Study objectives
The study’s primary objectives are to provide baseline surveillance of pneumococcal pneumonia in young children in Malaysia by determining the prevalence of SPN nasopharyngeal carriage among children aged 5 years and below with pneumonia and IPD. Secondary study objectives are: 1) to determine SPN serotypes by polymerase chain reaction (PCR) and whole-genome sequencing (WGS), 2) to evaluate the correlation of SPN detection between nasopharyngeal carriage and urine samples, and 3) to establish sensitivity and specificity cutoffs of BinaxNOW for the detection of SPN serotypes in children with pneumonia and IPD.
Study size power calculation
This is a prospective case-control study in which all children aged 5 years and below with clinically diagnosed pneumonia and healthy controls will be recruited at three study sites. As per assumptions of 95% confidence interval (CI), 80% power and detectable odds ratio (OR) of 1.5, we estimate 164 cases and 164 healthy controls per sentinel hospital site. Thus, we aim to recruit a total of 500 confirmed pneumonia/IPD cases and 500 healthy controls for this study.
Study sites and design
This prospective, hospital-based, multicentre case-control study is being conducted at three university hospitals in different states within Peninsular Malaysia – Kuala Lumpur, Pahang, and Kelantan (Figure 1). Kuala Lumpur serves as the capital city of Malaysia and is centrally located in the western part of Peninsular Malaysia. Pahang and Kelantan are located on the east coast of Peninsular Malaysia, along the South China Sea, providing them with access to the coastline. Kelantan is situated to the north of Kuantan and shares its eastern border with Thailand. Study enrolment is at 1) University Malaya Medical Centre (UMMC), Kuala Lumpur; 2) Hospital Universiti Sains Malaysia (HUSM), Kota Bharu, Kelantan; and 3) Sultan Ahmad Shah Medical Centre International Islamic University Malaysia (SASMEC IIUM), Kuantan, Pahang. Table 1 summarises the demographics and burden of pneumonia in each state of the participating sentinel site. Cases and controls are age-matched within the range of 6 months. Cases are age-matched children clinically diagnosed with pneumonia and attending the outpatient department or admitted as a hospital inpatient, Invasive Pneumococcal Disease (IPD) (without pneumonia), bacteraemic pneumococcal pneumonia, and chest radiograph (CXR) confirmed pneumonia (by WHO guideline) (10). Controls are healthy age-matched children without any intercurrent respiratory illness. Subject enrollment is over 24 months for each participating site, and a total sample of 400 subjects (200 cases and 200 controls) is targeted for UMMC and HUSM, respectively, and 200 subjects (100 cases and 100 controls) for IIUM. Male and female patients from the three major ethnic groups (Malay, Chinese, and Indian) are being recruited from each sentinel site. The study protocol workflow is as outlined in Figure 2.
Table 1 Demographic characteristics by states of participating university hospitals in MY-Pneumo study.
State
|
State population type
|
State population (‘000) a
|
State population of children <5 years old, (‘000) a
|
No. of deaths of children <5 years old b
|
Under-5 mortality rate per 1000 live births b
|
Deaths of under-5 due to pneumonia, % b
|
Kelantan
|
Urban/Rural
|
1829.4
|
173.9
|
285
|
8.2
|
4%
|
Pahang
|
Urban/Rural
|
1612.5
|
128.1
|
173
|
7.1
|
5.20%
|
Federal Territory of Kuala Lumpur
|
Urban
|
1945.3
|
115.9
|
130
|
6.2
|
1.60%
|
a : 2022 prediction by Department of Statistics, Malaysia. Vital Statistics, Malaysia, 2022
b : Statistics on Causes Of Death Malaysia, 2022, Department of Statistics, Malaysia
Study participants
The study population is comprised of children under 5 years of age who comply with protocol definitions and inclusion criteria. Eligible participants are identified by study paediatricians and research assistants at each participating site. The following criteria define cases: 1) hospitalised patients aged between 2 weeks and 59 months, 2) clinical features of pneumonia, as described below, 3) radiological confirmation of pneumonia based on CXR findings as per WHO guidelines(Cherian et al., 2005) (10), and 4) an informed consent statement signed by the children’s parents or legal guardian. The exclusion criteria for cases are the following: children who 1) do not meet the case definition, and 2) whose parents or legal guardian declined to sign the informed consent statement. Controls are defined by children aged 5 years and below, who are in good health, as determined by a brief medical history and/or clinical judgement of the investigator, and whose parent or legal guardian is willing and able to give informed consent. Exclusion criteria for the controls are: 1) any symptom suggestive of respiratory illness, 2) has nasal surgery, 3) has significant diseases or symptoms, such as febrile illness or a temperature ≥38°C on the day of the visit or in the preceding 72 hours that can place the patient at an increased risk of the disease, 4) has a history of antibiotic administration in the month prior to sampling, or 5) minors whose parents or legal guardian decline to sign the informed consent statement. Cases and controls are matched for study site and age (±6 months). General questionnaires are administered to the parents or legal guardians of the participants to obtain demographic and socio-economic data and their medical history. Subjects are identified based on an anonymised identifier. The master list is kept in a password-protected spreadsheet.
Definition of pneumonia
Pneumonia cases are defined as patients with a history of cough and/or difficulty/rapid breathing and/or intercostal recession, with or without fever, and radiological confirmation of pneumonia as per WHO guidelines (10).
Biological samples
Samples are collected in the first 24 hours of patient hospitalisation (Table 2). Nasopharyngeal swabs (NPS) and urine samples are collected from all pneumonia cases and controls following Centers for Disease Control and Prevention (CDC) Pneumococcal Carriage Protocols (11). Each sample is aliquoted as per protocols outlined in this study. Each NPS is inoculated into a medium containing skim milk, tryptone, glucose, and glycerol (STGG) and stored at -80°C before shipment to the International Medical University Advanced Microbiology Collaborative Research Laboratory (IMU-AMCRL) in Kuala Lumpur, Malaysia, every 4 months by a licensed/registered courier. One NPS and one 15 mL urine sample are collected from each subject at the time of enrolment. The collection procedure is performed by trained clinical staff, nurses, and the research assistant at the sentinel site. The NPS collection procedure involves inserting a nylon-tipped FLOQswab (COPAN Diagnostics Inc., USA) through the nostril into the cavity between the nose and mouth for 5 seconds and rotating it several times. The swab is then inserted into a cryovial with 1 mL of STGG media (2% (w/v) skim milk powder, 3% (w/v) tryptone soy broth powder, 0.5% (w/v) glucose and 10% (v/v) glycerol in water), and immediately vortexed briefly and frozen at -80 °C within 4 hours. Different methods of urine collection are used depending on the age and tolerability of the participant, such as clean catch or void, urine collection bag, and diaper. Briefly, for children using diapers, we used the “urine ball method”, which involves placing sterile cotton balls on the subject’s diaper. The urine is subsequently collected by placing the urine-soaked cotton balls into a sterile 20 mL syringe, and the fluid is extracted with a plunger. The collected urine is mixed with PIPES buffer and aliquoted into three tubes of 5 ml each. All urine sampling supplies, including tubes, urine bags, buffer and BinaxNOW test kits, are provided to sentinel sites by Merck Sharp & Dohme (MSD)
via Pharmaceutical Product Development (PPD). Urine samples are tested with BinaxNOW at sentinel sites, and the remaining aliquots are shipped to the MSD Central Lab on dry ice. The BinaxNOW® test kit includes a test device (strip or card), a specimen swab, and a buffer solution. The test device is placed on a clean, flat surface before a buffer solution is added from a dropper bottle. A specimen swab is dipped into the urine specimen, removed, and then inserted into the test card. The card is then closed, bringing the specimen into contact with the test strip. Pneumococcal antigen present in the specimen reacts to bind anti-S. pneumoniae-conjugated antibody. The resulting antigen-conjugate complexes are captured by immobilised anti-S. pneumoniae antibody, forming the Sample Line. Immobilised control antibody captures anti-species conjugate, forming the Control Line. Test results are interpreted by the presence or absence of visually detectable pink-to-purple coloured lines. A positive test result, read in 15 minutes, will include the detection of both a Sample and a Control Line. A negative test result, read in 15 minutes, will produce only a Control Line, indicating that S. pneumoniae antigen was not detected in the specimen. Failure of the Control Line to appear, whether the Sample Line is present or not, indicates an invalid assay. Additional blood, cerebrospinal fluid (CSF) and pleural fluid will be taken for IPD patients, if available, at each sentinel hospital site.
Table 2 Laboratory tests on case and control subject samples.
Samples
|
Collection method
|
Analyses
|
Nasopharyngeal swab
|
Flocked swab in 1 ml STGG cryovial*
|
Molecular detection using multiplex PCR for SPN serotyping.
|
Microbiological culturing and optochin sensitivity
|
Urine
|
Sterile cotton balls, Catheter
|
Pneumococcal urine antigen detection (BinaxNOW)
|
*Skim milk powder, Tryptone soy broth powder, Glucose, Glycerol medium.
Laboratory analysis
Laboratory isolation (including genomic deoxyribonucleic acid (DNA) extraction), identification and confirmation of SPN in each sample are carried out at the IMU-AMCRL. Aliquots of NPS in STGG media include volumes of 400 µl, 350 µl and 250 µl, are stored in -80 °C freezers. The 250 µl NPS-STGG aliquot is the working sample used for blood agar culture, optochin-sensitivity, and DNA extraction for each participant. A 10 µl loopful of sample is taken from the 250 µl aliquot, plated onto Columbia agar with 5% sheep blood (CBA) (Oxoid), and incubated in 5% CO2 at 37 °C for 24 hours. The plate culture is observed after 24 hours of incubation, and colonies exhibiting SPN morphology are picked for replating onto fresh CBA plates with 5 mcg optochin disc (HIMEDIA), followed by incubation as in the previous step. SPN isolates are identified as small, greyish, alpha-hemolytic culture growths showing Draughtsman morphology, and are optochin sensitive. Genomic DNA from the remaining 240 µl NPS-STGG aliquot is extracted using the New England Biolabs (NEB) Monarch® Genomic DNA Purification Kit (USA). The extracted genomic DNA is then subjected to multiplex conventional PCR analysis according to the CDC protocol (US scheme protocol) to detect SPN serotypes by amplifying the capsular polysaccharide biosynthesis gene A (cpsA) targets. SPN isolates will be transferred to the University of Southampton (UoS), United Kingdom (UK), for WGS analysis. Isolates will be sequenced using a MiSeq (Illumina, UK) to generate 2 × 300 paired-end data. Assembly will be done using SPAdes with assembly improvement and QC as described previously (12).
Data sources and quality control
Clinical site monitoring for research conduct and management is conducted yearly by the Principal Investigator team from the International Medical University (IMU). The main purpose of clinical site monitoring is primarily to ensure protocol adherence, source data verification, investigator training, site performance, data quality assurance, and maintaining data integrity at each participating site. Tasks and responsibilities are based on standard research guidelines, including standard operating procedures. Data sources are monitored and evaluated for case and control definition conformity, errors, and missing data at each sentinel site. Vaccination records, underlying diseases, medical history, radiological findings, and demographic characteristics are recorded prospectively for each patient on a case report form (CRF) (Table 3). Data quality reporting is redacted for each site to ensure the confidentiality and conformity of all study data variables. This process will be applied to data analysis of each enrolled case and control subject. Data accuracy will be assessed by comparing the recorded values with source documents. The principal investigator at each site is contacted for queries regarding this quality assessment and is involved in resolution.
Table 3 Overview of questionnaire used in MY-Pneumo study.
Category
|
Information
|
Subject interview
|
Hospital records
|
Demographic
|
Ethnicity
|
Main caretaker
|
Date of Birth
|
|
Residence
|
|
Weight at Birth
|
|
Parents' Education Level
|
Feeding habit
|
Height at Birth
|
|
Parents/Family member smoking habit
|
|
Gestational term
Current Weight and Height
|
Underlying diseases
|
Immunodeficiency
|
Liver disease
|
|
|
Kidney disease
|
Respiratory disease
|
|
|
Cardiac disease
|
Malnutrition
|
|
|
Blood disease
|
|
|
Medical history
|
|
|
Prior ear infections
|
|
|
|
Prior respiratory illness
|
|
|
|
Prior antibiotic treatment
|
|
|
|
Prior hospitalisations
|
Vaccination history (Dates, Number of Doses)
|
BCG, HepB, DTap, Hib, MMR, IPV, Tetanus
|
PCV
|
|
Medical intervention
|
|
|
Date of admission
|
|
|
|
Date of discharge
|
|
|
|
Symptoms
|
|
|
|
Level of care
|
|
|
|
Antibiotic treatment
|
|
|
|
Radiological findings
|
Data analysis
An anonymous database will be built, and clinical data will be linked to laboratory data. Quantitative variables will be described and categorised according to their distribution in the study population. Descriptive analysis will address each covariate for the entire population and will be stratified by site. Patients’ characteristics and laboratory data for cases and controls will be compared. The associations between risk factors and carriage will also be examined by estimating the relative risk in the study population. Data analysis will be conducted using software such as R and SPSS. The absence/presence of SPN will be modelled using a mixed effects logistic regression model and random forest. The validity of the model will be tested using cross-validation. Sensitivity analysis will also be carried out to evaluate the impact of selected clinical and demographic parameters on the BinaxNOW test outcome.