RESUMEN
INTRODUCTION: Chagas disease caused by Trypanosoma cruzi (T cruzi) found in the Americas is often missed during the early stage of infection due to lack of sensitive diagnostic tests. The classic immunological and parasitological tests often fail in the acute phase due to the nonspecific and low antibody level nature of the infection and in the chronic phase due to low levels of trypanosomes in the blood. For successful control strategies, there must be a sensitive and specific diagnostic test. OBJECTIVE/METHODS: We have demonstrated the possibility (proof of concept) of detecting T cruzi-specific repeat DNA via polymerase chain reaction (PCR) by (1) spiking 15 urine samples collected from volunteers free of prior infection with 3 different concentrations of T cruzi (3 strains), Trypanosoma brucei, and Trypanosoma rhodesiense (African strain) genomic DNA and (2) from filtered collected clinical samples from Argentina. Three sets of primers were used. RESULTS: Our approach detected repeat DNA specific for T cruzi strains from 1 clinical sample by 2 sets of primer and from spiked urine by all 3 sets of primer but not the African species. A serial dilution (spiking) also was performed on T cruzi strains to detect sensitivities of the assay. One set of primers constantly detected satellite DNA for all T cruzi strains from 70 pg/µl to 175 fg/µl. CONCLUSIONS: We were able to demonstrate the feasibility of detecting T cruzi-specific DNA from filtered urine samples by sensitive and specific PCR assay. Besides the evident increased sensitivity and specificity of primers, our approach can be used to explore Chagas prevalence in endemic areas - especially in congenital Chagas newborn screening - and in the acute phase.
Asunto(s)
Enfermedad de Chagas , ADN Protozoario , Reacción en Cadena de la Polimerasa , Trypanosoma cruzi , Humanos , Enfermedad de Chagas/diagnóstico , Enfermedad de Chagas/orina , ADN Protozoario/orina , Trypanosoma cruzi/genética , Trypanosoma cruzi/aislamiento & purificación , Sensibilidad y Especificidad , Argentina/epidemiología , Especificidad de la EspecieRESUMEN
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.