key: cord-0705359-kj9dkgz2
authors: Baggett, Henry C; Watson, Nora L; Deloria Knoll, Maria; Brooks, W Abdullah; Feikin, Daniel R; Hammitt, Laura L; Howie, Stephen R C; Kotloff, Karen L; Levine, Orin S; Madhi, Shabir A; Murdoch, David R; Scott, J Anthony G; Thea, Donald M; Antonio, Martin; Awori, Juliet O; Baillie, Vicky L; DeLuca, Andrea N; Driscoll, Amanda J; Duncan, Julie; Ebruke, Bernard E; Goswami, Doli; Higdon, Melissa M; Karron, Ruth A; Moore, David P; Morpeth, Susan C; Mulindwa, Justin M; Park, Daniel E; Paveenkittiporn, Wantana; Piralam, Barameht; Prosperi, Christine; Sow, Samba O; Tapia, Milagritos D; Zaman, Khalequ; Zeger, Scott L; O’Brien, Katherine L
title: Density of Upper Respiratory Colonization With Streptococcus pneumoniae and Its Role in the Diagnosis of Pneumococcal Pneumonia Among Children Aged <5 Years in the PERCH Study
date: 2017-05-27
journal: Clinical Infectious Diseases
DOI: 10.1093/cid/cix100
sha: a5c17c1af17e4ff8b75ae721ad2b89286dafdc32
doc_id: 705359
cord_uid: kj9dkgz2

BACKGROUND: Previous studies suggested an association between upper airway pneumococcal colonization density and pneumococcal pneumonia, but data in children are limited. Using data from the Pneumonia Etiology Research for Child Health (PERCH) study, we assessed this potential association. METHODS: PERCH is a case-control study in 7 countries: Bangladesh, The Gambia, Kenya, Mali, South Africa, Thailand, and Zambia. Cases were children aged 1–59 months hospitalized with World Health Organization–defined severe or very severe pneumonia. Controls were randomly selected from the community. Microbiologically confirmed pneumococcal pneumonia (MCPP) was confirmed by detection of pneumococcus in a relevant normally sterile body fluid. Colonization density was calculated with quantitative polymerase chain reaction analysis of nasopharyngeal/oropharyngeal specimens. RESULTS: Median colonization density among 56 cases with MCPP (MCPP cases; 17.28 × 10(6) copies/mL) exceeded that of cases without MCPP (non-MCPP cases; 0.75 × 10(6)) and controls (0.60 × 10(6)) (each P < .001). The optimal density for discriminating MCPP cases from controls using the Youden index was >6.9 log(10) copies/mL; overall, the sensitivity was 64% and the specificity 92%, with variable performance by site. The threshold was lower (≥4.4 log(10) copies/mL) when MCPP cases were distinguished from controls who received antibiotics before specimen collection. Among the 4035 non-MCPP cases, 500 (12%) had pneumococcal colonization density >6.9 log(10) copies/mL; above this cutoff was associated with alveolar consolidation at chest radiography, very severe pneumonia, oxygen saturation <92%, C-reactive protein ≥40 mg/L, and lack of antibiotic pretreatment (all P< .001). CONCLUSIONS: Pneumococcal colonization density >6.9 log(10) copies/mL was strongly associated with MCPP and could be used to improve estimates of pneumococcal pneumonia prevalence in childhood pneumonia studies. Our findings do not support its use for individual diagnosis in a clinical setting.

High density of pneumococcal colonization (ie, high bacterial density in the nasopharynx) has been proposed as a more sensitive marker for pneumococcal pneumonia than blood culture [7] .

Previous studies, mostly in adults, have demonstrated an association between the density of pneumococcal colonization and pneumococcal pneumonia [8] [9] [10] [11] . Several other studies among children evaluated the use of pneumococcal colonization density as a marker of pneumococcal pneumonia [12] [13] [14] , but these studies used surrogate end points (ie, radiographic pneumonia) for true pneumococcal pneumonia rather than confirmed pneumococcal pneumonia cases for the density evaluation. Although these studies suggest the use of pneumococcal nasopharyngeal (NP) density as a potential diagnostic tool for pneumococcal pneumonia, additional data among children are needed to confirm the association and identify a density threshold with acceptable diagnostic accuracy. Therefore, we evaluated the utility of upper respiratory tract colonization density as a diagnostic tool for pneumococcal pneumonia in a large study of childhood pneumonia.

The Pneumonia Etiology Research for Child Health (PERCH) study is a multicountry, standardized case-control evaluation of the etiologic agents causing severe and very severe pneumonia among children in developing countries [15] . Enrollment occurred for 24 months between August 2011 and January 2014 at each of 9 study sites in 7 countries: Dhaka and Matlab, Bangladesh; Basse, The Gambia; Kilifi, Kenya; Bamako, Mali; Soweto, South Africa; Nakhon Phanom and Sa Kaeo, Thailand; and Lusaka, Zambia. Identification and selection of cases and controls have been described elsewhere [16] .

Cases were hospitalized children aged 1-59 months with World Health Organization-defined severe or very severe pneumonia [17] . Severe pneumonia was defined as the presence of cough or difficulty breathing and lower chest wall indrawing; very severe pneumonia, as cough or difficulty breathing and ≥1 of the following: central cyanosis, difficulty breastfeeding/ drinking, vomiting everything, convulsions, lethargy, unconsciousness, or head nodding. Exclusion criteria for cases were hospitalization within the previous 14 days, discharged as a PERCH case within the past 30 days, residence outside the study catchment area, or resolution of lower chest wall indrawing after bronchodilator therapy for children with wheezing.

Controls were randomly selected children from the community without severe or very severe pneumonia, were enrolled year round, and were frequency matched to cases by age group [16] . Controls were also matched for human immunodeficiency virus (HIV) status at the 2 sites (Zambia and South Africa) with high HIV prevalence. Controls with acute respiratory illness or other mild illnesses were included only if they did not have severe or very severe pneumonia.

Pneumococcal conjugate vaccine (PCV) was in use for the entire enrollment period in The Gambia, Kenya, Mali, and South Africa. PCV was introduced in July 2013 in Zambia, 18 months after enrollment started. In Bangladesh and Thailand, PCV was available only on the private market during the study period with almost no usage in the study areas.

All laboratory methods were standardized across sites [18] . A flocked NP swab (flexible minitip; Copan) and a rayon oropharyngeal (OP) swab specimen were collected from each case and control and were placed into the same vial. The NP/OP specimen was tested for pneumococcus (lytA gene target) as part of a multiplex real-time polymerase chain reaction (PCR) assay (FTD Respiratory Pathogens 33; Fast-track Diagnostics) performed using an Applied Biosystems 7500 (ABI-7500) platform. Standard curves for quantification were generated on an approximately 3-monthly basis and were used to calculate pathogen density (in copies per milliliter) from the sample cycle threshold values. Densities <10 4 or >10 8 copies/mL were outside the linear range of the PCR assay, limiting precise density estimation.

A second NP specimen for S. pneumoniae culture was collected simultaneously with the first swab specimen; pneumococcal isolates were serotyped using Quellung reaction or latex agglutination, as described elsewhere [18] . Testing was performed at each site, and all sites participated in external quality assurance programs for both pneumococcal PCR and serotyping [18] .

Cases, but not controls, had blood collected for culture. Some sites (Bangladesh, The Gambia, Mali, and South Africa) collected lung aspirates from children with consolidation on chest radiographs (CXRs) who met clinical and radiologic criteria for the procedure [19] . Pleural fluid was collected from cases when clinically indicated. Lung aspirate and pleural fluid specimens were tested for pneumococcus by means of culture and PCR; pleural fluid was also tested for pneumococcal antigen (Binax NOW; Alere).

Antibiotic pre-exposure was defined as either a positive serum bioassay result (cases and controls) or documentation of antibiotics administered at the referral or study hospital before specimen collection (cases only) [20] . Microbiologically confirmed pneumococcal pneumonia (MCPP) was defined, in PERCH cases, as detection of pneumococcus from a culture of blood, lung aspirate, or pleural fluid; by PCR of lung aspirate or pleural fluid; or by detection of pneumococcal antigen in pleural fluid. A control was considered to have a respiratory tract illness (RTI) if cough or runny nose were reported. RTI was also considered present if a child had (1) ear discharge, wheezing, or difficulty breathing and (2) either fever (temperature ≥38.0°C or reported fever in the past 48 hours) or sore throat.

CXRs were obtained at admission for cases, and each digital image was assessed by 2 members of a panel of 14 radiologists and pediatricians trained in the standardized interpretation of pediatric CXRs; films with discordant conclusions were adjudicated [21, 22] . Clinical characteristics, including oxygen saturation, were assessed on the day of enrollment. Case mortality was assessed at hospital discharge and by contact 30 days after discharge.

Demographic, clinical and laboratory characteristics were compared by subject group using the χ 2 test. Median pneumococcal colonization density was compared across groups with the Kruskal-Wallis test. Density histograms and comparisons by subject group were repeated among strata defined by antibiotic exposure before NP/OP specimen collection.

An optimal density threshold for discriminating cases with MCPP (MCPP cases) from all controls was identified using the Youden index [23] . The optimal density threshold was also calculated for MCPP cases versus the subset of controls without RTI (non-RTI controls), and among children who were HIV negative. To guard against bias in the estimates of sensitivity owing to a small number of MCPP cases, the Youden index was calculated using leave-one-out cross-validation. To characterize a potential trend in risk associated with increasing pneumococcal density, we used logistic regression models adjusted for age, sex, and site to evaluate associations of pneumococcal density categories with clinical and CXR indicators of pneumonia, and with case severity measures.

To evaluate whether elevated colonization density may identify cases with pneumococcal pneumonia among those without MCPP, we compared known clinical and laboratory correlates of bacterial pneumonia among cases without MCPP (non-MCPP cases) with colonization density above versus below the identified optimal threshold. The association of elevated pneumococcal colonization density with known correlates of pneumonia was evaluated using separate logistic regression models of density above versus below the threshold as a predictor of each characteristic, with adjustment for age, sex, and site. Analyses were repeated to extend comparison of characteristics among non-MCPP cases with density above the threshold versus all MCPP cases, and among MCPP cases above versus below the threshold.

The PERCH study protocol was approved by the institutional review board or ethical review committee at each of the study site institutions and at The Johns Hopkins Bloomberg School of Public Health. Parents or guardians of all participants provided written informed consent.

Of 4232 cases enrolled in the PERCH study, 4136 had available S. pneumoniae colonization and density data. Of those, data on MCPP status were available on 4091 cases (56 MCPP and 4035 non-MCPP cases); 45 cases were excluded owing to missing data required to define MCPP status. Of 5325 controls, the analysis included 1226 controls with and 3962 without RTI in whom S. pneumoniae colonization and density were measured by PCR analysis of the NP/OP specimen. An additional 3 MCPP cases, 82 non-MCPP cases, 11 cases with unknown MCPP status, and 137 controls did not have analyzable NP/OP PCR results because of missing or insufficient samples (2.3%).

Among the 56 MCPP cases, 21% were aged 1-5 months, 23% were 6-11 months, 30% were 12-23 months, and 25% were 24-59 months; 52% were male. Age and sex distribution were similar across MCPP, non-MCPP, and control groups (mean age, 14 months), except that a higher proportion of non-MCPP cases (41%) were aged <6 months compared with MCPP cases (21%). Cases with MCPP were identified at all 5 African sites (15 in The Gambia, 5 in Kenya, 24 in Mali, 5 in South Africa, and 7 in and Zambia) but at neither of the 2 Asian sites (Bangladesh and Thailand) ( , and were more likely to be HIV infected (23.2%) than non-MCPP cases (5.6%) (P ≤ .01 for each). Non-MCPP cases were more likely than those with MCPP to have received antibiotics before NP/OP specimen collection (46% vs 29%; P < .01). Antibiotic use before NP/ OP specimen collection occurred in 3 of 14 MCPP cases in The Gambia (data missing for 1), 2 of 5 in Kenya, 3 of 24 in Mali, 5 of 5 in South Africa and 3 of 6 in Zambia (data missing for 1).

Among children who had a positive density value, median S. pneumoniae colonization density was highest in MCPP cases (17.28 × 10 6 copies/mL) relative to non-MCPP cases (0.75 × 10 6 ) and controls (0.60 × 10 6 ) (P < .001 for each) (Table 1) . However, in South Africa, the only site where all MCPP cases had received prior antibiotics, MCPP cases had lower median density (0.25 × 10 6 ) than both non-MCPP cases (0.70 × 10 6 ) and controls (0.77 × 10 6 ), although differences were not statistically significant. For each case and control group, the median colonization density was lower in children with prior antibiotic use than in those without, and lower in those with NP culture negative versus positive for S. pneumoniae (Table 1) .

Density among MCPP cases varied by site (Table 1 and Figure 1 ; P < .001); median density differed by >100-fold between the site with the highest density, Mali (35.81 × 10 6 copies/mL), which that also had the highest proportion of MCPP cases (3.6%), and the sites with the lowest density, Kenya and South Africa (0.35 and 0.25 × 10 6 copies/mL), both with 5 MCPP cases (<0.2%). Among non-MCPP cases and controls, density distributions were similar across sites ( Figure  1 ). Median densities were lowest in Thailand in all groups. The all-site density distribution curves were shifted toward higher densities in MCPP cases versus controls, but the distributions of these groups overlapped substantially (Figure 2 ). The colonization density distribution among MCPP cases pretreated with antibiotics was shifted toward lower densities compared with MCPP cases without antibiotics before NP specimen collection. The optimal colonization density threshold for discriminating MCPP cases from controls was >6.9 log 10 copies/mL (sensitivity, 64.3%; specificity, 92.2%; age-, sex-, and site-adjusted odds ratio, 17.9 [95% confidence interval 9.9-32.4]). The threshold was unchanged when restricted to controls without RTI and when limiting the comparison to HIV-negative children. When restricted to those MCPP cases (n = 40) and controls (n = 5074) without prior use of antibiotics, the optimal threshold was 6.6 log 10 copies/mL (sensitivity, 77.5%; specificity, 85.3%), and it was 4.4 log 10 copies/mL when restricted to MCPP cases (n = 16) and controls (n = 114) exposed to antibiotics (sensitivity, 100%; specificity, 52.6%).

The proportion of cases and controls with densities >6.9 log 10 copies/mL among those positive varied by site (Figure 3) , sex, HIV status, antibiotic pre-exposure, and pneumococcal culture positivity ( Table 2 ). The proportion of MCPP cases with density >6.9 log 10 copies/mL ranged from 0 of 5 in Kenya to 21 of 24 (87.5%) in Mali. Across sites, this proportion was lower among MCPP cases who received antibiotic pretreatment than in those who did not (P = .04). The proportion of controls with density >6.9 log 10 copies/mL ranged from 1.2% in Thailand to 15.6% in Mali.

Among all PERCH cases, high colonization density was associated with clinical and severity measures considered suggestive of bacterial pneumonia (Table 3) . Increasing density was associated in a dose-dependent manner with very severe pneumonia, white blood cell count >15/μL, C-reactive protein (CRP) ≥ 40 mg/L. and coinfection with any virus for which testing was performed. CXRconfirmed pneumonia, consolidation on CXR, HIV infection, oxygen saturation <92% with room air, and respiratory syncytial virus coinfection were all associated with density >6.9 log 10 copies/ mL, but without clear evidence of increasing strength of association with increasing densities.

Compared with MCPP cases with density ≤6.9 log 10 copies/ mL, those with density >6.9 log 10 copies/mL had higher frequencies of very severe pneumonia and fatal outcome, and lower frequencies of prior antibiotic use, CXR-confirmed pneumonia, and consolidation on CXR (Table 4 ), but these differences were not statistically significant. Among non-MCPP cases, those with density >6.9 log 10 copies/mL (n = 500; 12.4%) were more likely than those below the threshold to have very severe pneumonia, CXR-confirmed pneumonia, consolidation on CXR, oxygen saturation <92%, HIV infection, CRP ≥40 mg/L, or any virus coinfection, and they were less likely to have been previously treated with antibiotics. MCPP cases, regardless of colonization density, were similar to non-MCPP cases with density >6.9 log 10 copies/mL, for frequency of elevated white blood cell count, oxygen saturation <92%, prior antibiotic use, or any virus coinfection, but they were more likely to be HIV positive or to have very severe pneumonia, CXR-confirmed pneumonia, alveolar consolidation on CXR, CRP ≥40 mg/L, or fatal outcomes, after adjustment for age, sex, and site.

The serotype of the invasive pneumococcal isolate was available for 46 (98%) of 47 culture-positive MCPP cases, and that of the NP isolate was available for all 44 NP culture-positive MCPP cases. One MCPP case infected with serotype 18C, although NP culture-positive, was PCR negative for pneumococcus, so density could not be determined. Of 43 with serotype data for both the NP and invasive isolate and PCR data, 32 (72.7%) had matching invasive and NP serotypes, with 18 serotypes represented, including both vaccine and nonvaccine serotypes (Figure 4) . Although the number of MCPP cases with each identified serotype was small (1-4 per serotype), the distribution of colonization densities seemed similar by serotype. However, the 2 MCPP cases infected with serotype 13 and serotype 14 had colonization densities ≤6.9 log 10 copies/ mL; neither had received prior antibiotics. For serotypes identified in ≥10 controls, the percentages of controls with density >6.9 log 10 copies/mL were similar across serotypes and ranged from 2.3% to 15.6% (Figure 4 ), equivalent to 84.4% to 97.7% specificity.

In the PERCH study, pneumococcal colonization density was significantly higher among children with MCPP than among other pneumonia cases or community controls. The strength of the association increased with increasing colonization density, with an optimal density threshold of >6.9 log 10 copies/mL (64% sensitivity, 92% specificity) to distinguish MCPP cases from controls, but performance varied by site. The optimal threshold was lower (≥4.4 log 10 copies/mL; 100% sensitivity, 52.6% specificity) for children treated with antibiotics before specimen collection. Pneumococcal colonization density was associated in a dose-dependent manner with characteristics regarded as suggestive of bacterial pneumonia (alveolar consolidation on CXR, very severe pneumonia, and elevated CRP levels).

Pneumococcal colonization density was also was found to divide PERCH cases along a spectrum of disease severity from MCPP cases; MCPP cases with density >6.9 log 10 copies/mL had the greatest proportion with very severe pneumonia and Figure 3 . Percentage of children with nasopharyngeal/oropharyngeal pneumococcal colonization density >6.9 log 10 copies/mL among positives, by site and case and control group; density was calculated by means of polymerase chain reaction for the lytA gene performed on nasopharyngeal/oropharyngeal specimens. Numbers above bars represent the number of microbiologically confirmed pneumococcal pneumonia (MCPP) cases at the site. RTI, respiratory tract illness. fatal outcomes, followed by MCPP cases with density ≤6.9 log 10 , non-MCPP cases with density >6.9 log 10 , and non-MCPP cases ≤6.9 log 10 , who had the lowest proportion with these characteristics. The association of colonization density with disease severity was observed in a previous study among HIV-infected adults with pneumonia in South Africa [24] but has not been reported among children.

Viral infections, especially influenza, have previously been associated with pneumococcal pneumonia and invasive pneumococcal disease in human studies [25, 26] and animal models [27, 28] . We found that high pneumococcal colonization density was associated with virus detection in the upper respiratory tract, and this finding was explained in part by respiratory syncytial virus coinfection. This finding may indicate that upper respiratory infection with viral pathogens enhanced the density of pneumococcal colonization, but it does not directly address whether these copathogen infections are themselves related to the lower respiratory tract disease. Our finding is consistent with a recent study in South Africa among hospitalized adults and children with acute lower respiratory tract infection (LRTI), which showed that pneumococcal colonization density was associated with the presence of respiratory viruses [10] . In a case-control study such as the PERCH study, we cannot assess the potential causal role of viral infection increasing pneumococcal density or even whether viral infection preceded pneumococcal colonization. Longitudinal cohort studies, such as the Drakenstein study [29] , are more suited to address this question.

Our findings in children are similar to the reported association between pneumococcal colonization density and confirmed pneumococcal pneumonia in adults [8, 9, 11, 30] . Studies among children have found that higher colonization density was associated with alveolar consolidation on CXR [12] [13] [14] , a proxy for pneumococcal pneumonia. In a study among 550 children hospitalized with LRTI in Vietnam [12] , cases with consolidation on CXR had higher median NP pneumococcal density at PCR (6.9 log 10 copies/mL) than others with LRTI (6.1 log 10 copies/mL) and community controls (5.9 log 10 copies/mL). These studies did not identify a colonization density threshold that reliably predicted radiographically confirmed pneumonia. Abbreviations: HIV, human immunodeficiency virus; MCPP, microbiologically confirmed pneumococcal pneumonia; NP, nasopharyngeal; OP, oropharyngeal; PCR, polymerase chain reaction for lytA gene; PCV, pneumococcal conjugate vaccine; RTI, respiratory tract illness. a Pneumococcal colonization density calculated by PCR for the lytA gene performed on NP/OP specimens in PCR-positive children.

b P < .05 for comparison of proportion with pneumococcal colonization density ≥6.9 log 10 copies/mL by sex (non-MCPP case group), HIV (non-MCPP case group, all controls, and non-RTI controls), prior antibiotic use (non-MCPP case group), and NP culture positive (MCPP and non-MCPP case groups, all controls, RTI controls, and non-RTI controls). c PCV vaccinated was defined as ≥1 dose (restricted to Kenya, Gambia, Mali, and South Africa).

A study among children and adults hospitalized with acute LRTI in South Africa found that invasive pneumococcal pneumonia was associated with increased colonization density; cases with density >1000 copies/mL had 18 times greater odds of invasive pneumococcal pneumonia than colonized cases with density <1000 copies/mL [10] . The South African study defined invasive pneumococcal pneumonia by detection of S. pneumoniae by PCR in the blood, a diagnostic not used in our study owing to poor specificity [31, 32] . We found that the best-performing threshold (6.9 log 10 [10 6.9 ] copies/mL) was much higher than that suggested by the South African study, but comparison of density thresholds between studies is limited by methodologic differences.

The association of pneumococcal colonization density with MCPP does not indicate its utility for patient care. Even in a population with a relatively high prevalence of pneumococcal disease (eg, children hospitalized with pneumonia), the positive predictive value would probably be too low to influence clinical decision making. In settings with lower pneumococcal disease prevalence (eg, countries using PCV), the positive predictive value would be even lower. Although the negative predictive value may be relatively high, it would not be high enough to justify withholding antibiotics in hospitalized children with clinical or radiographic evidence suggestive of bacterial pneumonia. Furthermore, to be useful in a clinical setting, local data on the pneumococcal colonization density distribution would be needed, and patient assessment would have to account for antibiotic pretreatment.

Although our findings are strengthened by the large study size, 7 country sites, and systematic enrollment of well-characterized cases and controls using standardized clinical criteria and laboratory procedures, there were limitations. The number of MCPP cases limited stratified analyses by study site and pneumococcal serotype and prevented calculation of site-specific density thresholds. The findings were largely driven by cases from the 3 sites with the most MCPP cases (The Gambia, Mali, and Zambia). Despite previous evidence of substantial pneumococcal disease burden in children in Bangladesh [33, 34] and Thailand [35, 36] , no MCPP cases were identified among enrolled PERCH cases in either of those sites, limiting the evaluation of this threshold at those sites. However, Bangladesh and Thailand did have cases with colonization density above the threshold, the proportion of which in Bangladesh exceeded that in Kenya and Zambia.

The association between pneumococcal pneumonia and colonization density was derived using MCPP cases, but the potential application as a diagnostic assay would be most important to identify cases without pneumococcal detection from blood or other sterile body fluid, which represent the majority of cases with pneumococcal pneumonia [6] . Therefore, the sensitivity of the 6.9 log 10 copies/mL threshold for detecting pneumococcal pneumonia may be lower than we estimated based on the MCPP cases. Finally, our study design did not allow assessment of the temporal relationship of colonization density with MCPP. Our analysis aimed not to assess causality but rather to identify a diagnostic adjunct to improve pneumococcal case detection over detection from invasive specimens alone. In addition to study limitations, there are limitations inherent to the measurement of pneumococcal colonization density. Although the PERCH study made great efforts to standardize specimen collection [37] there was no way to standardize the specimen volume taken from the NP/OP space. Higher specimen volume resulting from, for example, coryza could increase measured colonization density. Our findings provide strong evidence for the relationship between pneumococcal colonization density and pneumococcal pneumonia in children. Pneumococcal colonization density seems to improve detection of pneumococcal pneumonia beyond blood culture, which though highly specific, is insensitive and available only in settings with good microbiology 

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Acknowledgments. We offer sincere thanks to the patients and families who participated in this study. We recognize the efforts of the following groups during the development, study conduct, and analysis phases (see Supplemental Materials