Development of novel nucleic acid diagnostics technologies for the detection of predominant microorganisms associated with bacterial meningitis

Abstract

With an estimated 1.2 million cases annually worldwide, bacterial meningitis is a

medical emergency and primary prevention as well as accurate diagnosis and

treatment of this often fatal disease is paramount. The most common microorganisms

associated with bacterial meningitis are Haemophilus influenzae, Neisseria

meningitidis and Streptococcus pneumoniae.

Several vaccines are available for the prevention of bacterial meningitis caused by

these three microorganisms; however, these vaccines offer limited serotype coverage

and as a consequence bacterial meningitis cases as a result of non-vaccine serotypes

have emerged. Furthermore, limited vaccine availability in resource poor countries in

addition to vaccine failures is a major concern in the prevention of bacterial

meningitis worldwide. As such, patients with suspected meningitis require

immediate medical assessment and accurate diagnosis of the disease in order to

provide an optimal therapeutic regime. Moreover, as the incidence of bacterial

meningitis varies significantly by age and geographic location, the accurate

diagnosis of the causative microorganism would enable unambiguous

epidemiological studies to be carried out.

Presently, culture remains the gold standard for the diagnosis of bacterial meningitis,

however, this is limited in its ability to rapidly and accurately diagnose and nucleic

acid based diagnostic methods are more sensitive and specific for the diagnosis of

this fatal disease. The overall aim of this study was to design, develop, optimise and

validate robust, internally controlled nucleic acid based in-vitro amplification

methods for the rapid and accurate identification of H. influenzae, N. meningitidis

and S. pneumoniae. This was achieved using sequential experimental design

consisting of three main studies.

In the first study, RNA transcripts encoded for by the ssrA (tmRNA) and lepA (lepA

mRNA) genes were evaluated using real-time Nucleic Acid Sequence Based

Amplification (NASBA) as potential diagnostic targets for species specific

identification of H. influenzae, N. meningitidis and S. pneumoniae. This established

that tmRNA and lepA mRNA have potential as diagnostic targets for the species

specific identification of N. meningitidis and S. pneumoniae respectively. However,

neither the tmRNA transcript nor the lepA mRNA transcript can be used as a

diagnostic target to unequivocally differentiate H. influenzae from its most closely

related species Haemophilus haemolyticus.

In the second study, whole genome comparative analysis of H. influenzae and H.

haemolyticus identified two novel gene targets, phoB and pstA, present in H.

influenzae and absent in all other closely related species including H. haemoylticus.

Subsequently, two internally controlled real-time PCR diagnostic assays were

developed targeting both phoB and pstA and validated against an extensive panel of

H. influenzae isolates and non-H. influenzae closely related species for the specific

identification of H. influenzae. Analytical sensitivities of the real-time PCR

diagnostic assays developed were determined to be comparable to other published

diagnostic targets and more superior in terms of specificity than a previously

published diagnostics targets for the identification of H. influenzae.

In the third study, phoB was further validated as a diagnostic target for the species

specific detection of H. influenzae by targeting the phoB mRNA transcript in a

duplex real-time NASBA diagnostic assay. In addition, two duplex real-time

NASBA diagnostic assays were also developed targeting the RNA transcripts

encoded for by the ssrA gene and lepA gene for the species specific identification of

N. meningitidis and S. pneumoniae, respectively. All three duplex real-time NASBA

diagnostic assays were determined to be 100% specific for the target species tested

for and analytical sensitivities of less than 60 cell equivalents were determined for

each of the diagnostic assays in duplex format. Each real-time NASBA diagnostic

assay developed in this study includes an endogenous non-competitive Internal

Amplification Control (IAC) to amplify transcript 1 of the Homo Sapiens TBP gene

from human total RNA. By incorporating an endogenous internal amplification

control stably expressed at low levels in human blood, these duplex real-time

NASBA diagnostic assays have the potential to be used in a clinical setting for the

specific, sensitive and rapid (< 60 mins) detection and identification of the most

prominent microorganisms associated with bacterial meningitis in humans.

In summary, nucleic acid based in-vitro amplification diagnostic assays described in

this study are the first description of internally controlled real-time PCR diagnostic

assays and real-time NASBA diagnostic assay for the accurate, species specific

identification of H. influenzae, N. meningitidis and S. pneumoniae.