We propose the development and clinical validation of a rapid qualitative molecular test to detect Neisseria meningitidis DNA in patient specimens, allowing rapid (<2 hour) confirmation of meningococcal infection. There is currently no rapid diagnostic test which can be used to assist with clinical diagnosis of early-stage meningococcal disease, and significant turnaround times are required even for the fastest laboratory tests. In contrast, the proposed new test would be available for near-patient testing by staff without laboratory skills and without highly specialized or costly equipment (ie it could be used in hospital A&E departments, large primary care units or pharmacies).
Objectives: a] The first objective of this work is to develop a culture-independent molecular test that is rapid, can be used in near-patient settings and that can be operated by staff outside a laboratory, b] The second objective is to validate the test in a paediatric A&E setting against the current gold-standard methods (culture and laboratory PCR testing) offered by the routine laboratory service in this hospital, and c] Assessment of feasibility, acceptability and problems associated with implementing a near-patient test for meningococcal disease.
a) Test development
The proposed near-patient test will detect the same target genes (ctrA and porA) as our existing polymerase chain reaction (PCR) assay, but will use an alternative isothermal method known as ‘Loop-mediated Isothermal Amplification’ (LAMP). Unlike PCR, isothermal methods do not require expensive or complicated thermal cycling instruments, which makes them very attractive for point-of-care testing. LAMP assays typically allow amplification of target DNA sequences with higher sensitivity and specificity than PCR, with reaction times of between 30 and 60 minutes, which is equivalent to the fastest real-time PCR tests. Significantly, the amplified target DNA can be easily detected in the LAMP reaction allowing rapid discrimination between positive and negative specimens. As proof-of-principle, we already have a prototype LAMP assay working in the laboratory which can detect the ctrA gene in clinical specimens within 60 minutes. Following laboratory optimisation of the new assay, protocols for a sensitive, specific, simple and robust near-patient test will be developed.
b) Clinical validation
This study will be carried out in the A&E department of the Royal Belfast Hospital for Sick Children. The results of near-patient testing using the new LAMP assay will be compared with results from laboratory testing using the same assay, and with results from routine laboratory PCR tests and culture.
Group 1: All children with suspected meningococcal disease (MD) are entered into a ‘multiprofessional integrated care pathway’ and have a standardised set of investigations (to make diagnosis, assessment of severity and initial treatment) performed. In a recent one year study in this hospital, 104 suspected MD children were recruited and over 1/3 had proven MD. The study proposed here would test all children with suspected MD over at least a 2½ year period (giving ~250 patients, with at least 80 definite cases), allowing the clinical sensitivity of the near-patient test to be established.
Group 2. Seven hundred and fifty children presenting with non-specific febrile or upper respiratory tract illnesses (illnesses similar to the MD prodrome) and attending the A&E department will also be studied, in order to determine the specificity of the new test. These children will be followed up for 72 hours to record their final outcome.