Mucopolysaccharidosis IIIB and mild skeletal anomalies: coexistence of NAGLU and CYP26B1 missense variations in the same patient in a Chinese family

Jinliang Li
Han Xie
Yuwu Jiang
Mucopolysaccharidosis IIIB and mild skeletal anomalies: coexistence of NAGLU and CYP26B1 missense variations in the same patient in a Chinese family
10.1186/s12881-018-0562-4C A S E R E P O R T Open Access Case presentation: We found an infant with biallelic variation both in NAGLU-compound heterozygous c.1843C > T (p. R615C) and c.1224C > A (p. H408Q) as well as in CYP26B1-compound heterozygous c.529G > A (p. E177K) and c. 525C > A (p. H175Q). All variations were novel but predicted pathogenicity according to American College of Medical
Background: Sanfilippo type B syndrome (mucopolysac-charidosis type IIIB; MPS IIIB) is an autosomal recessive lysosomal storage disorder. It is caused by a critically reduced α-2-acetamido-2-deoxy-D-glucoside acetamidodeoxy glucohydrolase (α-N-acetylglucosaminidase or NAGLU) activity. Recently, an autosomal recessive disorder of skeletal dysplasia associated with CYP26B1 was reported in three families, in which the patients were all homozygous variations. However, the co-occurrence of two rare diseases in a person is very rare. Here, we reported one patient with two novel pathogenic missense variations in NAGLU and CYP26B1. Genetics and Genomics (ACMG) guidelines. The main phenotypes of the infant were quite different from those previously reported, and some were combinations of the two rare diseases, including epilepsy, early onset epileptic encephalopathy, hypermyotonia, skull deformity, dilatation of the lateral ventricles and premature closure of fontanel. His NAGLU enzyme activity was significantly decreased. Conclusions: NAGLU and CYP26B1 mutations were related to MPS IIIB and skeletal dysplasia, respectively. Here, we first reported the pathogenic mutations of two genes concurrent in one patient, which not only expands the phenotype and genotype spectra of NAGLU and CYP26B1, but more importantly indicates the possibility of simultaneous occurrence of two rare diseases in one patient. This interesting finding should be attributed to the use of whole exome sequencing (WES), which indicates that we should be aware of the importance of WES in diagnosing rare diseases.

Background

Mucopolysaccharidosis type III (MPS III) or Sanfilippo syndrome includes five subtypes, each of which results from a specific impaired lysosomal enzyme, including sulfamidase (MPS IIIA); α-N-acetylglucosaminidase (NAGLU, MPS IIIB); heparan acetyl CoA α-glucosaminide N-acetyltransferase (MPS IIIC); N-acetylglucosamine 6-sulfatase (MPS IIID) and N-glucosamine 3-O-sulfatase (MPS IIIE) [1]. The prenatal and early post-natal development are usually normal.

The clinical features are similar in all the subtypes, which are characterised by progressive mental deterioration and behavioural problems with more or less prominent dysmorphic facial features.

Thus far, only two studies reported a syndrome of craniosynostosis, cranium bifidum, multiple skeletal abnormalities, and dilatation of the lateral and third ventricles due to biallelic variations in CYP26B1 [2,3]. In the two families reported in the first article described, a more severe phenotype led to intrauterine death in the three siblings, while in a single patient in the second family, the phenotype was milder to allow survival until five months of age.

In the present study, we reported a case with biallelic variations both in NAGLU and CYP26B1, whose main phenotypes were quite different from those previously reported, and with some combination of the two rare diseases, including epilepsy, early onset epileptic encephalopathy, hypertonia, skull deformity, dilatation of the lateral ventricles and premature closure of fontanel. Furthermore, NAGLU enzyme activity was significantly decreased.

Case presentation

A 10-day-old new born was admitted to our unit due to limb hypertonia, pulling up with head lag, and repeated neonatal convulsions. From the second day after birth, he was presented with hypertonia, and four days later, he developed repeated convulsions.

On physical examination, umbilical hernia, inguinal hernia, kyphosis, hypertonia and skull deformity were found (Figs. 1a, b, c). He did not show facial coarseness, hypertrichosis and other positive signs reported to be related to NAGLU and CYP26B1 gene variation.

He was a full-term baby without any problem during his birth. He had an elder sister who died at the 30th day after birth. When the sister was still a foetus, her ultrasound examination indicated that her brain lateral ventricle was enlarged. She was naturally delivered and a full-term baby. From the 10th day after birth, she had lethargy and feeding difficulty but no seizure. Facial asymmetry was observed, and her brain magnetic resonance imaging (MRI) results indicated skull deformity, and the bilateral structure was asymmetrical (Figs. 2a, b).

After admission, the boy was treated with vitamin B6 and vitamin B12, but the treatment was ineffective. At the 16th day after birth, he was administered with Topamax. With the increase of the dosage, the convulsions decreased transiently. Unilateral facial muscles began to twitch at the 26th day after birth, which lasted several seconds every time, and the longest was 10 s. The convulsions and the facial twitches disappeared after the first month of life but spasms occurred. Levetiracetam and Vigabatrin were then gradually added.

Video-electroencephalograph showed gradual deterioration, followed by hypsarrhythmia and spasms ( Table 1).

The routine and biochemical laboratory test results of cerebrospinal fluid (CSF) were normal. Microbe examinations of the CSF, urine and blood were negative. The screening test results for metabolic diseases in blood and urine were normal. Mitochondrial genes were normal. The infant was diagnosed with epilepsy and early onset epileptic encephalopathy (EOEE).

The WES in family trios was conducted. The amplified DNA was captured using GenCap exome capture kit (MyGenostics). The enrichment libraries were sequenced on Illumina HiSeq X ten sequencer for paired read at 150 bp. Finally, we found the compound heterozygous c.1843C > T (p.R615C) and c.1224C > A (p.H408Q) variants in NAGLU. Through verification by Sanger sequencing, we confirmed that c.1843C > T was paternal and c.1224C > A was maternal (Fig. 3). The variations of the two sites were both novel. According to ACMG guidelines [4], both the variations were categorized to be "pathogenic"(c.1843C > T met PS3, PM1, PM2, PM3, PP3 and PP4; c.1224C > A met PS3, PM1, PM2, PM3, PP4).

At the same time, we found another compound heterozygous c.529G > A (p.E177K) and c.525C > A (p.H175Q) variants in CYP26B1. Through verification by Sanger sequencing, we confirmed that c.529G > A was paternal and c.525C > A was maternal (Fig. 3). Both the variations were categorized to be "likely pathogenic"(c.529G > A met PM2, PM3, PP1, PP2 and PP4; c.525C > A met PM2, PM3, PP1, PP2 and PP4) according to ACMG guidelines. Fortunately, the sister's blood specimen is available, and her CYP26B1 were compound heterozygous, which was the same as the younger brother. However, the sister had only one heterozygous mutation of NAGLU. No pathogenic Then we measured the lysosomal enzyme activity in this boy and found that NAGLU enzyme activity was significantly decreased (0.3 nmol/g/min, reference range, 7.3-32.4 nmol/g/min). Now, the boy is 8 months old, and his weight is 9. 25 kg. The height and head circumference seem normal. However, he experienced frequent epileptic spasms and repeated respiratory infection are happening to him. Anterior fontanelle has been closed. Facial asymmetry and skull deformity were apparent (Figs. 1d, e). Cry is rare and weak, and sleep disturbance is obvious. The reexamined brain MRI (Figs. 2c, d) showed bilateral ventricle enlargement, asymmetry of brain structure, decreased white matter and thin corpus callosum.

Discussion and conclusions

Five subtypes of MPS III and their clinical features are similar among all the subtypes. Pre-natal and early stages of post-natal development are usually normal. The severe phenotype is usually more common, and it can be divided into three phases [5]. The first phase may begin between the ages of 1 and 3 years, and the patients mainly manifested as delayed cognitive development and/or aggressive behavioural problems, as well as hindered speech development [6]. The second phase starts at approximately 3-4 years of age and is followed by progressive cognitive deterioration and the emergence of behavioural difficulties and sleep disturbances [7]. The third phase begins, usually in the teenage years, with the onset of severe dementia and motor function decline. All motor functions start to decline, and the patient deteriorates into a vegetative state. Death usually occurs early in the third decade of life. However, patients with attenuated phenotypes surviving for 69 years were reported [8]. The patient in our study indicates that the age of onset of MPS IIIB may be early in the neonatal period, and early onset epileptic encephalopathy may have been related to NAGLU biallelic mutations because no other mutation was found in trio WES analysis, including the EOEE-related genes, and epilepsy did not occur in the older sister who had only one heterozygous mutation of NAGLU.

Diagnostic delay in the population of MPS III is very common, particularly in patients with a slowly progressing or mild phenotype. The boy in this study can obtain an earlier diagnosis because of the help of Nextgeneration sequencing (NGS), especially WES. This study found not only the variations of NAGLU, but also compound heterozygous variations of CYP26B1, which was associated with skeletogenesis. With the development of NGS technique, gene testing becomes more convenient. When encountering a rare disease of unknown origin, we should perform a comprehensive genetic test to confirm the diagnosis.

NAGLU contains six exons, and the cDNA coding for a 720-amino acid mature protein has six potential N- Fig. 2 Head MRI of the siblings. a, b Head MRI of the sister at 25 days old showed skull deformity and asymmetrical bilateral structure. c, d Bilateral ventricle enlargement, asymmetry of brain structure, decreased white matter and thin corpus callosum of the proband at 8 months old Table 1 Evolution of process of V-EEG Age V-EEG

days

Moderately abnormal neonatal EEG with moderately delayed maturity of electricity of the brain (alternate and discrete graphics during QS period). Multiple focal and interhemispheric sharp wave and slow wave activated asynchronously, which is primarily in the left temporal region. Unilateral or bilateral front head was distributed with intermittent theta rhythms.

days

Abnormal infant EEG with multiple focal spike waves, sharp waves, and irregular slow waves discharged non-synchronously, which mainly at the right central-parietal region. The lateral occipital areas showed intermittent low wave amplitude and fast activities. There were multiple focal episodes during waking and sleeping stages, and the suspected local myoclonus and spasms.

months and days

Indicated multifocal spike waves, sharp waves, spike slow wave complex, sharp slow wave complex and slow waves which primarily distributed at the frontal and temporal regions and detected multiple spasms during waking and sleeping stages.

months and 18 days

Hypsarrhythmia which were primarily at occiput glycosylation sites [9]. No common pathogenic variations in MPS IIIB was observed, and most of the known pathogenic variations in MPS IIIB patients occur at low frequencies or not more than once [10]. Over 160 variations in NAGLU gene by far have been identified as disease-causing, and most of them were missense/nonsense type (HGMD Professional). The variations we found in NAGLU in this study were both novel. According to ACMG guidelines, both the variations were categorized to be "pathogenic". Variations in NAGLU can cause MPS IIIB or Charcot-Marie-Tooth disease (CMT). However, the siblings in the present study had no any phenotypes of CMT disease. Moreover, NAGLU enzyme activity was significantly decreased. Although some unusual presentations were observed, such as neonatal epileptic seizure and very early developmental delay, according to the main phenotypes, enzyme activity assay and pathogenic NAGLU mutations, the diagnosis of MPS IIIB is definite. CYP26B1 encodes a cytochrome P450 enzyme responsible for the catabolism of retinoic acid (RA) in a very restricted fashion during embryonic development [3]. Both the deficiency and excess of RA have considerable impacts during vertebrate embryogenesis and organogenesis. Some studies showed that abnormalities of limb and craniofacial morphogenesis can result from exposure to exogenous RA at critical periods during foetal skeletal development [11]. In the three siblings and an unrelated Turkish patient with radiohumeral fusions and other skeletal and craniofacial anomalies, Laue et al. identified homozygous missense variations in the CYP26B1 gene (R363L and S146P) [11]. The severe phenotypes of the three siblings led to intrauterine death, and the baby in the second family survived up to five months of age. However, the patient in another article survived into adulthood, and she had dilatation of brain ventricles [3]. In this study, compound heterozygous variations of CYP26B1 were found in the two siblings. The phenotypes of the older sister exhibited facial asymmetry, encephalomalacia of the white matter, and asymmetrical bilateral structure. The proband of this study showed facial asymmetry, skull deformity, ventricle enlargement, asymmetry of brain structure and premature closure of fontanel. According to the malformations of the skull and the ACMG guidelines, those compound heterozygous variations of CYP26B1 were probably pathogenic.

In summary, in this case, the phenotypes of MPS IIIB were developmental delay, sleep disturbance, epilepsy, repeated respiratory infections, navel hernia, inguinal hernia and decreased NAGLU enzyme activity; whereas, phenotypes related to variant CYP26B1 included facial asymmetry, skull deformity, bilateral ventricle enlargement and asymmetry of brain structure, which were presented in both kids. Kyphosis, in this case, might be the common manifestation of both diseases.

The estimated incidence of MPS IIIB is very low and varied among the countries. Some studies found that MPS IIIA was the most frequent type in Northern Europe [10,12,13], whereas MPS IIIB is more frequent in Southern Europe [1,14,15]. The most frequent type in Chinese Taiwan was MPS IIIB, according to a study (0.28 per 100,000 live births) [16]. However, epidemiologic investigations about this disease in mainland China were not conducted. Skeletal dysplasia associated with the variations of CYP26B1 might be rare, and only two related reports have been published.

Given the lack of a specific therapy, the present efforts are generally relieving strategies. However, promising therapies for MPS III are continuously being developed and evaluated, such as enzyme replacement therapy (ERT), gene therapy, genistein and substrate deprivation therapy. The ability for the enzyme NAGLU to be secreted from the lysosomes within cells has provided the basis for the foundation of therapies for MPS IIIB. However, the major obstacle of ERT is the inability of the enzyme to cross the blood brain barrier. The use of gene therapy is an extension of ERT as it attempts to introduce the coding sequence of the protein of interest into the cells of the patient using a viral vector, rather than synthesize it and purify it from another host [1].

The co-occurrence of two rare diseases in a patient is very rare. We diagnosed a patient with MPS IIIB due to compound heterozygous pathogenic variations of NAGLU, as well as skeletal dysplasia, associated with the compound heterozygous pathogenic variations of CYP26B1. We also found two novel missense variations in NAGLU and CYP26B1 respectively. This report will provide a better understanding of MPS IIIB due to NAGLU variation and skeletal dysplasia associated with CYP26B1. The simultaneous discovery of two rare diseases in one patient reminded us to value the importance of WES in precisely diagnosing complex cases.  Availability of data and materials All data and materials mentioned in this manuscript, except patient's private information, can be promptly available to readers by a reasonable request to the corresponding author.

Authors' contributions YJ: study design, analysis and revision of the manuscript; JL: follow-up patient's information, analyse WES data and draft of the first manuscript; and HX: follow-up patient's information and contribute to Sanger sequencing. All authors read and approved the final manuscript.

Ethics approval and consent to participate This study was approved by the Ethics Committee of Peking University First Hospital. The parents of the patients signed written informed consents and agreed themselves and their children to participate in this study and allowed the use of the relevant data and information for scientific research.

Consent for publication

Written informed consent was obtained from the parents for publication of this case report and any accompanying tables/images. A copy of the written consent is available for review by the Editor of this journal.

Fig. 1
1Serial photographs of the patient. a Hypertonia at one month old, b inguinal hernia and c kyphosis at 100 days old. d Skull deformity and e facial asymmetry at 8 months old mutation of known EOEE-related genes had been found in this trios WES.

Fig. 3
3Pedigree of the family. Arrow indicates the proband. Brown represents variants of CYP26B1, and yellow represents variants of NAGLU. The variants c.1843C > T (p.R615C) in NAGLU and c.529G > A (p.E177K) in CYP26B1 were paternal, while the variants c.1224C > A (p.H408Q) in NAGLU and c.525C > A (p.H175Q) in CYP26B1 were maternal

Abbreviations ACMG: American College of Medical Genetics and Genomics; CMT: Charcot-Marie-Tooth disease; CSF: Cerebrospinal fluid; ERT: Enzyme replacement therapy; MPS III: Mucopolysac-charidosis type III; MRI: Magnetic resonance imaging; NAGLU: α-Nacetylglucosaminidase; NGS: Next-generation sequencing; RA: Retinoic acid; WES: Whole exome sequencingAcknowledgmentsThis work was supported by the company of MyGenostics for providing HGMD professional database and DNA sequencing results. We also thank the families who participated in this study.

Funding
This work was supported by the National Natural Science Foundation of China (Grant number: 81501123), Beijing Natural Science Foundation (Grant number: 7151010), Beijing Municipal Science & Technology Commission (Grant number: Z161100000216133, Z161100004916169), Beijing Institute for Brain Disorders Foundation (grant number: BIBDPXM2014_014226_000016), Beijing Key Laboratory of Molecular Diagnosis and Study on Pediatric Genetic Diseases (Grant number: BZ0317) and National Key Research and Development Program of China (Grant number: 2016YFC1306201).
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Sanfilippo syndrome: causes, consequences, and treatments. A O Fedele, Appl Clin Genet. 8Fedele AO. Sanfilippo syndrome: causes, consequences, and treatments. Appl Clin Genet. 2015;8:269-81.

Craniosynostosis and Multiple Skeletal Anomalies in Humans and Zebrafish Result from a Defect in the Localized Degradation of Retinoic Acid. K Laue, H-M Pogoda, Daniel Philip, B Van Haeringen, A Alanay, Y Von Ameln, S Rachwalski, M Morgan, T , Gray Mary, J , Breuning Martijn, H , Am J Hum Genet. 895Laue K, Pogoda H-M, Daniel Philip B, van Haeringen A, Alanay Y, von Ameln S, Rachwalski M, Morgan T, Gray Mary J, Breuning Martijn H, et al. Craniosynostosis and Multiple Skeletal Anomalies in Humans and Zebrafish Result from a Defect in the Localized Degradation of Retinoic Acid. Am J Hum Genet. 2011;89(5):595-606.

Biallelic mutations in CYP26B1: A differential diagnosis for Pfeiffer and Antley-Bixler syndromes. J E Morton, S Frentz, T Morgan, A J Sutherland-Smith, S P Robertson, Am J Med Genet A. 17010Morton JE, Frentz S, Morgan T, Sutherland-Smith AJ, Robertson SP. Biallelic mutations in CYP26B1: A differential diagnosis for Pfeiffer and Antley-Bixler syndromes. Am J Med Genet A. 2016;170(10):2706-10.

Standards and guidelines for the interpretation of sequence variants: a joint consensus recommendation of the American College of Medical Genetics and Genomics and the Association for Molecular Pathology. S Richards, N Aziz, S Bale, D Bick, S Das, J Gastier-Foster, W W Grody, M Hegde, E Lyon, E Spector, Gen Med. 175Richards S, Aziz N, Bale S, Bick D, Das S, Gastier-Foster J, Grody WW, Hegde M, Lyon E, Spector E, et al. Standards and guidelines for the interpretation of sequence variants: a joint consensus recommendation of the American College of Medical Genetics and Genomics and the Association for Molecular Pathology. Gen Med. 2015;17(5):405-24.

Inborn metabolic diseases. J-M Saudubray, G Van Den Berghe, J H Walter, SpringerBerlin Heidelberg5th ed.Saudubray J-M, Van den Berghe G, Walter JH. Inborn metabolic diseases. 5th ed. Verlag Berlin Heidelberg: Springer; 2012.

Clinical and genetic spectrum of Sanfilippo type C (MPS IIIC) disease in The Netherlands. G J Ruijter, M J Valstar, J M Van De Kamp, R M Van Der Helm, Durand S Van Diggelen, O P Wevers, R A Poorthuis, B J Pshezhetsky, A V Wijburg, F A , Mol Genet Metab. 932Ruijter GJ, Valstar MJ, van de Kamp JM, van der Helm RM, Durand S, van Diggelen OP, Wevers RA, Poorthuis BJ, Pshezhetsky AV, Wijburg FA. Clinical and genetic spectrum of Sanfilippo type C (MPS IIIC) disease in The Netherlands. Mol Genet Metab. 2008;93(2):104-11.

Cognitive development in patients with Mucopolysaccharidosis type III (Sanfilippo syndrome). M J Valstar, J P Marchal, M Grootenhuis, V Colland, F A Wijburg, Orphan J Rare Dis. 643Valstar MJ, Marchal JP, Grootenhuis M, Colland V, Wijburg FA. Cognitive development in patients with Mucopolysaccharidosis type III (Sanfilippo syndrome). Orphan J Rare Dis. 2011;6:43.

Mucopolysaccharidosis type IIIB may predominantly present with an attenuated clinical phenotype. M J Valstar, H T Bruggenwirth, R Olmer, R A Wevers, F W Verheijen, B J Poorthuis, D J Halley, F A Wijburg, J Inherit Metab Dis. 336Valstar MJ, Bruggenwirth HT, Olmer R, Wevers RA, Verheijen FW, Poorthuis BJ, Halley DJ, Wijburg FA. Mucopolysaccharidosis type IIIB may predominantly present with an attenuated clinical phenotype. J Inherit Metab Dis. 2010;33(6):759-67.

Cloning and expression of the gene involved in Sanfilippo B syndrome (mucopolysaccharidosis III B). B Weber, L Blanch, P R Clements, H S Scott, J J Hopwood, Hum Mol Genet. 56Weber B, Blanch L, Clements PR, Scott HS, Hopwood JJ. Cloning and expression of the gene involved in Sanfilippo B syndrome (mucopolysaccharidosis III B). Hum Mol Genet. 1996;5(6):771-7.

Cumulative incidence rates of the mucopolysaccharidoses in Germany. F Baehner, C Schmiedeskamp, F Krummenauer, E Miebach, M Bajbouj, C Whybra, A Kohlschutter, C Kampmann, M Beck, J Inherit Metab Dis. 286Baehner F, Schmiedeskamp C, Krummenauer F, Miebach E, Bajbouj M, Whybra C, Kohlschutter A, Kampmann C, Beck M. Cumulative incidence rates of the mucopolysaccharidoses in Germany. J Inherit Metab Dis. 2005; 28(6):1011-7.

Craniosynostosis and multiple skeletal anomalies in humans and zebrafish result from a defect in the localized degradation of retinoic acid. K Laue, H M Pogoda, P B Daniel, A Van Haeringen, Y Alanay, S Von Ameln, M Rachwalski, T Morgan, M J Gray, M H Breuning, Am J Hum Genet. 895Laue K, Pogoda HM, Daniel PB, van Haeringen A, Alanay Y, von Ameln S, Rachwalski M, Morgan T, Gray MJ, Breuning MH, et al. Craniosynostosis and multiple skeletal anomalies in humans and zebrafish result from a defect in the localized degradation of retinoic acid. Am J Hum Genet. 2011;89(5):595-606.

Mucopolysaccharidosis type III (Sanfilippo disease) in Sweden: clinical presentation of 22 children diagnosed during a 30-year period. G Malm, J E Mansson, Acta Paediatr. 998Malm G, Mansson JE. Mucopolysaccharidosis type III (Sanfilippo disease) in Sweden: clinical presentation of 22 children diagnosed during a 30-year period. Acta Paediatr. 2010;99(8):1253-7.

The frequency of lysosomal storage diseases in The Netherlands. B J Poorthuis, R A Wevers, W J Kleijer, J E Groener, J G De Jong, S Van Weely, K E Niezen-Koning, O P Van Diggelen, Hum Genet. 1051-2Poorthuis BJ, Wevers RA, Kleijer WJ, Groener JE, de Jong JG, van Weely S, Niezen-Koning KE, van Diggelen OP. The frequency of lysosomal storage diseases in The Netherlands. Hum Genet. 1999;105(1-2):151-6.

Prevalence of lysosomal storage diseases in Portugal. R Pinto, C Caseiro, M Lemos, L Lopes, A Fontes, H Ribeiro, E Pinto, E Silva, S Rocha, A Marcao, Eur J Hum Genet. 122Pinto R, Caseiro C, Lemos M, Lopes L, Fontes A, Ribeiro H, Pinto E, Silva E, Rocha S, Marcao A, et al. Prevalence of lysosomal storage diseases in Portugal. Eur J Hum Genet. 2004;12(2):87-92.

Biochemical and molecular analysis of mucopolysaccharidoses in Turkey. S Emre, M Terzioglu, T Coskun, A Tokath, I Ozalp, V Muller, J Hopwood, Turk J Pediatr. 441Emre S, Terzioglu M, Coskun T, Tokath A, Ozalp I, Muller V, Hopwood J. Biochemical and molecular analysis of mucopolysaccharidoses in Turkey. Turk J Pediatr. 2002;44(1):13-7.

Incidence of the mucopolysaccharidoses in Taiwan. H Y Lin, S P Lin, C K Chuang, D M Niu, M R Chen, F J Tsai, M C Chao, P C Chiu, S J Lin, L P Tsai, Am J Med Genet A. 1495Lin HY, Lin SP, Chuang CK, Niu DM, Chen MR, Tsai FJ, Chao MC, Chiu PC, Lin SJ, Tsai LP, et al. Incidence of the mucopolysaccharidoses in Taiwan, 1984- 2004. Am J Med Genet A. 2009;149a(5):960-4.


Trauma Cases and Reviews
January 13, 2016

International Library
MDNoboru Harada email@example.com
Department of Surgery
Hiroshima Red Cross Hospital and Atomic Bomb Survivors Hospital
HiroshimaJapan

Ken Shirabe
Department of Surgery and Medical Science
Kyushu University
FukuokaJapan

Shinji Itoh
Department of Surgery and Medical Science
Kyushu University
FukuokaJapan

Hideaki Uchiyama
Department of Surgery and Medical Science
Kyushu University
FukuokaJapan

Motoyuki Yamagata
Department of Surgery
Saiseikai Karatsu Hospital
SagaJapan

Fumiaki Kishihara
Department of Surgery
Nakatsu Municipal Hospital
OitaJapan

Takashi Maeda
Department of Surgery
Hiroshima Red Cross Hospital and Atomic Bomb Survivors Hospital
HiroshimaJapan

Nao Kinjo
National Hospital Organization Kyushu Cancer Center
FukuokaJapan

Kensaku Sanefuji
Oita Red Cross Hospital
OitaJapan

Yosuke Kuroda
Matsuyama Red Cross Hospital
EhimeJapan

Kazutoyo Morita
Fukuoka City Hospital
FukuokaJapan

Tomoharu Yoshizumi
Department of Surgery and Medical Science
Kyushu University
FukuokaJapan

Toru Ikegami
Department of Surgery and Medical Science
Kyushu University
FukuokaJapan

Yo-Ichi Yamashita
Department of Surgery and Medical Science
Kyushu University
FukuokaJapan

Yoshihiko Maehara
Department of Surgery and Medical Science
Kyushu University
FukuokaJapan

Department of Surgery
Hiroshima Red Cross Hospital and Atomic Bomb Survivors Hospital
1-9-6 Senda-machi Naka-ku730-8619HiroshimaJapan

Trauma Cases and Reviews
2469-5777January 13, 2016C04E5AC8B4CC5D9A64B5B8508A23827EReceived: December 04, 2015: Accepted: January 11, 2016:SOFASeptic shockSurgical critically illPrognostic factors
Purpose: This study was designed to identify factors prognostic of in-hospital deaths of surgical, critically ill patients with sepsis and to evaluate the effects of treatments for sepsis on in-hospital deaths.Methods: This retrospective cohort study involved 103 patients with sepsis who were hospitalized in the surgical intensive care units of 20 hospitals.Clinical, microbiologic, and laboratory factors, as well as treatments, were compared between patients who survived hospitalization and those who died in-hospital.Results:The in-hospital mortality and septic shock rates were 24.3% and 19.4%, respectively.Multivariate logistic regression analysis showed that Sequential Organ Assessment (SOFA) score was the only independent predictor of in-hospital death (P = 0.027).Receiver operating characteristic curve analysis of in-hospital death showed that the optimal SOFA score cutoff at admission to the surgical intensive care unit was 8, with in-hospital death rate being significantly higher in the 22 patients with SOFA score > 8 than in the 81 patients with SOFA score ≤ 8 (P = 0.0039).Cox regression analyses by inverse probability treatment weighting to control for selection bias showed that in-hospital death rates were not significantly altered by treatment with intravenous immunoglobulins, renal replacement therapy, or endotoxinabsorbing therapy using polymyxin B.Conclusions: SOFA score may be prognostic of in-hospital deaths of surgical critically ill patients with sepsis.SOFA score > 8 was associated with a significantly higher in-hospital death rate and should be regarded as a cut-off for intensive treatment in surgical patients with sepsis.

Introduction

Sepsis, defined as infection-induced systemic inflammatory response syndrome (SIRS), is the leading cause of death in noncardiac critically ill patients [1].In the United States, nearly 200,000 deaths per year are attributed to sepsis [2].Worldwide, as many as 20 million people may experience sepsis annually, with a mortality rate of about 35% [3].Sepsis involves multiple mechanisms, including the release of cytokines and the activation of the complement, coagulation and fibrinolytic systems [4].

The first internationally accepted guidelines to improve outcomes in patients with severe sepsis and septic shock were adopted in 2004 [5,6] and updated in 2008 [7] and 2012 [8,9].Current guidelines recommend a specific anatomical diagnosis of infection as rapidly as possible, with intervention for source control started within the first 12 hours after diagnosis, if feasible [8,9].Few studies to date have described the treatment of surgical, critically ill patients with sepsis [10,11,12].The results of treatments of sepsis in surgical patients requiring other types of treatment, including burn care, catheter drainage of the source of infection and abdominal surgery under general anesthesia, remain unclear.It is therefore of interest to clarify factors prognostic of survival in surgical, critically ill patients with sepsis.

Moreover, although adjunctive therapies, including intravenous immunoglobulins (IVIG) [13,14], renal replacement therapy [15,16], and endotoxin-absorbing therapy using polymyxin B (PMX) [17], have been reported to reduce the risks of death in septic patients, these treatments have not been universally accepted.Inverse probability of treatment weighting (IPTW) was therefore used to balance the underlying distributive covariates among patients who did and did not receive each adjunctive therapy.IPTW weights the samples using propensity score to reduce the confounding that frequently occurs in cohort studies of the effects of treatment on outcome, and enables estimation of marginal or population-average treatment effects [18].

This study was therefore designed to identify factors prognostic of in-hospital deaths in surgical, critically ill patients with sepsis and to evaluate the effects of treatment of sepsis on patient survival.

Materials and Methods

This retrospective cohort study involved 110 patients who were hospitalized with sepsis in the surgical intensive care units of 20 hospitals affiliated with the Department of Surgery and Medical Science, Graduate School of Medical Sciences, Kyushu University, between January 2012 and December 2013 (Supplemental Table 1).Patients lacking adequate clinical data were excluded.

Sepsis was defined as an infection with SIRS, defined as the occurrence of at least two of the following criteria: (1) body temperature > 38˚C or < 36˚C, (2) heart rate > 90 beats per minute, (3) respiratory rate > 20 breaths a minute or PaCO 2 < 32 mmHg, and (4) white blood cell count > 12,000/ mm 3 or < 4000/mm 3 or < 10 % immature forms [4].Blood samples were drawn when patients first fulfilled the criteria for SIRS.Septic shock was defined as sepsisinduced hypotension, consisting of systolic blood pressure below 90 mmHg, which persisted despite adequate fluid resuscitation [8].Ileus was defined as any impairment, arrest, or reversal of the normal flow of intestinal contents toward the anal canal.

All patients diagnosed with sepsis and admitted to the surgical intensive care unit before and after surgery were enrolled in this study.Demographic and clinical data retrieved from their medical records included sex, age, underlying disease, location of the primary infection, bacterial species, Acute Physiology and Chronic Health Evaluation II (APACHE II) score, Sequential Organ Assessment (SOFA) score, blood pressure, heart rate, hematocrit, white blood cell (WBC) count, platelet count, C-reactive protein (CRP), total bilirubin, serum creatinine, fibrinogen, prothrombin activity, fibrinogen degradation products (FDP), D-dimer, and type of surgical intervention for sepsis, including removal or drainage of sites of infection.

Treatment of sepsis, including surgical intervention and antimicrobial therapy, was initiated as soon as possible according to the Surviving Sepsis Campaign guidelines [8,9].If primary antibiotics were not effective, as determined by WBC count, CRP concentration, and clinical signs such as high fever, IVIG (5 g/kg body weight) was administered for 3 days along with antibiotics [13,14].Patients with septic shock who experienced acute renal failure, defined as anuria and/or serum creatinine concentration > 4.0 mg/dl, were administered renal replacement therapy, including continuous hemodiafiltration (CHDF) or intermittent hemodialysis, to protect renal function and remove inflammatory cytokines [15,16].If hemodynamic stability could not be restored by adequate fluid resuscitation and vasopressor therapy, patients were administered PMX [16].In addition, mechanical ventilation was administered to patients with sepsisinduced acute lung injury (ALI)/acute respiratory distress syndrome (ARDS).Fluid resuscitation and catecholamine were administered to maintain circulation in patients with septic shock, thus preventing the development of a more critical condition that could result in multiple organ failure (MOF) and death [8,9].

Statistical Analysis

For continuous variables, nonparametric analyses were performed using Wilcoxon rank-sum tests.Categorical variables were compared using chi-squared or Fisher's exact tests.The Kaplan-Meier method was used to construct cumulative survival curves.To identify factors independently predictive of in-hospital death, the factors found to be significant on univariate analysis were assessed by multivariate logistic regression analyses.The SOFA score cut-off value for in-hospital death was calculated using the receiver operator characteristic (ROC) curve method [19].

IPTW analysis was performed to overcome possible biases from differences in distribution among patients who did and did not receive each treatment [18,20].Propensity scores by IPTW were calculated using a Cox regression model to predict the probability of each patient receiving each treatment on the basis of eight clinical variables: age; sex; APACHE II score; SOFA score; positive (vs.negative) blood culture; and treatment including IVIG, renal replacement therapy, and PMX.Following balancing by IPTW, the between group differences in in-hospital death rates were evaluated by the log-rank test.Statistical analyses were performed using the R statistical programming environment (the Comprehensive R Archive Network, http://cran.md.tsukuba.ac.jp) and JMP 9.0 software (SAS Institute, Cary, NC).Four basic R programming packages were used for IPTW analyses: Rcmdr, survival, RcmdrPlugin, survival, and Epi.Statistical significance was defined as a P value < 0.05.

Results

Of the 110 patients enrolled initially, seven were excluded owing to uncertainties about initial recognition of SIRS.The primary causes of sepsis in the 103 analyzed patients included acute peritonitis (n = 50), acute cholecystitis (n = 17), acute cholangitis (n = 9), ileus (n = 7), gangrene of the lower limbs (n = 4), liver abscess (n = 2), postoperative catheter infection (n = 2), postoperative acute enteritis (n =2), postoperative pneumonitis (n = 2), postoperative urinary tract infection (n = 2), postoperative infectious endocarditis (n = 1), extensive burn (n = 1), abdominal compartment syndrome (n = 1), esophageal perforation (n = 1), postoperative leakage of esophageal anastomosis (n = 1), and postoperative pancreas fistula (n = 1).Table 1 shows the relationships between the primary cause of sepsis and surgical treatment in these patients.Of the 103 patients, 60 (58.3%) underwent surgery under general anesthesia, and 24 ( The mean age of the 103 patients (65 men and 38 women) was 72.3 ± 1.2 years (range, 37-99 years).Twenty-five (24.3 %) patients died in-hospital and 20 (19.4 %) experienced septic shock.The mean hospital stay was 36.7 ± 2.5 days (range, 2-100 days).The mean APACHE II and SOFA scores at admission were 15.5 ± 0.8 and 5.9 ± 0.4, respectively.

Univariate analysis identified four variables as risk factors for inhospital deaths in these patients: heart failure as a comorbidity (P = 0.036), APACHE II score (P = 0.002), SOFA score (P < 0.0001), and prothrombin activity (P = 0.006).Of the 78 in-hospital survivors, nine had chronic renal failure, including two on hemodialysis, with no differences in rates of chronic renal failure between survivors and non-survivors (P = 0.109).Multivariate logistic regression analysis showed that SOFA score was the only independent predictor of inhospital death (P = 0.027, odds ratio 1.24; 95% confidence interval 1.03-1.52,Table 2 and Table 3).

ROC curve analysis and determination of the area under the ROC curve (AUC) showed that SOFA score was effective in distinguishing surgical, critically ill patients with sepsis who did and did not survive in-hospital.The AUC of SOFA score was 0.76.ROC curve analysis for in-hospital deaths showed that the optimal SOFA score cutoff at admission to the surgical intensive care unit was 8 (sensitivity; 0.64, 1-specificity; 0.25, Figure 1).The in-hospital death rate was significantly higher in the 22 patients with SOFA score > 8 than in the 81 patients with SOFA score ≤ 8 (P = 0.0039, Figure 2).

The microorganisms isolated from these patients are shown in table 4. Gram-positive microorganisms were cultured from 37.9 % of these patients; Gram-negative organisms from 34.0 %; and Candida species from 2.9%.In-hospital deaths were not associated with either Gram-positive or Gram-negative organisms.Escherichia coli was associated with the main source of infection in patients with septic shock (P = 0.021), but not with in-hospital deaths.None of the other microorganisms was associated with either septic shock or inhospital death rate.Antimicrobial treatment was administered to all patients.Only six patients (7.8%) received inadequate antimicrobial treatment, but this was not related to in-hospital mortality (P = 0.620).Of these six patients, three were infected with methicillin-resistant Staphylococcus aureus (MRSA), two with Candida glabrata, and one with Enterococcus faecium; four (67%) of these patients received empirical treatment with carbapenems.

We also assessed the effects of adjunctive treatment with IVIG, renal replacement therapy, and PMX in patients with sepsis.Following IPTW using eight variables: age, sex, positive blood culture, APACHE score, SOFA score, and treatment with IVIG, renal replacement therapy, and PMX, we found that the weighted in-hospital death rates were similar in patients who did and did not receive each treatment (Table 5).

Discussion

Sepsis is the leading cause of death of surgical patients pre-and postoperatively [21][22][23].Once the sources of infection are identified, surgical intervention plus antibiotics are recommended as treatment [8,9].However, the in-hospital death rate remains high, even following these guidelines [8,9].The mortality rate in surgical patients with sepsis was reported to be 25.5%, similar to the rate in our study (24.3%) [22].However, the effectiveness of adjunctive treatments, such as IVIG, renal replacement therapy, and PMX, remains unclear.Moreover, few reports have assessed surgical treatments associated with sepsis, risk factors associated with mortality of surgical, critically ill patients, and microbes associated with sepsis [23].Although epidemiology and outcomes before and after admission to the surgical intensive care unit have been analyzed [10,11], prognostic factors and treatment results in septic surgical patients remain unclear.The duration of SIRS has been reported to be significantly prognostic of sepsis in medical and surgical units (P = 0.015) [22].Our study showed that SOFA score may be potentially prognostic of in-hospital deaths in surgical, critically ill patients with sepsis.These results are in accordance with a study that found an association between SOFA score and outcomes in patients admitted to the intensive care unit [24].Serial measurements of SOFA score during the first week were very useful in predicting the outcome of sepsis, whereas APACHE II scores on the day of admission were not reliable in predicting mortality [25].Abbreviations: HR: hazard ratio; CI: confidence interval; PMX: endotoxinabsorbing therapy using polymyxin B.  SOFA score is simple and objective, allowing the calculation of the number of dysfunctional organ systems and their severity among six organ systems (respiratory, coagulatory, liver, cardiovascular, renal, and neurologic) [26].Moreover, SOFA score can measure dysfunction of individual and aggregate organs [27].SOFA score has been used to evaluate patients with multiple organ dysfunction syndromes (MODS) and their survival [26].Organ failure may worsen outcomes in surgical critically ill patients with sepsis.Indeed, we found that in-hospital death rates were significantly higher in patients with SOFA scores > 8 than ≤ 8 at admission.SOFA score at admission may therefore suggest the type of primary treatment, including adjunctive therapy.In surgical patients, organ dysfunction at sites other than that of surgery may be more strongly associated with death than transient vital signs and serum chemistry included in the APACHE II score on admission [28].

Although our univariate analysis found that APACHE II score was prognostic of in-hospital deaths, this finding was not observed on from multivariate analyses.APACHE II score is used in intensive care units to assess disease severity.However, SOFA score may be superior to APACHE II score in predicting in-hospital deaths of surgical, critically ill patients with sepsis, because SOFA score, which includes platelet count, may objectively evaluate failure of individual and aggregate organs [26].Coagulopathy, such as low platelet count, has been associated with sepsis-induced disseminated intravascular coagulation (DIC) [29] and may lead to a severe disease condition or in-hospital death.A prospective survey in Japan also demonstrated that SOFA and DIC scores were consistently higher in nonsurvivors than survivors on the day of admission and 3 days later [23].

Microbial assessment found that E. coli infection was frequently associated with septic shock, including in patients with communityacquired bloodstream infection [30].Because none of the other microorganisms assayed was associated with septic shock or inhospital death and only 7.8% of patients received inadequate antimicrobial treatment, surgical, critically ill patients with sepsis should be treated with a broad empirical antimicrobial agent such as a carbapenem.Infections with MRSA and Candida species are of particular concern.The frequency of invasive fungal infections in developed countries has increased because of advances in medical management [31].Although invasive Candidiasis is frequently intraabdominal in critically ill surgical patients, prompt antifungal therapy and adequate source control may yield good outcomes [32].Resistance to azoles, particularly fluconazole, should be considered when starting an empirical treatment [32].

Although we attempted to assess the effects of adjunctive treatment, including IVIG, renal replacement therapy, and PMX, in patients with sepsis by IPTW analysis, we found that none had a significantly positive effect on this patient population.However, our study had several limitations.First, relatively few patients received each type of adjunctive treatment.Second, this was a retrospective cohort study assessing the causes of sepsis in Japanese patients.Thus, the results were not externally validated and were limited to patients of Japanese ethnicity.Third, we could not plan a prospective study to clarify the effectiveness of each adjunctive therapy in surgical critically ill patients with sepsis.However, well-designed randomized controlled studies and/or meta-analyses may result in external validity, as well as assessing these adjunctive treatments in patients of varying ethnicity.

In conclusion, this study found that SOFA score may be prognostic of in-hospital deaths of surgical, critically ill patients with sepsis.SOFA > 8 was associated with significantly higher in-hospital death rate and may be a cut-off for the necessity of intensive treatment in these patients.

Figure 1 :
1
Figure 1: Receiver operating characteristic (ROC) curve analysis of SOFA score distinguishing in-hospital survivors and non-survivors among surgical, critically ill patients with sepsis.The area under the ROC curve (AUC) was 0.76.

Figure 2 :
2
Figure 2: Kaplan-Meier survival curves of surgical, critically ill patients with sepsis having SOFA scores ≤ 8 and > 8.The 22 patients with SOFA score > 8 had a significantly higher in-hospital death rate than the 81 patients with SOFA score ≤ 8 (P = 0.0039).

Table 1 :
1
23.3%) underwent surgical drainage under local anesthesia.Adjunctive Hospitals included in this Study
Supplemental National Hospital Organization Kyushu Cancer CenterKyushu Central Hospital of the Mutual Aid Association of Public SchoolTeachersSaiseikai General HospitalFukuoka City HospitalFukuoka Higashi Medical CenterSocial Insurance Nakabaru HospitalMunakata Medical Association HospitalSaiseikai Yahata General HospitalSteel Memorial Yahata HospitalShinnakama HospitalAso Iizuka HospitalTagawa Municipal HospitalOnga HospitalOita Red Cross HospitalOita Prefectural HospitalNakatsu Municipal HospitalSaiseikai KaratsuImari Arita Kyoritsu Hospital

Table 2 :
2
Univariate analysis of risk factors for in-hospital deaths among surgical, critically ill patients with sepsis Results reported as mean ± standard error
VariablesHospitalHospitalP-valueSurvivorsNon-survivors(n = 78)(n = 25)Age (years)72.5 ± 1.471.7 ± 2.30.869Gender (Male)47 (60.3%)18 (72.0%)0.347Body weight (kg)52.1 ± 1.357.0 ± 3.60.299ComorbiditiesDiabetes mellitus11 (14.1%)4 (16.0%)0.755Hypertension18 (23.1%)3 (12.0%)0.271Heart failure4 (5.1%)5 (20.0%)0.036Chronic renal failure9 (11.5%)0 (0.0%)0.109Neoplasm21 (26.9%)9 (36.0%)0.450Positive blood culture30 (38.5%)7 (28.0%)0.473Localization of infectionRespiratory2 (2.6%)0 (0.0%)1.000Heart1 (1.3%)0 (0.0%)1.000Upper digestive tract11 (14.1%)2 (8.0%)0.730Hepatobiliary-pancreas25 (32.0%)8 (32.0%)1.000Lower digestive tract32 (41.0%)13 (52.0%)0.362Urinary tract1 (1.3%)1 (4.0%)0.428Soft tissue6 (7.7%)1 (4.0%)1.000APACHE II score (points)14.0 ± 0.820.4 ± 2.00.002SOFA score (points)5.0 ± 0.48.8 ± 0.8< 0.0001Body temperature (centigrade)37.6 ± 0.237.0 ± 0.30.145Heart rate (/minute)101.8 ± 2.0106.6 ± 4.90.508Hematocrit (%)33.6 ± 0.835.1 ± 1.60.263White blood cell count (/mm 3 )13920 ± 1184 15296 ± 99680.661Platelet (×10 4 /mm 3 )20.4 ± 1.317.2 ± 2.40.162C-reactive protein (mg/dl)16.2 ± 1.314.4 ± 3.20.222Total bilirubin (mg/dl)1.3 ± 0.12.3 ± 0.80.205Serum creatinine (mg/dl)1.7 ± 0.21.9 ± 0.30.147Fibrinogen (mg/dl)476.9 ± 30.9351.0 ± 72.20.054Prothrombin activity (%)66.6 ± 2.755.2 ± 3.40.006FDP (μg/ml)24.9 ± 3.762.2 ± 34.00.983D-dimer (μg/ml)10.6 ± 1.517.4 ± 7.50.480Surgical intervention66 (84.6%)18 (72.0%)0.234

Table 3 :
3
Multivariate analysis of risk factors for in-hospital deaths among surgical, critically ill patients with sepsis
VariablesMultiple logistic regression analysisOdds ratio95% ConfidenceP-valueIntervalHeart failure1.710.25-11.90.575APACHE II score (points)1.020.94-1.110.617SOFA score (points)1.241.03-1.520.023Prothrombin activity (%)0.990.96-1.020.521

Table 1 :
1
Causes of sepsis and types of treatment in surgical critically ill patients with sepsis
Cause of sepsisPatientsPrimary therapyAdjunctive therapySurgeryDrainageIVIGRRTPMX(n = 103)(n = 60)(n = 24)(n = 33)(n = 18)(n = 21)Acute peritonitis50424191115Acute cholecystitis17510411Acute cholangitis904310Ileus761110Lower limbs gangrene440211Liver abscess202000Postop. catheter infection202000Postop. acute enteritis200111Postop. Pneumonitis200101Postop. UTI201000Postop. infectious endocarditis100000Extensive burn110111Abd. compartment syndrome110111Esophageal perforation110000Postop. leakage of esophageal Anastomosis100000Postoperative pancreas fistula100000Abbreviations: IVIG: intravenous immunoglobulins; RRT: renal replacement therapy; PMX: endotoxin-absorbing therapy using polymyxin B; UTI: urinary tractinfection; Postop: postoperative; Abd: abdominal.

Table 4 :
4
Microorganisms associated with in-hospital deaths among surgical, critically ill patients with sepsis
Gram positive3180.34450.211Enterococcus species103110.454Staphylococcus aureus1020.72610.453Streptococcus species710.67620.651Clostridium species210.5710.481Staphylococcus epidermidis10110.454Bacillus species10101Corynebacterium species01101Gram negative25100.476100.116Escherichia coli1160.35270.021Bacteroides species61110.454Klebsiella pneumoniae22111Pseudomonas aeruginosa210.5710.481Enterobacter cloacae20101Citrobacter freundii10101Proteus vulgaris10101Candida species210.5701Unknown206-5-

Table 5 :
5
Analyses of the effects of adjunctive treatments of surgical, critically ill patients with sepsis by inverse probability of treatment weighting
TreatmentsMultiple Cox regression analyses for in-hospital deathnAdjusted HR95% CIP-valueIntravenous immunoglobulins332.8341.085-7.4040.059Renal replacement therapy180.8550.361-2.0250.733PMX212.3560.872-6.3710.229
AcknowledgmentsThe authors thank Dr. Y. Fujinaka (Matsuyama Red Cross Hospital), Dr. R. Nakanishi (Matsuyama Red Cross Hospital), Dr. T. Iso (Social Insurance Nakabaru Hospital), Dr. Y Ikeda (National Hospital Organization Kyushu Cancer Center), Dr. T, Rikimaru (Munakata Medical Association Hospital), Dr. K. Nomoto (Saiseikai Yahata General Hospital), Dr. H. Hasegawa (Saiseikai Yahata General Hospital), Dr. S. Maehara (Matsuyama Red Cross Hospital), Dr. E. Adachi (Oita Prefectural Hospital), Dr. K. Tada (Oita Prefectural Hospital), Dr. M. Noda (Oita Prefectural Hospital), Dr. K. Umeda (Oita Prefectural Hospital), Dr. K. Yamamoto (Imari Arita Kyoritsu Hospital), Dr. H. Sonoda (Imari Arita Kyoritsu Hospital), Dr. G. Anegawa (Imari Arita Kyoritsu Hospital), Dr. T. Nishizaki (Matsuyama Red Cross Hospital), Dr. Y. Soejima (Matsuyama Red Cross Hospital), Dr. T. Honbo (Matsuyama Red Cross Hospital), Dr. K. Iwaki (Oita Red Cross Hospital), Dr. S. Kai (Oita Red Cross Hospital), Dr. K. Fukuzawa (Oita Red Cross Hospital), Dr. T. Ezaki (Fukuoka Higashi Medical Center), Dr. T. Ohga (Fukuoka Higashi Medical Center), Dr. H. Uehara (Nakatsu Municipal Hospital), Dr. T. Okada (Nakatsu Municipal Hospital), and Dr. Y. Nakaji (Saiseikai Karatu Hospital) for helping with data collection.Conflict of InterestAll authors declare no conflicts of interest.Compliance with Ethical RequirementsThe study protocol was approved by the ethics committees of all participating hospitals, and study information was disclosed to the patients or their survivors by members of the Department of Surgery and Medical Science, Graduate School of Medical Sciences, Kyushu University.
The natural history of the systemic inflammatory response syndrome (SIRS). A prospective study. M S Rangel-Frausto, D Pittet, M Costigan, T Hwang, C S Davis, JAMA. 2731995

Epidemiology of severe sepsis in the United States: analysis of incidence, outcome, and associated costs of care. D C Angus, W T Linde-Zwirble, J Lidicker, G Clermont, J Carcillo, Crit Care Med. 292001

Surviving the first hours in sepsis: getting the basics right (an intensivist's perspective). R Daniels, J Antimicrob Chemother. 662011

M M Levy, M P Fink, J C Marshall, E Abraham, D Angus, SCCM/ESICM/ACCP/ATS/SIS International Sepsis Definitions Conference. 2003. 200131

Surviving Sepsis Campaign guidelines for management of severe sepsis and septic shock. R P Dellinger, J M Carlet, G Ramsay, J L Zimmerman, J L Vincent, Crit Care Med. 322004

Surviving Sepsis Campaign guidelines for management of severe sepsis and septic shock. R P Dellinger, J M Carlet, G Ramsay, J L Zimmerman, J L Vincent, Intensive Care Med. 302014

Surviving Sepsis Campaign: international guidelines for management of severe sepsis and septic shock. R P Dellinger, M M Levy, J M Carlet, J Bion, M M Parker, Intensive Care Med. 342008. 2008

Surviving sepsis campaign: international guidelines for management of severe sepsis and septic shock: 2012. R P Dellinger, M M Levy, S A Webb, R J Beale, J L Vincent, Crit Care Med. 4122013

Surviving Sepsis Campaign: international guidelines for management of severe sepsis and septic shock. R P Dellinger, M M Levy, A Rhodes, D Annane, H Gerlach, Intensive Care Med. 392013. 2012

Epidemiology and outcome following post-surgical admission to critical care. A Rhodes, R P Moreno, B Metnitz, H Hochrieser, P Bauer, Intensive Care Med. 372011

Incidence and risk factors for sepsis in surgical patients: a cohort study. A C Elias, T Matsuo, C M Grion, L T Cardoso, P H Verri, J Crit Care. 272012

How to treat infections in a surgical intensive care unit. J De Waele, BMC Infect Dis. 141932014De Bus L

Intravenous immunoglobulin for treating sepsis and sepotic shock. M M Alejandria, M A Lansang, L F Dans, J B Mantaring, Cochrane Database Syst Rev. 2002. CD001090

Meta-analysis: intravenous immunoglobulin in critically ill adult patients with sepsis. A F Turgeon, B Hutton, D A Fergusson, L Mcintyre, A A Tinmouth, Ann Intern Med. 1462007

A randomized clinical trial of continuous versus intermittent dialysis for acute renal failure. R L Mehta, B Mcdonald, F B Gabbai, M T Pascual, A Farkas, Kidney Int. 2469-57776062001

. Harada, Trauma Cases Rev. 2262016

Effects of different doses in continuous veno-venous haemofiltration on outcomes of acute renal failure: a prospective randomised trial. C Ronco, R Bellomo, P Homel, M Dan, P Piccinni, Lancet. 3562000

Effectiveness of polymyxin B-immobilized fiber column in sepsis: a systematic review. D N Cruz, M A Perazella, R Bellomo, M De Cal, N Polanco, Crit Care. 11R472007

The performance of different propensity score methods for estimating marginal hazard ratios. P C Austin, Stat Med. 322013

Lusted LB (1971) Signal detectability and medical decision-making. Science. 171

Blood transfusion is associated with recurrence of hepatocellular carcinoma after hepatectomy in Child-Pugh class A patients. N Harada, K Shirabe, T Maeda, H Kayashima, T Ishida, World J Surg. 392015

Secondary peritonitis -evaluation of 204 cases and literature review. S K Doklestic, D D Bajec, R V Djukic, V Bumbasirevic, A D Detanac, J Med Life. 72014

The duration of SIRS before organ failure is a significant prognostic factor of sepsis. H Sugita, Y Kinoshita, H Baba, Int J Emerg Med. 5442012

Epidemiology of severe sepsis in Japanese intensive care units: a prospective multicenter study. H Ogura, S Gando, D Saitoh, N Takeyama, N Yamashita, J Infect Chemother. 202014

Application of the sequential organ failure assessment (SOFA) score to bacteremic ICU patients. C Routsi, M Pratikaki, C Sotiropoulou, E Platsouka, V Markaki, Infection. 352007

Utility of SOFA and APACHE II score in sepsis in rural set up MICU. S Desai, J D Lakhani, J Assoc Physicians India. 612013

The SOFA (Sepsis-related Organ Failure Assessment) score to describe organ dysfunction/failure. On behalf of the Working Group on Sepsis-Related Problems of the European Society of Intensive Care Medicine. J L Vincent, R Moreno, J Takala, C K Reinhart, P M Suter, Intensive Care Med. 221996

Prognostic factors of mortality in patients with community-acquired bloodstream infection with severe sepsis and septic shock. A Artero, R Zaragoza, J J Camarena, S Sancho, R González, J Crit Care. 252010

Is worsening multiple organ failure the cause of death in patients with severe sepsis?. J L Vincent, D R Nelson, M D Williams, Crit Care Med. 392011

A randomized, controlled, multicenter trial of the effects of antithrombin on disseminated intravascular coagulation in patients with sepsis. S Gando, D Saitoh, H Ishikura, K Mashiko, T Tsubota, Crit Care. 7R2972013

The epidemiology of intra-abdominal flora in critically ill patients with secondary and tertiary abdominal sepsis. J De Ruiter, J Weel, E Manusama, W P Kingma, P H Van Der Voort, Infection. 372009

Genomic Analysis of Single Nucleotide Polymorphisms Asp299Gly and Thr399Ile in Japanese Patients with Invasive Aspergillosis. K Aki, Y Okubo, H Nanjo, T Ishiwatari, H Masuda, Jpn J Infect Dis. 682015

Epidemiology of invasive candidiasis in a surgical intensive care unit: an observational study. G Aguilar, C Delgado, I Corrales, A Izquierdo, E Gracia, BMC Res Notes. 84912015


META-ANALYSIS Adding Blood to St Thomas Solution Does Not Improve Mortality in Pediatric Cardiac Surgery: A Meta-analysis of a Homogenous Population

Mark Awori firstname.lastname@example.com
University of Nairobi

Nikita Mehta
University of Nairobi

Naomi Kebba
University of Nairobi

Enoch Makori
University of Nairobi

META-ANALYSIS Adding Blood to St Thomas Solution Does Not Improve Mortality in Pediatric Cardiac Surgery: A Meta-analysis of a Homogenous Population
10.4314/aas.v17i2.460 Correspondence to: Dr. Mark Awori;
Background: Cardioplegia is the gold standard for providing ideal operating conditions while effecting myocardial protection. Some studies suggest that adding blood to St Thomas cardioplegia solution improves efficacy; this is generally accepted as true. However, the few meta-analyses conducted on children have pooled heterogeneous populations; this raises concern about the validity of their conclusions. Methods: PUBMED, the Cochrane Library and Google Scholar were searched systematically until March 2019 using the search terms "cardioplegia"; "myocardial protection"; "pediatric" "pediatric"; "children"; "infants"; "neonates". Full text articles were examined if abstracts revealed that the studies possibly contained a blood cardioplegia arm and a crystalloid cardioplegia arm. Studies were included in the meta-analysis if they had a 4:1 blood cardioplegia arm and a St Thomas solution arm. Meta-analysis was performed using Meta-Mar software.Results:The search retrieved 423 articles; 5 were included in the meta-analysis, representing 324 patients. The risk ratio for operative mortality was 0.77(95% CI 0.24-2.5; p=0.66). Little evidence was seen of heterogeneity of the pooled patients. Conclusion: Adding blood to St Thomas cardioplegia solution did not improve in-hospital operative mortality; this may have implications for use of blood cardioplegia.

Introduction

The attainment of optimal cardiac surgical results requires the heart to be still, flaccid, lying in a bloodless field and protected from irreversible ischemic damage. The gold standard for achieving this is the use of cardioplegia, which employs three principle concepts for myocardial protection: chemical arrest, hypothermia and additional protection such as manipulating ionic imbalance (1). The merits and demerits of the use of various types of cardioplegia have been examined; however, ambiguity still remains regarding which is best (2,3). Although the exact mechanism of action remains unknown, the addition of blood to a crystalloid cardioplegia base is thought to improve myocardial protection by prolonging aerobic metabolism (4). In comparing clinical outcomes between blood and nonblood cardioplegia, most randomized clinical trials have been on adult patients; pediatric trials are few (2). Some systematic reviews with meta-analyses have been conducted; however, the populations included have been significantly heterogeneous (5). Meta-analysis is a powerful and useful tool when correctly applied; elemental to proper application is the analysis of populations that are as homogenous as possible (6). Available studies examining surgical outcomes related to cardioplegia type are too heterogeneous for conducting meta-analyses (3). To optimize homogeneity, answers to the following questions may guide decisions on which studies to include in a meta-analysis: 1. Is the study on pediatric or adult patients? 2. Did cardioplegia achieve depolarized or hyperpolarized arrest? 3. Was blood added to the cardioplegia solution? 4. Was warm or cold cardioplegia used? 5. What cardioplegia dosing regimen was used? 6. Were other modifications to or variants of cardioplegia used?

Studies for inclusion should differ in as few parameters as possible, preferably in only one (6). The current study aimed to determine whether blood-based cardioplegia was associated with a lower in-hospital operative mortality than non-blood-based cardioplegia in pediatric cardiac surgery. The results of this study may have implications for the use of blood cardioplegia in general.

Methods

Search strategy and selection criteria

Results of search 1 and search 2 combined using "AND"

Titles and abstracts were interrogated and full-text articles were obtained when the abstract showed a possibility that the study included a group of pediatric patients exposed to blood cardioplegia and a group of pediatric patients exposed to non-blood cardioplegia (St Thomas solution). The outcome of interest was inhospital operative mortality. Non-English language studies and non-human studies were excluded. Only randomized clinical trials were included. The six questions mentioned above were used to refine the choice of studies for inclusion: only studies that used intermittent cold cardioplegia (blood or non-blood) were included and only after all authors were in agreement about their inclusion. Two reviewers performed the search and data extraction; when necessary, discrepancies were resolved by discussion and consensus with a third reviewer. Figure 1 shows the search flow.

Statistical analysis

The meta-analysis used Meta-Mar free online metaanalysis service (V1.8.0) (8); risk ratio (RR) was used for analysis. Fixed and random effects moDels were employed; I2-statistic was used to assess for heterogeneity (6).

Results

The search yielded 423 articles; 11 relevant full-texts were obtained after examining titles and abstracts. Six full-text articles were rejected on grounds of unacceptable clinical heterogeneity (according to the six questions). Table 2 shows the important details of the 5 studies included in the meta-analysis and Table 3 lists the full text articles rejected.    Table 4 shows the key findings of the meta-analysis and Figure 2 the forest plot.

Discussion

Del Nido solution is composed of 1-part blood and 4 parts base solution (i.e., it is 20% blood). It is generally administered as single dose and Delivers arrest for up to 90 minutes. Del Nido solution, and blood cardioplegia in general, is the preferred option for some practitioners in pediatric cardiac surgery based on evidence such as that provided by a relatively recent randomized clinical trial by Talwar et al. (20). This study found that the Del Nido solution improved postoperative outcomes in cardiac output, duration of mechanical ventilation, troponin-1 release and electron microscopic evidence of myocardial damage (20). Although these are probably more sensitive outcome measures, we choose not to examine metabolic markers of myocardial protection as they were not measured in a homogenous fashion. For similar reasons we did not examine postoperative inotrope use. Simplicity improves efficiency and reduces the chance of error. In this regard, minimizing the steps required to execute any stage of cardiac surgery has a cumulative effect, and marginal gains such as these, have been shown to result in significant improvements in outcomes in other disciplines (21). If adding blood to cardioplegia does not improve outcomes, it should not be added. The ultimate measure of the success of cardiac surgery is long-term mortality. Before long-term mortality can be considered, operative mortality must be examined. Although outcomes such as postoperative cardiac index and release of metabolic markers are considered to be more sensitive markers of the degree of myocardial preservation, they cannot be assumed to translate into improvements in the ultimate outcome. Furthermore, the best quality evidence on the effect of adding blood to cardioplegia, on operative outcome, must be documented.

Our work attempts to document the available evidence of the highest quality in pediatric cardiac surgery. As we worked to achieve this, we remained aware that the immature heart responds differently to ischemia and that the transition phase is around 3 months of age (22). Results of clinical studies should be easily applied to patient care; we chose to examine risk ratio (RR) instead of odds ratio (OR) as it is a more intuitive parameter for clinicians (23). We found that there was no significant difference, in in-hospital operative mortality, between blood and non-blood (St Thomas crystalloid solution) cardioplegia in pediatric cardiac surgery. A relatively large meta-analysis examining 5576 patients from 36 RCTs found that blood cardioplegia was not superior to crystalloid (non-blood) cardioplegia for myocardial protection with respect to in-hospital operative mortality (2). However, the meta-analysis included a highly heterogenous group of patients; no distinction was made between children and adults; use of warm cardioplegia and cold cardioplegia; and use of continuous cardioplegia Delivery, intermittent Delivery and single dose Delivery. Two relatively recent meta-analyses (5,24) comparing blood and non-blood cardioplegia in pediatric cardiac surgery found no difference in clinical outcomes following the use of either strategy. Of particular interest to us was that Mylonas et al. found no difference in 30day mortality (5). Fang et al. examined metabolic markers of myocardial protection, length of postoperative ventilation and length of intensive care unit (ICU) stay; they found that these outcomes were essentially the same irrespective of strategy used (18). The study by Mylonas was solely on pediatric patients; however, it included studies using warm cardioplegia, cold cardioplegia, intermittent dosing and single dosing, and it also included studies with other significant potential confounders. For example, it included a study by Mimic et al. (25) which had a high concentration of glucose in one arm (25). The study by Fang et al. was more clinically homogenous, but it included a study that compared warm blood cardioplegia with cold non-blood cardioplegia (24). The heterogeneity in these two meta-analyses could have affected the pooled outcome. When the six questions are used to assess study inclusion in these two meta-analyses, it can be argued that the studies included were too heterogeneous for meta-analysis. This sentiment was echoed recently by Drury et al. who concluded that cardioplegia studies in pediatric patients were too heterogeneous for meaningful meta-analyses (3). Study heterogeneity may be fundamentally classified as heterogeneity caused by chance and heterogeneity caused by systematic factors; the degree of heterogeneity may simply be assessed clinically or statistically (6). Ensuring clinical homogeneity is a pragmatic method for appropriately selecting studies for inclusion in metaanalyses; the six questions mentioned above facilitate this for cardioplegia strategy comparison (26). It is standard practice to assess statistical heterogeneity when conducting meta-analyses. In the current study, we sought to minimize the clinical heterogeneity as much as possible by only including studies using intermittent cold cardioplegia. For the same reason, we excluded studies that looked at endpoints other than mortality. By doing so, we tried to reduce any confounding resulting from differences in cardioplegia dosing, cardioplegia temperature, and cardioplegia additives other than blood.

In particular, we included only studies that had a blood cardioplegia ratio of 4:1; blood to crystalloid. Statistically, our studies heterogeneity was low (I 2 = 0%). The validity of this finding is strengthened by the similar RR results obtained by the fixed and random effect moDels in our study (27). Meta-analyses are susceptible to bias; of particular concern are selection bias and small study bias (28).

Although it is impossible to determine with certainty the extent to which bias has affected pooled results, a sensible attempt to quantify bias using a funnel plot is performed when appropriate (29). We did not construct a funnel plot as its benefit is questionable when less than 10 studies are included in the analysis (29). We used the meta-mar online calculator which has been validated against RevMan and is significantly easier to use (8).

Four out of five of the studies that we included had no mortality (zero-event) in at least one of their arms; three studies had no mortality in both arms. A continuity correction is usually used for analysis when there is no event (e.g. mortality) in an arm. It has been shown that a continuity correction of 0.5 reduces bias when there is no event (e.g. mortality) in both arms, particularly if there is a greater likelihood of there being no significant difference in outcomes between arms (27). A large observational study of over 7000 pediatric patients (greater than 3000 patients in each arm) found no difference in in-hospital operative mortality between Del Nido (single-dose blood-based cardioplegia) and St Thomas II solution (crystalloid cardioplegia) (30); in view of this we opted to use a continuity correction of 0.5. It has been recommended that zero-event studies be included in meta-analyses when it is likely that there is no difference between interventions. We think that the findings of this large observational study provide further justification for including zero-event (i.e. no mortality) studies in our meta-analysis (27). Modi et al. suggested that blood cardioplegia offers better results in cyanotic patients (11). There was no significant difference in the proportion of cyanotic patient, in our study, when all cases, except those in the study by Romolo et al. (13), were pooled together. Although Romolo et al. did not indicate the proportion of cyanotic patients in each arm, their study was on patients with tetralogy of Fallot. The mean arterial oxygen saturation was less than 90% in both arms and was not significantly different; the operative mortality was the same in both arms. When this fact, and the fact that the combined proportions of cyanotic patients in the other four studies were not significantly different in the two arms, it would seem that there is no advantage of using blood cardioplegia in cyanotic patients.

Conclusion

Our results suggest that the in-hospital operative mortality associated with the addition of blood to St Thomas I or II solutions in pediatric cardiac surgery is not significantly different from that associated with the use of non-blood (St Thomas solution I or II) cardioplegia. Appropriately conducted meta-analyses on well-designed RCTs remain the highest form of evidence with respect to clinical practice. We conducted a metaanalysis, in accordance with best practice guiDelines, of available RCTs that were pragmatically clinically homogenous. We ensured that both cardioplegia arms used cardioplegia that caused depolarized arrest and that the only difference between the two arms was the addition of blood in equal concentrations to one arm. Our results may have implications for the use of blood cardioplegia. RCTs with larger populations are required to confirm our finding. We recommend that additional well-designed RCTs be conducted to systematically and robustly examine the role of adding blood to cardioplegia.

Figure 1 :
1Search flow diagram

St-Th-1(4:1) B: St-Th-1(4:1) B: St-Th-1(4:1) B: St-Th-1 OR 2(4:1) B: St-Th-1(4

Table 1 :
1Search Strategy
Search
step

Search strategy

PubMed
Google
Scholar

Cochrane
Library

1
"cardioplegia"
OR
"myocardial
protection"

Table 2 :
2Studies included on meta-analysis
Study
n (324)
Cardioplegia type
Dose
timing
(min)
Young 1997 (9)
Caputo 2002 (10)
Modi 2004 (11)
Amark 2005 (12)

Table 3 :
3List of excluded full text papers Authors Year 1. Sobieraj et al. (14) 2. O'Brien et al. (15) 3. Sinha et al. (16) 4. Zhu et al. (17) 5. Modi et al. (18) 6. Toyoda et al.(19) 2018
2009
2008
2006
2006
2003

Table 4 :
4Meta-analysis resultsMoDel
Risk
ratio

SE
95%CI
p-value
I 2

Fixed effect
Random effect

0.77
0.51

0.6
0.59

0.24-2.5
0.16-1.63

0.66
0.25

0%
0%

Developments in cardio-protection: "Polarized" arrest as an alternative to "depolarized" arrest. D J Chambers, D J Hearse, Ann Thorac Surg. 68Chambers DJ, Hearse DJ. Developments in cardio-protection: "Polarized" arrest as an alternative to "depolarized" arrest. Ann Thorac Surg 1999; 68:1960-6.

Is there any difference between blood and crystalloid cardioplegia for myocardial protection during cardiac surgery? A meta-analysis of 5576 patients from 36 randomized trials. Mpbo Sá, F G Rueda, P E Ferris, Perfusion. 27Sá MPBO, Rueda FG, Ferris PE, et al. Is there any difference between blood and crystalloid cardioplegia for myocardial protection during cardiac surgery? A meta-analysis of 5576 patients from 36 randomized trials. Perfusion. 2012; 27:535-46.

Cardioplegia in paediatric cardiac surgery: A systematic review of randomized controlled trials. N E Drury, I Yam, A J Patel, Interact CardioVasc Thorac Surg. 28Drury NE, Yam I, Patel AJ, et al. Cardioplegia in paediatric cardiac surgery: A systematic review of randomized controlled trials. Interact CardioVasc Thorac Surg. 2019; 28:144-50.

Del Nido versus St. Thomas cardioplegia solutions: A single-center retrospective analysis of post cross-clamp defibrillation rates. S T Buel, C W Striker, O 'brien, J E , JECT. 48Buel ST, Striker CW, O'Brien JE. Del Nido versus St. Thomas cardioplegia solutions: A single-center retrospective analysis of post cross-clamp defibrillation rates. JECT. 2016; 48:67-70.

Blood versus crystalloid cardioplegia in pediatric cardiac surgery: A systematic review and meta-analysis. K S Mylonas, A Tzani, P Metaxas, Pediatr Cardiol. 38Mylonas KS, Tzani A, Metaxas P, et al. Blood versus crystalloid cardioplegia in pediatric cardiac surgery: A systematic review and meta-analysis. Pediatr Cardiol. 2017; 38:1527-39.

The effects of clinical and statistical heterogeneity on the predictive values of results from meta-analyses. W G Melsen, M C Bootsma, M M Rovers, Clin Microbiol Infect. 20Melsen WG, Bootsma MC, Rovers MM et al. The effects of clinical and statistical heterogeneity on the predictive values of results from meta-analyses. Clin Microbiol Infect. 2014; 20:123-9.

Preferred reporting items for systematic review and meta-analysis protocols (PRISMA-P) 2015 statement. D Moher, L Shamseer, M Clarke, Systematic Reviews. 41Moher D, Shamseer L, Clarke M, et al. Preferred reporting items for systematic review and meta-analysis protocols (PRISMA-P) 2015 statement. Systematic Reviews. 2015; 4:1.

Meta-Mar free online meta-analysis service. Meta-Mar free online meta-analysis service. Available at http://www.meta-mar.com . Accessed April 11, 2019.

Antegrade cold blood cardioplegia is not demonstrably advantageous over cold crystalloid cardioplegia in surgery for congenital heart disease. J N Young, I O Choy, N K Silva, J Thorac Cardiovasc Surg. 114Young JN, Choy IO, Silva NK, et al. Antegrade cold blood cardioplegia is not demonstrably advantageous over cold crystalloid cardioplegia in surgery for congenital heart disease. J Thorac Cardiovasc Surg. 1997; 114:1002-9.

Cold blood versus cold crystalloid cardioplegia for repair of ventricular septal defects in pediatric heart surgery: A randomized controlled trial. M Caputo, P Modi, H Imura, Ann Thorac Surg. 74Caputo M, Modi P, Imura H, et al. Cold blood versus cold crystalloid cardioplegia for repair of ventricular septal defects in pediatric heart surgery: A randomized controlled trial. Ann Thorac Surg. 2002; 74:530-5.

Myocardial metabolic changes during pediatric cardiac surgery: A randomized study of 3 cardioplegic techniques. P Modi, M S Suleiman, B Reeves, J Thorac Cardiovasc Surg. 128Modi P, Suleiman MS, Reeves B, et al. Myocardial metabolic changes during pediatric cardiac surgery: A randomized study of 3 cardioplegic techniques. J Thorac Cardiovasc Surg. 2004; 128: 67-75.

Blood cardioplegia provides superior protection in infant cardiac surgery. K Amark, H Berggren, K Bjork, Ann Thorac Surg. 80Amark K, Berggren H, Bjork K, et al. Blood cardioplegia provides superior protection in infant cardiac surgery. Ann Thorac Surg. 2005; 80:989-94.

Comparison between blood and non-blood cardioplegia in tetralogy of Fallot. H Romolo, L Hernisa, D Fakhri, Asian Cardiovasc Thor Annals. 272Romolo H, Hernisa L, Fakhri D, et al. Comparison between blood and non-blood cardioplegia in tetralogy of Fallot. Asian Cardiovasc Thor Annals. 2019; 27(2):75-9.

Type of cardioplegic solution as a factor influencing the clinical outcome of open-heart congenital procedures. M Sobieraj, M Kilanowska, P Ładziński, Kardiochir Torakochirurgia Pol. 152Sobieraj M, Kilanowska M, Ładziński P, etal. Type of cardioplegic solution as a factor influencing the clinical outcome of open-heart congenital procedures. Kardiochir Torakochirurgia Pol. 2018; 15(2):86-94.

Pediatric cardioplegia strategy results in enhanced calcium metabolism and lower serum troponin T. J D O'brien, S E Howlett, H J Burton, Ann Thorac Surg. 875O'Brien JD, Howlett SE, Burton HJ, et al. Pediatric cardioplegia strategy results in enhanced calcium metabolism and lower serum troponin T. Ann Thorac Surg. 2009; 87(5):1517-23.

Comparison of two cardioplegia solutions using thermodilution cardiac output in neonates and infants. P Sinha, D Zurakowski, R A Jonas, Ann Thorac Surg. 865Sinha P, Zurakowski D, Jonas RA. Comparison of two cardioplegia solutions using thermodilution cardiac output in neonates and infants. Ann Thorac Surg. 2008; 86(5):1613-9.

Seven years' experience of pediatric cardiopulmonary bypass: 8685 cases in Shanghai Children's Medical Center. D M Zhu, W Wang, Z W Xu, ASAIO J. 525Zhu DM, Wang W, Xu ZW et al. Seven years' experience of pediatric cardiopulmonary bypass: 8685 cases in Shanghai Children's Medical Center. ASAIO J. 2006; 52(5):556-8.

Changes in myocardial free amino acids during pediatric cardiac surgery: A randomized controlled trial of three cardioplegic techniques. P Modi, S M Suleiman, B C Reeves, Eur J Cardiothorac Surg. 301Modi P, Suleiman SM, Reeves BC, et al. Changes in myocardial free amino acids during pediatric cardiac surgery: A randomized controlled trial of three cardioplegic techniques. Eur J Cardiothorac Surg. 2006; 30(1):41-8.

Cardioprotective effects and the mechanisms of terminal warm blood cardioplegia in pediatric cardiac surgery. Y Toyoda, M Yamaguchi, N Yoshimura, J Thorac Cardiovasc Surg. 1256Toyoda Y, Yamaguchi M, Yoshimura N, et al. Cardioprotective effects and the mechanisms of terminal warm blood cardioplegia in pediatric cardiac surgery. J Thorac Cardiovasc Surg. 2003; 125(6):1242-51.

Comparison of Del Nido and St Thomas cardioplegia solutions in pediatric patients: A prospective randomized clinical trial. S Talwar, A Bhoje, V Sreenivas, Semin Thorac Cardiovasc Surg. 293Talwar S, Bhoje A, Sreenivas V, et al. Comparison of Del Nido and St Thomas cardioplegia solutions in pediatric patients: A prospective randomized clinical trial. Semin Thorac Cardiovasc Surg. 2017; 29(3):366-74.

This coach improved every tiny thing by 1 percent and here's what happened. J Clear, Clear J. This coach improved every tiny thing by 1 percent and here's what happened. https://jamesclear.com/marginal-gains.

Myocardial cytochrome oxidase activity increases with age and hypoxemia in patients with congenital heart disease. M Onwugbufor, R J Levy, D Zurakowski, Perfusion. 324Onwugbufor M, Levy RJ, Zurakowski D, et al. Myocardial cytochrome oxidase activity increases with age and hypoxemia in patients with congenital heart disease. Perfusion. 2017; 32(4):306-12.

Interpretation of odds and risk ratios. A M O'connor, J Vet Intern Med. 27O'Connor AM. Interpretation of odds and risk ratios. J Vet Intern Med. 2013; 27:600-3.

Blood versus crystalloid cardioplegia for pediatric cardiac surgery: A meta-analysis. Y Fang, C Long, S Lou, Perfusion. 30Fang Y, Long C, Lou S, et al. Blood versus crystalloid cardioplegia for pediatric cardiac surgery: A meta-analysis. Perfusion. 2015; 30:529-36.

Comparison of high glucose concentration blood and crystalloid cardioplegia in paediatric cardiac surgery: A randomized clinical trial. B Mimic, S Ilic, I Vulicevic, Interact Cardiovasc Thorac Surg. 225Mimic B, Ilic S, Vulicevic I, et al. Comparison of high glucose concentration blood and crystalloid cardioplegia in paediatric cardiac surgery: A randomized clinical trial. Interact Cardiovasc Thorac Surg. 2016; 22(5):553-60.

A guide to understanding meta-analysis. H Israel, R R Richter, J Orthop Sports Phys Ther. 41Israel H, Richter RR. A guide to understanding meta-analysis. J Orthop Sports Phys Ther. 2011; 41:496-504.

Impact of including or excluding both-armed zero-event studies on using standard meta-analysis methods for rare event outcome: A simulation study. J Cheng, E Pullenayegum, J K Marshall, BMJ Open. 610983Cheng J, Pullenayegum E, Marshall JK, et al. Impact of including or excluding both-armed zero-event studies on using standard meta-analysis methods for rare event outcome: A simulation study. BMJ Open. 2016; 6:e010983.

Meta-analysis: Pitfalls and hints. Heart, Lung and Vessels. T Greco, A Zangrillo, G Biondi-Zoccai, 5Greco T, Zangrillo A, Biondi-Zoccai G, et al. Meta-analysis: Pitfalls and hints. Heart, Lung and Vessels. 2013; 5(4):219-25

Recommendations for examining and interpreting funnel plot asymmetry in metaanalyses of randomized controlled trials. J A Sterne, A J Sutton, J P Ioannidis, BMJ. 3434002Sterne JA, Sutton AJ, Ioannidis JP. Recommendations for examining and interpreting funnel plot asymmetry in meta- analyses of randomized controlled trials. BMJ. 2011; 343: d4002.

Seven years' experience of pediatric cardiopulmonary bypass: 8685 cases in Shanghai Children's Medical Center. D Zhu, W Wang, Z Xu, ASAIO Journal. 52Zhu D, Wang W, Xu Z, et al. Seven years' experience of pediatric cardiopulmonary bypass: 8685 cases in Shanghai Children's Medical Center. ASAIO Journal. 2006; 52:556-8.