International journal of infectious diseases : IJID : official publication of the International Society for Infectious Diseases · 2012
ECC-RT-PCR: a new method to determine the viability and infectivity of Giardia cysts.
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Alum Absar, Sbai Basel, Asaad Hamas, Rubino Joseph R, Khalid Ijaz M
Аннотация
BACKGROUND: Giardia sp is a major cause of diarrheal illness worldwide, and millions of people are infected each year. Rapid methods to determine the infectivity and virulence of isolates are critical for the development of intervention strategies to control the transmission of Giardia sp cysts, which occurs through contaminated surfaces, food, and water. However, determining the viability, infectivity, and virulence of Giardia sp cysts using molecular methods is a technical challenge because of the lack of a cell culture model.
METHOD: This study was designed to evaluate mRNA expression in trophozoites and to assess trophozoite attachment to cell monolayer and changes in transcellular resistance as an indicator of Giardia sp viability and infectivity. Heat shock mRNA in Giardia cysts and variant-specific protein (VSP) mRNA in trophozoites were quantified by reverse transcription polymerase chain reaction (RT-PCR). C2bb (Caco-2) cells were grown on transwell chambers to study the attachment of trophozoites, changes in transcellular resistance, and expression of VSP in trophozoites.
RESULTS: The results of these molecular and cell culture studies indicate a direct linear correlation between the viability and infectivity of fresh stocks of Giardia sp cysts. The attachment of trophozoites to cell monolayer, expression of VSP, and change in the transcellular resistance was directly correlated with their infectivity in neonatal mice. PCR was successfully combined with the electrophysiological analysis of cell culture (ECC-RT-PCR) post-trophozoite attachment.
CONCLUSION: This study shows that the ECC-RT-PCR, a new integrated cell culture assay, can be used as a rapid and cost-effective tool for assessing the viability and infectivity of environmental isolates of Giardia sp cysts.
METHOD: This study was designed to evaluate mRNA expression in trophozoites and to assess trophozoite attachment to cell monolayer and changes in transcellular resistance as an indicator of Giardia sp viability and infectivity. Heat shock mRNA in Giardia cysts and variant-specific protein (VSP) mRNA in trophozoites were quantified by reverse transcription polymerase chain reaction (RT-PCR). C2bb (Caco-2) cells were grown on transwell chambers to study the attachment of trophozoites, changes in transcellular resistance, and expression of VSP in trophozoites.
RESULTS: The results of these molecular and cell culture studies indicate a direct linear correlation between the viability and infectivity of fresh stocks of Giardia sp cysts. The attachment of trophozoites to cell monolayer, expression of VSP, and change in the transcellular resistance was directly correlated with their infectivity in neonatal mice. PCR was successfully combined with the electrophysiological analysis of cell culture (ECC-RT-PCR) post-trophozoite attachment.
CONCLUSION: This study shows that the ECC-RT-PCR, a new integrated cell culture assay, can be used as a rapid and cost-effective tool for assessing the viability and infectivity of environmental isolates of Giardia sp cysts.