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Biomarkers of Systemic Inflammation in Ugandan Infants and Children Hospitalized With Respiratory Syncytial Virus Infection

Sawatzky, Julia BSc*; Soo, Jeremy BSc*; Conroy, Andrea L. PhD; Bhargava, Ravi MD; Namasopo, Sophie MBChB, MPH§; Opoka, Robert O. MBChB, MSc; Hawkes, Michael T. MD, PhD‖,**,††

The Pediatric Infectious Disease Journal: August 2019 - Volume 38 - Issue 8 - p 854–859
doi: 10.1097/INF.0000000000002343
Translational Medicine Reports
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Background: Optimizing outcomes in respiratory syncytial virus (RSV) pneumonia requires accurate diagnosis and determination of severity that, in resource-limited settings, is often based on clinical assessment alone. We describe host inflammatory biomarkers and clinical outcomes among children hospitalized with RSV lower respiratory tract infection (LRTI) in Uganda and controls with rhinovirus and pneumococcal pneumonia.

Methods: 58 children hospitalized with LRTI were included. We compared 37 patients with RSV, 10 control patients with rhinovirus and 11 control patients with suspected pneumococcal pneumonia.

Results: Patients in the RSV group had significantly lower levels of C-reactive protein (CRP) and chitinase-3-like protein 1 (CHI3L1) than the pneumococcal pneumonia group (P < 0.05 for both). Among children with RSV, higher admission levels of CRP predicted prolonged time to resolution of tachypnea, tachycardia and fever. Higher levels of CHI3L1 were associated with higher composite clinical severity scores and predicted prolonged time to resolution of tachypnea and tachycardia, time to wean oxygen and time to sit. Higher levels of lipocalin-2 (LCN2) predicted prolonged time to resolution of tachypnea, tachycardia and time to feed. Higher admission levels of all 3 biomarkers were predictive of a higher total volume of oxygen administered during hospitalization (P < 0.05 for all comparisons). Of note, CHI3L1 and LCN2 appeared to predict clinical outcomes more accurately than CRP, the inflammatory biomarker most widely used in clinical practice.

Conclusions: Our findings suggest that CHI3L1 and LCN2 may be clinically informative biomarkers in childhood RSV LRTI in low-resource settings.

*Department of Pediatrics, University of Alberta, Edmonton, Canada

Ryan White Center for Pediatric Infectious Diseases and Global Health, Indiana University School of Medicine, Indianapolis

Department of Radiology and Diagnostic Imaging, University of Alberta, Edmonton, Canada

§Department of Pediatrics, Jinja Regional Referral Hospital

Department of Pediatrics and Child Health, Mulago Hospital and Makerere University, Kampala, Uganda

Department of Pediatrics

**Department of Medical Microbiology and Immunology, University of Alberta, Edmonton, Canada

††Department of Global Health, School of Public Health, University of Alberta, Edmonton, Canada.

Accepted for publication March 14, 2019.

This work was supported by Grand Challenges Canada, Women and Children’s Health Research Institute and Association of Medical Microbiology and Infectious Disease Canada.

The authors have no conflicts of interest to disclose.

Supplemental digital content is available for this article. Direct URL citations appear in the printed text and are provided in the HTML and PDF versions of this article on the journal’s website (www.pidj.com).

Sophie Namasopo, MBChB, MPH is currently at Kabale District Hospital, Kabale, Uganda., Address for correspondence: Michael T. Hawkes, Department of Pediatrics, University of Alberta, 3-588D Edmonton Clinic Health Academy, 11405 87 Ave NW, Edmonton, AB T6G 1C9, Canada. E-mail: mthawkes@ualberta.ca.

Respiratory syncytial virus (RSV) is a leading cause of lower respiratory tract infection (LRTI) globally. In 2015, RSV resulted in approximately 3.2 million childhood hospital admissions and 118,200 deaths, 99% of which occurred in low and middle-income countries.2 RSV is an enveloped RNA paramyxovirus that is highly infectious via droplet transmission. The severity of RSV respiratory infections can be attributed largely to the host immune response, which is characterized by neutrophil-mediated inflammation of the airways.1

There is significant overlap between the clinical manifestations of RSV LRTI and bacterial pneumonia, which may lead to the inappropriate use of antimicrobials in patients with RSV. The current World Health Organization and United Nations International Children’s Emergency Fund integrated management of childhood illness guidelines for the management of childhood LRTI in low-resource settings include cough, difficulty breathing, tachypnea and chest indrawing as clinical criteria for the administration of empiric dispersible amoxicillin.3 These criteria are sensitive and will capture most children with bacterial pneumonia; however, a large number of patients meeting these clinical criteria will have RSV infection and will not benefit from antibiotic therapy. Effective management of pediatric LRTI in low-resource settings also requires determination of severity and prognosis to guide resource allocation, prioritize the sickest patients and prevent fatality.4 Although clinical characteristics and outcomes of children with RSV infection have been well documented in resource-intensive settings,5 fewer data are available from low-resource settings where access to viral diagnostics is limited.

Biomarkers of host response to infection may have clinical utility in distinguishing between infectious etiology and determining disease severity in LRTI.respiratory syncytial virus-associated pneumonia among children aged <5 years in the PERCH study. Clin Infect Dis. 2017;64(suppl 3):S378–S386.','400');" onMouseOut="javascript:ImageWrapperControl_ImageMouseOut();">6 Proteomic signatures from blood samples have been shown to provide rapid and actionable clinical information capable of guiding treatment decisions.7 Several candidate biomarkers of systemic inflammation have been examined in LRTI, including C-reactive protein (CRP),respiratory syncytial virus-associated pneumonia among children aged <5 years in the PERCH study. Clin Infect Dis. 2017;64(suppl 3):S378–S386.','400');" onMouseOut="javascript:ImageWrapperControl_ImageMouseOut();">6 chitinase-3-like protein 1 (CHI3L1)8 and lipocalin-2 (LCN2).9 While some studies have examined these biomarkers in pediatric LRTI in low-resource settings,10,11 none have examined their potential clinical utility in RSV patients or as predictors of clinical course and outcome.

The objective of this study was to describe admission characteristics, radiographic and laboratory abnormalities, inflammatory biomarker levels at admission, clinical course and outcomes of children hospitalized with RSV LRTI in Uganda. We compared cases with RSV LRTI to 2 control groups:1 rhinovirus respiratory tract infection (RTI) and2 suspected pneumococcal (Streptococcus pneumoniae, Sp) lobar pneumonia.

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MATERIALS AND METHODS

Study Design and Participants

We conducted a prospective study of children hospitalized with signs of pneumonia at 2 resource-limited hospitals in Uganda: Jinja Regional Referral Hospital and Kambuga District Hospital. Children were included if they:1 were under 13 years of age;2 required admission to hospital; and3 presented with cough or difficulty breathing and one or more of the following signs: tachypnea, chest indrawing and/or hypoxemia. Of note, all children included in this study met the World Health Organization and United Nations International Children’s Emergency Fund integrated management of childhood illness clinical definition of pneumonia.3 Children with clinically suspected tuberculosis were excluded.

Enrolled children underwent history and physical examination, had blood and nasopharyngeal (NP) swabs collected and were followed over the course of admission until discharge, death or transfer to another facility with frequent monitoring of vital signs. Cases of RSV LRTI were defined as patients meeting the above clinical inclusion criteria, plus detection of RSV from the nasopharynx by multiplex polymerase chain reaction (PCR). Two control groups were chosen:1 rhinovirus RTI, defined as patients meeting the clinical inclusion criteria, plus rhinovirus detected in the nasopharynx and absence of lobar consolidation on CXR; and2Sp pneumonia, defined as patients meeting the clinical inclusion criteria, plus lobar consolidation on CXR and Sp detected in the blood or at high genomic load (>6.9 log10 copies/mL) in the nasopharynx. Of note, high-density colonization with Sp in the nasopharynx in children with clinical pneumonia, above a threshold of 6.9 log10 copies/mL, has previously been associated with invasive pneumococcal infection.pneumonia among children aged <5 years in the PERCH study. Clin Infect Dis. 2017;64(suppl 3):S317–S327.','400');" onMouseOut="javascript:ImageWrapperControl_ImageMouseOut();">12

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Specimen Collection and Analysis

Staff were trained in correct procedures for flocked NP swab collection (FLOQSwabs, Copan Diagnostics, Murrieta, CA). NP swabs were placed into viral transport medium (UTM, Universal Transport Medium, Copan Diagnostics, Murrieta, CA) and 1-mL aliquots of viral transport medium were stored at −80°C before shipment to Canada. A semiautomated nucleic acid extraction protocol was used to perform nucleic acid extraction with a KingFisher mL Purification System (Thermo Fisher Scientific Inc, Waltham, Maria) and the MagaZorb Total RNA Mini-Prep Kit (Promega, Madison, WI). Quantitative real-time PCR (qPCR) with FTDResp33 (Fast-Track Diagnostics, Esch-sur-Alzet, Luxembourg) was used to identify respiratory pathogens in the NP swab. We ran these samples using an Applied Biosystems 7500 Real-Time PCR System (Applied Biosystems, Foster City, CA). To quantify the number of genome copies of Sp present in each sample, we used the standard curves relating the real-time polymerase chain reaction cycle threshold to plasmid DNA concentrations provided by the manufacturer.

Ethylenediaminetetraacetic acid plasma was collected in the field and stored at −80°C until shipment to Canada. ELISAs were performed according to the manufacturer’s instructions and blinded to all associated clinical data to quantify the following biomarkers: CRP, CHI3L1, LCN2 (R&D Systems, Minneapolis, MN). Dilutions for CRP ranged from 1:500,000 to 1:5000 to achieve an assay range of 500–0.16 μg/mL. Dilutions for CHI3L1 ranged from 1:62.5 to 1:1800 to achieve a range of 3600–2 nm/mL. Dilutions for LCN2 ranged from 1:20 to 1:200 to achieve a range of 500–1.6 ng/mL. Background signal was determined from blank wells on each plate and subtracted from all samples and standards prior to analysis. A 4-parameter logistic regression curve fitted to data was used to determine biomarker concentrations from the ELISA optical density.

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Statistical Analysis

Continuous data were presented as medians (interquartile range) and analyzed nonparametrically, with relationships assessed using the Mann-Whitney U test. Categorical data were analyzed using Pearson χ2 test. Time-to-event analysis was performed using Kaplan-Meier plots and log-rank test for differences between factor levels. Cases were right censored at the time of last encounter. Where multiple P-values were calculated, we used the Bonferroni correction for multiple statistical comparisons, to avoid family-wise inflation of type 1 statistical error.

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Ethics Statement

The study was approved by the School of Biomedical Sciences Research and Ethics Committee (Makerere University, Kampala, Uganda), the Uganda National Council of Science and Technology and the Human Research Ethics Board of the University of Alberta. Accompanying legal guardians provided informed written consent for participants at the time of enrollment.

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RESULTS

Fifty-eight children hospitalized with RTIs between February 25, 2014 and July 3, 2015 were included: 37 patients with RSV LRTI, 11 with rhinovirus RTI and 10 with Sp pneumonia. Table 1 shows clinical characteristics of the study cohort. Patients in the control groups were significantly older and weighed more than the children in the RSV case group. Codetection of multiple pathogens was common, such that 1 of 37 patients with RSV LRTI also met inclusion criteria for the rhinovirus control group, and 4 of 37 also met inclusion criteria for the Sp pneumonia control group. We accounted for this overlap in microbial classification in a subsequent sensitivity analysis.

TABLE 1

TABLE 1

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Clinical Course and Outcomes

Recovery times for clinically relevant endpoints are shown in Table 2. Of note, time to resolution of fever and tachypnea and time to feed were significantly prolonged in patients with Sp pneumonia compared with those with RSV LRTI.

TABLE 2

TABLE 2

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Host Inflammatory Biomarkers Distinguish RSV From Suspected Pneumococcal Pneumonia

Table 3 and Figure 1 show plasma concentrations of several host biomarkers. Among patients with RSV LRTI, inflammatory biomarkers were correlated with each other (CHI3L1 with LCN2 ρ = +0.82, P < 0.001; CHI3L1 with CRP ρ = +0.62, P < 0.001; LCN2 with CRP, ρ = +0.75, P < 0.001). Markers of systemic inflammation (CRP, CHI3L1 and LCN2) were significantly higher in the Sp pneumonia group than the RSV LRTI and rhinovirus RTI groups. Differences in CRP, CHI3L1, but not LCN2, remained statistically significant following correction for multiple comparisons. In linear regression models adjusting for the potentially confounding effect of age, Sp pneumonia remained independently associated with higher CRP (P = 0.002) and CHI3L1 (P = 0.034), relative to RSV LRTI. Some patients had multiple organisms detected simultaneously in the NP, such that one patient in the RSV LRTI group also fulfilled criteria for rhinovirus RTI, and 4 patients in the RSV LRTI group also fulfilled criteria for Sp pneumonia. We performed a sensitivity analysis, excluding those patients with overlapping microbial classifications, and found that differences in CRP and CHI3L1 between patients with RSV LRTI and Sp pneumonia remained statistically significant (P < 0.01 for both comparisons).

TABLE 3

TABLE 3

FIGURE 1

FIGURE 1

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Predictive Value of Inflammatory Biomarkers Among Children With RSV LRTI

Higher levels of CHI3L1 were associated with higher composite clinical severity scores (Respiratory Index of Severity in Children), r = 0.41, P = 0.019). Admission biomarker levels directly correlated with recovery times within the RSV LRTI group (n = 37): higher admission levels of CHI3L1 were associated with prolonged time to resolution of tachypnea and tachycardia, time to wean oxygen and time to sit (P < 0.05 for all comparisons). Similarly, higher admission levels of LCN2 at admission were associated with prolonged time to resolution of tachypnea, tachycardia and time to feed. In contrast, higher admission CRP levels were not significantly associated with any of these endpoints. Higher admission levels of CHI3L1, LCN2 and CRP, however, were all predictive of a higher total volume of oxygen administered over the course of hospitalization (P < 0.05 for all comparisons). Prognostic biomarkers are illustrated in Figure 2, with the cohort dichotomized according to median admission biomarker values (CHI3L1, 32 ng/mL; LCN2, 94 ng/mL and CRP, 18 μg/mL). The biomarkers outperformed clinical variables, including tachypnea and Respiratory Index of Severity in Children score, as predictors of recovery times. For full results see Table, Supplementary Digital Content, http://links.lww.com/INF/D473.

FIGURE 2

FIGURE 2

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DISCUSSION

In sub-Saharan Africa, RSV is commonly detected in children with severe acute respiratory illness13 and is a significant predictor of hospitalization.14 This study provided a unique examination of clinical features and host inflammatory biomarkers in Ugandan children hospitalized with RSV LRTI. Noteworthy, strengths of this study include the use of a sensitive molecular virology assay and host biomarker measurement in patients from a low-resource setting where such laboratory data are not usually available, and detailed longitudinal follow-up of hospitalized children. Our main findings included:1 RSV-infected children had significantly lower CRP and CHI3L1 levels compared with an Sp pneumonia control group; and2 admission levels of CHI3L1 and LCN2 were predictive of clinical outcomes in children with RSV LRTI.

The clinical characteristics of our cohort were broadly consistent with prior studies of children with RSV LRTI; however, children in our study had evidence of greater disease severity, including higher respiratory rates, lower oxygen saturation and a higher proportion with chest indrawing.15–17 Control children with Sp pneumonia were more frequently lethargic and tachycardic.

CRP is an acute phase protein produced in response to inflammatory cytokines and is widely used as a biomarker of systemic inflammation in clinical practice.18 We found that CRP was lower in patients with RSV LRTI compared with those with suspected Sp pneumonia, consistent with previous reports that demonstrated the diagnostic utility of CRP as a marker of serious bacterial infection.respiratory syncytial virus-associated pneumonia among children aged <5 years in the PERCH study. Clin Infect Dis. 2017;64(suppl 3):S378–S386.','400');" onMouseOut="javascript:ImageWrapperControl_ImageMouseOut();">6

We found that the admission levels of CHI3L1 were lower in children with RSV LRTI than Sp pneumonia, but similar between RSV LRTI and rhinovirus RTI. Furthermore, among children with RSV LRTI, higher levels of CHI3L1 and LCN2 predicted longer recovery times. CHI3L1 is a secreted glycoprotein expressed by immune and respiratory epithelial cells that promotes bacterial clearance and augments host tolerance to infection in pneumococcal pneumonia.8 In past studies, CHI3L1 was elevated in RSV respiratory infection19,20 and higher CHI3L1 was associated with radiographic consolidation in African children with clinical signs of pneumonia.11 LCN2 is a secreted transport protein produced by neutrophils in the respiratory mucosa that inhibits bacterial uptake of iron and propagates inflammation via interleukin-8-mediated induction of neutrophils.21 In previous studies, LCN2 distinguished between pneumonia etiology9 was associated with pneumonia severity9 and predicted poor outcomes in childhood pneumococcal pneumonia.10 Our findings build on these observations, suggesting that CHI3L1 and LCN2 are clinically informative biomarkers in childhood RSV LRTI in a low-resource setting. They may have utility as diagnostic markers to distinguish viral (RSV and rhinovirus) RTI from Sp pneumonia and/or prognostic markers of prolonged recovery time in children with RSV LRTI.

Several factors limit the validity and generalizability of these data. The statistical power of this study was restricted by a small sample size. Diagnostic workup was incomplete, because we did not have access to blood culture or lung aspirates at our low-resource hospital. Further studies would be needed to determine the clinical utility of these biomarkers used in real time for pneumonia triage and management. Codetection of multiple pathogens complicated the comparison between RSV LRTI cases and controls; however, we performed sensitivity analyses and demonstrated that our conclusions were robust to exclusion of patients with multiple codetected microorganisms.

Findings from the current study may have several implications. Protein biomarkers that could be adapted to a rapid test platform might be used to discriminate bacterial from viral etiology to help guide responsible antibiotic usage. Analogous to the HRP2 rapid diagnostic test for malaria, such a test could be inexpensive and achieve rapid penetration to clinical settings in low-resource settings. In sub-Saharan Africa, the combination of a high burden of communicable diseases and lack of diagnostic capacity is leading to increased rates of antimicrobial resistance,22 which may be combatted by limiting the use of antimicrobials for patients with viral illness.23 Second, prognostic biomarkers such as CHI3L1 and LCN2 may help to guide resource allocation and supportive management decisions. This finding is relevant for resource-scarce healthcare settings where triage to high-dependency units should prioritize cases of the greatest severity to prevent fatality. Finally, our study shows that high levels of CHI3L1 and LCN2 are associated with either bacterial infection or more severe viral infection but are not elevated in mild viral infection. In clinical practice, low levels of these biomarkers could be used to rule out serious infection and identify children with mild viral illness who could be managed as outpatients without antibiotics. Further studies to examine the utility of CHI3L1 and LCN2 in guiding clinical decision-making are warranted.

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Keywords:

respiratory syncytial virus; pneumonia; Africa; child; biomarkers

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