Emilie Frobert
email: emilie.frobert@chu-lyon.fr

V Escuret

E Javouhey

J S Casalegno

M Bouscambert-Duchamp

C Moulinier

Y Gillet

B Lina

D Floret

F Morfin

G Paranhos-Baccala

Respiratory viruses in children admitted to hospital intensive care units: 1 evaluating the CLART® Pneumovir DNA array 2 3 4

Keywords: Respiratory viruses, DNA array, paediatric intensive care unit DNA array, pediatric intensive care unit 29 30

Viruses play a significant part in children's respiratory infections, sometimes leading 32 to hospitalization in cases of severe respiratory distress. The aim of this study was to 33 investigate respiratory infections in children treated in a hospital intensive care unit. Assays 34 were performed using the CLART® Pneumovir DNA array assay (Genomica, Coslada, 35 Madrid, Spain), which makes it possible to detect 11 genus of respiratory viruses 36 simultaneously. During the winter of 2008-2009, 73 respiratory specimens collected from 53 37 children under 2 years of age and admitted to an intensive care unit were tested. At least one 38 virus was detected in 78% (57/73) of the samples. The virological diagnosis was based on 39 single infections in 65% (37/57) and on multiple infections in 35% (20/57) of cases. The array 40 assay revealed respiratory syncytial virus (RSV) in 73.6 % (42/57) of the samples and 41 rhinovirus (hRV) in 24.6% (14/57), either on their own or in co-infections. All viruses 42 identified in single and multiple infections were tested, taking into account clinical features, 43 risk factors and severity criteria. Children with no risk factors presented more multiple 44 infections, up to 42% of cases, than children with at least one risk factor. RSV seemed to 45 induce severe symptoms by itself as no difference in intubation needs was observed when 46 RSV was detected on its own or in co-infection. The CLART® Pneumovir DNA array was 47 useful for examining severe viral respiratory infections, when other viruses than those 48 detected by conventional methods could be involved, particularly in an intensive care unit. 49 Page 6 of 18 John Wiley & Sons Journal of Medical Virology bocaviruses (HBoV) can now be detected by multiplex assays. These assays are based on 71 different types of technology, such as ligation-dependent probe amplification (MLPA®), dual 72 priming oligonucleotide (DPO) technology, target specific primer extension (TSPE) or target 73 specific extension (TSE) [

60% R S V R h V O t h e r v i r u s e s R S V A / R h V R S V A / R S V B R h V / B o c a V R S V A / R h V / A D V R S V A / A D V R S V A / E V R S V A / B o c a V R S V A / R S V B / A D V R S V A / R h V / F l u A F l u A / B o c a V P I V / A D V / B o c a V B o c a V / M P V Single infections Multiple infections F o r P e e r R e v i e w INTRODUCTION 50
Acute respiratory diseases are very common in children and a viral etiology can be 51 identified in up to 80% of cases [START_REF] Mahony | Detection of respiratory viruses by molecular methods[END_REF]. These infections can lead to damage to the 52 lower respiratory tract, including bronchitis, bronchiolitis, pneumonia and bacterial 53 superinfections. In some cases, particularly for children under 2 years of age, hospitalization 54 in an intensive care unit may be required in severe cases.

55

Respiratory syncytial virus (RSV) is the most frequently reported virus in infants 56 admitted to hospital, but many other viral agents can be associated with acute respiratory 57 infections [START_REF] Freymuth | Comparison of multiplex PCR assays and conventional 294 techniques for the diagnostic of respiratory virus infections in children admitted to 295 hospital with an acute respiratory illness[END_REF]Mentel et al., 2005]. Rhinovirus, influenza virus and 58 metapneumovirus (hMPV) are also now well known to be involved in such pathologies, with 59 prevalence of around 30%, 10% and 5%, respectively [START_REF] Bouscambert-Duchamp | 283 Detection of human metapneumovirus RNA sequences in nasopharyngeal aspirates of 284 young French children with acute bronchiolitis by real-time reverse transcriptase PCR 285 and phylogenetic analysis[END_REF] 60 [START_REF] Freymuth | Comparison of multiplex PCR assays and conventional 294 techniques for the diagnostic of respiratory virus infections in children admitted to 295 hospital with an acute respiratory illness[END_REF]. In pediatric intensive care units (ICU), the prevalence of viruses may 61 differ, with a larger proportion of dual or mixed infections [Paranhos-Baccala et al., 2008]. et al., 2008]. Other techniques are based on RT-PCR amplifications followed by 75 microarray analysis, as performed in the present study [START_REF] Li | Simultaneous detection and high-throughput identification of a panel 308 of RNA viruses causing respiratory tract infections[END_REF]. Broadening viral 76 diagnosis in this way may help clinicians to decrease prescriptions of antibiotics, implement 77 early antiviral treatments when available and prevent virus transmission.

78

The aim of the present study was to investigate respiratory infections in children 3). [START_REF] Calvo | Multiple simultaneous viral infections in infants with acute respiratory tract 288 infections in Spain[END_REF]Miron et al., 2010] and 209 were associated with higher fever, longer hospitalization and more frequent use of antibiotics 210 than in the case of infection with RSV alone [START_REF] Calvo | Multiple simultaneous viral infections in infants with acute respiratory tract 288 infections in Spain[END_REF]. Infants with multiple 211 infections are at higher risk of being admitted to a pediatric ICU [Paranhos-Baccala et al., 212 2008;Richard et al., 2008]. In the present study, multiple infections were detected by arrays 213 in 35% of the positive cases. In bronchiolitis, viral co-infection was found in 27.3% of the 214 cases with a predominance of the RSV and hRV co-infection, as described previously 215 [Richard et al., 2008]. Respiratory syncytial virus seems to induce severe symptoms on its 216 own. Viral co-infections with RSV effectively did not increase the need for intubation in 217 comparison to infections with RSV as a single agent, as recently reported in a study 218 considering duration of ventilation and length of hospitalization [START_REF] Marguet | In very young infants 320 severity of acute bronchiolitis depends on carried viruses[END_REF].

219

Interestingly, children admitted to a pediatric ICU with no risk factor were found to present 220 more co-infections than children with risk factors, even if the difference was not significant. [START_REF] Freymuth | Comparison of multiplex PCR assays and conventional 294 techniques for the diagnostic of respiratory virus infections in children admitted to 295 hospital with an acute respiratory illness[END_REF]]. In the 223 population studied, the array assay revealed RSV in 73% of the samples either alone or in co-224 infection. This prevalence of RSV may be explained by a higher sensitivity of array assay 225 versus RT-PCR, but this point needs to be evaluated in more detail. It may also suggest that

226

RSV may be responsible for a higher risk of hospitalization in a pediatric ICU. The detection 227 of hRV has been greatly improved by molecular techniques [START_REF] Mahony | Detection of respiratory viruses by molecular methods[END_REF]. The prevalence 228 of hRV was previously reported as being around 30%-35% with multiplex RT-PCR 229 [START_REF] Freymuth | Comparison of multiplex PCR assays and conventional 294 techniques for the diagnostic of respiratory virus infections in children admitted to 295 hospital with an acute respiratory illness[END_REF]. This study reported hRV in 24% of the samples. This could suggest et al., 2001;van den Hoogen et al., 2003].

234

hMPV outbreaks occur predominantly in the winter and spring months, overlapping or 235 following RSV outbreaks [START_REF] Mahony | Detection of respiratory viruses by molecular methods[END_REF]. However, there can be significant differences in 236 the detection of hMPV across seasons from year to year. Overall prevalence is around 7%, 237 ranging from 2 to 25% [START_REF] Gaunt | Incidence, molecular 300 epidemiology and clinical presentations of human metapneumovirus; assessment of its 301 importance as a diagnostic screening target[END_REF][START_REF] Mahony | Detection of respiratory viruses by molecular methods[END_REF]. Only 2 hMPV infections were , 2007;[START_REF] Kesebir | 303 Human bocavirus infection in young children in the United States: molecular 304 epidemiological profile and clinical characteristics of a newly emerging respiratory 305 virus[END_REF][START_REF] Manning | Epidemiological profile and clinical associations of human bocavirus and other 317 human parvoviruses[END_REF] and acute wheezing [Allander et al.,

62

  Viral respiratory diagnosis has traditionally relied on antigen detection and virus 63 isolation. But the lack of sensitivity of antigen detection, the delay in the results with virus 64 isolation and the limitations of detecting only cultivable viruses make the diagnosis of 65 respiratory viruses incomplete. As a consequence, some clinical cases remain virologically 66 negative. 67 Molecular technology has better sensitivity and the development of multiplex 68 amplifications makes it possible to detect a broader panel of viruses. Adenoviruses (ADV), 69 parainfluenza viruses (PIV), enteroviruses (EV) but also coronaviruses (HCoV) and

79

  admitted to a hospital pediatric ICU. Assays were performed with the CLART® Pneumovir 80 DNA array assay (Genomica, Coslada, Madrid, Spain) which makes it possible to detect 11 81 genus of respiratory viruses simultaneously. Technical aspects, virological and clinical data 82 respiratory samples from 53 children under 2 years of age and admitted 89 to a hospital pediatric ICU in Lyon were collected between December 1, 2008 and March 30, 90 2009. Some children were sampled more than once but the samples corresponded to distinct 91 episodes of respiratory disease. In most cases, the samples were nasopharyngeal or tracheo-92 bronchic aspirates. Samples were collected in different clinical contexts: bronchiolitis (30 93 samples), pneumonia (14 samples), bronchiolitis associated with pneumonia (3 samples). 94 Twenty samples were collected in other clinical contexts including isolated apnea (10 95 samples), sepsis (1 sample), apnea associated with sepsis (3), convulsions associated with 96 fever (5), and asthma (1). No clinical information was available for 6 samples. Risk factors 97 were considered for each patient. Of the 53 children, 12 were less than 42 days old (22.6%), 4 98were born prematurely (7.5%), 2 had a cardiopathy (3.8%), one child had chronic respiratory 99 child was immunodepressed. Twenty-one children presented more than 100 one risk factor (39.6%) and 12 children had no risk factors at all (22.6%). The severity of the 101 respiratory disease was related to intubation requirements. samples were treated with 10 µl of proteinase K at 20 mg/ml (Proteinase K 105 PCR grade, Roche). RNA was then extracted using the automated NucliSens easyMAG 106 system (Biomerieux). Elution of the extracted nucleic acids was performed in 70 µl. 107 108 DNA array assay 109 The CLART® Pneumovir DNA array assay (Genomica, Coslada, Madrid, Spain) 110 detects and characterizes the most frequent human viruses causing respiratory symptoms in a 111 total of 8 hours after nucleic acid extraction. The viruses analyzed include: Respiratory 112 syncytial virus (RSV) A and B; Influenza (Flu) virus A, B, C; Parainfluenza virus (PIV) 1, 2, 113 3, 4A, 4B; Metapneumovirus A and B (hMPV); Adenovirus (ADV) (33 subtypes), 114 Enterovirus (EV) (50 subtypes), Rhinovirus (hRV) (65 subtypes); Coronavirus (HCoV) 115 (subtype 229E); Bocavirus (HBoV). This kit is based on the amplification of specific 116 fragments of the viral genome by means of 2 multiplex RT-PCR or PCR and a subsequent 117 detection by hybridization with specific binding probes. During the 5-hour RT-PCR / PCR 118 amplification, the amplified products were labeled with biotin. Following amplification, 119 hybridization with specific probes immobilized in sites of the micro-array was performed in a 120 second step lasting 1h30. After incubation with a streptavidin-peroxidase conjugate, the t-test and Person Chi 2-test were used to assess intergroup differences. Statistical 127 analyses were performed on EpiInfo software (V 3.5.1 CDC). Odds ratio (OR) and 95% 128 confidence interval (CI) were calculated for the likelihood of co-detection and to test the 129 association between multiple infections and risk factors with distress severity. A test was 130 considered to be significant when the p value was less than 0.05. with CLART® Pneumovir in a pediatric intensive care unit 135 Of the 73 samples collected in this study, 57 samples contained at least one virus 136 (78%) (

  children, 20 presented one risk factor (37.8%), 21 presented more than one 164 risk factor (39.6%) and 12 children had no risk factors at all (22.6%). For the children with no 165 risk factors, viral factors that could have led to the severe respiratory infection were 166 investigated. Children with no risk factors presented more multiple infections, up to 42% of 167 cases, than children with at least one risk factor, even if the difference was not significant, 168 probably because of the low number of patients (n = 12) (p = 0.23) (Table

  intubation was used as a surrogate for the severity of the respiratory distress.172Nineteen episodes of respiratory disease required intubation and 52 did not. Information

  missing in 2 cases. There was no difference in the severity of the 174 distress between single and multiple infections (p = 0.62). In addition, the detection of RSV 175 in a co-infection did not worsen the respiratory distress, as intubation was required in 23.1% 176 of the cases when RSV was detected alone and in 14.3% of the cases when detected in co-177 infection with another viral agent. No association was observed between RSV in either single 178 or multiple infections and distress severity (p = 0.4). RSV seemed to induce severe symptoms 179 on its own. In RSV negative samples, no difference was observed in the distress severity 180 between single and multiple infections (p = 0.35), probably because of the low number of 181 RSV negative samples. 182 HBoV was detected in eight clinical cases (1 in single, 7 in multiple infections) with 183 intubation required in 3 cases (Table 4). Interestingly, detection of HBoV resulted in an 184 increased likelihood of co-detecting another virus as reflected by an odds ratio of > 1 (3.15; 185 248 IC95) p = 0.0003. But there was no difference in distress severity between single and 186 multiple infections when HBoV was detected (p = 0.5), probably because of the low number 187 of cases (n = 8).

  found to be highly sensitive and made it possible to detect 192 viruses that are not detected otherwise, either with the standard multiplex RT-PCR (PIV, 193 ADV, EV, HBoV, hMPV, HCoV) or in culture (HBoV, hMPV, HCoV) and which may be 194 involved in multiple infections. From a practical point of view, the CLART Pneumovir 195 array assay is easy to use even if technical training is necessary to avoid false positives 196 because of insufficient washing or overexposure of the arrays. Nevertheless, after nucleic acid . The main advantages are its high sensitivity and broader detection 199 of respiratory viruses, improving viral diagnosis. However, the detection of multiple 200 infections raises certain questions regarding the pathogenicity of the different viruses. Are all 201 the viruses detected responsible for the clinical signs presented by the patient? These multiple 202 viruses can be successive infections and molecular tests could detect a persistent genome in 203 the absence of virus activity. This array assay is only a qualitative technique. However, 204 quantitation should help to identify the predominant viral agent believed to be responsible for 205 an acute respiratory syndrome. 206 Different studies have described previously the clinical characteristics of multiple viral 207 infections versus RSV single infection. Multiple infections varied from 17.4% to 30% in 208 children under 2 years of age admitted to hospital

  previously been detected at around 40% using multiplex RT-PCR 222 in children admitted to the emergency room during winter

  230 that infections caused by hRV are less represented in pediatric ICU than in other pediatric 231 units. 232 hMPV is an RNA virus discovered in 2001 and is known to induce symptoms very 233 similar to those caused by RSV [van den Hoogen

  238detected in this study, probably because the study period missed potential hMPV circulation 239 during the spring season.240HBoV was discovered in 2005 using large-scale molecular viral techniques[Allander 241 et al., 2005]. Prevalence, ranging from 2 to 11% in respiratory samples from symptomatic 242 patients, has been reported worldwide. In most cases, as in this study, HBoV was detected in 243 co-infections with other known viral agents raising the question of its actual pathogenic role 244[START_REF] Mahony | Detection of respiratory viruses by molecular methods[END_REF]. However, HBoV has been associated with low respiratory infections[Fry et

  Even if HBoV can be found in asymptomatic patients, HBoV genome quantitation 247 could help to identify the implication of HBoV in lower respiratory tract infections, as a high 248 HBoV load has been reported in patients with lower respiratory tract infections [van de Pol et 249 al., 2009]. In this study, HBoV prevalence was around 14%, higher than the prevalence 250 usually reported [van de Pol et al., 2009], almost certainly because of the differences in 251 detection techniques, patient recruitment and the seasonal study period. Nevertheless, Fry et 252 al. have previously reported up to 12% of HBoV in children under 5 years of age with 253 pneumonia[START_REF] Fry | Human bocavirus: a novel parvovirus epidemiologically associated 298 with pneumonia requiring hospitalization in Thailand[END_REF].254In conclusion, microarrays have a high level of sensitivity and make broader viral 255 detection possible. However, the relevance of the positive results obtained with this highly 256 sensitive array is not easy to determine, particularly in cases of multiple infections caused by 257 the lack of quantitation. DNA array may be useful regarding severe viral respiratory 258 infections, particularly in pediatric ICU when no viral diagnosis has been established using 259 conventional techniques that detect the most common viral respiratory pathogens.

Table 1 Virological results on 73 respiratory samples Number of positive samples (%) detected by CLART® Pneumovir Virus

 1

					Number	Percentage
	Positive samples					57	78%
	Negative samples					16	22%
	Single infections
		RSV				28	49%
				(RSV A)		20	35%
				(RSV B)		8	14%
	hRV Flu A F Flu B o ADV r PIV			4 0 1 1 1	7% 0 1.7% 1.7% 1.7%
	Total	hMPV HBoV	P e		1 1 37	1.7% 1.7% 65%
	Multiple infections	RSV A + hRV	e r		4	7%
		RSV A + RSV B hRV + HBoV RSV A + hRV + ADV RSVA + ADV RSV A + EV RSVA + HBoV RSV A + RSV B + ADV R e v i e	3 3 2 1 1 1 1	5.3% 5.3% 3.5% 1.7% 1.7% 1.7% 1.7%
		RSVA + hRV + FluA FluA + HBoV	w	1 1	1.7% 1.7%
		PIV + ADV + HBoV		1	1.7%
		HBoV + hMPV			1	1.7%
	Total					20	35%
						1

Table 2 Viral distribution correlated to respiratory syndrome Number of positive samples (%) detected by CLART® Pneumovir

 2

	Bronchiolitis* n = 33	Virus	Number	Percentage
	Single	RSV	19	57.6%
	infections	RSV A	13
		RSV B	6
		hRV	0
		Total	19	57.6%
	Multiple	RSV A + hRV	3	9%
	infections	RSV A + RSV B	2	6%
		RSV A + EV	1	3%
		RSV A + HBoV	1	3%
		hRV + HBoV	1	3%
		RSV A + hRV + ADV	1	3%
	Total		9	27.3%
	Global			85%
	positivity rate Pneumonia*
	n = 17
	Single	RSV	4	23.5%
	infections	RSV A	4
		RSV B	0
		hRV	1	5.9%
		Flu B	1	5.9%
		ADV	1	5.9%
		PIV	1	5.9%
	Total		8	47.1%
	Multiple	RSV A + hRV	1	5.9%
	infections	RSV A + RSV B	1	5.9%
		hRV + HBoV	2	11.8%
		RSV A + hRV + ADV	1	5.9%
	Total		5	29.5%
	Global			76.4%
	positivity rate * 3 samples were collected in case of bronchiolitis associated to pneumonia

Table 3 Risk factors associated to single or multiple infections

 3

	Risk factors	Single infection	Multiple infection	No virus detected
	Age < 42 days	67%	20%	13%
	Prematurity	48%	15%	27%
	Chronical respiratory disease	42%	25%	33%
	Cardiopathy	38%	25%	37%
	No risk factor	42%	42%	16%

Table 4 Clinical cases involving HBoVirus

 4

	Virus	Clinical data	Ventilation	Risk factor
	HBoV	Apnea, convulsions	IV	Age < 42 days
	HBoV + hRV	Bronchiolitis	NIV	None
	HBoV + hRV (n=2)	Pneumonia	IV	CRD, cardiopathy
	HBoV + RSV A	Bronchiolitis, apnea	NIV	Age < 42 days
	HBoV + influenza A	Apnea	NIV	Prematurity, CRD
	HBoV + hMPV	Apnea	NIV	None
	HBoV + ADV + PIV	Apnea, convulsions	IV	Age < 42 days
	CRD = Chronical respiratory Disease
	IV = invasive ventilation
	NIV = non-invasive ventilation

Distribution of virus detected in single and multiple infections by Clart®pneumovir in pediatric intensive care unit

	50%
	40%
	30%
	20%
	10%
	0%

Table 1 )

 1 . The virological diagnosis revealed single infections in 65% (37/57) and 137 multiple infections in 35 % (20/57) of the cases (Table1). Of the single infections, RSV was PIV, hMPV and HBoV were also detected. In multiple infections, RSV was found in 140 co-infection with hRV in 7% of cases, RSV A with RSV B in 5.3% of cases, hRV with HBoV 141 in 5.3% of cases, and RSV A associated with both hRV and ADV in 3.5% of cases. Eight CLART® Pneumovir DNA array made it possible to diagnose at least one virus in 149 85% of the bronchiolitis cases (Table2). In 57.6% of the cases, RSV was detected as a single 150 infection. Multiple infections were also detected in around 27% of the bronchiolitis cases. Other viruses were involved, such as hRV, influenza B, ADV and PIV in single 157 infections. Two cases of hRV associated with HBoV were also reported.

	138
	found in 49% (28/37) of cases and hRV in 7% (4/37). Other viruses, such as influenza B,

139

ADV,

DF received research grants from Biomerieux and Merieux Foundation 267 EY, YG declare no conflict of interest.

Conflict of interest


Ana ; Mota

Jorge Areias

Margarida Fonseca

Salazar Cardoso

Margarida Fonseca Cardoso

Chronic liver disease and cirrhosis among patients with hepatitis B virus infection in northern Portugal with reference to the viral genotypes RUNNING TITLE: Liver disease and cirrhosis among patients with HBV in Portugal

Keywords: Hepatitis B, genotypes, liver disease, cirrhosis, Portugal

Chronic liver disease and cirrhosis among patients with hepatitis B virus infection in northern Portugal with reference to the viral genotypes

 (450,584.1; 384,269.7 (60,657.6;2,434,373.5)
INTRODUCTION

Cirrhosis occurs in 20-30% of those patients who are infected chronically with the hepatitis B virus (HBV). About 25% of these may develop hepatocellular carcinoma (HCC). In patients with chronic HBV and HCC, the genetic material of HBV is frequently found to be part of the genetic material of the cancer cells. The HBV genetic material enters the normal liver cells and disrupts their function, and may lead to transformation into cancerous cells. Because of the genetic mutation caused by HBV, a patient with HBV infection may develop HCC with or without cirrhosis.

Persistent hepatitis B infection affects 350-400 million people worldwide [START_REF] Lee | Hepatitis B virus infection[END_REF][START_REF] Yuen | Independent risk factors and predictive score for the development of hepatocellular carcinoma in chronic hepatitis B[END_REF]). These patients are at least 100 times more likely to develop hepatocellular carcinoma (HCC) compared with uninfected individuals [START_REF] Beasley | Hepatocellular Carcinoma and Hepatitis B Virus: A Prospective Study of 22 707 Men in Taiwan[END_REF]. HCC is the leading cause of death in patients with cirrhosis (Varela et al. 2009). Several potential factors have been linked with a higher risk of developing HCC, including male gender, advanced age, virological factors such as hepatitis B e antigen positivity and high serum HBV DNA levels [START_REF] Sherman | Hepatocellular Carcinoma: Epidemiology, Risk Factors, and Screening[END_REF].

The association between HBV genotypes and severity of liver disease remains debatable, although one study has found more fibrosis in patients with genotype A than with genotype D [START_REF] Halfon | Multicentre study of hepatitis B virus genotypes in France: correlation with liver fibrosis and hepatitis B e antigen status[END_REF].

Portugal is populated by approximately 10 million inhabitants, with an immigrant community of around 4%. Immigrants are concentrated in the south with an epicentre in Lisbon while the north of the country has relatively fewer immigrants [START_REF] Mai | Foreign Population in Portugal[END_REF]. At present, there is no information on the prevalence of HBV infection in Portugal. According to the most representative research in this field, patients chronically infected with HBV comprise approximately 0.36 to 1% of the Portuguese population [START_REF] Lecour | Prevalence of viral hepatitis markers in the population of Portugal[END_REF][START_REF] Santos | Prevalence of markers of hepatitis B infection in the adult population of the district of Coimbra[END_REF]Ministério da Saúde 2004). The results of the study are part of a project in progress at a Portuguese university to characterize the epidemiological profile of the hepatitis B virus in northern Portugal [START_REF] Mota | Epidemiological Study of Genotypes of Hepatitis B virus in Northern Portugal[END_REF][START_REF] Mota | Alcohol consumption among patients with hepatitis B infection in northern Portugal considering gender and hepatitis B virus genotype differences[END_REF]). This study, within the ambit of a larger project, focuses on the epidemiological profile of liver damage, particularly cirrhosis, in patients infected chronically with hepatitis B virus.

Clinical findings

Information related to the demographics, clinical, histological and medical history of the patients was retrieved from medical files.

Biochemical tests of liver function and other blood parameters

The blood tests considered when assessing liver function included: alanine aminotransferase (ALT) (reference values 10 to 36 U/L at 37ºC) and aspartate aminotransferase (AST) (reference values 10 to 30 U/L at 37ºC), which are contained within liver cells, gamma- less than 10.9 ng/ml). Haematological parameters like mean corpuscular volume (MCV), and platelets were also evaluated using an Advia 120 System (Bayer, New York, USA).

Prothrombin time (PT) measurement was performed using an ACL TOP system (Instrumentation Laboratory Company, Lexington, USA).

Detection of virological markers in serum

HBsAg, anti-HBs and anti-HBc, HBeAg, and anti-HBe antibodies were evaluated using a

Vitros ECI assays (Ortho-Clinical Diagnostics, Amersham, Buckinghamshire, UK).

HBV DNA quantitation

HBV DNA levels were determined using the Versant ® HBV DNA 3.0 assay (bDNA) (Bayer, New York, USA). The assay quantitation range is 357-17,900,000 HBV DNA IU/ml.

HBV Genotyping

HBV genotypes were identified by the Trugene ® HBV genotyping kit in conjunction with the Open Gene ® DNA Sequencing System (Bayer, New York, USA).

Cirrhosis

Cirrhosis was evaluated by liver biopsy.

Child-Pugh classification

This classification was used to assess the prognosis of cirrhosis. Child's grading of severity in chronic liver disease was classified along with Pugh's 1973 modifications [START_REF] Pugh | Transection of the oesophagus for bleeding oesophageal varices[END_REF] and based upon information recorded from medical files.

RESULTS

Liver biopsy was performed on 249 patients for histological assessment. Cirrhosis was found in 59 patients (23.7%).

Genders were analyzed separately, and patients with and without cirrhosis were compared.

Genotypes A and D were predominant, and no significant differences between the study groups were observed with respect to genotype distribution, in both genders (Table 1). The distribution of the presumed sources of transmission of viral infection was similar among female patients, while sexual transmission was an increased factor in male patients with cirrhosis (p=0.013). Alcohol intake levels (less or more than 20g/day) were similar between the two groups with or without cirrhosis. On average, male patients with cirrhosis were older than patients without cirrhosis (p=0.001), while the same was true with female patients the relationship was not significant.

(Table 1)

In patients with cirrhosis, liver disease severity was evaluated using Child's grading of disease severity with Pugh's modifications (Table 2).

(Table 2)

Evaluation of the 59 patients, 32.2% female, was carried out according to the Child-Pugh classification. In the female group, Child's grade A classification was the most frequent (78.9%) and only one woman was classified as Child´s grade C. On the other hand, an increased proportion of Child's grade B classification was observed in men (35% versus 15.8% in women) while two were classified as Child's grade C.

Child's grade equivalent C

Only three patients scored sufficiently to receive a Child-Pugh classification level C, two of them being male. All patients were genotype D, and aged between 66 and 80 years old. The presumed source of infection is unknown in two patients while the third patient had a reported intrafamilial transmission of HBV infection. All patients (n=3) were HBeAg negative, and all of them also presented viral load of HBV greater than 357-HBV DNA IU/ml.

Biochemical tests revealed ALT was elevated in two individuals, AST in all three, while only one occurrence of elevated alkaline phosphatase, GGT and alpha-fetoprotein was found.

Child's grade equivalent A and B

Individuals classified as Child's grade A (n=39) and B (n=17) were compared by gender, genotype, the presumed source of infection, alcohol intake, place of birth, median age, viral markers, viral load and biochemistry parameters (Table 3).

(Table 3)

When patients with cirrhosis, were compared by gender, genotype and the presumed source of infection, no significant differences between Child's grade A and B were found. Individuals classified as Child's grade B were older and had a significantly higher proportion of patients with alcohol intake above 20g/day (p≤0.004). Patients classified as Child's grade B showed a tendency to have higher proportions of elevated biochemistry parameters like AST, alkaline phosphatase, GGT and alpha-fetoprotein when compared with individuals classified as grade A.

Haematological parameters were also compared (Table 3). Patients classified as Child's grade B had significantly lower levels of platelets (p<0.001) and significant higher levels of prothrombin time (p=0.013) than patients classified as Child's grade equivalent A. All patients in the study were from the north of Portugal. In the sample of patients infected with HBV who underwent histological evaluation (n=249) the results showed that there was a considerable proportion with an increased incidence of liver damage, while 23.7% of the patients had cirrhosis. Genotypes A and D were predominant in patients with or without cirrhosis. A previous study in the north of Portugal [START_REF] Mota | Epidemiological Study of Genotypes of Hepatitis B virus in Northern Portugal[END_REF] found genotypes A and D to be predominant, with genotype D accounting for 60.1% and genotype A accounting for 30.1% of the patients evaluated. In Spain, the only country that borders Portugal, genotypes A and D have been also found to be prevalent [START_REF] Echevarria | Hepatitis B virus genotypes identified by a Line Probe Assay (LiPA) among chronic carriers from Spain[END_REF][START_REF] Basaras | Characterization of hepatitis B virus genotypes in chronically infected patients[END_REF]), although the relationship with the stage of cirrhosis is undocumented. A study in India found that genotype D was associated with more severe liver diseases when compared with genotype A. Genotype D was more prevalent in HCC patients less than 40 years of age, when compared with asymptomatic carriers [START_REF] Thakur | Profile, spectrum and significance of HBV genotypes in chronic liver disease patients in the Indian subcontinent[END_REF]. Similar results are likely to be found in the north of Portugal, given a suitably large sample size.

In this study a statistical association was not found between cirrhosis and genotypes of HBV (p≤0.554), or alcohol consumption level (p≤0.850), for both male and female patients.

Comparison between patients without and with cirrhosis showed a tendency for patients with cirrhosis to be older. Recent studies have shown that, older age, male gender and cirrhosis were the major risk factors associated with the development of HCC [START_REF] Lok | Hepatitis B: liver fibrosis and hepatocellular carcinoma[END_REF].

In this study a classification system developed to assess the severity of liver cirrhosis, the Child-Pugh grade, was chosen to evaluate patients. In the study sample, 3 out of the 59 studying the relationship between the severity of liver disease and age and alcohol consumption [START_REF] Mota | Alcohol consumption among patients with hepatitis B infection in northern Portugal considering gender and hepatitis B virus genotype differences[END_REF]. While this previous study reported a relationship between alcohol consumption and liver disease severity, a relaxed definition of liver disease was used, including patients with and without histological evaluation, and patients without disease reports. The present study, restricted only to patients with cirrhosis, achieved the same positive association between age, alcohol intake and severity of liver disease. Age and alcohol consumption were also reported as risk factors associated with HCC in many studies from different countries, including Portugal [START_REF] Pereira | The effect of ethanol intake on the development of hepatocellular carcinoma in HBsAg carriers[END_REF][START_REF] Oliveira | Carcinoma hepatocelular: Casuística do Serviço de Medicina III dos Hospitais da Universidade de Coimbra[END_REF][START_REF] Wang | Risk of hepatocellular carcinoma and habits of alcohol drinking, betel quid chewing and cigarette smoking: a cohort of 2416 HBsAg-seropositive and 9421 HBsAgseronegative male residents in Taiwan[END_REF][START_REF] Chung | A comparative cross-sectional study of the development of hepatocellular carcinoma in patients with liver cirrhosis caused by hepatitis B virus, alcohol, or combination of hepatitis b virus and alcohol][END_REF]).

Hepatitis B infection may influence the development of liver disease in heavy alcohol drinkers in several ways. Firstly, persistent infection with HBV might encourage alcohol induced liver damage, and evidence exists that this may occur at fairly low levels of alcohol intake [START_REF] Villa | Susceptibility of chronic symptomless HBsAg carriers to ethanol-induced hepatic damage[END_REF]. On the other hand, chronic active hepatitis associated with HBsAg may coexist with alcoholic liver disease, and the two disease processes would probably result in more rapid progression to cirrhosis. For this reason a vaccination program would be of great benefit to excessive drinkers, as infection with HBV and Hepatitis C virus (HCV) are major contributing factors towards the development of HCC [START_REF] Nalpas | ESBRA 1997 Award lecture: relationship between excessive alcohol drinking and viral infections[END_REF]. Alcohol dependent patients under treatment or attended to by emergency hospital services, are opportunities for preventive intervention. Such patients should be checked for viral diseases like hepatitis C, human immunodeficiency virus (HIV) and HBV infection. In all individuals without evidence of infection with HBV, a vaccination should be implemented.

The pattern observed for patients without hepatitis B e antigen and viral load, in patients with chronic hepatitis B, seems to be predominant in Mediterranean countries Additionally some investigators have found that HBV infection remained active after seroconversion to anti-HBe in nearly 40% of patients [START_REF] Rodriguez-Frias | Hepatitis B virus infection: precore mutants and its relation to viral genotypes and core mutations[END_REF][START_REF] Grandjacques | Rapid detection of genotypes and mutations in the pre-core promoter and the pre-core region of hepatitis B virus genome: correlation with viral persistence and disease severity[END_REF]. In France, an increasing prevalence of HBeAg negative was observed [START_REF] Zarski | Characteristics of patients with chronic hepatitis B in France: predominant frequency of HBe antigen negative cases[END_REF], in accordance with this study of Portugal, with probable basal promoter core or a precore region mutations (two individuals presented anti-HBc negative). The knowledge of this characteristic is very important for optimizing clinical management and future therapeutic trials for chronic hepatitis B. Particularly, with respect to HBV replication status, HBV genotype and mutations in the basal core promoter region play an important role in the HCC development [START_REF] Lok | Hepatitis B: liver fibrosis and hepatocellular carcinoma[END_REF].

Many factors seem to contribute to the development of HCC: host and viral risk factors associated with increased risk of cirrhosis or HCC include older age (longer duration of infection), alcohol consumption, smoking, HBV genotype C, high levels of HBV DNA and concurrent infection with hepatitis like HCV, hepatitis D virus (HDV) or HIV [START_REF] Fattovich | Occurrence of hepatocellular carcinoma and decompensation in western European patients with cirrhosis type B. The EUROHEP Study Group on Hepatitis B Virus and Cirrhosis[END_REF][START_REF] Mcmahon | Serologic and clinical outcomes of 1536 Alaska Natives chronically infected with hepatitis B virus[END_REF][START_REF] Fattovich | Natural history and prognosis of hepatitis B[END_REF][START_REF] Yim | Natural history of chronic hepatitis B virus infection: what we knew in 1981 and what we know in 2005[END_REF]. In the evaluation of cirrhosis, liver biopsy continues to be the preferred diagnostic method, but the appearance of 'stiffness' measurement techniques will probably be the eventual future diagnosis of cirrhosis. However, additional studies are needed for the evaluation of liver fibrosis possibly based on liver 'stiffness' measurements, and cutoff values must be defined for categorizing the different stages of cirrhosis.

  as part of an epidemiological study of the hepatitis B virus in northern Portugal[START_REF] Mota | Epidemiological Study of Genotypes of Hepatitis B virus in Northern Portugal[END_REF]. From a group of 358 individuals with chronic hepatitis B virus infection, serum samples were tested for HBsAg, antibodies to HBsAg (anti-HBs), antibodies to the HBV core antigen (anti-HBc), HBeAg, and antibodies to HBeAg (anti-HBe). HBV genotypes were determined along with other biochemical and haematological parameters. The present study includes the 249 patients who underwent histological evaluation.PatientsPatients with hepatitis B infection were identified by the presence of HBsAg in their serum for at least 6 months. The majority of patients from the north of Portugal, are treated in three hospitals in the city of Oporto: Hospital de Santo António, Hospital Joaquim Urbano and Hospital de São João. The patients in this study were examined in 2008 and 2009 in the Hospital de Santo António and Hospital Joaquim Urbano. The study was approved by the Health Ethics Committee of Hospital de Santo António, and written consent was obtained from all participants.

  ) (reference values 10 to 66 U/L at 37ºC), alkaline phosphatase (AP) (reference values 45 to 122 U/L at 37ºC), and alpha-fetoprotein (AFP) (reference values

  one of the first attempts to describe the epidemiological profile of liver damage, particularly cirrhosis, in patients infected chronically with hepatitis B virus in Portugal. Both patients with and without cirrhosis formed part of the study.

  as Child-Pugh grade C, the most severe, 17 (28.8%) as grade B and 39 (66.2%) as grade A.To understand better what factors are associated with the severity of liver disease those patients with cirrhosis classified as Child-Pugh grade B were compared with the group classified with the less severe grade, A, in terms of HBV genotypes, patient demographics and alcohol intake, transmission routes, virological markers for HBV, viral load, liver enzymes, and hematological parameters. In this study a statistical association was not found between severity of liver disease and gender (p=0.222), genotypes, (p=0.844), eventual transmission routes of infection, HBeAg negativity (p=0.222), anti-HBe positive (p=0.119) and viral load (p=0.212). All patients were anti-HBc positive. The analysis showed that patients classified as Child-Pugh grade B were older (p=0.001) and had a significantly higher percentage of alcohol intake above 20g/day (p=0.004) when compared to individuals classified as Child-Pugh grade A. The same trend has been observed previously when

  showed that ALT levels in patients with Child's grade A and B had similar values. On the other hand, Child's grade B patients, showed a higher proportion with elevated levels of AST and alkaline phosphatase, when compared with individuals classified as grade A. Even in this Portuguese study, patients classified as Child's grade equivalent B had significantly lower levels of platelets (p<0.001) and significant higher levels of PT (p=0.013) compared to the Child's grade equivalent A. Thrombocytopaenia is considered as a valid surrogate of cirrhosis and a valid marker for the identification of individuals at high-risk for HCC[START_REF] Lu | Thrombocytopenia as a surrogate for cirrhosis and a marker for the identification of patients at high-risk for hepatocellular carcinoma[END_REF]. Although not significant, Child's grade B individuals presented elevated values for GGT and AFP.In conclusion, the present study examined the associations between cirrhosis, and an extended number of patient's characteristics, providing useful information on the profile of patients infected with hepatitis B virus in northern Portugal. The group of patients with cirrhosis, classified as Child-Pugh grade B, was older and showed elevated levels of alcohol intake.Comparison of patients classified as Child-Pugh grade B with individuals classified as Child-Pugh grade A, showed a higher proportion of biochemical parameters particularly, AST, AP, GGT and elevated AFP, with hematological parameters showing lower platelet levels and higher PT values.

  between HBV genotypes and severity of liver disease was observed with the sample evaluated, it would be important in future investigations to determine the impact of genotypes on liver disease, particularly cirrhosis.

Table 1 :

 1 Characteristics of patients with and without cirrhosis

	2
		Without cirrhosis	With cirrhosis
			(n=190)	(n=59)
		n a	(%)	n a	(%)	P
						Value
	Female
	Genotype					0.554 c
	A	20/83	(24.1)	7/19	(36.8)
	C	0/83	(0.0)	0/19	(0.0)
	D	55/83	(66.3)	12/19	(63.2)
	E	4/83	(4.8)	0/19	(0.0)
	F	4/83	(4.8)	0/19	(0.0)
	D and F	0/83	(0.0)	0/19	(0.0)
	Presumed source of					d
	HBV infection
	Perinatal	20/83	(24.1)	4/19	(21.1)
	Sexual	13/83	(15.7)	2/19	(10.5)
	Intrafamilial	29/83	(34.9)	6/19	(31.6)
	Others b	21/83	(25.3)	7/19	(36.8)
	Alcohol intake >	25/70	(35.7)	6/18	(33.3)	0.850
	20g/day
	Mean Age (± SD)	44.1	(±12.9)	48.5	(± 19.1)	0.354
	Male
	Genotype					0.742 c

a Total for each variable may not sum to the sample size due to missing data.

3

b Other sources, including at least one incidence of intravenous drug use, blood transfusion, occupational 4 exposure, iatrogenic exposure (for example, endoscopy or coloscopy, history of surgery, acupuncture or

Table 2 -

 2 Patients with cirrhosis (n=59) classified by Child's grade

	9
		n	(%)
	Female
	Child's grade A	15/19	(78.9)
	Child's grade B	3/19	(15.8)
	Child's grade C	1/19	(5.3)
	Male
	Child's grade A	24/40	(60.0)
	Child's grade B	14/40	(35.0)
	Child's grade C	2/40	(5.0)

Table 3 :

 3 Comparison of the characteristics of patients with cirrhosis classified as Child's

	11
	12	grade A and Child's grade B (n= 56)
		Characteristics	Child-Pugh A (n=39)	Child-Pugh B (n=17)	P
			n a	(%)	n a	(%)	Value
		Gender					0.222
		Female	15/39	(38.5)	3/17	(17.6)
		Male	24/39	(61.5)	14/17	(82.4)
		Genotypes
		A	15/39	(38.5)	5/17	(29.4)	0.844 c
		C	0/39	(0.0)	0	(0.0)
		D	24/39	(61.5)	11/17	(64.7)
		E	0	(0.0)	0	(0.0)
		F	0	(0.0)	1/17	(5.9)
		D and F	0	(0.0)	0	(0.0)
		Presumed source of HBV
		infection
		Perinatal	7/39	(17.9)	0	(0.0)	c
		Sexual	10/39	(25.6)	4/17	(23.5)
		Intrafamilial	10/39	(25.6)	5/17	(29.4)
		Others b	12/39	(30.8)	8/17	(47.1)
		Alcohol intake > 20g/day	12/36	(33.3)	13/16	(81.2)	0.004
		Mean age (± SD)	44.5	(±16.6)	59.9	(±11.4)	0.001
		Viral markers
		Anti-HBc positive	39/39	(100.0)	17/17	(100.0)	d
		HBeAg negative	24/39	(61.5)	14/17	(82.4)	0.222
		Anti-HBe positive	22/39	(56.4)	14/17	(82.4)	0.119
		Viral load Geom. Mean	1,400,024.1

  The classification has three grades, from A to C with grade C being the severest. The respective overall 5-year mortality rates for grades C to A are 88%, 38%, and 29%.Qualitative variables were described as percentages and quantitative variables as means, or geometric means where necessary. The distribution of viral load was logarithmically transformed and the results are given as geometric means with 95% confidence intervals. For the other quantitative variables, results were given as arithmetic means (±standard deviation).

	Statistical analysis
	Statistical analysis was performed with SPSS (version 16.0) and two-sided significance tests
	at the 5% level were used throughout.
	F o r F o r
	P P
	e e
	e r e r
	R R
	e e
	v i e v i e
	w w
	7 8

Proportions were compared using the Pearson chi-square test (with continuity correction) as appropriate, or by Fisher's exact test. Continuous variables were compared using the t-test, after log transformation so that assumptions were met, if necessary. Multivariable analysis was not considered due to the reduced number of individuals when subdivided by cirrhosis, gender and the Child's grading of disease severity for chronic liver disease.

b Other sources, including at least one incidence of intravenous drug use, blood transfusion, occupational

exposure, iatrogenic exposure (for example, endoscopy or coloscopy, history of surgery, acupuncture or

haemodialysis), or unknown.

c Evaluated only with genotypes A and D.

Acknowledgments:

This study was supported by a grant from Forum Hematológico do Norte, Porto, Portugal.

The authors would like to thank J.H. Tice for scientific English editing.

Conflict of interest

The authors disclose that there are no financial or personal relationships with other people or organizations that could inappropriately influence their work during the submission process.


Daniel Rodriguez
email: daniel@greve-solvang.dk

Daniel R Greve

Kornvaenget

Keywords: Homoaromaticity, Semibullvalene, Azasemibullvalene, Phosphasemibullvalene

  L'archive ouverte pluridisciplinaire

 46in bold.

Introduction

Semibullvalene (1) undergoes a rapid, degenerate Cope rearrangement, where the experimental enthalpy difference ∆H ‡ between the localized C s symmetric structure 1a and the delocalized bishomoaromatic C 2v symmetric transition state structure 1b is 4.8-5.2 kcal/mol. [1,2] Over the last decades researchers have discovered and explored a wealth of structures based on 1, [3,4] the intriguing fundamental goal being the experimental identification of a semibullvalene having a delocalized bishomoaromatic ground state. Strategies towards this goal have mainly been twofold: a) introduction of substituents [5][6][7][8][9][10][11][12][13][14][15] or coordinating entities [16,17] aiming at electronic stabilization of 1b relative to 1a and b) annelation of small rings [7,[18][19][20][21][22][23][24] which by strain destabilizes 1a relative to 1b. Both strategies have been pursued by experimental and computational means, the latter proving to be a valuable tool in the search for homoaromatic structures. [5][6][7][8][11][12][13][14][15][16][17][19][20][21]23] From strategy a) Dewar et al. furthermore proposed [5,8,25] that introduction of hetero atoms in the semibullvalene skeleton could lead to a delocalized ground state.

Based on semiempirical MINDO/2 and AM1 calculations, they estimated that both diazasemibullvalenes 2 and 4 would have a 'nonclassical' delocalized bishomoaromatic ground state, while tetraazasemibullvalene 12 should have a classical localized structure. Experimentally, derivatives of 2,6diazasemibullvalene (4) are known. [26][27][28] The 1,5-dimethyl-3,7-diphenyl derivative of 4 was shown to have a lower barrier of the Cope rearrangement as compared to 1, although still retaining a localized ground state. [26] Upon heating to 90 o C this compound rearranges to the 1,5-diazocine. [27] Attempted synthesis of derivatives of tetraazasemibullvalene 12 failed, giving 1,3,5,7-tetrazocines instead. [29] Derivatives of diphosphasemibullvalene [30] and tetraphosphasemibullvalene [31,32] have been synthesized, but none of them were observed to undergo a Cope rearrangement. However, these phosphasemibullvalenes had a P atom in the bridge, i.e. at position 1 or 5 (Scheme 1). Heteroatoms are unfavorable in these positions, since the weaker N-N and P-P bonds could result in easier breakdown of the semibullvalene skeleton, favouring valence tautomeric structures, i.e. the delocalized structure of 1,5-diazasemibullvalene was shown to be the valence tautomeric diazapentalene. [7] Analogously to 1, introduction of heteroatoms in the related fluxional molecule barbaralane has been investigated. Experimentally, 2,4,6,8-tetraazasubstitution slightly reduces the barrier of the Cope rearrangement, [33] and two heterobarbaralenes were shown to have homoaromatic ground states using ab initio computational methods. [34,35] Hence, on basis of experimental findings and early semiempirical computational studies, a systematic study of how introduction of two or more hetero atoms in the semibullvalene skeleton affects the height of the barrier to the Cope rearrangement could lead to identification of a hetero semibullvalene based bishomoaromatic ground state structure. In this paper, the results of such a study using correlated ab initio computational methods are presented. The hetero semibullvalenes investigated here, 2 -15 (Scheme 1), are generated by replacing two, three and four carbon atoms with nitrogen or phosphorus in the semibullvalene skeleton. How these replacements affects the height of the barrier to the Cope rearrangement is addressed by calculation of ∆H ‡ , while the aromatic character of 2 -15 is discussed in terms of their calculated Nucleus Independent Chemical Shift (NICS) [36] as well as calculated geometric characteristics like interallylic distances R and selected bond lengths.

Computational Methodology

All molecular geometries were optimized at the MP2 level of theory using analytical gradients, imposing the symmetries shown in Scheme 1. Using these geometries, energies where evaluated at the CCSD(T) and MP4(SDTQ) levels of theory. Vibrational frequencies and harmonic zero-point energies (ZPE) were calculated at the MP2 level of theory and are unscaled. All calculations were performed with the cc-pVDZ [37] basis set and all correlated calculations were performed with the frozen core approximation. Additionally, for 1a and 1b the cc-pVTZ [37] basis set was used, as were the Becke3LYP, MP3, MP4, CASSCF and CCSD computational methods. Calculations were performed using GAMESS (US) QC package [38] , either as PC GAMESS [START_REF] Granovsky | PC Gamess[END_REF] or as WinGamess. [START_REF] Bandeira | [END_REF] The cc-pVDZ and cc-pVTZ basis sets were obtained from the Extensible Computational Chemistry Environment Basis Set Database. [START_REF]Basis sets were obtained from the Extensible Computational Chemistry Environment Basis Set Database[END_REF] Calculation of shielding tensors for determination of NICS were done at the GIAO-HF/cc-pVDZ level of theory on MP2 optimized geometries using NWChem 4.5. [START_REF] Bylaska | NWChem, A Computational Chemistry Package for Parallel Computers, Version 5.1[END_REF]

Results and discussion

In order to select an appropriate combination of basis set and computational level of theory, which would give calculated data of 2 -15 with reasonable predictive value, the well-described native semibullvalene 1 was first investigated using different methods and basis sets.

Semibullvalene

The calculated values of ∆H ‡ for 1 at different levels of theory are presented in Table 1. Like all applied methods, the HF and (6,6)CASSCF methods correctly predict 1 to have a localized 1a ground state, but they far from reproduce the experimental ∆H ‡ (compare entries 1 and 13 with 17), in agreement with earlier findings. [23] From Becke3LYP density functional theory a barrier lower than experiment is found with the cc-pVDZ basis (entry 11), which is lowered still further using cc-pVTZ (entry 12). Jiao et al. [23] (using Becke3LYP/6-31G*) and Wu et al. [15] (using Becke3LYP/6-311+G(d,p)) made similar observations. The MP2 and MP3 methods (entries 2, 3, 7 and 8) similarly give values of ∆H ‡ far from experimental result, while the calculated MP4 values (entries 4 and 9) are starting to get close. However, oscillating values of ∆H ‡ are observed in the HF, MP2, MP3 and MP4 series of calculations, using both cc-pVDZ (entries 1-4) and cc-pVTZ (entries 7-9): HF overestimates, MP2 underestimates, MP3 overestimates (but less than HF) while MP4 again underestimates ∆H ‡ , but much less than MP2. Actually, the MP4 results are close to experiment (compare entries 4 and 9 with 17), although the MP4/cc-pVTZ energy at first glance appears too low. The MP results clearly converge to a limiting "MP-∞" value of ∆H ‡ , which is estimated to 4.9 kcal/mol for cc-pVDZ (from entries 1-4) and 4.7 kcal/mol for cc-pVTZ (from entries 7-9), both in good agreement with the experimental value of 4.8-5.2 kcal/mol. [1,2] Such oscillating behaviour of MP calculations are well known, and often the correct answer is to be found somewhere between the MP3 and MP4 results. [START_REF] Jensen | Introduction to Computational Chemistry[END_REF] For 1, this is indeed true. When comparing experimental and calculated values, it should be noted that the values presented here are calculated in vacuum, while experimental ∆H ‡ were measured in CF 2 Cl 2 /CD 2 Cl 2 , [1] and furthermore, that significant solvent effects have been reported [START_REF] Seefelder | [END_REF]45] for substituted derivatives of 1.

The coupled cluster methods CCSD and CCSD(T) were applied using the cc-pVDZ basis. By including non-iterative triples 3.3 kcal/mol is gained in ∆H ‡ (compare entries 14 and 15) relative to the CCSD result. The CCSD(T) value is around 2 kcal/mol higher than experiment. This is in accordance with Wu et al. [15] using the 6-311+G(d,p) basis.

In an attempt to include MP4 and CCSD(T) correlation effects in the interallylic interaction, and because it was not possible to fully optimize 1b at these levels of theory, a fitting approach was employed: Single-point MP4/cc-pVDZ and CCSD(T)/cc-pVDZ energies were calculated at several C 2v symmetric 1b geometries. These geometries were optimized at the MP2/cc-pVDZ level with fixed values of the <456 angle (Figure 2 right) which controls the interallylic distance R. Fitting of the MP4 and CCSD(T) energies to quadratic potentials gave optimized <456 angles of 1b at the MP4 and CCSD(T) levels (Figure 1). The optimal <456 angle extrapolated at the MP4 level is 89.0 degrees and at the CCSD(T) level it is 89.6 degrees (Figure 1). Hence, by including higher order correlation effects in the interallylic interaction, the two allylic subunits are moved away from each other, compared to the MP2/cc-pVDZ result of 86.9 degrees. The calculated ∆H ‡ at these angle optimized geometries are 4.91 kcal/mol for MP4 and 7.10 kcal/mol for CCSD(T) (Table 1 entries 5 and 16, Hence, around 0.2 kcal/mol are gained for both MP4 (compare entries 5 and 6) and CCSD(T) (compare entries 15 and 16) by including these correlation levels in the optimization of the interallylic interaction. The MP4 result is similar to the earlier estimated "MP-∞" value.

From the above discussion, the fact that the MP4/cc-pVDZ//MP2/cc-pVDZ value of ∆H ‡ (entry 4) agrees well with experimental values (entry 17), is not coincidental. It is clearly supported by the extrapolated "MP-∞" values (cc-pVDZ and cc-pVTZ). Hence, the MP4/cc-pVDZ//MP2/cc-pVDZ method serves as the compromise of choice with good predictive value of ∆H ‡ . Additionally, the CCSD(T)/cc-pVDZ//MP2/cc-pVDZ method was applied, since this method is increasingly gaining popularity and often is referred to as an effective way of including electron correlation. The geometry of 1a and 1b optimized at various levels of theory are presented in Figure 2 and compared with available experimental values. [46] As judged by Jiao, [23] the experimental [46] bond lengths r 15 and r 23 (Figure 2 left) in 1a should be interchanged. Very good agreement across the different applied computational methods is observed for 1a and the calculated bond lengths furthermore compare well with experimental values (Figure 2 left). The r 23 and r 34 bonds are clearly localized single and double bonds, respectively, while for 1b the corresponding bond length is of intermediate value, around 1.40 Å, predictive of a delocalized structure. For 1b (Figure 2 right), comparable bond lengths are found across the applied methods, but variations in the interallylic distances R are observed. The MP4 and CCSD(T) angle <456 optimized geometries (as described earlier) have interallylic distances of R = 2.11 Å and R = 2.12 Å, respectively. These methods are expected to give the best results, and therefore the other methods are compared to these. The B3LYP method, with cc-pVDZ and cc-pVTZ basis sets, gives interallylic distances similar to the highly correlated methods. So the B3LYP method appears to result in accurate geometries of 1b, but the corresponding ∆H ‡ is too low, as discussed earlier. The MP2 method, with cc-pVDZ and cc-pVTZ basis sets, gives interallylic distances of 2.07 Å and 2.04 Å, respectively, which are shorter than the MP4 and CCSD(T) values. However, the very good results for ∆H ‡ obtained using MP4/cc-pVDZ//MP2/cc-pVDZ, made MP2/cc-pVDZ the method chosen for geometry optimizations, with the notion that the method could underestimate the interallylic distance.

The aromaticity of the investigated molecules is addressed using NICS, which is defined as the negative of the absolute magnetic shielding [36] and is a simple and efficient measure of aromaticity. A negative NICS, like -9.7 at the ring center of benzene, [36] indicates an aromatic structure, while a positive value, like 27.6 in cyclobutadiene, [36] indicates an antiaromatic structure. A negligible value, like -2.2 in cyclohexane, [36] indicates a non-aromatic structure.

The NICS(0) of 1 is calculated at the geometric center (nonweighted mean of heavy atom coordinates) of the 6 active atoms (i.e. atoms 2,3,4,6,7 and 8, see Scheme 1). However, as discussed by Jiao, [23] this geometric center is only around 1.1 Å from the cyclopropane ring, hence the special shielding effect encountered for cyclopropane 16 needs to be taken into account when NICS is used to address aromaticity in 1. The part of the NICS(0) at the geometric center of 1 which arises from the cyclopropane ring 1.1 Å away, is taken into account by calculating NICS 1.1 Å above the face of 16. In Table 2 the calculated GIAO-HF/cc-pVDZ NICS values are listed. The NICS(1.1) value of -7.5 (entry 1 rigth) for 16, is close to the -10.8 NICS(0) calculated for 1a (entry 1 left). Hence, the cyclopropane unit is largely responsible for the calculated NICS(0) of 1a. The calculated NICS(0) of the delocalized 1b structure is between -22.1 and -22.9 (entry 2-4 left), which is not due to the cyclopropane unit alone, but must have a significant contribution from homoconjugation. The calculated NICS(0) of 1b clearly shows that it is a bishomoaromatic structure. The NICS values for 1 reported herein calculated using GIAO-HF/cc-pVDZ//MP2/cc-pVDZ are in good accordance with the values reported by Jiao [23] obtained using GIAO-HF/6-31G*//Becke3LYP/6-31G*.

Similarly, the magnetic properties of the corresponding three membered rings found in 2 -15, namely aziridine 17, phosphirane 18, diaziridine 19, and diphosphirane 20, are relevant parameters to address when using NICS(0) to discuss the aromaticity in 2 -15. The calculated NICS values are given in Table 2. For 17 a NICS(1.1) value of -4.2 is calculated for the planar C 2v symmetric structure, while -6.1 to -6.2 is calculated at each side of the non-planar C s symmetric structure. Similar for 18, a NICS(1.1) value of -2.8 is calculated for the planar C 2v symmetric structure, and NICS(1.1) at each side of the nonplanar C s symmetric structure is -8.1 to -9.1. For both 17 and 18 the nonplanar structures are the most stable ones, and NICS(1.1) at the two possible 1.1 Å points (above and below the ring centers) are close and hence considered identical for all practical purposes. For 19 and 20, one planar, one cis and one trans structure can be envisaged. The planar and the trans structures will each have a single point 1.1 Å above the ring centers, while the cis structures each will have two different points (above and below) 1.1 Å from the ring centers. The trans structures are found to be the most stable ones, closely followed by the cis structures and then the planar ones.

Almost identical NICS values are calculated for the cis and trans structures of 19 (entry 4) and 20 (entry 5), while their planar structures have negligible NICS values.

Aza-and phosphasemibullvalenes

The calculated MP4 and CCSD(T) values of ∆H ‡ for 2 -15 are presented in Table 3, while the calculated NICS(0), interallylic distances R and selected bond lengths are given in Table 4.

From geometry optimizations at the MP2/cc-pVDZ level, the delocalized, symmetric b structures of 5, 7, 9, 11, 12, 13 and 15 were found to be ground state structures. Vibrational frequency calculations gave only positive frequencies, confirming that they are energy minima. Full re-optimization of these delocalized 5b, 7b, 9b, 11b, 12b, 13b and 15b geometries, but now removing all symmetry constraints, in all cases gave structures that were indistinguishable from the originally optimized symmetry constrained structures. Energy minima for the localized azasemibullvalene 12a, the phosphasemibullvalenes 5a, 7a, 9a, 11a, 13a and 15a could not be located at the MP2/cc-pVDZ level.

Using the symmetry constrains shown in Scheme 1, these localized, low-symmetry a structures all optimize to structures that are indistinguishable from their corresponding higher symmetry b structures. Calculated MP4 and CCSD(T) energies, as well as MP2 zero-point energies, were identical for the a and b structures of 5, 7, 9, 11, 12, 13 and 15 (Table 3). From this it follows that these molecules are predicted not to undergo a Cope rearrangement, but have single well potentials with highly symmetric ground states. Hence, the only energy minimum that could be located at the MP2/cc-pVDZ surface for the hetero semibullvalenes 5, 7, 9, 11, 12, 13 and 15 are the delocalized b structures. At the computational levels applied herein, the early finding by Dewar [8] that 12 should be a localized structure is hence not corroborated. The calculated C-C bond lengths ( 2). When the calculated NICS(1.1) value of 18, up to -9.1 (Table 2), is taken into account when considering the values calculated for 5, 7, 9, 11 and 15, evidently there remains major contributions attributably only to these molecules being homoaromatic. Similarly, considering the calculated NICS(1.1) of 19 and 20 (Table 2) clearly leaves 12 and 13, respectively, homoaromatic based on their calculated NICS(0) values.

The NICS method has previously been applied to a tetraphosphabarbaralane related to 13, although in a more advanced molecular dynamics framework, [35] where the homoaromatic nature was confirmed when addressing temporal evolution and temperature dependency.

The interallylic distances R in 5, 7, 9, 11 and 15 are between 2.20 and 2.28 Å. They are C-P bonds and are hence expected to be somewhat longer than R in 1b. For 13, the interallylic P-P bond length is calculated to be 2.49 Å, which is in good agreement with the time dependent average of 2.53 Å calculated for the related tetraphosphabarbaralane. [35] For 12 the N-N interallylic R is 2.10 Å, comparable to R in 1b. These short N-N and P-P interallylic distances indicate good through-space interactions between the two allylic fragments. However, Brown et al. [13] discussed that substituted semibullvalenes with C 2v geometries not necessarily benefit from bishomoaromatic stabilization. This was observed in derivatives of 1 having large interallylic distances (>2.5Å), reduced interallylic through-space interaction and wavefunctions with large amount of diradical character or even triplet groundstates. To address this issue, the geometries of 5, 7, 9, 11, 12, 13 and 15 where optimized at the UHF-MP2/cc-pVDZ level. All wavefunctions were found to be pure singlets with <S 2 >=0.00, and the UHF-MP2 geometries were indistinguishable from the RHF-MP2 geometries. This further corroborates that 5, 7, 9, 11, 12, 13 and 15 are delocalized and bishomoaromatic. For the azasemibullvalenes 2, 4, 6, 8, 10 and 14, and the phosphasemibullvalene 3 energy minima for both the localized a and delocalized b structures could be identified at the MP2/cc-pVDZ surface.

Vibrational frequency analyses showed that all localized a structures had positive frequencies only, while delocalized b structures had 1 imaginary frequency, except for 4, 8 and 14. The latter b structures had all positive vibrational frequencies, indicating that these structures could also be energy minima. The calculated activation enthalpy for 2 is comparable to semibullvalene, while it is somewhat higher for 3. For 4, 6, 8, 10 and 14 the calculated activation enthalpies are several kcal/mol lower than semibullvalene. Notably 4 and 6 have very low calculated barriers to the Cope rearrangement, below 1 kcal/mol at MP4 and around 3 kcal/mol at CCSD(T). For 4 and 6, and possibly also 8, 10 and 14, the calculated ∆H ‡ values are so small that these molecules in reality could be delocalized ground states structures. At the computational levels applied herein, the early finding by Dewar [25] that 3,7-diazasemibullvalene 2 should be 'nonclassical' is hence not corroborated. Both 2 and the phospha derivative 3 have clear barriers to the Cope rearrangement.

The localized ground states 2a, 3a, 4a, 6a, 8a, 10a and 14a all have calculated NICS(0) values that are negative but clearly smaller than the corresponding delocalized b structures (Table 4). When NICS(1.1) of 16 is taken into account for 2a and 3a, their remaining NICS are -5.4 and 1.3, respectively, clearly suggesting non-aromatic ground state structures. Similarly, when NICS(1.1) of 17 is accounted for, the remaining NICS(0) for 6a and 10a is -4.9 and -4.8, respectively, pointing at non-aromatic structures. However, for 4a, 8a and 14a NICS(0) values after accounting for NICS(1.1) of 17, are -10.4, -7.6 and -9.4, respectively, suggesting aromatic structures. As discussed earlier 4, 8 and 14 have very low ∆H ‡ values, which together with their large negative NICS(0) suggests that they could be more delocalized than localized. This is in line with Dewar's [8,25] finding that 4 should actually be 'nonclassical'. The calculated ∆H ‡ for 4 is clearly lower than ∆H ‡ calculated for 1. This trend is supported by the experimental observations [26][27][28] on a 3,7-diphenyl derivative as well as on a 4,8-dicyano-3,7-diphenyl derivative of 4. In both cases, a drastic increase in the rate to the Cope rearrangement was observed.

The actual nature of the ground state of molecules based on structures like 4, 8 and 14, that is to which extend homoconjugation is present, is highly influenced by parameters like additional substituents, solvent and temperature [START_REF] Seefelder | [END_REF]45] .

Conclusions

Using correlated ab initio methods the tetraazasemibullvalene 12, the phosphasemibullvalenes 5, 7, 9, 11, 13 and 15 were shown to have highly symmetric, delocalized minima, possessing large, negative NICS values, which together with calculated bond lengths suggests that these molecules are highly bishomoaromatic. Furthermore, 4, 8 and 14 could also be bishomoaromatic structures due to their calculated NICS(0) values and very low ∆H ‡ . Appropriate introduction of two, three and four N or P atoms in the semibullvalene sceleton appears as a succesfull design strategy towards homoaromatic semibullvalene structures.

Supplementary material

Calculated energies and coordinates are available online as supplementary material. 1 HF/cc-pVDZ [b] 15.70 7 MP2/cc-pVTZ [e] 0.94 13 (6,6)CASSCF/cc-pVDZ [h] 23.54 2 MP2/cc-pVDZ [c] 2.25 8 MP3/cc-pVTZ [e] 9.76 14 CCSD/cc-pVDZ [c] 10.61 3 MP3/cc-pVDZ [c] 10.80 9 MP4/cc-pVTZ [e] 3.67 15 CCSD(T)/cc-pVDZ [c] 7.31 4 MP4/cc-pVDZ [c] 5.10 10 "MP-∞"/cc-pVTZ 4.73 16 CCSD(T)/cc-pVDZ [i] 7.10 5 MP4/cc-pVDZ [d] 4.91 11 Becke3LYP/cc-pVDZ [f] 4.30 17 Experiment [1,2] 4.8-5.2 6 "MP-∞"/cc-pVDZ 4.95 12 Becke3LYP/cc-pVTZ [g] 3.80 [a] ∆H ‡ = ∆E + ∆ZPE, where ∆E is the energy difference between the C 2v (1b) and C s (1a) semibullvalene, and ∆ZPE = -0.95 kcal/mol is the corresponding difference in zero-point vibrational energies taken from MP2/cc-pVDZ calculations.

F

[b] HF/cc-pVDZ optimized geometries. [c] MP2/cc-pVDZ optimized geometries. [d] MP4/cc-pVDZ optimized <456 geometries, see text. [e] MP2/cc-pVTZ optimized geometries. [f] Becke3LYP/cc-pVDZ optimized geometries. [g] Becke3LYP/cc-pVTZ optimized geometries. [h] (6,6)CASSCF/cc-pVDZ optimized geometries. [i] CCSD(T)/cc-pVDZ optimized <456 geometries, see text.

Table 2. Calculated GIAO-HF/cc-pVDZ NICS(0) (ppm) for 1a, 1b and NICS(1.1) (ppm) for 16 -20.

Molecule NICS(0) [a] Molecule NICS(1.1) [b] 1a [c] -10.8 16 [c] -7.5 1b [c] -22.9 17 [c] -4.2 [f] , -6.1 [g] , -6.2 [g] 1b [d] -22.4 18 [c] -2.8 [f] , -8.1 [g] , -9.1 [g] 1b [e] -22.1 19 [c] -0.6 [f] , -4.7 [h] , -5.6 [h] , -5.3 [i] 20 [c]
-0.3 [f] , -8.8 [h] , -9.2 [h] , -9.9 [i] [a] NICS calculated at ring center. [b] NICS calculated 1.1 Å above ring center. [c] MP2/cc-pVDZ optimized geometries. [d] MP4/cc-pVDZ optimized angle geometries, see text.

[e] CCSD(T)/cc-pVDZ optimized angle geometries, see text. [a] For localized structures R is taken as r 46 . [b] For these structures no localized energy minimum could be located. The a structures, optimized without any symmetry constraints, are indistinguishable from the symmetry constrained b structures.

  of planar structure.[g] NICS(1.1) of non-planar structure.

  of cis structure.[i] NICS(1.1) of trans structure.

Table 4

 4 ) r 34 and r 78 for 5, and r 67 and r 78 for 7 and 9, are between 1.40 and 1.41 Å, which is intermediate of classical single and double bond lengths. Similarly, the C-P distances r 23 and r 67 for 5 of 1.75 Å, and r 23 and r 34 for 7 of 1.74 Å, are all shorter than the C-P single bond r 23 and longer than the C-P double bond r 34 observed in the bond localized 3a. For 11, 13 and 15 all C-P bond lengths are found to be between 1.74 to 1.76 Å, which is of intermediate length like in bond delocalized 3b. For 12, a C-N bond length of 1.35 Å is calculated, which is in between the C-N single and double bonds found for 2a. These geometric findings all points at delocalized bonds in 5, 7, 9, 11, 12, 13 and 15. The calculated NICS(0) values of the delocalized ground states 5, 7, 9, 11, 12, 13 and 15 are all large and negative (Table4). They span from -19.3 for 15 to -22.8 for 7. The values are comparable to NICS(0) calculated for delocalized semibullvalene 1b (Table

Table 1 .

 1 Calculated activation enthalpies ∆H ‡ (kcal/mol)[a] of 1 at different levels of theory.

	o r
	P
	e
	e r
	R
	e
	v i e
	w

Table 3 .

 3 Calculated MP4 and CCSD(T) energy differences, ∆E MP4 and ∆E CC respectively (kcal/mol), MP2 Zero-Point Energy differences ∆ZPE MP2 (kcal/mol) and MP4 and CCSD(T) activation enthalpies, ∆H ‡MP4 and ∆H ‡CC respectively (kcal/mol).[a] Molecule ∆E MP4 ∆H

Table 4 .

 4 Calculated Nucleus Independent Chemical Shifts NICS(0) (ppm), interallylic distances R[a] (Å), bond lengths r 23 , r 34 , r 67 and r 78 (Å).

	Molecule NICS(0)	R	r 23	r 34	r 67	r 78	Molecule NICS(0)	R	r 23	r 34	r 67	r 78
	2a	-12.9	2.25	1.42	1.30	1.30	1.42	9a [b]	-21.4	2.25	2.12	2.12	1.41	1.41
	2b	-24.9	1.95	1.35	1.35	1.35	1.35	9b [b]	-21.4	2.25	2.12	2.12	1.41	1.41
	3a	-6.2	2.36	1.85	1.71	1.71	1.85	10a	-11.0	2.25	1.41	1.30	1.30	1.42
	3b	-18.8	1.98	1.76	1.76	1.76	1.76	10b	-20.4	2.00	1.35	1.35	1.34	1.34
	4a	-16.6	2.27	1.42	1.36	1.31	1.46	11a [b]	-20.8	2.23	1.74	1.74	1.76	1.76
	4b	-23.3	2.08	1.35	1.40	1.35	1.40	11b [b]	-20.8	2.23	1.74	1.74	1.76	1.76
	5a [b]	-22.4	2.28	1.75	1.40	1.75	1.40	12a [b]	-22.1	2.10	1.35	1.35	1.35	1.35
	5b [b]	-22.4	2.28	1.75	1.40	1.75	1.40	12b [b]	-22.1	2.10	1.35	1.35	1.35	1.35
	6a	-11.1	2.28	1.41	1.30	1.37	1.47	13a [b]	-21.7	2.49	1.75	1.75	1.75	1.75
	6b	-17.8	2.05	1.35	1.35	1.40	1.40	13b [b]	-21.7	2.49	1.75	1.75	1.75	1.75
	7a [b]	-22.8	2.26	1.74	1.74	1.40	1.40	14a	-15.6	2.19	1.41	1.31	1.28	1.41
	7b [b]	-22.8	2.26	1.74	1.74	1.40	1.40	14b	-27.3	1.98	1.33	1.34	1.33	1.34
	8a [b]	-13.8	2.24	1.41	1.27	1.37	1.46	15a [b]	-19.3	2.20	2.14	1.76	2.14	1.76
	8b [b]	-23.8	2.01	1.33	1.33	1.40	1.40	15b [b]	-19.3	2.20	2.14	1.76	2.14	1.76

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[a] ∆H ‡ = ∆E + ∆ZPE, where ∆E is the energy difference between the more symmetric b and less symmetric a structures and ∆ZPE is the corresponding difference in zero-point vibrational energies taken from MP2/cc-pVDZ calculations. [b] For these structures no localized energy minimum could be located. The a structures, optimized without any symmetry constraints, are indistinguishable from the symmetry constrained b structures.