Urogenital <i>Chlamydia trachomatis</i> multilocus sequence types and genovar distribution in chlamydia infected patients in a multi-ethnic region of Saratov, Russia

<div><p>Background</p><p>This is the first report to characterize the prevalence and genovar distribution of genital chlamydial infections among random heterosexual patients in the multi-ethnic Saratov Region, located in Southeast Russia.</p><p>Methods</p><p>Sixty-one clinical samples (cervical or urethral swabs) collected from a random cohort of 856 patients (7.1%) were <i>C</i>. <i>trachomatis</i> (CT) positive in commercial nucleic acid amplification tests (NAATs) and duplex TaqMan PCRs.</p><p>Results</p><p>Sequence analysis of the VDII region of the <i>ompA</i> gene revealed seven genovars of <i>C</i>. <i>trachomatis</i> in PCR-positive patients. The overall genovars were distributed as E (41.9%), G (21.6%), F (13.5%), K (9.5%), D (6.8%), J (4.1%), and H (2.7%). CT-positive samples were from males (n = 12, 19.7%), females (n = 42, 68.8%), and anonymous (n = 7, 11.5%) patients, with an age range of 19 to 45 years (average 26.4), including 12 different ethnic groups representative of this region. Most patients were infected with a single genovar (82%), while 18% were co-infected with either two or three genovars. The 1156 bp-fragment of the <i>ompA</i> gene was sequenced in 46 samples to determine single nucleotide polymorphisms (SNP) among isolates. SNP-based subtyping and phylogenetic reconstruction revealed the presence of 13 variants of the <i>ompA</i> gene, such as E (E1, E2, E6), G (G1, G2, G3, G5), F1, K, D (D1, Da2), J1, and H2. Differing genovar distribution was identified among urban (E>G>F) and rural (E>K) populations, and in Slavic (E>G>D) and non-Slavic (E>G>K) ethnic groups. Multilocus sequence typing (MLST) determined five sequences types (STs), such as ST4 (56%, 95% confidence interval, CI, 70.0 to 41.3), ST6 (10%, 95% CI 21.8 to 3.3), ST9 (22%, 95% CI 35.9 to 11.5), ST10 (2%, 95% CI 10.7 to 0.05) and ST38 (10%, 95% CI 21.8 to 3.3). Thus, the most common STs were ST4 and ST9.</p><p>Conclusion</p><p><i>C</i>. <i>trachomatis</i> is a significant cause of morbidity among random heterosexual patients with genital chlamydial infections in the Saratov Region. Further studies should extend this investigation by describing trends in a larger population, both inside and outside of the Saratov Region to clarify some aspects for the actual application of <i>C</i>. <i>trachomatis</i> genotype analysis for disease control.</p></div

Clonal grouping of sequence types (STs) of <i>C</i>. <i>trachomatis</i> strains found in our study (marked with gray ovals).

<p>The division into Group I (A) and Group III (B) was based on the EBURST analysis with other STs of these clonal complexes obtained from PubMLST/Chlamydiales database.</p

Phylogenetic tree demonstrating the relationship of Saratov <i>ompA</i> sequences to references strains from Table 1.

<p>Thick branches, subtending all large clades, represent 100% bootstrap support. The scale at the bottom represents one substitution per site.</p

Phylogenetic analyses of concatenated sequences of 7 housekeeping gene fragments of representative <i>C</i>. <i>trachomatis</i> strains.

<p><b>Concatenated sequences were aligned and analyzed in MEGA 7</b>. Phylogenetic tree was constructed using the UMPGA hierarchical clustering method model. Relevant STs & genotypes including in the clustering analyses are presented in Tables <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0195386#pone.0195386.t001" target="_blank">1</a> & <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0195386#pone.0195386.t002" target="_blank">2</a>, respectively.</p

The <i>ompA</i> and seven housekeeping genes MLST sequence types of Saratov and reference strains of <i>C</i>. <i>trachomatis</i> obtained from the PubMLST database (https://pubmlst.org).

<p>The <i>ompA</i> and seven housekeeping genes MLST sequence types of Saratov and reference strains of <i>C</i>. <i>trachomatis</i> obtained from the PubMLST database (<a href="https://pubmlst.org" target="_blank">https://pubmlst.org</a>).</p

Distribution of genovars in CT-positive patients.

<p><b>(A) According to their gender, place of residence and ethnic origin. (B) According to their age in mono-infected and multiple-infected CT-positive samples</b>. Analysis was done by Fisher’s exact test (two-tailed). Statistically significant differences are indicated by * (<i>p</i><0.05) or by ** (<i>p</i><0.01) or by *** (<i>p</i><0.001). Slavic cohort included Russians, Byelorussians and Ukrainians, Non-Slavic cohort was presented by Caucasians, Jews, Kyrgyzs, Koreans, Moldavians, Germans, Mordovians, and anonymous.</p