Point-of-care immunoassay for early diagnosis of pertussis

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Project Summary Pertussis is rapidly re-emerging as a serious public health threat in the United States. Despite high vaccine coverage nationally, annual reported cases in the U.S. have been increasing recently, with a 57-year high of 48,000 in 2012. Moreover, reported cases represent a large underestimate of pertussis infections. Diagnosis of early pertussis (catarrhal stage; prior to paroxysmal cough) is particularly challenging because its symptoms are non-specific and because there are no assays that can diagnose pertussis at the point of patient care. There is a critical need to develop improved pertussis diagnostics to fill this gap because pertussis treatment reduces disease severity and duration, but only if treatment begins prior to paroxysmal cough. The goal of this project is to develop a rapid (<15min), point-of-care (POC) immunoassay to detect Bordetella pertussis during early disease. The target population will be infants with the non-specific, respiratory tract infection symptoms of early-stage pertussis. The approach will be detection of B. pertussis antigens from nasopharyngeal (NP) samples by lateral flow immunoassay (LFI). The POC diagnostic that we propose is innovative because it will change the current clinical status quo: a pertussis LFI will give healthcare providers immediate access to actionable and relevant information, which will enable rapid and appropriate patient care. In Phase I, we demonstrated the feasibility of our LFI approach and achieved every milestone of our Phase I Specific Aims. Specifically, we used an innovative bioinformatics-based strategy to develop epitope-specific polyclonal antibodies (pAbs) against a B. pertussis antigen that has both cell-associated and secreted isoforms. We then validated our pAbs for reactivity with both isoforms in sensitive and specific LFI prototypes. The limit of detection of our current pAb-based LFI is 1.6 x 105 CFU, which is well below the typical bacterial burden of infant NP washes (107 to 1010 CFU/ml) or swabs (106 CFU). In Phase II, we will build on our Phase I results and develop an advanced monoclonal antibody (mAb) based LFI commercial diagnostic. We will focus on developing mAbs against the validated biomarker epitopes (Aim 1) and incorporating these mAbs into an LFI with sensitivity and commercialization potential superior to that of the Phase I pAb-based prototypes. We will i) optimize the mAb-based LFI for analytical sensitivity in NP samples (Aim 2), ii) determine clinical sensitivity and specificity at different disease stages in a clinically relevant, baboon infection model (Aim 3), and iii) determine the LFI's limit of detection with patient samples (Aim 4). Together, this data will guide our FDA 510(k) Pre-Submission at the end of Phase II. Successful completion of our milestones will ultimately yield a rapid, affordable, POC immunoassay that will dramatically increase early pertussis diagnosis, which will i) initiate prompt treatment, ii) reduce disease severity and duration, iii) limit outbreaks by preventing unnecessary transmission, and iv) save infant lives.