Evaluation of New Biomarker-based Approaches for Improving the Diagnosis of Childhood Tuberculous Meningitis
Conditions: Tuberculous Meningitis
Sponsor: University of Stellenbosch

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Evaluation of new biomarker-based approaches for improving the diagnosis of 
childhood tuberculous meningitis  
 
Protocol version: 3.0, 21 April 2020 
 
Principal Investigators: 
Prof. Novel N Chegou 
Division of Molecular Biology and Human Genetics, Department of Biomedical Sciences, 
Faculty of Medicine and Health Sciences, Stellenbosch University 
 
 
Prof. Regan Solomons 
Department of Paediatrics and Child Health, Faculty of Medicine and Health Sciences, 
Stellenbosch University / Ward G10, Tygerberg Children’s Hospital, Francie van Zijl avenue, 
Parow Valley 
 
Co-investigators: 
Prof. Gerhard Walzl  
Division of Molecular Biology and Human Genetics, Department of Biomedical Sciences, 
Faculty of Medicine and Health Sciences, Stellenbosch University 
 
Prof. Willem Perold 
Department of Electrical and Electronic Engineering, Faculty of Engineering, Stellenbosch 
University 
 
Project Manager:  
Mr Vinzeigh Leukes 
Division of Molecular Biology and Human Genetics, Department of Biomedical Sciences, 
Faculty of Medicine and Health Sciences, Stellenbosch University  
 
Postgraduate students: 
 

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Mr Manyelo Masilo Charles 
Division of Molecular Biology and Human Genetics, Department of Biomedical Sciences, 
Faculty of Medicine and Health Sciences, University of Stellenbosch 
 
Miss Georgina Nyawo 
Division of Molecular Biology and Human Genetics, Department of Biomedical Sciences, 
Faculty of Medicine and Health Sciences Stellenbosch University 
 
Miss Isabel Pretorius 
Department of Electrical and Electronic Engineering, Faculty of Engineering, Stellenbosch 
University 
Miss Stephanie Minnies 
Division of Molecular Biology and Human Genetics, Department of Biomedical Sciences, 
Faculty of Medicine and Health Sciences, University of Stellenbosch 
 
Literature review 
 
Introduction 
Tuberculous meningitis (TBM) is the most common type of bacterial meningitis in the Western 
Cape Province (WCP) of South Africa 1. The tuberculosis (TB) incidence in South Africa has risen 
from 301 new cases/100,000 population in 1990 to 948 new cases/100,000 population in 2007 2, 
and although the world-wide incidence of the disease has fallen by an average of 1.5% per year 
since 2000, the incidence of the disease in South Africa is still 834 per 100, 000 population as 
indicated by recent World Health Organization (WHO) reports 3. The TB burden is worsened by 
the human immunodeficiency virus (HIV) pandemic. TBM represents the most severe 
manifestation of TB with high morbidity and mortality, despite adequate anti-TB therapy. This is 
mainly the result of delayed diagnosis and initiation of appropriate therapy 4. The pathogenesis of 
TB is still poorly understood. Despite ongoing research, early, cost-effective diagnostic tools are 
lacking 5. Demonstration of acid-fast bacilli and/or culture of Mycobacterium tuberculosis (Mtb) 
from cerebrospinal fluid (CSF) represents the gold standard for diagnosing TBM, but unfortunately 
the sensitivity of both these tests is low 6, 7. The sensitivity of the recently developed GeneXpert 

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test (Cepheid Inc., USA) for TBM is ˜50-60%, but only improves to 72% when CSF is centrifuged 
as demonstrated in a recent study 8. The diagnosis of TBM is therefore based on a combination of 
clinical, laboratory and radiological findings. However, a clinical diagnosis is often delayed, 
especially in the early stages of the disease when few neurological signs may be present. In 
addition, the CSF picture can be highly variable 9. Although various case definitions for TBM have 
been reported in the literature, diagnostic criteria vary widely, so that no standardized case 
definition exists 6, 7, 9-11. An international workshop on TBM was held during May 2009 in Cape 
Town, South Africa. This was attended by international experts on TBM, and a uniform case 
definition for TBM was proposed for future research. 
 
Uniform research case definition for TBM 12 
It is proposed that patients suspected of having TBM be categorized into definite, probable, 
possible and no TBM. This is proposed to obtain standardization for research purposes, for adults 
and children, and was not primarily devised as a clinical tool. The clinical diagnosis of TBM 
(probable or possible) is based on clinical findings and special investigations (Table 1). The 
boundaries between groups are not fixed, and patients can interchange between groups, based on 
results of laboratory and radiological investigations. Definite TBM requires demonstration of acid- 
fast bacilli in the CSF, Mycobacterium tuberculosis culture from CSF, a positive nucleic acid 
amplification test (PCR) of CSF or histopathological evidence of Mycobacterium tuberculosis 
from a central nervous system site. 
 
Patients must have a clinical diagnosis of meningitis including one or more of the following 
symptoms: headache, irritability, vomiting, fever and neck stiffness (Table 1). The diagnosis of 
probable or possible TBM is based on 1) clinical findings 2) CSF results 3) neuroimaging findings 
4) evidence for TB outside the central nervous system and 5) additional laboratory criteria. A 
scoring system then determines whether a patient falls in the probable or possible TBM category 
(Table1). Points are allocated for a positive finding in each of the categories, with a maximum 
score for each category. A total score of at least 10 is compatible with probable TBM, while a total 
score of at least 6 equates with a possible TBM diagnosis. A certain number of points have to come 
from the CSF or neuro-imaging criteria to ensure that the patient is indeed suffering from 
meningitis and not some other chronic type of tuberculosis. It is also suggested by the consensus 

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group that all patients should have additional CSF investigations to exclude alternative causes for 
the clinical presentation. 
 
In practice, a clinical case definition of bacterial meningitis is needed because most patients 
admitted to a tertiary setting such as Tygerberg Hospital, will already have had antibiotics, which 
usually results in negative blood and CSF cultures 13. Similarly, a definite or clinical diagnosis of 
viral meningitis as possible cause for aseptic meningitis is critical, since aseptic meningitis is 
frequently caused by viruses 14. If an alternative cause is found for the clinical presentation, a 
diagnosis of not-TBM should be made, excepting where dual infection is confirmed. 
 
CSF diagnosis of TBM 
It has been shown in adults that the bacterial yield of Mycobacterium tuberculosis can be improved 
by obtaining larger volumes of CSF and optimizing centrifugation 15, 16. Additional measures for 
improving sensitivity in identification of the organism include, increasing the amount of time taken 
with microscopy and alternative staining methods 16. Based on calculations of Den Hertog et al., 
optimal CSF centrifugation in case of mycobacterium tuberculosis infection should be at a 
minimum speed and duration of 3200x g for 22 minutes 17. 
 
Acid-fast staining is the most rapid method for the detection of mycobacteria in clinical samples. 
Acid-fast staining methods include the Ziehl-Nielsen (ZN) and Auramine O stains. Since CSF is 
sterile, as a rule, the demonstration of acid-fast bacilli in the CSF is highly suggestive of TBM. 
Fluorescence microscopy uses a lower power objective lens than conventional microscopy. This 
enables a more rapid and cost-effective assessment of the full area of a slide. A disadvantage is the 
possibility of false-positive results due to inorganic objects incorporating the fluorochrome dyes 
18. Unfortunately, the use of fluorescence microscopy has been limited in resource-poor settings 
by the short half-life and high cost of the traditional mercury vapour lamp. Light-emitting diode 
(LED) technology provides a cost-efficient alternative; it is cheap, robust and has very low energy 
requirements 19. 
 
In response to the need for more rapid diagnosis of TB and the identification of resistant strains, 
nucleic acid amplification tests specific for Mtb have been developed. In a comparative study 

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between the Genotype Mycobacteria direct assay, the Gen-Probe Mycobacterium tuberculosis 
amplified direct test and the Genotype MTBDRplus for direct detection of mycobacterium 
tuberculosis complex in clinical samples, the Genotype MTBDRplus had the highest sensitivity 
(97.9%) 20. In a study evaluating the Mtb complex detection rate using the Genotype MTBDRplus 
assay on acid-fast bacillus (AFB) smear positive sputum and PCR positive non- sputum specimens, 
the sensitivities were 93.8% and 77.8% respectively 21. For the Genotype MTBDRplus assay, the 
sensitivity (98.1%) and specificity (98.7%) for rifampicin resistance is very high. The accuracy for 
isoniazid is variable, with a lower sensitivity (84.3%) but very high specificity (99.5%) 22. PCR is 
usually only done on culture. PCR on fresh CSF could potentially offer earlier confirmation of 
TBM diagnosis and resistance. 
 
The GeneXpert MTB/RIF (Xpert) assay targets the rifampin resistance-associated rpoB gene 
region by heminested PCR with three specific primers and combines the sensitive detection of M. 
tuberculosis DNA and determination of rifampicin resistance using molecular beacons 23. 
Processing time is short due to automation of bacterial lysis, DNA extraction, real-time PCR 
amplification, and amplicon detection in a single system. High sensitivity and specificity of the 
Xpert assay has been shown on sputa with the assay correctly identifying 77.3% of all culture- 
positive specimens 24. Similar results are not applicable to extrapulmonary specimens, though, 
where the amount of mycobacterial DNA is much less. The test runs on the GeneXpert platform 
(Cepheid, Sunnyvale, CA) using a disposable plastic cartridge with all required reagents 25. 
Although the WHO currently recommends Xpert as an initial diagnostic test for TBM, the 
sensitivity of the test on CSF samples is not as high as would be obtained with sputum samples 
and increased to 72% when CSF was centrifuged 8. 
 
Use of host biomarkers in the diagnosis of TBM 
Recent technological advances have made it possible to screen for many biomarkers in as little as 
3 µl of sample using the Luminex multiplex cytokine beaded arrays. Using the Luminex platform, 
it is now possible to simultaneously detect the levels of 100 biomarkers in 3 to 50 µl (if using the 
Luminex/Bio-Plex 200 platform) and up to 500 markers if using the Luminex FlexMap 3D system. 
This platform has enabled the identification of novel host protein biomarkers for several diseases 
including TB. In previous studies, some of which were pioneered by our research group at 

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Stellenbosch University, novel biomarkers were detected in adult Quantiferon supernatants, which 
allowed for discrimination between TB disease and latent infection 26. When the biomarkers 
identified in adults were investigated in children, a biosignature comprising of four markers 
diagnosed paediatric TB disease with an accuracy of 84% 27. 
 
In a recently concluded large multi-centred pan-African study (the African European Tuberculosis 
Consortium –www.ae-tbc.eu), we observed that some of the markers that frequently showed 
potential in our previous TB-antigen stimulation studies26-30 performed best either individually or 
in multi-marker models when measured in unstimulated supernatants. Further work on ex vivo 
serum biomarkers in 716 individuals that presented signs and symptoms suggestive of TB, prior 
to clinical diagnosis in five African countries (Namibia, Uganda, Malawi, The Gambia and South 
Africa), resulted in the identification of a seven-marker serum biosignature, which diagnosed TB 
disease in adults with high accuracy 31. 
 
There are not many studies in literature that specifically employed multiplex cytokine detection 
platforms in the search of host biomarkers specifically for TBM. In a recently patented and 
published study conducted by our research group 32, we investigated the usefulness of some of the 
host markers that we identified in our previous studies as diagnostic candidates for TBM. A three-
marker CSF biosignature comprising of IL-13, VEGF and cathelicidin LL-37, diagnosed TBM 
with a sensitivity of 52%, specificity of 95%, with positive and negative predictive values of 91% 
and 66% respectively 33. In other biomarker studies, specifically investigating the use of whole 
blood RNA biosignatures in the diagnosis of TB disease, it was shown that host blood-based RNA 
transcripts might be useful in the diagnosis of active TB disease in both adults 34 and children 35. 
Furthermore, a recent study identified blood RNA signature that showed potential as a biosignature 
for the risk of developing active TB disease 36. However, none of these studies have been 
conducted specifically in children with TBM and it is not known, whether simple blood RNA 
signatures such as the ones already identified for pulmonary TB might be useful in this disease. 
The overall aim of the current study is therefore to assess the utility of ex vivo blood and CSF 
based host cytokine biomarkers and RNA transcripts as diagnostic tools for TBM. We would 
specifically be validating the diagnostic accuracy of our previously established 3-marker TBM 
CSF biosignature, evaluate other new host markers which have shown potential in adult studies 

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but which have not been investigated in children, in more children with TBM and also RNA 
signatures in blood samples collected from the same children. At the conclusion of the proposed 
study, the validated blood-based biosignatures identified in the present study shall be ready for 
possible incorporation into rapid, point-of-care diagnostic tools such as the one currently being 
developed for adult pulmonary TB by our research group (www.screen-tb.eu). 
 
Hypothesis 
CSF and blood samples obtained from children with TBM will possess distinct host biosignatures 
in comparison to CSF and blood samples from children with other forms of meningitis and these 
biosignatures can be used as a tool for the rapid diagnosis of TBM. 
 
Aims 
The main aims of the proposed project are as follows: 
 
1) To validate a previously identified and patented three-marker CSF biosignature (IL-13, VEGF, 
cathelicidin LL37) for the diagnosis of TBM, in a new cohort of children with suspected TBM 
 
2) To evaluate the usefulness of a recently identified and patented adult seven-marker serum 
protein biosignature (CRP, serum amyloid A, apolipoprotein A-1, transthyretin, complement 
factor H, interferon-gamma and IP-10) and other recently identified host markers, as a tool for 
the diagnosis of TBM 
 
3) To investigate the usefulness of whole blood RNA transcripts as biosignatures for the diagnosis 
of TBM 
 
4) To characterize the site of disease (CSF) versus peripheral blood immune profile of children 
with TBM, the response of immune cells to stimulation with M.tb antigens and explore 
differences between such responses in children with and those without TBM as part of student 
projects. 
 
5) To develop a prototype point-of-care test based on host inflammatory protein biomarkers for 

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the rapid diagnosis of TBM (e.g. at the bedside) in children suspected of having TBM. 
 
Patients and methods 
1. Study design 
A prospective study 
 
2. Study setting 
The study will be conducted at Tygerberg Children’s Hospital, Parow Valley, Cape Town. The 
hospital is the tertiary academic hospital of the Faculty of Medicine and Health Sciences, 
University of Stellenbosch. Children with tuberculous meningitis are referred from primary care 
day hospitals, district and secondary level hospitals from our drainage area. Children with 
suspected TBM are referred to our institution to establish the diagnosis of TBM and to treat the 
complications associated with advanced disease (stage 2 and 3 TBM, e.g. hydrocephalus). 
Research samples collected for the purposes of the current study shall be processed at the 
Stellenbosch University Immunology Research Group (SUN-IRG) laboratory, Department of 
Biomedical Sciences, Faculty of Medicine and Health Sciences. The SUN-IRG laboratory is 
accredited by the South African National Accreditation Agency as a specialist Immunology 
Research laboratory. 
 
3. Inclusion and exclusion criteria: 
All children between the ages of 3 months and 13 years with suspected meningitis, and who require 
CSF examination for routine diagnostic purposes at Tygerberg Children’s Hospital, where written 
consent is obtained will be included. If possible, assent will be obtained in those children older 
than 7 years who have a normal level of consciousness, i.e. a Glasgow Coma Score (GCS) of 
15/15. Children 13 years and older will be excluded from the study. Failure to obtain written 
consent will also exclude children from the study. 
 
The anticipated study period is November 2016 to December 2025. The estimated sample size, 
using a two-sample comparison of proportions (STATA®-Statistics/Data Analysis), required for 
each of the diagnostic aims of the study includes, at least, 72 children with definite or probable 
TBM and a total sample size of up to 460 patients (This includes children with TBM, bacterial 

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meningitis, viral meningitis and normal CSF). An average of 40 new TBM cases is admitted to our 
wards annually. 
 
4. Diagnostic work-up 
All patients will undergo a comprehensive clinical evaluation (Addendum1). Routine special 
investigations will be performed. CT of the brain will be done in all probable TBM cases and in 
other cases of meningitis when indicated. Air- encephalography will be performed in all TBM 
cases with hydrocephalus. MR imaging will be done as clinically indicated. Lumbar puncture will 
be performed and the following CSF investigations performed: appearance and colour 
determination; differential cell count determination by standard methods; protein, glucose and 
chloride determination by standard methods; centrifugation with Gram stain and India ink 
examination of the deposit; culture of the centrifuged deposit on blood agar plates for pyogenic 
bacteria; 
Auramine 
“O” 
staining 
& 
fluorescence 
microscopy, 
culture 
using 
the 
BBL™MGIT(Mycobacterium Growth Indicator Tubes)™ of Becton and Dickinson and detection 
of Mtb DNA using the HAIN Genotype MTBDRplus kit. Samples will be tested using Xpert Ultra 
and newer technologies e.g. AlereQ TB and the Savannah MTB assay. Microbiomic analysis will 
be performed on microbial DNA extracted from patient specimens and sequenced on the Illumina 
MiSeq platform. All data will be recorded on a pre-designed clinical data capture sheet (see 
addendum) and entered into a computerized database. Finally, data analysis will be carried out 
using appropriate software and statistical packages. 
 
The diagnosis of TBM will be made based on the following criteria: 
A diagnosis of probable TBM will be made if two or more of the following criteria are present in 
the setting of a characteristic history and CSF changes associated with TBM: A positive history of 
contact with an adult TB case, a positive tuberculin skin test, a chest x-ray suggestive of pulmonary 
tuberculosis (hilar lymphadenopathy, miliary tuberculosis or cavitation, CT or MRI demonstrating 
the characteristic features of TBM (ventricular dilatation, meningovascular enhancement and/or 
granuloma/s), poor weight gain or weight crossing percentiles documented on the Road to Health 
Card or positive identification of acid fast bacilli from gastric washings (microscopy or culture). 
 
A diagnosis of definite TBM will be made if acid-fast bacilli is present in the CSF, Mtb is cultured 

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from CSF, a nucleic acid amplification test of CSF is positive or there is histopathological evidence 
of Mtb from another central nervous system site. The degree of TBM will be classified according 
to the proposed modified British Medical Research Council (BMRC) classification of 1948. -Stage 
I: Glasgow Coma Scale (GCS) of 15 and no focal neurology, Stage IIa: GCS of 15 plus focal 
neurology, Stage IIb: GCS of 11-14 with focal neurology, Stage III: GCS <11. All investigations 
required for establishing a diagnosis (TBM or no TBM) are usually done routinely and shall 
therefore not specifically be as part of the current study. 
 
5. Treatment 
All patients admitted with a diagnosis of TBM will be treated with standard regimen, four anti- 
tuberculous drugs for 6 months and prednisone (2mg/kg body weight; maximum dose 60mg daily) 
for the first month of treatment. All patients with ventriculomegaly on computed tomography will 
undergo an air encephalogram. Children with non-communicating hydrocephalus will undergo 
immediate ventriculoperitoneal (VP) shunting. Response to treatment will be monitored clinically 
and by a repeat CT scan of the brain after the first month of treatment. 
 
All patients admitted with a diagnosis of bacterial meningitis will be treated with standard regimen 
third generation cephalosporin. Viral meningitis will be treated symptomatically; cases of 
suspected or proven herpes encephalitis will be treated with acyclovir. All HIV co-infected patients 
will be offered antiretroviral therapy (ART). Patients treated for TBM will only be started on ART 
after 4-6 weeks of anti-tuberculous therapy to minimize the risk of immune reconstitution 
inflammatory syndrome (IRIS). An exception will be made in cases who are severely immune- 
suppressed; they may be started on ART immediately. ART will consist of first-line therapy (for 
drugs &dosages per kg see addendum 2). Baseline CD4 counts and viral loads will be done before 
initiation of ART and repeated at 6 monthly intervals. 
 
Collection and processing of samples for the proposed study 
All samples required for the current study shall be collected during specimen collection for routine 
diagnosis. That is, specimens shall not specifically be collected from any study participant solely 
for the purposes of this study. During the collection of samples (CSF and whole blood) for routine 
clinical investigations, the alcohol swab used to sterilize the puncture site will be collected and 

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used to subtract background DNA found on the skin, during sequence analysis for the microbiome 
aspects of the study (aim 4). About 5 mL of CSF (3-7ml would be aimed for dependent on age and 
ease of lumbar puncture), shall be collected into sterile cryo tubes (Greiner bio-one) for use for 
research purposes, depending on how much is left over after specimens required for routine 
diagnosis have been collected. It must be stressed that although it is known that collection of up to 
10ml of CSF in children with TBM may be beneficial in relieving of the pressure on the brain, 
hence help with alleviation of the symptoms, the amount of CSF collected will depend on the age, 
clinical presentation of the child, and whether collection of the specimen (3 to 7mL) will be 
beneficial to the child as determined by the specialist neurologist or medical practitioner treating 
the child. This will be followed by the collection of 1ml of whole blood into a serum blood tube 
and 2.5ml into a paxgene blood RNA tube, for the isolation of messenger RNA for future gene 
expression studies (aim 3). Furthermore, an additional 1 ml of blood will be collected from a subset 
of children for investigation of immune cells (whole blood intracellular staining by Flow 
cytometry; aim 7). Whether such an additional blood specimen is collected will depend on the 
judgement of the treating clinician and specialist paediatric neurologist, with the child’s wellbeing 
always taking priority over the research. Where possible, mid-stream urine will be collected into 
sterile collection tubes for diagnostic analysis as part of aims 5 and 6. Therefore, depending on the 
judgement of the clinician and specialist paediatric neurologist,  a total of up to 5mL (3 to 7ml) of 
extra CSF and up to 4.5ml of whole blood and up to 5 mL of urine (where possible) shall be 
collected from each consenting study participant for the purposes of this study. The proposed 
procedure (blood and CSF collection) was used in previously approved and now completed studies 
which provided samples for the pilot studies leading to this validation study (HREC ref no: 
N11/01/006, Principal Investigator, Dr Regan Solomons; and Ref no: N09/10/265, Principal 
Investigator, Dr D Visser). 
 
After collection of specimens for routine diagnostic purposes, the proposed research specimens 
shall be collected from consenting study participants and sent to the SUN Immunology Research 
laboratory, Fourth Floor, Education Building, where processing of these specimens shall occur. 
The CSF samples shall be centrifuged, aliquoted into 250µl amounts and frozen at minus 80 
degrees until used for biomarker analysis using the Luminex technology, as was done in the pilot 
study (33). If more than 1mL of CSF is available, aliquots of the CSF will be used for immune cell 

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analysis by flow cytometry as mentioned above. Similarly, the 1ml of blood collected into a serum 
tube shall be centrifuged and serum harvested and stored at minus 80 degrees until used for 
biomarker analysis. The additional 1ml of blood, if collected, will also be used for flow cytometry 
as part of a student project. For the Paxgene blood, the tubes shall be frozen and when needed for 
use, RNA shall be isolated using the Paxgene blood RNA isolation kit. We will then use 
downstream techniques including quantitative Real Time PCR or other suitable technique to look 
at the usefulness of the RNA blood biosignatures already described in publications such as 
references (34-36), and also new signatures in the diagnosis of TBM. All sample processing shall 
be done according to standard laboratory procedures which are currently used in the SANAS 
accredited SUN-IRG laboratory.  
 
Both the CSF and urine for metabolomics, up to 2ml and 5ml respectively, will be transported to 
the metabolic laboratory at Northwest University in Potchefstroom for analysis of metabolites. 
This will be done by performing mass spectroscopy, coupled with chromatography, followed by 
complex data analysis. The investigations at the metabolic laboratory will be performed and 
interpreted by Dr S Mason, from the biochemistry division at Northwest University. Stellenbosch 
University students will use aliquots of the CSF in future for unbiased evaluation of novel protein 
biomarkers that may be used in the diagnosis of TBM, for optimization of the test that will be 
developed as part of this project or biomarkers that may guide clinical patient management. 
 
Statistical methods and analysis plan 
Differences in the concentrations of individual host biomarkers between participants with TBM 
disease and those without TBM shall be evaluated by the Mann–Whitney U-test for non-parametric 
data analysis or the students t test, if data is normally distributed. P-values shall be deemed 
significant if less than 5%. The diagnostic accuracy of individual host biomarkers shall be 
investigated by receiver operator characteristics (ROC) curve analysis. The predictive abilities of 
combinations of analytes (biosignatures) shall be investigated by General discriminant analysis 
(GDA), random forests, and other biomarker analysis techniques, following the training/test set 
approach as done in previous studies 27, 31-33. For Flow cytometry data, cell gating and data analysis 
will first be carried out on FlowJo software (Oregon, USA), followed by parametric (t-test) or non-
parametric (Mann-Whitney U-) tests to assess the differences in quantities of immune cell 

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subpopulations, intra and extracellular cytokines and other biomarkers among children with TBM 
and those with other meningitis. Kruskall-Wallies test or one-way ANOVA with post-hoc analysis 
will be performed for comparison of more than 2 groups as advised by bioinformaticians from the 
Division of Molecular Biology and Human Genetics. 
 
Ethical concerns and consent 
Informed consent will be obtained from all parents whose children are entered into the study. 
Only patients, whose legal care-givers give written consent for the study will be included. The 
information and consent document will be explained in the 3 main languages spoken in our area 
and obtained in the home language of parents/legal guardians. All consent forms will be back 
translated into English, to ensure clarity and accuracy, in accordance with South African Good 
Clinical Practice guidelines. As mentioned above, assent will be obtained in those children older 
than 7 years who have a normal level of consciousness, i.e. a Glasgow Coma Score (GCS) of 
15/15. Urine, CSF and blood samples shall be collected from consenting study participants only 
where possible, and as deemed possible by the treating clinician and specialist paediatric 
neurologists. It is important to note that about 10ml of CSF may be collected from children 
diagnosed with TBM and collection of the CSF helps relieve the pressure on the brain caused by 
the fluid, thus alleviating symptoms. Ethical clearance for this study was already obtained from 
the Health Research Ethics Committee of the Faculty of Medicine and Health Sciences, 
University of Stellenbosch (Project Reference #: 4130 and HREC Reference #: N16/11/142). 
However, we are hereby requesting an amendment to the ethics to indicate that the project is now 
funded by the European and Developing Countries Clinical Trials Partnership (EDCTP), who 
have provided funds for us to validate the biomarkers, followed by development of a rapid point-
of-care diagnostic test that may assist in the early management of TBM in children with the 
assistance of the Engineering Faculty at Stellenbosch University (Prof Willem Perold and 
students). The Engineers have developed a promising prototype platform to which validated 
biomarkers from this project shall be incorporated, followed by evaluation of the usefulness of 
the test during the course of the project. These later aspects (test development and evaluation) 
were not included in the version of the proposal that was previously approved by the HREC. 
 All information/data collected on the patients will remain confidential. All information/data 

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collected will be kept in a separate password protected file and this will only be known to and 
accessed by the Clinical Principal Investigator (Prof R Solomons). All samples sent to the 
Immunology Research Laboratory shall be anonymized and only identifiable by RedCap 
generated bar codes. Patients/legal guardians shall be informed that their refusal to participate in 
the present study shall not influence their care in any way. Good clinical practice guidelines will 
be adhered to. 
 
Funding 
Most of the special investigations are part of the routine workup of TBM, bacterial and viral 
meningitis patients at Tygerberg Children’s Hospital. The costs for the extra tubes for collection 
of the additional specimens for research purposes and for all the research experiments including 
biomarker validation studies, development and evaluation of the point-of-care test shall be funded 
by an EDCTP (European and Developing Countries Clinical Trials Partnership) Senior Fellowship 
that was recently awarded. The project has been funded for a period of five years. 
 
 
 
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