Between August 2011 and January 2014, each site enrolled children 1–59 months old into PERCH during 24 consecutive months. Study sites included Dhaka and Matlab, Bangladesh; Basse, The Gambia; Kilifi, Kenya; Bamako, Mali; Soweto, South Africa; Sa Kaeo and Nakhon Phanom, Thailand; and Lusaka, Zambia. Sites, selected to represent pneumonia epidemiology in low- and middle-income settings, contained a mix of urban and rural populations, high and low human immunodeficiency virus (HIV) prevalence rates, and varying infant mortality [4]. Identification and enrollment of cases hospitalized with severe and very severe pneumonia have been described [5]. Severe pneumonia was defined as presence of cough or difficulty breathing plus lower chest wall indrawing; very severe pneumonia was defined as cough or difficulty breathing, plus at least 1 danger sign (ie, central cyanosis, difficulty breastfeeding/drinking, vomiting everything, convulsions, lethargy, reduced consciousness, or head nodding) [6]. Cases were excluded if they were hospitalized within the previous 14 days, discharged as a PERCH case within the past 30 days, did not reside in the study catchment area, or recovered from lower chest wall indrawing following bronchodilator therapy for those with wheeze.

Control children were enrolled year round, randomly selected from the community serving as the catchment area for cases, and frequency-matched to cases by enrollment date and age within the following strata: 28 days to 5 months, 6–11 months, 12–23 months, and 24–59 months, as described [5]. Those found to have WHO-defined severe or very severe pneumonia were ineligible and referred for medical care; those with nonsevere respiratory illness were eligible for enrollment.

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

Cases and controls underwent a standardized clinical assessment that included review of pertinent medical history, respiratory findings, WHO danger signs, comorbidities, and possible risk factors (eg, lack of immunization and breastfeeding). Height, weight, and pulse oximetry were measured during a focused physical examination. Thirty days after enrollment, cases were reevaluated to ascertain vital status and clinical outcomes. We defined underweight as weight for age at enrollment <−2 SD of the median age- and sex-specific WHO reference; prematurity as gestational age <37 weeks by parental report; low birth weight as <2.5 kg or small size at birth by parental report; HIV-infected as either positive virological test or HIV seropositive if >12 months old. We defined HIV uninfected-exposed as an infant or child with a negative virologic test for HIV who had evidence of HIV exposure, defined as HIV seropositive (if <12 months of age), or seronegative with a maternal history of HIV infection (for all ages), with the caveat that maternal exposure must be confirmed by maternal serology for seronegative infants aged <7 months.

Pertussis vaccine administration dates were collected from vaccination cards, when available, or by maternal history when the card was unavailable. Whole-cell pertussis vaccine was used at all sites except South Africa where acellular pertussis vaccine was used. The recommended schedule was 6, 10, and 14 weeks at all sites except The Gambia (2, 3, and 4 months) and Thailand (2, 4, and 6 months), with a booster at 18 months in South Africa (Supplementary Table 1A). Vaccination status was determined by the number of age-appropriate vaccine doses that were received at least 2 weeks before enrollment or 2 weeks before cough onset (for children with cough); the child was deemed undervaccinated if fewer doses were received than recommended for age, and unvaccinated if no doses were received in this time frame. Children were considered too young for vaccination if they were unvaccinated and were enrolled or had onset of cough before 8 weeks (5 sites) or 10 weeks (The Gambia and Thailand) of age. The child's age used in this definition was based on the child's age 2 weeks before enrollment or 2 weeks before cough onset (for children with cough).

Chest radiographs (CXRs) were performed on cases and were read and adjudicated according to WHO-standardized interpretation procedures by trained readers who were blinded to the clinical or laboratory findings [7]. CXR positivity was defined as consolidation and/or other infiltrate.

A blood sample was collected for a complete blood count (cases only) and HIV antibody testing (cases [all sites except Bangladesh] and controls [at all sites except Thailand, Bangladesh, and The Gambia]). Using standardized methods, induced sputum (IS) was collected from cases only, and a flocked nasopharyngeal (NP) swab (flexible minitip, Copan) and a rayon oropharyngeal (OP) swab specimen were collected from each case and control; the NP and OP swabs from each child were placed into the same vial with 3 mL of universal transport media (UTM; Copan) [8, 9].

NP/OP and IS specimens were tested in-country by multiplex polymerase chain reaction (PCR) for 33 pathogens, including the B. pertussis insertion sequence IS481 (FTD Resp 33, Fast-track Diagnostics, Sliema, Malta) [10]. When >1 IS specimen was available from a child, only the first was included; results were considered analyzable regardless of sputum quality indicators such as density of epithelial cells by microscopy [11]. Standard PCR curves were generated every 3 months during the testing phase to calculate pathogen load from PCR cycle threshold values. All samples positive for B. pertussis by multiplex PCR underwent uniplex B. pertussis IS481 and Bordetella holmesii recA PCR assays at Canterbury Health Laboratories (Christchurch, New Zealand) [12]. Children with at least 1 sample that tested positive for B. pertussis IS481 and negative for B. holmesii recA were regarded as having pertussis infection.

Data collected at each site were entered into an electronic data capture system maintained by the Data Coordinating Center (Emmes Corporation, Rockville, Maryland). To be analyzable as a case or a control in this analysis, at least 1 respiratory sample had to be tested. The Kruskal–Wallis test was used to compare quantitative PCR data between pertussis-positive cases and controls to evaluate whether higher median density was associated with clinical infection. To compare continuous and binary characteristics between pertussis-positive and -negative cases and controls, as applicable, multiple logistic regression adjusted for site, and age where appropriate, was used. Analyses involving case fatality ratios were restricted to sites with pertussis-positive cases. Two-sided P values <.05 were considered significant. Analyses were performed using Stata software version 12.1 and SAS version 9.4.

A total of 4232 cases and 5325 control children were enrolled in PERCH during the 24-month study period at each site; data from 4200 cases (99.2%) and 5196 controls (97.6%) were considered analyzable for pertussis. Pertussis was detected by PCR in 53 cases (1.3%; range by site, 0%–2.5%) and 11 control children (0.2%; range by site, 0%–0.6%) (adjusted odds ratio [aOR] for cases vs controls: 5.1; 95% confidence interval [CI], 2.6–9.8; P < .01) (Table 1). An additional 6 cases and 5 controls had samples that were PCR positive for B. holmesii, and were not included in this analysis. Pertussis-positive cases were detected at all sites except Thailand; positive controls were found in Mali, South Africa, and Thailand. The preponderance of pertussis cases (52/53 [98.1%]) and controls (8/11 [72.7%]) came from Africa. Adjusting for differences in the age distribution of cases, continent (Africa vs Asia: aOR, 8.8; P = .03) was significantly associated with pertussis positivity. Within Africa, site was not a significant determinant of positivity. Among the 300 PERCH cases 1–59 months of age who died in-hospital and had pertussis testing at all sites combined, 8 (2.7%) tested positive for pertussis. Table 1.

Study Participants Enrolled and Tested for Bordetella pertussis, Percentage Positive by Age and Site, and Case Fatality Ratio Among Pertussis-Positive Cases

Cases 1–5 months of age were significantly more likely to be pertussis positive compared with cases aged 6–59 months (2.3% vs 0.5%; aOR, 4.4; 95% CI, 2.4–8.4; P < .01); similarly, controls aged 1–5 months were more likely to be pertussis positive than their 6- to 59-month-old counterparts (0.5% vs 0.1%; aOR, 5.9; 95% CI, 1.6–22.3; P = .01) (Table 1). Among the 43 pertussis-positive cases with both NP/OP and IS specimens available, 14 were positive by IS but not NP/OP (32.6%), whereas 2 were positive in the NP/OP but not IS (4.7%); 27 (62.8%) were positive by both. Similar results were seen when restricting to cases aged 1–5 months and when restricting to children with high-quality IS specimen (defined as <10 squamous epithelial cells per low-power field) [11]. Figure 1.

Cumulative age distribution of children with severe or very severe pneumonia by pertussis infection status as determined by polymerase chain reaction (PCR) results. A, Children with a positive PCR result for pertussis (n = 53). B, Children whose PCR results were negative (n = 4120). Pertussis-positive children were significantly younger than pertussis-negative children (median, 2 vs 7 months; P = .01, logistic regression adjusted for site, restricted to sites in Africa and Bangladesh where positive cases were found).

No pertussis-positive cases aged 1–5 months were identified at the 2 Asian sites where the proportion of controls undervaccinated for age was lower (12.5%) compared with African sites (23.5%; P < .0001; Table 2). Thus, to identify potential risk factors for pertussis-associated hospitalized severe and very severe pneumonia, we compared pertussis-positive cases to controls (regardless of pertussis status) only at African sites. Pertussis-positive cases were significantly younger than controls (mean, 10.3 vs 15.8 weeks; P < .01) and were more likely to be HIV uninfected-exposed (31.6% vs 15.2%; aOR, 2.2; 95% CI, .99–4.76; P = .053), underweight (35.0% vs 7.6%; aOR, 6.3; 95% CI, 3.0–13.0; P < .01), unvaccinated (66.7% vs 21.5%; aOR, 3.7; 95% CI, 1.5–9.2; P < .01), and too young to be immunized (66.7% vs 14.8%; aOR, 16.1; 95% CI, 4.1–62.6; P < .001) compared to controls (Table 2). Most (9/14 [64.3%]) of the underweight children were considered either premature or low birth weight; however, the association between underweight and pertussis case status remained significant after adjusting for prematurity and low birth weight. Pertussis-positive cases were more likely to have never been breastfed compared with controls (25.0% vs 9.0%; aOR, 2.5; 95% CI, 1.0–6.0; P = .05), but this association was no longer significant when HIV infection/exposure was included in the model (OR, 1.82; P = .24). Most pertussis-positive cases (26/40 [66.7%]) were unvaccinated, all because they were age-ineligible 2 weeks before cough onset (Table 2). Pertussis-positive cases had a trend toward higher median density of B. pertussis than pertussis-positive controls (P = .07; Table 3; Supplementary Figure 1). Table 2.

Characteristics of Cases and Controls Aged 1–5 Months by Pertussis Status

We also performed case-case comparisons at the African sites to identify factors that might distinguish pertussis-positive from negative cases of severe or very severe pneumonia. Compared to cases testing negative, pertussis-positive cases were significantly younger, more likely to be unvaccinated (66.7% vs 39.8%; aOR, 2.8; P = .02; Figure 1; Table 2) and more likely to have findings classically associated with pertussis disease (prolonged cough longer than 7 or 14 days, no audible wheeze, vomiting, leukocytosis ≥20 000 cells/µL, and lymphocytosis ≥10 000 cells/µL; Table 3). Fifty-nine percent of pertussis-positive cases and 47.0% of pertussis-negative cases had radiographic pneumonia (P = .23).

A small fraction (1.1% [8/701]) of controls aged 1–5 months in Mali, South Africa, and Thailand tested positive for pertussis; none were detected at the other sites. Compared to pertussis-negative controls at those sites in this age strata, pertussis-positive controls were significantly more likely to have fever, vomiting, or tachypnea (all P < .05; Table 3; Supplementary Table 2), conceivably manifestations of clinically mild pertussis.

The in-hospital case fatality ratio of pertussis-positive cases aged 1–5 months (12.5%; 95% CI, 4.2%–26.8% [5/40]) was not significantly different than that of pertussis-negative cases (8.8%; 95% CI, 7.4%–10.3% [132/1507]; P = .43). Deaths of pertussis-positive cases occurred in Zambia, Mali, and South Africa (Table 1). Among cases discharged alive, 30-day vital status was available for 60.0% (21/35) of pertussis-positive (with no additional deaths documented) compared with 84.6% of 1375 pertussis-negative cases. A sensitivity analysis that takes into consideration cases not tested for B. pertussis and the possible outcomes of cases who did not complete a 30-day follow-up visit is included in the Supplementary Appendix.

The 5 pertussis-positive deaths accounted for 3.7% of the 137 in-hospital PERCH deaths at the 5 African sites among infants 1–5 months of age who were tested for pertussis. All 5 fatal cases had an infiltrate on CXR, elevated white blood cell (WBC) count (mean, 50.2 × 10³ cells/µL; range, 14.2–70.0 × 10³ cells/µL), including 4 cases with WBC > 40 × 10³ cells/µL; most were very young (mean age, 13.0 weeks; range, 7–25 weeks), and 1 was HIV positive. All had multiple potential etiologic pathogens detected on both NP/OP and IS, as did most infants without pertussis infection (Supplementary Table 3). Only 1 of the pertussis-positive infants who died had received 3 doses of pertussis vaccine at least 2 weeks prior to cough onset. In contrast to the other infants who died, this infant did not manifest a leukemoid reaction (WBC count was 14.2 × 10³ cells/µL in the single sample tested); this infant presented with cough for 15 days, and was HIV exposed and severely underweight.

Among PERCH cases (n = 2477) and controls (n = 3564) 6–59 months of age with specimens tested for pertussis, 13 cases (0.5%) and 3 controls (0.1%) were positive. Compared with pertussis-negative cases, pertussis-positive cases were more likely to report cough of ≥7 days, inability to feed, leukocyte count ≥20 000 cells/µL, and lymphocyte count ≥10 000 cells/µL (all P < .05) (Supplementary Table 4). Three of the 13 pertussis-positive cases 6–59 months died (23.1%); 1 child was fully vaccinated, the second was HIV-positive and had received only a single pertussis vaccination, and the third child was unimmunized (clinical findings are shown in Supplementary Table 3). These 3 cases accounted for 1.9% of the 158 in-hospital deaths at the African sites in this age group.