Efectividad de los métodos diagnósticos para la detección de ehrlichiosis monocítica humana y canina / Effectivity of diagnostic methods for the detection of human and canine monocytic ehrlichiosis

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.

Ehrlichiosis is a disease transmitted by tick's bite that affect dogs and humans caused by the species Ehrlichia canis and E. chaffeensis, respectively. These bacteria are obligated intracellular gram negatives, with a cocoid to pleomorph aspect and can infect monocytes and trigger symptoms such as high fever, anorexia, thrombocytopenia, hemorrhages, anemia, and some serious problems such as splenomegaly, hepatomegaly and meningitis. There are several diagnostic tests for ehrlichiosis such as the hematological ones that evaluate the morphology of the monocytes in search of morulae; serological tests that includes the search of anti-Ehrlichia antibodies, although they might be limited due to cross reaction with other species. In other hand, the culture of Ehrlichia species is an effective method to obtain antigens and even develop indirect immunofluorescence assays (IFA). The polymerase chain reaction offers a definitive diagnosis associated to the use of genus-specific and species-specific primers, as well as its increased sensibility and specificity, compared to the others methods. Thus, in this review, we will discuss various methods applied to the diagnosis of this disease, as well as the advantages and disadvantages that these present.

Tick-borne rickettsial pathogens in questing ticks, removed from humans and animals in Mexico

Tick-borne rickettsial diseases (TBRD) are commonly encountered in medical and veterinary clinical settings. The control of these diseases is difficult, requiring disruption of a complex transmission chain involving a vertebrate host and ticks. The geographical distribution of the diseases is related to distribution of the vector, which is an indicator of risk for the population. A total of 1,107 ticks were collected by tick dragging from forests, ecotourism parks and hosts at 101 sites in 22 of the 32 states of Mexico. Collected ticks were placed in 1.5 mL cryovials containing 70% ethanol and were identified to species. Ticks were pooled according to location/host of collection, date of collection, sex, and stage of development. A total of 51 ticks were assayed by polymerase chain reaction (PCR) to confirm species identification using morphological methods. A total of 477 pools of ticks were assayed using PCR techniques for selected tick-borne pathogens. Anaplasma phagocytophilum was the most commonly detected pathogen (45 pools), followed by, Ehrlichia (E.) canis (42), Rickettsia (R.) rickettsii (11), E. chaffeensis (8), and R. amblyommii (1). Rhipicephalus sanguineus was the tick most frequently positive for selected pathogens. Overall, our results indicate that potential tick vectors positive for rickettsial pathogens are distributed throughout the area surveyed in Mexico.

Primer caso de ehrlichiosis monocítica humana reportado en Colombia / First case of human monocytic ehrlichiosis reported in Colombia

La ehrlichiosis es una infección no reconocida ni sospechada en nuestro medio, sin embargo, ciertas características epidemiológicas hacen que deba pensarse en esta entidad a la hora de abordar a un paciente con un cuadro clínico compatible. Presentamos el caso de un soldado joven con factores de riesgo claros para zoonosis, con fiebre, erupción petequial, trombocitopenia, hepatitis y disfunción orgánica múltiple a quien se le confirmó infección por Ehrlichia chaffeensis por medio de serología luego de descartar infecciones comunes y otras infecciones por vectores. Existen limitaciones en los registros de este tipo de enfermedades pues no son de notificación obligatoria y un alto número cursan de manera subclínica, lo que hace probable un elevado subregistro. La importancia de tener en mente esta condición radica en que el retraso del tratamiento se asocia a mayor incidencia de complicaciones, incluyendo desenlaces fatales.

Human ehrlichiosis is an infection that is neither suspected or recognized in our country. Certain epidemiologic clues should arouse suspicion for this entity when the symptoms are suggestive. We present a case of a young soldier with clear risk factors for zoonotic infection, with fever, a petechial rash, thrombocytopenia, hepatitis and multiple organ failure, for whom a diagnosis of Ehrlichia chaffeensis was confirmed by serology after ruling out common infections and other infections caused by vectors . There are important limitations in documenting these cases, because there is no mandatory public reporting and a significant proportion of these cases are asymptomatic, leading to underdocumentation. It is however, of critical importance to raise awareness about this entitysince delays in treatment portend a higher incidence of complications, including death.

Genetic Identification and Phylogenetic Analysis of Anaplasma and Ehrlichia Species in Haemaphysalis longicornis Collected from Jeju Island, Korea

A total of 1,395 Haemaphysalis longicornis ticks collected from Jeju Island of Korea were examined by 16S rRNA gene-based nested PCR for the presence of infection with Anaplasma and Ehrlichia species. Template DNAs to detect the tick-borne pathogens were prepared from a total 506 tick pools. Eight genera of Anaplasma and six Ehrlichia by 16S rRNA gene PCR and sequencing analysis were identified. A. phagocytophilum was the most prevalent (27 [1.9%]) by nested PCR, followed by A. bovis (5 [0.4%]), E. chaffeensis (4 [0.2%]), and A. centrale (1 [0.1%]). In the phylogenetic analysis based on 16S rRNA sequences, eight genera of Anaplasma group (> 99.4% homology) and six Ehrlichia group (> 99.5% homology) were close to deposited A. marginale strains (AF309867, AF414874, and FJ226454) and Ehrlichia sp. (DQ324547), respectively. Three Anaplasma species groups A. phagocytophilum (group A), A. bovis (group B), and A. centrale (group C) and one Ehrlichia species E. chaffeensis (group D) were determined by comparing with Anaplasma and Ehrlichia related sequences. First, twenty-eight A. phagocytophilum clones belonging to group A were divided into 7 genotypes. The sequence similarity among genotypes A1 to A4 was very high (> 99.6%). Genotype B2 was close to A. bovis from Korea (99.7%). Genotype D1 was close to known E. chaffeensis strains (M73222, AF147752, and AY350424) and their similarity value was 99.7%. In conclusion, the genera of Anaplasma/Ehrlichia, A. phagocytophilum, and E. chaffeensis identified in predominant H. longicornis ticks were ubiquitous throughout the Jeju Island. The various native groups have been found through sequence identities and phylogenetic analysis.

Tick-borne ehrlichiosis infection in human beings.

Human monocytic ehrlichiosis is a tick-borne infectious disease transmitted by several tick species, especially Amblyomma spp caused by Ehrlichia chaffeensis. E. chaffeensis is an obligatory intracellular, tick-transmitted bacterium that is maintained in nature in a cycle involving at least one and perhaps several vertebrate reservoir hosts. Two additional Ehrlichia spp, Anaplasma (formerly Ehrlichia) phagocytophila (the agent of human granulocytic ehrlichiosis [HGE]) and E. ewingii (a cause of granulocytic ehrlichiosis in dogs) act as human pathogens. Human E. chaffeensis infections have generally been reported in North America, Asia and Europe, but recently human cases have been reported in Brazil only. Human monocytic ehrlichiosis is diagnosed by demonstration of a four-fold or greater change in antibody titer to E. chaffeensis antigen by IFA in paired serum samples, or a positive PCR assay and confirmation of E. chaffeensis DNA, or identification of morulae in leukocytes and a positive IFA titer to E. chaffeensis antigen, or immunostaining of E. chaffeensis antigen in a biopsy or autopsy sample, or culture of E. chaffeensis from a clinical specimen.

More about human monocytotropic ehrlichiosis in Brazil: serological evidence of nine new cases

Human Ehrlichia chaffeensis infections have been reported in North America, Asia and Europe, but only recently have human cases been reported in Brazil. Nine new human cases of E. chaffeensis infection diagnosed on a clinical and serological basis are reported. Serological tests were performed with indoor slides prepared with CDC stock DH-82 cells infected with E. chaffeensis (Arkansas strain). All but two patients were adults. Seven patients were male and two female. The fever duration varied from 4 to 120 days with a median of 6 days. All patients recalled previous tick attack. IgM was detected in four cases. Influenza like syndrome was the most frequent clinical form affecting five patients. Two patients had fever of unknown origin (FUO), one patient had blood culture-negative endocarditis and one had encephalitis. All patients except one recovered. Two patients were correctly treated. One patient with FUO had AIDS and unexplained pancytopenia. The occurrence of human ehrlichiosis by E. chaffeensis remains to be proved in Brazil; the cases reported here highlight the possibility of such disease occurrence in Brazil.

Antibodies to Rickettsia rickettsii, Rickettsia typhi, Coxiella burnetii, Bartonella henselae, Bartonella quintana, and Ehrlichia chaffeensis among healthy population in Minas Gerais, Brazil

Rickettsial diseases except those belonging to spotted fever group rickettsioses are poorly studied in South America particularly in Brazil where few epidemiological reports have been published. We describe a serosurvey for Rickettsia rickettsii, R. typhi, Coxiella burnetii, Bartonella henselae, B. quintana, and Ehrlichia chaffeensis in 437 healthy people from a Brazilian rural community. The serum samples were tested by indirected micro-immunoflourescence technique and a cutoff titer of 1:64 was used. The seroprevalence rates for R. rickettsii, R. typhi, C. burnetii, B. henselae, B. quintana, and E. chaffeensis were respectively 1.6 percent (7 samples); 1.1 percent (5 samples); 3.9 percent (17 samples); 13.7 percent (60 samples); 12.8 percent (56 samples), and 10.5 percent (46 samples). Frequent multiple/cross-reactivity was observed in this study. Age over 40 years old, urban profession, and rural residence were significantly associated with some but not all infections rate. Low seropositivity rates for R. rickettsii, R. typhi, and C. burnetii contrasted with higher rates of seropositivity for B. quintana, B. henselae, and E. chaffeensis. These results show that all tested rickettsial species or antigenically closely related possible exist in this particular region.

Identification and prevalence of Ehrlichia chaffeensis infection in Haemaphysalis longicornis ticks from Korea by PCR, sequencing and phylogenetic analysis based on 16S rRNA gene

Genomic DNAs extracted from 1,288 Haemaphysalis longicornis ticks collected from grass vegetation and various animals from nine provinces of Korea were subjected to screening by genus-specific (Ehrlichia spp. or Anaplasma spp.) real-time TaqMan PCR and speciesspecific (E. chaffeensis) nested-PCR based on amplification of 16S rRNA gene fragments. In all, 611 (47.4%) ticks tested positive for genus-specific amplification of 116 bp fragment of 16S rRNA of Ehrlichia spp. or Anaplasma spp. Subsequently, 396 bp E. chaffeensis-specific fragment of 16S rRNA was amplified from 4.2% (26/611) tick samples. The comparison of the nucleotide sequence of 16S rRNA gene from one tick (EC-PGHL, GeneBank accession number AY35042) with the sequences of 20 E. chaffeensis strains available in the database showed that EC-PGHL was 100% identical or similar to the Arkansas (AF416764), the Sapulpa (U60476) and the 91HE17 (U23503) strains. The phylogenetic analysis also revealed that the E. chaffeensis EC-PGHL formed a single cluster with the above strains. This is the first study to report molecular detection and phylogenetic analysis of E. chaffeensis from H. longicornis ticks in Korea. The implicit significance of E. chaffeensis infection in H. longicornis ticks in Korea is discussed.

Human ehrlichioses in Brazil: first suspect cases

Brazilian spotted fever (BSF) rickettsiosis is the most common and recognized of the human rickettsioses in Brazil. It is difficult to establish the diagnosis of human rickettsiosis infection by routine microbiologic methods, creating a false idea that Rickettsia and Ehrlichia infections are rare and without importance. New tick-borne diseases, like Human Granulocytic Anaplasmosis (HGA) and Human Monocytic Ehrlichiosis (HME), have been described in many countries. These diseases can present symptoms similar to rickettsioses of the spotted fever group, and they are transmitted by ixodid ticks. The first two suspected cases of human ehrlichiosis in Brazil were first considered to be cases of BSF. The differential diagnosis was made at the Minas Gerais Rickettsiosis Public Health Laboratory. The clinical and laboratory findings, with positive serology for the HME agent, indicated suspected cases of human ehrlichioses in Brazil.