Unravelling Chlamydia trachomatis Diversity in Amhara, Ethiopia: MLVA-ompA Sequencing as a Molecular Typing Tool for Trachoma

Trachoma is the leading infectious cause of blindness worldwide and is now largely confined to around 40 low- and middle-income countries. It is caused by Chlamydia trachomatis (Ct), a contagious intracellular bacterium. The World Health Organization recommends mass drug administration (MDA) with azithromycin for treatment and control of ocular Ct infections. To understand the molecular epidemiology of trachoma, especially in the context of MDA and transmission dynamics, the identification of Ct genotypes is a necessity. While many studies have used the Ct major outer membrane protein (ompA) for genotyping, it has limitations. Our study applies a novel typing system, Multiple Loci Variable Number Tandem Repeat Analysis combined with ompA (MLVA-ompA). Ocular swabs were collected post-MDA from four trachoma-endemic zones in Ethiopia between 2011-2017. DNA from 300 children with high Ct polymerase chain reaction (PCR) loads was typed using MLVA-ompA, utilizing three variable number tandem repeat (VNTR) loci within the Ct genome. Results show that MLVA-ompA exhibited high discriminatory power (0.981) surpassing the recommended threshold for epidemiological studies. We identified 87 MLVA-ompA variants across 26 districts. No significant associations were found between variants and clinical signs or chlamydial load. Notably, overall Ct diversity significantly decreased after additional MDA rounds, with a higher proportion of serovar A post-MDA. Despite challenges in sequencing one VNTR locus (CT1299), MLVA-ompA demonstrated cost-effectiveness and efficiency relative to whole genome sequencing, providing valuable information for trachoma control programs on local epidemiology. The findings suggest the potential of MLVA-ompA as a reliable tool for typing ocular Ct and understanding transmission dynamics, aiding in the development of targeted interventions for trachoma control. Author SummaryTrachoma is the leading infectious cause of blindness worldwide and is largely confined to low- and middle-income countries. It is caused by Chlamydia trachomatis (Ct), a contagious intracellular bacterium. The World Health Organization recommends mass drug administration (MDA) with the antibiotic azithromycin for treatment of ocular Ct infections. In most regions MDA is successfully reducing trachoma prevalence to the point where it is no longer a public health issue, however in some places trachoma persists despite multiple rounds of treatment. To investigate why trachoma persists, especially in the context of MDA and transmission dynamics, the identification of Ct genotypes is necessary. Our study applies a novel Ct typing system, which augments the standard method by adding three loci with high mutation rates. Results show that the novel typing system was able to discriminate between variants with greater resolution than the standard method, and was both cost-effective and more efficient relative to the gold-standard of whole genome sequencing. The findings suggest that this novel method is a reliable tool for typing ocular Ct, which can aid in the development of targeted interventions for trachoma control.