Anaemia in solitary acyanotic ventricular septal defect in comorbid with pneumonia or pulmonary hypertension : A retrospective study of 75 paediatric cases

Ventricular septal defects (VSD) are the second commonest Background: congenital heart defects after bicuspid aortic valve. When left unrepaired, they can undergo spontaneous closure or elicit a spectrum of complications including pneumonia (PNA) or pulmonary hypertension (PH) with subsequent anaemia. In this retrospective study, we aim to establish and compare the prevalence of anaemia in patients with solitary acyanotic VSD in comorbid with PNA or PH. A total of 75 case files of patients with solitary acyanotic VSD, who Methods: underwent surgical closure or device occlusion had haemoglobin level analysed prior to the procedure. The cohort included patients with (history of) PNA and PH, and asymptomatic. The cohort included 27 females and 48 males with mean age and weight of 8.3±5.72 (3-24) months and 5.9±3.9 (2.7-17.8) kilograms, respectively. Depending on associated complication and age, the cohort was divided: PNA (A), PH (B) and Control (C); and (I) young children (≥3-6≤) and (II) older children (>6-≤24) months. We used 95 and 105 grams per litre as haemoglobin lower threshold level for (I) and (II), respectively. According to data analysis 27 patients (36%) in total had anaemia. Of Results: the anaemia cohort 16 (59.3%) had PNA, 9 (33.3%) PH and 2 (7.4%) were asymptomatic. Of the cohort, 42 were young children, with anaemia prevalence of 19/42 (45.2%), while 24.2% of the older children had anaemia. Intergroup ANOVA independent sample t-test was significant (p<0.05). In addition, intergroup test for haemoglobin: A/B (p>0.05), A/C (p<0.01), B/C Tukey HSD (p<0.01). Paediatric patients with acyanotic VSD in comorbid with PNA or Conclusion: PH are 8 and 4 times more susceptible to develop anaemia compared to asymptomatic counterparts. Susceptibility is even higher among young children (3-6months). However, a prospective study is needed to validate our findings.


Introduction
Anaemia is a common complication of a myriad medical conditions often met in general ward. Its etiology is complex and multifactorial, encompassing intrinsic and extrinsic factors [1][2][3] . Additionally, large intracardiac defects (cyanotic ventricular septal defects) can cause pulmonary vascular overload, infection and anaemia 2 . Further, pulmonay hypertension (PH) due to pulmonary vascular overload can elicit a cascade of events leading to poor quality of life, morbidity and mortality 4,5 . In the developing world with no heart surgery centers, pneumonia (PNA) due to large intracardiac defect(s) (cyanotic VSDs) is responsible for retardation, persistent morbidity and mortality 2,6 .
Nevertheless, the effect(s) of small to moderate VSD's on the occurrence of both PNA and PH amongst paediatric patients, especially infants has not been fully explored. Adults and older children may tolerate and survive the effects. However, infants with limited iron storage and supply (exclusive breast milk) may not with stand 7,8 . For these reasons, we hypothesize that paediatric patients with solitary acyanotic VSD coexisting with PNA or PH retain a risk of developing anaemia. In this retrospective study, we aim to establish the prevalence of anaemia in patients with solitary acyanotic VSD in comorbid with PNA or PH.

Case file details and classification
Between February 2014 and September 2018, 90 case files of patients with solitary acyanotic-VSD, who underwent either surgical or minimal invasive closure in our Department of Cardiac Surgery, Shandong Provincial Hospital Affiliate of Shandong University were primarily selected for this study.
However, only 75 case files met study criteria, which included patients with recurrent (history of...) pneumonia of proven PNA by chest radiography with positive bacterial culture of trans-tracheal aspirate or polymerize chain reaction from nasopharyngeal swab. Pulmonary hypertension diagnosis was echocardiography based, except in 5 patients from PNA group, who presented in heart failure state. Excluded from this study were 15 files of patients: 7, sickle cell; 4, β-Thalassemia; 4, blood transfusion.
Data variables Statistical analysis. Data was analysed using SPSS-IBM-21 software (one-way-ANOVA) and all statistics expressed as mean ± standard deviation. Intergroup haemoglobin level was compared using independent samples student's t-test. Statistical comparison of proportions was analysed using Tukey HSD Test, and the probability value of less than 0.05 was considered significant. Patient proportions are expressed in number and percentage (n, %).

Amendments from Version 1
On behalf of co-authors, I wish to pass our heartfelt gratitude to the reviewer for his professionalism and sincerity, and the raised points deserve clarification. The main differences between the old and revised version are put forward as per reviewer concerns: The introduction section has been summarized and repair techniques clarified with references. Diagnosis modality of PH has been clarified and reference cited. While we understand the variation in etiologies of pneumonia, cardio-pulmonary hemodynamic changes provide a fertile ground for persistent infections (dereferences available). SPSS version has been given in full. Discussion has been expanded by two paragraphs and references provided. Conclusion has been furnished. We have changed the addresses to suit institutional requirement Its our sincere hope that our revised version answers the few but cardinal concerns raised by our reviewer. Your time and dedication to reviewing our manuscript is highly appreciated.

Results
In this case study, we used hemoglobin reference ranges based on age as follow: (I) young children (95-135) and (II) older children were (105-135) gram per liter, as per local protocol. According to data analysis reflected in Table 3

Discussion
Anaemia, defined as haemoglobin (Hb) concentration below the 5 th percentile for age at sea-level, is a common complication of a myriad medical conditions often met in the general ward 1 .
Its aetiology is complex and multifactorial, encompassing intrinsic and extrinsic factors. Both pneumonia (PNA) and pulmonary hypertension (PH) due to cyanotic congenital heart defect (CHD) have been implicated in the occurrence of anaemia 2,3,6 . In addition, sporadic reports linking anaemia to PNA or PH amongst patients with acyanotic ventricular septal defect (VSD) have been publish.
VSD is the second commonest CHD after bicuspid aortic valve 4,9 , and solitary cases account for almost 20%. One of the most common defects associated with elevated pulmonary artery pressure is a large VSD. Elevated pulmonary artery pressure in CHD can be due to pulmonary hyper-circulation, pulmonary vasoconstriction, and pulmonary vascular disease, either alone or in combination. In an infant, despite pulmonary pressure being at systemic level, pulmonary vascular resistance is low; therefore, minor shunt easily elicits hyper-circulation 2,4,6,10 .
PH, defined as mean pulmonary artery pressure of ≥25mmHg at rest as measured by cardiac catheterization in children aged ≥3months, is a serious disorder with a high morbidity and mortality rate 2 . Blood shunt may cause haemolysis due to shear stress and produce free haemoglobin, which in turn depletes nitric oxide leading to endothelial dysfunction, vasoconstriction, pulmonary oedema and hypoxia. Furthermore, haemolysis produces arginase, which converts L-arginine to ornithine; therefore, bypassing nitric oxide production 2,8,11 .
PNA as defined by Ozdemir and colleagues is a serious reason for morbidity and mortality in children (≤2years) with hemodynamic significant VSD 5,12 . Both PNA and PH share a common interface; inflammation, homostasis, hypoxia, and subsequent upregulation of erythropoiesis 7,13 . Prolonged upregulated erythropoiesis in young children with low iron store and limited iron supplement leads to anaemia 1 . In addition, microangiopathic hemolytic anaemia in CHD and PH has been reported 3 , a complication commonly observed in primary PH. Unlike in PH, Mycoplasma Pneumonia and Plebsiella are known to cause anaemia in PNA 14-16 .
This study shows that acyanotic VSD within mean sizes: 1.2±0.3 and 0.89±0.2 centimeters, thus, defect measured from the left ventricular septal side are prone to pulmonary vascular infection/dysfunction. Both transthoracic and transoesophageal echocardiography were employed in the diagnosis and delineation of VSD and PH 17 . Although, right heart catheterization (RHC) is regarded as gold standard, our center favor echocardiography due to less vascular and technical challenges, especially in clinically compromised infants. According to 2018 guidelines issued by British Society of Echocardiography, aforementioned is recommended and RHC superiority is insignificant 18 . Young infants (3moths old) with small defects were considered for closure if defect(s) showed no trait of spontaneous closure in the presence of symptoms after 2 consective follow-up at 2-month interval. Apical VSDs (Swiss cheese) seldom achieved closure, hence, inclusion.
Although this study is not focused on closure techniques, suffice to mention that surgical and minimally invasive. i.e. 1. perventricular 19 , 2. peratrial 20 and 3. percutaneous (<10%) device closure were used. Surgical was employed when device implation proved futile, while percutaneous was limited to a small potion due to vascular limitation and possible complications. Recent publication cited small weight and age as recipe for complication during percutaneous intervention 21 . In addition, surgical technique was employed with utmost care due to bypass related complication and blood transfusion complications related in PH subjects. Its worthy mentioning that this study does not include prevalence of anaemia post intervention. Symptomatic subjects became asymptomatic at dismissal, and both aforementioned and asymptotic ones progressively improved anthropometric parameters during sequential follow-ups 22 .

Conclusion
Paediatric patients without hematologic disorders, diagnosed with hemodynamic significant acyanotic VSD in comorbid with pneumonia or pulmonary hypertension are 8 and 4 times susceptible to develop anaemia compared to asymptomatic counterparts. Susceptibility is even high amongst young children (3-6 months). However, a long post closure follow-up study is required to exclude possibly missed intrinsic (genetical/gastro-intestinal) and extrinsic (economical) etiologies and validate findings.

Ethical considerations
The Shandong Provincial Hospital Ethics Committee approved this study, and waived individual patient consent as the study was based on archived data.

Open Peer Review
The statistical analysis section must be reorganized. Please find an example below: 'Statistical analysis. Statistical analyses were performed using the Statistical Package for the Social Sciences Statistics (SPSS), version 21 for Macintosh (IBM, Armonk, NY). Categorical variables were reported as frequency and percentage. Continuous variables are represented as mean with standard deviation or median with the range depending on normality of distribution. Intergroup haemoglobin level was compared using independent samples student's t-test. Statistical comparison of proportions was analyzed using Tukey HSD test. A p-value < 0.05 was considered statistically significant. All reported p values are two-sided.' ○ I don't recommend indexing with this form before all comments are addressed appropriately.
○ the authors to mention minimum and maximum values especially in each of the 3 groups for the PHT row as it may seems that some of Group A patients had PHT.
Statistics: SPSS version must reported. 3. Table 1 and 2 should be briefly described in the results sections. 4. Table 3 must be just reported in the text. 5.
The first 4 paragraphs of the discussion are pure literature review without any relevant discussion with the study results. In fact excessive review could impose more stratified results: should authors report the etiology on the PNA since the described the possible implications of infectious organisms in anemia. Therefore, discussion should focus in some part on the prevalence of anemia in repaired VSD (and if possible discussing the timing of closure and its possible effect of the results) while highlighting what is has been previously written by the authors.

6.
No changes are needed for the limitations and conclusions section. 7.

If applicable, is the statistical analysis and its interpretation appropriate? Yes
Are all the source data underlying the results available to ensure full reproducibility? Partly Are the conclusions drawn adequately supported by the results? Partly https://doi.org/10.5256/f1000research.19583.r44745 expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above.
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