Accelerating the Development of an Extended-specificity Multiplex Urine Immunoassay for the Diagnosis and Serotyping of Pneumococcal Pneumonia in High Carriage and Disease Burden Settings Like Malawi
Conditions: Pneumonia, Pneumonia - Bacterial
Sponsor: Liverpool School of Tropical Medicine

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Accelerating the development of an extended-specificity multiplex urine immunoassay 
for the diagnosis and serotyping of pneumococcal pneumonia in the high carriage and 
disease burden setting of Blantyre, Malawi 
Protocol V2.0 10th August 2025 
Host 
Malawi Liverpool Wellcome Research Programme (MLW) 
 
Funder  
Wellcome  
 
Sponsor 
Liverpool School of Tropical Medicine (LSTM) 
 
 
 
 
 
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Principal Investigator 
Dr. Brenda Kwambana-Adams, Wellcome International Intermediate Fellow, MLW, LSTM, 
KUHeS 
Principal  Investigator signature: 
                       Date: 23 June 2025 
Co-Investigators 
Akuzike Kalizang’oma, MBBCH, PhD- Post-Doctoral Research Associate, MLW, LSTM 
Dr. Lucy O’Connor-  Clinical PhD Fellow, LSTM 
Charles B. Nkhata, BSc-Pre- MSc Trainee, MLW 
Comfort Brown, BSc-  Senior Laboratory Technician, MLW 
Collaborators 
Prof. Wei Shen Lim - NIHR Nottingham Biomedical Research Centre / Nottingham University 
Hospitals NHS Trust, UK 
Dr Seyi D. Eletu- Respiratory and Vaccine Preventable Bacteria Reference Unit, UK Health 
Security Agency, London, UK 
Dr David Litte- Respiratory and Vaccine Preventable Bacteria Reference Unit, UK Health 
Security Agency, London, UK 
Prof. Neil French- Infectious Diseases and Vaccinology, UoL 
Prof. Robert Heyderman-Infectious Diseases  and Global Health, UCL 
Dr. Todd Swarthout – University Medical Center Utrecht, Netherlands 
Dr. Yamikani Chimalizeni, Paediatric Cardiology, MLW, QECH 
 
 
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List of Abbreviations 
Abbreviation 
Term 
24ssUAD  
24-plex Serotype-specific urinary antigen detection 
ARI 
Acute Respiratory Infection 
AUC 
Area under the curve 
ARGs 
Antibiotic resistance genes 
COMREC  
College of Medicine Research and Ethics Committee 
CRSU  
Clinical Research Support Unit 
GPCC 
Gateway Primary Care Centre 
LMICs 
 Low and Middle Income Countries 
LSTM 
Liverpool School of Tropical Medicine 
MLW 
Malawi Liverpool Wellcome Research Programme 
NHC 
Ndirande Health Centre 
NHS 
National Health Service 
NPS  
Nasopharyngeal Swab 
PCVs 
Pneumococcal conjugate vaccines 
PERCH 
Pneumonia Etiology Research for Child Health 
QECH 
Queen Elizabeth Central Hospital 
QC  
Quality control 
ROC 
Receiver Operating Characteristic 
sSA  
Sub Saharan Africa 
ssUAD 
Serotype-specific urinary antigen detection 
SOPs 
Standard Operating Procedures 
UCL 
University College London 
UKHSA 
UK Health Security Agency 
 
 
 
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Table of Contents 
Executive Summary ............................................................................................. 6 
1.0 
Background .............................................................................................. 8 
1.1. 
Literature Review ........................................................................................... 9 
1.2. 
Rationale...................................................................................................... 10 
1.2.1. 
Summary of NP Resistome study .............................................................................. 10 
1.3. 
Hypothesis ................................................................................................... 11 
1.4. 
Study Objectives ........................................................................................... 11 
1.4.1. 
Broad objective ....................................................................................................... 11 
1.4.2. 
Specific objectives .................................................................................................. 11 
2.0 
Methodology ........................................................................................... 12 
2.1. 
Study Design and Setting: .............................................................................. 12 
2.2. 
Study Population: ......................................................................................... 12 
2.3. 
Study Period: ................................................................................................ 12 
2.4. 
Inclusion and Exclusion criteria: .................................................................... 14 
2.5. 
Sample collection and handling: .................................................................... 14 
2.6. 
Laboratory analysis: ...................................................................................... 14 
2.7. 
Sample size: ................................................................................................. 15 
2.8. 
Data management: ....................................................................................... 15 
2.9. 
Data analysis: ............................................................................................... 16 
2.9.1. 
Prevalence and distribution (objective 1): .................................................................. 16 
2.9.2. 
Association with carriage dynamics (objective 2): ...................................................... 16 
2.9.3. 
Defining diagnostic thresholds (objective 3): ............................................................. 16 
2.10. 
Data sharing: ............................................................................................ 17 
3.0 
Quality assurance and control ................................................................. 17 
4.0 
Dissemination plan ................................................................................. 17 
5.0 
Ethical consideration .............................................................................. 17 
6.0 
Participant compensation ....................................................................... 18 
7.0 
Project risks and constraints ................................................................... 18 
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7.1. 
Dependency on NP Resistome recruitment timeline ........................................ 18 
7.2. 
Difficulty in collecting adequate urine volumes ............................................... 18 
7.3. 
Laboratory assay limitations or technical errors .............................................. 18 
7.4. 
Interpretation challenges due to high carriage prevalence ............................... 19 
8.0 
Budget .................................................................................................... 19 
8.1. 
Budget justification ....................................................................................... 19 
8.1.1. 
Urine Analysis: ........................................................................................................ 19 
8.1.2. 
Shipping: ................................................................................................................ 20 
8.1.3. 
Reagents and consumables: .................................................................................... 20 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
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Executive Summary 
Study type: This is a nested case-control sub-study within the multi-site, observational NP 
Resistome study (COMREC reference P.10/24-1200). 
Problem: Pneumococcal pneumonia is a leading cause of morbidity and mortality among 
children under five, especially in sub-Saharan Africa. Accurate diagnosis remains a challenge 
due to the need for invasive specimen collection and poor sensitivity of standard culture-based 
diagnostic tests, particularly after antibiotic use. Serotype-specific urinary antigen detection 
(ssUAD) offers a promising, non-invasive alternative for serotype surveillance and diagnosis 
of pneumococcal disease. Serotype-specific identification provided by ssUAD is crucial for 
monitoring the impact of vaccines and informing public health interventions. The ssUAD test 
is a Luminex-based urine antigen capture assay developed by the UK Health Security Agency 
(UKHSA) that targets 24 pneumococcal serotypes, with good sensitivity and specificity among 
adults with community-acquired pneumonia in the UK. Further investigation is required to 
determine its ability/utility to identify invasive disease among children, particularly in settings 
like sub-Saharan Africa, where high rates of asymptomatic carriage may affect diagnostic 
accuracy. 
Objectives 
Broad: 
To evaluate the performance of the ssUAD test in detecting pneumococcal carriage and 
distinguishing it from invasive disease among children under five years old in Blantyre,  
Malawi. 
Specific: 
1. To determine the prevalence of serotype-specific pneumococcal antigens in urine among 
children under five years of age with pneumonia compared to their healthy counterparts.  
2. To characterise the association between the detection of serotype-specific antigens in 
urine and nasopharyngeal pneumococcal carriage among children. 
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3. To define revised ssUAD thresholds that may distinguish pneumococcal carriage from 
disease, to support field evaluations and inform diagnostic and surveillance strategies in 
Malawi and similar settings.  
Methodology: We will test 350 existing urine samples that have already been collected from 
children as part of the NP Resistome study (Protocol v5.0 COMREC reference P.10/24-1200), 
including healthy children in the community, children with pneumonia in the community, and 
children hospitalised with pneumonia at the time of recruitment. Participants of the NP 
Resistome study will be recruited from Ndirande Health Centre (NHC), Gateway Primary Care 
Centre (GPCC), and Queen Elizabeth Central Hospital(QECH) in Blantyre, Malawi. Urine 
samples will be aliquoted into 1.8 mL cryotubes and stored at -80°C at Malawi Liverpool 
Wellcome Research Programme (MLW) laboratory in Malawi. Aliquots from each urine 
sample will be tested using the ssUAD in the UK, as the assay is not currently available in 
Malawi. However, we are working towards developing and evaluating the assay locally as part 
of future implementation efforts. Urinary detection of pneumococcal serotypes will be 
compared with both culture-based and metagenomic sequencing results from nasopharyngeal 
swab (NPS) samples taken as part of the main study. 
Expected results and dissemination: This sub-study will generate key data on the prevalence 
of pneumococcal serotype-specific urinary antigens in children with pneumonia and healthy 
controls. It will improve understanding of ssUAD performance as a surveillance tool in this 
setting, help distinguish antigenuria due to carriage from disease, and refine diagnostic 
thresholds for use in high-carriage and disease-burden settings. Study findings will inform 
surveillance, pneumonia diagnostics, and vaccine impact assessments in LMICs. Results will 
be shared with COMREC, the Blantyre District Health Office, at scientific conferences, both 
local and international, and in peer-reviewed publications. 
 
 
 
 
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1.0 Background 
Streptococcus pneumoniae (the pneumococcus) is a common commensal of the human 
nasopharynx, transmitted primarily through respiratory secretions1. While typically 
asymptomatic during carriage, it serves as a prerequisite for the development of invasive 
disease2. The pneumococcus causes a range of life-threatening infections, including 
pneumonia, meningitis, and bacteraemia, with a disproportionately high burden among 
children in sub-Saharan Africa (sSA)3. It remains a leading cause of childhood pneumonia 
worldwide. It contributes significantly to morbidity and mortality in children under five years 
of age, particularly in low- and middle-income countries (LMICs)4,5. Although the introduction 
of pneumococcal conjugate vaccines (PCVs) has significantly reduced the burden of vaccine-
type disease, pneumonia caused by both vaccine and non-vaccine serotypes continues to 
present significant diagnostic challenges6. These challenges stem from the reliance on invasive 
specimen collection, such as blood or endotracheal aspirates and the limited availability of 
sensitive diagnostic tools in resource-limited settings. Conventional culture-based methods are 
further hampered by low sensitivity, especially when antibiotics have been administered before 
specimen collection7. These limitations undermine disease surveillance, leading to 
underestimation of the true burden of pneumococcal disease and the overall impact of 
vaccination programmes 
Serotype-specific urinary antigen detection (ssUAD) has emerged as a promising non-invasive 
tool for diagnosing and serotyping pneumococcal pneumonia8. The ssUAD test is a 25-plex 
Luminex-based extended-range urine antigen capture assay developed by UKHSA, which has 
shown favourable results for diagnosing pneumococcal disease among adult patients in the UK. 
The 24-serotype-specific urine antigen detection (24ssUAD) assay can detect pneumococcal 
capsular polysaccharide antigens in urine, offering greater diagnostic sensitivity than the 
culture-based method9. The current version of ssUAD targets 24 pneumococcal serotypes and 
has a sensitivity of 96.2% and specificity of 89.9% among adults with community-acquired 
pneumonia or invasive pneumococcal disease in the UK8. However, there is limited data on the 
surveillance and diagnostic performance of ssUAD in high-carriage and disease-burden 
settings, such as Malawi, where asymptomatic or healthy children may also shed pneumococcal 
antigens in urine, making it more challenging to distinguish invasive disease. Unpublished data 
from our testing of the 24ssUAD assay on historical urine samples from Malawian children 
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with acute respiratory infection (ARI) indicate that pneumococcal antigens can be detected in 
urine, suggesting the assay’s potential utility in such settings and highlighting the need for 
further evaluation to distinguish between carriage and disease. The data also revealed that pre-
hospital antibiotic exposure significantly reduced the sensitivity of culture-based methods, 
decreasing pneumococcal detection rates from 81% to 21%, highlighting the potential 
diagnostic advantage of urine antigen assays in such contexts. 
1.1. Literature Review 
The accurate diagnosis of pneumococcal pneumonia remains a persistent challenge in both 
clinical and surveillance contexts, especially among young children in high-burden settings10. 
Several studies have demonstrated that reliance on conventional microbiological diagnostics, 
such as blood culture, results in significant under-detection of pneumococcal infections due to 
their low sensitivity and the frequent use of antibiotics before hospital admission11,12. In a 
systematic review by the Pneumonia Etiology Research for Child Health (PERCH) project, the 
limitations of blood culture sensitivity were particularly evident in children, where pathogen 
recovery was often below 10%13.  
These diagnostic limitations have prompted interest in antigen detection technologies, which 
are not reliant on viable organisms14. Among these, the ssUAD assay has gained prominence8. 
The ssUAD platform detects pneumococcal capsular polysaccharides in urine and allows for 
simultaneous serotyping of up to 24 pneumococcal serotypes using a multiplex Luminex-based 
platform8,15. Studies conducted in adult populations in high-income countries have reported 
high sensitivity and specificity for the assay, particularly in cases of community-acquired 
pneumonia16.  
A critical advantage of ssUAD is its performance in patients who have received antibiotics. 
Multiple studies have reported that urinary antigen tests retain high diagnostic sensitivity even 
after antibiotic treatment, in contrast to sputum and blood cultures, which show significantly 
reduced positivity. Similarly, in studies involving hospitalised adults, both the BinaxNOW ICT 
and ssUAD assays were able to detect pneumococcal antigens in urine despite negative cultures 
due to prior antibiotic exposure. However, ssUAD appears to offer superior specificity and 
serotype information. In contrast, BinaxNOW does not provide serotype information and has 
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been associated with false positives, particularly in settings with high pneumococcal carriage18. 
This property makes ssUAD a particularly attractive diagnostic tool in LMICs, where empiric 
antibiotic treatment is often initiated before specimen collection. 
Available evidence primarily comes from adult populations where nasopharyngeal 
colonisation is relatively uncommon and less likely to interfere with urine-based antigen 
detection19. In contrast, children in sSA, including Malawi, often exhibit high nasopharyngeal 
carriage rates20,21, which may make it difficult to distinguish between antigenuria due to 
invasive disease and antigen spillover from asymptomatic carriage. 
Limited data exist on the specificity of ssUAD in high-carriage paediatric populations, 
particularly in low-resource settings where distinguishing between pneumococcal disease and 
antigenuria from asymptomatic carriage remains challenging. As such, there is an urgent need 
for context-specific data to define serotype-specific thresholds that can accurately differentiate 
carriage from disease. Addressing this gap is critical for the optimal use of ssUAD in clinical 
diagnostics, disease surveillance, and vaccine impact evaluations in high-burden LMIC 
settings. 
1.2. Rationale 
There is a critical need to improve pneumococcal pneumonia diagnostics and surveillance in 
LMICs where high nasopharyngeal carriage rates and frequent antibiotic use limit the utility of 
traditional culture-based methods22,23.The ssUAD assay has the potential to fill this gap, but its 
diagnostic thresholds must be tailored to high-burden settings. By evaluating the prevalence 
and distribution of serotype-specific urinary antigens in children with and without pneumonia, 
and assessing how nasopharyngeal carriage influences antigen detection, this study will 
generate the evidence necessary to define thresholds that could distinguish carriage from 
disease. These data will guide the use of ssUAD in diagnostics, surveillance, and vaccine 
impact studies in LMICs. 
1.2.1. Summary of NP Resistome study 
This parent study is a multi-site observational extended observational case–control study with 
a preliminary cohort component designed to examine nasopharyngeal resistome (NPR) 
evolution following antibiotic selective pressure and healthcare exposure for pneumonia in 
children in Blantyre, and whether this is associated with adverse health outcomes. The 
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nasopharynx is a reservoir for bacteria that cause pneumonia in children, and their associated 
antimicrobial resistance. The NPR encompasses all the genetic determinants of antibiotic 
resistance found in this microbial niche. The study will recruit 350 children aged 12–24 months 
in four groups over an 18-month period; healthy children in the community, children with 
pneumonia in the community, children hospitalised with pneumonia and children re-
hospitalised with pneumonia. The study will be conducted at Ndirande Community Health 
Centre and Queen Elizabeth Central Hospital. Both nasopharyngeal swabs (NPS) and urine 
samples will be taken at recruitment; only NPS will be taken at follow-up study visits. NPS 
will undergo metagenomic sequencing to identify the nasopharyngeal microbiome and NPR, 
and a viral PCR to identify viral pathogens associated with acute respiratory infections 
requiring hospitalisation in this age group; urine samples will be used for an antibiotic bio-
assay. Demographics, co-morbidities, health outcomes and antibiotic exposure will be recorded 
at recruitment for all participants, and subsequent study visits where applicable. 
The diversity and relative abundance of antibiotic resistance genes (ARGs) in the NPR will be 
compared between the study groups to determine whether a hospital-acquired NPR persists 
after discharge, and is associated with adverse health outcomes, particularly recurrent 
hospitalisation. The NP Resistome study will also provide contemporary data on AMR carriage 
in the community, hospital admission and re-admission rates with pneumonia, and antibiotic 
use patterns. 
1.3. Hypothesis 
We hypothesise that the ssUAD assay will show high sensitivity for detecting pneumococcal 
serotype-specific antigens in children with pneumonia, but background antigenuria due to 
asymptomatic carriage will necessitate revised thresholds for accurate disease attribution in 
this high-burden setting. 
1.4. Study Objectives 
1.4.1. Broad objective 
To evaluate the performance of the ssUAD test in detecting pneumococcal carriage and 
distinguishing it from invasive disease among children aged 12-24 months with pneumonia, 
compared to healthy children, in Blantyre, Malawi. 
1.4.2. Specific objectives 
1. Determine the prevalence of serotype-specific pneumococcal antigens in urine 
among children under five years of age with pneumonia compared to healthy 
counterparts.  
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2. To characterise the association between the detection of serotype-specific antigens 
in urine and nasopharyngeal pneumococcal carriage among children. 
3. To define revised ssUAD thresholds that distinguish pneumococcal carriage from 
disease, to support field evaluations and inform diagnostic and surveillance 
strategies in Malawi and similar settings. 
2.0 Methodology 
2.1. Study Design and Setting:  
This case-control sub-study is nested within the NP Resistome study (Protocol v5.0 COMREC 
reference P.10/24-1200), a prospective observational cohort conducted in Blantyre, Malawi. 
Recruitment will take place at Ndirande Health Centre (NHC), Gateway Primary Care Centre 
(GPCC), and Queen Elizabeth Central Hospital (QECH). The sub-study will involve the 
analysis of urine samples that have already been collected from children aged 12–24 months 
across four defined participant groups in the parent NP Resistome study. 
Study Population: 
Participants will be children aged 12–24 months enrolled in the NP Resistome study 
(COMREC reference P.10/24-1200) in the following groups; 
• 
Children hospitalised with pneumonia at QECH. 
• 
Children with pneumonia treated at primary health centres (i.e NHC). 
• 
Healthy community controls with and without recent antibiotic exposure. 
2.2. Study Period:  
This sub-study is planned over an 18-month period, from July 2025 to January 2027, running 
alongside the NP Resistome study (Protocol v5.0 COMREC reference P.10/24-1200). 
Activities include sample aliquoting, international shipment, laboratory testing using the 
ssUAD assay, data integration, statistical analysis, and dissemination of findings. 
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These activities will be carried out in sequential and overlapping phases, aligned with the 
timelines and logistics of the parent study. Community and hospital-based participant samples 
collected through NP Resitome will be utilised throughout. 
A detailed timeline of the sub-study activities is presented in the Gantt chart below, outlining 
the expected duration and sequencing of each phase. 
July -August 
2025 
September-
October 2025 
November- 
December 2025 
January- 
February 2026 
March – April 
2026 
June-  July 2026 
August – 
September 2026 
October- 
November 2026 
December 2026 – 
January 2027 
Urine sample 
aliquoting and 
storage 
  
 
 
 
 
 
 
 
 
Shipping of 
urine samples to 
the UK 
  
  
  
ssUAD testing at 
Nottingham/UK
HSA 
Compile carriage 
and 
metagenomic 
data 
Data cleaning 
and linkage 
  
  
  
  
Data analysis 
(prevalence, 
regression, 
ROC) 
  
  
Write-up and 
report 
preparation 
 
  
  
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Figure 1. Study timeline 
All urine samples collected as part of this sub-study will be stored at the Malawi-Liverpool-
Wellcome (MLW) Programme for up to five years following study completion, in accordance 
with institutional and ethical guidelines. Upon conclusion, final reports will be submitted to 
COMREC, the Blantyre District Health Office, and the study sponsor (LSTM), alongside 
dissemination to scientific and policy stakeholders. 
2.3. Inclusion and Exclusion criteria:  
This sub-study will use existing urine samples that have already been collected as part of the 
NP Resistome study (COMREC reference P.10/24-1200). The inclusion criteria applied 
during that study were: children aged 12–24 months residing in Blantyre District whose 
parents or legal guardians provided written informed consent. 
As no new participant recruitment or sample collection will be undertaken, no additional 
inclusion or exclusion criteria are applicable for this sub-study. 
2.4. Sample collection and handling:  
This sub-study will use existing urine specimens that have already been collected as part of NP 
Resistome study. Up to 5 ml of urine was collected per participant using sterile paediatric urine 
collection bags. Each specimen was aliquoted into three 1.5 ml cryovials and stored at –80 °C 
until analysis. All specimens were Pseudonymised and linked to participants only through 
study ID numbers, in accordance with data protection protocols. 
2.5. Laboratory analysis:  
Urine samples will be analysed using the ssUAD, a Luminex-based immunoassay designed to 
detect pneumococcal polysaccharide capsules. Testing will follow validated protocols and will 
be conducted at an accredited reference laboratory with expertise in multiplex immunoassay 
Dissemination 
(thesis, papers, 
conferences) 
  
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techniques at the University of Nottingham NHS Trust, UKHSA and PATH based in Seattle, 
USA. 
2.6. Sample size: 
The minimum required sample size for this study is 147 urine specimens, accounting for a 10% 
allowance for non-response or data loss. This estimate is based on detecting a significant 
difference in pneumococcal serotype-specific antigen detection between children hospitalised 
with pneumonia, those treated at health centres, and healthy community controls. 
The core sample size calculation indicates that at least 49 specimens per group are needed, 
assuming antigen detection rates of 70% in hospitalised children, 53% in health centre cases, 
and 35% in healthy controls, based on prior studies8,9. Using a two-sided significance level of 
0.05 and 80% statistical power, the estimated minimum total sample size is 147 participants 
across the three groups. 
To address the limitation of a small sample size faced in our preliminary analysis of 214 urine 
specimens from hospitalised children, we plan to test all 350 urine specimens collected under 
the parent NP Resistome study. This expanded approach will improve the precision of our 
estimates, allow for subgroup analyses, and provide broader insights into pneumococcal 
antigen detection patterns across the study groups 
2.7. Data management:  
All data generated from this sub-study will be managed in alignment with the main NP 
Resistome study (COMREC reference P.10/24-1200). Urine specimens will be labelled using 
the same barcoded identifiers already assigned under the NP Resistome framework. No new 
study identifiers will be created. 
Data will be entered into a secure, password-protected electronic database with access 
restricted to authorised study personnel. To ensure data accuracy and completeness, double 
data entry procedures and regular validation checks will be implemented.  
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All data will be used solely for the objectives described in this protocol and handled in 
accordance with institutional data governance policies and COMREC ethical approvals. No 
personally identifiable information will be included in shared datasets or reported results. 
2.8. Data analysis:  
The data analysis plan is structured to address the study’s three specific objectives: 
2.8.1. Prevalence and distribution (objective 1):  
Descriptive statistics (frequencies, means, medians) will be used to summarise participant 
characteristics across study groups. Serotype-specific prevalence of urinary pneumococcal 
antigens will be calculated for each group, hospitalised pneumonia, community-managed 
pneumonia, and healthy controls. Differences between groups will be assessed using chi-square 
tests or Fisher’s exact test, as appropriate. 
2.8.2. Association with carriage dynamics (objective 2):  
Logistic regression models will be employed to assess the association between urinary antigen 
detection (the outcome variable) and nasopharyngeal carriage (the predictor), adjusting for 
potential confounders such as age, antibiotic use, and HIV status. Subgroup analyses will 
examine the influence of carriage density and serotype co-carriage on antigen detection. 
Multivariable regression and interaction terms will help explore effect modification. 
2.8.3. Defining diagnostic thresholds (objective 3):  
Signal intensity data (median fluorescence intensity or equivalent) from the 24ssUAD assay 
will be analysed to compare antigen profiles between cases (pneumonia) and carriers (healthy 
children). Receiver Operating Characteristic (ROC) curve analysis will be used to define 
optimal cut-off values for distinguishing pneumococcal disease from carriage. Area under the 
curve (AUC) values will quantify diagnostic performance. Findings will be cross-referenced 
with previously published UK datasets to support broader applicability. 
All analyses will be conducted using R software, with statistical significance defined at p < 
0.05 and confidence intervals at 95%. Graphical data presentation will include bar charts, ROC 
curves, and stratified prevalence plots. 
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2.9. Data sharing: 
Data generated from this sub-study will be shared with collaborators under restricted-access 
data sharing agreements. All shared datasets will be de-identified and used solely for the 
purposes outlined in this protocol, in accordance with institutional and ethical guidelines. 
3.0 Quality assurance and control 
All laboratory analyses will be conducted using standardised protocols at the UK Health 
Security Agency (UKHSA) and NIHR Nottingham Biomedical Research Centre. Internal 
quality control (QC) procedures and reproducibility assessments will be implemented 
throughout the testing process to ensure data integrity and reliability. 
4.0 Dissemination plan 
Study findings will be disseminated through multiple channels, including submission to the 
College of Medicine Research and Ethics Committee (COMREC), presentations at national 
conferences, such as the Research Dissemination Conference (RDC), and international 
scientific conferences, as well as publication in peer-reviewed journals. A summary of findings 
will also be shared with local stakeholders, including clinical and public health partners in 
Malawi. 
5.0 Ethical consideration 
The parent NP Resistome study (COMREC reference P.10/24-1200) has obtained ethical 
approval from all relevant institutional review boards. Written informed consent for participant 
enrollment will be obtained from parents or guardians. Informed consent will be obtained from 
the parents or guardians of participants for the testing of stored urine samples (collected as part 
of the NP Resistome study) using urinary pneumococcal antigen assays, with the assistance of 
a separate patient information leaflet and informed consent form. All procedures will be 
conducted in accordance with the approved ethical protocol and institutional research 
governance policies. 
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6.0 Participant compensation 
Participants in this sub-study will receive no additional compensation beyond what is already 
provided for participation in the NP Resistome study (COMREC reference P.10/24-1200). All 
participants in the NP Resistome study receive 17,000 MWK as compensation, which covers 
all study-related procedures, including urine sample collection. 
7.0 Project risks and constraints 
This sub-study is embedded within the NP Resistome study (COMREC reference P.10/24-
1200), which will obtain all relevant ethical approvals and is poised to begin participant 
recruitment. While leveraging the infrastructure and participant pool of the parent study offers 
significant advantages, there remain potential risks and constraints specific to the successful 
implementation of the urinary pneumococcal antigen sub-study. 
7.1. Dependency on NP Resistome recruitment timeline 
The sub-study relies on timely and sufficient recruitment within the NP Resistome study 
(COMREC reference P.10/24-1200) to prospectively collect urine samples from children with 
pneumonia and healthy controls. Any delays or slower-than-expected enrolment in the parent 
study could limit the number of available urine specimens. To mitigate this, the sub-study team 
will maintain close coordination with the NP Resistome field team to ensure that urine 
collection is integrated into the enrolment process for each eligible participant from the outset. 
7.2. Difficulty in collecting adequate urine volumes 
Obtaining sufficient urine volumes, particularly from young children, may pose practical 
challenges in both community and hospital settings. Missed collections due to dehydration or 
discomfort could affect sample availability. To mitigate this, caregivers will be supported by 
trained staff using child-friendly collection protocols. 
7.3. Laboratory assay limitations or technical errors 
The ssUAD assay, although robust, involves complex multiplex detection that requires strict 
standardisation and quality control to minimise risks such as batch variability, technical 
failures, or low signal intensity. All testing will be conducted at the NIHR Nottingham 
Biomedical Research Centre and UKHSA under validated SOPs, with internal quality control 
and pilot validation runs to ensure assay reliability prior to full sample processing. While a 
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Luminex platform is available locally at MLW, current capacity is limited due to a lack of 
validated reagents, technical expertise, and established protocols specific to ssUAD testing. 
Therefore, testing will be performed in UK laboratories with the necessary infrastructure and 
experience. Plans are underway to build local capacity and eventually introduce and evaluate 
the assay in Malawi as part of future implementation efforts. 
7.4. Interpretation challenges due to high carriage prevalence 
In a high-carriage setting like Malawi, interpreting the clinical significance of urinary 
pneumococcal antigen detection, especially among asymptomatic individuals, may be 
challenging. If urinary antigen is detected in individuals without pneumococcal carriage, or 
vice versa, this could complicate efforts to set reliable thresholds for distinguishing disease 
from carriage. To address this, the sub-study will incorporate detailed nasopharyngeal carriage 
data and apply ROC curve analysis to refine diagnostic interpretation. 
8.0 Budget 
Table 1. Estimated study budget (1 GBP = 2200 MWK) 
 
Budget Item 
Sub Item 
Cost (£) 
Cost (MWK) 
1. Urine analysis 
Testing of 350 samples at 
£30/sample 
£10,500.00 MWK23,100,000 
2. Shipping costs 
International courier to Nottingham 
University 
£2,500.00 
MWK5,500,000 
Packaging and cold chain materials 
like dry ice 
£1,500.00 
MWK3,300,000 
3. Laboratory 
consumables 
Pipette tips, cryoboxes and gloves 
£2,363.00 
MWK5,198,600 
Total 
 
£16,863.00 MWK37,098,600 
8.1. Budget justification 
8.1.1. Urine Analysis:  
This will support the urine analysis and optimisation of the ssUAD assay at the University of 
Nottingham NHS Trust and UKHSA. 
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8.1.2. Shipping:  
This will contribute to the costs of shipping the urine samples to the UK for further analyses as 
outlined in the protocol.  
8.1.3. Laboratory consumables:  
This will support the purchase of laboratory consumables in Malawi for the preparation of urine 
specimens.  
References 
1. Du Q, Liu Z, Wang H, et al. Nasopharyngeal carriage of Streptococcus pneumoniae among 
children aged 30 days to <60 months in Beijing and Shenzhen, China (2018-2021) during 
pneumococcal  conjugate vaccine introduction and the coronavirus disease (COVID-19) 
pandemic. Front Pediatr. 2024;12:1382165. doi:10.3389/fped.2024.1382165 
2. Bogaert D, De Groot R, Hermans PWM. Streptococcus pneumoniae colonisation: the key 
to pneumococcal disease. Lancet Infect Dis. 2004;4(3):144-154. doi:10.1016/S1473-
3099(04)00938-7 
3. Seidu AA, Dickson KS, Ahinkorah BO, Amu H, Darteh EKM, Kumi-Kyereme A. 
Prevalence and determinants of Acute Lower Respiratory Infections among children 
under-five years in sub-Saharan Africa: Evidence from demographic and health  surveys. 
SSM - Popul Health. 2019;8:100443. doi:10.1016/j.ssmph.2019.100443 
4. Wahl B, O’Brien KL, Greenbaum A, et al. Burden of Streptococcus pneumoniae and 
Haemophilus influenzae type b disease in children in the era of conjugate vaccines: global, 
regional, and national  estimates for 2000-15. Lancet Glob Health. 2018;6(7):e744-e757. 
doi:10.1016/S2214-109X(18)30247-X 
5. Chewapreecha C, Harris SR, Croucher NJ, et al. Dense genomic sampling identifies 
highways of pneumococcal recombination. Nat Genet. 2014;46(3):305-309. 
doi:10.1038/ng.2895 
6. Santé WHO= O mondiale de la. Pneumococcal conjugate vaccines in infants and children 
under 5 years of age: WHO position paper –February 2019 ─ Vaccins 
antipneumococciques conjugués chez les nourrissons et les enfants de moins de 5 ans: note 
de synthèse de l’OMS – février 2019. Wkly Epidemiol Rec Relevé Épidémiologique Hebd. 
2019;94(08):85-103. 
7. Harris AM, Bramley AM, Jain S, et al. Influence of Antibiotics on the Detection of 
Bacteria by Culture-Based and Culture-Independent Diagnostic Tests in Patients 
Hospitalized With  Community-Acquired Pneumonia. Open Forum Infect Dis. 
2017;4(1):ofx014. doi:10.1093/ofid/ofx014 
05/09/2025

05/09/2025
 
 
 
 
Urinary Pneumo Antigen Study.        Version 2.0 10/08/2025 
Page 21
8. Eletu SD, Sheppard CL, Rose S, et al. Re-validation and update of an extended-specificity 
multiplex assay for detection of Streptococcus pneumoniae capsular serotype/serogroup-
specific antigen and  cell-wall polysaccharide in urine specimens. Access Microbiol. 
2020;2(3):acmi000094. doi:10.1099/acmi.0.000094 
9. Eletu Seyi D., Sheppard Carmen L., Thomas Elizabeth, et al. Development of an 
Extended-Specificity Multiplex Immunoassay for Detection of Streptococcus pneumoniae 
Serotype-Specific Antigen in Urine by Use of Human Monoclonal Antibodies. Clin 
Vaccine Immunol. 2017;24(12):e00262-17. doi:10.1128/CVI.00262-17 
10. 
Goodman D, Crocker ME, Pervaiz F, et al. Challenges in the diagnosis of paediatric 
pneumonia in intervention field trials: recommendations from a pneumonia field trial 
working group. Lancet Respir Med. 2019;7(12):1068-1083. doi:10.1016/S2213-
2600(19)30249-8 
11. 
Saukkoriipi A, Palmu AA, Jokinen J, Verlant V, Hausdorff WP, Kilpi TM. Effect of 
antimicrobial use on pneumococcal diagnostic tests in elderly patients with community-
acquired pneumonia. Eur J Clin Microbiol Infect Dis Off Publ Eur Soc Clin Microbiol. 
2015;34(4):697-704. doi:10.1007/s10096-014-2278-5 
12. 
Carugati M, Aliberti S, Reyes LF, et al. Microbiological testing of adults hospitalised 
with community-acquired pneumonia: an international study. ERJ Open Res. 
2018;4(4):00096-02018. doi:10.1183/23120541.00096-2018 
13. 
Deloria Knoll M, Morpeth SC, Scott JAG, et al. Evaluation of Pneumococcal Load in 
Blood by Polymerase Chain Reaction for the Diagnosis of Pneumococcal Pneumonia in 
Young Children in the PERCH Study. Clin Infect Dis Off Publ Infect Dis Soc Am. 
2017;64(suppl_3):S357-S367. doi:10.1093/cid/cix149 
14. 
Asalkhou M, Alem N, Ahmadi N, Hamedi N, Alem M. Evaluation of an Innovative 
Diagnostic Method for Detection of Antibodies and Antigens. Int J Clin Med. 
2017;08:306-321. doi:10.4236/ijcm.2017.85029 
15. 
Pride MW, Huijts SM, Wu K, et al. Validation of an Immunodiagnostic Assay for 
Detection of 13 Streptococcus pneumoniae Serotype-Specific Polysaccharides in Human 
Urine. Clin Vaccine Immunol. 2012;19(8):1131-1141. doi:10.1128/CVI.00064-12 
16. 
Kalina WV, Souza V, Wu K, et al. Qualification and Clinical Validation of an 
Immunodiagnostic Assay for Detecting 11 Additional Streptococcus pneumoniae 
Serotype-specific Polysaccharides in Human  Urine. Clin Infect Dis Off Publ Infect Dis 
Soc Am. 2020;71(9):e430-e438. doi:10.1093/cid/ciaa158 
17. 
Chen M, Zhou M, Xiao W, Ai B, Liu X, Li Y. The urinary antigen tests have high 
sensitivity in diagnosis of Pneumococcus caused community-acquired pneumonia 
posterior to antimicrobial therapy. Cell Biochem Biophys. 2014;70(2):1029-1034. 
doi:10.1007/s12013-014-0015-4 
18. 
Bradley JS, Byington CL, Shah SS, et al. The Management of Community-Acquired 
Pneumonia in Infants and Children Older Than 3 Months of Age: Clinical Practice 
05/09/2025

05/09/2025
 
 
 
 
Urinary Pneumo Antigen Study.        Version 2.0 10/08/2025 
Page 22
Guidelines by the Pediatric Infectious Diseases Society and the Infectious Diseases 
Society of America. Clin Infect Dis. 2011;53(7):e25-e76. doi:10.1093/cid/cir531 
19. 
Yasuo S, Murata M, Nakagawa N, et al. Diagnostic accuracy of urinary antigen tests 
for pneumococcal pneumonia among patients with acute respiratory failure suspected 
pneumonia: a systematic review  and meta-analysis. BMJ Open. 2022;12(8):e057216. 
doi:10.1136/bmjopen-2021-057216 
20. 
Mzumara G, Chirombo J, Swarthout TD, et al. Radiographically confirmed 
pneumonia in Malawian children and associated pneumococcal carriage after introduction 
of the 13-valent pneumococcal conjugate  vaccine. Pneumonia Nathan Qld. 
2024;16(1):23. doi:10.1186/s41479-024-00147-7 
21. 
Heinsbroek E, Tafatatha T, Phiri A, et al. Pneumococcal carriage in households in 
Karonga District, Malawi, before and after introduction of 13-valent pneumococcal 
conjugate vaccination. Vaccine. 2018;36(48):7369-7376. 
doi:10.1016/j.vaccine.2018.10.021 
22. 
Nyeko R, Otim F, Obiya EM, Abala C. Pre-hospital exposures to antibiotics among 
children presenting with fever in northern Uganda: a facility-based cross-sectional study. 
BMC Pediatr. 2022;22(1):322. doi:10.1186/s12887-022-03375-2 
23. 
Bebell LM, Muiru AN. Antibiotic use and emerging resistance: how can resource-
limited countries turn the tide? Glob Heart. 2014;9(3):347-358. 
doi:10.1016/j.gheart.2014.08.009 
 
 
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