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Acta Médica Costarricense
versão On-line ISSN 0001-6002versão impressa ISSN 0001-6012
Acta méd. costarric vol.55 supl.1 San José Jul. 2013
Conferencias Magistrales
Los desafíos en el diagnóstico, la investigación y la concientización sobre las rickettsiosis en América Latina
The challenges of rickettsial diagnosis, research, and awareness in
David H. Walker
Resumen
Los retos para establecer el diagnóstico de una enfermedad rickettsial específica, desarrollar un programa de investigación clínica o científica que se base en métodos eficaces de laboratorio, y promulgar la conciencia y el conocimiento de las rickettsiosis entre los médicos de atención primaria y los organismos de salud pública son hechos sustanciales. El logro de estas metas es una misión mancomunada. En esta mini revisión se presentan los principales desafíos en estos aspectos y se proponen algunos métodos para superarlos.
Descriptores: Infecciones por Rickettsiaceae, Rickettsia, América Latina, zoonosis
Abstract
The challenges to establish the diagnosis of a specific rickettsial disease, to develop a clinical or scientific research program that relies upon effective laboratory methods, and to promulgate awareness and knowledge of rickettsial diseases among primary care physicians and public health agencies are substantial. Achieving these goals is our mission. This minireview delineates the challenges and proposes some approaches to surmount them.
Keywords: Rickettsiaceae infections, Rickettsia,
Challenges of Diagnosis
Establishing a laboratory-confirmed diagnosis is the cornerstone of the foundation upon which the study of rickettsial diseases depends. The standard diagnostic tool, serologic demonstration of antibodies to rickettsiae, remains the major approach to document the diagnosis of rickettsial diseases. The deficiencies of serologic diagnosis include frequent absence of diagnostic antibodies early in the clinical course when critical therapeutic decisions are needed, inability to distinguish among the etiologic agents within the spotted fever (SFG) or typhus group (TG) owing to shared antigens, and presence of preexisting antibodies to the test antigens during the acute phase of illness owing to prior stimulation by crossreactive antigens. Currently in the
A major challenge to the performance of confirmatory serologic diagnosis of rickettsial infection on the basis of seroconversion between acute and convalescent sera is the availability of reagents, namely rickettsial antigens. Commercially available antigens are expensive and generally would be imported. Few laboratories in the world cultivate Rickettsia, Ehrlichia, or Orientia. The methods for cultivation require antibiotic-free cell culture or propagation in yolk sacs of embryonated eggs of chickens from flocks maintained on antibiotic-free feed. This approach demands skilled expertise and is threatened by contamination with bacteria and fungi. Motivated scientists can become proficient if trained by an experienced rickettsiologist. There are laboratories in Latin America and the
Another challenge is the broad classification of Rickettsia as requiring biosafety level-3 (BSL-3) biocontainment by
It has been arbitrarily considered that reactivity of a serum sample at four-fold or greater titer with one antigen than the rest indicates that species is the causative agent. Frequently the antibody titers do not differ by four-fold dilutions, and etiologically proven infections have occasionally stimulated antibodies reactive at a higher titer with antigens of another Rickettsia species than the etiologic agent. A serologic assay that has detected species-specific antibodies is a method developed by Jorge Zavala-Castro that demonstrates antibodies to a fragment of outer membrane protein A of R. felis.7 This achievement suggests that further research could identify species-specific peptides that might serve as effective serologic antigens. Another promising approach that could be pursued is the whole genome protein array developed by Felgner.8,9 The goal would be to identify which antigens are recognized early in the course and most strongly by a high proportion of patients and to determine whether any of the antigens detect reactivity to only the causative Rickettsia species8,9 so that serological assays could be manufactured using the ideal combination of antigens capable of yielding highly sensitive and specific results.
Molecular diagnosis by polymerase chain reaction (PCR) seems deceptively easy after one has obtained a thermal cycler and a source of primers. However, positive results that are not supported strongly by clinical, epidemiologic, and other laboratory data are viewed skeptically. Contamination of PCR by target DNA, particularly amplicons generated in previous PCR runs, can occur despite extensive precautions. Amplification and sequencing of multiple gene targets increases the strength of support for a PCR diagnosis, but not as much as seroconversion and an appropriate clinicoepidemiologic history would support the PCR result. Real time PCR and isothermal amplification methods based on transcription-mediated techniques are much less likely to suffer target DNA contamination.
Blood is not the ideal sample for diagnosis of rickettsial diseases by PCR because of the low concentration of circulating organisms. Rickettsiae are located predominantly in endothelial cells in the tissues. The eschar scab and a swab from its base are excellent samples for patients who have this lesion at the tick feeding site and should be examined with suspected infection by R. parkeri, R. massiliae, and R. akari10.For patients such as those infected by R. rickettsii, R. typhi, and R. prowazekii in whom most of the bacteria are located in the lesions rather than in peripheral blood, approaches such as needle aspiration of the rash could be evaluated. Low cost multiplex instrument-free point-of-care nucleic acid amplification devices with built-in lyophilized reagents and microfluidic processing have been developed that could be applied to the diagnosis of rickettsioses and ehrlichioses.
Challenges of Rickettsial Research
The definitive and most convincing evidence for the presence of an infectious disease is isolation of the pathogen from a patient with compatible clinical manifestations. This goal has been achieved in very few Latin American laboratories. The obstacles are similar to those related to the production of antigens for serologic diagnosis, namely cultivation of rickettsiae in antibiotic-free cell culture without bacterial or fungal contamination and manipulating potentially highly pathogenic organisms without accidental infection of personnel in the laboratory or its nearby environment. Appropriate use of a biosafety cabinet, laboratory safety precautions, and personal protective equipment in a facility engineered or arranged to prevent escape of aerosolized bacteria is the ideal situation. Under any circumstances it is necessary to institute surveillance of febrile disease in laboratory personnel and to treat illness suspected to possibly represent laboratory-acquired infection early in the course. Many rickettsiae such as R. parkeri and all Ehrlichia can be cultivated in a BSL-2 laboratory. Cultivation of R. felis, R. massiliae, E. chaffeensis, Orientia tsutsugamushi, and any novel member of the order Rickettsiales from patients in
Other topics related to rickettsial diseases that would be major research achievements include active prospective clinical studies of acute undifferentiated febrile illness that determined the actual incidence of rickettsioses and ehrlichioses in a defined population, development of effective species-specific serologic methods, and extensive characterization of human immune responses to rickettsiae. Determination of the likely mechanism(s) of greater virulence could include comparison of the differences between the more severe Latin American and less severe North American infections with R. rickettsii in terms of rickettsial genome comparisons, differential rickettsial gene expression during infection, rickettsial growth rates, and host immune responses as could be revealed by network analysis of several cytokines, chemokines and growth factors. Recognizing the superior achievements of Marcelo Labruna and his colleagues in the elucidation of the natural ecologic cycles of R. rickettsii in vertebrate reservoirs and tick vectors in Brazil, they could be challenged to use their experience in transmission by ticks to animals to approach the vector biology of identification of the initial target cells of vertebrate infection and the effects of tick saliva on experimental infections. This foundation of knowledge could be a prelude to elucidating the mechanisms of immune modulation by tick saliva and identification of the tick salivary effector molecules.
Challenges of Increasing Awareness of Rickettsial Diseases
The challenges of achieving increased awareness of rickettsial diseases in
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