Rickettsia rickettsii and Rickettsia typhi in inhabitants from a rural community of southeast Mexico

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To the editor.The genus Rickettsia includes bacteria of the phylum Proteobacteria, of subclass a-1 of the order Rickettsiales.Traditionally, this genus is divided into two major clades: the typhoid group (TG) which includes Rickettsia typhi and Rickettsia prowazekii, transmitted by fleas and lice, and the spotted fever group (SFG) which includes more than 20 species, including Rickettsia rickettsii, mainly transmitted by "hard" ticks.These bacteria are characterized by causing emergent or re-emergent infection (depending on the infecting species) in people and animals around the world, known as rickettsial diseases or rickettsiosis (1) .In southeastern Mexico, several Rickettsia species have been reported in humans, animals, and ectoparasites of numerous species (2) , making it an endemic area and a "hot spot" for these bacteria.The aim of this study was to describe the frequency of Rickettsia spp.DNA in asymptomatic inhabitants of a rural community in southeastern Mexico and to identify the species involved in the infection through the use of bioinformatic analysis.
This research was approved by the Research Ethics Committee of the Centro de Investigaciones Regionales "Dr.Hideyo Noguchi", Universidad Autónoma de Yucatán (act: CEI-007-2018).Additionally, we considered the statutes of the World Medical Assembly of Helsinki and the International Code of Medical Ethics for the collection of biological samples and safeguarding of personal data.
From March to April 2018, we worked with 128 (asymptomatic) inhabitants of Maxcanú, Yucatán, Mexico.For each participant, we collected peripheral blood (5 mL in adults, 3 mL in children) in BD-Vacutainer® tubes with anticoagulant (EDTA-K2) that were kept in a cooler with refrigerants for transfer to the laboratory.The samples were centrifuged (15,000 revolutions per minute for 15 minutes at 24 °C) to collect the white blood cell package that was used for total DNA extraction using the QIAamp DNA Mini Kit® (QIAGEN®), "DNA Purification from liquids and fluids" protocol.The extracted DNA samples were evaluated in a NanoDrop-2000® spectrophotometer (Thermo Scientific®) to determine their concentration and purity.
Two PCRs were performed to identify Rickettsia DNA in blood samples, one nested and one multiplex semi-nested, for detection of the 17kDa and sca5 genes, respectively, using the previously described methodologies (3) .Positive (Rickettsia conorii DNA) and negative (sterile water) controls were considered in all reactions.Electrophoresis of the products of both PCRs was carried out on agarose gels (1%) stained with ethidium bromide (8%).
The sca5 amplicons (17kDa amplicons were not considered) with the expected sizes (420 and 230 base pairs for R. rickettsii and R. typhi, respectively) were purified with the Gel DNA Recovery kit (ZymocleanTM®) according to standardized specifications, and sent to the DIMYGEN® laboratory (Merida, Mexico) for sequencing (sense and antisense) by the Sanger method.The obtained sequences were edited and aligned with MEGA version 7.0® software (https://www.megasoftware.net/), and subsequently compared, using the Megablast algorithm, with partial sca5 sequences from Rickettsia previously deposited in GenBank (https://blast.ncbi.nlm.nih.gov/Blast.cgi), in order to determine their identity and coverage percentages.
Rickettsia DNA was identified in 34.4% (44/128) of the blood samples.The results of the GenBank analysis are shown in Table 1.As can be confirmed, seven sequences showed identity and coverage for R. rickettsii and two for R. typhi.
Isolated cases of infection with R. typhi (4,5) and R. rickettsii (6) have been reported in inhabitants and animals of Yucatan; nevertheless, our findings are important because of the evidence of infection (bacteremia) with two species of Rickettsia in the same asymptomatic studied population, which represents advances in the epidemiological study and clinical approach to cases of rickettsiosis in southeastern Mexico.Changes in the ecological niche, the continuous circulation of vectors (ticks and fleas), the presence of reservoirs (animals) and accidental hosts (humans) that maintain the transmission cycles of the different Rickettsia species (2,3) in Yucatan, could be associated with our report.
One of the limitations of our study is that only nine positive products were sent for sequencing, which could limit the identification of more Rickettsia species involved in the infection.Another limitation is the lack of clinical follow-up and subsequent generation of data from Rickettsia DNA-positive individuals.These data are relevant to improve the approach and diagnosis of rickettsial diseases in Yucatan.
Two Rickettsia species were found in asymptomatic individuals in southeastern Mexico.Active surveillance and vector control (ticks and fleas) are recommended to reduce the risk of Rickettsia transmission in the inhabitants and domestic animals of Yucatan communities (3) , as well as to conduct studies to better understand the protagonists in the rickettsial cycle and reduce the incidence of these diseases in southeastern Mexico.

Table 1 .
Coverage and identity percentages shown by bioinformatic analysis with Megablast, homologous Rickettsia species and GenBank access number for homologous sequences with sequences obtained from sca5 amplicons in an asymptomatic population from southeastern Mexico.