Childhood pneumonia and meningitis in the Eastern Highlands Province, Papua New Guinea in the era of conjugate vaccines: study methods and challenges
Blyth et al. Pneumonia
Childhood pneumonia and meningitis in the Eastern Highlands Province, Papua New Guinea in the era of conjugate vaccines: study methods and challenges
Christopher C. Blyth 0 1 2
Rebecca Ford 5
Joycelyn Sapura 5
Tonny Kumani 5
Geraldine Masiria 5
John Kave 5
Lapule Yuasi 5
Andrew Greenhill 4 5
Ilomo Hwaihwanje 3
Amanda Lang 5
Deborah Lehmann 0 5
William Pomat 0 5
on behalf of the Papua New Guinea Pneumonia
Meningitis Etiology Study Team
0 Wesfarmers Centre for Vaccines and Infectious Diseases, Telethon Kids Institute, University of Western Australia , PO Box 855, West Perth 6872, WA , Australia
1 School of Paediatrics and Child Health, The University of Western Australia, Princess Margaret Hospital for Children , Roberts Road, Subiaco 6008, WA , Australia
2 Department of Infectious Diseases and PathWest Department of Microbiology, Princess Margaret Hospital for Children , Roberts Road, Subiaco 6008, WA , Australia
3 Eastern Highlands Provincial Hospital , PO Box 392, Goroka 441, Eastern Highlands Province , Papua New Guinea
4 School of Applied and Biomedical Sciences, Federation University Australia , Gippsland Campus, Northways Road, Churchill 3842, VIC , Australia
5 Papua New Guinea Institute of Medical Research , PO Box 60, Goroka 441, Eastern Highlands Province , Papua New Guinea
Background: Pneumonia and meningitis are common causes of severe childhood illness in Papua New Guinea (PNG). The etiology of both clinical conditions in PNG has not been recently assessed. Changes in lifestyle, provision and access to healthcare, antimicrobial utilization and resistance, and the national childhood vaccination schedule necessitate reassessment. Methods: A prospective case-control study was undertaken, enrolling children <5 years of age to determine the contemporary etiology of clinically defined moderate or severe pneumonia or suspected meningitis. Cases were identified following presentation for inpatient or outpatient care in Goroka town, the major population centre in the Eastern Highlands Province. Following enrolment, routine diagnostic specimens including blood, nasopharyngeal swabs, urine and (if required) cerebrospinal fluid, were obtained. Cases residing within one hour's drive of Goroka were followed up, and recruitment of healthy contemporaneous controls was undertaken in the cases' communities. Results: 998 cases and 978 controls were enrolled over 3 years. This included 784 cases (78.6%) with moderate pneumonia, 187 (18.7%) with severe pneumonia and 75 (7.5%) with suspected meningitis, of whom 48 (4.8%) had concurrent pneumonia. The median age of cases was 7.8 months (Interquartile range [IQR] 3.9-14.3), significantly lower than community controls, which was 20.8 months (IQR 8.2-36.4). Half the cases were admitted to hospital (500/998; 50.1%). Recruitment of cases and controls and successful collection of diagnostic specimens improved throughout the study, with blood volume increasing and rates of blood culture contamination decreasing. The overall case fatality rate was 18/998 (1.8%). Of cases eligible for follow-up, outcome data was available from 76.7%. Low but increasing coverage of Haemophilus influenzae type B conjugate vaccines on the national schedule was observed during the study period: three dose DTPw-HepB-Hib coverage in children >3 months increased from 14.9 to 43.0% and 29.0 to 47.7% in cases and controls (both p < 0.001). Despite inclusion in the national immunization program in 2014, 2015 PCV13 three-dose coverage in cases and controls >3 months was only 4.0 and 6.5%. (Continued on next page) © The Author(s). 2017 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
(Continued from previous page)
Conclusions: Recruitment of large numbers of pediatric pneumonia and meningitis cases and community
controls in a third-world setting presents unique challenges. Successful enrolment of 998 cases and 978 controls
with comprehensive clinical data, biological specimens and follow up was achieved. Increased vaccine coverage
remains an ongoing health priority.
Pneumonia and meningitis are common childhood
diseases, resulting in significant morbidity and mortality
among young children . It is estimated that acute
lower respiratory tract infection results in approximately
2.7 million deaths per annum, with 50% occurring in
children <5 years of age. Of childhood pneumonia
deaths, 33 and 16% are attributable to Streptococcus
pneumoniae and Haemophilus influenzae, respectively
. Meningitis is estimated to result in >300,000 deaths
(all ages) globally, with 26 and 21% estimated to be
caused by S. pneumoniae and H. influenzae .
Papua New Guinea (PNG) was previously identified as
a high priority country for the achievement of the
United Nations Millennium Development Goals because
the baseline child mortality rate is among the highest in
the Western Pacific Region (<5 year old mortality rate in
2015: 57 per 1000 live births ). Pneumonia and
meningitis are the most common causes for childhood
deaths reported to the National Health Information
Systems [3, 4]. Studies conducted in the Asaro Valley,
including Goroka town, the provincial capital of Eastern
Highlands Province (EHP), have repeatedly
demonstrated the importance of S. pneumoniae and H.
influenzae in the etiology of pneumonia and meningitis [5–11].
There are a paucity of contemporary data examining the
bacterial causes of pneumonia and meningitis: the only
recent study used opportunistically collected
cerebrospinal fluid (CSF) samples from children admitted to
Goroka General Hospital (GGH; 1996–2005) . The
current contribution of respiratory viruses to pneumonia
also remains uncertain; apart from a single study
assessing respiratory viruses in infants enrolled in a neonatal
pneumococcal conjugate vaccine trial , prospective
studies have not been undertaken since 1985 .
Conjugate H. influenzae type B (Hib) vaccine and
13valent S. pneumoniae vaccine (13vPCV) were introduced
into the national vaccination schedule in 2008 and 2014,
respectively. From 2014, PNG infants were
recommended neonatal BCG and hepatitis B vaccines followed
by diphtheria, tetanus, whole-cell pertussis, hepatitis B
and Hib vaccine (DTPw-HepB-Hib), 13vPCV and oral
polio vaccines at 1, 2 and 3 months of age. Routine
measles vaccines were recommended at 6 and 9 months
. In 2015, the PNG National Department of Health
launched a Special Integrated Routine EPI Strengthening
Program (SIREP Plus), a multimodal program aimed at
improving coverage of vaccines already provided free
under the national immunization program as well as
introducing measles-rubella (MR, recommended at 6, 9
and 18 months) and inactivated polio vaccine (IPV, at
3 months) to all PNG children, and PCV13 to provinces
yet to introduce the vaccine. Despite the 2014 launch,
PCV13 was not available for use in children in the PNG
highlands until 2015.
Given these changes, determining vaccine coverage in
the PNG highlands and assessing the contemporary
etiology of pneumonia and meningitis is a research priority.
Commencing in 2013, we conducted a prospective
casecontrol study enrolling children with clinically-defined
moderate or severe pneumonia or suspected meningitis
(cases) and contemporaneous community controls. This
article describes the study methodology, population,
vaccine coverage, and some of the operational and logistical
challenges encountered throughout this study.
We performed a prospective case-control study
recruiting children with clinical pneumonia and/or suspected
meningitis presenting either to the Eastern Highlands
Provincial Hospital (EHPH, previously known as the
Goroka General Hospital, GGH) or community
healthcare clinics elsewhere in Goroka town and
contemporaneous healthy community controls (January 2013 to
January 2016). The date of commencement was chosen
to ensure sufficient recruitment prior to the national
introduction of the 13vPCV.
The study aims were to: (i) determine the etiology of
pneumonia and meningitis in a setting with previously
reported high rates of both diseases; (ii) identify the
serotypes of S. pneumoniae causing invasive disease; (iii)
determine the antimicrobial susceptibility profiles of
isolated bacterial pathogens; (iv) examine the utility of
molecular and other diagnostic assays in PNG children with
pneumonia and meningitis and (v) explore clinical and
laboratory variables associated with specific pathogens
and severe diseases.
The study was conducted in the EHP of PNG, an area of
11,157 km2, located at 6030’S 145040’E with a population
of 579,825 (2011 census) . Goroka, the provincial
capital of EHP (population of 23,277) is located 1600 m
above sea level, is only accessible by air from the
national capital Port Moresby, and by a narrow
mountainous highway to the nearest sea port, Lae. Sealed roads
exist between major centres, but are often in poor
condition. Feeder roads to villages are generally unsealed
tracks and many villages and hamlets are only
accessible on foot. Living standards vary significantly. In rural
areas people live in hamlets in traditional thatched
houses and are primarily subsistence farmers, but may
obtain cash through sales of coffee and market produce.
In the urban and peri-urban areas, many houses are
built with strong and long-lasting materials such as an
iron roof and cement walls and floors, although houses
in the poorer settlements are more traditional and
made of bush materials. Modern houses are connected
to the town water and electricity supply (though
interruptions are common) but this is less common in the
EHPH is the only tertiary healthcare facility in EHP
and caters for all age groups. EHPH has a dedicated
pediatric ward and children’s outpatient facility staffed
by pediatricians, junior medical officers and pediatric
nurses. In addition, urban clinics located in and around
Goroka offer outpatient nurse-led care to children and
adults. EHPH is the only site for inpatient hospital care
in Goroka and surrounding villages and the EHPH
children’s Outpatient Clinic, North Clinic and Lopi Clinic
are the busiest outpatient facilities in the region.
Children presenting to the Children’s Outpatient
Clinic, EHPH or two smaller urban clinics (North
Clinic, Lopi Clinic) were eligible for recruitment. In
addition, children presenting to the PNG Institute of
Medical Research (PNGIMR) research clinic (located at
the PNGIMR facility in Goroka, next to EHPH) or
already admitted to the EHPH children’s ward were
eligible for recruitment. Research was undertaken by
Recruitment of children with pneumonia and/or
Unwell children <5 years of age meeting the inclusion
criteria were eligible for enrolment (see definitions of
moderate and severe pneumonia and clinical meningitis
as described in the PNG clinical manual and used
nationwide [Table 1] ). Children not eligible for
enrolment included those with severe congenital
abnormalities, those hospitalized in the preceding 14 days
and those previously enrolled within 30 days. Prior
receipt of antibiotics was not an exclusion criteria.
Criteria Classification Definitions
Inclusion Moderate Presence of all three symptoms and signs:
Criteria pneumonia cough
(cases) tachypnoea rate (≥60/min if <2 months,
≥40/min if ≥2 mo of age)
lower chest wall indrawing
Moderate pneumonia with one of the
following added features:
pulse >160/min with hepatomegaly
>2 cm below costal margin
cyanosed or restless
inability to breastfeed/drink or vomiting.
The following children were excluded
from enrolment as controls
age ≥ 5 years
symptoms or signs of active infection
including cough, tachypnoea, lower chest
wall indrawing, fever (>37.5 °C), altered
consciousness, drowsy or “staring eyes”,
irritability, bulging fontanelle and neck
stiffness or signs of meningeal irritation
severe congenital abnormality
hospitalization in preceding 14 days
previous enrolment in the study as a controla
exposed to antibiotics in the past 30 days
parent unable or unwilling to give consent
Eligible children were identified by clinically-trained
research staff using a screening questionnaire. Upon
determining eligibility and obtaining informed consent,
research staff undertook a brief history and
examination before laboratory specimens were obtained and
treatment provided. Verbal consent and minimal data
were obtained initially ensuring timely provision of
appropriate medical care. This was followed by a more
in-depth interview exploring risk factors for disease,
recent antibiotic exposure, vaccination history and
symptoms, examination including oximetry (recorded
using LifeBox Pulse Oximeter, Acare Technology Co.
Ltd, Taiwan), written consent and further specimen
collection as required. In addition to parental interview,
individual health records were examined for perinatal
history, co-morbidities and vaccination status.
Children meeting the inclusion criteria yet deemed
well enough for outpatient management were still
eligible for enrolment. In addition to children enrolled
through outpatient clinics, inpatients presenting
afterhours were enrolled following parental consent. This
was restricted during the first year of the study to those
identified <48 hours following admission, but was
extended to 96 h to enhance recruitment, particularly for
children with suspected meningitis perceived to be too
sick to undergo an early lumbar puncture.
All admitted cases were reassessed daily during their
hospital stay by clinically trained research staff. Children
deemed well enough to be managed as outpatients were
discharged and reviewed within 48 h. Contact mobile
phone numbers, if available, were collected from all
parents prior to discharge. Review of all children residing
within one hour (by vehicle and/or foot) from Goroka
was attempted, 2 to 4 weeks post-discharge. At review,
their health status was assessed and the presence of
ongoing symptoms and/or signs confirmed.
Recruitment of controls
Healthy children <5 years of age were enrolled to assist
in determining community vaccination coverage and
for further nasopharyngeal bacterial colonization and
viral studies. Children were eligible if they met the
inclusion/exclusion criteria (Table 1) and were recruited
during community follow-up visits for discharged cases.
PNGIMR staff conducted community clinics to provide
primary healthcare and vaccination, thereby facilitating
further identification and recruitment of controls.
Given the logistical challenges of recruiting matched
controls, formal case-control matching was not
undertaken although contemporaneous controls were
recruited from the same communities as cases. In an
attempt to reduce bias, the number of cases recruited
and median age were regularly assessed and used to
guide targeted control recruitment. Following informed
consent, a brief history and examination was undertaken,
individual health and immunization records were
examined and two nasopharyngeal swabs were collected.
Consent was sought to collect blood, nasopharyngeal
specimens, urine and (if clinically indicated) CSF from
all cases (Additional file 1: Table S1), and
nasopharyngeal specimens from controls. Although recommended
in cases of suspected meningitis, a decision to collect
CSF was left to the treating pediatric staff. Using
appropriate aseptic technique (iodine solution followed by
70% alcohol, ensuring adequate drying time prior to
procedure), PNGIMR research staff collected up to 5 ml of
whole blood for culture (inoculated directly into BD
BACTEC™ PedsPlus bottles; BD, Franklin Lakes, New
Jersey, United States [US]), PCR and antimicrobial
activity. Blood volume was recorded by clinical staff prior to
blood culture inoculation. Additional blood tests, if
clinically indicated (e.g. malaria blood slides,
haemoglobin, human immunodeficiency virus [HIV] serology)
were also obtained. If obtainable, urine was collected
for antimicrobial activity and storage. Two
deepflocked nasopharyngeal swabs (Copan Diagnostics,
Murrieta, California, US) were collected on all children:
the first placed immediately in 1 ml of skim milk
tryptone glucose glycerol broth (STGGB) and the second in
1 ml of viral transport media (VTM). Following initial
processing, STGGB samples were stored at −80 °C
whilst EDTA, serum, urine and VTM samples were
stored at −20 °C. Blood and CSF culture contamination
rates were monitored throughout the study and a
review of collection technique and additional staff
training was undertaken regularly during periods of
All laboratory testing was undertaken at the PNGIMR
Bacteriology and Molecular Laboratories, Goroka. Blood
cultures were incubated for up to 5 days at 35 °C using a
BD BACTEC™ automated blood culture system (BD,
Franklin Lakes, NJ) or when unavailable, a 35–37 °C
incubator. Positive bottles (using the BACTEC system)
were subcultured onto standard and selective media and
incubated with/without 5% CO2. During periods when
the BACTEC system was unavailable, manual daily
subculture from inoculated BACTEC™ bottles was
undertaken using methods previously described . All CSF
underwent macroscopic examination, manual cell count
and Gram stain. In the setting of CSF pleocytosis, CSF
was cultured on standard media and incubated with/
without 5% CO2, bacterial antigen detection for
S.pneumoniae and H.influenzae using latex agglutination was
performed and further staining for acid-fast bacilli and
yeast undertaken. CSF biochemistry (protein and
glucose) was undertaken using Uristix® reagent urinalysis
strips (Siemens, Victoria, Australia).
Bacterial isolates were identified using colony
morphology and standard confirmatory tests (S.pneumoniae:
optochin sensitivity, bile solubility, catalase;
H.influenzae: differential growth on blood/chocolate agar, X&V
disks). Serotyping was undertaken by the Quellung
reaction using commercially obtained antisera (Staten
Serum Institut, Denmark) and H.influenzae
agglutinating sera (Remel, Dartford, United Kingdom).
Antimicrobial susceptibility testing was conducted on all
clinically relevant isolates by disc diffusion with
suspected resistance confirmed by E-tests® (bioMérieux,
Durham, North Carolina, US) using Clinical and
Laboratory Standards Institute (CLSI) breakpoints .
For real-time PCR on blood and CSF samples, DNA
was extracted from 200 μl blood/CSF using
commercially available kits (Qiagen DNease Blood & Tissue Kit,
Valencia, California, US) and an in-house real-time
PCR for S.pneumoniae (lytA) and H. influenzae (hpd)
performed [18, 19]. In general, replicate samples within
0.5 Cq of each other and with a Cq value ≤35 (but
within the limit of detection as determined by the
standards) were deemed to be positive.
Respiratory virus testing was undertaken using the
dedicated VTM sample and an in-house multiplexed
real-time quantitative PCR assay. DNA/RNA were
extracted using commercially available kits (Qiagen
DNease Blood & Tissue Kit, Valencia, California, US).
Viral targets are described in Additional file 2: Table S2
[20–22]. Semi-quantitative bacterial culture of STGGB
was performed using blood, chocolate, gentamicin-blood
and bacitracin-chocolate agar plates, incubated with 5%
CO2. Bacterial isolates were identified and typed and
susceptibility testing performed as described above.
Antibiotic activity was measured in blood, urine and/
or CSF to better understand the yield of blood and CSF
cultures. This was performed by placing a
specimensoaked filter paper disk on agar inoculated with a 0.5
MacFarland suspension of Staphylococcus aureus ATCC
25923. Following incubation at 37 °C overnight, a zone
of inhibition was examined and measured.
When a chest X-ray was requested as part of clinical
care, digital copies were extracted and stored on a
dedicated hard drive. These will be reviewed by international
experts using standardised methods .
Ethics, data and study management and analysis
Ethical approval was obtained from the Institutional
Review Board of PNGIMR (IRB no 1204), the Medical
Research Advisory Committee of PNG (MRAC number
11.29) and the University of Western Australia (RA/4/1/
7960). Clinical and laboratory data were collected on
paper forms, subsequently checked by a second staff
member and entered in a dedicated Filemakerpro
database 11 (DB services, Indianapolis, IN, US). Monthly
teleconferences and regular data checks were
undertaken to ensure the accuracy of all data collected.
Analysis was undertaken using SPSS 20.0.0 (IBM Corp,
New York, NY, US). Continuous variables were
compared with the Student’s t test, and categorical variables
were compared with χ2 or Fisher’s Exact test. A p value
of ≤0.05 was considered significant.
From January 2013 to January 2016, 1001 children with
pneumonia or meningitis and 994 heathy children were
consented to the study. Three cases and 16 controls
were excluded from the analysis as they did not meet
the inclusion criteria (cases: not meeting clinical case
definitions [n = 2]; >5 years of age [n = 1]; controls:
evidence of acute respiratory infection [n = 5]; >5 years of
age [n = 11], Table 1). Thirty-four children were enrolled
as cases more than once and 14 controls were
subsequently enrolled as cases.
Pneumonia and meningitis cases
The median age of enrolled cases was 7.8 months
(interquartile range [IQR] 3.9–14.3) and 562/998 cases were male
(56.3%; Table 2). Parents infrequently reported chronic
health concerns (12/984; 1.2%), the most frequent being
chronic respiratory disease (n = 6) and seizures (n = 4).
Ninety-six (9.6%) and 7 (0.7%) cases reported a history of
pneumonia and meningitis, respectively.
Case enrolment during the study increased from 286
cases in 2013 to 421 in 2015 (Table 2). Despite the
seasonal nature of pneumonia (peak activity during the
southern hemisphere winter), cases were recruited year
round: the average number of cases recruited per month
was 36 (range 9–61; Fig. 1). A significant proportion of
cases were recruited following admission to EHPH (404/
998; 40.5%) with the remainder recruited from the
EHPH outpatient clinic (313; 31.4%), community (201;
20.1%) or PNGIMR research clinics (75; 7.5%). The
proportion of children recruited prior to admission to
hospital (i.e. from EHPH outpatients or community clinics,
as opposed to the EHPH children’s ward) increased
over the duration of the study. This shift in recruitment
was part of a dedicated focus to enhance recruitment of
children prior to antibiotic delivery (2013: 30.8%
recruited prior to admission to EHPH, 2014: 58.1%, 2015:
80.1%; p < 0.0001).
The most frequent diagnosis was moderate
pneumonia (n = 784, 78.6%), followed by severe pneumonia
(n = 187, 18.7%) and suspected meningitis (with or
without pneumonia; n = 75, 7.5%). Nasopharyngeal
samples were collected on all cases with blood
obtainable from 987 children (98.9%; Table 2). The volume
of blood collected per patient increased throughout
the study period: mean volume in 2013, 1.36 ml; 2014,
1.49 ml; 2015, 1.95 ml; difference in means; 0.12 ml,
(95% confidence interval [CI]: 0.016–0.224; p <0.03)
and 0.46 ml (95%CI: 0.32–0.61; p < 0.0001),
respectively. In addition, the number of children with
dedicated EDTA samples increased over time: 2013, 89/
286 (31.1%); 2014, 150/291 (51.5%) and 2015, 273/421
(64.8%), p < 0.001. The rate of contaminated blood
cultures decreased during the study: 2013, 63/281
Table 2 Cases and controls: demographics, recruitment,
diagnosis, diagnostic specimens and follow up
Median age (months; IQR)
Children’s ward, EHPH
Diagnostic specimens collected
Successful follow up
(of eligible children) by year
156/223b (69.9%) NA
Abbreviations: IQR interquartile range, EHPH Eastern Highlands Provincial
Hospital, NA not applicable
a inclusive of the first 3 weeks of January 2016
b denominator is children residing within 1 h by vehicle from Goroka
(22.4%); 2014, 41/285 (14.4%) and 2015, 48/421
(11.4%), p < 0.001. Collection of blood prior to
antibiotics was also more frequently achieved over the
course of the study: 2013, 139/275 (50.5%); 2014, 198/
282 (70.2%) and 2015, 246/415 (83.4%), p < 0.001.
Both CSF and urine samples were infrequently
collected during the study. In children with a clinical
Moderate pneumonia 784 (78.6%)
Without suspected meningitis 753
With suspected meningitis 31
Severe pneumonia 187 (18.7%)
Without suspected meningitis 170
With suspected meningitis 17
diagnosis of suspected meningitis (n = 75), only 29
underwent a CSF examination. Of the remainder, 24
cases were deemed by EHPH medical staff not to have
clinical evidence of meningitis, seven cases had
contraindication to lumbar puncture and eight had a lumbar
puncture without CSF being obtained. In a further seven
cases with suspected meningitis, it is uncertain why
sampling was not performed. A further 2 CSF samples were
taken from children not meeting the clinical definition
Half the patients were admitted to hospital (500/998;
50.1%) with the remainder managed as outpatients. Less
than 25% of children had an X-ray performed. The
median length of hospital stay was 2 days (IQR 2–5).
Eighteen deaths were identified (overall case fatality
rate (CRF): 18/998, 1.8% overall; pneumonia, 1.5%;
meningitis, 8.0%). The in-hospital CFR was 14/500 (2.8%
overall: pneumonia, 2.3%; meningitis 8.7%). Follow-up
was attempted in children residing within 1 h by vehicle
from Goroka (n = 742). Outcome data was obtained on
569 children (76.7%). During the period of the study, the
proportion of eligible children that were successfully
followed up increased from 69.9% (2013) to 81.0%
(2015), p < 0.02.
The median age of enrolled controls was 20.8 months
(IQR 8.2–36.4) and 522/983 enrolled controls were male
(53.2%; Table 2). Successful recruitment of controls
increased during the study period, particularly following
the institution of dedicated clinics in 2014 and 2015.
Despite ongoing attempts to recruit younger controls,
controls remained significantly older than cases
throughout the study; median ages cases vs. controls: 6.6 vs.
14.3 months (2013), 9.3 vs. 22.7 months (2014) and 8.4
vs. 21.0 months (2015).
Child health books that included vaccination status were
assessable in 991 cases (99.3%) and 976 controls (99.8%).
Coverage for one dose of DTPw-HepB-Hib in children
>1 month of age was 65.5% in cases and 71.9% in
controls (p < 0.01, Fig. 2), falling for three doses in children
>3 months of age to 31.6% in cases and 43.3% in
controls (p < 0.001). The significant difference in vaccine
coverage between cases and controls remained when
restricted to children < 12 months of age at enrolment.
The overall median age of the first, second and third
dose of DTPw-HepB-Hib was 1.4 months (IQR 1.1–3.0),
3.1 months (IQR 2.1–6.1) and 4.7 months (IQR 3.1–8.0).
During the study, vaccine coverage significantly
improved: three dose DTPw-HepB-Hib coverage in
children >3 months of age increased from 14.9 to 43.0% in
cases and 29.0 to 47.7% in controls (p < 0.001 in both).
Fig. 1 Enrolment of cases and controls by month: January 2013 to January 2016
Despite the launch of the PCV13 program in 2014, the
vaccine was not available in the EHP until 2015. The
2015 coverage of PCV13 was low in both cases and
controls with only 25.4 and 24.4% of cases and controls
>1 month receiving one dose, and 4.0 and 6.5% of cases
and controls >3 months receiving three doses (no
significant difference between cases and controls).
The ongoing burden of childhood disease in PNG
necessitates a contemporary reassessment of pneumonia and
meningitis etiology and outcomes. Given the lack of
access to and (if present) underutilisation of the
microbiological laboratory in PNG, targeted projects using
routine and contemporary diagnostics are required to
assess the current pathogen-specific burden of disease.
These data are essential to assess potential benefits of
childhood vaccination and to identify potential changes
Undertaking such studies, particularly in locations
such as the highlands of PNG, pose unique challenges in
study design, recruitment, specimen handling and
laboratory practice. A number of these challenges are
unique to both PNG and to other isolated highland
communities, while others are frequent with any large
prospective study. This paper outlines the methods and
challenges observed in recruiting nearly 2000 children
over 3 years.
Based on existing EHPH service data, the study
proposed to recruit >1000 children with clinical pneumonia
and meningitis over 2 years. Prior to commencement,
concern was raised by the community about the invasive
Fig. 2 Vaccine coverage by case-control status and age (*PCV13 data for 2015 only)
nature of the specimens proposed. PNGIMR has a long
history of community engagement developed over many
successful research studies. Regular visits to
communities to discuss this study, explaining the routine nature
of specimen collection in other healthcare settings and
the collaborative nature of healthcare provided by
PNGIMR and hospital staff was essential in the early
phase of the study. The provision of non-monetary
incentives such as transport from hospital to home for
study participants and future access to the PNGIMR
clinic are also likely to have assisted with recruitment,
which improved throughout the study.
Barriers to recruitment included frequent and
sometimes prolonged industrial action leading to closure of
health facilities. As a result, lower numbers of children
were admitted to the ward and at times, PNGIMR staff
needed to focus on providing healthcare rather than the
research project. Outbreaks of vaccine preventable
diseases (e.g. an outbreak of measles in April to June 2015)
required additional healthcare resources to be diverted
away from research activities. Hospital senior and junior
medical staff changed several times during the 3-year
study period and a lack of familiarity with the study
required regular re-engagement by the research team with
the EHPH paediatric staff. Significant variation in
practice, especially with CSF collection and utilisation of
radiology were noted throughout the study. As both
procedures were not mandated, rather recommended based
on clinical need, it is likely that physician preference
contributed to variable utilisation. Provision of a
dedicated study doctor may have assisted with the variation
in practice observed.
Compared with previous pneumonia studies
conducted at EHPH , a number of differences should be
noted: a lower proportion of enrolled children had
severe pneumonia (19% of the pneumonia cohort in this
study vs 46% in Barker et al. ). A higher number were
deemed well enough to manage as outpatient following
initial treatment (50% vs 0% in Barker et al. ). Forty
per cent of children were enrolled from the ward
following initial management by EHPH staff, albeit in
decreasing numbers over the duration of the study. A
significant number of children had been administered
antibiotics even prior to their first interview at EHPH
(data not shown). It is expected that these factors will
reduce the rate of detection of bacteraemia and invasive
bacterial isolates from the current cohort. The
inhospital mortality rate (2.8%) is significantly lower than
Barker et al. (8%)  and the rate reported nationally
(5.2%) by the Paediatric Hospital Reporting (PHR)
system, yet comparable to 2013 PHR data collected from
EHPH (2.4%) . These severity of illness and mortality
data are encouraging and suggest that, compared with
past studies, improvements in access to health services,
earlier recognition of childhood pneumonia with earlier
institution of antimicrobial therapy and supportive care
are leading to better outcomes for children with
Recruitment of controls was challenging, particularly
in the early phases of the study. Despite regular visits to
rural and peri-urban communities, well children
(particularly those < 6 months of age) were often absent,
frequently accompanying their parents to the fields early in
the morning. There was an attempt to overcome this by
scheduling PNGIMR community clinics and establishing
age specified targets. Following institution of these
clinics, larger numbers of controls were recruited, yet
recruitment of younger controls remained an ongoing
challenge. Given the uneven balance between cases and
controls, assessment of biological samples (e.g.
nasopharyngeal samples) will be undertaken in cases and
controls matched post-enrolment by month of illness, age
PNG has a long history of utilization of a hand-held
child health record. Documentation of vaccination status
is routinely recorded in the health book (on the reverse
of front cover and in the continuous clinical records).
The health record was assessable in more than 99% of
cases and controls. The variable nature of recording
required additional training and care to ensure accuracy of
the data collected by the research team. This may have
resulted in underestimation of the vaccine coverage early
in the study. Replacement of lost hand-held records or
an older child having more than one book may result in
underestimation of vaccine uptake (data not presented).
Despite this, the record provides a unique opportunity
to assess uptake of vaccination status in a country where
access to electronic or manually collected vaccine
distribution data remains a challenge. Vaccine coverage in the
highlands remains lower than the reported national
average : the reasons for this are likely to be multifactorial
and may include reduced access to health services given
the remoteness of highland villages, problems with
consistent immunization supply and parental education with
ongoing mistrust of infant immunization. Increased
vaccine coverage is encouraging and suggests that targeted
programs including the Special Integrated Routine EPI
Strengthening Program (SIREP) is having an impact in
the PNG highlands.
Undertaking a study dependent on key infrastructure
in isolated locations poses unique challenges. For much
of the study the BD BACTEC™ Instrumented Blood
Culture System was not working, despite multiple attempts
by the laboratory staff and the manufacturer to repair
the machine both in Goroka and Port Moresby. The
laboratory was reliant on manual subculture of blood
cultures increasing the time and risk of contamination of
critical specimens. Higher rates of contamination are
likely to impact on the ability to detect significant
bacteraemia. For significant periods of time, the hospital X-ray
facility required repairs, meaning that physician-requested
X-rays were not available. These key challenges must be
considered when designing studies in third-world settings,
particularly in locations outside the nation’s capital city
with difficult access by road or by air.
Achieving a follow up rate of >75% in an environment
where formal town maps and permanent phone
connections are largely non-existent is a testament to both
study participants and staff. This was achieved by
meticulous documentation of village, hamlet and house
location and use of PNGIMR drivers to return children
and their parents to their homes. This was also
enhanced by the provision of ongoing healthcare clinics in
larger villages and access for study participants to the
PNGIMR clinic. Despite these incentives, numerous
attempts were often required for follow up.
In summary, recruitment of large numbers of pediatric
pneumonia and meningitis cases and community controls
in a third-world setting with limited health services, health
and other infrastructures presents unique challenges. The
study successfully enrolled 998 cases and 978 controls,
obtained comprehensive clinical data and biological
specimens, and was able to follow up >75% following discharge.
Furthermore, staff documented increasing coverage of
pneumococcal and Hib conjugate vaccines in PNG children.
Key aspects to overcoming the challenges of conducting
research in third-world settings include a pragmatic and
flexible study design and strong collaborative relationships
with healthcare providers. Prior to commencing research,
the infrastructure needs of the study (and alternative plans
should key equipment be unavailable) must be considered.
Ongoing community engagement is paramount to optimize
recruitment and translation. In addition, the provision of
ongoing training for both research and clinical staff ensures
staff retention and facilitates translation of research findings
into changes in clinical practice.
Additional file 2: Table S2. Nasopharyngeal specimens collected into viral
transport media: Gene targets for viruses and bacteria [20–22]. (DOC 44 kb)
13vPCV: 13-valent Pneumococcal conjugate vaccine; CSF: Cerebrospinal fluid;
EHP: Eastern Highlands Province; EHPH: Eastern Highlands Provincial Hospital;
GGH: Goroka General Hospital; HIV: Human immunodeficiency virus;
PCR: Polymerase chain reaction; PHR: Paediatric Hospital Reporting;
PNG: Papua New Guinea; PNGIMR: Papua New Guinea Institute of Medical
Research; STGGB: Skim milk tryptone glucose glycerol broth; VTM: Viral
The investigators would like to thank the study participants and the Eastern
Highlands Province Hospital and urban clinic staff, without whom this study
would not have been possible.
Members of the Papua New Guinea Pneumonia and Meningitis Etiology
Study Team include: Yazid Abdad, Celestine Aho, Christopher Blyth, Trevor
Duke, Rebecca Ford, Andrew Greenhill, Paul Horwood, Ilomo Hwaihwanje,
John Kave, Wendy Kirarock, Lea-Ann Kirkham, Tonny Kumani, Sarah Javati,
Dorcas Joseph, Joe Jude, Amanda Lang, Deborah Lehmann, Geraldine
Masiria, Barry Nagepu, Tilda Orami, William Pomat, Peter Richmond, Joycelyn
Sapura, Peter Siba, Johanna Wapling, Mition Yoannes, and Lapule Yuasi.
The study was co-funded by an investigator-initiated Pfizer Global Research
Grant (WS1801400) and PNGIMR funds provided through the Internal
Competitive Research Award Scheme (ICRAS). The sponsor had no role in
the design of the study, collection, analysis, interpretation of the data and
in writing the manuscript. CCB is supported by an Australian National
Health and Medical Research Council Career Development Fellowship
Availability of data and materials
Please contact author for data requests.
CB, AG, DL and WP conceived and designed the study, sought
funding and supervised the study. JS, TK, GM and JK recruited cases
and controls and undertook community engagement necessary to
conduct this study. RF, LY, AG and AL performed microbiological
studies on specimens collected. CB analysed data for this manuscript
and wrote the draft manuscript. All authors have reviewed the
manuscript prior to submission. All authors read and approved the
DL has previously been a member of the GSK Australia
PneumococcalHaemophilus influenzae-Protein D conjugate vaccine (“Phid-CV”) Advisory
Panel, has received support from Pfizer Australia and GSK Australia to attend
conferences, has received an honorarium from Merck Vaccines to attend a
conference/seminar. WP has received support from Pfizer Australia to attend
conferences. No other authors have competing interests.
Ethics approval and consent to participate
Ethical approval was obtained from the Institutional Review Board of
PNGIMR (IRB no 1204), the Medical Research Advisory Committee of PNG
(MRAC number 11.29) and the University of Western Australia (RA/4/1/7960).
Verbal and written consent was sought from all parents of eligible children
prior to enrolment.
1. GBD Mortality and Causes of Death Collaborators . Global, regional, and national age-sex specific all-cause and cause-specific mortality for 240 causes of death, 1990-2013: a systematic analysis for the Global Burden of Disease Study 2013 . Lancet. 2015 ; 385 ( 9963 ): 117 - 71 .
2. GAVI: Papua New Guinea . In. http://www.gavi. org/country/papua-newguinea/ (Accessed 8 Dec 2016 ): GAVI publications; 2016.
3. PNG Ministry of Health: Papua New Guinea Child Health Plan 2008 - 2015 . 2008 http://www.rhtu.org.pg/wp-content/uploads/2013/06/PNG_Child_Health_ Plan_ 2008 - 2015 .pdf. Accessed 24 Feb 2017 .
4. Child Health Advisory Committee: Child Morbidity and Mortality, Annual Report 2013 . Edited by PNG National Department of Health and the PNG Paediatric Society; 2013 . http://pngpaediatricsociety.org/wp-content/uploads/2013/05 /2013- Annual-Child-Morbidity-and-Mortality-Report .pdf. Accessed 24 Feb 2017 .
5. Gratten M , Montgomery J. The bacteriology of acute pneumonia and meningitis in children in Papua New Guinea: assumptions, facts and technical strategies . P N G Med J. 1991 ; 34 ( 3 ): 185 - 98 .
6. Lehmann D. Epidemiology of acute respiratory tract infections, especially those due to Haemophilus influenzae, in Papua New Guinean children . J Infect Dis . 1992 ;165 Suppl 1: S20 - 5 .
7. Lehmann D , Michael A , Omena M , Clegg A , Lupiwa T , Sanders RC , Marjen B , Wai'in P , Rongap A , Saleu G , et al. Bacterial and viral etiology of severe infection in children less than 3 months old in the highlands of Papua New Guinea . Pediatr Infect Dis J . 1999 ; 18 (10 Suppl): S42 - 9 .
8. Barker J , Gratten M , Riley I , Lehmann D , Montgomery J , Kajoi M , Gratten H , Smith D , Marshall TF , Alpers MP . Pneumonia in children in the Eastern Highlands of Papua New Guinea: a bacteriologic study of patients selected by standard clinical criteria . J Infect Dis . 1989 ; 159 ( 2 ): 348 - 52 .
9. Greenhill AR , Phuanukoonnon S , Michael A , Yoannes M , Orami T , Smith H , Murphy D , Blyth C , Reeder J , Siba P , et al. Streptococcus pneumoniae and Haemophilus influenzae in paediatric meningitis patients at Goroka General Hospital, Papua New Guinea: serotype distribution and antimicrobial susceptibility in the pre-vaccine era . BMC Infect Dis . 2015 ; 15 : 485 .
10. Lehmann D , Yeka W , Rongap T , Javati A , Saleu G , Clegg A , Michael A , Lupiwa T , Omena M , Alpers MP . Aetiology and clinical signs of bacterial meningitis in children admitted to Goroka Base Hospital , Papua New Guinea, 1989 - 1992 . Ann Trop Paediatr . 1999 ; 19 ( 1 ): 21 - 32 .
11. Kirkham LAS , Smith-Vaughan HC , Greenhill AR . Improving aetiological diagnosis of bacterial pneumonia and meningitis in PNG . PNG Med J . 2010 ; 53 ( 3-4 ): 139 - 46 .
12. Chidlow GR , Laing IA , Harnett GB , Greenhill AR , Phuanukoonnon S , Siba PM , Pomat WS , Shellam GR , Smith DW , Lehmann D. Respiratory viral pathogens associated with lower respiratory tract disease among young children in the highlands of Papua New Guinea . J Clin Virol . 2012 ; 54 ( 3 ): 235 - 9 .
13. Phillips PA , Lehmann D , Spooner V , Barker J , Tulloch S , Sungu M , Canil KA , Pratt RD , Lupiwa T , Alpers MP . Viruses associated with acute lower respiratory tract infections in children from the eastern highlands of Papua New Guinea (1983- 1985) . Southeast Asian J Trop Med Public Health . 1990 ; 21 ( 3 ): 373 - 82 .
14. PNG Department of Health. Standard Treatment for Common Illness of Children in Papua New Guinea: a manual for nurses, community heatlh workers, health extension officers and doctors . 10th ed. Port Moresby: Papua New Guinea Department of Health and Paediatric Society of Papua New Guinea ; 2016 .
15. National Statistical Office PNG. National Population & Houseing Census 2011 : “ Count Me IN ”. In. Edited by National Statistical Office PNG. Port Moresby: PNG Government ; 2011 .
16. Phuanukoonnon S , Reeder JC , Pomat WS , Van den Biggelaar AH , Holt PG , Saleu G , Opa C , Michael A , Aho C , Yoannes M , et al. A neonatal pneumococcal conjugate vaccine trial in Papua New guinea: study population, methods and operational challenges . PNG Med J . 2010 ; 53 ( 3-4 ): 191 - 206 .
17. Clinical and Laboratory Standards Institute . Performance standards for antimicrobial susceptibility testing: approved standard, 23rd ed CLSI document M100-AS23 . Wayne: Clinical and Laboratory Standards Institute ; 2013 .
18. Resti M , Moriondo M , Cortimiglia M , Indolfi G , Canessa C , Becciolini L , Bartolini E , Benedictis FM , de Martino M , Azzari C. Community-acquired bacteremic pneumococcal pneumonia in children: diagnosis and serotyping by real-time polymerase chain reaction using blood samples . Clin Infect Dis . 2010 ; 51 ( 9 ): 1042 - 9 .
19. Wang X , Mair R , Hatcher C , Theodore MJ , Edmond K , Wu HM , Harcourt BH , Carvalho MD , Pimenta F , Nymadawa P , et al. Detection of bacterial pathogens in Mongolia meningitis surveillance with a new real-time PCR assay to detect Haemophilus influenzae . Int J Med Microbiol . 2011 ; 301 ( 4 ): 303 - 9 .
20. Chidlow GR , Harnett GB , Shellam GR , Smith DW. An economical tandem multiplex real-time PCR technique for the detection of a comprehensive range of respiratory pathogens . Viruses . 2009 ; 1 ( 1 ): 42 - 56 .
21. Gunson RN , Collins TC , Carman WF . Real-time RT-PCR detection of 12 respiratory viral infections in four triplex reactions . J Clin Virol . 2005 ; 33 ( 4 ): 341 - 4 .
22. Klemenc J , Asad Ali S , Johnson M , Tollefson SJ , Talbot HK , Hartert TV , Edwards KM , Williams JV . Real-time reverse transcriptase PCR assay for improved detection of human metapneumovirus . J Clin Virol . 2012 ; 54 ( 4 ): 371 - 5 .
23. Cherian T , Mulholland EK , Carlin JB , Ostensen H , Amin R , de Campo M , Greenberg D , Lagos R , Lucero M , Madhi SA , et al. Standardized interpretation of paediatric chest radiographs for the diagnosis of pneumonia in epidemiological studies . Bull World Health Organ . 2005 ; 83 ( 5 ): 353 - 9 .