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      Isolation of Rickettsia typhi from Human, Mexico

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          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, Nuaillé, 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 ( search of the GenBank database; the 17-kDa (accession no. JX198507) and gltA (accession no. KC469611) gene fragment sequences showed 100% identity with R. typhi strain Wilmington (accession no. 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.

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          Most cited references 8

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          Genotypic identification of rickettsiae and estimation of intraspecies sequence divergence for portions of two rickettsial genes.

          DNA sequences from specific genes, amplified by the polymerase chain reaction technique, were used as substrata for nonisotopic restriction endonuclease fragment length polymorphism differentiation of rickettsial species and genotypes. The products amplified using a single pair of oligonucleotide primers (derived from a rickettsial citrate synthase gene sequence) and cleaved with restriction endonucleases were used to differentiate almost all recognized species of rickettsiae. A second set of primers was used for differentiation of all recognized species of closely related spotted fever group rickettsiae. The procedure circumvents many technical obstacles previously associated with identification of rickettsial species. Multiple amplified DNA digest patterns were used to estimate the intraspecies nucleotide sequence divergence for the genes coding for rickettsial citrate synthase and a large antigen-coding gene of the spotted fever group rickettsiae. The estimated relationships deduced from these genotypic data correlate reasonably well with established rickettsial taxonomic schemes.
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            Typhus and typhuslike rickettsiae associated with opossums and their fleas in Los Angeles County, California.

            The recent discovery of cat fleas (Ctenocephalides felis) infected with a typhuslike rickettsia (designated the ELB agent) raises the question of whether similar rickettsial infections exist in wild cat flea populations. We verified the presence of the ELB agent and Rickettsia typhi in urban and suburban areas of Los Angeles, Calif. Opossums trapped in close proximity to the residences of human murine typhus cases in Los Angeles county and other areas within the city of Los Angeles were tested for the presence of typhus group rickettsiae by the polymerase chain reaction (PCR). The presence of rickettsiae in the spleen tissues of three opossums (n = 9) and in 66 opossum fleas (n = 205) was determined by PCR and was verified by dot blot and Southern transfer hybridization. Further analysis of the amplified PCR products generated by a series of primer pairs derived from either the 17-kDa antigen gene or the citrate synthase gene revealed that both R. typhi and the ELB agent were present in the tested samples. Dual infection was not noted in the samples; however, the fleas were infected with either R. typhi or the ELB agent. The presence of the ELB agent in the cat flea population may have implications for public health. Whether this agent is responsible for the mild cases of human murine typhus in urban and suburban areas of Los Angeles or in other endemic foci remains to be determined.
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              Detection of murine typhus infection in fleas by using the polymerase chain reaction.

              Polymerase chain reaction (PCR) amplification of DNA was used to detect the etiologic agent of murine typhus, Rickettsia typhi, in experimentally infected adult fleas. A primer pair derived from the 17-kilodalton antigen sequence of typhus and spotted fever group rickettsiae was used to amplify a 434-base-pair (bp) fragment of the genome of the murine typhus rickettsiae. The amplified 17-kilodalton protein antigen-specific sequence was detected in ethidium bromide-stained agarose gels in individual fleas as early as 2 days after exposure to rickettsemic rats (two of six tested). The 434-bp sequence was not detected in uninfected control fleas. A dot hybridization assay used to detect the 434-bp fragment was also specific and about 100-fold more sensitive than the agarose gel PCR assay. Since the PCR assay employed a boiled extract of triturated fleas, both PCR and an antigen capture enzyme-linked immunosorbent assay (ELISA) could be performed on the same individual flea homogenate. The ELISA identified 12 infected fleas out of 29 randomly selected fleas, compared with 14 specimens which were positive by PCR. The PCR assay detected rickettsiae in samples in which no viable rickettsiae were detected by plaque assay. Like the ELISA, the PCR assay sensitivity was due in part to its suitability for detecting small numbers of both live and dead R. typhi in fleas.

                Author and article information

                Emerg Infect Dis
                Emerging Infect. Dis
                Emerging Infectious Diseases
                Centers for Disease Control and Prevention
                August 2014
                : 20
                : 8
                : 1411-1412
                Universidad Autónoma de Yucatán, Mérida, México
                Author notes
                Address for correspondence: Jorge E. Zavala-Castro, Centro de Investigaciones Regionales Dr. Hideyo Noguchi, Universidad Autónoma de Yucatán, Av Itzaés no. 490 x Calle 59 Colonia Centro, Mérida, Yucatán 97000, México; email: zcastro@
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