MULTIPLEX STI AND NEONATE PATHOGEN TEST CLINICAL VALIDATION
Research type
Research Study
Full title
Formulation of Multiplex PCR test using TaqMan low-density arrays for simultaneous detection of multiple STI and Neonate Pathogens
IRAS ID
202273
Contact name
Victoria Chalker
Contact email
Sponsor organisation
Public Health England
Duration of Study in the UK
1 years, 0 months, 1 days
Research summary
Research Summary
We are proposing to use surplus DNA from specimen from NHS patients received by PHE that are referred to the reference laboratory for bacterial testing to validate a bacterial pathogen Multiplex PCR. This will enable the validation of two multiplex tests for sexually transmitted pathogens and neonate pathogens that contain in some cases the same primer design for specific pathogens.. Sexually transmitted pathogens are of major and increasing concern, with 450,000 diagnoses (PHE, 5/6/13). Group B streptococci, is the major cause of mortality in babies less than 1 month old, causing meningitis, septicemia and long term disability. In countries (USA, France, Canada) all pregnant women are screened for GBS prior to treatment. Taqman® low density array cards (TLDA) have recently been developed by PHE to enable the detection of several pathogens simultaneously, including microbes involved in liver disease and respiratory disease. We wish to perform clinical evaluation of the TLDA cards which might be used as a rapid diagnostic tool to inexpensively supply evidence of complex infections particularly in neonates, to detect 48 targets simultaneously. Current reference laboratory methods for several pathogens have been combined into two single assays to give detection of pathogens in under 2 hours, alleviating the requirment for culture. This application is to enable clincial validation of the new multiplex tests on anonomysed specimens submitted to PHE for pathogen detection and if successful could enable rapid detection of mixed infections and multiple targets in babies with septicemia and meningitis.Summary of Results
This study was initiated to develop a multiplex PCR for the detection of STI and neonate pathogens.
Group B streptococci, is the major cause of mortality in babies less than 1 month old, causing meningitis, septicaemia and long term disability. In countries (USA, France, Canada) all pregnant women are screened for GBS prior to treatment. Taqman® low density array cards (TLDA) have recently been developed by PHE/UKHSA to enable the detection of several pathogens simultaneously, including microbes involved in liver disease and respiratory disease. This test might be used as a rapid diagnostic tool to inexpensively supply evidence of complex infections, to detect 48 targets simultaneously. In clinical settings, diagnostic test selection may be based on limited information and therefore screening for many relevant pathogens may not occur, simply because they were not requested by the referring clinician, consequently infectious aetiologies can remain unidentified especially if less common pathogens such as ureaplasma are implicated. Delays may occur if reference laboratories are required to run tests for less common pathogens where specimen transport time and several tests are performed, greatly increasing the turn-around time for results. The patient may remain for extended periods on broad-spectrum therapy pending the results of different tests, with the associated increased drug toxicity, unwarranted selective pressure for antibiotic resistance, cost and length of hospital stay. The detection takes place within an analyser (such as ViiA7) with results generated in less than 90 minutes. Often normal PCR and culture results are available after 48 h.
Aims To ensure that this assay is appropriately validated and fit for purpose as a diagnostic assay, according to the UKHSA document Validation of assays, in order to comply with section F1.2 of the CPA Standards, Section 5.5 of ISO15189 and with section 8 of the Joint Code of Practice for Research.Results The test was developed to detect multiple pathogens and validated. This assay is intended to combine current reference laboratory tests and enable detection of mixed infections simultaneously, resistance (such as inclusion of mecA) and virulence marker detection (such as GBS gene indicated in increased meninges adhesion). Sensitivity was determined the limit of detection (LOD) for Array cards as dilution 10-9 with an equivalent of three plasmid copies/µl reaction. The assay was reproducible. The multiplex was tested for exclusivity against 106 different species of bacteria and viruses. (All results were negative for all 45 targets on the TLDA cards). Inclusivity panels were tested to confirm that the TLDA assays detected all the species of microorganisms at their corresponding targets and that there was no cross reaction amongst targets. This test is intended to detect a wide variety of microorganisms in neonate respiratory, CSF or sera specimens, simultaneously detecting several pathogens. The sensitivity on stored DNA was lower for M. hominis and Ureaplasma when compared with the in house PCR tests and therefore it is recommended that the assay is used as a primary screen for neonate specimens and negatives are subject to additional PCR testing for a trial period in parallel.
Status Insufficient staffing and funding prevented implementation to service and completion of the validation. The main investigator left the organisation in June 2022 No pharmaceutical formulation was used in the study.
Results were published in two paediatric conferences. As the work was not supported to completion final publication has not yet been undertaken
REC name
London - Camden & Kings Cross Research Ethics Committee
REC reference
16/LO/0551
Date of REC Opinion
9 May 2016
REC opinion
Favourable Opinion