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Original Studies

Clinical Characteristics and Histopathology of Coronavirus Disease 2019-Related Deaths in African Children

Mabena, Fikile C. MMed*,†; Baillie, Vicky L. PhD*,‡; Hale, Martin J. FCPath*,§; Thwala, Bukiwe N. MPH*,‡; Mthembu, Nonhlanhla MSc*,‡; Els, Toyah MSc*,‡; Serafin, Natali MSc*,‡; du Plessis, Jeanine BScHons*,‡; Swart, Peter MMed§; Velaphi, Sithembiso C. PhD; Petersen, Karen L. MMed; Wadula, Jeannette FCPath; Govender, Nelesh P. MMed; Verwey, Charl MMed*,†; Moore, David P. PhD*,†; Moosa, Fatima Y. MMed; Nakwa, Firdose L. MMed; Maroane, Basetsana V. FCPaed; Okudo, Grace FCPaed; Mabaso, Theodore M. MMed; Dangor, Ziyaad PhD*,†; Nunes, Marta C. PhD*,‡; Madhi, Shabir A. PhD*,**

Author Information
The Pediatric Infectious Disease Journal: September 2021 - Volume 40 - Issue 9 - p e323-e332
doi: 10.1097/INF.0000000000003227

Abstract

The coronavirus disease 2019 (COVID-19) pandemic, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has caused at least 3 million deaths as of April 30, 2021.1 SARS-CoV-2 infection in children is more often associated with asymptomatic infection (80%) than in adults (40%–60%).2,3 COVID-19–associated deaths are uncommon in children. Only 121 (0.06%) of the 190,000 COVID-19 fatalities in United States of America by July 2020,4 and none of 30,000 COVID-19 deaths in Italy during their first wave of the outbreak were documented in children.5 Similarly, only 17 (0.29%) of 5733 COVID-19–associated deaths reported in India occurred in children less than 18 years of age.6 Although multiorgan inflammatory syndrome is a feature of COVID-19 in children, the associated case fatality risk is low (0.69%).7,8

Attributing causality of illness or death solely by identification of respiratory viruses, including SARS-CoV-2, in the upper airways secretions is confounded by the possibility of coincidental detection. Notably, identification of the endemic coronaviruses (HKU1, NL63, OC43, 229E) in upper airway secretions was not causally associated with severe lower respiratory tract infection in children.9 Therefore, adjunct diagnostic investigations are required to determine whether the identification of SARS-CoV-2 infection in the deceased is causally related to the death.

The postmortem lung histopathologic features attributed to COVID-19 in adults were determined based on samples obtained by complete diagnostic autopsy or endoscopy.10 Postmortem minimally invasive tissue sampling (MITS), which is more culturally acceptable and pragmatic in resource-constrained, low- and middle-income countries, has been validated as having high concordance compared with complete diagnostic autopsy for attributing cause of death (CoD) in children, particularly for infectious diseases.11,12 The use of MITS has been adopted by the multicountry Child Health and Mortality Prevention Surveillance (CHAMPS) program, in which postmortem biologic investigations are undertaken to assist in determining the cause of stillbirths and childhood deaths.13

The present case series systematically evaluated the CoD in deceased children among whom SARS-CoV-2 was identified from respiratory tract samples by a polymerase chain reaction (PCR) either antemortem or postmortem.

METHODS

The study was undertaken at Chris Hani Baragwanath Academic Hospital (CHBAH), Soweto, South Africa, from April 14, 2020, to August 31, 2020, which coincided with the peak of the first wave of the COVID-19 outbreak in South Africa.14 The hospital provides secondary- and tertiary-level hospital care and is 1 of only 2 public hospitals serving the population of 1.3 million in Soweto, the majority (>90%) of who do not have private medical insurance and are dependent on the public health service. Details of the population of Soweto and standard of care practices at the CHBAH have been described.15

This study leveraged upon an established surveillance program for deaths undertaken as part of the CHAMPS program, of which Soweto (South Africa) is 1 of 7 participating sites.13 The CHAMPS surveillance includes identification of all stillbirth, neonatal and childhood deaths from specified localities in Soweto presenting at CHBAH (see Text, Supplemental Digital Content 1, http://links.lww.com/INF/E439). Fatal cases from birth to 14 years in whom SARS-CoV-2 was identified by PCR either antemortem or postmortem were included in this study. Antemortem nasopharyngeal specimen testing for SARS-CoV-2 was done at the discretion of attending physicians who had a policy of screening all children hospitalized during the study period irrespective of diagnosis. Also, postmortem nasopharyngeal sampling was undertaken by attending physicians on children who presented either dead on arrival at the emergency department and at time of death of a child during the course of their hospitalization.

Study-specific postmortem sampling was undertaken by trained study-staff on neonatal and childhood deaths whose families resided in Soweto, after the parents had provided consent. This included a subset of children whose families lived in a Health Demographic Surveillance in Soweto and a surrounding informal settlement that forms part of the CHAMPS program.

The standard operating postmortem MITS procedures, undertaken with 24 to 48 hours of death, are detailed in Table (Supplemental Digital Content 2, http://links.lww.com/INF/E440). Briefly, MITS procedures included a midturbinate nasal swab for SARS-CoV-2 nucleic acid amplification test (NAAT); nasopharyngeal swab for identification of other organisms (see Table, Supplemental Digital Content 3, http://links.lww.com/INF/E441). Blood obtained by cardiac puncture was submitted for bacterial culture, HIV-1 and molecular detection of bacteria (18 species). In addition, multiple lung, heart and liver core biopsy tissue samples were obtained. Children eligible for enrollment into the CHAMPS program also had brain tissue samples sent for histopathology. Histopathology examination was undertaken on all tissue samples; lung samples were sent for bacterial culture and molecular identification of SARS-CoV-2 and bacteria (n = 13), viruses (n = 18) and nonbacterial organisms (n = 3) (see Table, Supplemental Digital Content 4, http://links.lww.com/INF/E442).

Nucleic Acid Extraction

Nucleic acid extraction followed a protocol optimized for whole blood samples.16 Briefly, 200 μL of whole blood samples and 400 μL of respiratory specimens were extracted and eluted to a final volume of 100 μL of total nucleic acids using the NucliSENS “specific B” extraction protocol for blood and “Generic” protocol for respiratory specimens (BioMérieux, Marcy l’Etoile, France).

SARS-CoV-2 Testing

Reverse transcriptase PCR using the Emergency Use Authorization assay developed by the US Centers for Diseases Control and Prevention was used to detect SARS-CoV-2 in respiratory and blood samples.17 Results were classified as positive for SARS-CoV-2 when both the N1 and N2 targets of the nucleocapsid gene were detected and cycle threshold (Ct) values were <40. Results were classified as indeterminate if only N1 or N2 was detected with Ct values <40. If a repeat swab test yielded an indeterminate result, the child was considered to be infected with SARS-CoV-2 per Centers for Diseases Control and Prevention guidelines.

Real-time TaqMan Open Array in Blood and Nasopharyngeal Swab Samples

Real-time PCR was performed using the Open Array platform. The TaqMan Open Array Assay was spotted with 56 assays for the respiratory panel and 18 assays for the blood sepsis panel (see Table, Supplemental Digital Content 3, http://links.lww.com/INF/E441 and Table, Supplemental Digital Content 4, http://links.lww.com/INF/E442). Both panels included multiple internal controls including ribonucleoprotein 3 to ensure the integrity of clinical samples, the TaqMan Universal DNA extraction control to ensure optimal extraction of bacterial targets and the TaqMan universal RNA spike in reverse transcription control to ensure optimal extraction of RNA targets and that the reverse transcription step was carried out correctly. The analysis was performed on the TaqMan Open Array PCR with a QuantStudio 12k Flex Real-Time PCR System according to manufacturer’s instructions (Thermo Fisher Scientific, Waltham, MA).

Histopathology

Following biopsy, all tissues were immediately placed in 10% neutral buffered formalin and fixed for a minimum period of 6 hours, but no longer than 24 hours. After fixation, the biopsies were dehydrated in graded ethanol concentrations, cleared in xylene and infiltrated with wax, using the Tissue-Tek Vacuum Infiltration Processor manufactured by Sakura. Tissues were then embedded in wax blocks from which 4-micron sections were cut and stained with hematoxylin and eosin (H&E). Protocols for the tissue processing and H&E staining were based on widely established protocols and methodology.18,19

Stained tissue sections were examined by a histopathologist using an Olympus BX41 light microscope. The use of special histochemical stains was guided by the clinical history, including signs and symptoms and pathologic findings identified in the H&E sections. Established protocols were used for these stains.18,19

Determining Cause of Death Attribution

The determining cause of death attribution (DeCoDe) panel was made up of a multidisciplinary team including 5 pediatricians, 2 microbiologists and 2 pathologists (see Acknowledgment section). The panel reviewed all available antemortem clinical data, laboratory data and postmortem findings (histology, molecular and microbiologic results). The causal pathway to death was determined and reported by the panel using the World Health Organization standardized framework for CoD attribution.20 This included identifying the “underlying condition” considered to have initiated or predisposed to the causal pathway of death, any other antecedent medical events that may have contributed to the death and the “immediate” illness that led to death. This method of attributing the CoD is also used for the CHAMPS program.13 Only cases in whom SARS-CoV-2 was identified either on antemortem or postmortem sampling had a complete DeCoDe evaluation for the purpose of generating this report. Histopathology evaluation of cases in whom SARS-CoV-2 was not identified and their CoD have not been completed.

ETHICS CONSIDERATIONS

The study was approved by the Human Research Ethics Committee at the University of the Witwatersrand (HREC approval numbers: CHAMPS M1604102, MITS-lite M200265 and COVID-MITS M200313). Informed consent was obtained from parents/legal guardians of the deceased.

RESULTS

During the study period, 171 childhood deaths were recorded at CHBAH (Fig. 1). MITS was undertaken in 47.2% (58/123), 67.6% (25/37) and 72.8% (8/11) of deaths in age groups ≤28 days, 29 days to 12 months and 13 to 168 months, respectively. Reasons for MITS not been done included inability to obtain consent within 48 hours of death (39/171; 22.8%), refusal of consent (20/171; 11.7%) and child not being resident in Soweto (21/171; 12.3%) (Fig. 1).

FIGURE 1.
FIGURE 1.:
Study participants flow diagram.

Overall, SARS-CoV-2 was detected by NAAT in 20 (11.7%) of 171 deceased children. In 10 cases, SARS-CoV-2 was only identified on postmortem sampling. This included 4 children who were sampled upon presenting dead at time of hospital arrival (cases iii, iv, v and vii; Table 1), 4 cases not investigated antemortem (cases 1, 2, 3 and 10; Table 2) and 2 who had tested negative within 2 days before death (cases 4 and 7; Table 2).

TABLE 1. - Eight SARS-CoV-2 Positive Cases in Whom Postmortem Minimal Invasive Tissue Sampling Was Not Done
Case No. Age Calendar Week of Death Gender Timing of SARS-CoV-2 Testing Reason MITS Was Not Done Underlying Cause of Death Attribution by Attending Clinicians (C) and DeCoDe Panel (D) Antecedent Condition Attribution by Attending Clinicians (C) and DeCoDe Panel (D) Immediate Cause of Death Attribution by Attending Clinicians (C) and DeCoDe Panel (D) Other Conditions Attribution by Attending Clinicians (C) and DeCoDe Panel (D)
i. 10 d 28 Female Antemortem Parent refused to give consent Prematurity/low birth weight (C) COVID-19 (D) Acinetobacter baumannii septicemia (C) COVID-19 (C)
Prematurity/low birth weight (D) A. baumannii septicemia (D) Acute kidney injury (D)
ii. 19 d 26 Female Antemortem Unable to contact parents for consent Streptococcus pyogenes septicemia and meningitis (C) S. pyogenes S. pyogenes septicemia and meningitis (C) COVID-19 (C)
COVID-19 (D) Meningitis (D) S. pyogenes septicemia (D) Acute kidney injury (D)
iii. 21 d 26 Male Postmortem Out of surveillance catchment area COVID-19 (C) COVID-19 pneumonia (C) Prematurity/low birth weight (C)
COVID-19 (D) COVID-19 pneumonia (D)
iv. 31 d 26 Male Postmortem Unable to contact parents for consent COVID-19 (D) Suspected neonatal sepsis (C) COVID-19 (C)
Suspected neonatal sepsis (D)
v. 35 d 26 Male Postmortem Out of surveillance catchment area COVID-19 pneumonia(D) COVID-19 pneumonia (C)
vi. 10 mo 29 Female Antemortem Unable to contact parents for consent Burns (C) Staphylococcus aureus (D) A. baumannii septicemia (C) COVID-19 (D)
Burns (D) A. baumannii septicemia
Enterococcus faecalis (D)
vii. 20 mo 26 Female Postmortem Unable to contact parents for consent COVID-19 (C) Acute gastroenteritis (C)
COVID-19 (D)
viii. 8 yr 27 Female Antemortem Unable to contact parents for consent COVID-19 (C) Diabetic ketoacidosis (C) COVID-19 pneumonia (C) Suspected nosocomial sepsis (C)
Type 1 diabetes (D) Diabetic ketoacidosis (D) COVID-19 pneumonia (D)
Comments on the clinical presentation of above cases in Table (Supplemental Digital Content 5, http://links.lww.com/INF/E443).

TABLE 2. - Demographic Information, Presentation and SARS-CoV-2 Status of 12 Cases in Whom Postmortem Minimal Invasive Tissue Sampling Was Done
Case No. Age (d) Sex Gestational Age at Birth and Birth Weight Anthropometry at Time of Death, Z Score WFL Duration of Ventilation (d) Antemortem Nasopharyngeal Swab SARS-CoV-2 N1 and N2 Ct Values Postmortem Nasal Turbinate SARS-CoV-2 N1 and N2 Ct Values Postmortem SARS-CoV-2 N1 and N2 Ct Values on Lung Biopsy Specimens Postmortem SARS-CoV-2 N1 and N2 Ct Values on Blood
1. 1 Male Term BW = 3000 g 1 Not done N1 = 29.9; N2 = 30.2 Negative Negative
Normal
2. 3 Male 28 wk ELBW = 900 g 2 Not done N1 = 37.5; N2 = 35.7 Negative Negative
3. 4 Female Term BW = 3200 g 3 Not done N1 = negative; N2 = 39.3* Negative Negative
Normal
4. 10 Female Term W = 2555 g 4 Negative N1 = 32.7; N2 = 32.9 Negative Negative
Wasting, WFL = –2.3 SD
5. 12 Male Term BW = 3800 g 1 N1 negative; N2 = 38.4* Not done Negative Negative
Normal, WFL = 0.1 SD
6. 13 Female Term W = 2000 g 3 N1 = 17.4; N2 = 16.4 N1 = 15.8; N2 = 15.1 N1 = 11.8; N2 = 11.4 N1 = 20.9; N2 = 20.9
Wasting
WFL = –2.5 SD
7. 22 Male 35 wk W = 2115 g 2 Negative N1 = negative; N2 = 38.6* N1 = 38.9; N2 = negative Negative
Wasting
WFL = –2.0 SD
8. 35 Male Term W = 2600 g 18 N1 = 25.6; N2 = 25.1 N1 = 25.2; N2 = 24.6 N1 = 22.8; N2 = 23.7 Negative
Wasting
WFL = –2.9 SD
9. 52 Male 28 wk VLBW = 1440 g 7 N1 = 31.4; N2 = 31.3 N1 = 27.0; N2 = 26.4 N1 = 16.6; N2 = 16.7 N1 = 22.7; N2 = 20.7
10. 53 Female 27 wk LBW = 1795 g 5 Not done N1 = 16.5; N2 = 15.6 N1 = 13.6; N2 = 14.9 N1 = 19.2; N2 = 19.6
11. 90 Male Term W = 4000 g 1 N1 = 23.3; N2 = 23.4 N1 = 20.8; N2 = 20.3 N1 = 18.7; N2 = 19.1 N1 = 32.5; N2 = 33.2
Normal WFL = –1.0 SD
12. 120 Female Term W = 5700 g 3 N1 = 30.82; N2 = 32.3 N1 = negative; N2 = 37.6 N1 = 38.4; N2 = negative Negative
Normal
WFL = –0.3 SD
*N1 and N2 targets detected with Ct values < 40 classified as positive for SARS-CoV-2. Results are classified as inconclusive if only N1 or N2 detected with Ct values < 40, but if clinical features suggestive of COVID-19 then viewed as positive result.
BW indicates birth weight; ELBW, extremely low birthweight (BW < 1000 g); LBW, low birthweight (BW < 2500 g); VLBW, very low birthweight (BW < 1500 g); W, weight; WFL, weight-for-length, wasting = WFL<–2 SD.
Case 7 and case 12 labeled as SARS-CoV-2 positive with 2× indeterminate postmortem results (case 12 has SARS-CoV-2 positive antemortem result).
Comments on the clinical presentation of above cases in Table (Supplemental Digital Content 6, http://links.lww.com/INF/E444).

MITS was not undertaken in 8 cases in whom SARS-CoV-2 was identified, including 5 whose parents could not be contacted for consenting, 2 who resided outside of Soweto and 1 in whom consent was declined (Table 1).

Of 12 cases with SARS-CoV-2 infection detected antemortem and/or postmortem sampling in whom MITS was done; their median age was 35 days (range: 1–120 days), 7 (58.3%) were male and all had a nonreactive HIV-1 PCR (including 2 born to women living with HIV). Included among these 12 cases were 8 infants born at full term and 4 who were born prematurely at 27, 28 (n = 2) and 35 weeks of gestational age. Five (41.7%) of the 12 cases (cases 1, 2, 3, 9 and 10; Table 2) were hospitalized since birth and acquired SARS-CoV-2 in hospital.

The clinical symptomatology among cases in whom MITS was undertaken included respiratory illness in 7 (58.3%), gastroenteritis in 3 (25.0%) and 3 (25.0%) with seizures (2 of whom had concurrent respiratory illness or gastroenteritis). All 12 infants were on mechanical ventilation for median of 4 days (range: 1–18 days) before death (Table 2).

COVID-19 was attributed in the causal pathway of death in 11 (91.7%) of the 12 cases in whom MITS was done. The single case (case 3; Table 2) in whom COVID-19 was not included in the causal pathway had a single indeterminate SARS-CoV-2 test on postmortem nasal swab sample only.

In a further 2 cases (cases 5 and 7; Tables 2 and 3), although SARS-CoV-2 NAAT was reactive for only 1 of the 2 N targets in any of the tested samples, COVID-19 was still attributed to being in the causal pathway to death by the DeCoDe panel. In case 5, only the antemortem sample tested positive for N2 (Ct = 38.4) on PCR, but histopathology of lung, liver and heart samples was highly suggestive of COVID-19 pathology. In case 7, the SARS-CoV-2 test was negative 2 days before death, but the postmortem PCR was positive for N2 (Ct = 38·6) on nasal swab and for N1 (Ct = 38·9) on lung biopsy.

TABLE 3. - Antemortem Blood, Postmortem Blood, Tissue Results and CoD Attribution for 12 Cases Who Had Minimal Invasive Tissue Sampling Procedure Done
Case No. Antemortem Blood Culture Postmortem Blood Culture Lung Culture Blood PCR Lung PCR Histopathology Lung Features Histopathology Liver Features Histopathology Heart Features Underlying CoD Attribution by DeCoDe Panel Antecedent Conditions Attribution by DeCoDe Panel Other Condition Attribution by DeCoDe Panel Immediate CoD Attribution by DeCoDe Panel
1. Not done Escherichia coli Negative Negative Negative ARDS EMH Not done Diaphragmatic hernia with lung hypoplasia COVID-19 Intrauterine hypoxia Diaphragmatic hernia with lung hypoplasia
Acinetobacter baumannii Interstitial pneumonitis
Type II pneumocyte proliferation
HMD
2. Staphylococcus haemolyticus CNS Negative E. coli (Ct = 37) Negative HMD (severe) Not done Normal ELBW HMD AKI Pulmonary hemorrhage
Premature lung Prematurity COVID-19
Type II pneumocyte proliferation
Alveolar hemorrhage
3. A. baumannii A. baumannii Negative A. baumannii (Ct = 33) A. baumannii (Ct = 34) Premature lung, type II pneumatocyte hyperplasia EMH, Kupffer cell hyperplasia Not done Intra uterine Hypoxia Neonatal encephalopathy
HMPV (Ct = 35)
4. Negative Negative S. haemolyticus Negative Ureaplasma spp. (Ct = 37) Resolving bleed, hemosiderin-laden macrophages Interstitial pneumonitis Normal Not done COVID-19 AKI Pulmonary hemorrhage
Hypovolemic shock
5. Negative CNS Not done Negative E. coli (Ct = 37) BPN EMH Endocarditis myocarditis, fibrin-platelet thrombi S. pyogenes sepsis COVID-19 Endocarditis, omphalitis S. pyogenes meningitis (D)
Intravascular fibrin, microthrombi, congestion, macrophages Portal, sinusoidal inflammation, lobular hepatitis, lympho-erythro phagocytosis in Kupffer cells
6. Negative Enterococcus faecium Klebsiella pneumoniae K. pneumoniae (Ct = 32) E. coli (Ct = 27) HMD Not done Not done COVID-19 AKI K. pneumoniae
E. faecium E. faecium (Ct = 32) K. pneumoniae (Ct = 31) Interstitial pneumonitis E. faecium
E. coli (Ct = 37) S. aureus (Ct = 34) Alveolar hemorrhage, fibrin, meconium Sepsis
Streptococcus pneumoniae (Ct = 35)
7. Negative Negative E. coli Negative E. coli (Ct = 30) HMD, alveolar hemorrhage Not done Not done COVID-19 AKI E. coli pneumonia and sepsis
E. faecium Type II pneumocyte proliferation
8. Negative CNS Not done K. pneumoniae (Ct = 37) ARDS Submassive Myocarditis indeterminate COVID-19 AKI COVID-19 pneumonia
A. baumannii (Ct = 34) Hyaline membranes Necrosis congestion Myocarditis
S. aureus (Ct = 36) Interstitial pneumonitis Hepatic necrosis
Severe type II proliferation & atypia, alveolar collapse (Bronchiolitis obliterans organizing pneumonia), septal fibrosis, honeycombing, fibrin thrombi
9. Negative A. baumannii A. baumannii ARDS, neutrophils, congestion, septal edema, fibrin thrombi, hyaline membranesHM, alveolar collapse & necrosis, severe type II pneumocyte proliferation EMH Normal ELBW COVID-19 A. baumannii pneumonia nosocomial sepsis
E. faecium Portal inflammation Prematurity
Sinusoidal leukocytosis
Acute hepatitis lympho-erythro phagocytosis
10. S. hominis A. baumannii A. baumannii Ureaplasma spp. (Ct = 27) Ureaplasma spp. (Ct = 23) HMD, diffuse alveolar damage, BPN, premature lungs alveolar hemorrhage, type II pneumocyte proliferation Not done Not done VLBW COVID-19 AKI A. baumannii sepsis nosocomial sepsis
E. faecalis E. coli (Ct = 30) E. coli (Ct = 24) Prematurity
A. baumannii (Ct = 32) A. baumannii (Ct = 30)
Candida albicans (Ct = 33) C. albicans (Ct = 33)
S. aureus (Ct = 36)
11. Not done Pseudomonas aeruginosa K. pneumoniae Negative E. coli (Ct = 28) ARDS Steatosis, cholestasis, bile duct paucity Normal Familial congenital nephrotic syndrome None COVID-19
EBV (Ct = 31) BPN fibrin thrombi, septal collagen Cirrhosis
A. baumannii (Ct = 33)
12. Negative Not done E. faecium C. alb S. pneumoniae (Ct = 19) HiB (Ct = 30) Indeterminate ARDS fibrin thrombi Droplet steatosis Normal COVID-19 None S. pneumoniae meningitis
CMV (Ct = 34) Sinusoidal leukocytosis, lympho-erythro phagocytosis
HHV6 (Ct = 36)
AKI indicates acute kidney injury; ARDS, acute respiratory distress syndrome; BPN, bronchopneumonia; CMV, cytomegalovirus; CNS, coagulase-negative Staphylococcus; EBV, Epstein-Barr virus; ELBW, extremely low birthweight < 1000 g; EMH, extramedullary hematopoiesis; HHV6, human herpes virus 6; HiB, Haemophilus influenzae type B; HMD, hyaline membrane disease; HMPV, human metapneumovirus; VLBW, very low birthweight < 1500 g.
PCR refers to Real-time TaqMan Open Array (Thermofisher Scientific, Waltham, MA, USA), antecedent conditions, underlying and immediate CoD attribution done by determination of CoD panel.
Brain tissue samples obtained from cases 1, 5, 9 and 11, with 3 being normal and 1 (case 5) with evidence of cerebral abscess, vasculitis and Gram-positive cocci on staining (Fig. 2D), in whom S. pyogenes meningitis was the immediate CoD (Table 3).

Among the 11 cases with MITS in whom COVID-19 was attributed in the causal pathway to death, it was attributed by the DeCoDe panel as the underlying cause in 5 (45.5%), antecedent cause in 5 (45.5%) and immediate cause in 1 case (9.0%; case 11 who had familial congenital nephrotic syndrome as an underlying cause).

The immediate CoD in 5 cases where COVID-19 was attributed as the underlying cause included 2 deaths (cases 4 and 8) due to COVID-19 attributed multiorgan complications, while the immediate cause in the other 3 cases was culture-confirmed invasive bacterial disease (cases 6 and 7) and concurrent pneumococcal meningitis (serotype 6C; case 12) (Table 3).

Among the 5 cases in whom COVID-19 was attributed as an antecedent illness in the causal pathway to death, 4 were infants hospitalized since birth due to being born prematurely (cases 2, 9 and 10) and 1 with diaphragmatic hernia (case 1) (Table 3). In the 3 preterm neonates with COVID-19 as an antecedent illness, 2 died from nosocomial-acquired Acinetobacter baumannii sepsis (cases 9 and 10) and 1 from pulmonary hemorrhage (case 2) (Table 3). The immediate CoD in the fifth infant (case 5) was Streptococcus pyogenes sepsis and meningitis (Table 3).

Overall, 6 (54.5%) of the 11 COVID-19–associated deaths had culture-confirmed bacterial infections attributed as the immediate CoD. In addition to postmortem culture of bacteria from blood and lung, other organisms identified in blood and lung samples on Open Array PCR testing are reported in Table 3, albeit not necessarily attributed in the causal pathway to death by the DeCoDe panel.

Among the 11 cases in which COVID-19 was included in the causal pathway to death, histopathology lung findings (n = 11) included features of diffuse alveolar damage (DAD), Figure 2A (n = 6, 54.5%; see Figures, Supplemental Digital Content 7, http://links.lww.com/INF/E445), interstitial pneumonitis (n = 4, 36.4%; see Figures, Supplemental Digital Content 8, http://links.lww.com/INF/E446), fibrin thrombi (n = 4, 36.4%; see Figures, Supplemental Digital Content 9, http://links.lww.com/INF/E447), hyaline membrane formation (n = 5, 36.4%; see Figures, Supplemental Digital Content 10, http://links.lww.com/INF/E448), type 2 pneumocyte proliferation (n = 6, 54.5%; see Figures, Supplemental Digital Content 11, http://links.lww.com/INF/E449) and bronchopneumonia (n = 3, 27.3%; see Figures, Supplemental Digital Content 12, http://links.lww.com/INF/E450) (Table 3). Bacteria cultured from the lung in 3 cases of histologically diagnosed bronchopneumonia were Klebsiella pneumoniae and A. baumannii in 1 case each, while being negative for the third case (Table 3).

FIGURE 2.
FIGURE 2.:
Histopathologic findings. A: Lung showing severe histopathologic changes of acute respiratory distress syndrome (diffuse alveolar damage) including lymphocytic interstitial inflammation and hyaline membrane formation (long arrows). An intraluminal fibrin-platelet thrombus is seen in a pulmonary arteriole (short arrow and inset top right). Marked, bizarre, type II pneumocyte hyperplasia lines the inner aspect of many alveoli (inset top left), and in some areas, syncytia of type II pneumocytes is seen (inset lower left). B: Liver biopsy showing submassive hepatocyte necrosis with pan-zonal loss of hepatocytes between PTs and CVs. Bile ductular proliferation indicative of hepatocyte regeneration is seen adjacent to the PT (long arrow), together with residual islands of surviving hepatocytes (short arrow). C: Section of heart showing a focus of myocarditis with a mononuclear inflammatory cell infiltrate and myocyte necrosis (arrow). D: Meningeal vessel showing septic vasculitis resulting in destruction of the vessel wall. The mononuclear inflammatory infiltrate is associated with abundant karyorrhectic debris and interspersed cocci (arrow). CV indicates central vein; PT, portal tract.

Liver samples were available in 7 of 11 COVID-19–associated deaths. Hepatic histopathologic changes included submassive necrosis (case 8; Fig. 2B), steatosis (cases 11 and 12; see Figures, Supplemental Digital Content 13, http://links.lww.com/INF/E451), lympho-erythro phagocytosis (cases 5 and 12, see Figures, Supplemental Digital Content 14, http://links.lww.com/INF/E452) and cirrhosis (case 11; see Figures, Supplemental Digital Content 13a, http://links.lww.com/INF/E451).

Heart samples were available in 6 (54.5%) of the COVID-19–associated deaths, with endocarditis, myocarditis and fibrin-platelet thrombi identified in case 5 (Fig. 2C), indeterminate myocarditis in case 8 (see Figures, Supplemental Digital Content 15, http://links.lww.com/INF/E453), while being unremarkable in the remaining cases (Table 3).

Brain tissue samples were obtained from 4 cases, with 3 being normal and 1 (case 5) with evidence of cerebral abscess, vasculitis and Gram-positive cocci on staining (Fig. 2D), in whom S. pyogenes meningitis was the immediate CoD (Table 3).

Cases in Whom MITS Was Not Done

The DeCoDe panel also adjudicated on the CoD for the 8 cases in whom SARS-CoV-2 was detected but in whom MITS was not done (Table 1). One of these cases (case vi) was a 10-month-old child who suffered accidental burns and subsequently developed and likely demised due to nosocomial-acquired sepsis, in whom the antemortem identification of SARS-CoV-2 was considered to be coincidental by the DeCoDe panel and attending physician (Table 1). In 3 cases (case i, ii and iv), the DeCoDe panel but not the attending physician (who did not have access to any postmortem data) attributed COVID-19 in the causal pathway to death. Among the 3 cases in whom identification of SARS-CoV-2 was considered coincidental by the attending physician, death was attributed to culture-confirmed sepsis (cases i and ii) or suspected bacterial sepsis (cases iv). In addition, case ii (Table 1) was a 19-day-old child with disseminated S. pyogenes invasive disease, in whom the DeCoDe panel attributed COVID-19 as the underlying cause that possibly predisposed to the invasive bacterial disease, premised on findings from case 5 in whom MITS was done (Table 2). In the remaining 3 children (cases iii, v, viii), the attending physician and DeCoDe panel concurred that COVID-19 was likely involved in the causal pathway to death, including an 8-year-old child who was known to have poorly controlled type 1 diabetes and presented with diabetic ketoacidosis (Table 1).

Prematurity (n = 4; 22.2%) was the most common underlying comorbid condition in the 18 cases for whom COVID-19 was attributed in the causal pathway to death. Other underlying comorbid conditions were type 1 diabetes (n = 1), familial congenital nephrotic syndrome (n = 1) and congenital diaphragmatic hernia (n = 1).

DISCUSSION

Although deaths due to COVID-19 have rarely been reported in children under 18 years of age globally,5,6 this study provides clinical and histopathologic evidence of COVID-19–related deaths in children. In our study, a multidisciplinary DeCoDe panel using a standardized World Health Organization framework for evaluating the causal pathway20 to death attributed COVID-19 contributing to the death in 18 of 20 (90%) children in whom SARS-CoV-2 infection was identified on antemortem or postmortem sampling. Earlier studies on COVID-19 in children suggested that infants have more severe disease than older children, albeit there being more recent contradictory reports.21–23 Nevertheless, in our setting where surveillance included deaths occurring through to 14 years age, 16 (88.9%) of the 18 COVID-19–associated deaths were in infants less than 6 months of age. In the 11 of 12 cases in whom MITS was done, and COVID-19 was attributed in the causal pathway, 6 were neonates (54.5%), 5 (45.5%) 1–6 months of age; notably, 7 (63.6%) were born at term without any underlying comorbidity. Speculatively, the dominance of COVID-19 deaths in younger than older children may possibly be due to differential expression of angiotensin-converting enzyme 2 receptors. Significant fluctuation of angiotensin-converting enzyme 2 expression has been found in a murine model, being lower in the fetus, highest 1–3 days after birth and dropping thereafter.24 This may in part explain the median age of 35 days in our cohort.

The lung histopathologic features of COVID-19–related deaths in our study were consistent with histopathology features associated with COVID-19 attributed deaths in adults.10 A systematic review of postmortem lung histopathologic features of COVID-19 deaths in adults (n = 45) reported DAD, hyaline membrane formation and vascular congestion with occasional inflammatory cells. Other features included damaged pneumocytes with focal sloughing and formation of syncytial giant cells, type II pneumocyte hyperplasia, fibrinoid necrosis of the small vasculature and focal infiltration of lymphocytes, monocytes and increased stromal cells. Abundant intra-alveolar neutrophil infiltration was suggestive of superimposed bacterial bronchopneumonia.10 In our study, the major lung histopathologic features were acute respiratory distress syndrome/DAD in 6 (54.5%), hyaline membrane formation in 4 (36.4%) and bronchopneumonia in 3 (27.3%) cases. The histopathologic findings in liver and heart, also observed in fatal COVID-19 cases in adults, suggest extrapulmonary involvement in children with COVID-19–associated deaths.

A systemic review of hospitalized adult patients with COVID-19 found bacterial coinfection to be uncommon at an overall proportion of 6.9% (3.5% community-acquired and 14.3% for hospital-acquired infections).25 Despite being infrequent overall, bacterial coinfections were more common in critically ill patients (8.1%).25 Notably, in our case series, culture-confirmed invasive bacterial disease was evident in 54.5% of the COVID-19–associated deaths on postmortem sampling. Although 3 (50.0%) of the 6 culture-confirmed invasive bacterial cases were likely hospital-acquired infections, the DeCoDe panel adjudicated that SARS-CoV-2 infection could have predisposed to these invasive bacterial disease episodes. Hence, our findings indicate a possible causal role of bacterial infections in fatal COVID-19 in children.

Notable in our case series, there were also cases of community-acquired invasive bacterial disease attributed to S. pyogenes and S. pneumoniae, with histopathologic evidence of lung pathology suggestive of COVID-19. In the cases with community-acquired bacterial disease, the SARS-CoV-2 and blood cultures were both positive at admission for 2 cases (cases 5 and 12; Table 3) and positive on postmortem specimens for 1 case (case 7; Table 3). This supported the DeCoDe deliberations in attributing COVID-19 in the causal pathway to death, rather than identification of SARS-CoV-2 only being coincidental asymptomatic colonization in these children. This was similarly observed for cases in whom COVID-19 was associated with nosocomial-acquired bacterial invasive disease (n = 3), among whom all had histologic features suggestive of COVID-19 pathology. The positive SARS-CoV-2 test preceded the positive blood culture in 2 cases with nosocomial bacterial coinfection and was only positive on postmortem sampling in the third case. Furthermore, sepsis was also attributed as the immediate CoD in 2 of the deaths not investigated by MITS. Implications of these findings include the need to maximize infection control practices to prevent hospital-acquired infections and to consider appropriate empiric antibiotic treatment in children requiring admission with severe COVID-19.

Although this is to our knowledge, the first case series to report on COVID-19–associated histopathology features in children, limitations of our study include being a single-center study with a limited sample size. Nevertheless, there have only been few COVID-19 childhood deaths reported globally and postmortem investigation is rarely undertaken in low- and middle-income countries. Furthermore, sampling was limited mainly to the lung, hence, preventing a fuller characterization of multiorgan histopathologic features in fatal COVID-19 cases. Also, we have not completed histopathology and CoD analysis of deaths not associated with SARS-CoV-2 infection, which would provide insight into whether the observed histopathology findings differ to deaths not attributed to COVID-19. Although the histopathology features identified in the deaths attributed to COVID-19 in our study are unlikely to be pathognomonic for COVID-19, the DeCoDe panel used the standardized World Health Organization framework for attributing CoD supporting that the observed histopathology features are COVID-19–related. Another limitation is interpreting the discordance in attributing COVID-19 in the causal pathway to death in SARS-CoV-2 infection cases without MITS between the attending physician and DeCoDe panel. The DeCoDe panel adjudication of the cases in whom MITS was not undertaken was done after they reviewed the cases in who MITS was done. This could have led the DeCoDE panel being more likely to attribute a role for COVID-19 in these cases, based on their prior experience with the MITS cases.

This study highlights the utility of MITS as a practical, culturally and socially acceptable means to providing granular insight into the role of SARS-CoV-2 infection in the causal pathway of death in children. The supporting histopathology findings dispel any notion that identification of SARS-CoV-2 in infants who demise is likely to be only coincidental, despite 80% of SARS-CoV-2 infections in young infants possibly being asymptomatic.

ACKNOWLEDGMENTS

The authors thank determining cause of death attribution (DeCoDe) panel (specialization in parenthesis) included Nelesh P. Govender (microbiology), Martin J. Hale (histopathology), Fikile C. Mabena (pediatrics and infectious diseases), Shabir A. Madhi (pediatrics, infectious diseases and vaccinology), Karen L. Petersen (pediatrics and nephrology), Gillian Sorour (pediatrics and infectious diseases), Peter Swart (histopathology), Sithembiso C. Velaphi (neonatologist), Janet Wadula (microbiology).

REFERENCES

1. Dong E, Du H, Gardner L. An interactive web-based dashboard to track COVID-19 in real time. Lancet Infect Dis. 2020;20:533–534.
2. Davies NG, Klepac P, Liu Y, et al.; CMMID COVID-19 working group. Age-dependent effects in the transmission and control of COVID-19 epidemics. Nat Med. 2020;26:1205–1211.
3. Bi Q, Wu Y, Mei S, et al. Epidemiology and transmission of COVID-19 in 391 cases and 1286 of their close contacts in Shenzhen, China: a retrospective cohort study. Lancet Infect Dis. 2020;20:911–919.
4. Bixler D. SARS-CoV-2–associated deaths among persons aged 21 years—United States, February 12–July 31, 2020. MMWR Morb Mortal Wkly Rep. 2020;69:1324–1329.
5. Parri N, Magistà AM, Marchetti F, et al. Characteristic of COVID-19 infection in pediatric patients: early findings from two Italian Pediatric Research Networks. Eur J Pediatr. 2020;179:1315–1323.
6. Laxminarayan R, Wahl B, Dudala SR, et al. Epidemiology and transmission dynamics of COVID-19 in two Indian states. Science. 2020;370:691–697.
7. Feldstein LR, Rose EB, Horwitz SM, et al. Multisystem inflammatory syndrome in US children and adolescents. New Engl J Med. 2020;383:334–346.
8. Götzinger F, Santiago-García B, Noguera-Julián A, et al.; ptbnet COVID-19 Study Group. COVID-19 in children and adolescents in Europe: a multinational, multicentre cohort study. Lancet Child Adolesc Health. 2020;4:653–661.
9. O’Brien KL, Baggett HC, Brooks WA, et al. Causes of severe pneumonia requiring hospital admission in children without HIV infection from Africa and Asia: the PERCH multi-country case-control study. Lancet. 2019;394:757–779.
10. Deshmukh V, Motwani R, Kumar A, et al. Histopathological observations in COVID-19: a systematic review. J Clin Pathol. 2021;74:76–83.
11. Maixenchs M, Anselmo R, Zielinski-Gutiérrez E, et al. Willingness to know the cause of death and hypothetical acceptability of the minimally invasive autopsy in six diverse African and Asian settings: a mixed methods socio-behavioural study. PLoS Med. 2016;13:e1002172.
12. Bassat Q, Castillo P, Martínez MJ, et al. Validity of a minimally invasive autopsy tool for cause of death determination in pediatric deaths in Mozambique: an observational study. PLoS Med. 2017;14:e1002317.
13. Taylor AW, Blau DM, Bassat Q, et al.; CHAMPS Consortium. Initial findings from a novel population-based child mortality surveillance approach: a descriptive study. Lancet Glob Health. 2020;8:e909–e919.
14. Blumberg L, Jassat W, Mendelson M, et al. The COVID-19 crisis in South Africa: protecting the vulnerable. S Afr Med J. 2020;110:825–826.
15. Chawana R, Baillie V, Izu A, et al. Potential of minimally invasive tissue sampling for attributing specific causes of childhood deaths in South Africa: a pilot, epidemiological study. Clin Infect Dis. 2019;69(suppl 4):S361–S373.
16. Driscoll AJ, Karron RA, Morpeth SC, et al. Standardization of laboratory methods for the PERCH study. Clin Infect Dis. 2017;64(suppl_3):S245–S252.
17. Lee A, Kothare A, Dollar F, et al. CDC guidelines Info sheet TEST W. Evidence-based medicine InfoSheet: COVID-19 diagnostics and testing. 2020:1–15.
18. Suvarna S, Layton C. Bancroft’s. Theory and Practice of Histological Techniques. Churchill Livingstone, Elsevier. 2018
19. Aughey E, Frye FL. Comparative. Veterinary Histology. Manson Publishing; 2013:173–186.
20. World Health Organization. The WHO Application of ICD-10 to Deaths During Pregnancy, Childbirth and Puerperium: ICD-MM. World Health Organization; 2012.
21. Dong Y, Mo X, Hu Y, et al. Epidemiology of COVID-19 among children in China. Pediatrics. 2020;145:e20200702.
22. Ouldali N, Yang DD, Madhi F, et al.; investigator group of the PANDOR study. Factors associated with severe SARS-CoV-2 infection. Pediatrics. 2021;147:e2020023432.
23. Panetta L, Proulx C, Drouin O, et al. Clinical characteristics and disease severity among infants with SARS-CoV-2 infection in Montreal, Quebec, Canada. JAMA Netw Open. 2020;3:e2030470.
24. Li M, Chen L, Zhang J, et al. The SARS-CoV-2 receptor ACE2 expression of maternal-fetal interface and fetal organs by single-cell transcriptome study. PLoS One. 2020;15:e0230295.
25. Langford BJ, So M, Raybardhan S, et al. Bacterial co-infection and secondary infection in patients with COVID-19: a living rapid review and meta-analysis. Clin Microbiol Infect. 2020;26:1622–1629.
Keywords:

coronavirus disease 2019; pediatrics; histopathology

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