Zoonotic Chlamydiaceae Species Associated with Trachoma, Nepal

Deborah Dean

, James Rothschild, Anke Ruettger, Ram Prasad Kandel, and Konrad Sachse

Author affiliations: Children's Hospital Oakland Research Institute, Oakland, California, USA (D. Dean, J. Rothschild); University of California, San Francisco, California, USA (D. Dean); University of California, Berkeley, California, USA (D. Dean); Friedrich-Loeffler-Institut, Jena, Germany (A. Ruettger, K. Sachse); Lumini Eye Hospital, Bhairahawa, Nepal (R.P. Kandel)

Abstract

Trachoma is the leading cause of preventable blindness. Commercial assays do not discriminate among all Chlamydiaceae species that might be involved in trachoma. We investigated whether a commercial Micro-ArrayTube could discriminate Chlamydiaceae species in DNA extracted directly from conjunctival samples from 101 trachoma patients in Nepal. To evaluate organism viability, we extracted RNA, reverse transcribed it, and subjected it to quantitative real-time PCR. We found that 71 (70.3%) villagers were infected. ArrayTube sensitivity was 91.7% and specificity was 100% compared with that of real-time PCR. Concordance between genotypes detected by microarray and ompA genotyping was 100%. Species distribution included 54 (76%) single infections with Chlamydia trachomatis, C. psittaci, C. suis, or C. pecorum, and 17 (24%) mixed infections that includied C. pneumoniae. Ocular infections were caused by 5 Chlamydiaceae species. Additional studies of trachoma pathogenesis involving Chlamydiaceae species other than C. trachomatis and their zoonotic origins are needed.

Trachoma was first recognized as an ocular disease in the 27th century BC in China (1). Subsequent reports documented the disease among the Egyptians and Greeks in the 19th and 1st centuries BC, respectively. The word trachoma derives from the Greek word for rough swelling, referring to the follicles that appear on the tarsal conjunctiva. Epidemic trachoma was spread from the Middle East to Europe during the Crusades and was a major cause of blindness during the Napoleonic era (1). The disease was eliminated from most industrialized countries after the industrial revolution, which heralded the institution of improved sanitation, hygiene, and nutrition. Currently, trachoma prevalence is hypoendemic, mesoendemic, and hyperendemic among populations residing in tropical developing countries.
During the past few decades, rates of trachoma have increased; in response, at the end of the 1990s, the World Health Organization developed the SAFE program with the goal of eliminating blinding trachoma by the year 2020. SAFE refers to Surgery, Antibiotics, Facial cleanliness, and Environmental improvements, specifically, surgery to correct trichiasis (in-turned eyelashes), oral antimicrobial drugs to treat Chlamydia trachomatis infections, facial cleanliness to decrease ocular infections, and environmental improvements such as latrines and wells to provide clean water. Unfortunately, most efforts have focused on the surgery and antimicrobial drug components and had disappointing results. Trichiasis often recurs months to years after surgery for 25%–75% of patients (2,3) and can be a result of reinfection (3). Infection often returns to pretreatment levels 6–24 months after termination of treatment (4,5). The recurrence of infection and disease is probably multifactorial. There is evidence that oral treatment of C. trachomatis infection blunts the immune response, increasing the patient’s susceptibility to reinfection (4). Furthermore, additional species of Chlamydiaceae, namely Chlamydia pneumoniae and C. psittaci, have been implicated in trachomatous disease by our group (6) and by another independent research group from Paris working in Guinea, Africa (7). To eliminate infections with species other than C. trachomatis, longer treatment intervals might be required (8).
Although some Chlamydiaceae screening tests and strain-typing methods exist, they are expensive, are time-consuming, require trained personnel, and are available only in specialized laboratories; most do not discriminate among species of Chlamydiaceae. The tests or methods include serotyping of the major outer membrane protein by using monoclonal or polyclonal antibodies that are species or genus specific; commercial nucleic acid amplification tests for C. trachomatis only (9); conventional species-specific and genus-specific PCRs (10); direct sequence analysis of ompA, 16S rRNS, or 23S rRNA genes (11,12); multilocus sequence typing for C. trachomatis (13, 14) and other species (14); real-time (RT)-PCR (6,15), multilocus variable number tandem repeat analysis (16); and the commercial micro ArrayTube or ArrayStrip (Alere Technologies, Jena, Germany) (17). Serotyping requires a cultured isolate, and techniques that involve sequencing might not be able to detect mixed-strain or mixed-species infections unless multiple strain–specific or species-specific primers are used, which require sufficient quantities of DNA. The advantage of the ArrayTube or ArrayStrip is that minimal DNA is required for amplification, and the hybridization patterns indicate species-specific nucleotide polymorphisms in regions of high sequence similarity.
The commercial ArrayTube assay has been successfully used to identify mixed infections among animals infected with multiple species of Chlamydiaceae (18,19). Because of these benefits, we investigated whether the ArrayTube could discriminate among Chlamydiaceae species in DNA that was extracted directly from conjunctival samples from trachoma patients residing in a trachoma-endemic region of Nepal. We also evaluated the correlation of the ArrayTube test with ompA genotypes. As an independent test for viability of Chlamydiaceae organisms, RNA was isolated from the same samples and tested by quantitative RT-PCR (qRT-PCR).