Abstract
Objective:
To examine the association between bacterial-viral co-occurrence in the nasopharynx and the risk of community acquired pneumonia (CAP) in young children living in resource-limited settings.
Methods:
A case-control study was conducted between January and December 2017 in Moshi, Tanzania. Children 2–59 months with CAP and healthy controls were enrolled. RSV and Influenza A/B were detected with a standardized polymerase chain reaction (PCR) method, and a simplified real-time quantitative PCR method, without sample pre-processing, was developed to detect bacterial pathogens in nasopharyngeal samples.
Results:
A total of 109 cases and 324 healthy controls were enrolled. Co-occurrence of H. influenzae and S. pneumoniae was linked with higher odds of CAP (aOR=3.2, 95% CI=1.1-9.5). The majority of the H. influenzae isolated in cases and controls (95.8%) were non-typeable. Of the viruses examined, respiratory syncytial virus (RSV) was most common (n=31, 7.2%) in cases and controls. Children with RSV had 8.4 times higher odds to develop pneumonia than healthy children (aOR=8.4, 95%CI= 3.2 – 22.1).
Conclusion:
H. influenzae and S. pneumoniae were primarily co-detected in patients and were associated with CAP. The high prevalence of non-typeable H. influenzae might be a sign of replacement as a consequence of Haemophilus influenzae type b vaccination.
Keywords: bacterial and viral colonization, children under five years of age, community-acquired pneumonia, Tanzania
Introduction
Pneumonia is the leading cause of under-five morbidity and mortality worldwide.1 In 2017 alone, pneumonia caused around 808,694 deaths accounting for 15.0% of all under-five deaths, with increased risk among children under two years of age.2 Viruses, particularly respiratory syncytial virus (RSV), account for 15 – 40% of pneumonia episodes, followed by Influenza virus A and B.3 However, bacteria—particularly Streptococcus pneumoniae (S. pneumoniae) and Haemophilus influenzae (H. influenzae)—are linked with severe forms of pneumonia, accounting for more than 35% of all pneumonia mortality.3,4 The co-infection of bacterium and virus is also common (30 – 41%) in pneumonia etiology.5
The nasopharynx is the reservoir for these bacteria, with colonization being a pre-requisite step in the pathophysiology of pneumonia.6 The colonization rates in children living in low-income countries are usually higher.7 S. pneumoniae, H. influenzae, Moraxella catarrhalis (M.catarrhalis), and Staphylococcus aureus (S. aureus) colonize up to 50% of healthy children.8 But how normal flora and viruses interact and whether there is a link with these patterns and the cause of pneumonia remains unclear. Vaccines targeting S. pneumoniae and H. influenzae can reduced the carriage and consequently reduce the risk of disease, although not all vaccines are equally effective against carriage.7,9
Tanzania, like other countries, adopted the WHO recommendation of including vaccines against H. influenzae and S. pneumoniae in the National Vaccination Program. In 2009 and 2013 the H. influenzae type b vaccine (Hib) and 13-valent pneumococcal conjugate vaccine (PCV) were introduced, respectively.10,11 In other African countries a decline in nasopharyngeal colonization of some of the vaccine-targeted pathogens of the respiratory tract was observed.12 In addition to the direct benefit, the impact of the PCV has extended to other non-target pathogens. For instance, the decline of pneumonia incidence due to vaccination was found to be associated with a decline in RSV-linked hospitalization after the introduction of PCV.13
Despite the high vaccine coverage in 2016, in Tanzania pneumonia still caused 17,624 deaths in children younger than five years.14 The contribution of nasopharyngeal pathogens, both bacterial and viral, to the etiology of pneumonia in the era of Hib and PCV vaccination is poorly understood. Therefore, the current study was designed to examine the role of bacterial-viral co-detection in community-acquired pneumonia etiology among children under five years of age in resource-limited settings.
Material and methods
Design and settings
A case-control study was conducted between January and November 2017 in Moshi municipality in the Kilimanjaro region of Tanzania, as previously described.15 Cases were recruited from the three hospitals in Moshi municipality: Kilimanjaro Christian Medical Centre, Mawenzi Hospital (a regional referral hospital), and St. Joseph Hospital (a district designated hospital). These hospitals represent three different levels of health facilities in Tanzania, from district level to tertiary hospital.
Population
Children aged 2 to 59 months admitted with X-ray–confirmed pneumonia were eligible for participation as cases. The controls were healthy children with no reported respiratory tract infection sign or symptom within 28 days preceding the enrollment from the same community. The incidence cases were matched with controls on the basis of sex and age (± 1 month). Following the enrollment of incidence cases, monthly frequency matching was performed, and controls were enrolled from the community within 28 days of the case being identified, as previously described.15 The required sample size was estimated using the case-control formula,16 assuming 20% of the controls are exposed with a desired 80% power to detect an odds ratio of 2, we analyzed 433 in the ratio of 1:3 cases to controls, respectively.
Procedure
The trained research nurses based in the three facilities reviewed the admission log and identified children admitted with CAP. Children with an acute infection of the lungs within 14 days after the onset of respiratory symptoms were considered having CAP.18 Cases were defined as hospitalized children aged 2 – 59 months who met the WHO case definition for pneumonia in this age category17 and a chest X-ray film suggestive of pneumonia. Children with reported hospital admission within 14 days were excluded. The parents of children meeting eligibility criteria were invited to participate. In addition to the interview to collect demographic information and prior antibiotic use (reported use of antibiotic within a month before enrollment), clinical information and vaccination status were extracted from the file and the child growth and monitoring card. Children were considered to have completed vaccination if they had received the PCV and Hib vaccine according to the schedule of the Tanzanian Expanded Programme for Immunization. Measuring the child’s current weight using the Seca weight scale followed the interviews.
Following the interview, nasopharyngeal swabs were collected from the participants. Briefly, a nasopharyngeal flocked swab (cat. No. HCPN305C, Copan Italy) was carefully and gently inserted into the nasopharynx and gently rotated. Then samples were immediately transported with universal transport medium to the Kilimanjaro Clinical Research Institute Biotechnology laboratory and stored in −80°C freezers. Later these samples were transported on dry ice to the pediatric infectious diseases section of the Radboud University Medical Centre in the Netherlands for analysis.
Real-time quantitative PCR (qPCR) for the detection of bacteria
The stored swab samples were thawed on ice then vortexed to release the bacteria from the swab into the medium, and 200 μl from each sample was aliquoted into a Hard-Shell High-Profile 96-Well Semi-Skirted PCR Plate (cat. no. HSS9601, Bio-Rad USA). The plate was incubated for 15 minutes at 95°C to lyse the bacteria. DNA for positive controls (S. pneumoniae, strain: TIGR4, S. aureus, strain: NCTC8178, M. catarrhalis, strain: BBH18, H. influenzae, strain: R2866, Mycoplasma pneumoniae (M. pneumoniae), strain: DSM23979 and Klebsiella pneumoniae (K. pneumoniae), strain: DSM30104) were extracted using the Qiagen DNeasy Blood and Tissue kit following the protocol for gram-positive bacteria according to the manufacturer’s instructions (cat. No. 69506, Qiagen German). Following extraction, the DNA of positive controls were diluted tenfold starting at 10ng/μl. The serial dilution was used to quantify the amount of DNA in the clinical samples. Five sets of primers (Table 1) specific to these five bacteria were selected for the RT-qPCR. For S. pneumoniae identification, lytA encoding the autolysin was used.19 For H. influenzae, the gene encoding protein D (hpd) was selected.20 In the case of S. aureus, the thermostable nuclease (nuc) gene was used.21 Gene encoding an outer member protein (copB) was selected for the identification of M. catarrhalis,22 gene encoding for a large integral membrane protein of M. pneumoniae (mpn 141) was selected for the identification of M. pneumoniae, and for K. pneumoniae the phoE gene coding for a conserved outer membrane protein was used.
Table 1.
Primers and probes used for the qPCR assays to detect S. pneumoniae, H. influenzae, S. aureus, M. catarrhalis, K. pneumoniae and M. pneumoniae and Multiplex PCR to detect H. influenzae capsule gene
| Organism | Target gene | Primers and probes |
|---|---|---|
| S. pneumoniae | lytA | |
| Forward primer | lytA-CDC-f, 5’-ACGCAATCTAGCAGATGAAGCA-3’ | |
| Reverse primer | lytA-CDC-r, 5’-TCGTGCGTTTTAATTCCAGCT-3’ | |
| Probe | lytA-CDC-pr, 5’-FAM-GCCGAAAACGCTTGATACAG GGAG-3-BHQ1 | |
| H. influenzae | Hpd | |
| Forward primer | hpdF729, 5’-AGATTGGAAAGAAACACAAGAAAAAGA-3’ | |
| Reverse primer | hpdR819, 5’-CACCATCGGCATATTTAACCACT-3’ | |
| S. aureus | nuc | |
| Forward primer | nucF, 5′-GTTGCTTAGTGTTAACTTTAGTTGTA-3′ | |
| Reverse primer | nucR, 5′-AATGTCGCAGGTTCTTTATGTAATTT-3′ | |
| M. catarrhalis | copB | |
| Forward primer | copBF, 5’-CGTGTTGACCGTTTTGACTTT-3’ | |
| Reverse primer | copBR, 5’-TAGATTAGGTTACCGCTGACG-3’ | |
| K. pneumoniae | phoE | |
| Forward primer | phoE_F, 5’- TGCCCAGACCGATAACTTTA-3’ | |
| Reverse primer | phoE_R, 5’- CTGTTTCTTCGCTTCACGG-3’ | |
| M. pneumoniae | ||
| Forward primer | mpn141 | Mpn141_F, 5’-AAGCACGAGTGACGGAAACAC-3’ |
| Reverse primer | Mpn141_R, 5’-CACCACATCATTCCCCGTATTAG-3’ | |
| Probe | Mpn141_P, 5’-FAM-CTCCACCAACAACCTCGCGCCTA-3-TAMRA | |
| H. influenzae | bexA | |
| Forward primer | Hi-1, 5’-CGTTTGTATGATGTTGATCCAGAC-3’ | |
| Reverse primer | Hi-2, 5’-TGTCCATGTCTTCAAAATGATG-3’ | |
Real-time PCR for the detection of RSV, influenza virus A and B
For detection of RSV and influenza A and B virus, the Diagnose assay was performed using the primer and probe design validated and described by Templeton and co-authors.23 Briefly, nucleic acids were extracted from 200 μl clinical samples using the MagNA Pure and the MagNA-Pure LC Total Nucleic Acid Isolation Kit (Roche Diagnostics) according to the instructions of the manufacturer. An internal control consisting of Phocine Herpesvirus (PhPV, IC DNA control) and Equine Arthritis Virus (EAV, IC RNA control) was included in the assay. RNA was reverse transcribed to cDNA using the TaqMan Reverse Transcription Reagents kit (Applied Biosystems) in a 50 μl reaction mix containing 20 μl of nucleic acid isolate and random hexamers as primers, according to the manufacturer’s instructions. PCR was performed on the LightCycler 480 instrument using LightCycler 480 Probes Master Mix (Roche Diagnostics). Cycling conditions were 95°C for 5 min, followed by 50 cycles of 95°C for 15 sec, 55°C for 15 sec, and 72°C for 20 sec.
H. influenzae multiplex PCR for the detection of capsule genes
The presence of capsule gene bexA and H. influenzae hpd gene was determined for real-time PCR samples with H. influenzae, Cq value <36. The multiplex PCR was performed in 20 μl reactions containing 4 μl 5x GC buffer, 2 μl 4 pmol/μl primer HI-1, 2 μl 4 pmol/μl primer HI-2, 2 μl 2.5 mM dNTP mix, and 0.2 μl Phusion polymerase (New England Biolabs). Cycling conditions were 98°C for 30 sec, followed by 50 cycles of 98°C for 10 sec, 60°C for 30 sec, and 72°C for 30 sec. PCR products were visualized by agarose gel electrophoresis for analysis.
Statistical analysis
The data processing and analysis were done in STATA version 15 (StataCorp. 2017. Stata Statistical Software: Release 15. College Station, TX: StataCorp LLC). Descriptive analysis was performed to determine the distribution of the participants. The proportions of various variables were computed and compared using the Chi-square test. Logistic regression analysis was carried out to examine the association between CAP and the nasopharyngeal isolates. The crude odds ratios (cORs), adjusted odds ratios (aORs), and their corresponding 95% confidence intervals (CIs) were calculated using unconditional logistic regression. Differences with a p-value of less than 0.05 were considered statistically significant.
Ethical consideration
The Kilimanjaro Christian Medical University College Research Ethics Review Committee and the Tanzanian National Health Research Ethics Committee approved the study. Before enrollment, parents or a legal guardian of eligible children signed a written consent.
Results
Characteristics of study participants
A total of 433 children were enrolled in this study, of which 109 children had X-ray–confirmed pneumonia, and 324 were age- and sex-matched with healthy controls. Of the cases, 13.1% of children had not completed vaccination according to their age while the proportion in controls was 5.9%. Reported history of antibiotic use within a month was common among control (12.7%) and cases (7.3%). Cases were also different from controls in terms of being underweight. The majority (73.4%) of cases had fever and severe pneumonia at admission (Table 2).
Table 2:
General characteristics of the study participants (N=433)
| Variable | Cases (n=109) n (%) |
Healthy controls (n=324) n (%) |
p value |
|---|---|---|---|
| Age in months* | 14 (7 – 26) | 15 (7 – 26) | 0.761 |
| Sex | |||
| Male | 66 (60.6) | 187 (57.7) | |
| Female | 43 (39.4) | 137 (42.3) | 0.603 |
| Completed vaccination** | |||
| Yes | 93 (86.9) | 304 (94.1) | |
| No | 14 (13.1) | 19 (5.9) | 0.015 |
| Number of doses of PCV and Hib vaccines | |||
| 1st PCV and Hib | 91 (97.9) | 263 (99.6) | 0.107 |
| 2nd PCV and Hib | 89 (98.0) | 252 (99.2) | 0.777 |
| 3rd PCV and Hib | 86 (98.9) | 210 (96.3) | 0.240 |
| WAZ score | |||
| Underweight | 22 (20.6) | 21 (6.8) | |
| Normal | 85 (79.4) | 288 (93.2) | < 0.001 |
| Exclusive breastfeeding | |||
| Yes | 37 (35.2) | 162 (51.4) | |
| No | 68 (64.8) | 153 (48.6) | 0.004 |
| Prior antibiotic use** | |||
| Yes | 8 (7.3) | 39 (12.7) | |
| No | 101 (92.7) | 267 (87.3) | 0.126 |
| Attending day-care/school** | |||
| Yes | 13(12.2) | 20(6.5) | |
| No | 94(87.8) | 287(93.5) | 0.064 |
| Cooking fuel | |||
| Clean | 31 (29.0) | 140 (44.0) | |
| Unclean | 76 (71.0) | 170 (56.0) | 0.006 |
| Other children under 5 years of age | |||
| 0 | 71 (65.7) | 234 (73.6) | |
| ≥1 | 37 (34.3) | 84 (26.4) | 0.118 |
| Household crowding | |||
| Yes | 43 (39.5) | 111 (34.6) | |
| No | 66 (60.5) | 210 (65.4) | 0.360 |
| Hospitals | |||
| KCMC | 46 (42.2) | - | |
| Mawenzi | 43 (39.5) | - | |
| St. Joseph | 20 (18.3) | - | |
| Classification of pneumonia | |||
| Non-severe pneumonia | 29 (26.6) | - | |
| Severe pneumonia | 80 (73.4) | - | |
| Cyanosis | |||
| Yes | 17 (16.0) | - | |
| No | 89 (84.0) | - | |
| Fever at admission | |||
| Yes | 76 (73.1) | - | |
| No | 28 (26.9) | - | |
| Other co-morbidities | |||
| Malaria | 6 (5.6) | - | |
| Diarrhoea | 4 (3.7) | - | |
| Urinary tract infection | 14 (13.1) | - | |
Median
The total does not add to either 109 cases or 324 controls because some participants had missing information.
WAZ= WHO Weight for Age Z-score
UTI=Urinary tract infection
Completed vaccination= received age appropriate PCV and Hib vaccine
Bacterial and viral pathogens in the upper respiratory tract
In a total of 433 subjects with and without pneumonia, bacterial pathogens were detected. The common isolates were M. catarrhalis (55.7%, n=241), S. pneumoniae (45.0%, n=195), and H. influenzae (43.0%, n=186), see Table 3. M. pneumoniae was not detected in any of the samples. More than a quarter of the children had two bacteria, and another quarter of the sample had three bacterial pathogens in the nasopharynx. Viruses were isolated from 50 children with and without pneumonia, and the most commonly detected virus was RSV (7.2%, n=31) followed by Influenza A virus (4.2%, n=18).
Table 3.
Distribution of bacterial and viral isolated from the nasopharynx of all study participants (pneumonia patients and controls).
| Bacteria/viruses | Combinations of bacterial and viral species | n (%) |
|---|---|---|
| Bacteria | ||
| Present | 337 (77.8) | |
| Absent | 96 (22.2) | |
| 1 bacterial species | ||
| H. influenzae | 186 (43.0) | |
| S. pneumoniae | 195 (45.0) | |
| M. catarrhalis | 241 (55.7) | |
| K. pneumoniae | 4 (0.8) | |
| S. aureus | 45 (10.4) | |
| 2 bacterial species | H. influenzae and S. pneumonia | 122 (28.2) |
| H. influenzae and M. catarrhalis | 134 (31.0) | |
| H. influenzae and S. aureus | 16 (3.7) | |
| H. influenzae and K. pneumoniae | 3 (0.6) | |
| S. pneumonia and S. aureus | 13 (3.0) | |
| S. pneumoniae and M. catarrhalis | 148 (34.2) | |
| S. pneumoniae and K. pneumoniae | 1 (0.2) | |
| S. aureus and M. catarrhalis | 16 (3.7) | |
| S. aureus and K. pneumoniae | 0 | |
| M. catarrhalis and K. pneumoniae | 0 | |
| 3 bacterial species | H. influenzae, S. pneumoniae and S. aureus | 8 (1.9) |
| H. influenzae, S. pneumoniae and M. catarrhalis | 102 (23.6) | |
| H. influenzae, S. pneumoniae and K. pneumoniae | 1 (0.2) | |
| H. influenzae, S. aureus and M. catarrhalis | 7 (1.6) | |
| S. pneumoniae, S. aureus and M. catarrhalis | 8 (1.9) | |
| S. pneumonia, S. aureus and K. pneumoniae | 0 | |
| 4 bacterial species | S. pneumonia, S. aureus, H. influenzae and M. catarrhalis | 6 (1.4) |
| Viruses | ||
| Present | 50 (11.6) | |
| Absent | 383 (88.4) | |
| 1 viral species | RSV | 31 (7.2) |
| Influenza A | 18 (4.2) | |
| Influenza B | 2 (0.5) | |
| 2 viral species | RSV and Influenza A | 1 (5.6) |
| RSV and Influenza B | 0 | |
| Influenza A and B | 0 | |
Of the 207 children that carried H. influenzae at relatively high density (i.e. Cq value <36) were serotyped by multiplex PCR, only 9 (4.2%) had a capsule; 6 of those were derived from pneumonia cases. Two of the H. influenzae with capsule were negative for the hpd gene in the multiplex PCR.
Bacterial and viral-bacterial (co-)-occurrence in the upper respiratory tract
Many children with S. pneumoniae also carried H. influenzae (aOR=4.2, 95% CI= 2.8 – 6.4) and M. catarrhalis (aOR=4.6, 95% CI=3.0 – 7.0). The combination of M. catarrhalis and H. influenzae was also found to be common (aOR=3.1, 95% CI= 2.1 – 4.7). Children co-colonized with M. catarrhalis and H. influenzae had increased odds for being colonized with S. pneumoniae (aOR=6.7, 95% CI 4.1 – 10.8). Children with M. catarrhalis were 60% less likely to carry S. aureus in the nasopharynx (aOR=0.4, 95% CI= 0.2 - 0.8). This negative association was also observed for the co-occurrence of H. influenzae and M. catarrhalis with S. aureus (aOR=0.4, 95% CI= 0.2 – 0.9) (Table 4). Furthermore, the co-occurrence between bacteria and viruses was only significant for the combination Influenza A virus and S. pneumoniae (aOR=4.2, 95% CI=1.2 – 10.0).
Table 4.
Bacterial and viral nasopharyngeal co-occurrence among children under five years of age.
| S. pneumoniae | H. influenzae | S. aureus | M. catarrhalis | RSV | Influenza A | |
|---|---|---|---|---|---|---|
| S. pneumoniae | n.a. | 4.2 (2.8 – 6.4) | 0.5 (0.2 – 1.0) | 4.6 (3.0 – 7.0) | 1.6 (0.8 – 3.4) | 3.4 (1.2 – 10.0) |
| H. influenzae | 4.2 (2.8 – 6.4) | n.a. | 0.7 (0.4 – 1.4) | 3.1 (2.1 – 4.7) | 1.8 (0.8 – 3.7) | 1.2 (0.4 – 3.0) |
| S. aureus | 0.5 (0.2 – 1.0) | 0.7 (0.4 – 1.4) | n.a. | 0.4 (0.2 – 0.8) | n.a. | n.a. |
| K. pneumoniae | 0.5 (0.04 – 5.6) | n.a. | n.a. | 0.4 (0.2 – 0.8) | 12.3 (0.7– 209.6) | n.a. |
| M. catarrhalis | 4.6 (3.0 – 7.0) | 3.1 (2.1 – 4.7) | 0.4 (0.2 – 0.8) | n.a. | 0.6 (0.3 – 1.3) | 0.7 (0.3 – 1.9) |
| RSV | 1.6 (0.8 – 3.4) | 1.8 (0.8 – 3.7) | n.a. | 0.6 (0.3 – 1.3) | n.a. | 1.0 (0.1 – 7.9) |
| Influenza A | 4.2 (1.2 – 10.0) | 1.2 (0.4 – 3.0) | n.a. | 0.7 (0.3 – 1.9) | 0.9 (0.1 – 7.7) | n.a. |
| H. influenzae and M. catarrhalis | 6.7 (4.1 – 10.8) | n.a. | 0.4 (0.2 – 0.9) | n.a. | 1.1 (0.5 – 2.4) | 0.7 (0.3 – 2.2) |
| H. influenzae and S. pneumoniae | n.a. | n.a. | 0.5 (0.2 – 1.2) | 5.9 (3.5 – 10.2) | 2.1 (1.0 – 4.5) | 1.2 (0.4 – 3.3) |
| H. influenzae and S. aureus | 1.2 (0.4 – 3.3) | n.a. | n.a. | 0.6 (0.2 – 1.6) | n.a. | n.a. |
| S. pneumoniae and S. aureus | n.a. | 2.2 (0.7 – 6.9) | n.a. | 1.3 (0.4 – 4.1) | n.a. | n.a. |
| S. aureus and M. catarrhalis | 1.3 (0.5 – 3.5) | 1.0 (0.4 – 2.8) | n.a. | n.a. | n.a. | n.a. |
| H. influenzae and M. catarrhalis and S. aureus | 7.7 (0.9 – 64.7) | n.a. | n.a. | n.a. | n.a. | n.a. |
| S. pneumoniae, M. catarrhalis and S. aureus | n.a. | 4.0 (0.8 – 20.3) | n.a. | n.a. | n.a. | n.a. |
| H. influenzae and M. catarrhalis and S. pneumoniae | n.a. | n.a. | 0.5 (0.2 – 1.2) | n.a. | 1.7 (0.8 – 3.8) | 0.9 (0.3 – 2.7) |
| H. influenzae and S. aureus and S. pneumoniae | n.a. | n.a. | n.a. | 2.5 (0.5 – 12.5) | n.a. | n.a. |
aOR: adjusted odds ratio
CI: confidence interval
n.a.: not applicable
Adjusted for age, sex and prior antibiotic use
Viral-bacterial (co-)occurrence patterns in the upper respiratory tract of pneumonia patients
In this population, the co-occurrence of S. pneumoniae and H. influenzae and the colonization with RSV were independently associated with CAP. Children with CAP had higher odds for S. pneumoniae and H. influenzae co-detection as compared with healthy children (aOR=3.2, 95%CI=1.1 – 9.5) and had 8.4 times higher odds for nasopharyngeal detection of RSV than healthy children (aOR=8.4, 95%CI=3.2 – 22.1). Furthermore, in children with CAP it was 70% less likely to detect S. aureus in the nasopharynx compared to healthy children and 90% less likely to detect the combination S. pneumoniae, H. influenzae, and M. catarrhalis. Other bacteria had no significant association with CAP (Table 5).
Table 5:
Bacterial and viral isolates and the odds of radiological confirmed community-acquired pneumonia among children under five years of age
| Bacteria | Cases n (%) |
Controls n (%) |
cOR (95%CI) | p-value | aOR* (95%CI) | p-value |
|---|---|---|---|---|---|---|
| S. pneumoniae | ||||||
| Present | 44 (40.4) | 151 (46.6) | 0.8 (0.5 – 1.2) | 0.258 | - | - |
| Absent | 65 (59.6) | 173 (53.4) | 1 | - | - | - |
| H. influenzae | ||||||
| Present | 50 (45.9) | 136 (42.0) | 1.2 (0.8 – 1.8) | 0.477 | - | - |
| Absent | 59 (54.1) | 188 (58.0) | 1 | - | - | - |
| S. aureus | ||||||
| Present | 4 (3.7) | 41 (12.6) | 0.3 (0.1 – 0.8) | 0.013 | 0.3 (0.1 – 0.9) | 0.026 |
| Absent | 105 (96.3) | 283 (87.4) | 1 | - | 1 | - |
| M. catarrhalis | ||||||
| Present | 51 (46.8) | 190 (58.6) | 0.6 (0.4-1.0) | 0.032 | - | - |
| Absent | 58 (53.2) | 134 (41.4) | 1 | - | - | - |
| K. pneumoniae | ||||||
| Present | 2 (1.8) | 1 (0.3) | 6.0 (0.5- 67.2) | 0.143 | 0.8 (0.1 – 14.0) | 0.907 |
| Absent | 107 (98.2) | 323 (99.7) | 1 | - | 1 | - |
| S. pneumoniae and H. influenzae | ||||||
| Present | 31 (28.4) | 91 (28.1) | 1.0 (0.6 – 1.6) | 0.943 | 3.2 (1.1 – 9.5) | 0.032 |
| Absent | 78 (71.6) | 233 (71.9) | 1 | - | 1 | - |
| S. pneumoniae, H. influenzae and M. catarrhalis | ||||||
| Present | 20 (18.3) | 82 (25.3) | 0.7 (0.4 – 1.1) | 0.140 | 0.1 (0.04 – 0.5) | 0.002 |
| Absent | 89 (81.6) | 242 (74.7) | 1 | - | 1 | - |
| RSV | ||||||
| Present | 22 (20.2) | 9(2.8) | 8.9 (3.9 – 20.0) | <0.001 | 8.4 (3.2 – 22.1) | <0.001 |
| Absent | 87(79.8) | 315(97.2) | 1 | 1 | ||
| S. pneumoniaeand Influenza A | ||||||
| Present | 2 (1.8) | 11 (3.4) | 0.5 (0.1 – 2.4) | - | - | - |
| Absent | 107 (98.2) | 313 (96.6) | 1 | - | - | - |
| Influenza A | ||||||
| Present | 6(5.5) | 12 (3.7) | 1.5 (0.6 – 4.1) | 0.418 | - | - |
| Absent | 103 (94.5) | 312(96.3) | 1 | - | - | - |
| Influenza B | ||||||
| Present | 2(1.8) | 0 | - | - | - | - |
| Absent | 107(98.2) | 324(100) | - | - | - | - |
cOR: crude odds ratio
aOR: adjusted odds ratio
CI: confidence interval
Adjusted for age, sex, prior antibiotic use, day care attendance, WAZ, exclusive breast-feeding status, household crowding, cooking fuel and vaccination status
Discussion
We found that co-occurrence of bacteria, particularly S. pneumoniae, M. catarrhalis, and H. influenzae, was common in this population. Also, children colonized with S. pneumoniae were more likely to harbour Influenza A virus in the upper respiratory tract than children without S. pneumoniae. Those with CAP had higher odds of H. influenzae and S. pneumoniae co-detection. Furthermore, children with CAP had higher odds of having RSV. S. aureus was commonly detected in the nasopharynx of healthy children and S. aureus co-occurrence with the other bacterial species included in this study was rare.
We found a high PCV coverage in this population, compared to other regions of Sub-Saharan African like Addis Ababa, Ethiopia, and Kilifi, Kenya, which in the same period reported a country coverage of 91.4% and 84%, respectively.24,25 Despite a high PCV-13 vaccine coverage in this population, high S. pneumoniae colonization rates were found. However, it is not known whether these were vaccine or non-vaccine serotypes. Our results are similar to another study in this population that reported 32% carriage for S. pneumoniae.26 Although about half of the children were carrying H. influenzae, the carriage was common in children with pneumonia compared with healthy children. Despite vaccination, many children still carry H. influenzae bacteria, however the majority were non-typable H. influenzae (NTHi). These results confirm the effectiveness of Hib vaccine as previously reported.27 However, more importantly, it also strongly indicates that NTHi is emerging, which might be an effect of Hib vaccination. Non-typeable H. influenzae are frequently carried and are known to cause pneumonia in children.28 Because the 10-valent PCV contains protein D derived from NTHi as a carrier protein, to which 8 of the 10 polysaccharides are conjugated, vaccination with PCV10 might lead to partial protection against H. influenzae, however solid evidence for broad protection against NTHi is lacking.29 This observation strongly demands better surveillance of this important pathogen.
Children with H. influenzae were more likely to also carry S. pneumoniae or M. catarrhalis. Our finding supports the growing evidence for frequent co-occurrence of S. pneumoniae and H. influenzae.31,32 A synergistic link between S. pneumoniae and H. influenzae has earlier been suggested.33 S. pneumoniae and H. influenzae are living together in biofilms,33 offering passive protection against antibiotics.34 Further, the presence of S. pneumoniae in the nasopharynx enables the invasion of H. influenzae.35 In contrast to the theory of synergism a few studies have reported a negative association between S. pneumoniae and H. influenzae,36 suggesting that hydrogen peroxide produced by S. pneumoniae blocks the growth of H. influenza.37 Furthermore, the S. pneumoniae neuraminidase enhances H. influenzae clearance by the complement system through the desialylation of lipopolysaccharide from H. influenza.38 In our study we found that this co-occurrence was linked with higher odds for the development of pneumonia. This confirmed the findings of an earlier study conducted in Greece where a link was found between S. pneumoniae and H. influenzae co-occurrence in the upper respiratory tract and acute lower respiratory tract infections in young children.39 Our findings also confirm the conclusion from a previous study conducted in Tanzania, in which this specific co-occurrence was commonly found in febrile children with pneumonia.31 Despite the evidence for synergism, only a few studies investigated its relationship in pneumonia.
We also found that M. catarrhalis concurrently colonize the upper respiratory tract with S. pneumoniae and H. influenzae. This supports a positive association between M. catarrhalis and either S. pneumoniae or H. influenzae or both as previously described.31 There is one study that reported a negative association between these three bacteria, however when both S. pneumoniae and H. influenzae were co-occurring they were positively associated with M. catarrhalis.36 The contradiction suggests a more complex interplay between these bacteria in the nasopharynx, requiring more research to fully understand the interaction. Surprisingly, in this population, the co-occurrence of M. catarrhalis with S. pneumoniae and H. influenzae had a negative association with CAP. The finding contradicts current knowledge that M. catarrhalis has a role in lower respiratory tract infection.31,40 However, these studies were conducted before the PCV vaccine was widespread and how the vaccine has affected the colonization is not clear.
Unlike the previously reported positive association, S. aureus was negatively associated with S. pneumonia, H. influenzae, and M. catarrhalis. This negative association has been reported by many investigators.31,32,39 It has been suggested that the negative association is linked to the hydrogen peroxide production by S. pneumoniae—hydrogen peroxide is toxic to S. aureus and inhibits its growth, hence protecting the host from S. aureus colonization.41 The negative association of S. aureus and the other potential pathogens causing CAP might explain the unexpectedly high proportion of S. aureus isolated from controls compared to cases in this population.
RSV alone was found significantly more often in pneumonia patients than in controls. Previous and recent studies have also reported the association of RSV with pneumonia. For example, the PERCH multi-country study reported that RSV is commonly associated with childhood pneumonia more than any other pathogens, while other viruses had no link with CAP.42 In reducing pneumonia deaths, RSV link with CAP should be considered in prevention and also treatment. Although virus infections, including RSV, are challenging to treat, immunization against this important pathogen might be more promising.
In this population, none of the enrolled children (both cases and controls) had M. pneumoniae. This is contrary to other previous studies from different regions.43,44 Although the sensitivity of nasopharyngeal swab samples used in this study is considered low, further studies are needed to investigate the sensitivity of detection, the geographical distribution, seasonality, and their importance for the management of CAP.
Strength and limitation
We have shown that bacterial pathogens can directly be detected in crude, unprocessed swab samples using a highly sensitive qPCR method This allows quick determination of the most likely causative agents, which can help in the decision on the most appropriate treatment. A limitation of the study was that antibiotic use could not be verified in all cases, though this likely has an impact on carriage.
Conclusions
Co-detection of H. influenzae and S. pneumoniae in the nasopharynx was strongly associated with CAP. The majority of H. influenzae found in the URT was NTHi, for which no vaccine is available. It was confirmed that RSV is playing an important role in the etiology of CAP in young children. These results could be applied to the improvement of diagnosis of young children with pneumonia, allowing targeted treatment leading to a decline in morbidity and mortality in this vulnerable population.
Highlights.
Co-occurrence of H. influenzae and S. pneumoniae in the nasopharynx is associated with pneumonia
The majority of H. influenzae found in the nasopharynx are the non-typable H. influenzae
Respiratory syncytial virus plays an important role in the aetiology of community-acquired pneumonia in children
Real time PCR on nasopharyngeal swab allows rapid detect of the likely causative agents
Acknowledgement
The authors would like to acknowledge the children who participated in this study and the parents who allowed their children to take part. Furthermore, the authors are grateful to the laboratory technicians who helped with sample analyses. Last, the authors would like to thank the staff at all health facilities and the authorities for their support.
Funding
This research was supported by the Fogarty International Centre of the National Institutes of Health under Award Number D43TW010138 and partly by D43TW006578 and German Academic Exchange Service (DAAD). The funding bodies had no role in the design of the study, collection, analysis, and interpretation of the data nor in writing the manuscript.
Footnotes
Competing interest
The authors declare that they have no competing interests.
Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
References
- 1.Liu Li, Oza Shefali, Hogan Daniel, Perin Jamie, Rudan Igor, Lawn Joy E, et al. Global, regional, and national causes of child mortality in 2000-13, with projections to inform post-2015 priorities: an updated systematic analysis. Lancet (London, England) 2015;385(9966):430–40. Doi: 10.1016/S0140-6736(14)61698-6. [DOI] [PubMed] [Google Scholar]
- 2.WHO. Pneumonia. Available at https://www.who.int/news-room/fact-sheets/detail/pneumonia. Accessed September 23, 2019, 2019.
- 3.Rudan Igor, Boschi-Pinto Cynthia, Biloglav Zrinka, Mulholland Kim, Campbell Harry. Epidemiology and etiology of childhood pneumonia. Bull World Health Organ 2008:408–16. Doi: 10.2471/BLT.07.048769. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 4.Rudan Igor, O’Brien Katherine L, Nair Harish, Liu Li, Theodoratou Evropi, Qazi Shamim, et al. Epidemiology and etiology of childhood pneumonia in 2010: estimates of incidence, severe morbidity, mortality, underlying risk factors and causative pathogens for 192 countries. J Glob Health 2013;3(1):010401 Doi: 10.7189/jogh.03.010401. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 5.Bezerra Patrícia GM, Britto Murilo C a, Correia Jailson B., Duarte Maria Do Carmo MB, Fonceca Angela M., Rose Katie, et al. Viral and atypical bacterial detection in acute respiratory infection in children under five years. PLoS One 2011;6(4):e18928 Doi: 10.1371/journal.pone.0018928. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 6.Siegel Steven J, Weiser Jeffrey N. Mechanisms of Bacterial Colonization of the Respiratory Tract. Annu Rev Microbiol 2015;69:425–44. Doi: 10.1146/annurev-micro-091014-104209. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 7.WHO Publication. Pneumococcal vaccines WHO position paper - 2012 - recommendations. Vaccine 2012;30(32):4717–8. Doi: 10.1016/j.vaccine.2012.04.093. [DOI] [PubMed] [Google Scholar]
- 8.Dunne Eileen M, Murad Chrysanti, Sudigdoadi Sunaryati, Fadlyana Eddy, Tarigan Rodman, Indriyani Sang Ayu Kompiyang, et al. Carriage of Streptococcus pneumoniae, Haemophilus influenzae, Moraxella catarrhalis, and Staphylococcus aureus in Indonesian children: A cross-sectional study. PLoS One 2018; 13(4):e0195098 Doi: 10.1371/journal.pone.0195098. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 9.Loughlin Anita M, Hsu Katherine, Silverio Amy L, Marchant Colin D, Pelton Stephen I. Direct and indirect effects of PCV13 on nasopharyngeal carriage of PCV13 unique pneumococcal serotypes in Massachusetts’ children. Pediatr Infect Dis J 2014;33(5):504–10. Doi: 10.1097/INF.0000000000000279. [DOI] [PubMed] [Google Scholar]
- 10.AMC Secretariat at GAVI, in collaboration with the World Bank and UNICEF’s Supply Division approved by the Independent Assessment Committee. PNEUMOCOCCAL AMC ANNUAL REPORT – 1 APRIL 2013 TO 31 MARCH 2014; 2014. [Google Scholar]
- 11.WHO/UNICEF. United Republic of Tanzania: WHO and UNICEF estimates of immunization coverage: 2017 revision. 2018.
- 12.Hammitt Laura L, Akech Donald O, Morpeth Susan C, Karani Angela, Kihuha Norbert, Nyongesa Sammy, et al. Population effect of 10-valent pneumococcal conjugate vaccine on nasopharyngeal carriage of Streptococcus pneumoniae and non-typeable Haemophilus influenzae in Kilifi, Kenya: findings from cross-sectional carriage studies. Lancet Glob Heal 2014;2(7):e397–405. Doi: 10.1016/S2214-109X(14)70224-4. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 13.Weinberger Daniel M, Klugman Keith P, Steiner Claudia A, Simonsen Lone, Viboud Cécile. Association between respiratory syncytial virus activity and pneumococcal disease in infants: a time series analysis of US hospitalization data. PLoS Med 2015;12(1):e1001776 Doi: 10.1371/journal.pmed.1001776. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 14.IVAC International Vaccine Access Center. Pneumonia and Diarrhea Progress Report 2018. 2018. [Google Scholar]
- 15.Ngocho James Samwel, de Jonge Marien Isaäk, Minja Linda, Olomi Gaudencia Alois, Mahande Michael Johnson, Msuya Sia Emmanueli, et al. Modifiable risk factors for community-acquired pneumonia in children under 5 years of age in resource-poor settings: a case-control study. Trop Med Int Health 2019;24(4):484–92. Doi: 10.1111/tmi.13211. [DOI] [PubMed] [Google Scholar]
- 16.Kirkwood Betty R, Sterne Jonathan AC Essential medical statistics 2nd ed. Blackwell Science; 2003. [Google Scholar]
- 17.WHO/UNICEF. Revised WHO Classification and Treatment of Pneumonia in Children at Health Facilities. World Health Organization; 2014. [PubMed] [Google Scholar]
- 18.Bartlett John G, Dowell Scott F, Mandell Lionel A, File Thomas M, Musher Daniel M, Fine Michael J. Practice guidelines for the management of community-acquired pneumonia in adults. Infectious Diseases Society of America. Clin Infect Dis 2000;31(2):347–82. Doi: 10.1086/313954. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 19.Thomas Greve, Møller Jens Kjølseth. Accuracy of using the lytA gene to distinguish Streptococcus pneumoniae from related species. J Med Microbiol 2012;61(Pt 4):478–82. Doi: 10.1099/jmm.0.036574-0. [DOI] [PubMed] [Google Scholar]
- 20.Binks Michael J, Temple Beth, Kirkham Lea-Ann, Wiertsema Selma P, Dunne Eileen M, Richmond Peter C, et al. Molecular surveillance of true nontypeable Haemophilus influenzae: an evaluation of PCR screening assays. PLoS One 2012;7(3):e34083 Doi: 10.1371/journal.pone.0034083. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 21.Maes N, Magdalena J, Rottiers S, De Gheldre Y, Struelens MJ. Evaluation of a triplex PCR assay to discriminate Staphylococcus aureus from coagulase-negative Staphylococci and determine methicillin resistance from blood cultures. J Clin Microbiol 2002;40(4):1514–7. Doi: 10.1371/journal.pone.0034083. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 22.Gadsby N J, McHugh M P, Russell C D, Mark H, Conway Morris A, Laurenson I F, et al. Development of two real-time multiplex PCR assays for the detection and quantification of eight key bacterial pathogens in lower respiratory tract infections. Clin Microbiol Infect 2015;21(8):788.e1–788.e13. Doi: 10.1016/j.cmi.2015.05.004. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 23.Templeton Kate E, Scheltinga Sitha A, Beersma Matthias F C, Kroes Aloys C M, Claas Eric C J. Rapid and sensitive method using multiplex real-time PCR for diagnosis of infections by influenza a and influenza B viruses, respiratory syncytial virus, and parainfluenza viruses 1, 2, 3, and 4. J Clin Microbiol 2004;42(4): 1564–9. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 24.Abrham Wondimu, Qi Cao, Wilschut Jan C., Postma Maarten J. Factors associated with the uptake of newly introduced childhood vaccinations in Ethiopia: the cases of rotavirus and pneumococcal conjugate vaccines. BMC Public Health 2019; 19(1): 1656 Doi: 10.1186/s12889-019-8002-8. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 25.Hammitt Laura L, Etyang Anthony O, Morpeth Susan C, Ojal John, Mutuku Alex, Mturi Neema, et al. Effect of ten-valent pneumococcal conjugate vaccine on invasive pneumococcal disease and nasopharyngeal carriage in Kenya: a longitudinal surveillance study. Lancet 2019;393(10186):2146–54. Doi: 10.1016/S0140-6736(18)33005-8. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 26.Matilda Emgård, Msuya Sia E, Nyombi Balthazar M, Mosha Dominic, Gonzales-Siles Lucia, Nordén Rickard, et al. Carriage of penicillin-non-susceptible pneumococci among children in northern Tanzania in the 13-valent pneumococcal vaccine era. Int J Infect Dis 2019;81:156–66. Doi: 10.1016/j.ijid.2019.01.035. [DOI] [PubMed] [Google Scholar]
- 27.Charlotte Jackson, Andrea Mann, Punam Mangtani, Paul Fine. Effectiveness of Haemophilus influenzae Type b Vaccines Administered According to Various Schedules. Pediatr Infect Dis J 2013;32(11): 1261–9. Doi: 10.1097/INF.0b013e3182a14e57. [DOI] [PubMed] [Google Scholar]
- 28.Slack Mary P E The evidence for non-typeable Haemophilus influenzae as a causative agent of childhood pneumonia. Pneumonia (Nathan Qld) 2017;9:9 Doi: 10.1186/S41479-017-0033-2. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 29.Leach Amanda Jane, Wigger Christine, Kim Hare, Vanya Hampton, Jemima Beissbarth, Ross Andrews, et al. Reduced middle ear infection with nontypeable Haemophilus influenzae, but not Streptococcus pneumoniae, after transition to 10-valent pneumococcal non-typeable H. influenzae protein D conjugate vaccine. BMC Pediatr 2015; 15(1): 1–13. Doi: 10.1186/s12887-015-0483-8. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 30.Roman Prymula, Lode Schuerman. 10-valent pneumococcal nontypeable Haemophilus influenzae PD conjugate vaccine: Synflorix. Expert Rev Vaccines 2009;8(11): 1479–500. Doi: 10.1586/erv.09.113. [DOI] [PubMed] [Google Scholar]
- 31.Sopio Chochua, Valérie D’Acremont, Christiane Hanke, David Alfa, Joshua Shak, Mary Kilowoko, et al. Increased Nasopharyngeal Density and Concurrent Carriage of Streptococcus pneumoniae, Haemophilus influenzae, and Moraxella catarrhalis Are Associated with Pneumonia in Febrile Children. PLoS One 2016;11(12):e0167725 Doi: 10.1371/journal.pone.0167725. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 32.Chien Yu-Wen, Vidal Jorge E, Grijalva Carlos G, Bozio Catherine, Edwards Kathryn M, Williams John V, et al. Density interactions among Streptococcus pneumoniae, Haemophilus influenzae and Staphylococcus aureus in the nasopharynx of young Peruvian children. Pediatr Infect Dis J 2013;32(1):72–7. Doi: 10.1097/INF.0b013e318270d850. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 33.Alexandra Tikhomirova, Kidd Stephen P. Haemophilus influenzae and Streptococcus pneumoniae: living together in a biofilm. Pathog Dis 2013;69(2): 114–26. Doi: 10.1111/2049-632X.12073. [DOI] [PubMed] [Google Scholar]
- 34.Weimer Kristin ED, Juneau Richard A., Murrah Kyle A., Pang Bing, Armbruster Chelsie E., Richardson Stephen H., et al. Divergent mechanisms for passive pneumococcal resistance to β-lactam antibiotics in the presence of Haemophilus influenzae. J Infect Dis 2011;203(4):549–55. Doi: 10.1093/infdis/jiq087. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 35.Elisa Margolis, Andrew Yates, Levin Bruce R. The ecology of nasal colonization of Streptococcus pneumoniae, Haemophilus influenzae and Staphylococcus aureus: the role of competition and interactions with host’s immune response. BMC Microbiol 2010; 10(1):59 Doi: 10.1186/1471-2180-10-59. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 36.Pettigrew Melinda M, Gent Janneane F, Revai Krystal, Patel Janak A., Chonmaitree Tasnee. Microbial Interactions during Upper Respiratory Tract Infections. Emerg Infect Dis 2008; 14(10): 1584–91. Doi: 10.3201/eid1410.080119. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 37.Pericone C D, Overweg K, Hermans P W, Weiser J N. Inhibitory and bactericidal effects of hydrogen peroxide production by Streptococcus pneumoniae on other inhabitants of the upper respiratory tract. Infect Immun 2000;68(7):3990–7. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 38.Shakhnovich Elizabeth A, King Samantha J, Weiser Jeffrey N. Neuraminidase expressed by Streptococcus pneumoniae desialylates the lipopolysaccharide of Neisseria meningitidis and Haemophilus influenzae: a paradigm for interbacterial competition among pathogens of the human respiratory tract. Infect Immun 2002;70(12):7161–4. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 39.Skevaki Chrysanthi L, Tsialta Paraskevi, Trochoutsou Aikaterini I, Logotheti Ioanna, Makrinioti Heidi, Taka Styliani, et al. Associations Between Viral and Bacterial Potential Pathogens in the Nasopharynx of Children With and Without Respiratory Symptoms. Pediatr Infect DIs J 2015;34(12): 1296–301. Doi: 10.1097/INF.0000000000000872. [DOI] [PubMed] [Google Scholar]
- 40.Murphy Timothy F, Parameswaran G. Iyer. Moraxella catarrhalis, a human respiratory tract pathogen. Clin Infect DIs 2009;49(1): 124–31. Doi: 10.1086/599375. [DOI] [PubMed] [Google Scholar]
- 41.Regev-Yochay Gili, Trzcinski Krzysztof, Thompson Claudette M, Malley Richard, Marc Lipsitch. Interference between Streptococcus pneumoniae and Staphylococcus aureus: In vitro hydrogen peroxide-mediated killing by Streptococcus pneumoniae. J Bacteriol 2006; 188(13):4996–5001. Doi: 10.1128/JB.00317-06. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 42.Pneumonia Etiology Research for Child Health (PERCH) Study Group. Causes of severe pneumonia requiring hospital admission in children without HIV infection from Africa and Asia: the PERCH multi-country case-control study. Lancet (London, England) 2019;394(10200)757–79. Doi: 10.1016/S0140-6736(19)30721-4. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 43.Zhao Meng-Chuan, Wang Le, Qiu Fang-Zhou, Zhao Li, Guo Wei-Wei, Yang Shuo, et al. Impact and clinical profiles of Mycoplasma pneumoniae co-detection in childhood community-acquired pneumonia. BMC Infect Dis 2019;19(1):835 Doi: 10.1186/s12879-019-4426-0. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 44.Maimuna Carrim, Nicole Wolter, Benitez Alvaro J., Tempia Stefano, du Plessis Mignon, Walaza Sibongile, et al. Epidemiology and Molecular Identification and Characterization of Mycoplasma pneumoniae, South Africa, 2012-2015. Emerg Infect Dis 2018;24(3):506–13. Doi: 10.3201/eid2403.162052. [DOI] [PMC free article] [PubMed] [Google Scholar]