Carbapenem-resistant Enterobacteriaceae (CRE) pose a serious threat to public health. These organisms cause infections that are associated with high mortality rates, have the potential to spread widely, and are resistant to all ?-lactam agents and most other antimicrobials leaving virtually no treatment options. Much CRE increase has been due to the spread of CRE that produce the carbapenemase ?Klebsiella pneumoniae Carbapenemase? (KPC) enzyme. KPC in health care settings is a significant challenge as mortality for patients infected with KPC was 23 percent in 7 days, 42 percent in 30 days, and 60 percent by the end of their hospitalization. To reduce the risk of KPC outbreaks and minimize associated healthcare costs, we propose to develop an ultra-sensitive point-of-care (POC) test for quantifying KPC in about 4 hours from rectal swabs using a POC cartridge. The test will confirm that Klebsiella pneumoniae is present, viable and capable of producing KPC enzymes when exposed to a carbapenem, and it will also indicate the KCE concentration to medical professionals which can provide unique insight for treatment options. The proposed KPC guanine- amplified immunoassay will quantify ultra-low levels of KPC in ~4 hours from rectal swabs using a POC cartridge. Rectal swab samples will be exposed to a carbapenem antibiotic along with nutrients, and incubated for 2.5 hours at 35O C to produce KPC if Klebsiella is present, viable and carbapenemase-producing. The test will quantify low levels of KPC using a novel signal amplification platform in a sandwich immunoassay that replaces single optical labels with millions of electroactive guanine-rich oligonucleotide tags bound to microbeads. Capture antibodies bound to immunomagnetic beads will magnetically separate targets from nonspecific materials. Detection limits can be adjusted by changing the size of the microbeads and/or the length of the oligonucleotides. KPC enzyme levels will be quantified from voltammetry signals using a commercial-off-the-shelf 96-well biosensor micro-plate and electrochemical reader (potentiostat). The feasibility of the Guanine-amplified immunoassay is supported by a US Navy project which detected 3 cfu/mL of E.coli O157:H7 and a US Army project for simultaneously detecting E.coli, cryptosporidium and norovirus. Independent testing by demonstrated a 3 - 5 log reduced level in detecting C. parvum antigens from Guanine?s immunoassay compared with commercial ELISA kits. In Phase I we will develop a Guanine-amplified immunoassay for quantifying low level KPC enzymes produced by CP-Klebsiella pneumoniae exposed to carbapenem antibiotics within 4 hours from rectal swab samples (Mo 0-8), adapt the KPC Guanine-amplified immunoassay to operate in a cartridge for automated POU (Mo 6 - 10), and evaluate the KPC Guanine- amplified immunoassay versus Modified Hodge Test, PCR and culture (Mo 10 -12). In Phase 2, expanded testing will be used in applications to the FDA for certification. An automated instrument will also be developed to operate the cartridge at the POC for rapid, ultra-sensitive, multiplex quantification of KPC and other CRE. Guanine, Inc Confidential and Proprietary Page 1
Carbapenem-resistant Enterobacteriaceae (CRE) cause infections associated with high mortality rates, have the potential to spread widely, and are resistant to all ?-lactam agents and most other antimicrobials leaving virtually no treatment options. Most common CRE tests involve time-intensive culturing techniques which can take 2 ? 6 days to confirm the presence of KCE. PCR tests are not widely available leaving healthcare settings vulnerable. The proposed project will develop a POC for quantifying KPC from rectal swabs in 4 hours Guanine, Inc Confidential and Proprietary Page 1
