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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.

Clinical or serological coinfections of Rickettsia and Leptospira are uncommon but should be included in differential diagnosis when poor sanitation and cohabitation with infected animals may converge. Rickettsial and leptospiral infections have been continuously increasing throughout the past decade in Yucatan, Mexico. Leptospirosis is a zoonotic disease caused by aerobic spirochetes, while rickettsiosis is an arthropod-borne disease. In 2020, 16% of all rickettsiosis cases and 10% of leptospirosis in the country originated in Yucatan. The objective of the present case report was to document an unusual case of probable coinfection with Rickettsia and Leptospira with emphasis on clinical manifestations and the epidemiological context that may orient future multidisciplinary measures. Here, we presented the case of a 12-year-old female whose mother had recently recovered from a rickettsial infection. The patient presented with fever and developed unspecific signs and symptoms of infection; however, her condition quickly deteriorated with gastrointestinal, hepatic, renal, and neurological dysfunction. After discounting rabies and identifying infection with Rickettsia and Leptospira, antibiotic treatment was indicated, but it was too late to prevent death. Simultaneous infections of Rickettsia and Leptospira may be considered in endemic regions when environmental, epidemiological, and clinical conditions converge.

Resumen La ehrlichiosis es una enfermedad transmitida por la picadura de garrapatas que afecta a perros y humanos, causada por las especies Ehrlichia canis y E. chaffeensis, respectivamente. Estas bacterias son gramnegativas, intracelulares obligadas, de aspecto cocoide a pleomorfo, que infectan los monocitos y desencadenan síntomas como fiebre elevada, anorexia, trombocitopenia, hemorragias, anemia y problemas graves como esplenomegalia, hepatomegalia y meningitis. Para diagnosticar esta enfermedad existen diversos métodos, entre los que se encuentran los hematológicos que evalúan la morfología de los monocitos en búsqueda de mórulas y la serología, que incluye la búsqueda de anticuerpos anti-Ehrlichia, pero que se encuentra limitado debido a la reactividad cruzada que presenta. Por otra parte, el cultivo de especies de Ehrlichia ha resultado ser un método efectivo para la obtención de antígenos y así desarrollar ensayos por inmunofluorescencia indirecta (IFI). El método por reacción de polimerasa en cadena ofrece un diagnóstico definitivo por tener una mayor sensibilidad y especificidad que los otros métodos, al haberse desarrollado cebadores género-específicos, así como especie-específicos. En esta revisión, se discutirán los diversos métodos aplicados al diagnóstico de esta enfermedad, así como las ventajas y desventajas que estos presentan.

Ticks are obligate haematophagous ectoparasites that are associated with a wide range of vertebrate hosts, among them also reptiles and amphibians. They have dynamic ecological interactions with multiple microorganisms, ranging from endosymbionts to pathogens, such as the members of the genus Rickettsia. The aim of this work was to detect Rickettsia in ticks from amphibians and reptiles from southern Mexico by the amplification, sequencing and phylogenetic reconstruction of the gltA and ompA genes, and also to compile all the published records worldwide of Rickettsia associated with ticks attached to reptiles and amphibians, in order to elucidate the host-parasite relationships, and to identify the geographical distribution of each bacterial species. We record for the first time the presence of Rickettsia sp. strain Colombianensi and Rickettsia amblyommatis in ticks from several reptiles and amphibians collected in three new localities from the states of Guerrero and Veracruz, Mexico. Additionally, we here report 23 Rickettsia taxa associated with 18 tick species attached to 42 host taxa of amphibians and reptiles in 36 countries. Our findings increase the inventory of rickettsia reported in Mexico and summarizes the knowledge of these bacteria associated with ticks of this particular group of vertebrate host worldwide.

The genus Rickettsia encompasses 35 valid species of intracellular, coccobacilli bacteria that can infect several eukaryotic taxa, causing multiple emerging and re-emerging diseases worldwide. This work aimed to gather and summarise the current knowledge about the genus Rickettsia in Mexico, updating the taxonomy of the bacteria and their hosts by including all the records available until 2020, to elucidate host-parasite relationships and determine the geographical distribution of each Rickettsia species present in the country. Until now, 14 species of Rickettsia belonging to four groups have been recorded in Mexico. These species have been associated with 26 arthropod species (14 hard ticks, three soft ticks, two sucking lice, and seven fleas) and 17 mammal species distributed over 30 states in Mexico. This work highlights the high biological inventory of rickettsias for Mexico and reinforces the need to approach the study of this group from a One Health perspective.

Objective. To determine the presence of Rickettsia typhi in synanthropic rodents captured in the rural community of Bolmay, Yucatan, Mexico, an area with history of murine typhus cases. Materials and methods. Thirty Mus musculus, 6 Rattus rattus and 1 Heteromys gaumeri were examined. Total DNA was obtained of rodent’s spleen. The identification of Rickettsia typhi was performed through the fragment’s amplification of the htrA (17-kDa gene protein) by endpoint PCR. The positive products were purified and sent for sequencing and analysis with the BLAST tool. Results. Rickettsial DNA was identified in 27% (10/37) of the rodents: seven M. musculus and 3 R. rattus. The alignment analyses obtained identity and coverage percentages of 97 - 99% for R. typhi. Conclusions. The present study suggests the participation of synanthropic rodents in the murine typhus cycle in the region. We described the first molecular evidence of R. typhi in M. musculus of Yucatan, Mexico., Objetivo. Determinar la presencia de Rickettsia typhi en roedores sinantrópicos capturados en la comunidad rural de Bolmay, Yucatán, México, con antecedentes de tifo murino en sus habitantes. Materiales y métodos. Se examinaron 37 roedores: 30 Mus musculus, 6 Rattus rattus y 1 Heteromys gaumeri. Se obtuvo ADN total del bazo de los roedores. La identificación de R. typhi se realizó a través de la amplificación de un fragmento del gen htrA (proteína 17-kDa) por PCR punto final. Los productos positivos fueron purificados y enviados a secuenciación para su análisis de alineamiento con la herramienta BLAST. Resultados. La identificación de ADN rickettsial se logró en un 27% (10/37) de los roedores: siete M. musculus y 3 R. rattus. El análisis de alineamiento obtuvo porcentajes de identidades y coberturas del 97 - 99% para R. typhi, respectivamente. Conclusiones. El presente estudio sugiere la participación de los roedores sinantrópicos en el ciclo de transmisión del tifo murino en la región. Se describe la primera evidencia molecular de R. typhi en M. musculus de Yucatán, México.

Key Clinical Message Domestic animals can carry ticks or fleas, which constitute common vectors of rickettsial infections. The contact with them should be considered as suggestive of rickettsial infections in symptomatic patients. Misdiagnosis might occur in regions where other vector‐borne diseases are endemic. Anamnesis is essential for an accurate clinical diagnosis.

During the second half of the twentieth century, neurologic sequelae associated with central nervous system impairment caused by Rickettsia rickettsii were studied widely and exclusively in the United States. We present the case of a Mexican pediatric patient with neurologic sequelae 10 years after an acute infection by R. rickettsii. Keywords: Rocky mountain spotted fever, Rickettsia rickettsii, Neurologic manifestations

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.

Rickettsia typhi is the causal agent of murine typhus; a worldwide zoonotic and vector-borne infectious disease, commonly associated with the presence of domestic and wild rodents. Human cases of murine typhus in the state of Yucatán are frequent. However, there is no evidence of the presence of Rickettsia typhi in mammals or vectors in Yucatán. The presence of Rickettsia in rodents and their ectoparasites was evaluated in a small municipality of Yucatán using the conventional polymerase chain reaction technique and sequencing. The study only identified the presence of Rickettsia typhi in blood samples obtained from Rattus rattus and it reported, for the first time, the presence of R. felis in the flea Polygenis odiosus collected from Ototylomys phyllotis rodent. Additionally, Rickettsia felis was detected in the ectoparasite Ctenocephalides felis fleas parasitizing the wild rodent Peromyscus yucatanicus. This study’s results contributed to a better knowledge of Rickettsia epidemiology in Yucatán. Rickettsia typhi es el agente causal del tifo murino; una enfermedad zoonótica transmitida por vector mundialmente distribuida, comúnmente asociada con la presencia de roedores domésticos y silvestres. Los casos humanos de tifo murino en el Estado de Yucatán son frecuentes. Sin embargo, no existe evidencia de la presencia de Rickettsia typhi en mamíferos o vectores en Yucatán. En la búsqueda de vectores y reservorios de Rickettsia typhi, evaluamos la presencia de bacterias del género Rickettsia en roedores y sus ectoparásitos de un pequeño municipio del estado de Yucatán por medio de técnicas de PCR convencional y secuenciación de ADN. Se identificó la presencia de Rickettsia typhi en muestras de sangre obtenidas de Rattus rattus y reportamos por primera vez la presencia de Rickettsia felis en la pulga Polygenis odiosus colectado de Ototylomys phyllotis. Complementariamente, Rickettsia felis fue detectado en la pulga Ctenocephalides felis parasitando al roedor Peromyscus yucatanicus. No se identificó especie de Rickettsia en las muestras de sangre de O. phyllotis y P. yucatanicus analizados. Nuestros resultados contribuyen también en el conocimiento de ciclo de vida biológico del género Rickettsia.

This work emphasizes the detection of Candidatus "Rickettsia amblyommii" in questing Haemaphysalis juxtakochi and Amblyomma mixtum. From February 2009 to December 2012, questing ticks were collected from the vegetation and leaf-litter of four protected forests and two grassy areas around the Panama Canal basin. DNA was extracted from Amblyomma mixtum, Amblyomma naponense, Amblyomma oblongoguttatum, Amblyomma pecarium, Amblyomma tapirellum, Haemaphysalis juxtakochi, and unidentified immature Amblyomma. Specific primers of citrate synthase gene gltA were used to detect and identify the rickettsiae. Amplicons with the expected band size were purified and sequenced. DNA of C. "R. amblyommii" was found in A. mixtum, H. juxtakochi and Amblyomma immatures. To our knowledge, these finding represent the first report of C. "R. amblyommii" in free-living ticks in the wilderness of Central America.

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.

In this work we present a new option to identify 11 rickettsial species that cause human rickettsioses, with some advantages over the previous methods described. Using rickettsial isolates from 11 Rickettsia species as a sample, we used the polymerase chain reaction to amplify a 990- to 1,000-bp DNA fragment from the ompB gene, common for the 11 Rickettsia species analyzed in this study, which were digested with AluI restriction enzyme to obtain different digestion patterns. This restriction pattern can be visualized using a polyacrylamide gel electrophoresis technique. Using this method we could differentiate between the 11 Rickettsia species analyzed regardless of the group to which the Rickettsia belonged. We developed a simple method to identify 11 Rickettsia species which cause human rickettsioses using polymerase chain reaction and restriction fragment length polymorphism techniques with the advantage that it only needs one amplicon and only one restriction enzyme to obtain the restriction pattern. The identification of the species infecting vectors, reservoirs, and humans is essential to establish the ecological and behavioral ecosystem involved in its maintenance and transmission in nature in the specific region where the pathogen is circulating. This method is very helpful to identify Rickettsia species in a short time.

Rickettsia felis is an emergent pathogen and the causative agent of a typhus-like rickettsiosis in the Americas. Its transmission cycle involves fleas as biological vectors (mainly Ctenocephalides felis) and multiple domestic and synanthropic mammal hosts. Nonetheless, the role of mammals in the cycle of R. felis is not well understood and many efforts are ongoing in different countries of America to clarify it. The present study describes for the first time in Mexico the infection of two species of opossum (Didelphis virginiana and D. marsupialis) by R. felis. A diagnosis was carried out from blood samples by molecular methods through the gltA and 17 kDa genes and sequence determination. Eighty-seven opossum samples were analyzed and 28 were found to be infected (32.1%) from five out of the six studied localities of Yucatan. These findings enable recognition of the potential epidemiological implications for public health of the presence of infected synanthropic Didelphis in households.

Fungus from the Aspergillus genus mainly affects lung tissue, occurring when the integrity of the host immune system is compromised. The human body uses immunocompetence conditions from mechanical and enzymatic defenses and the action of the innate immune system cells and also uses adaptive responses to control infection. Neutrophils, macrophages, and dendritic cells are critical as antifungal effector cells possess surface receptors that recognize fungal structures and trigger specific responses. TLRs and Dectin-1 the most studied for this interaction. TLRs are responsible for the production and release of cytokines and Dectin-1 is essential in the phagocytosis of the particle recognition and production of ROS. The best-studied cytokines and its crucial role in the response to Aspergillus spp. are TNF-α, IFN-γ, and IL-12. In this work, we reviewed the main mechanisms related to molecular receptors on phagocytic cells involved in the recognition of Aspergillus spp. Understanding the immune response in situations of immunocompetence and its comparison in immunodeficient organisms could provide alternatives to control invasive aspergillosis.

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.

The first human case of infection caused by Rickettsia in Yucatan was detected in 1996, and it was determined that the species was R. felis. Since then, passive epidemiologic surveillance was implemented to search for human cases in the public hospitals of the state, and in 2005, the first human case of Rocky Mountain spotted fever was detected. During the following 2 years, eight new confirmed cases and one probable case were identified. Seven cases involved children younger than 12 years of age, with a fatal outcome in three of the cases. Children are a particularly vulnerable population for this serious emerging infection.

Rickettsia felis infection in humans has been detected worldwide, causing an illness that could be confused with other viral and bacterial infections such as dengue fever. Despite the high incidence of infection in humans, reservoirs, and vectors, the definitive diagnosis of R. felis infection currently requires polymerase chain reaction and sequencing because its antigens are closely related to other rickettsiae, making serologic diagnosis problematic. In this work, we report the immune reactivity of sera from four patients infected with R. felis with recombinant peptides representing regions of outer membrane protein A of R. felis.

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.

Background & objectives: The nature of the rickettsial antigens and the immune response generated by them, have been the subject of exhaustive research so that a suitable vaccine can be developed. Till date evaluations of Rickettsia rickettsii antigens that induce both humoral and cellular responses in animal models have only shown partial protection and short-term immunological memory. This study was aimed to evaluate the immune response induced by DNA plasmids generated from the OmpA and OmpB genes of R. rickettsii in peripheral blood mononuclear cells of rickettsial (sensitized) patients compared to healthy subjects. Methods: Plasmids OmpA-49, OmpB-15 and OmpB-24 were generated in the pVAX vector. Macrophages derived from the THP-1 cell line were transfected in vitro with the plasmids and were co-cultured with T-lymphocytes from sensitized subjects and healthy subjects to evaluate cell proliferation and cytokine production. Results: The OmpB-24 plasmid induced proliferative response in human lymphocytes, with production of IL-2, IFN-γ, IL-12p70, IL-6 and TNF-α, likely due to the presence of conserved epitopes among R. rickettsii, R. typhi and R. felis (differing from 1 to 3 amino acids) during the construction of the plasmids. Interpretation & conclusion: DNA sequences of rickettsial epitopes can be cloned into the pVAX vector. Constructed plasmids can generate a proliferative response and produce cytokines in vitro, in co-culture of transfected macrophages with sensitized human lymphocytes. Plasmid OmpB-24 proved to be the most immunogenic with respect to plasmids OmpA-49 and OmpB-15.

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.

Rickettsia akari is the causative agent of rickettsialpox, a primarily urban mite–borne rickettsiosis that is encountered in the United States and in a few countries around the world. Its vector is the mite Liponyssoides sanguineus, which is found on rats and mice, which serve as reservoirs for the disease. In this work we report a severe animal case of R. akari infection with two unusual features: R. akari was found in a dog, and its potential vector was a tick.

is an intracellular bacteria who causes murine typhus. His importance is reflected in the high frequency founding specific antibodies against Rickettsia typhi in several worldwide seroepidemiological studies, the seroprevalence ranging between 3-36%. Natural reservoirs of R. typhi are rats (some species belonging the Rattus Genus) and fleas (Xenopsylla cheopis) are his vector. This infection is associated with overcrowding, pollution and poor hygiene. Typically presents fever, headache, rash on trunk and extremities, in some cases may occur organ-specific complications, affecting liver, kidney, lung or brain. Initially the disease is very similar to other diseases, is very common to confuse the murine typhus with Dengue fever, therefore, ignorance of the disease is a factor related to complications or non-specific treatments for the resolution of this infection. This paper presents the most relevant information to consider about the rickettsiosis caused by Rickettsia typhi.es una bacteria intracelular causante del tifo murino. Su importancia queda reflejada en la elevada frecuencia con que se encuentran anticuerpos específicos frente a

The first fatal Rickettsia rickettsii infection was diagnosed in the southwest of Mexico. The patient had fever, erythematous rash, abdominal pain, and severe central nervous system involvement with convulsive crisis. The diagnosis of R. rickettsii infection was established by immunohistochemistry and specific polymerase chain reaction.

To the Editor: Rickettsia typhi is the causal agent of murine typhus, a febrile illness affecting humans worldwide (1). In Mexico, recent studies demonstrated a 14% prevalence of antibodies against typhus group rickettsiae in healthy adult blood donors in Mexico City, and a recent nonfatal case of endemic typhus was reported in Yucatan (2,3). In May 2011, a 42-year-old woman and her 12-year-old son sought care at the clinical service of the Autonomous University of Yucatan. They had malaise, headache, fever (39°C), fibromyalgia, sore throat, and fatigue and an erythematous rash on the chest that after 6 days spread to the abdomen and extremities. Dengue fever was diagnosed, and the patients were treated empirically with acyclovir, methanesulfonamide, N-(4-nitro-2-phenoxyphenyl) and clarithromycin. Dengue could not be confirmed by laboratory testing. Murine typhus was diagnosed on the basis of PCR amplification and immunofluorescent assay for antibodies to R. typhi. Rickettsia species was determined by sequencing of rickettsial genes. Three serum samples were collected from the woman (8, 12, and 16 days after illness onset) and 1 from the boy (8 days) in 3.8% sodium citrate as anticoagulant, and DNA was extracted immediately by QIAamp DNA Blood Mini Kit (QIAGEN Valencia, CA, USA) in accordance with the manufacturer’s instructions. Single-step PCR amplification was performed by using genus-specific primers for the rickettsial 17-kDa protein and citrate synthase (gltA) genes as reported (4) Sequences of the citrate synthase and 17-kDa PCR products were compared at the National Center for Biotechnology Information BLAST software (5). Three PCR amplicons of both genes were fully sequenced and compared with sequences in GenBank. The 17-kDa and citrate synthase fragment sequences (GenBank accession nos. {"type":"entrez-nucleotide","attrs":{"text":"JX198507","term_id":"402230109"}}JX198507 and {"type":"entrez-nucleotide","attrs":{"text":"JX458814","term_id":"406654549"}}JX458814) showed 99% and 100% identity, respectively, with R. typhi strain Wilmington strain (GenBank accession no. {"type":"entrez-nucleotide","attrs":{"text":"AE017197.1","term_id":"51459527"}}AE017197.1) (Table). Table Ig detection by immunofluorescent assay and molecular results by PCR and sequence identity of the amplicons of gltA and 17-kDa genes of Rickettsia spp. from 2 patients with murine typhus, Yucatan, Mexico Immunofluorescent assay was performed by using R. rickettsii and R. typhi antigen fixed on slides. We examined the serum samples for IgG and IgM, assessing reactivity of γ chain–specific and μ heavy chain–specific secondary conjugates, respectively, with rickettsial antigens. All 3 samples from the woman and the sample from the boy contained antibodies to R. typhi (Table). Both patients were treated with 100 mg of oral doxycycline 2×/day for 7 days (boy), and 10 days (woman); symptoms improved in 72 hours for the child. The woman’s symptoms resolved completely in 5 days. Typhus has been endemic in Mexico since before the conquest period (6). Socioeconomic aspects play a major role in zoonotic diseases, such as rickettsioses, especially in their distribution in urban and suburban areas because of factors such as marginalized communities, animal breeding, education levels, poverty, and social exclusion from health systems. Overcrowding resulting from migration from rural areas to large urban centers contributes to increased zoonoses in urban areas. Also contributing is the ecologic imbalance of flora and fauna associated with deteriorating sanitary conditions in areas where mammals involved in the cycle of R. typhi, such as rodents and opossum, may live in the same habitat as humans and colonize backyards, waste deposit area, and areas around the neighborhoods where they can find food. The concurrence and presence of mammals, vectors, and humans may contribute to maintaining transmission of endemic typhus in a reduced area, with the possibility to cause outbreaks (7,8). Housing conditions and culture, such as courtyards with vegetation and presence of pets, in several suburban areas of Yucatan encourage close contact between humans and possible reservoirs for several infectious diseases, such as mice, rats, opossums, dogs and cats, and their ectoparasites (9). Because housing and cultural conditions are similar in all countries of Latin America, endemic typhus probably is transmitted in the same way: by coexistence with domestic animals and close contact with wild reservoirs. Epidemiologic control should be closely linked to education aimed at encouraging villagers to interrupt the cycle of transmission and thus prevent the disease. We have confirmed the presence of murine typhus in Yucatan. The finding of human cases demonstrates the need to consider R. typhi infection in the differential diagnosis of febrile illnesses considered endemic in Yucatan, such as dengue fever and leptospirosis (10). Early and accurate diagnosis should enable physicians to treat this disease appropriately and early in the clinical course to prevent increased illness and death.