B-237 Analytical and Clinical Performance Evaluation of the Panther Fusion® GI Parasite Assay (In Development)
Authors: Moberly J , Dong V , Dockter B , Marsh Z , Higgins M , Miller D , Laffer K , Lopez D , Kirkconnell B , Carvallo Pinto M 
Published: September 2023

Abstract

Background
Acute diarrhea from gastrointestinal (GI) infections is the leading cause of outpatient visits, hospitalizations, and loss of quality of life, with an estimated global impact of 500 million illnesses and 230 000 deaths annually. Most GI infections from bacteria, viruses, and parasites present similar symptoms, but successful treatment is dependent on accurate pathogen identification. For identification of parasites, microscopic testing is often used, but is laborious and often results in inconclusive or inaccurate diagnoses. Clinicians now rely on rapid and accurate molecular diagnostics to correctly identify the causative organism, which leads to optimal infection control and appropriate treatment. The Panther Fusion (PF) GI Parasite Assay is a multiplex qualitative real-time PCR assay that addresses the need for accurate and sensitive identification and detection of the most common parasites that are known to cause gastroenteritis: Cryptosporidium, Entamoeba histolytica, Giardia lamblia and Cyclospora cayetanensis.

Methods
Stool specimens collected in Cary-Blair (CB) are transferred to the AptimaTM Multitest collection tube (Hologic). Multitest tubes are loaded on Panther Fusion where nucleic acid isolation, amplification, and detection are performed. Analytical sensitivity was assessed using cell suspension of Cryptosporidium, Giardia or E. histolytica spiked in negative CB Stool (CBS) matrix and the limit of detection (LoD) was determined using probit. Since Cyclospora cayetanensis cannot be cultured, in-vitro transcribed RNA (IVT) was spiked in CBS matrix for LoD evaluation. Inclusivity was evaluated using cell suspensions for 20 Giardia and 15 E. histolytica strains spiked in CBS matrix. Cryptosporidium inclusivity was evaluated with synthetic nucleic acid. Specificity was evaluated by spiking cells suspensions or synthetic nucleic acid of various microbes in CBS in the presence or absence of GI targets. For clinical performance, 119 archived frozen stool specimens in CB and 40 contrived specimens were tested on Film Array GI Panel (BioFire) and PF GI Parasite Assay and results were compared.

Results
The LoD for Cryptosporidium, Giardia and E. histolytica was determined to be &lt; 1 cell/mL in the Aptima Multitest tube. For C. cayetanensis, the LoD was found to be 1032 copies/mL. The PF GI Parasite Assay detected 20 strains of Giardia lamblia and 15 E. histolytica strains. In-silico and initial testing of synthetic nucleic acid demonstrated detection of 10 Cryptosporidium species. No cross-reactivity to closely related organisms or other microorganisms commonly found in the gastrointestinal tract was observed, except for cross-reactivity to Entamoeba nuttalli, which rarely infects humans. In testing of clinical remnant specimens, the PF GI Parasite Assay had a 97% PPA (35/36) for Cryptosporidium, 100% PPA (58/58) for Giardia lamblia, 100% PPA (42/42) for E. histolytica (included 40 contrived specimens) and 100% PPA (8/8) for Cyclospora with the Film Array GI Panel.

Conclusion
The PF GI Parasite Assay demonstrated excellent analytical and clinical performance in the detection and differentiation of Cryptosporidium, Entamoeba histolytica, Giardia lamblia and Cyclospora cayetanensis. Together with its companion Panther Fusion assays that detect bacterial and viral GI pathogens (also in development), this assay will provide a sensitive and accurate molecular tool for the automated detection of parasites in human stool.