John Wiley

L Piñeiro

D Vicente

M Montes

U Hernández-Dorronsoro

Dr G Cilla

Human Parechoviruses in Infants with Systemic Infection 4 5

Keywords: Human parechovirus, systemic infection, febrile syndrome, meningitis

Human parechoviruses (HPeVs) are RNA viruses related to neonatal sepsis, 2 meningoencephalitis and other infections in young children. Little clinical and 3

epidemiological information is available on these viruses. HPeVs were sought in 4 cerebrospinal fluid from 397 infants aged less than 12 months from whom a sample was 5 obtained to exclude meningitis or encephalitis from 2006 to 2009. HPeV infections 6 were also tested in stool samples from 271 children aged less than 3 years old with 7 gastroenteritis from November 2008 to March 2009. HPeV detection was by real-time 8 polymerase chain reaction assay (region 5´UTR), followed by genotyping (region 9 VP3/VP1). HPeVs were detected in the cerebrospinal fluid of nine infants (2.3%), one 10 aged 6 months and eight aged 14-55 days old. All were admitted to hospital for febrile 11 syndrome with abrupt clinical deterioration and suspected systemic infection without 12 clear laboratory signs of meningeal inflammation. The same virus was detected in all 13 the available nasopharyngeal aspirates, stool and/or serum samples from each patient. 14 At least eight of the nine cases were caused by HPeV3. HPeVs were detected in stool 15 samples from 17 children (6.3%), the most prevalent types being types 1 and 3. In 16 conclusion, HPeV infection is common in the Basque Country (Spain) and HPeV3 is a 17 significant cause of hospital admissiondue to systemic infection in the first few months 18 of life. In these patients, HPeVs should be investigated as part of routine tests for 19 enterovirus. 20 21 KEY WORDS: Human parechovirus, systemic infection, febrile syndrome, meningitis 22

INTRODUCTION 1
Human parechoviruses (HPeVs) are RNA viruses belonging to the 2 Picornaviridae family and have been associated with distinct types of acute infection, 3 mainly systemic, gastrointestinal, respiratory, and central nervous system (CNS) 4 infections in infants and young children [START_REF] Harvala | Human parechoviruses: biology, epidemiology and 24 clinical significance[END_REF]. Initially 5 identified as enteroviruses because of their clinical and biological characteristics, in 6 1999 HPeVs were reassigned to the new Parechovirus genus on the basis of 7 phylogenetic studies. The two oldest members of the genus, HPeV types 1 and 2 8 (HPeV1 and HPeV2), previously known as echovirus 22 and 23 respectively, were 9 isolated in 1956 and have mainly been detected in children with gastrointestinal and 10 respiratory infections, as well as in asymptomatic children [START_REF] Stanway | Human parechoviruses--biology and 4 clinical significance[END_REF]. Since 11 2004, eight new types have been discovered and at least a further four are under 12 investigation. Consequently, this genus appears to be highly diverse 13 [www.picornaviridae.com/parechovirus/hpev/hpev.htm; Kim [START_REF] Pham | Novel human parechovirus, 13 Sri Lanka[END_REF]. 14 HPeV3 was described in Japan in 2004 and has been related to severe disease, 15 especially sepsis-like illness, meningitis and encephalitis in the first few months of life, 16 although this virus has also been detected in other types of infections [START_REF] Ito | Isolation and identification 5 of a novel human parechovirus[END_REF]17 Harvala et al., 2009] 2009 to exclude CNS infection. These samples represented 90% of the patients of this 7 age in whom CSF was extracted during the study period. The CSF or cDNA aliquot 8 were stored at -80ºC and were studied retrospectively. From January 2009 diagnosis 9 was made prospectively. These samples were taken for routine investigation of 10 enterovirus. 11

The second group comprised 271 stool samples from a distinct group of children 12 aged less than 3 years old with acute gastroenteritis (median age 13.8 months), obtained 13 between November 2008 and March 2009. These samples were suspended in B199 14 medium (Sigma-Aldrich, St Louis MO, USA) and were refrigerated at 4ºC for a 15 maximum of 14 days until processing. To do this, the samples were pooled in groups of 16 nine samples each, and samples from groups with a positive result were studied 17 individually. The aim of the study of this second group was to obtain preliminary 18 information on the presence of HPeVs and their distinct types in the Basque Country 19 (Spain), since the gastrointestinal tract is believed to be one of the main sites of 20 replication [START_REF] Harvala | Human parechoviruses: biology, epidemiology and 24 clinical significance[END_REF]. 21

22

Molecular Methods 23

HPeV detection was by a nucleic acid amplification technique, with reverse 24 transcription of viral RNA followed by polymerase chain reaction (RT-PCR). RNA 25 HPeVs were detected in CSF from nine infants aged less than 12 months old 10 (2.3% [9/397]; range 14-185 days), this percentage being 3.0% in infants aged less than 11 2 months old (8/265) (Table I). In these age groups, the percentages of enterovirus 12 detection were 16.1% (64/397) and 18.9% (50/265). HPeV strains were detected in all 4 13 years of the study, mainly (7/9) between June and November (Table II). The nine 14 infants with HPeV in CSF had been discharged from hospital after birth and were 15 readmitted due to HPeV infection. Eight of these infants were born at term (≥37 weeks) [START_REF] Goldman | Fever in Infants Group Research, 18 Pediatric Emergency Research of Canada. Practice variations in the treatment of febrile 19 infants among pediatric emergency physicians[END_REF] and one was premature (week 34 of pregnancy). At admission to the pediatric 17 emergency department, the infants showed febrile syndrome (temperature >38ºC) 18 accompanied by abrupt clinical deterioration and suspected infection (sepsis-like 19 illness), with no clear laboratory signs of meningeal inflammation in CSF (low 20 cellularity [1-12 leukocytes/mm 3 ] and slightly elevated proteinorrachia in three patients 21 with normal glucorrachia). In three patients no focus was found, five showed mild 22 respiratory symptoms and three showed gastrointestinal symptoms. In all patients, 23 concentrations of C reactive protein and procalcitonin in the acute phase were <10 24 mg/dl and <0.5 ng/ml, respectively, and the results of blood and urine cultures were 25 Eight patients were treated with antibiotics for 4-14 days. In the nine infants, HPeVs 2 were also detected in all the samples available from other sites: in nasopharyngeal 3 aspirates in four (in one of these, the serum sample was also positive), in stool samples 4 and nasopharyngeal aspirates in three, and in stool samples only in two. Eight cases 5 were attributed to HPeV3 infection based on the amplicon sequence of the VP3/VP1 6 region in the CSF or other samples (Table II). The nucleotide sequence identity among 7 samples from the same patient was ≥99%, while that among the eight cases and the 8 prototype HPeV3 strain A308/99 (GenBank accession number AB084913) was 94-96% 9 (Fig. 1). The amplicon sequence of the 5´UTR region of the patient without 10 amplification of the VP3/VP1 region had 94% nucleotide identity with the prototype 11 HPeV3 strain A308/99. 12 13 Human Parechoviruses in Stool Samples 14 HPeVs were detected in stool samples from 17 children with gastroenteritis 15 (6.3% [17/271]; median age 210 days). Of these, the nested RT-PCR for genotyping 16 was positive in 14, corresponding to type 1 (n=5), type 3 (n=6) and type 4 (n=2) strains. 17

One strain (two isolates, Spa250229/08 and Spa254577/08) could not be allocated to 18 any HPeV type (nucleotide sequence similarity <75% with known types) (Fig. 1). 19

Another enteropathogenic agent, norovirus, was found only in a stool sample of one of 20

3

 3 . Knowledge of the pathogenicity, geographical distribution and 18 other epidemiological features of these viruses is limited, since the tests required for 19 their detection are not routinely undertaken in most clinical microbiology laboratories. 20 The aims of the present study were to analyze the presence of HPeVs in the Basque 21 Country (Spain) and their possible association with systemic infection in infants aged This study was performed in two groups of samples. The first group consisted of 4 424 samples from 397 infants aged less than 12 months (median 1.1 months) in whom 5 cerebrospinal fluid (CSF) samples were taken between January 2006 and September 6

  in the NucliSENS Easy-Mag system (bio-Mèrieux SA, 1 Marcy l`Etoile, France) with an input volume of 200 µL of CSF and 400 µL of the other 2 samples. Reverse transcription of RNA to cDNA was performed with M-MuLV reverse 3 transcriptase (USB Corp., Cleveland, OH, USA), using random primers. PCR was 4 performed in real time (Ligthcycler, Roche) by amplifying a fragment of the 5'UTR 5 gene [van der Sanden et al., 2008]. Each PCR run included a positive control (a 6 previously positive clinical sample, GenBank accession number GU477688) and a 7 negative control (water control), which were treated identically to the clinical samples 8 throughout. The presence of amplification inhibitors in the clinical samples was 9 excluded through amplification of a fragment of the human β-globin gene, or the 10 GAPDH gene from 2009 onwards. All procedures were carried out using routine 11precautions to avoid contamination. In patients with positive CSF, HPeVs were 12 investigated in other samples related to the episode (nasopharyngeal aspirates, faeces 13 and/or serum). For parechovirus genotyping, in the HPeV-positive samples, a nested 14 RT-PCR was performed, which amplified a 300 bp fragment of the VP3/VP1 region 15[START_REF] Harvala | Epidemiology and clinical associations of human parechovirus 22 respiratory infections[END_REF]. The amplicons obtained from both regions (5'UTR and 16 VP3/VP1) were sequenced in an ABIPRISM 3100 Genetic Analyzer (Applied 17 Biosystems, Foster City, CA, USA) and the sequences were analyzed in the BasicLocal 18 Alignment and Search Tool (http://www.ncbi.nlm.nih.gov/blast/Blast.cgi). A 19 phylogenetic analysis was performed using the ClustalW2 and Mega4.1 programs. 20 Detection of enterovirus RNA in CSF was performed with the cDNA sample by a real-21 time PCR method, based on amplification of the 5´UTR gene[START_REF] Rotbart | Diagnosis of enteroviral meningitis by using PCR with a 2 colorimetric microwell detection assay[END_REF]. 22 described in the present study were deposited in the 3 GenBank database under accession numbers GU477688-GU477711 and GU574202-

  cultures and investigation of enterovirus in CSF were also negative. 1

23 Few

 23 studies have investigated the presence of HPeVs in CSF samples requested 24 for viral diagnosis. In the present study, HPeVs were found in 3.0% (8/264) of CSF 25 aged less than 2 months under investigation for possible CNS 1 infection, representing a 16% increase in diagnostic confirmation with respect to the 50 2 samples positive for enterovirus. These data are similar to those reported in a large 3 study performed in Edinburgh between 2006 and 2008, in which HPeV3 was detected 4 in 2.9% of CSF samples sent for virological analysis in infants aged less than 2 months 5 old (n=278)[START_REF] Harvala | Human parechoviruses: biology, epidemiology and 24 clinical significance[END_REF]. Likewise, in Amsterdam, HPeVs were found in 4.6% 6 of CSF samples from children investigated in 2004-2006, who were mainly aged less 7 than 2 years old[START_REF] Wolthers | Human parechoviruses as an important viral cause of 20 sepsislike illness and meningitis in young children[END_REF]. HPeVs also accounted for most of the isolates 8 that could not be typed in a Dutch enterovirus surveillance program between 2000 and 9 2007, representing 12.2% of the total number of viruses obtained, mostly from young 10 children with systemic infections [van der Sanden et al., 2008]. Therefore, HPeVs can 11 be a significant cause of severe illness in young children, especially in the first few 12 months of life. 13 Eight of the nine HPeV strains detected in the present study in CSF samples 14 were confirmed as type 3, and the ninth was probably also HPeV3, providing evidence 15 that, among HPeVs, this type is the main agent of CNS infections, in line with recent 16 observations [Boivin et al., 2005; Benschop et al., 2006; van der Sanden et al., 2008; 17 Harvala et al., 2009]. In contrast, HPeV1, the most prevalent type in respiratory and 18 stool samples, is rarely detected in CSF and is normally found in slightly older children, 19 aged 3 to 24 months [Abed and Boivin, 2006; Benschop et al., 2006; van der Sanden et 20 al., 2008; Harvala et al., 2009]. In the present study, HPeV1 was also detected in stool 21 samples, but not in CSF. The reason for the differences observed between these types is 22 unknown, although HPeV3 lacks the RGD motif involved in cell entrance of HPeV1 23 and other viruses, which could translate into differences in tissue tropism and 24 pathogenicity [van der Sanden et al., 2008]. Another hypothesis is that the differences 25 to the lower prevalence of HPeV3 antibodies observed in adults, making 1 passive transfer of antibodies to the newborn less frequent [Benschop et al., 2006; 2 Harvala and Simmonds, 2009]. 3 The nine patients with HPeV detection in CSF were admitted to hospital due to 4 febrile syndrome and suspected severe acute infection (one patient in the pediatric 5 intensive care unit), with a mean stay of 6.8 days, and eight were treated with 6 antibiotics. Febrile syndrome with no apparent source of infection in the first few 7 months of life is a common reason for consulting hospital emergency services, as well 8 as for hospital admission for microbiological analysis and antibiotic therapy, since this 9 syndrome may be caused by potentially life-threatening bacterial infections [Goldman et 10 al., 2009]. However, the most frequent cause of these episodes are viral infections, 11 mainly due to enterovirus, which are sometimes serious [Verboon-Maciolek et al., 12 2003; Jordán et al, 2009]. The results of this study and other recent observations support 13 the causal role of HPeV3 in this type of episodes (febrile syndrome, sepsis-like illness) 14 in the first few months of life [Boivin et al 2005; Benschop et al, 2006; Verboon-15 Maciolek et al, 2008a; Harvala et al., 2009; Levorson et al., 2009]. 16 A notable finding in this series was the poor or absent cellular reaction and the 17 frequent normal values of CSF protein concentrations despite HPeV detection in the 18 CNS. This finding, recently reported in another study [Verboon-Maciolek et al., 2008a], 19 is also observed in enterovirus infections [Ritichier et al., 2005], and could be due to the 20 systemic nature of some HPeV3 infections rather than to localization of the infection in 21 the CNS. HPeV3 detection in the samples investigated in each patient in this series 22 (nasopharyngeal aspirate, stools, serum) supports the disseminated nature of these 23 infections [Harvala et al., 2009]. Although most of the HPeV3 isolates in CSF reported 24 to date correspond to children with sepsis-like illness [Wolthers et al., 2008; Harvala et 25 2009], as in our series, cases of neonatal meningoencephalitis with white matter 1 injury have also been reported, sometimes with neurological sequelae and almost 2 always without pleocytosis or biochemical alterations in CSF [Verboon-Maciolek et al., 3 2008b]. 4 In contrast with previous findings, the present series showed no predominance of 5 cases among males [Benschop et al., 2006] or a biannual circulation pattern for HPeV3 6 (paired years), as reported in Holland and Scotland [van der Sanden et al., 2008; 7 Benschop et al., 2008a; Harvala et al., 2009]. The present data suggest that viral 8 circulation was higher in autumn 2006 and summer 2009, an observation that is 9 coherent with variations in the annual incidence and indicates the possibility of 10 geographical differences in the circulation of this virus. However, a preferential 11 distribution in the warmest months (between late spring and autumn) was found, as 12 reported by other authors [van der Sanden et al., 2008; Wolthers et al., 2008] and as 13 usually occurs with enterovirus [Khetsuriani et al., 2006]. 14The non-coding 5'UTR region of HPeV shows preserved areas in the distinct 15 types of HPeVs and consequently this region is useful for Parechovirus genus detection.[START_REF] Goldman | Fever in Infants Group Research, 18 Pediatric Emergency Research of Canada. Practice variations in the treatment of febrile 19 infants among pediatric emergency physicians[END_REF] However, because antigenicity lies in the VP region, this region is chosen for 17 genotyping, even though VP is sometimes difficult to amplify because of its greater 18 variability, especially in clinical samples such as CSF, which normally have a low viral 19 load[Benschop et al., 2008a]. As with other genera of the Picornaviridae family, 20 recombination events resulting in the exchange of non-coding regions among HPeV are 21 frequent[Benschop et al., 2008b]. In the present series, among the nine HPeV3 strains 22 causing systemic infection, eight (88.9%) could be genotyped based on direct 23 sequencing of the VP3/VP1 region, since other samples obtained simultaneously with 24 the CSF sample were available. In the remaining strain, the similarity observed in the 25 the A308/99 reference strain and the fact that HPeV3 is less variable 1 and less frequently recombines than other HPeV types[Benschop et al., 2008b] 2 suggests that it probably also belongs to type 3. 3 Clinical microbiology laboratories do not test routinely for HPeVs in biological 4 samples due to lack of awareness of the role of these viruses in human disease and 5 technical difficulties. However, current real-time PCR methods allow rapid etiological 6 diagnosis of HPeV infections, which could aid the management of children with febrile 7 syndromes, thus avoiding unnecessary treatments and shortening length of hospital stay. 8 The results of the present study, the first to demonstrate the presence of HPeV3 in 9 southern Europe, indicate that investigation of HPeVs in CSF should be undertaken as 10 part of routing tests for enterovirus in young infants admitted to hospital for sepsis-like 11 syndrome and/or meningoencephalitis. 12 13 With regard to the issue of Ethical Committee approval and informed consent, 14 none of the human samples were obtained for research purposes. The study involves 15 only samples taken for routine diagnosis of meningeal, acute respiratory, or acute 16 enteric infections. For this reason, informed consent from the parents or guardians was 17 not obtained (most of the study was retrospective). Nevertheless, a statement by the 18 Ethical Committee for the study of new potentially pathogenic viruses in samples taken 19 for routine diagnosis was obtained in December 2007

Figure 1 .

 1 Figure 1. Phylogenetic analysis of 26 nucleotide sequences of human parechovirus (HPeV) (region

Table I .

 I Distribution by age and detection of parechoviruses and enteroviruses in 1 infants aged less than 12 months in whom a cerebrospinal fluid sample was taken to 2

				Journal of Medical Virology		Page 16 of 20
	3	exclude central nervous system infection
	4	Parechovirus Nº (%) 0 -4 (5.8) 4 (5.3) 0 -0 -0 -0 -1 (1.8) r e P Nº investigated 121 69 75 25 18 17 15 57 F <14 days Age 14-30 days 1 month 2 months 3 months 4 months 5 months 6-11 months o r e F o r P e e r R e v i e w Enterovirus Nº (%) 13 (10.7) 21 (30.4) 16 (21.3) 6 (24.0) 1 (5.6) 1 (5.9) 0 6 (10.5) -
		Total	397	9	(2.3) R	64	(16.1)
					e
					v i e
						w
				John Wiley & Sons

Table II .

 II Main clinical characteristics and laboratory data of nine infants with systemic infection by human parechovirus type 3 1

										19
	1
	2
	3									CSF		Positive samples
	4 5 6 7	Case Nº -year (month)	Sex and age (days)	Main diagnosis	Other diagnoses Coinfections	Antibiotic therapy (days)	Hospital stay (days)	(leukocytes/mm 3 and proteins	CRP (mg/dL) and PCT (ng/mL)	(amplified region ) (5'UTR) (VP3/VP1)
	8									mg/dL)
	9
	10 11 12	1 -2006 (October)	M (45)	Febrile syndrome	Irritability, skin rash (face and trunk ),	Rhinovirus in NPA	CTX+AM (5) and CFX (5)	5	1 and 43	0.3 and <0.5	CSF, NPA, FAE	FAE
	13				URTS
	14
	15 16 17 18 19 20 21 22 23	2 -2006 (October) 3 -2006 (November)	F (14) M (55)	Febrile syndrome Febrile syndrome F o r P Irritability Irritability		--	CTX+AM (9) CTX+AM (4)	8 5	8 and 53 2 and 22	2.6 and 0.5 7 and <0.5	CSF, FAE CSF, FAE	-FAE
	24 25 26 27 28	4 -2007 (June)	M (43)	Febrile syndrome	URTS e e r	-	CTX+AM (5)	5	5 and 35	1.8 and <0.5	CSF, NPA, FAE NPA, FAE
	29 30			Febrile	R
	31 32 33 34 35 36 37 38	5 -2008 (December)	F (16)	syndrome	Irritability, URTS, vomiting	-e v i CTX+AM (4) e w	4	10 and 42	9.6 and 0.12	CSF, NPA, FAE NPA, FAE
	39
	40
	41
	42
	43
	44

Journal of Medical Virology

fever in young infants, as identified by direct typing of cerebrospinal fluid samples. J

John Wiley & SonsJournal of Medical Virology

VP3/VP1, positions 2159-2458) of isolates of cerebrospinal fluid (csf), faeces (fae) and nasopharyngeal

John Wiley & Sons

Journal of Medical Virology F o r P e e r R e v i e w


MD Dimitriou Gabriel
email: gdimitriou@med.upatras.gr

MD Dimitriou Gabriel

MD Papakonstantinou Despina

MSC Stavrou F Eleana

MD Tzifas Sotirios

MD Sotirios

Angiotensin converting enzyme gene polymorphism and respiratory muscle function in infants

Keywords: ACE gene, respiratory muscles, pressure-time index, infants ACE gene, respiratory muscles, pressure-time index, infants ACE gene, respiratory muscles, pressure-time index, infants

Objective: Angiotensin converting enzyme (ACE) gene contains a polymorphism consisting of either the presence (I) or absence (D) of a 287 base pair fragment.

Recent studies have suggested that the I-allele may be associated with superior exercise endurance; respiratory muscle function may be similarly influenced. The pressure-time index of inspiratory muscles (PTImus) is a measure of the loadcapacity ratio of the inspiratory muscles. The objective of this study was to determine whether infants homozygous for the I-allele have lower PTImus compared to infants homozygous for the D-allele or heterozygous I/D. Patients and Methods: One hundred thirty two infants were studied. ACE genotyping was performed by polymerase chain reaction amplification, using DNA from peripheral blood. PTImus was calculated as (Pi mean /Pi max )x(Ti/Ttot), where Pi mean was the mean inspiratory pressure estimated from airway pressure, generated 100 milliseconds after an occlusion (P 0.1 ), Pi max was the maximum inspiratory pressure and Ti/Ttot was the ratio of the inspiratory time to the total respiratory cycle time. Pi max was the largest pressure generated during brief airway occlusions performed at the end of a spontaneous crying effort.

Results: Infants with I/I genotype had significantly lower PTImus than infants with either D/D or I/D genotypes (p=0.000007). ACE genotype was significantly related (p=0.005) to PTImus measurements, independent of other factors that may affect respiratory muscle function.

Conclusion:

These results suggest that an association of ACE genotypes with PTImus measurements may exist in infants.

INTRODUCTION

Angiotensin I-converting enzyme (ACE) converts angiotensin I to vasoactive and aldosterone-stimulating peptide angiotensin II and it is an important component of the local renin-angiotensin systems (RASs). 1,2 There is a significant variation in circulating ACE activity among individuals, which may be genetically determined.

The human ACE gene is located on chromosome 17 (17q23) and contains a two allele polymorphism, consisting of either the presence (insertion, I) or absence (deletion, D) of a 287 base pair (bp) fragment. 3 The deletion is associated with increased ACE activity in both tissue and circulation and accounts for about 44% of the intra-individual variation in plasma ACE activity in Caucasians. 3 Recent studies have suggested that I-allele may be associated with superior exercise endurance, 4,5 partly through improved skeletal muscle metabolic efficiency. 6 Respiratory muscle function may be similarly influenced.

Several methods have been used to assess the endurance properties of respiratory muscles. Diaphragmatic pressure-time index (PTIdi) is a measure of the load-capacity ratio of the diaphragm. 7 In adults, a PTIdi greater than 0.15-0.18 may indicate impending diaphragm fatigue. 7 Determination of PTIdi, however, is invasive, requiring the use of an esophageal catheter. A non-invasive method of measuring pressure-time index of inspiratory muscles (PTImus) has been validated in both adults 8 and infants. [START_REF] Dimitriou | A non-invasive tension-time index of respiratory muscles in neonates[END_REF] The aim of this study was to test the hypothesis that infants homozygous for the I-

METHODS

Patients

Infants cared for at the Neonatal Intensive Care Unit-Pediatric Department of the University General Hospital of Patras, Greece, were eligible for the study. Infants were entered into the study if parents gave informed written consent. The study was approved by the local Research Ethics Committee. Part of the studied population was recruited from a study examining the association of ACE genotypes and circulating blood ACE on diaphragmatic function in infants. All infants were studied awake, before discharge, in supine position and at least one hour after a feed.

Infants had no respiratory symptoms for at least 3 days before measurement. Furthermore, infants were on full oral feeds, had serum electrolytes, calcium, magnesium and phosphates within normal range and did not receive any methylxanthines.

ACE genotype determination

ACE genotyping was performed on DNA extracted from whole blood, collected from an indwelling catheter or via peripheral venipuncture during routine blood sampling.

The method has been previously described. [START_REF] Lindpaintner | A prospective evaluation of an angiotensinconverting-enzyme gene polymorphism and the risk of ischemic heart disease[END_REF] Briefly, DNA was extracted from 200 µl blood using Qiamp spin columns (Blood mini kit-Qiagen, QIAGEN Inc., Germantown, U.S.A). DNA was analyzed by electrophoresis on an agarose gel. DNA amplification of the 16 th ACE intron was performed using two sets of primers flanking the polymorphic site, (outer and inner primers), because mistyping of the D/D genotype is reported to occur using the conventional amplification with insertion/deletion (I/D) flanking primer.

Measurement of PTImus

Airflow was measured using a pneumotachograph (Mercury F10L, GM Instruments, Kilwinning, Scotland) connected to a differential pressure transducer (DP45, range ± 2 cm H 2 O, Validyne Corp, Northridge, CA, USA). Airway pressure (Paw) was measured from a side port on the pneumotachograph using a differential pressure transducer (DP45, range ± 100 cm H 2 O, Validyne Corp, Northridge, CA, USA). The signals from the pressure transducers were amplified using a carrier amplifier (Validyne CD 280, Validyne Corp, Northridge, CA, USA). The pressure and flow signals were recorded and displayed in real time on a computer (Dell Optiplex GX620, Dell Inc., Texas, U.S.A) running Labview TM software (National Instruments, Austin, Texas, U.S.A) with analog-to-digital sampling at 100 Hz (16-bit NI PCI-6036E, National Instruments, Austin, Texas, U.S.A). P 0.1 was calculated as the airway pressure generated 100 milliseconds after an occlusion, using a face mask and a unidirectional valve which allowed expiration but not inspiration, while the infant was quietly breathing. At least four airway occlusions were performed and the average P 0.1 was calculated. Pressure-time index of the inspiratory muscles (PTImus), was calculated as: PTImus= (Pi mean /Pi max ) x (Ti/Ttot) where Pi mean was the average airway pressure during inspiration, obtained from the formula Pi mean = 5 x P 0.1 x Ti. [START_REF] Gaultier | Inspiratory force reserve of the respiratory muscles in children with chronic obstructive pulmonary disease[END_REF] Pi max was the maximum inspiratory airway pressure, Ti was the inspiration time and Ttot was the total time for each breath calculated from the airway flow signal. To measure Pi max, a facemask (total deadspace, 4.5 mL) was held firmly over the infant's nose and mouth and the airway was occluded at the end of a spontaneous crying effort using a unidirectional valve attached to the pneumotachograph, which allowed expiration, but not inspiration. The occlusion was Pi max measurements were not statistically different between the three groups (table 2). Stepwise regression analysis revealed that ACE genotypes were significantly related to the PTImus measurements after logarithmic transformation, independent of GA, BW, PNA, gender and support from mechanical ventilation (p=0.005) (table 3).

RESULTS

Between

In the subgroup of infants that never required any form of ventilatory support, 10 infants (13.5%) were homozygous I/I, 20 ( Furthermore, the ACE polymorphism was related to the PTImus measurements, independent of other factors, which could affect respiratory muscle performance. The association between ACE genotypes and PTImus measurements was replicated on the subgroup of infants that required ventilation in the initial stage of their illness and on the subgroup of infants that never required any form of ventilatory support.

One limitation of our study was the fact that measurements in an age-matched control group, consisting of infants in the postnatal ward, were not performed. The recruitment of such babies is extremely difficult. To overcome this issue, we measured infants in the neonatal unit that are free of factors that could possibly interfere with respiratory muscle function such as requirement of respiratory support. One part of the whole population consisted of infants that never required oxygen supplementation, indeed, and a separate analysis was performed in this subgroup.

Fatigue of respiratory muscles may result in an inability to maintain adequate alveolar ventilation and respiratory failure. A simple method of studying the endurance properties of inspiratory muscles, is the measurement of the duration that a specific submaximal task (maximum sustainable ventilation, external mechanical load) can be maintained (time limit or endurance time, TLim), referred to as the point where the inspiratory muscles failed to maintain the task, despite maximal effort. 7 However, this kind of test is difficult to be applied to infants. Diaphragmatic pressuretime index (PTIdi), the product of the ratio of mean inspiratory transdiaphragmatic pressure per breath (Pdi mean ) to maximum transdiaphragmatic pressure (Pdi max ) and the ratio of the inspiratory time (Ti) to the total respiratory cycle time (Ttot), is closely related to the Tlim. [START_REF] Bellemare | Effect of pressure and timing of contraction on human diaphragm fatigue[END_REF] The determination of PTIdi, however, is rather invasive, since it requires the placement of an esophageal catheter. Analysis of inspiratory muscle function by measurement of PTImus was first described by Gaultier et al. [START_REF] Gaultier | Inspiratory force reserve of the respiratory muscles in children with chronic obstructive pulmonary disease[END_REF] As measurement of P 01 is non-invasive, measurement of PTImus has the advantage of being relatively easy to be performed. This index was validated in adults. 8 Studies in healthy children, demonstrated that PTImus ranged between 0.036 and 0.090. [START_REF] Hayot | Determinants of the tension-time index of inspiratory muscles in children with cystic fibrosis[END_REF][START_REF] Hahn | Noninvasive tension time index in relation to severity of disease in children with cystic fibrosis[END_REF] In spontaneously breathing infants, PTImus was compared to PTIdi and an agreement between the two methods, using Bland and Altman analysis, was found. [START_REF] Dimitriou | A non-invasive tension-time index of respiratory muscles in neonates[END_REF] In this study, PTImus measurements in infants that never required any oxygen supplementation ranged between 0.028 and 0.119, something that is close to the values observed in healthy children. Several studies have suggested that there is an association of the ACE I/D polymorphism with exercise performance. British Army recruits were examined before and after an identical 10-week general physical training program. 5 Although, pre-training performance was independent of genotype, the improvement in performance was 11-fold greater among those of I/I, compared with D/D genotype. 5 Consistently, a significant excess of the I/I genotype was found in Australian national rowers attending their pre-Olympics selection trial, compared to matched healthy controls. [START_REF] Gayagay | Elite endurance athletes and the ACE I allele--the role of genes in athletic performance[END_REF] The frequency of I-allele was higher in British Olympic-standard distance runners. 4 Furthermore, genotype distribution and I-allele frequency differed significantly between elite mountaineers, who had ascended beyond 7000 m without the use of supplemental oxygen and controls. 5 All such studies are prone to difficulties in comparison, as the definition of the "elite athlete" and the heterogeneity of the elite athlete phenotype may be variable. The polymorphism of the ACE gene has been examined in infants, but not in association with muscle endurance. 17,[START_REF] Harding | Angiotensin-converting enzyme DD genotype is associated with worse perinatal cardiorespiratory adaptation in preterm infants[END_REF] The association of the ACE genotype and respiratory muscle function, has not been widely examined yet. In a study of COPD (Chronic Obstructive Pulmonary Disease) patients and healthy control subjects, the ACE genotype was related to the regulation of skeletal muscle aerobic work efficiency, but it was not associated with the ventilatory responses to exercise. [START_REF] Zhang | Association between angiotensinconverting enzyme gene polymorphisms and exercise performance in patients with COPD[END_REF] The relationship between circulating ACE activity, ACE I/D genotype, mechanical efficiency and the maximal rate of oxygen uptake has been assessed in untrained women. [START_REF] Day | No correlation between circulating ACE activity and VO2max or mechanical efficiency in women[END_REF] Neither circulating ACE activity nor ACE genotype showed a statistically significant association with any of the endurance phenotypes measured. However, ACE inhibition in heart failure increases respiratory muscle strength [START_REF] Coirault | Angiotensin-converting enzyme inhibitor therapy improves respiratory muscle strength in patients with heart failure[END_REF] and exercise tolerance. [START_REF] Mancini | Dependence of enhanced maximal exercise performance on increased peak skeletal muscle perfusion during long-term captopril therapy in heart failure[END_REF][START_REF] Drexler | Contrasting peripheral short-term and long-term effects of converting enzyme inhibition in patients with congestive heart failure. A double-blind, placebocontrolled trial[END_REF] The enhanced endurance performance in the ACE I/I genotype may be due to higher oxygen delivery, conversion of fast-twitch, type II, to slow-contracting, type I, muscle fibers, increased mitochondrial numbers and density, and raised myoglobin content of the muscle. 24 ACE I-allele compared to D-allele is associated with an increased percentage of fatigue-resistant type I skeletal muscle fibres [START_REF] Zhang | The I allele of the angiotensin-converting enzyme gene is associated with an increased percentage of slow-twitch type I fibers in human skeletal muscle[END_REF] and hence with enhanced mechanical 6 and metabolic [START_REF] Montgomery | Angiotensinconverting-enzyme gene insertion/deletion polymorphism and response to physical training[END_REF] efficiency of the trained muscle. The effect of ACE genotype on energy balance, might be mediated through several different pathways, such as systemic endocrine or nutritional mechanisms. [START_REF] Montgomery | Angiotensinconverting-enzyme gene insertion/deletion polymorphism and response to physical training[END_REF] Local RASs exist in several tissues. The adipose RAS may alter mobilization of substrate from fat stores and affect metabolism. [START_REF] Montgomery | Angiotensinconverting-enzyme gene insertion/deletion polymorphism and response to physical training[END_REF] Finally, the local skeletal muscle RAS may modify the use of substrate and enhance the metabolic efficiency of the muscles. [START_REF] Montgomery | Angiotensinconverting-enzyme gene insertion/deletion polymorphism and response to physical training[END_REF] Several factors may affect respiratory muscle endurance such as nutrition, [START_REF] Arora | Effect of body weight and muscularity on human diaphragm muscle mass, thickness, and area[END_REF] prolonged ventilatory support, [START_REF] Tobin | Discontinuation of mechanical ventilation[END_REF] drugs, [START_REF] Aubier | Dopamine effects on diaphragmatic strength during acute respiratory failure in chronic obstructive pulmonary disease[END_REF][START_REF] Vires | Effects of aminophylline on diaphragmatic fatigue during acute respiratory failure[END_REF]31 as well as phosphate, 32 calcium 33 and magnesium [START_REF] Dhingra | Hypomagnesemia and respiratory muscle power[END_REF] blood levels. All infants were measured prior to discharge, being on full enteral feeds, they did not receive any medication and their biochemistry blood tests were within the normal range during measurements.

PTImus integrates all components that may affect inspiratory muscles while breathing at rest, i.e, timing of breathing cycle, inspiratory demand at rest and maximal inspiratory force reserve. When we considered these components, the timing of the breath cycle (Ti/Ttot) and the maximal inspiratory force reserve (Pi max )

were not statistically different between infants with I/I, D/D and I/D ACE genotype. Indeed, infants with non-I/I ACE genotypes have been suggested to be more likely to develop bronchopulmonary dysplasia, perhaps as a consequence of the role of ACE activity in the inflammatory cascade. 17 However, in this study, no difference in regards to bronchopulmonary dysplasia rate between infants with I/I, I/D and D/D ACE genotypes was noted. In addition, the same association between ACE genotypes and Pi mean was replicated in a subgroup of infants that never required any form of respiratory support and in infants that never required any oxygen supplementation.

Fatigue threshold of inspiratory muscles has not yet been established in infants.

All infants measured, were spontaneously breathing on air. Therefore, the PTImus results of all infants would be expected to lie far from the fatigue threshold. These results, however, indicate an increased effectiveness of inspiratory muscles of infants with the I/I ACE genotype, due to a lower energy demand and thus to a lower risk of respiratory muscle fatigue, under conditions that increase inspiratory load. [START_REF] Roussos | Inspiratory muscle fatigue[END_REF][START_REF] Vassilakopoulos | Neuromuscular respiratory failure[END_REF] The findings of this study suggest that in clinical practice, infants with I/I ACE genotype, if supported by mechanical ventilation, may succeed extubation more often than infants with I/D and D/D ACE genotypes or may have a lower risk of respiratory failure under conditions that increase inspiratory load, such as respiratory infections.

In conclusion, these results suggest that an association of ACE genotypes with 1,2 There is a significant variation in circulating ACE activity among individuals, which may be genetically determined.

The human ACE gene is located on chromosome 17 (17q23) and contains a two allele polymorphism, consisting of either the presence (insertion, I) or absence (deletion, D) of a 287 base pair (bp) fragment. 3 The deletion is associated with increased ACE activity in both tissue and circulation and accounts for about 44% of the intra-individual variation in plasma ACE activity in Caucasians. 3 Recent studies have suggested that I-allele may be associated with superior exercise endurance, 4,5 partly through improved skeletal muscle metabolic efficiency. 6 Respiratory muscle function may be similarly influenced.

Several methods have been used to assess the endurance properties of respiratory muscles. Diaphragmatic pressure-time index (PTIdi) is a measure of the load-capacity ratio of the diaphragm. 7 In adults, a PTIdi greater than 0.15-0.18 may indicate impending diaphragm fatigue. 7 Determination of PTIdi, however, is invasive, requiring the use of an esophageal catheter. A non-invasive method of measuring pressure-time index of inspiratory muscles (PTImus) has been validated in both adults 8 and infants. [START_REF] Dimitriou | A non-invasive tension-time index of respiratory muscles in neonates[END_REF] The aim of this study was to test the hypothesis that infants homozygous for the I- Infants had no respiratory symptoms for at least 3 days before measurement. Furthermore, infants were on full oral feeds, had serum electrolytes, calcium, magnesium and phosphates within normal range and did not receive any methylxanthines.

ACE genotype determination

ACE genotyping was performed on DNA extracted from whole blood, collected from an indwelling catheter or via peripheral venipuncture during routine blood sampling.

The method has been previously described. [START_REF] Lindpaintner | A prospective evaluation of an angiotensinconverting-enzyme gene polymorphism and the risk of ischemic heart disease[END_REF] Briefly, DNA was extracted from 200 µl blood using Qiamp spin columns (Blood mini kit-Qiagen, QIAGEN Inc., Germantown, U.S.A). DNA was analyzed by electrophoresis on an agarose gel. DNA amplification of the 16 th ACE intron was performed using two sets of primers flanking the polymorphic site, (outer and inner primers), because mistyping of the D/D genotype is reported to occur using the conventional amplification with insertion/deletion (I/D) flanking primer. P 0.1 was calculated as the airway pressure generated 100 milliseconds after an occlusion, using a face mask and a unidirectional valve which allowed expiration but not inspiration, while the infant was quietly breathing. At least four airway occlusions were performed and the average P 0.1 was calculated. Pressure-time index of the inspiratory muscles (PTImus), was calculated as: PTImus= (Pi mean /Pi max ) x (Ti/Ttot) where Pi mean was the average airway pressure during inspiration, obtained from the formula Pi mean = 5 x P 0.1 x Ti. [START_REF] Gaultier | Inspiratory force reserve of the respiratory muscles in children with chronic obstructive pulmonary disease[END_REF] Pi max was the maximum inspiratory airway pressure, Ti was the inspiration time and Ttot was the total time for each breath calculated from the airway flow signal. To measure Pi max, a facemask (total deadspace, 4.5 mL) was held firmly over the infant's nose and mouth and the airway was occluded at the end of a spontaneous crying effort using a unidirectional valve attached to the pneumotachograph, which allowed expiration, but not inspiration. The occlusion was Pi max measurements were not statistically different between the three groups (table 2). Stepwise regression analysis revealed that ACE genotypes were significantly related to the PTImus measurements after logarithmic transformation, independent of GA, BW, PNA, gender and support from mechanical ventilation (p=0.005) (table 3).

RESULTS

Between

In the subgroup of infants that never required any form of ventilatory support, 10 Furthermore, the ACE polymorphism was related to the PTImus measurements, independent of other factors, which could affect respiratory muscle performance. The association between ACE genotypes and PTImus measurements was replicated on the subgroup of infants that required ventilation in the initial stage of their illness and on the subgroup of infants that never required any form of ventilatory support.

One limitation of our study was the fact that measurements in an age-matched control group, consisting of infants in the postnatal ward, were not performed. The recruitment of such babies is extremely difficult. To overcome this issue, we measured infants in the neonatal unit that are free of factors that could possibly interfere with respiratory muscle function such as requirement of respiratory support. One part of the whole population consisted of infants that never required oxygen supplementation, indeed, and a separate analysis was performed in this subgroup.

Fatigue of respiratory muscles may result in an inability to maintain adequate alveolar ventilation and respiratory failure. A simple method of studying the endurance properties of inspiratory muscles, is the measurement of the duration that a specific submaximal task (maximum sustainable ventilation, external mechanical load) can be maintained (time limit or endurance time, TLim), referred to as the point where the inspiratory muscles failed to maintain the task, despite maximal effort. 7 However, this kind of test is difficult to be applied to infants. Diaphragmatic pressuretime index (PTIdi), the product of the ratio of mean inspiratory transdiaphragmatic pressure per breath (Pdi mean ) to maximum transdiaphragmatic pressure (Pdi max ) and the ratio of the inspiratory time (Ti) to the total respiratory cycle time (Ttot), is closely related to the Tlim. [START_REF] Bellemare | Effect of pressure and timing of contraction on human diaphragm fatigue[END_REF] The determination of PTIdi, however, is rather invasive, since it requires the placement of an esophageal catheter. Analysis of inspiratory muscle function by measurement of PTImus was first described by Gaultier et al. [START_REF] Gaultier | Inspiratory force reserve of the respiratory muscles in children with chronic obstructive pulmonary disease[END_REF] As measurement of P 01 is non-invasive, measurement of PTImus has the advantage of being relatively easy to be performed. This index was validated in adults. 8 Studies in healthy children, demonstrated that PTImus ranged between 0.036 and 0.090. [START_REF] Hayot | Determinants of the tension-time index of inspiratory muscles in children with cystic fibrosis[END_REF][START_REF] Hahn | Noninvasive tension time index in relation to severity of disease in children with cystic fibrosis[END_REF] In spontaneously breathing infants, PTImus was compared to PTIdi and an agreement between the two methods, using Bland and Altman analysis, was found. [START_REF] Dimitriou | A non-invasive tension-time index of respiratory muscles in neonates[END_REF] In this study, PTImus measurements in infants that never required any oxygen supplementation ranged between 0.028 and 0.119, something that is close to the values observed in healthy children. Several studies have suggested that there is an association of the ACE I/D polymorphism with exercise performance. British Army recruits were examined before and after an identical 10-week general physical training program. 5 Although, pre-training performance was independent of genotype, the improvement in performance was 11-fold greater among those of I/I, compared with D/D genotype. 5 Consistently, a significant excess of the I/I genotype was found in Australian national rowers attending their pre-Olympics selection trial, compared to matched healthy controls. [START_REF] Gayagay | Elite endurance athletes and the ACE I allele--the role of genes in athletic performance[END_REF] The frequency of I-allele was higher in British Olympic-standard distance runners. 4 Furthermore, genotype distribution and I-allele frequency differed significantly between elite mountaineers, who had ascended beyond 7000 m without the use of supplemental oxygen and controls. 5 All such studies are prone to difficulties in comparison, as the definition of the "elite athlete" and the heterogeneity of the elite athlete phenotype may be variable. The polymorphism of the ACE gene has been examined in infants, but not in association with muscle endurance. 17,[START_REF] Harding | Angiotensin-converting enzyme DD genotype is associated with worse perinatal cardiorespiratory adaptation in preterm infants[END_REF] The association of the ACE genotype and respiratory muscle function, has not been widely examined yet. In a study of COPD (Chronic Obstructive Pulmonary Disease) patients and healthy control subjects, the ACE genotype was related to the regulation of skeletal muscle aerobic work efficiency, but it was not associated with the ventilatory responses to exercise. [START_REF] Zhang | Association between angiotensinconverting enzyme gene polymorphisms and exercise performance in patients with COPD[END_REF] The relationship between circulating ACE activity, ACE I/D genotype, mechanical efficiency and the maximal rate of oxygen uptake has been assessed in untrained women. [START_REF] Day | No correlation between circulating ACE activity and VO2max or mechanical efficiency in women[END_REF] Neither circulating ACE activity nor ACE genotype showed a statistically significant association with any of the endurance phenotypes measured. However, ACE inhibition in heart failure increases respiratory muscle strength [START_REF] Coirault | Angiotensin-converting enzyme inhibitor therapy improves respiratory muscle strength in patients with heart failure[END_REF] and exercise tolerance. [START_REF] Mancini | Dependence of enhanced maximal exercise performance on increased peak skeletal muscle perfusion during long-term captopril therapy in heart failure[END_REF][START_REF] Drexler | Contrasting peripheral short-term and long-term effects of converting enzyme inhibition in patients with congestive heart failure. A double-blind, placebocontrolled trial[END_REF] The enhanced endurance performance in the ACE I/I genotype may be due to higher oxygen delivery, conversion of fast-twitch, type II, to slow-contracting, type I, muscle fibers, increased mitochondrial numbers and density, and raised myoglobin content of the muscle. 24 ACE I-allele compared to D-allele is associated with an increased percentage of fatigue-resistant type I skeletal muscle fibres [START_REF] Zhang | The I allele of the angiotensin-converting enzyme gene is associated with an increased percentage of slow-twitch type I fibers in human skeletal muscle[END_REF] and hence with enhanced mechanical 6 and metabolic [START_REF] Montgomery | Angiotensinconverting-enzyme gene insertion/deletion polymorphism and response to physical training[END_REF] efficiency of the trained muscle. The effect of ACE genotype on energy balance, might be mediated through several different pathways, such as systemic endocrine or nutritional mechanisms. [START_REF] Montgomery | Angiotensinconverting-enzyme gene insertion/deletion polymorphism and response to physical training[END_REF] Local RASs exist in several tissues. The adipose RAS may alter mobilization of substrate from fat stores and affect metabolism. [START_REF] Montgomery | Angiotensinconverting-enzyme gene insertion/deletion polymorphism and response to physical training[END_REF] Finally, the local skeletal muscle RAS may modify the use of substrate and enhance the metabolic efficiency of the muscles. [START_REF] Montgomery | Angiotensinconverting-enzyme gene insertion/deletion polymorphism and response to physical training[END_REF] Several factors may affect respiratory muscle endurance such as nutrition, [START_REF] Arora | Effect of body weight and muscularity on human diaphragm muscle mass, thickness, and area[END_REF] prolonged ventilatory support, [START_REF] Tobin | Discontinuation of mechanical ventilation[END_REF] drugs, [START_REF] Aubier | Dopamine effects on diaphragmatic strength during acute respiratory failure in chronic obstructive pulmonary disease[END_REF][START_REF] Vires | Effects of aminophylline on diaphragmatic fatigue during acute respiratory failure[END_REF]31 as well as phosphate, 32 calcium 33 and magnesium [START_REF] Dhingra | Hypomagnesemia and respiratory muscle power[END_REF] blood levels. All infants were measured prior to discharge, being on full enteral feeds, they did not receive any medication and their biochemistry blood tests were within the normal range during measurements.

PTImus integrates all components that may affect inspiratory muscles while breathing at rest, i.e, timing of breathing cycle, inspiratory demand at rest and maximal inspiratory force reserve. When we considered these components, the timing of the breath cycle (Ti/Ttot) and the maximal inspiratory force reserve (Pi max )

were not statistically different between infants with I/I, D/D and I/D ACE genotype. Indeed, infants with non-I/I ACE genotypes have been suggested to be more likely to develop bronchopulmonary dysplasia, perhaps as a consequence of the role of ACE activity in the inflammatory cascade. 17 However, in this study, no difference in regards to bronchopulmonary dysplasia rate between infants with I/I, I/D and D/D ACE genotypes was noted. In addition, the same association between ACE genotypes and Pi mean was replicated in a subgroup of infants that never required any form of respiratory support and in infants that never required any oxygen supplementation.

Fatigue threshold of inspiratory muscles has not yet been established in infants.

All infants measured, were spontaneously breathing on air. Therefore, the PTImus results of all infants would be expected to lie far from the fatigue threshold. These results, however, indicate an increased effectiveness of inspiratory muscles of infants with the I/I ACE genotype, due to a lower energy demand and thus to a lower risk of respiratory muscle fatigue, under conditions that increase inspiratory load. [START_REF] Roussos | Inspiratory muscle fatigue[END_REF][START_REF] Vassilakopoulos | Neuromuscular respiratory failure[END_REF] The findings of this study suggest that in clinical practice, infants with I/I ACE genotype, if supported by mechanical ventilation, may succeed extubation more often than infants with I/D and D/D ACE genotypes or may have a lower risk of respiratory failure under conditions that increase inspiratory load, such as respiratory infections.

In conclusion, these results suggest that an association of ACE genotypes with

  allele may have lower PTImus compared to infants homozygous for the D-allele or heterozygous I/D.

11

 11

  maintained for at least four inspiratory efforts. At least three airway occlusions were performed and the maximum Pi max achieved for individual, was recorded.Statistical analysisDatawas tested for normality using the Shapiro-Wilk and D'Agostino skewness tests. Differences between ACE genotype groups were assessed for statistical significance using the Kruskal-Wallis and Dunn's post-hoc non-parametric tests and Cramer's V test, as appropriate. Stepwise multiple regression analysis was performed to determine whether the polymorphism of the ACE gene is related to PTImus measurements, independently to gestational (GA) and postnatal (PNA) age, birthweight (BW), gender and support from mechanical ventilation. Statistical analysis was performed using StatView 5.0 (SAS Institute, Inc., NC, USA), NCSS 2007 (NCSS, Utah, USA) and SPSS version 17 (SPSS Inc, Chicago, IL, USA).

  January 2007 and August 2008 from the 532 infants admitted in the Neonatal Intensive Care Unit of the University of Patras, one hundred thirty two infants were included in the study. Fifty eight infants required ventilation in the initial stage of their illness. The characteristics of the study population are presented in table 1. Eighteen infants (13.6%) were homozygous for the I-allele (I/I), 41 (31.1%) homozygous for the D-allele (D/D) and 73 infants (55.3%) were heterozygous I/D. A Hardy-Weinberg equilibrium (HWE) calculation was performed to examine whether genotype frequencies remain constant in the studied population. Participants were in HWE (Chi square for HWE 2.613, p=0.106). Overall, there were no significant differences in the characteristics of the infants with I/I, D/D and I/D ACE genotypes (table 2). Only four infants were oxygen dependent at 36 weeks postmenstrual age (1 infant with I/I, 2 with I/D and 1 with D/D ACE genotypes). Infants with I/I genotype had lower PTImus than infants with either D/D or I/D genotypes (Kruskal-Wallis, p=0.000007; Dunn's test, z-value= 3.58, p<0.01 and z-value= 4.88, p<0.01, respectively) (table 2), (figure). No difference in regards to PTImus values was found between infants with D/D and I/D genotypes (Dunn's test, z-value=1.39, n.s). Analysis of the components of the PTImus has shown that only the ratio of Pi mean to Pi max was lower in infants with the I/I genotype, compared to infants with either the D/D or I/D genotypes (Kruskal-Wallis, p= 0.000005; Dunn's test, z-value= 3.33, p<0.01 and z-value= 4.91, p<0.01, respectively). Lower values of Pi mean in infants with I/I genotype compared to infants with either D/D or I/D genotypes, were also observed (Kruskal-Wallis, p= 0.0009; Dunn's test, z-value= 2.61, p<0.05 and z-value= 3.73, p<0.01, respectively) (table 2).

  In the subgroup of the infants who required ventilation in the initial stage of their illness, 8 infants (13.8%) were homozygous I/I, 21(36.2%) homozygous D/D and 29 infants (50%) were heterozygous I/D. Participants were in HWE (Chi square for HWE 0.1622, p=0.687). Infants with I/I ACE genotype had lower PTImus than infants with either D/D or I/D genotypes (Kruskal-Wallis, p= 0.0024; Dunn's test, z-value= 1.98, p<0.05 and z-value= 3.37, p<0.01, respectively). DISCUSSION In this study, an association between ACE genotypes and PTImus measurements in infants, was demonstrated. Infants homozygous for the I-allele had lower PTImus values than infants homozygous for the D-allele and heterozygous I/D. No difference in regards to PTImus values was found between infants with D/D and I/D genotypes.

  However, the Pi mean /Pi max ratio and the Pi mean (inspiratory demand at rest), were lower in infants with the I/I compared with infants with D/D and I/D genotypes. A higher inspiratory demand at rest (Pi mean ) in infants with D/D and I/D, compared with infants with I/I ACE genotypes, could suggest that a higher elastic and/or resistive load (existing lung disease) imposed on the respiratory muscles of infants with I/D and D/D genotypes compared with infants with I/I ACE genotypes, may exist.

9 .

 9 PTImus measurements may exist in infants, independent of other factors, which could affect respiratory muscle function. Dimitriou G, Patel S, Patel J, Rafferty GF, Greenough A. A non-invasive tension-time index of respiratory muscles in neonates. European Respiratory Society Annual Congress. Glasgow, UK; 2004. 10. Lindpaintner K, Pfeffer MA, Kreutz R, Stampfer MJ, Grodstein F, LaMotte F, Buring J, Hennekens CH. A prospective evaluation of an angiotensinconverting-enzyme gene polymorphism and the risk of ischemic heart disease. N Engl J Med 1995;332(11):706-711. 11. Shanmugam V, Sell KW, Saha BK. Mistyping ACE heterozygotes. PCR Methods Appl 1993;3(2):120-121. 12. Gaultier C, Boule M, Tournier G, Girard F. Inspiratory force reserve of the respiratory muscles in children with chronic obstructive pulmonary disease. Am Rev Respir Dis 1985;131(6):811-815. 13. Bellemare F, Grassino A. Effect of pressure and timing of contraction on human diaphragm fatigue. J Appl Physiol 1982;53(5):1190-1195. 14. Hayot M, Guillaumont S, Ramonatxo M, Voisin M, Prefaut C. Determinants of the tension-time index of inspiratory muscles in children with cystic fibrosis. Pediatr Pulmonol 1997;23(5):336-343. 15. Hahn A, Ankermann T, Claass A, Mann M, Lindemann H, Neubauer BA. Noninvasive tension time index in relation to severity of disease in children with cystic fibrosis. Pediatr Pulmonol 2008;43(10):973-981. 16. Gayagay G, Yu B, Hambly B, Boston T, Hahn A, Celermajer DS, Trent RJ. Elite endurance athletes and the ACE I allele--the role of genes in athletic performance. Hum Genet 1998;103(1):48-50. SR, Johnson RH, Park DM, Rennie MJ, Sulaiman WR. Differences in the metabolic and hormonal response to exercise between racing cyclists and untrained individuals. J Physiol 1976;258(1):1-18. 25. Zhang B, Tanaka H, Shono N, Miura S, Kiyonaga A, Shindo M, Saku K. The I allele of the angiotensin-converting enzyme gene is associated with an increased percentage of slow-twitch type I fibers in human skeletal muscle. Clin Genet 2003;63(2):139-144. 26. Montgomery H, Clarkson P, Barnard M, Bell J, Brynes A, Dollery C, Hajnal J, Hemingway H, Mercer D, Jarman P, Marshall R, Prasad K, Rayson M, Saeed N, Talmud P, Thomas L, Jubb M, World M, Humphries S. Angiotensinconverting-enzyme gene insertion/deletion polymorphism and response to physical training. Lancet 1999;353(9152):541-545. 27. Arora NS, Rochester DF. Effect of body weight and muscularity on human diaphragm muscle mass, thickness, and area. J Appl Physiol 1982;52(1):64-70. 28. Tobin MJ, Alex C. Discontinuation of mechanical ventilation. In: Tobin M, editor. Principles and Practice of Mechanical Ventilation. New York: McGraw-Hill; 1994. p 1177-1206. 29. Aubier M, Murciano D, Menu Y, Boczkowski J, Mal H, Pariente R. Dopamine effects on diaphragmatic strength during acute respiratory failure in chronic obstructive pulmonary disease. Ann Intern Med 1989;110(1):17-23. 30. Vires N, Aubier M, Murciano D, Fleury B, Talamo C, Pariente R. Effects of aminophylline on diaphragmatic fatigue during acute respiratory failure. Am Rev Respir Dis 1984;129(3):396-402.

  Figure legend

  Figure 39x36mm (300 x 300 DPI)

  allele may have lower PTImus compared to infants homozygous for the D-allele or heterozygous I/D. at the Neonatal Intensive Care Unit-Pediatric Department of the University General Hospital of Patras, Greece, were eligible for the study. Infants were entered into the study if parents gave informed written consent. The study was approved by the local Research Ethics Committee. Part of the studied population was recruited from a study examining the association of ACE genotypes and circulating blood ACE on diaphragmatic function in infants. All infants were studied awake, before discharge, in supine position and at least one hour after a feed.

11

 11

  Airflow was measured using a pneumotachograph (Mercury F10L, GM Instruments, Kilwinning, Scotland) connected to a differential pressure transducer (DP45, range ± 2 cm H 2 O, Validyne Corp, Northridge, CA, USA). Airway pressure (Paw) was measured from a side port on the pneumotachograph using a differential pressure transducer (DP45, range ± 100 cm H 2 O, Validyne Corp, Northridge, CA, USA). The signals from the pressure transducers were amplified using a carrier amplifier (Validyne CD 280, Validyne Corp, Northridge, CA, USA). The pressure and flow signals were recorded and displayed in real time on a computer (Dell Optiplex GX620, Dell Inc., Texas, U.S.A) running Labview TM software (National Instruments, Austin, Texas, U.S.A) with analog-to-digital sampling at 100 Hz (16-bit NI PCI-6036E, National Instruments, Austin, Texas, U.S.A).

  maintained for at least four inspiratory efforts. At least three airway occlusions were performed and the maximum Pi max achieved for individual, was recorded.Statistical analysisDatawas tested for normality using the Shapiro-Wilk and D'Agostino skewness tests. Differences between ACE genotype groups were assessed for statistical significance using the Kruskal-Wallis and Dunn's post-hoc non-parametric tests and Cramer's V test, as appropriate. Stepwise multiple regression analysis was performed to determine whether the polymorphism of the ACE gene is related to PTImus measurements, independently to gestational (GA) and postnatal (PNA) age, birthweight (BW), gender and support from mechanical ventilation. Statistical analysis was performed using StatView 5.0 (SAS Institute, Inc., NC, USA), NCSS 2007 (NCSS, Utah, USA) and SPSS version 17 (SPSS Inc, Chicago, IL, USA).

  January 2007 and August 2008 from the 532 infants admitted in the Neonatal Intensive Care Unit of the University of Patras, one hundred thirty two infants were included in the study. Fifty eight infants required ventilation in the initial stage of their illness. The characteristics of the study population are presented in table 1. Eighteen infants (13.6%) were homozygous for the I-allele (I/I), 41 (31.1%) homozygous for the D-allele (D/D) and 73 infants (55.3%) were heterozygous I/D. A Hardy-Weinberg equilibrium (HWE) calculation was performed to examine whether genotype frequencies remain constant in the studied population. Participants were in HWE (Chi square for HWE 2.613, p=0.106). Overall, there were no significant differences in the characteristics of the infants with I/I, D/D and I/D ACE genotypes (table 2). Only four infants were oxygen dependent at 36 weeks postmenstrual age (1 infant with I/I, 2 with I/D and 1 with D/D ACE genotypes). Infants with I/I genotype had lower PTImus than infants with either D/D or I/D genotypes (Kruskal-Wallis, p=0.000007; Dunn's test, z-value= 3.58, p<0.01 and z-value= 4.88, p<0.01, respectively) (table 2), (figure). No difference in regards to PTImus values was found between infants with D/D and I/D genotypes (Dunn's test, z-value=1.39, n.s). Analysis of the components of the PTImus has shown that only the ratio of Pi mean to Pi max was lower in infants with the I/I genotype, compared to infants with either the D/D or I/D genotypes (Kruskal-Wallis, p= 0.000005; Dunn's test, z-value= 3.33, p<0.01 and z-value= 4.91, p<0.01, respectively). Lower values of Pi mean in infants with I/I genotype compared to infants with either D/D or I/D genotypes, were also observed (Kruskal-Wallis, p= 0.0009; Dunn's test, z-value= 2.61, p<0.05 and z-value= 3.73, p<0.01, respectively) (table 2).

infants ( 13 .

 13 5%) were homozygous I/I, 20 (27.0%) homozygous D/D and 44 infants (59.5%) were heterozygous I/D. Infants in this subgroup were in HWE (Chi square for HWE 3.304, p=0.069). No significant difference in the characteristics of the infants with I/I, D/D and I/D ACE genotypes, was found (table 4). Infants with I/I genotype had lower PTImus than infants with either D/D or I/D genotypes (Kruskal-Wallis, p= 0.0022; Dunn's test, z-value= 3.07, p<0.01 and z-value= 3.39, p<0.01, respectively), (table 4). No difference regarding PTImus values was found between infants with D/D and I/D genotypes (Dunn's test, z-value= 0.0008, n.s). The ratio of Pi mean to Pi max was lower in infants with the I/I genotype, compared to infants with either the D/D or I/D genotypes (Kruskal-Wallis, p= 0.0015; Dunn's test, z-value= 2.95, p<0.01 and z-value= 3.58, p<0.01, respectively). Pi max measurements were not statistically different between the three groups (table 4), but Pi mean was lower in infants with I/I genotype, compared to infants with either D/D or I/D genotypes (Kruskal-Wallis, p= 0.003; Dunn's test, z-value= 3.31, p<0.01 and z-value= 3.00, p<0.01, respectively). In this subgroup, forty six infants never required any oxygen supplementation; 6 infants (13.0%) were homozygous I/I, 13 (28.3%) homozygous D/D and 27 infants (58.7%) were heterozygous I/D. Infants in the subgroup that never required any oxygen supplementation were in HWE (Chi square for HWE 1.872, p=0.171). The median (range) of PTImus of the whole subgroup was 0.078 (0.028-0.119). Infants with I/I ACE genotype had lower PTImus than infants with either D/D or I/D genotypes [median (range), 0.040 (0.028-0.083) versus 0.073 (0.052-119) and 0.080 (0.040-117), respectively; Kruskal-Wallis, p= 0.0075; Dunn's test, z-value= 2.55, p<0.05 and z-value= 3.11, p<0.01, respectively]. The ratio of Pi mean to Pi max and Pi mean were lower in infants with the I/I ACE genotype, compared to infants with either D/D or I/D genotypes (Kruskal-Wallis, p= 0.0047; Dunn's test, z-value= 2.42, p<0.05 and z-value= 3.27, p<0.01, respectively and Kruskal-Wallis, p= 0.019; Dunn's test, z-value= 2.46, p<0.05 and z-value= 2.74, p<0.05, respectively).

  In the subgroup of the infants who required ventilation in the initial stage of their illness, 8 infants (13.8%) were homozygous I/I, 21(36.2%) homozygous D/D and 29 infants (50%) were heterozygous I/D. Participants were in HWE (Chi square for HWE 0.1622, p=0.687). Infants with I/I ACE genotype had lower PTImus than infants with either D/D or I/D genotypes (Kruskal-Wallis, p= 0.0024; Dunn's test, z-value= 1.98, p<0.05 and z-value= 3.37, p<0.01, respectively). DISCUSSION In this study, an association between ACE genotypes and PTImus measurements in infants, was demonstrated. Infants homozygous for the I-allele had lower PTImus values than infants homozygous for the D-allele and heterozygous I/D. No difference in regards to PTImus values was found between infants with D/D and I/D genotypes.

  However, the Pi mean /Pi max ratio and the Pi mean (inspiratory demand at rest), were lower in infants with the I/I compared with infants with D/D and I/D genotypes. A higher inspiratory demand at rest (Pi mean ) in infants with D/D and I/D, compared with infants with I/I ACE genotypes, could suggest that a higher elastic and/or resistive load (existing lung disease) imposed on the respiratory muscles of infants with I/D and D/D genotypes compared with infants with I/I ACE genotypes, may exist.

  Figure legend

Table 1 .

 1 Characteristics of the study population. Data are demonstrated as n (%) or median (range).

	Pediatric Pulmonology	Page 22 of 47
	Number of studied infants	132
	Gestational age (weeks)	35.5 (27-40)
	Birth weight (g)	2680 (900-4540)
	Gender (male)	76	(57.6)
	Preterm	80	(60.6)
	Duration of mechanical ventilation (days)	0	(0-59)
	Diagnoses on admission
	Respiratory distress syndrome (RDS)	45	(34.1)
	Transient tachypnea of the newborn (TTN)	23	(17.4)
	Meconium aspiration syndrome	5	(3.8)
	Infection	17	(12.9)
	Congenital pneumonia	6	(4.5)
	Prematurity (low birth weight)	26	(19.7)
	Birth depression	7	(5.3)
	Intrauterine growth retardation	3	(2.3)
	At the time of measurement
	Postnatal age (days)	9	(1-107)
	Postmenstrual age (PMA) (weeks)	37.1 (32.4-46.7)
	Preterm (PMA<37 weeks)	61	(46.2)
	Weight (g)	2550 (1850-4610)
	John Wiley & Sons, Inc.

Table 2 .

 2 Characteristics and PTImus results of infants with I/I, I/D and D/D ACE genotypes. Data are demonstrated as n (%) or median (range in parentheses).

	p value

Table 3 .

 3 Results of stepwise regression analysis

	Partial Correlation
	Coefficient	t Value	Significance
	Dependent variable: PTImus
	ACE gene polymorphism	0.245	2.870	0.005
	Birth weight	-0.176	-2.035	0.044
	Gestational age	0.011	0.125	0.901
	Postnatal age	0.020	0.222	0.825
	Gender	-0.033	-0.376	0.708
	Duration of mechanical
	ventilation	-0.061	-0.694	0.489

Table 4 .

 4 Characteristics and PTImus results of infants with I/I, I/D and D/D ACE genotypes that never required respiratory support. Data are demonstrated as n (%) or median (range in parentheses).

	p value

a Kruskal-Wallis test b Cramer's V test

John Wiley & Sons, Inc.Pediatric Pulmonology

ACKNOWLEDGMENTS

None

ACKNOWLEDGMENTS

None

FUNDING

None

ETHICS COMMITTEE APPROVAL

The study was approved by the Ethics Committee of the University Hospital of Patras, Patras, Greece Page 15 of 47 John Wiley & Sons, Inc. Pediatric Pulmonology FUNDING None ETHICS COMMITTEE APPROVAL The study was approved by the Ethics Committee of the University Hospital of Patras, Patras, Greece Page 41 of 47 John Wiley & Sons, Inc. Pediatric Pulmonology

COMPETING INTERESTS

The authors declare that they have no financial or other relationships that might lead to a conflict of interest.

COMPETING INTERESTS

The authors declare that they have no financial or other relationships that might lead to a conflict of interest.


T Lagergård
email: teresa.lagergard@microbio.gu.se

R Hadad

P Tunbäck

L Lindholm

G.-B Löwhagen

M Unemo

: M Unemo

Distribution of Chlamydia trachomatis ompA genovars and the new variant of C. trachomatis in the Göteborg area, Sweden

The aims of this study were to assess the proportion of the new variant of Chlamydia trachomatis (nvCT) and the distribution of ompA genovars among C. trachomatis-positive patients in the Göteborg area, Sweden. Consecutive urine samples positive for C. trachomatis using BD ProbeTec ET (177 patients, 88 men and 89 women) were collected. An nvCT-specific real-time polymerase chain reaction (PCR) assay was used to investigate the nvCT prevalence. To identify the genovars, a 990-bp ompA DNA segment from 105 specimens was sequenced. Seventeen percent (30/177) of all specimens contained nvCT. Nine different genovars were identified. About 50% were of genovar E, followed by F 16%, G 11%, K 8%, and D 5%, representing about 90% of the specimens in Göteborg. The occurrence of nvCT and the dominance of genovar E in Göteborg is similar to those in other areas of Sweden. To cover about 90% of the C. trachomatis infections in Sweden, the serovars D, E, F, G, and K should be included in future vaccines based on the major outer membrane protein.

Introduction

Chlamydia trachomatis genital infections, commonly asymptomatic, are the most prevalent worldwide bacterial sexually transmitted infection (STI), with considerable morbidity and sequelae, leading to socioeconomic consequences [START_REF] Persson | The role of serology, antibiotic susceptibility testing and serovar determination in genital chlamydial infections[END_REF][START_REF] Hafner | Chlamydia trachomatis infection: host immune responses and potential vaccines[END_REF].

Laboratory diagnosis based on nucleic acid amplification tests (NAATs) provide high sensitivity, high specificity, and are routinely used for the diagnosis of C. trachomatis infections [START_REF] Persson | The role of serology, antibiotic susceptibility testing and serovar determination in genital chlamydial infections[END_REF][START_REF] Pedersen | Typing Chlamydia trachomatis: from egg yolk to nanotechnology[END_REF].

C. trachomatis strains are, nowadays, classified into genovars using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) or sequencing of the ompA gene, which encodes the major outer membrane protein (MOMP). Serotypes A-C cause primarily trachoma/ blindness, D-K cause urogenital infections, and L1-3 the invasive lymphogranuloma venereum (LGV) [START_REF] Persson | The role of serology, antibiotic susceptibility testing and serovar determination in genital chlamydial infections[END_REF][START_REF] Hafner | Chlamydia trachomatis infection: host immune responses and potential vaccines[END_REF]. In most countries worldwide, the most common genovars among patients with urogenital infections are E, F, D, G (D and G are particularly prevalent in men who have sex with men [MSM)], and K, accounting for 60 to 80% of positive patients. The remaining genovars are less frequent, and Ba and Ia are rare. The urogenital genovars distribution shows some variations, especially among countries on different continents [START_REF] Persson | The role of serology, antibiotic susceptibility testing and serovar determination in genital chlamydial infections[END_REF][START_REF] Pedersen | Typing Chlamydia trachomatis: from egg yolk to nanotechnology[END_REF][START_REF] Millman | Population-based genetic epidemiologic analysis of Chlamydia trachomatis serotypes and lack of association between ompA polymorphisms and clinical phenotypes[END_REF][START_REF] Gao | Distribution study of Chlamydia trachomatis serovars among high-risk women in China performed using PCR-restriction fragment length polymorphism genotyping[END_REF][START_REF] Ngandjio | Restriction endonuclease patterns of the omp1 gene of reference Chlamydia trachomatis strains and characterization of isolates from Cameroonian students[END_REF]. In Sweden, the most prevalent genovars found have been E (47-60%) and F, followed by D, K/G [START_REF] Persson | The role of serology, antibiotic susceptibility testing and serovar determination in genital chlamydial infections[END_REF]. Genovar determination of C. trachomatis can be important in descriptive epidemiology and in basic research aiming to develop future vaccines. No effective vaccine has, so far, been developed, but some MOMP-based vaccines may be promising [START_REF] Hafner | Chlamydia trachomatis infection: host immune responses and potential vaccines[END_REF].

In 2006, a new variant of C. trachomatis (nvCT) was discovered in Sweden, with a 377-bp deletion in the cryptic plasmid that included the genetic targets for the diagnostic systems from Roche and Abbott [START_REF] Herrmann | Emergence and spread of Chlamydia trachomatis variant, Sweden[END_REF][START_REF] Ripa | A variant of Chlamydia trachomatis with deletion in cryptic plasmid: implications for use of PCR diagnostic tests[END_REF]. The nvCT strain, thus, escaped the detection and spread significantly (proportions of 7 to 64%) in many Swedish counties [START_REF] Herrmann | Emergence and spread of Chlamydia trachomatis variant, Sweden[END_REF][START_REF] Ripa | A variant of Chlamydia trachomatis with deletion in cryptic plasmid: implications for use of PCR diagnostic tests[END_REF][9]. In Göteborg, the BD ProbeTec ET (Becton Dickinson) NAAT, which detects the nvCT, has been used since 2001. The nvCT has not been frequently found in other European countries [START_REF] Herrmann | Emergence and spread of Chlamydia trachomatis variant, Sweden[END_REF]9]; however, few recent studies have been performed and many laboratories in several countries still cannot detect the nvCT.

The aims of this study were to investigate, for the first time, the proportion of the nvCT and the distribution of different genovars among patients with C. trachomatis infections in the Göteborg area, Sweden.

Materials and methods

Consecutive urine samples from 89 women (age: 16-49 years) and 88 men (age: 17-44 years) positive for C. trachomatis in routine testing using the BD ProbeTec ET test (Becton Dickinson, Franklin Lakes, NJ, USA) were collected from October 2008 to January 2009. For nvCT detection, an nvCT-specific real-time PCR was used on all primary samples (n=177) as previously described [START_REF] Ripa | A Chlamydia trachomatis strain with a 377-bp deletion in the cryptic plasmid causing false-negative nucleic acid amplification tests[END_REF]. To assess the genovar distribution, a 990-bp ompA segment of the first 112 selected primary specimens (from 44 women and 61 men) were sequenced as previously described [START_REF] Jurstrand | Characterization of Chlamydia trachomatis omp1 genotypes among sexually transmitted disease patients in Sweden[END_REF]. However, seven of these failed to amplify, probably due to the low initial positivity in BD ProbeTec ET, and were excluded. For the identification of genovars, phylogenetic analysis, as well as GenBank blasting, were performed as previously described [START_REF] Jurstrand | Characterization of Chlamydia trachomatis omp1 genotypes among sexually transmitted disease patients in Sweden[END_REF].

Results and discussion

The nvCT proportion in the Göteborg area, Sweden, has not been previously studied. The nvCT was detected in 17% of all the specimens (n = 177), showing that the nvCT proportion is relatively similar to those in other Swedish counties that used BD ProbeTec (7-19%) for the diagnosis of C. trachomatis, which, in general, is lower than the proportions in the Swedish counties using Roche and Abbott systems for diagnostics [START_REF] Herrmann | Emergence and spread of Chlamydia trachomatis variant, Sweden[END_REF].

The frequencies for the predominant ompA genovars identified in the Göteborg area were approximately E 50%, followed by F 16%, G 10%, K 8%, and D 5% (Table 1). These genovars represented approximately 90% of all the C. trachomatis-positive specimens. We did not find any unpreviously reported single nucleotide polymorphisms (SNPs) or other polymorphisms in the ompA gene within the genovars, or any significant differences in genovar distribution related to the gender or age of the patients. Inclusion of only the three most common serotypes in a vaccine based on the MOMP will cover about 76%, and inclusion of the five serotypes D, E, F, G, and K will account for almost 90% of all urogenital strains in this geographic area. The proportions of different genovars/serovars, especially concerning the dominance of genovar E, are similar to those found in other studies performed in Sweden, especially in the Örebro area, where the same 990-bp ompA segment was used for the study [START_REF] Jurstrand | Characterization of Chlamydia trachomatis omp1 genotypes among sexually transmitted disease patients in Sweden[END_REF]. Genovar E represents from 39 to 60% of isolates [START_REF] Persson | The role of serology, antibiotic susceptibility testing and serovar determination in genital chlamydial infections[END_REF][START_REF] Pedersen | Typing Chlamydia trachomatis: from egg yolk to nanotechnology[END_REF][START_REF] Jurstrand | Characterization of Chlamydia trachomatis omp1 genotypes among sexually transmitted disease patients in Sweden[END_REF]. Similar dominance of genovars, and especially E, has also been reported from many other European countries [START_REF] Pedersen | Typing Chlamydia trachomatis: from egg yolk to nanotechnology[END_REF]. However, in different populations, such as, e.g., in MSM from Sweden, the occurrence of other genovars such as D/Da (25-54%) and G (25-45%) was higher compared with a heterosexual population, which is also in concordance with previous studies from other countries [START_REF] Pedersen | Typing Chlamydia trachomatis: from egg yolk to nanotechnology[END_REF][START_REF] Millman | Population-based genetic epidemiologic analysis of Chlamydia trachomatis serotypes and lack of association between ompA polymorphisms and clinical phenotypes[END_REF].

MOMP is perhaps the most promising antigen candidate for a vaccine [START_REF] Hafner | Chlamydia trachomatis infection: host immune responses and potential vaccines[END_REF], and the D, E, F, G, and K strains account for approximately 90% of the C. trachomatis infections in Sweden and Denmark. It is notable that, in one study from Cameroon, a similar prevalence as in European countries was noted and strains D, E, F, and G accounted for 94% of isolates [START_REF] Ngandjio | Restriction endonuclease patterns of the omp1 gene of reference Chlamydia trachomatis strains and characterization of isolates from Cameroonian students[END_REF]. Among patients with an STI in Asia, e.g., China, genotype E is not dominating, but the most common serovars D, E, F, and G are more equally distributed and together account for about 75% of all serotypes [START_REF] Gao | Distribution study of Chlamydia trachomatis serovars among high-risk women in China performed using PCR-restriction fragment length polymorphism genotyping[END_REF]. The results indicated that the most common worldwide serovars D, E, F, and G should be considered in the development of an effective MOMP-based vaccine. There was no significant difference in the type distribution between sexes (χ 2 test)

Table 1

 1 Chlamydia trachomatis ompA genovar distribution by gender of 105 urogenital isolates in the Göteborg area, Sweden

	Genovar	Women (n=44)	Men (n=61)	Total (n=105)
		No. (%)	No. (%)	No. (%)
	Ba	1 (2.3)	0	1 (1.0)
	D	2 (4.55)	3 (4.9)	5 (4.8)
	E	23 (52.3)	29 (47.6)	52 (49.5)
	F	6 (13.6)	11 (18.0)	17 (16.2)
	G	4 (9.05)	8 (13.1)	12 (11.4)
	H	2 (4.55)	2 (3.3)	4 (3.8)
	Ia	2 (4.55)	0	2 (1.9)
	J	2 (4.55)	2 (3.3)	4 (3.8)
	K	2 (4.55)	6 (9.8)	8 (7.6)

Acknowledgments This study was supported by the Swedish Research Council (K2007-58X-20365-01-3), ALF/LUA, Göteborg, and The Royal Society of Arts and Sciences in Göteborg (KVVS).