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Localized cutaneous leishmaniasis (LCL) is an endemic disease in several Mexican States with the main endemic areas located in the South-Southeast region of the country, where 90% of Leishmania (Leishmania) mexicana cases are registered. The Southeast region is located in the Yucatan Peninsula, including Campeche, Quintana Roo and Yucatan States. Campeche and Quintana Roo register more than 60% of the cases in the country each year, while in Yucatan the reports are of imported cases due to residents traveling to endemic areas. However, since 2015, autochthonous cases have been diagnosed by health authorities in municipalities with no previous transmission records. We aimed to identify Leishmania parasite species involved in autochthonous cases by means of the PCR technique. The present study included 13 autochthonous cases of LCL with clinical and parasitological diagnoses during 2018 and 2019 by health authorities, without specific identification of the causal agent. Tissue samples were taken by scraping the margins of active lesions and then they were spotted onto an FTATM Elute Microcard. Next, DNA was eluted and used for PCR amplification of specific Leishmania genus and L. (L.) mexicana species-specific fragments. Molecular analysis showed evidence that L. (L.) mexicana was the causal agent of LCL in 12 of the 13 patients; in one patient, PCR was not performed due to the patient’s refusal to participate in the study. Identifying Leishmania species that cause LCL is necessary to define efficient treatment schemes and control strategies for the disease in vulnerable and susceptible areas of the Yucatan State’s municipalities.

The aim of the study was to describe the epidemiological factors associated with the risks of recent Rickettsia exposure in inhabitants of a rural population from Yucatan, Mexico. The study included 130 inhabitants from Maxcanu, Yucatan. Blood samples were collected to detect IgM and IgG antibodies against Rickettsia typhi and Rickettsia rickettsii by an indirect immunofluorescence antibody test. Additionally, nested polymerase chain reaction was performed to amplify fragments of the 17kDa and sca5 genes. Previously, an epidemiological questionnaire was applied focused on collecting information on personal and housing exposure variables related to the recent infection with Rickettsia to determine epidemiological associations. Results that exhibited a p-value < .25 were included in a generalized multinomial logistic linear model to determine the variables involved with the risk of contact or Rickettsia infection. In all, 76% (99/130) of the participants presented with immunoglobulin titres against the Rickettsia species evaluated, while rickettsial DNA was detected in 35.4% (46/130) of the participants. The association analysis with the personal exposure variables showed that the productive age group (OR = 0.32; 95% CI = 0.10-1.03) and the elders group (OR = 0.12; 95% CI = 0.01-0.83) were protective factors for recent infection with R. typhi, taking as reference the school group. The presence of a family orchard in the home (OR = 7.56; 95% CI = 1.62-35.29) was a risk factor for recent infection with R. rickettsii. Presumably, the presence of ectoparasites (OR = 2.71; 95% CI = 0.90-8.09) at home was a risk factor for recent infection with both Rickettsia species. Recent infection was demonstrated in inhabitants from Maxcanu, Yucatan. A high seropositive frequency was obtained. The results highlight the importance of the family garden and the presence of ectoparasites in the home as risk factors associated with recent infection with Rickettsia in inhabitants from Maxcanu.

Rickettsioses are vector-borne zoonotic diseases that occur in urban environments. Currently, they are associated with the presence of domestic and synanthropic animals, the ectoparasites that they harbor, and their local habitat. The implementation of prevention actions relies on the understanding of the local ecology of interactions between hosts, vector species, and the etiologic agents. In this context, this study aimed to explore and describe the occurrence of infected mammals and their ectoparasites in human urban dwellings, and those characteristics of urban dwellings associated to the presence of Rickettsia infected animals in groups of households where at least one human case of rickettsiosis has occurred in the previous year of the study. Briefly, blood-samples and ectoparasites from synanthropic and domestic animals, were obtained from groups of households from different areas of an urban settlement. Serologic and molecular diagnostics helped to identify Spotted Fever Group (SFG) and TG (Typhus Group) Rickettsia in animal and ectoparasite samples. A total of 99 mammals were sampled, 29 opossums (Didelphis virginiana), 13 house mice (Mus musculus), seven black rats (Rattus rattus) and 50 dogs. Infection occurrence in opossums was 8.3% of SFG, 50% for TG, and 4.2% of undetermined group. For house mice 46.2% for SFG and 30.8% were undetermined. Black rats 28.6% of SFG and 57.1% undetermined. Finally, dogs were 19.1% of SFG, 57.4% to TG, and 23.4% belonged to undetermined group. A total of 424 ectoparasites were collected from the mammals. In opossums occurred the ticks Ambyomma sp., Ornithodoros (Alecterobius) nr. talaje, and the flea Ctenocephalides felis. In dogs we found the ticks Rhipicephalus sanguineus s. l., Amblyomma sp., O. (A.) nr. talaje, and the flea Ct. felis. No ectoparasites were collected from rodents. The occurrence of infected animals was associated primarily with the material of the backyard floor, the type of sanitary system in the household, the presence of garbage in the backyard, presence of firewood storage, stored polyethylene terephthalate (PET) containers for sale to recyclers, and the store of construction supplies in the backyard. Nonetheless a generalized linear model showed that the household with a backyard with a dirt floor or other non-concrete material has more chances of harboring infected animals (RR= 1.74, 95% CI= 1.07-2.84 and RR= 1.03, 95% CI= 0.39-2.32 respectively). In contrast, when the house has a sanitary system of urban sewer system or a latrine outside de house, the chances of having infected animals decreased significantly (RR= 0.39, 95% CI= 0.12-0.94 and RR= 0.46, 95% CI= 0.03-2.22). We conclude that both SFG and TG rickettsioses occur in animals and their ectoparasites in peridomiciles of urban households were at least one human rickettsiosis case had occurred.

Rocky Mountain spotted fever is a disease caused by Rickettsia rickettsii, a bacteria transmitted by infected ticks. It is characterized by fever, exanthema, arthralgias and myalgias; but sometimes its clinical presentation is non specific. Due to its similarities with other exanthematic diseases like dengue or chikungunya, Rocky Mountain spotted fever is not a first line diagnosis, even though countries like Mexico show the ecologic and socioeconomic characteristics that favor its transmission, with a 30% mortality rate among pediatric patients. This mortality rate has been associated to a delayed diagnosis and therapy, due to a poor knowledge among physicians regarding this disease; this favors the occurrence of atypical and fulminant cases. The objective of this work is to describe a fulminant case of Rocky Mountain spotted fever, expecting that this disease could be later considered among the differential diagnosis which could directly impact its mortality rate.

Rocky Mountain spotted fever is a disease caused by Rickettsia rickettsii, a bacteria transmitted by infected ticks. It is characterized by fever, exanthema, arthralgias and myalgias; but sometimes its clinical presentation is non specific. Due to its similarities with other exanthematic diseases like dengue or chikungunya, Rocky Mountain spotted fever is not a first line diagnosis, even though countries like Mexico show the ecologic and socioeconomic characteristics that favor its transmission, with a 30% mortality rate among pediatric patients. This mortality rate has been associated to a delayed diagnosis and therapy, due to a poor knowledge among physicians regarding this disease; this favors the occurrence of atypical and fulminant cases. The objective of this work is to describe a fulminant case of Rocky Mountain spotted fever, expecting that this disease could be later considered among the differential diagnosis which could directly impact its mortality rate.La fiebre manchada de las Montañas Rocosas es una enfermedad ocasionada por Rickettsia rickettsii, una bacteria transmitida por garrapatas infectadas, y que se caracteriza por fiebre, exantema, artralgias y mialgias, aunque, ocasionalmente, su presentación es inespecífica. Debido a que su evolución asemeja otras enfermedades exantemáticas, como dengue o chikungunya, su diagnóstico no es de primera intención, a pesar de que países como México tienen las características ecológicas y socioeconómicas propicias para su transmisión, con índices de mortalidad hasta de 30% en pacientes pediátricos. Esta elevada mortalidad se asocia a diagnósticos y terapia retrasados debido al desconocimiento médico acerca de la enfermedad, lo que propicia la aparición de formas atípicas y fulminantes de fiebre manchada de las Montañas Rocosas. El objetivo del presente trabajo es describir un caso clínico fulminante de fiebre manchada de las Montañas Rocosas para que sea considerada en el diagnóstico diferencial, lo cual impactaría directamente en los índices de mortalidad

Rickettsia are intracellular vector-borne bacteria, which are the etiologic agent of severe infections that could inflict death to their host. The intracellular behaviour of Rickettsia makes the study of its genetics, proteomics and cellular processes very difficult. Hence, isolation remains an important experimental technique that permits the obtention of important yields of bacteria, useful for a broad range of experiments. Isolation of Rickettsia using passages in animals or embryonated eggs has been described for long time; however, it was until the 1990s that faster and more feasible approaches for cell culture were developed. Current isolation approaches are mainly based on shell vial culture, that varies according to the media, atmosphere or temperature conditions. These variations have allowed the establishment of isolates from different pathogenic and non-pathogenic Rickettsia species, using arthropod, animal or human samples. Purification method of bacteria has also witnessed changes alongside the quantification of its load in the resulting isolates, from the laborious and time consuming plaque assays, to the routinary use of real-time polymerase chain reaction (qPCR), which is faster and more accurate. This review discusses various approaches that have been used for the isolation and purification of different Rickettsia species along with the mention of some successful examples. It indicated that a successful strategy for the isolation of Rickettsia requires a careful selection of media, cell lines and culture conditions which now are not as time consuming as used to be.

Murine typhus is a rickettsiosis caused by Rickettsia typhi, whose transmission is carried out by rat fleas in urban settlements as classically known, but it also has been related to cat fleas in a sub-urban alternative cycle that has been suggested by recent reports. These studies remarks that in addition to rats, other animals like cats, opossums and dogs could be implied in the transmission of Rickettsia typhi as infected fleas obtained from serologically positive animals have been detected in samples from endemic areas. In Mexico, the higher number of murine typhus cases have been detected in the Yucatan peninsula, which includes a great southeastern region of Mexico that shows ecologic characteristics similar to the sub-urban alternative cycle recently described in Texas and California at the United States. To find out which are the particular ecologic characteristics of murine typhus transmission in this region, we analyzed blood and Rhipicephalus sanguineus ticks obtained from domestic dogs by molecular approaches, demonstrating that both samples were infected by Rickettsia typhi. Following this, we obtained isolates that were analyzed by genetic sequencing to corroborate this infection in 100% of the analyzed samples. This evidence suggests for the first time that ticks and dogs could be actively participating in the transmission of murine typhus, in a role that requires further studies for its precise description.

The aim of this study was to provide information of the occurrence of Rickettsia felis in wild mammals from three municipalities in Yucatan, Mexico. The reactivity of rodent serum to Rickettsia antigens was detected in 80.9% (17 of 21) samples using immunofluorescence assay. Polymerase chain reaction identified rickettsial DNA in spleens of 43.5% (10 of 23) rodents and 57.1% (4 of 7) opossums. The identification of the rickettsial DNA was confirmed as R. felis by restriction fragment length polymorphism and DNA sequencing. This study comprises the first report of R. felis detection in wild mammals in Yucatan.

To the Editor: Murine typhus is a febrile illness caused by Rickettsia typhi. The clinical manifestations are nonspecific, and the signs and symptoms resemble those of several other febrile illnesses. Murine typhus can be a self-limiting infection; however, it should be diagnosed and treated because complications and even death can result (1). In Mexico, particularly in Yucatan State, cases of murine typhus in humans and high prevalence of antibodies in healthy blood donors have been reported (2,3). In 2012, we isolated R. typhi from a human patient in southeastern Mexico by using a simple and effective method, an adaptation of the centrifugation shell vial method to cell culture plates. The patient, a 23-year-old man from Dzibzantun (21°15′00″N, 89°03′00″W), in the northeastern part of Yucatan State, was referred for possible diagnosis of rickettsial infection. He had a low-grade fever (37.6°C) and a maculopapular rash on the thorax and upper and lower extremities. The patient reported having cats in the house, but no fleas or ticks were observed. Clinical laboratory findings were within reference ranges. Test results were negative for dengue virus, but the Weil-Felix (Proteus OX19) test result was positive (titer 1:164). Single-step PCR amplification was performed by using genus-specific primers for the 17-kDa lipoprotein and the citrate synthase gene (gltA), as described previously, to obtain amplicons of 434 bp and 380–385 bp (4). PCR was positive for R. typhi, and 100 mg of oral doxycycline 2 times per day for 7 days was prescribed; the rash cleared. We subjected 5 mL of blood to centrifugation for 1 hour at 1,000 rpm and then stored the plasma at −80°C. Blood samples from other patients were used as controls. A total of 50,000 Vero cells were grown in 8 central wells of a 24-well cell culture cluster (Corning Incorporated, Corning, NY, USA) with minimal essential medium (MEM; Biowest, Nuaille, France) supplemented with 10% fetal bovine serum (Biowest) and incubated at 37°C with 5% CO2 for 48 hours to obtain 95% confluence. We then thawed 700 μL of the plasma in a 37°C water bath. The MEM was discarded, and the wells were refilled with 250 μL each of a mixture of the plasma and fresh medium at a 1:3 ratio. The plaque was covered with parafilm and centrifuged at 700 g for 60 minutes at 22°C. The supernatant was discarded and replaced with 1 mL of MEM supplemented with 5% fetal bovine serum, 100 U penicillin, 100 μg streptomycin, and 250 ng amphotericin B (Sigma Aldrich, St. Louis, MO, USA) and incubated at 33°C with 5% CO2. On day 3 after sample inoculation, the antimicrobial drug–containing medium was removed and replaced with MEM without antimicrobial drug and supplemented with 5% fetal calf serum (HyClone Laboratories, Inc., South Logan, UT, USA). Medium was changed every 3 days until day 15. A cell sample from each well was tested for infection at days 9 and 15 by using Gimenez stain and PCR with 17 kDa and gltA primers. Gimenez staining on day 15 yielded numerous red-stained bacteria in the cytoplasm of Vero cells in the 8 wells used. A single scraping of the cells from the positive wells was inoculated onto confluent layers of Vero cells, which enabled establishment of the isolate. Three PCR amplicons of the 17kDa– and gltA–specific primers (4–6) from positive wells were fully sequenced. After removing primer sequences, we compared amplicon sequences by conducting a gapped BLAST 2.0 (http://blast.st-va.ncbi.nlm.nih.gov/Blast.cgi) search of the GenBank database; the 17-kDa (accession no. {"type":"entrez-nucleotide","attrs":{"text":"JX198507","term_id":"402230109","term_text":"JX198507"}}JX198507) and gltA (accession no. {"type":"entrez-nucleotide","attrs":{"text":"KC469611","term_id":"478647817","term_text":"KC469611"}}KC469611) gene fragment sequences showed 100% identity with R. typhi strain Wilmington (accession no. {"type":"entrez-nucleotide","attrs":{"text":"AE017197.1","term_id":"51459527","term_text":"AE017197.1"}}AE017197.1). Murine typhus has been reemerging in southeastern Mexico for the past 6 years (3,7). Active epidemiologic surveillance led to early detection of human cases and opportune treatment, thereby decreasing the rate of severe illness. However, the prevalent social and cultural conditions in small villages, with close contact with domestic, peridomestic, and wild animals, facilitate the transmission of this fleaborne rickettsiosis; human infections, such as the case presented here, still occur. We replaced shell vials with cell culture plates and isolated rickettsiae from a biological sample from a patient with acute murine typhus. The method is as simple as the shell vial centrifugation technique and is highly sensitive and easy to perform, making it an excellent choice for rickettsiae isolation when shell vials are not available. In the United States, isolation of R. typhi from a human was last reported >50 years ago (8). The case reported here reinforces the need to extend surveillance to small towns and villages in Yucatan State. It also shows that a shell vial alternative method for R. typhi isolation is simple and effective.