis does occur and is more common among infants with pneumonia. Onset of shedding does not usually occur before 6 to 12 weeks of age and can be as late as 12 months. This condition likely represents organisms that are swallowed after coughing as opposed to those that are acquired at birth. The short- and long-term importance of rectal shedding remains unclear.

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Trachoma is a chronic follicular conjunctivitis persisting for 15 days or longer. It is characterized by follicles that cannot be distinguished from those caused by C. trachomatis STD strains, C. pneumoniae, or C. psittaci.7 Other entities also cause a follicular conjunctivitis including toxic follicular conjunctivitis resulting from cosmetics, drugs, or molluscum contagiosum; chronic follicular keratoconjunctivitis of Thygeson; folliculosis; Axenfeld's chronic follicular conjunctivitis; Vernal catarrh; Parinaud's oculoglandular syndrome; or other bacteria.57 The latter bacteria are H. influenzae, H. aegyptius, Moraxella spp., N. meningitidis, N. gonorrhoeae, and S. pneumoniae.1 The identification of C. trachomatis in suspected regions of trachoma endemicity provides good evidence that this is the etiologic agent despite the presence of other bacterial species or outbreaks of conjunctivitis.

The follicular stage of disease presents as a foreign body sensation and can resolve completely or progress to cicatricial conjunctivitis with superficial corneal vascularization. The lymphoid follicles are clear, yellowish or gray-white lesions of 0.2 to 2 mm in diameter in the subconjunctival epithelium primarily of the upper tarsal conjunctiva.57 Papillary hypertrophy can occur, which is a result of inflammatory infiltrates. This finding may be the only sign of disease in children younger than age 2 years because follicle formation is not as common in this age group. Furthermore, infection in infants and young children may go undetected, thereby providing a source for transmission within the household and community.

The corneal involvement of the disease includes lymphoid follicle formation at the limbus.1 This is a characteristic feature of trachoma and can lead to the development of Herbert's pits. Punctate epithelial erosions can occur anywhere on the cornea but are usually located at the superior corneal-scleral border. Other common corneal findings include anterior stroma infiltrates and shallow peripheral ulcers. These findings range from microscopic infiltrates to frank ulceration. Superficial corneal vessels, referred to as pannus, can also develop. Pannus can develop at any point along the limbal margin but is most pronounced at the superior limbal margin. Vision is not usually affected by pannus formation.

As the disease progresses, fine linear or stellate scars form horizontally across the upper tarsus. Children as young as 4 years can show signs of scarring. With age, the scars may expand into broad bands or, in severe cases, develop into synechial scars. As scarring progresses, especially toward the outer edge of the lid, the conjunctiva becomes distorted, resulting in trichiasis and entropion.1 Corneal abrasions can occur from the inturned lashes and become superinfected with bacteria. In these cases, healing usually produces fibrosis and scarring. Thus, mild to severe corneal opacification can develop that can result in visual deficits or blindness. Loss of lid apposition and subsequent exposure keratitis or trauma may also develop with time. A late complication of scarring is lacrimal duct stenosis, which may lead to dry eye syndrome. This can also contribute to corneal ulceration, because there are few if any tears to flush out bacteria or to act as a barrier to infection. Because the severe sequelae of trachoma do not occur for 10 to 40 years following the initial infections, most of the visual deficits and blindness occur in the adult and older adult populations of trachoma endemic areas.


Adults develop a follicular conjunctivitis that can be indistinguishable from that of trachoma. The follicles may be present on both the lower conjunctiva and upper tarsus. The onset is usually acute with preauricular lymphadenopathy on the involved side84 and a serosanguineous to mucopurulent discharge. After 2 weeks of infection, corneal involvement is more prominent and includes keratitis, subepithelial opacities, and infiltrates that are marginal and/or central. Occasionally there is mild scarring and corneal vascularization referred to as micropannus,74 but these are late findings, usually among cases that have not been treated. Otitis media is a common complication of chlamydial conjunctivitis.85 Although there can be prompt resolution of the disease, conjunctivitis may persist for as long as 12 months if left untreated. In addition, failure to treat the genital tract disease can result in recurrence of conjunctivitis.

Conjunctivitis resulting from the LGV serovars of C. trachomatis cause a disease referred to as Parinaud's oculoglandular conjunctivitis. This clinical syndrome includes severe inflammation and lymphadenopathy of the preauricular, submandibular, and cervical nodes. These infections are uncommon in the United States. However, there are a number of reports dating back to the 1930s and 1940s.78,86–89


Conjunctivitis in the neonate is characterized by swelling of both lids, a purulent discharge, and hyperemia. Without treatment, neonates are at risk for conjunctival scarring, keratitis, and superficial vascularization of the cornea.90 In addition, these infants are at risk for pneumonitis up to 6 months of age. The disease can persist for up to 12 months in untreated cases but does respond quickly to appropriate systemic treatment.

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The immunopathogenesis of chlamydial ocular diseases is not well understood but likely includes a combination of environmental, pathogen, and host-related factors. Early trachoma vaccine trials in humans followed by experiments in nonhuman primates suggested that immunity to C. trachomatis infections was serovar specific, dose dependent, and short lived.91–93 Both humoral and cell-mediated immunity (CMI) are considered important for this response. The severe sequelae of trachoma, scarring with trichiasis and entropion, are thought to result from repeated infection in childhood, either with the same or a different serovar. Support for this comes from evidence that children in trachoma communities are indeed repeatedly infected, and the rates of scarring tend to be higher in hyperendemic than in endemic communities. In one study that followed 32 households over 10 years in Taiwan, the children were noted to have numerous episodes of microbiologically proven infection.91 Furthermore, whole-organism vaccine studies revealed that Taiwanese children who received the vaccine developed hypersensitivity reactions on subsequent acquisition of natural infection in their communities.92 Severe inflammatory disease in childhood is also thought to predispose to scarring in adulthood. In one study, children with severe inflammation tended to have the same degree of severity on each subsequent episode of trachoma.94 In an early primate model of ocular disease, Taiwan monkeys that were repeatedly infected or those that were given a vaccine and subsequently challenged developed conjunctival scarring and pannus that resembled that seen in humans.93 Other animal models were able to corroborate these findings,95 but not all models resulted in development of pannus.96 Thus, it appeared that progression of disease was associated with an immunopathogenic response from recurrent infection.1

By adolescence or adulthood, the prevalence of active disease and the severity of the inflammatory response have diminished. For reasons that are not completely understood, some individuals develop conjunctival scarring that will progress to trichiasis and entropion years later, whereas others will not. This differential response to repeat infections is not explained by sociologic or environmental risk factors.97 Usually only about 10% of adults older than age 25 years will have active disease, and the chlamydial isolation rates are approximately 1% for these cases.94,98 Furthermore, many young adults harbor chlamydial antigens in their conjunctiva. Antigen presence is considered a risk factor for scarring disease.

Three theories are possible for the pathogenesis of trachomatous sequelae: (1) adults harbor persistent chlamydial organisms that usually cannot be isolated but provide a continuous or intermittent antigenic stimulus for deleterious host immune response (including the possibility that organisms may be associated with scarring but not causative); (2) after repeated infection, the organism is eliminated but chlamydial antigens (still present or now absent in tissue) trigger immune mediators that set up an initial inflammatory process that may or may not subside over the years and promote tissue destruction that results in scarring and trichiasis and entropion (includes the possibility that host genetic susceptibility plays a role in disease pathology); or (3) a combination of both.

Support for both theories comes from one study of American Indian school children in 1968 in whom active trachoma declined when the children attended a boarding school far from their endemic environment,99 but chlamydial antigens still persisted in the ocular tissue of these children despite an absence of cultivable organisms.99 Therefore, in addition to suggesting persistent infection, there may also be immune mediators that promote an immunopathogenic response that continues even when the individuals leave their endemic environment. In The Gambia, chlamydial antigens and secretory immunoglobulin A (sIgA) antibodies were found in 43% of adults who had conjunctival scarring but no evidence for active disease.98 Elevated sIgA antibodies are not usually found at mucosal sites when there is no antigenic stimulation, as there would be from active infection. In the same study,98 sIgA antibodies were present in 7% of adults who had no evidence for clinical disease and who resided in communities of low trachoma prevalence compared with 41% of adults living in high-prevalence communities. Also, tear IgG antibodies were significantly associated with antigen-positive adults compared with antigen-negative adults. These findings suggest that adults who are positive for chlamydial antigens have real yet unappreciated infection. These adults may harbor persistent organisms that intermittently replicate and produce antigen, thereby increasing the risk for fibrosis and scarring over subsequent years.

The ability of C. trachomatis to persist and evade the host immune response may be important in the pathogenesis of trachoma. Once infection is established, the organism can mutate at the gene and protein level in such a way that new immunogenic variant organisms (those derived from the infecting strain) arise that are able to evade the host immune response. In support of this is an in vitro study by Lampe and coworkers100 in which neutralizing antibodies were shown to prevent infection for prototype strains, but closely related ompA gene variants escaped neutralization. There are a few studies that have described the occurrence of ompA mutants or variants that appear to have arisen from immune selection over time in trachoma endemic regions. The first such report was from Tunisia where 15 variants of 23 B and Ba serovars arose over a 3-year period in the same village area.10 Although the original serovars persisted in the community, a new immunologic serotype evolved during this period.19 Similar studies in The Gambia have also shown mutations in MOMP that have arisen over a 6-month interval,101 and a study in Tanzania revealed a high degree of ompA polymorphisms among trachoma serovars along with evidence for persistent infections.63

Further evidence for in vivo persistence in trachoma populations comes from a study of conjunctival tissue obtained during surgery for trichiasis and for other types of corrective lid surgery.102 A significantly higher number of trichiasis patients were found to be positive for chlamydial DNA and RNA compared with the nontrichiasis patients, although all of the patients were from similar trachoma endemic regions of Vietnam. The tissue was examined for cell types by immunohistochemistry, and a predominance of macrophages was found in the trichiasis cases. Macrophages may function as cofactors both in inflammation and tissue injury. They induce tissue necrosis factor (TNF)-alpha but are also able to directly cause tissue injury through activation of complement (C3a) and hydrogen peroxide (H2O2) pathways and secretion of acid hydrolases.103 Resident macrophages may phagocytose but not kill chlamydiae, thereby harboring the organism for a long time. Indeed, it has recently been shown that apoptosis is inhibited in C. trachomatis infected cells via inhibition of cytochrome c release from the mitochondria and caspase activation.104 In another study, HeLa cells that were persistently infected with trachoma serovar A resisted apoptosis by mechanisms similar to those for acute infection.105 High levels of Hsp60 with very low levels of MOMP and LPS were found in this study, which is consistent with in vitro studies of chlamydial persistence.106 A recent study of chlamydial cervicitis revealed that approximately 5% of women had recurrent cervicitis, and 24% of these were with the same C. trachomatis serovar.107 OmpA genotyping of recurrent samples from the same women showed identical genotypes or a few nucleotide changes that encoded for amino acid substitutions. Many intervening culture-negative samples were positive by LCR 4 weeks after treatment. Because 15 days is considered the maximum time that DNA can remain in tissue after elimination of viable organisms by antibiotic treatment,108 these findings lend additional support to the growing body of evidence that chlamydial organisms can persist. Thus, chlamydiae may persist by using anti-apoptotic mechanisms, intermittently replicating, and producing antigen that elicits a deleterious host immune response.

As suggested previously, the host immune response to different antigens may also play a role in disease severity outcomes. Viable but not inactivated EBs, MOMP, or LPS can induce conjunctival inflammation within 24 hours. Another protein, the chlamydial Hsp60, which is a homologue of the 60 kDa GroEl Hsp of Escherichia coli, is also likely responsible for a direct inflammatory response.109 Chlamydial Hsp60 is surface exposed and stimulates a vigorous humoral immune response in trachoma patients, as well as in patients with tubal infertility and ectopic pregnancy.109–111 Furthermore, this protein has been associated with immunopathogenic responses in conjunctival105,109,112,113 and fallopian tube tissues.111,114 Interestingly, in vitro studies of persistence induced by gamma interferon (IFN-gamma), penicillin,115 or amino acid deprivation116 have shown that viable organisms cannot be recovered during this persistent state. Furthermore, these infections upregulate Hsp60 production.106 Because chlamydial Hsp60 has high homology with human Hsp60, it is possible that persistently infected cells trigger an autoimmune response to self-Hsp60.117 In a recent study of trachoma patients in Nepal,105 specific tear anti-Hsp60 IgG immunoreactivity in C. trachomatis-infected patients was significantly associated with scarring disease, whereas high rates of anti-Hsp60 sIgA antibodies were not. However, it is not entirely clear whether Hsp60 directly causes disease, elicits a specific host immune response that is damaging, represents prolonged exposure to many different chlamydial antigens, or is a combination of these factors. In another study of in vitro persistence, cytokines and chemokines were released by persistent cells that may induce severe inflammation and fibrosis.118 In addition, a depressed CMI response may prevent the host from clearing an infection,119 which may lead to persistence, inflammation, and conjunctival scarring.1 Thus, persistent organisms may chronically or intermittently produce immunogenic antigens or stimulate the release of cytokines/chemokines that precipitate the pathogenic responses seen in trachoma patients.

The role of genetic factors in determining severity of disease in trachoma populations is not well understood. In the mouse genital tract model of chlamydial STDs, the immune response to chlamydial Hsp60 was found to be genetically restricted.120 This may in part explain the wide range in disease severity observed within trachoma endemic populations.1 There are a few studies that suggest a genetic component to severity of the immune response in trachoma. An early study found that an important risk factor for scarring was evidence for maternal trichiasis.121 In another study, scarring was more common among individuals who had moderate to severe inflammation repeatedly in childhood.94 It has also been reported that siblings tend to have the same degree of inflammatory response.64 In the same study, persistently infected children had significantly more severe disease than children who were not persistently infected.64 However, this may have been partially because the persistently infected children had significantly higher loads of chlamydial DNA. Nonetheless, these findings suggest that there is likely a genetic component to the host immune response in trachoma.

Specific genetic markers for disease susceptibility are ill defined and limited in geographic scope. HLA class I allele A*6802 has been associated with trachomatous scarring in one study in The Gambia.122 But the mechanism(s) of this association are unknown, although the finding would suggest a role for CD8 T cells in trachoma pathogenesis. Trachomatous scarring has also been associated with high titers of TNF-alpha in tears and with mutations in the promotor for TNF-alpha.67 The potential mechanisms here include genetic determinants that control CD4 TH1 versus TH2 cytokine responses to chlamydiae and circulating antibody responses to chlamydial Hsp60. Other studies have shown elevated levels of IFN-gamma and interleukin (IL)-10 mRNA transcripts in severe trachomatous scarring,123 suggesting a combined TH1 and TH2 immune response. In twin studies in The Gambia,124 peripheral blood monocytes (PBMCs) were measured in response to chlamydial EBs and estimated at 39% (p = 0.07) for heritability. Additional studies of genetic markers in diverse trachoma populations worldwide will be needed to more fully estimate the role of host genetic determinants in chlamydial disease susceptibility.

The recently published sequence of the entire genome of C. trachomatis serovar D led to the identification of several ORFs predicted to encode outer membrane proteins, including a nine-member Pmp gene (pmp) family. Although it is not clear what role these membrane proteins may play in the pathogenesis of chlamydial ocular diseases, they are likely important because the pmp family is unique to the genus Chlamydia, comprises a surprising 10% of the genome, and interspecies amino acid sequence homology is less than 50% compared with 70% to 80% for other surface proteins. This will be an important and exciting area of research to define surface expression, antigenic determinants, and their potential role in host immune stimulation and pathogenesis.

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The histologic aspects of chlamydial infections include an array of features that change as the disease progresses. Initially, lymphocytes infiltrate the submucosa and deeper connective tissue. These form early follicles without germinal centers. Subsequently, there is progression to discrete follicles that have an outer layer composed primarily of lymphocytes with a central germinal center composed of macrophages in which the germinal center is displaced to the surface of the follicle.125–127 This feature distinguishes it from that the appearance of most lymph nodes. The conjunctiva is bound down to the tarsal plate, and, at these sites, villous processes develop and form papillae with vascular tufts in the center.128 Between the papillae are folds of epithelium. The lymphoid follicles reside primarily in the conjunctiva but can occur in the connective tissue below. In addition, the conjunctiva appears to be loosened over the follicle, which permits access of other cells and material into the follicle. The epithelium and subepithelium become infiltrated with polymorphonuclear cells (PMNs) in addition to lymphocytes. Inflammatory infiltrates and dilated papillary vessels can result in thickening of the entire conjunctivae. Plasma cells are present in both the epithelium and subepithelium and suggest the presence of chronic infection.1 The surface membrane of goblet cells can be altered, which may affect the normal absorptive surface of the epithelium127 and, thereby, disrupt the defense mechanism of the epithelium. At this point, the histopathology is similar to that of mucosal Peyer's patches in the intestine.

The follicles can become necrotic with connective tissue formation surrounding the follicles that results in fibrosis.1,129 As scarring occurs, posttrachomatous degeneration can develop. This is a condition in which the epithelium between papillae becomes trapped and forms cysts that are lined with epithelial cells. Follicles that form at the corneal-scleral junction do not cause ulceration or induce connective tissue formation, and, thus, there is no scar formation.1 However, Herbert's pits can develop, which are visualized as depressed points usually at the superior limbal border.130 Herbert's pits are the only pathognomonic feature of trachoma. Pannus formation can also occur at the limbal border and is characterized by a superficial vascularization of this tissue. It is usually localized to the superior limbus but can be appreciated anywhere along this border.1 Trichiasis develops later on from gradual contraction of both the superficial and deep scars located near the lid margin.

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Although there are no ideal models for chlamydial ocular infections, a few animal models have been developed that have advanced the understanding of ocular chlamydial diseases. These include guinea pigs that are naturally infected by C. psittaci131 and nonhuman primates who are experimentally infected with various C. trachomatis serovars. These two models comprise the bulk of the research that has been performed to date. Mouse strains have also been used as ocular models but for very limited studies in the past132 and recently.133 Initial guinea pig studies involved inoculation of the conjunctiva with guinea pig inclusion conjunctivitis (GPIC) strain of C. psittaci. The guinea pigs developed severe disease that took approximately 3 to 4 weeks to resolve and that stimulated the development of systemic and local antibodies.134,135 On challenge, the animals were resistant to reinfection, which was considered evidence for a protective immune response. This response appeared to be associated with elevated local sIgA and IgG antibodies. Furthermore, passive transfer of immune sera or tears to naïve guinea pig eyes (either preinfection or postinfection with GPIC) resulted in not only a delay in onset of disease but also lower levels of infection.134 Evidence for protective immune responses were also supported by experiments in which immunosuppressive treatment with cyclophosphamide, which targets the humoral immune response but not the CMI, resulted in delayed antibody responses at both sites, as well as persistent conjunctival infection that lasted 3 weeks beyond that of controls.136 This would also imply that CMI responses alone were insufficient to resolve disease. It should be noted that ocular challenge of guinea pigs subjected to passive transfer of immune sera were not protected.137,138 This provides further evidence that local sIgA and IgG, and not systemic IgG responses, are important in protection. There is also support for what has been referred to as “common circulation among mucosal sites.”139 In one study,139 guinea pigs were given viable GPIC orally and challenged at the genital and ocular mucosa; protection was observed at both sites regardless of the site of challenge. Repeated challenge with GPIC at 25 to 112 days has been noted to produce chronic inflammation with conjunctival scarring and pannus similar to that seen in humans.95 This response is considered a delayed hypersensitivity (DTH) response and is part of the CMI response.

Nonhuman primates have been experimentally inoculated with different strains of Chlamydia since the 1960s. Interestingly, the response to infection has varied depending on the primate species that was infected.140–142 Chlamydial STD strains caused a severe follicular conjunctivitis in Old World monkeys (baboons, langurs, and Macaca spp.) who, in some cases, developed scarring and pannus. However, the latter was appreciated only in repeatedly infected Taiwan monkeys.143 The trachoma serovars caused a milder form of the disease. The former infections resolved within 3 months at which time they were susceptible to reinfection. Trachoma serovar infections of New World monkeys (Aotus trivirgatus) resulted in acute conjunctivitis with disease resolution within 10 to 15 days and protective immunity to rechallenge.141

A model for chronic cicatrizing trachoma was produced in the cynomolgus monkey by weekly inoculations of either serovar A or E.96 Both serovars produced similar results: a chronic follicular conjunctivitis with few cases of scarring but no pannus. Viable organisms could be cultured for up to 8 weeks. Peak systemic IgG and tear IgA antibodies were produced at the time of peak infection, although the tear IgA responses tended to develop more slowly and decrease more rapidly compared with the serum IgG responses. On conjunctival challenge, viable organisms were present only up to the time when IgA antibodies developed. A similar model in rhesus monkeys was not as successful.

The effect of initial infection with subsequent rechallenge, similar to what is seen among children in endemic regions, was also explored in the cynomolgus monkey model.96 Following initial infection with serovar E, the monkeys developed a follicular conjunctivitis that persisted for 4 weeks with subsequent slow resolution during which time the organism could be cultured. At 15- to 30-weeks postinfection, the monkeys were rechallenged and found to develop a milder disease that resolved within 14 days. Thus, the animals were susceptible to reinfection, but the time to resolution was much shorter. The effect of immunosuppression on eye disease was also examined.144 In these cases, monkeys were treated with cyclosporine and then infected with chlamydial serovars. The follicular conjunctivitis persisted and was associated with infection and lower levels of tear IgA levels compared with immunocompetent monkeys. Thus, local production of IgA appears to be important in protection and resolution of infection.

Oral immunization protocols with subsequent ocular challenge of chlamydial serovars were also investigated. Serovar B was used in some experiments, and no difference was appreciated for duration of disease or presence of viable organisms recovered from the conjunctiva.145 Oral immunization with LGV followed by serovar B inoculation of the conjunctiva revealed a milder conjunctivitis but prolonged shedding of viable organisms. In contrast, use of ultraviolet-inactivated L2 for oral immunization resulted in prolonged disease and shedding. Thus, nonviable organisms were not able to stimulate an appropriate immune response. Immunization by different routes (oral, intramuscular, and rectal) resulted in early resolution of disease and shedding.

The impact of CMI response has also been studied in the cynomolgus monkey model. During peak infection, the conjunctiva of these animals were sectioned and stained for T-cell phenotypes.146 Both CD8+ and CD4 cells were present, but the former predominated, which suggested that a DTH response was likely involved.

In the early murine model of chlamydial ocular infection, limited duration of infection and lymphoproliferative responses were observed, although the mice did develop systemic IgG antibodies.132 More recently, three inbred strains of mice were infected with serovar C of C. trachomatis applied to the conjunctiva.133 Two strains of mice, BALB/c and C3H/HeN, were susceptible to infection. The organism could be recovered for up to 21-days postinfection, and histopathologic analyses revealed subepithelial mononuclear infiltration and loss of goblet cells within 1 week of infection. Furthermore, lymphoproliferative responses to chlamydial EBs were present for infected but not for uninfected mice. This model has been used recently to test an orally and systemically administered monoclonal anti-idiotypic antibody (anti-Id) to chlamydial exoglycolipid antigen (GLXA) as a vaccine to stimulate systemic immunity.147 Protection from ocular challenge with a C. trachomatis human serovar occurred, producing systemic anti-chlamydial neutralizing antibodies and anti-GLXA antibodies. However, there were no data regarding mucosal IgA responses, which are likely important in disease prevention and resolution. The mouse model of trachoma, however, is potentially exciting, because it can take advantage of the vast array of resources available for studies in mice, as well as the body of knowledge that has been accumulating over the last decade from the mouse model of chlamydial genital tract infections.

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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 important indicators for trachoma, and at least two must be present per person examined148: follicles in the upper tarsal conjunctiva, limbal follicles or their sequelae, Herbert's pits (which are pathognomonic for trachoma), typical conjunctival scarring, and vascular pannus. These signs along with conjunctival detection of C. trachomatis confirm the presence of endemic or hyperendemic trachoma in the respective area.

The World Health Organization (WHO) developed an extensive grading scale for trachoma, the MacCallan classification system,148 which has subsequently been modified for use in field applications.9 This modification has assisted in the understanding of the epidemiology of clinical disease, risk factors for disease, and disease prevalence.61 The simplified trachoma grading scale includes

  TF: trachoma follicles: 5 or more follicles in the central upper tarsal conjunctiva
  TI: intense trachoma: diffuse infiltration of the upper tarsal conjunctiva that obscures the deep tarsal vessels over 50% or more of the tarsal surface
  TS: trachomatous scarring of the conjunctiva
  TT: trichiasis (inturned eyelashes) that touches the eyeball
  CO: corneal opacity that impairs vision
  Active trachoma: presence of TF or TI
  Intense trachoma: presence of TI
  Cicatricial trachoma: presence of TS
  Healed trachoma: TS without TF or TI

Although this grading scale retains some features of the older system, including intensity of inflammation (TF, TI), trichiasis (TT), and sequelae (CO), it does not go into detail, for example, on the degree of conjunctival scarring that is essential for predicting disease complications.61


Conjunctival smears or swabs are required for making a diagnosis. The tests available to detect C. trachomatis include Giemsa stain, Lugol's iodine, or DFA for smears and for swabs, culture, enzyme-linked immunosorbent assay (ELISA), DNA probes, or commercial PCR or LCR. 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. Conjunctival smears should first be examined for the quality of the specimen. Epithelial cells that are clearly separated and the presence of PMNs, lymphocytes, plasma cells, or Leber cells (giant macrophages that contain phagocytosed material) can denote an adequate sampling of the conjunctiva. The degree of inflammation and bacterial superinfection can also be appreciated from these smears. Thus, although not diagnostic for chlamydiae, these findings are suggestive of trachoma.149 Giemsa stain is inexpensive, and the test is easy to perform, which makes it attractive 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.1 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.149 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. The DFA test detects the EBs instead of the inclusion body. Commercially 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.150 The EBs are stained an apple green color and are visualized as extracellular round dots. Technical expertise is critical to differentiate debris from true EBs. The sensitivity for this test is approximately 80% to 90%.


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.151 Today, tissue culture has supplanted the use of eggs, which has made isolation of chlamydiae more widely available, although it is still only performed in specialized reference laboratories. Tissue culture remains the gold standard for C. trachomatis identification but is not 100% sensitive,152 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, the enthusiasm for this test is decreased. Furthermore, culture requires technical expertise, can take 3 to 6 days for results, and is very expensive. Many different 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 detection of chlamydial inclusion bodies but are less frequently used.


ELISA or enzyme immunoassay (EIA) commercial assays are available to detect chlamydiae, but the sensitivity is only 70% to 85%.1 However, these assays can be cost effective compared with other commercially available tests such as the DNA detection assays153(see later). These tests detect the EB via polyclonal or monoclonal antibodies directed against the genus-specific chlamydial LPS. The antibodies are conjugated with an enzyme that reacts with a substrate to produce 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. The chlamydial LPS is blocked by an antibody, and the sample is rerun; if a positive sample is now negative, then it is considered a true positive. However, if a positive sample is still positive, then it is a false positive, because the assay is probably detecting LPS from other bacterial species.


The commercial LCR (Abbott) and PCR (Roche Diagnostics) 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. There have been some recent reports of C. trachomatis strains that do not contain the plasmid.154 Exactly how many strains are missing the plasmid is not known; the numbers are likely to be small and so would not significantly affect the sensitivity of these tests, which are in the range of 92% to 99%. 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 assays that employ a DNA purification protocol that removes the inhibitors. However, the internal control plates do add to the cost of the assay, and not all labs have the capabilities to perform the in-house purification assays or PCR. The commercial tests have been used in very few trachoma studies to date59,155,156 but appear to have similar sensitivities and specificities as in STD populations.

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%.


There are two serologic tests for Chlamydia: the microimmunofluorescent (MIF) test157 and the complement fixation (CF) test. However, neither is specific for the organism because patient sera can crossreact with different serovars and species and may represent current or previous sexually transmitted infection as opposed to conjunctival infection. The highest antibodies 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.158 Furthermore, ocular chlamydial infections tend to be chronic and endemic. Thus, these assays cannot 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 applied 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 binding. The slides are screened under fluorescent microscopy for fluorescing EBs that represent 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 areas159 and 90% of adults with inclusion conjunctivitis160 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 proportional to the severity of disease and to the presence of chlamydial organisms in the conjunctiva. 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.159 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 titers 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.157

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Trachoma was initially treated using copper sulfate solutions or crystals. The introduction of sulfonamides allowed for their use in the treatment of American Indian populations, which resulted in a marked decline in trachoma prevalence rates.83 For many tribes, trachoma was eradicated by 1942. Most developed countries have experienced complete elimination of trachoma because of a combination of factors including improved personal hygiene, improved sanitation, and access to clean water. Because of the lack of these socioeconomic indicators in developing countries, where trachoma is endemic, the WHO initiated control programs that used topical treatment with tetracyclines and erythromycins. Topical treatment, which was applied as continuous or intermittent regimens over a period of weeks or months, resulted in disease reduction, but these effects were short lived. Longer term and more consistent success rates were achieved with systemic treatment using sulfonamides or tetracyclines.161 However, a major problem with these regimens was compliance with taking a drug multiple times for many days. Also, children and pregnant women could not be treated with tetracycline drugs because it is contraindicated. A newer azalide antibiotic, azithromycin, has recently been introduced and used to treat trachoma cases in Africa. One to six doses of oral azithromycin, administered either as a community-wide treatment program or to selected persons, resulted in reduction of active trachoma and infection rates for approximately 12 months.59,155,162,163 However, no long-term follow-up was done to determine the time interval at which infection rates began to rise or returned to baseline. It may be possible that multiple treatments of azithromycin over a few years will prove to lower the rates of infection so that the burden of disease is decreased and blindness no longer occurs. But more comprehensive studies will need to be done to determine if this is likely. Furthermore, studies will need to address whether resistance to azithromycin develops as a consequence of mass treatment programs. To date, resistance to tetracycline and erythromycin has been reported but only for sporadic cases of sexually transmitted strains of C. trachomatis.164

Because azithromycin and mass treatment programs are expensive, along with the unlikely possibility that the socioeconomic status of trachoma-endemic countries will change anytime soon, one is still faced with the challenge of controlling trachoma to prevent blinding disease. The number of persons who are blind from trachoma is estimated to be 12 million by the year 2020.46 The WHO initiated the Global Elimination of Trachoma 2020 program (GET 2020), which employs the SAFE strategy.2,165 The SAFE program encompasses four different arms of treatment and prevention of disease: surgery for TT; antibiotics for active disease; face washing and good personal hygiene; and environmental improvements, consisting of improved sanitation (waste disposal and better water delivery systems). A number of trachoma-endemic countries in Africa and Asia have instituted the SAFE program, but results of these programs will not be available for a few years.


The best form of treatment for adult inclusion conjunctivitis is to prevent chlamydial STDs. Unfortunately, most chlamydial STDs are asymptomatic for males (approximately 40%) and females (approximately 70%) and usually go undetected because routine diagnostic screening for C. trachomatis is not performed.1 Thus, it is important to recognize adult inclusion conjunctivitis that is caused by C. trachomatis and treat both the ocular and genital tract disease. Because chlamydial STDs cannot be resolved by topical ocular antibiotics, systemic therapy is recommended. Most cases infected with non-LGV serovars will respond to 2 to 3 weeks of oral tetracycline, doxycycline, or erythromycin.166 For LGV cases, 6 weeks is required for complete eradication.167 The best treatment regimen for inclusion conjunctivitis caused by C. psittaci and C. pneumoniae is unknown, although 6 weeks of oral antibiotics has been successful in some cases.7


Treatment of pregnant women is effective in preventing 90% of infants from acquiring conjunctival infection,168 although retreatment may be necessary in high-risk populations.169 Topical treatment of neonatal inclusion conjunctivitis does not eradicate nasopharyngeal carriage, which can result in pneumonia or recurrent ocular infection. Thus, systemic treatment is recommended. Oral erythromycin at 50 mg per kg of body weight in four divided doses is required for 10 to 14 days. Approximately 80% of cases will respond to this regimen, but additional treatment may be necessary for some cases.170 In addition, mothers should be treated for genital tract infection to prevent recurrence of chlamydial conjunctivitis in the neonate.

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The development of an efficacious vaccine for C. trachomatis that would prevent and resolve infection has been slow largely because of the intracellular nature of Chlamydia and lack of ability to genetically transform the organism. However, recent advances in the field have identified some requirements for vaccine design. It is now generally accepted that MOMP, possibly with other antigens, would be important for a vaccine. However, because of the diversity of MOMP sequences that define different C. trachomatis strains, more than one MOMP would be required. The immune response that must be induced comprises mucosal sIgA antibody and systemic antigen-specific CD 4 TH1 lymphocyte responses. Protection of mice against challenge with MoPn has been partially successful using vaccine strategies that include conformationally intact MOMP,171,172 naked DNA constructs of ompA,173,174 and intact, nonviable organisms carried by dendritic cells.175 Other attempts at vaccination were less successful and included recombinant poliovirus or Salmonella expressing MOMP, denatured MOMP, or MOMP peptides (summarized in Brunham176). It is likely that a composite vaccine that includes intact MOMP, as well as naked DNA representing ompA from various strains, may be required to stimulate appropriate B- and T-cell responses, respectively. It may also be that only a few MOMP and ompA DNA strain sequences or only specific conserved sequences from MOMP and ompA are required to elicit a protective immune response (17). The development of the ideal vaccine remains a significant challenge.
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