Review 1: "Unravelling Chlamydia Trachomatis Diversity in Amhara, Ethiopia: MLVA-ompA Sequencing as a Molecular Typing Tool for Trachoma"

Valentina Feodorova

doi:doi:10.1162/2e3983f5.9c0586ce

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

by Anna Harte, Ehsan Ghasemian, Harry Pickering, Joanna Houghton, Ambahun Chernet, Eshetu Sata, Gizachew Yismaw, Taye Zeru, Zerihun Tadesse, E. Kelly Callahan, Scott D. Nash, and Martin Holland

Show Description
Published on Feb 07, 2024
www.medrxiv.org

Description

Abstract 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 Summary Trachoma 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.

RR:C19 Evidence Scale rating by reviewer:

Potentially informative. The main claims made are not strongly justified by the methods and data, but may yield some insight. The results and conclusions of the study may resemble those from the hypothetical ideal study, but there is substantial room for doubt. Decision-makers should consider this evidence only with a thorough understanding of its weaknesses, alongside other evidence and theory. Decision-makers should not consider this actionable, unless the weaknesses are clearly understood and there is other theory and evidence to further support it.

***************************************

Review: The main goal of this manuscript was to evaluate the efficacy of MLVA-ompA and test its discriminatory power (DP) on the DNA samples derived from conjunctival specimens of children living in trachoma-endemic areas of Amhara, Ethiopia. In fact, this is the first report concerning the investigation of Chlamydia trachomatis (CT) diversity using the MLVA-ompA which resulted in identification of at least 87 MLVA-ompA variants across 26 districts of the country. The authors could present the detailed information of the cohort investigated. Additionally, I highly appreciate the detailed study protocol and analytical analysis presented by the authors that make this research very attractive for understanding all key conclusions and reproducible as well. The current research is a part of the global strategy of the World Health Organization known as the SAFE (surgery, antibiotics, facial cleanliness, and environmental improvement), the Trachoma Control Program, which was realized since 2007 in the Amhara Region, Ethiopia. The majority of clinical specimens were used for both researches, presented in the paper published (Pickering H, Chernet A, Sata E, Zerihun M, Williams CA, Breuer J, Nute AW, Haile M, Zeru T, Tadesse Z, Bailey RL, Callahan EK, Holland MJ, Nash SD. Genomics of Ocular Chlamydia trachomatis After 5 Years of SAFE Interventions for Trachoma in Amhara, Ethiopia. J Infect Dis. 2022 Mar 15;225(6):994-1004. doi: 10.1093/infdis/jiaa615. PMID: 33034349; PMCID: PMC8922003) and in the current study as well. Overall, the reviewed manuscript is logically continued but does not copy the research indicated.

However, there are some points that could be considered to make this study even more attractive for a research community worldwide.

Specific comments:

Line 22 - MLVA-ompA is a method that has been known in CT since 2008 (Pedersen et al., 2008). This is not a novel method.
Lines 158-160 - all Ct trachoma genotypes, A, B, and C were identified in the 99 DNA samples which were investigated in the research of Pickering et al., 2022, and, probably, in the current study as well. Only genotypes A, B and Ba are presented in the reviewed manuscript. It should be checked.
Lines 162-166 - reference sequences from serovars A, B, C (trachoma); D (genital chlamydiosis), and L (lymphogranuloma venereum) were included for the current research. It is unclear why only the sequence of a single genotype D while not a panel of genital CT genotypes, such as D, E, F, G. H. J, and K were included in the study. I think it should be clarified.
Table 3 is difficult to interpret as not only the trachoma genotypes are included. I think it should be clarified.
The abbreviation must be corrected. As the authors used in their analysis of the nucleotide sequences, it corresponds to the term genotype but not serotype when diagnostic sera are applied.
Lines 103-104 – the authors aimed to study the DNA samples collected during 2011-2017. However, according to the section’ Study population’, additionally the samples from all 11 districts in the South Gondar zone, collected between 2014 and 2017 as part of the second round of surveys were also tested (lines 130-131). The goal of the study and the abstract should be corrected to make clearer to the readers the period of the survey and study conducted.
Figure 4 is not informative. It should present some additional data such as either CT type(s) identified or other data in each year indicated. Currently, the readers may recognize only different amounts of trachoma cases in each of the years.
There are several papers published in which co-infection by several genotypes of chlamydia patients was proved. In fact, in different research, more than a single genotype(s) were detected in conjunctival DNA samples. Whether any other genotypes of Ct could be identified in the ocular samples or not? This analysis could be very useful and reasonable as the whole genome sequences are available for the authors. Also, all the genomes sequenced should be deposited in the NCBI, and the relevant links included in the manuscript as Supplemental Materials.
The authors of the reviewed manuscript described several samples that were not typed by the technique MLVA-ompA. Thus, the authors should include the data from other molecular typing methods, such as MLST and ompAto make the data obtained more informative for the main readers and research community worldwide.
The authors include in the reviewed manuscript only the comparative analysis of three regions of CT according to the MLVA-ompA protocol (Pedersen et al., 2008). It is not enough for the molecular epidemiology of CT.
The authors demonstrate the novel CT types that were proved by the data presented in Figures 5 and 6. The relevant alignments should be included to demonstrate the polymorphisms found in the novel CT types described. The findings must be presented in the section Results and carefully discussed in the section Discussion.
The conclusion should present more concrete information on the current findings following the next direction in trachoma control in Africa overall and in the trachoma-endemic areas of Amhara, Ethiopia investigated. What is the benefit of this research? What's next?