History and examination: A male neonate was born at 35 weeks gestation with a birth weight of 1016g to a primigravida mother with the antenatal history of oligohydramnios and intra-uterine growth retardation (IUGR). The baby was delivered by induced vaginal delivery secondary to meconium stained amniotic fluid. His APGAR scores were 6 and 8 at 1 and 5 minutes after birth. His physical examination revealed symmetrical (IUGR) [head circumference 25 cm (4.5 z-score), Ponderal index= 2], bilateral cataract and hepatosplenomegaly.

Investigations: His complete hemogram revealed thrombocytopenia; cranial ultrasonography (USG) was suggestive of increased echogenicity in bilateral basal ganglia and periventricular region, and USG abdomen was normal. Two-dimensional echocardiography (2D-ECHO) was suggestive of atrial septal defect (ASD) and 4 mm patent ductus arteriosus (PDA) with left to right shunt. Toxoplasma, rubella, cytomegalovirus (CMV) and herpes (TORCH) work-up revealed positive Rubella Immunoglobulin M (IgM). His renal and liver function tests were normal. His acute phase reactants were negative and blood/ cerebrospinal fluid (CSF) cultures were sterile.

Pre-operative echocardiography showed large PDA with moderate pulmonary arterial hypertension (PAH); pulmonary arterial pressures were 45 mm Hg with Qp:Qs (Pulmonary : Systemic blood flow) = 2:1. On day 48 of life, he underwent bed-side PDA closure surgery in the NICU, during which 5 mm PDA was closed with a titanium clip. The aortic arch was intact. Chest was closed with left pleural drain in-situ. After 2 hours, his ventilatory requirement increased. Echocardiography revealed closed PDA, moderate to severe PAH with poor cardiac contractility (ejection fraction 30%). The baby was initiated on dobutamine and milrinone infusions as inhaled nitric oxide was unavailable at that time. However, pulmonary pressures did not improve requiring initiation of sildenafil. Nevertheless, the baby gradually developed respiratory failure, refractory shock and expired on day 58 of life. His blood cultures obtained before death were sterile.

The reason for deterioration in the post-operative period seemed to be pulmonary arterial hypertension. It was secondary to either broncho-pulmonary dysplasia (BPD) or/and worsening due to surgery induced inflammation [3]. It is evident from the literature that a long-standing large sized PDA can damage pulmonary parenchyma and cause re-modeling of pulmonary vasculature, which possibly happened in our case [4]. Preoperative hemodynamic information does not correlate well with post-operative outcome for various reasons [4].

Treating unit senior resident: The case was challenging in terms of managing ventilation and medically refractory CCF which in presence of a large PDA required surgical closure of PDA. But on the hindsight closure actually worsened the preexistent pulmonary hypertension. In the autopsy of lungs, we expect findings concomitant with bronchopulmonary dysplasia and distortion of normal lung architecture in form of alveolar simplification, smooth muscular hyperplasia, peribronchiolar and interstitial fibrosis, areas of atelectasis and abnormal microvasculature. Also we have suspicion of CMV pneumonitis. The findings of CNS will be important due to USG head abnormalities in neonate. Whether some of findings in heart are missed will also be informative.

Cardiologist: Post PDA ligation syndrome is unlikely to lead to worse outcome in this neonate as it was well managed and cardiac functions improved echo-cardiographically after starting dobutamine. Pulmonary hypertension crisis is known in cases with only severe pulmonary vasoconstrictive disease and is less likely to be a cause of mortality as in an unpublished study of 500 neonates with moderate to large PDA, wherein cardiac functions improved after PDA ligation, mortality was decreased post operatively.

Treating unit consultant: Post PDA ligation syndrome does not seem to be a contributor of mortality in our neonate. We expect BPD changes in lungs that seem to be contributed by multiple intercurrent illnesses and by a large PDA. Also we failed to identify any infectious illness by means of cultures. Although we tried to exclude congenital CMV by means of investigations, but perinatally acquired CMV remains a possibility.

Pediatrician 1: Congenital Rubella syndrome can have findings of bilateral basal ganglia calcifications, peri-ventricular leucoencephalopathy and temporal cysts on brain.

Pediatrician 2: Chest x-rays were suggestive of hyperinflation in the patient, which due to increased lung volumes can worsen pulmonary hypertension.

A complete autopsy was performed. Weight of neonate was 1438 gm, crown heel length was 40 cm and occipitofrontal circumference (OFC) were 27 cm, All these parameters were below -2 z-score for age. There was 10 mL serous fluid in pericardial cavity. The weight of heart was 20 g with globular shape and blunt apex and situs solitus. The pulmonary trunk was grossly dilated. PDA was clipped and an ASD measuring 5 mm was identified. Right ventricular wall was hypertrophic and measured 5 mm (normal values 1.3-4 mm) [5]. Microscopy of the right ventricle revealed mild anisonucleosis of the cardiac muscles indicating right ventricular hypertrophy.

Both lungs weighed together 90 and were overweight. The pleurae were dull and fibrins tags were noted on lungs suggestive of pleuritis. Both the lungs showed areas of consolidation and hemorrhage. Microscopy showed areas of intra-alveolar hemorrhage and interstitial widening with no evidence of fibrosis. Interstitial widening was predominantly caused by pulmonary capillary proliferation. In addition, there was abundance of type 2 pneumocytes in alveoli. Reticulin stain demonstrated interstitial widening with pulmonary capillary proliferation. There was evidence of pulmonary arterial hypertension in the form of pulmonary arteriopathy. The pre-acinar and intra-acinar pulmonary arteries showed intimal hyperplasia with medial hypertrophy and adventitial thickening. A few arteries had complete occlusion of lumen due to intimal proliferation. These changes were better appreciated on elastic von- Gieson stain (Web Fig. 1). There was an area of consolidation with neutrophilic inflammatory infiltrate in bronchial lumen and surrounding alveoli confirming bronchopneumonia (Fig. 2). Stains for gram positive, gram negative bacteria and PAS stains for fungus were negative. No CMV inclusions were found.

Brain weighed 320 g and OFC was 27 cm suggestive of microcephaly. There was softening of white matter and focal calcifications in the white matter, periventricular and basal ganglia region (Web Fig. 2). Micronodules were appreciated in white matter and basal ganglia. Edema and pallor was found in periventricular area consistent with periventricular leucomalacia (Web Fig. 3).

Liver, spleen, kidney, urinary systems, esophagus, stomach. thymus, bone marrow, lymph nodes, adrenal and testes were normal.

Final autopsy diagnosis:

A 2-month old male with evidence of congenital rubella syndrome and surgically clipped PDA:

Pediatrician 3: Congenital rubella syndrome surveillance is being conducted in 5 centers since 2016 (Postgraduate Institute of Medical Education and Research, Chandigarh; All India Institute of Medical Sciences, Jodhpur; KEM Hospital,Pune; Indira Gandhi Institute for Child Health, Bengaluru; Christian Medical College, Vellore). In a period of 8 months, during December 2016-July 2017; 207 cases of CRS were identified as clinical cases with 72 laboratory confirmed cases (35% positivity rate). Among laboratory confirmed cases of CRS, 83% had congenital heart diseases, 62% had eye manifestations and 35% had hearing deficits on 1-year follow up. Most common associated condition is congenital heart disease, though cataract had been most common defect leading to suspicion of CRS. Sixty percent mothers, whose neonates developed CRS, were <26 years at age of conception [6].

Treating unit consultant: Pathological findings were quite interesting. As opposed to our expectations of significant fibrotic changes in lungs affected with BPD, there were predominant angio-dysplastic changes. At 35 weeks of gestation, as lungs are in saccular phase of development, exposure to risk factors leading to chronic lung disease (CLD) can predispose to development of capillary proliferation rather than fibrosis. Findings of calcifications in central nervous system were also of interest as toxoplasma and CMV are common congenital infections known to cause these findings rather than CRS.

The burden of congenital rubella in developing nations is much higher than developed countries, where annual incidence of congenital rubella is less than one per hundred thousand live births [7]. As per the latest WHO update, there has been a constant increase in number of cases of CRS over the last decade [8]. An infant with CRS can present with one or more of the following clinical features – cataract, congenital glaucoma, congenital heart disease (PDA or peripheral pulmonary stenosis), hearing impairment, hepatosplenomegaly, microcephaly, meningoencephalitis, radiolucent bone disease etc [9]. The clinical presentation of neonates with CRS depend on the gestational age of fetus at the time of maternal infection. Structural heart defects and eye abnormalities occur if maternal infection is acquired before eight weeks; while hearing defects are more common, if infections occurs till 18 weeks of pregnancy [10]. Maternal infection later in course of pregnancy might manifest with only intrauterine fetal growth restriction. The index neonate manifested with several clinical features suggestive of CRS and the diagnosis was also confirmed with presence of rubella specific IgM antibodies. In addition to the several common features of CRS, the index neonate also had calcifications in white matter, periventricular area and basal ganglia [11]. The most common finding in CRS patients is that of microcephaly as in our case and most consistent pathological findings are that of vascular destruction [12]. Intracranial calcifications are more frequently associated with congenital CMV and toxoplasmosis but are rarely reported in CRS [13].

There is no definitive management of CRS. The treatment is primarily symptomatic, based on organ system involvement [14]. Due to multiple organ involvement, a multidisciplinary team involvement is warranted [15]. The management of PDA was tricky in our case. PDA closure was warranted as the PDA size was big, CCF was not fully controlled with medical therapy and ventilator dependence [16]. However presence of concomitant pulmonary arterial hypertension and underlying BPD posed significant challenge for deciding surgical closure. Although PDA closure is contraindicated in severe pulmonary artery disease, it can be considered in the presence of reversible pulmonary disease [17]. PDA closure in neonates with PAH is recommended in cases with lower baseline pulmonary vascular resistance along with lower pulmonary arterial pressures, Qp:Qs ratio of >1.5, and a pulmonary arterial to systemic arterial pressure ratio <0.5 [18,19]. In this case, Qp: Qs ratio was 2:1 and pulmonary arterial pressure was 45 mm Hg (moderately elevated) prior to PDA ligation. Thus, after excluding irreversible pulmonary vascular disease, the surgical ligation was decided in multidisciplinary meeting.

The baby had persistent ventilator requirement after PDA closure, which worsened and eventually baby died of respiratory failure. BPD development is associated with the altered pro-inflammatory cytokines/chemokines profile as well as mediators of parenchymal and vascular remodeling. Waleh and colleagues reported that the surgical PDA closure led to increase in the expression of pro-inflammatory mediators (COX-2, TNF-alpha, and cells expressing CD14), and decreased expression of angiogenesis genes (angiopoietin-2 and TGF beta 3) [20]. Additionally, surgical PDA ligation decreased pulmonary alpha-ENaC containing channels expression, which is involved in trans-epithelial sodium transport related to clearance of alveolar fluid. Furthermore, these changes were observed in lung tissue taken from the side opposite to the ligation even after one week of surgery. Thus, in our case possibly the surgical ligation has escalated the underlying pro-inflammatory state which was already present due to BPD. The autopsy findings are also suggestive of the same. The histopathology of baby’s lung showed the areas of consolidation with neutrophilic inflammatory infiltrate without any evidence of bacteria, fungus or CMV. These findings clearly point towards a non-infectious pro-inflammatory state. It is also noteworthy that ante-mortem sepsis workup was negative. Similar observations have previously been reported by other authors [21]. These findings have clear implications in the clinical practice. The physicians should be aware of this phenomenon. All possible efforts should be done to provide gentler ventilation, improved nutrition to counter pro-inflammatory states in such setting. There is definite role of multidisciplinary meetings including neonatology, cardiology and cardiothoracic surgery to take decision in case to case basis considering physiology of these neonates. Further research is needed to optimize the timings of surgical ligation in such complex settings.

Acknowledgements: Prof Ashim Das for conducting autopsy, providing us with essential information regarding histopathological findings and modification and approval of pathology protocol.

Contributors: SK: clinical management of patient, preparation of initial draft of manuscript, AB: assisted in performing the autopsy, presentation of pathology protocol in CPC, provision and description of histo-pathological image findings; SSS: clinical management of patient, critically reviewed the manuscript for its contents, modification and approval of manuscript; AM: surgical management of patient and approval of manuscript; PM: clinical management of patient, modification and approval of manuscript.

Discussants: Clinical discussant pediatrician: Akhil Raj MS, Treating unit senior resident: Supreet Khurana; Cardiologist: Ashish Tiwari; Treating unit consultant: Shiv Sajan Saini; Pediatrician 1: Sumeet Dhawan; Pediatrician 2: Suresh Kumar Angurana; Pediatrician 3: Sanjay Verma, Clinical discussant pathologist: Akriti Bansal.

Funding: None; Competing interest: None stated.

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