Recombinase polymerase amplification-based assay to diagnose Giardia in stool samples.

Giardia duodenalis is one of the most commonly identified parasites in stool samples. Although relatively easy to treat, giardiasis can be difficult to detect as it presents similar to other diarrheal diseases. Here, we present a recombinase polymerase amplification-based Giardia (RPAG) assay to detect the presence of Giardia in stool samples. The RPAG assay was characterized on the bench top using stool samples spiked with Giardia cysts where it showed a limit-of-detection nearly as low as the gold standard polymerase chain reaction assay. The RPAG assay was then tested in the highlands of Peru on 104 stool samples collected from the surrounding communities where it showed 73% sensitivity and 95% specificity against a polymerase chain reaction and microscopy composite gold standard. Further improvements in clinical sensitivity will be needed for the RPAG assay to have clinical relevance.

[1]  Rebecca Richards-Kortum,et al.  Nucleic Acid Test to Diagnose Cryptosporidiosis: Lab Assessment in Animal and Patient Specimens , 2014, Analytical chemistry.

[2]  Inacio Mandomando,et al.  Burden and aetiology of diarrhoeal disease in infants and young children in developing countries (the Global Enteric Multicenter Study, GEMS): a prospective, case-control study , 2013, The Lancet.

[3]  J. Farrar,et al.  Enteropathogens and chronic illness in returning travelers. , 2013, The New England journal of medicine.

[4]  Robert E Black,et al.  Global burden of childhood pneumonia and diarrhoea , 2013, The Lancet.

[5]  Veronica Leautaud,et al.  A Lateral Flow Assay for Quantitative Detection of Amplified HIV-1 RNA , 2012, PloS one.

[6]  M. Beach,et al.  Cryptosporidiosis surveillance--United States, 2009-2010. , 2012, Morbidity and mortality weekly report. Surveillance summaries.

[7]  R. Richards-Kortum,et al.  A paper and plastic device for performing recombinase polymerase amplification of HIV DNA. , 2012, Lab on a chip.

[8]  D. Stark,et al.  Enteric Protozoa in the Developed World: a Public Health Perspective , 2012, Clinical Microbiology Reviews.

[9]  P. Craw,et al.  Isothermal nucleic acid amplification technologies for point-of-care diagnostics: a critical review. , 2012, Lab on a chip.

[10]  P. Martus,et al.  High Prevalence of Giardia duodenalis Assemblage B Infection and Association with Underweight in Rwandan Children , 2012, PLoS neglected tropical diseases.

[11]  E. Houpt,et al.  Dual probe DNA capture for sensitive real-time PCR detection of Cryptosporidium and Giardia. , 2012, Molecular and cellular probes.

[12]  Shihab U. Sobuz,et al.  High throughput multiplex PCR and probe-based detection with Luminex beads for seven intestinal parasites. , 2011, The American journal of tropical medicine and hygiene.

[13]  S. Svärd,et al.  Behind the smile: cell biology and disease mechanisms of Giardia species , 2010, Nature Reviews Microbiology.

[14]  C. Gorrini,et al.  Evaluation of a real-time polymerase chain reaction assay for the laboratory diagnosis of giardiasis. , 2010, Diagnostic microbiology and infectious disease.

[15]  Olaf Piepenburg,et al.  DNA Detection Using Recombination Proteins , 2006, PLoS biology.

[16]  A. White,et al.  An updated review on Cryptosporidium and Giardia. , 2006, Gastroenterology clinics of North America.

[17]  L. Garcia,et al.  Commercial Assay for Detection of Giardia lamblia and Cryptosporidium parvum Antigens in Human Fecal Specimens by Rapid Solid-Phase Qualitative Immunochromatography , 2003, Journal of Clinical Microbiology.

[18]  V. Demidov,et al.  Rolling-circle amplification in DNA diagnostics: the power of simplicity , 2002, Expert review of molecular diagnostics.

[19]  T. Notomi,et al.  Loop-mediated isothermal amplification of DNA. , 2000, Nucleic acids research.

[20]  E. Ottesen,et al.  Training Manual on Diagnosis of Intestinal Parasites , 1999 .

[21]  L. Garcia,et al.  Evaluation of nine immunoassay kits (enzyme immunoassay and direct fluorescence) for detection of Giardia lamblia and Cryptosporidium parvum in human fecal specimens , 1997, Journal of clinical microbiology.

[22]  F. Mégraud,et al.  Complex polysaccharides as PCR inhibitors in feces: Helicobacter pylori model , 1997, Journal of clinical microbiology.

[23]  J. Compton,et al.  Nucleic acid sequence-based amplification , 1991, Nature.