RESUMEN
Cystic echinococcosis (CE) is a major neglected tropical zoonotic disease caused by the tissue-dwelling larval stage of the cestode parasite Echinococcus granulosus. For individuals suspected of CE, the diagnostic standard is imaging using ultrasonography, X rays, or computed tomography. These resource-demanding and expensive procedures are rarely available in endemic rural areas where CE is most prevalent. There is a critical need for a new approach to identify CE patients so that they can be managed early in the course of their infection. This study reports on the results of a diagnostic approach that identifies E. granulosus-derived cell-free DNA (cfDNA) in the urine of CE patients. Utilizing PCR to amplify a fragment of a major tandem repeat element found in E. granulosus nuclear DNA, urine samples from all seven imaging-confirmed CE patients who harbored active liver cysts were positive. In addition, the urine samples from 2/4 patients who presented with non-viable/calcified liver cysts were also PCR positive for the repeat fragment. To our knowledge, this is the first report of using parasite cfDNA from urine to diagnose CE. This approach provides an easy to implement and cost-effective method to survey for the prevalence of E. granulosus in humans populations.
Asunto(s)
Ácidos Nucleicos Libres de Células/orina , Equinococosis/diagnóstico , Echinococcus granulosus/genética , Animales , ADN de Helmintos/orina , Equinococosis/epidemiología , Echinococcus granulosus/aislamiento & purificación , Humanos , Enfermedades Desatendidas/diagnóstico , Enfermedades Desatendidas/epidemiología , Perú/epidemiología , Reacción en Cadena de la Polimerasa/métodos , Prevalencia , Zoonosis/diagnóstico , Zoonosis/epidemiologíaRESUMEN
Neurocysticercosis (NCC), caused by Taenia solium larvae that reside in the central nervous system, results in serious public health and medical issues in many regions of the world. Current diagnosis of NCC is complex requiring both serology and costly neuroimaging of parasitic cysts in the brain. This diagnostic pipeline can be problematic in resource-constrained settings. There is an unmet need for a highly sensitive and clinically informative diagnostic test to complement the present diagnostic approaches. Here, we report that T. solium-derived cell-free DNA is readily detectable in the urine of patients with the subarachnoid and parenchymal forms of NCC, and discuss the potential utility of this approach in enhancing and refining T. solium diagnostics.
Asunto(s)
Ácidos Nucleicos Libres de Células/genética , Disfunción Cognitiva/parasitología , ADN de Helmintos/genética , Neurocisticercosis/parasitología , Taenia solium/genética , Animales , Ácidos Nucleicos Libres de Células/sangre , Ácidos Nucleicos Libres de Células/orina , Sistema Nervioso Central/parasitología , Sistema Nervioso Central/fisiopatología , Disfunción Cognitiva/diagnóstico por imagen , Disfunción Cognitiva/fisiopatología , ADN de Helmintos/sangre , ADN de Helmintos/orina , Humanos , Larva/genética , Neurocisticercosis/diagnóstico por imagen , Neurocisticercosis/fisiopatología , Neuroimagen/métodos , Perú , Reacción en Cadena de la Polimerasa/métodos , Taenia solium/aislamiento & purificaciónRESUMEN
For epidemiological work with soil transmitted helminths the recommended diagnostic approaches are to examine fecal samples for microscopic evidence of the parasite. In addition to several logistical and processing issues, traditional diagnostic approaches have been shown to lack the sensitivity required to reliably identify patients harboring low-level infections such as those associated with effective mass drug intervention programs. In this context, there is a need to rethink the approaches used for helminth diagnostics. Serological methods are now in use, however these tests are indirect and depend on individual immune responses, exposure patterns and the nature of the antigen. However, it has been demonstrated that cell-free DNA from pathogens and cancers can be readily detected in patient's urine which can be collected in the field, filtered in situ and processed later for analysis. In the work presented here, we employ three diagnostic procedures-stool examination, serology (NIE-ELISA) and PCR-based amplification of parasite transrenal DNA from urine-to determine their relative utility in the diagnosis of S. stercoralis infections from 359 field samples from an endemic area of Argentina. Bayesian Latent Class analysis was used to assess the relative performance of the three diagnostic procedures. The results underscore the low sensitivity of stool examination and support the idea that the use of serology combined with parasite transrenal DNA detection may be a useful strategy for sensitive and specific detection of low-level strongyloidiasis.
Asunto(s)
ADN de Helmintos/aislamiento & purificación , Reacción en Cadena de la Polimerasa/métodos , Strongyloides stercoralis/genética , Strongyloides stercoralis/aislamiento & purificación , Estrongiloidiasis/diagnóstico , Adolescente , Adulto , Animales , Teorema de Bayes , Estudios Transversales , ADN de Helmintos/sangre , ADN de Helmintos/genética , ADN de Helmintos/orina , Ensayo de Inmunoadsorción Enzimática/métodos , Heces/parasitología , Femenino , Humanos , Masculino , Microscopía , Modelos Estadísticos , Sensibilidad y Especificidad , Strongyloides stercoralis/ultraestructura , Estrongiloidiasis/sangre , Estrongiloidiasis/parasitología , Estrongiloidiasis/orina , Adulto JovenRESUMEN
Detecting infections of Strongyloides stercoralis is arduous and has low sensitivity. Clinically this is a major problem because chronic infections may disseminate in the host and lead to a life threatening condition. Epidemiologically, S. stercoralis is often missed in surveys as it is difficult to identify by standard stool examination procedures. We present, for the first time, evidence that the infection can be detected in filtered urine samples collected and processed in the field and subsequently assayed for the presence of parasite DNA. Urine specimens (â¼40mL) were collected from 125 test and control individuals living in rural and peri-urban regions of Northern Argentina. From the same individuals, fresh stool specimens were processed using three different copropological methods. Urine specimens were filtered in the field through a 12.5cm Whatman No. 3 filter. The filters were dried and packed individually in sealable plastic bags with desiccant and shipped to a laboratory where DNA was recovered from the filter and PCR-amplified with primers specific to a dispersed repetitive sequence. Prevalence of S. stercoralis infection by stool culture and direct examination was 35/125 (28%), In contrast, PCR-based detection of parasite-specific trans-renal DNA in urine indicated that 56/125 (44.8%) carried the parasite. Of the patients that tested positive for urine-based parasite DNA, approximately half also tested positive in their stool specimens. There were 6.4% of cases where parasite larvae were seen in the stool but no DNA was amplified from the urine. As proof of principle, DNA amplification from urine residue reveals significantly more cases of S. stercoralis infection than the current standard stool examination techniques. Additional work is required to establish the relative utility, sensitivity and specificity of urine-based analysis compared to parasitological and nucleic acid detection from stool for clinical and epidemiological detection for S. stercoralis infection.