Loïc Legrand
email: l.legrand@cg14.fr

Frank Duncombe

Astrid Vabret

Julia Dina

Joëlle Petitjean-Lecherbonnier

Delphine Cuvillon

Valérie Tripey

Jacques Brouard

François Freymuth
email: freymuth-f@chu-caen.fr

Epidemiological and phylogenic study of human metapneumovirus infections during three 1 consecutive outbreaks in Normandy, France. 2 3 4

Keywords: Human metapneumovirus, epidemiological study, M gene, Phylogenic study Human metapneumovirus, epidemiological study, M gene, Phylogenic study

Table 1: Virus repartition in positive respiratory samples from children hospitalized

Human metapneumovirus (hMPV) is responsible for respiratory tract disease, particularly in 28 the young and elderly population. An epidemiological and phylogenic study was performed 29 on children admitted to hospital with an acute lower respiratory tract infection. Data was 30 obtained and analyzed over three consecutive winters, from 2002-03 to 2004-05. 31 Each year during the winter period, from November to March, 2,415 nasal swabs were tested 32 by a direct immunofluorescence assay (DFA) for influenza virus A and B, respiratory 33 syncytial virus, parainfluenza viruses and adenoviruses. Rhinoviruses, enteroviruses and 34 coronaviruses OC43 and 229E were detected by RT-PCR. A RT-PCR designed for the M 35 gene was performed on negative samples for hMPV detection and phylogenic analyses. For 36 the three consecutive winters, hMPV represented 10%, 22.6% and 8.8% of virus-negative 37 samples, respectively. In most cases, clinical symptoms indicated a lower respiratory tract 38 shown that hMPV strains can be divided into two major groups: A and B, and 4 minor groups: 125 A1, A2, B1, B2 (Bastien et al., 2003b;[START_REF] Boivin | Global genetic diversity of human metapneumovirus fusion gene[END_REF]van den Hoogen et al., 2004). 126 This paper describes studies concerning hMPV infection and reports on a genetic analysis of 127 hMPV strains that were detected in infected children during three consecutive winters in 128 Normandy, France. 129 130

Materiel and methods 131

Patients and samples 132

The study was performed on 2,415 children admitted to the University hospital of Caen and 133 the hospital of Flers, for an acute lower respiratory tract infection from November to March 134 during three consecutive years (November 2002to March 2004). All patients underwent a 135 nasal aspirate which was processed immediately by conventional methods (see below). The 136 remaining samples were frozen at -70°C and used for subsequent molecular analyses. All 137 samples were collected and tested in conformation with the ethical legislation in place. 138

Conventional methods 139

The conventional methods included a direct immunofluorescence assay (DFA). This 140 immunofluorescence assay used commercially available monoclonal antibodies (Imagen ® , 141 Dako, Glostrup, Denmark) and permitted the detection of influenza virus types A and B, 142 respiratory syncytial virus (RSV), parainfluenza virus types 1, 2 and 3, and adenoviruses. 143

When the DFA was negative, samples were inoculated onto HuH7 cell lines grown in 48-well 144 microplates as described previously [START_REF] Freymuth | Replication of respiratory viruses, particularly influenza virus, 431 rhinovirus, and coronavirus in HuH7 hepatocarcinoma cell line[END_REF]. Briefly, after four days of 145 incubation, HuH7 cells were harvested and used for DFA with the same antiviral antibodies. 146 When a cytopathic effect was observed, supernatants of the relevant wells were harvested and 147 used in a multiplex PCR for the detection of rhinovirus, enterovirus and human coronaviruses 148 229E, OC43 [START_REF] Bellau-Pujol | Development of three multiplex RT-PCR assays for the 368 detection of 12 respiratory RNA viruses[END_REF]. 149 RNA was extracted from nasal aspirates with the QIAamp Viral RNA minikit (Qiagen,151 Hilden, Germany) according to the manufacturer's instructions. RT-PCR was likewise 152 performed with the one-step RT-PCR kit (Qiagen) using primers designed for the M gene. 153

The forward primer hMPV.M1 (5'-CCCTTTGTTTCAGGCCAAC-3') and the reverse primer 154 hMPV.M2 (5'-GCAGCTTCAACAGTAGCTG-3') provided a 416 pb PCR product. The 155 thermocycler conditions were 30 min at 50°C, 15 min at 95°C following by 40 cycles of 1 156 min at 95°C, 1 min at 60°C and 1 min at 72°C. Finally, an extension at 72°C was performed 157 during 10 min. The result was analysed on 2% agarose gel. 158

The PCR sensitivity for each group was established using two fragments of the M gene 159 containing the amplification product (Eurofin-MWG-Operon, Ebersberg, Germany) 160 representing NL00-1 (AF371337) and NL99-1 (AY525843), respectively. NL00-1 was 161 considered like group A prototype sequence and NL99-1 like group B prototype sequence. 162 Synthesized DNA were cloned in a pCR2.1 vector and transcription was performed using T7 163 polymerase (Applied Biosystems/Ambion, Courtaboeuf, France). After DNase treatment 164 (Applied Biosystems/Ambion, Courtaboeuf, France), RNA concentration was determined by 165 measuring the absorbance at 260 nm with the Nanodrop spectrophotometer system. Serial 10-166 fold dilutions were performed to determine the sensitivity of the assay in copy of the viral 167 RNA per reaction. 168

Sequencing and phylogenic analysis 169

An hemi-nested PCR was performed with the reverse primer MPV.M2 and a new forward 170 primer MPV.M3 (5'-AGGCCAACACACCACCAG-3'). The amplification was realized 171 through the use of an AmpliTaq (Applied Biosystem, Foster City, CA) with the following 172 program: 5 min at 95°C followed by 40 cycles of 30 sec at 95°C, 30 sec at 60°C and 45 sec at 173 72°C and a final elongation at 72°C for 10 min. The 405 pb product obtained was first 174 8 purified with the enzyme ExoSap-IT (Amersham Biosciences, Uppsala, Sweden) according to 175 the manufacturer's instructions and then quantified on a spectrometer. Subsequently, the 176 purified PCR product was sequenced on a Beckman Ceq TM 8000 genetic analysis system with 177 the CEQ DTCS Quick start kit (Beckman coulter, Brea, CA). The sequences were assembled 178 and analyzed with BioEdit and ClustalW software. A phylogenic study was carried out using 179 Mega 3.1 software with the neighbour joining method. A bootstrap was performed with a 180 replicate rate of 1000. 181

Nucleotide Sequence Accession Numbers 182

The nucleotide sequences reported in this study were submitted to the GenBank under 183 accession numbers H384124 to H384159. 184

Results

hMPV infections 186

The RT-PCR allowed detection of about 30 and 50 copies per reaction for the A and B group, 187 respectively. This method was performed on virus-negative nasal aspirates. The frequency of 188 hMPV infection in hospitalized children could be assessed only during the first and last 189 winters because the study performed during the winter of 2003-04 was limited to children 190 under 2 years of age; this study was described by [START_REF] Freymuth | Comparison of multiplex PCR assays and conventional 435 techniques for the diagnostic of respiratory virus infections in children admitted to hospital 436 with an acute respiratory illness[END_REF]. In order to compare 191 the hMPV prevalence to the other common cold viruses, the period from November to March 192 was studied exclusively. In 2002 and 2004, there were 498 and 642 samples, respectively, 193 that contained a respiratory virus out of the 1040 and 1112 nasal aspirates tested (Table 1). 194

Human metapneumovirus was detected in 59 (10%) and 52 (8.8%) of the virus-negative 195 specimens analysed, respectively. As a function of age, there were 34. 2%, 22.5%, 16.4% and 196 26.9% cases of infected children between 0-6 months, 6-12 months, 12-24 months and older 197 than 24 months, respectively (Table 2). 198 commonly detected virus (8.8%). Human respiratory syncytial virus (hRSV) was the most 201 predominant and represented 45.6% of virus-positive specimens followed by rhinoviruses 202 (12.7%) and the influenza A virus (11.3%). The other detected viruses were parainfluenza 203 viruses 1, 2 and 3 (5.6%); adenoviruses (5.3%); coronaviruses (4.8%); enteroviruses (3%) and 204 the influenza B virus (2.8%). In children under two year old, hMPV (8.5%) became the third 205 most frequently encountered virus after hRSV (50.7%) and rhinoviruses (14.2%). Influenza A 206 virus represented about 7.7% of the cases studied; adenoviruses 5.5%; parainfluenza viruses 207 1, 2 and 3 5%; coronaviruses 4.4%; enteroviruses 2.8% and influenza B virus 1.2% (Table 1). 208

Clinical information was available from 46 out of the 52 hMPV-infected children during the 209 winter of 2004. The mean age of the infected children was 21 months (Table 2) and the 210 male/female ratio was equal. Children were hospitalized about 3.5 days after the onset of 211 symptoms. The most frequent clinical signs at admission were rhinitis (84.8%), cough 212

Phylogenetic analysis 226

A phylogenetic analysis was carried out on the M gene of 107 hMPV strains isolated during 227 the three consecutive outbreaks (Figure 2). The hMPV strains were distributed into two 228 groups, A and B, and into four relevant subgroups: A1, A2, B1, and B2. The percentages of 229 identity in the M gene between the hMPV group A and B strains varied from 84.2% to 88.8%. 230

The percentages of genetic similitude between the strains of subgroups A1 and A2 and 231 between the strains of B1 and B2 subgroups were 94.1% to 96.3% and 93.8% to 95.7%, 232 respectively. 233

Distribution of hMPV groups and subgroups 234

Human metapneumovirus strains of different groups and subgroups were associated with each 235 outbreak (Figure 3a). However, there was often one dominant subgroup in each epidemic. 236

Human metapneumovirus belonging to the A2 subgroup and B1 subgroup were predominant 237 during the winters of 2002-03 and 2004-05, respectively (Figure 3b). For both these periods, 238 two strains (Cae03-6-31 and Cae05-40-27) were more prominent and represented greater than 239 50% of the isolated hMPV. During the winter of 2003-04, the four subgroups were present 240 equally (Figure 3c). 241

Discussion 242

The present study reports on an epidemiological, clinical and phylogenic study of hMPV 243 outbreak during three consecutive winters in Normandy, France. Human metapneumovirus 244 infection was assessed in nasal aspirates received in the laboratory during the months of 245 November to March in order to perform a comparison of hMPV rate with the other common 246 cold viruses. It was reported that, the hMPV infection season is longer than that of hRSV or 247 influenza viruses (van den Hoogen et al., 2003;[START_REF] Esper | A 1-412 year experience with human metapneumovirus in children aged <5 years[END_REF][START_REF] Mackay | 476 Genetic diversity of human metapneumovirus over 4 consecutive years in Australia[END_REF]. As 248 previously that hMPV circulates predominantly during the winter-spring period (van den 250 Hoogen et al., 2003;Bastien et al., 2003a;[START_REF] Williams | Human metapneumovirus and lower respiratory tract disease 551 in otherwise healthy infants and children[END_REF][START_REF] Esper | A 1-412 year experience with human metapneumovirus in children aged <5 years[END_REF]Pitoiset et 251 al., 2010). The number and the percentage of hMPV detected in virus-negative samples 252 during the epidemics of 2002-03, 2003-04 and 2004-05 were 59 (10%), 17 (22.6%) and 52 253 (8.8%), respectively. The incidence of hMPV infections are reported in many other studies 254 [START_REF] Boivin | Virological features and clinical manifestations associated with human 372 metapneumovirus: a new paramyxovirus responsible for acute respiratory-tract infections in 373 all age groups[END_REF][START_REF] Van Den Hoogen | Antigenic and genetic variability of human metapneumoviruses[END_REF][START_REF] Williams | Human metapneumovirus and lower respiratory tract disease 551 in otherwise healthy infants and children[END_REF]. It is also suggested 255 that the number of hMPV infections is variable from one winter to another (Falsey et al., 256 2003;[START_REF] Mackay | 476 Genetic diversity of human metapneumovirus over 4 consecutive years in Australia[END_REF][START_REF] Williams | The 555 role of human metapneumovirus in upper respiratory tract infections in children: a 20-year 556 experience[END_REF]. 257

Human metapneumovirus was the fourth most prominent virus encountered during the present 258 study and became the third most prominent if the results are focused only on children under 259 two years old. Three-quarters of the hMPV were detected on children under two years old and 260 more than one third on those less than six months old. Human respiratory syncytial virus was 261 the most predominant virus observed with an increase in prevalency in children under two 262 years old. Influenza A virus was more commonly encountered in the population of over two 263 year olds. Unlike the hMPV and the other isolated viruses, the influenza A and B viruses 264 show a distinct prevalence between children under two years old and those over two years 265 old. 266

In comparison with hRSV and influenza viruses A and B, the outbreak of hMPV occurs over 267 the same period and appears to be closely related to the hRSV epidemic profile. In fact, 268 hMPV and hRSV outbreaks showed the same variation during the winter months (Boivin et 269 al., 2003;[START_REF] Esper | A 1-412 year experience with human metapneumovirus in children aged <5 years[END_REF]. The influenza epidemic profile, which might be a more 270 independent entity, did not correlate with hMPV or hRSV. During these three winters, the 271 influenza A virus is more prominent than influenza B virus and the percentage was 272 appreciatively the same for that described in Europe [START_REF] Paget | Heterogeneous influenza activity across 504 Europe during the winter of 2002-2003[END_REF][START_REF] Meijer | 485 Epidemiological and virological assessment of influenza activity in Europe, during the 2004-486 2005 winter[END_REF]. 273 13 subgroup in predominance. The last study [START_REF] Pitoiset | Human metapneumovirus 518 genotypes and severity of disease in young children (n = 100) during a 7-year study in Dijon 519 hospital, France[END_REF] reports on a phylogenic 299 analysis carried out on 100 strains isolated over eight years, from 2002 to 2009 with 300 alternative subgroup predominance. During the first winter 2002-03, the A group was the 301 most prevalent, with 87.5% of A2 subgroup being the most prominent. This repartition was 302 also found in many other parts of the world [START_REF] Boivin | Global genetic diversity of human metapneumovirus fusion gene[END_REF][START_REF] Esper | A 1-412 year experience with human metapneumovirus in children aged <5 years[END_REF]303 Ludewick et al., 2005. Pitoiset et al., 2010). Two studies described another pattern of 304 circulation of the virus during the winter of 2002-03 in which a prevalent B1 subgroup was 305 noted [START_REF] Gerna | Changing circulation rate of human metapneumovirus strains and types 444 among hospitalized pdiatric patients during three consecutive winter-spring seasons[END_REF][START_REF] Gray | Multi-year study of human metapneumovirus infection at a large US 449 Midwestern Medical Referral Center[END_REF]. For the winter of 2003-04, no predominant group 306 could be identified. The incidence of groups A and B were approximately the same, although 307 the number of strains belonging to group B and A were nine and eight, respectively. The B2 308 and A2 subgroup were the most frequently identified with five strains for each cluster. During 309 this same period in Italy (winter of 2003-04), the A2 subtype was also the most prominent 310 strain, even if other strains were present [START_REF] Gerna | Changing circulation rate of human metapneumovirus strains and types 444 among hospitalized pdiatric patients during three consecutive winter-spring seasons[END_REF][START_REF] Gerna | Changing circulation rate of human metapneumovirus strains and types 444 among hospitalized pdiatric patients during three consecutive winter-spring seasons[END_REF]. The rate of 311 occurrence of group B was confirmed during the winter of 2004-05 and represented 96% of 312 all hMPV detected, including one hMPV B2 strain. Over the three winter periods, 46.5% of 313 B1, 37.5% of A2, 8% of A1 and 8% of B2 strains were observed. These data are closely 314 related to those proposed by [START_REF] Mackay | 476 Genetic diversity of human metapneumovirus over 4 consecutive years in Australia[END_REF] in a study carried out in Australia during 315 the same period. In addition, both predominant subgroups found in 2002 and 2004 were the 316 same (viz., A2 and B1). 317

The RNA fragment studied is small (405bp), but some mutations that were found in this gene 318 allowed us to discriminate between, and compare, the different isolates. The sequence 319 analysis for the three epidemic periods showed two distinct genetic profiles. The first profile, 320 found during the winter of 2002-03 and 2004-05, seemed to be composed of two predominant 321 strains showing exactly the same sequence (2002-03 and 2004-05), the foremost strains belonged to the A2 and B1 subgroups, 324 respectively. This finding corroborates previous observations [START_REF] Mackay | 476 Genetic diversity of human metapneumovirus over 4 consecutive years in Australia[END_REF]. For the 325 winter period 2003-04, the isolated strains belonged to the four subgroups and, at the most, 326 two identical sequences were found. Even if it is impossible to consider these isolates like 327 different strains because the analysis was assessed on too short a RNA fragment, such 328 heterogeneity was not observed during the other winter periods and might be considered as 329 the beginning of a shift from group A to group B as confirmed by the data of 2004. A shift of 330 this nature was reported previously [START_REF] Agapov | Genetic 351 variability of human metapneumovirus infection: evidence of a shift in viral genotype without 352 a change in illness[END_REF][START_REF] Mackay | 476 Genetic diversity of human metapneumovirus over 4 consecutive years in Australia[END_REF]Oliveira et al., 331 2009). However, no evolution can be established from the heterogenic composition of hMPV 332 infection during the winter of 2003-04 to the subgroup B1 predominance during the winter of 333 2004-05. The B1 strain responsible for 52% of hMPV infection during the winter of 2004-05 334 was not found during the previous winter of 2003-04. 335 Despite the fact that the 2003-04 study is incomplete and that the period studied was only 336 from November to March, the rate of hMPV can still be evaluated each year and the 337 molecular evolution of the viruses can be analyzed. Human metapneumovirus is circulating in 338 Normandy at a rate equivalent to that described previously [START_REF] Peret | Characterization of human metapneumoviruses isolated from patients in North 515 America[END_REF]Bastein et al., 339 2003;Bovin et al., 2003;[START_REF] Esper | A 1-412 year experience with human metapneumovirus in children aged <5 years[END_REF]. The present clinical study demonstrates that 340 hMPV is responsible for lower respiratory tract infection in the majority of cases studied. All 341 the reported hMPV subgroups are present in France but not with the same incidence each 342 year. The results confirm the genetic evolution of hMPV in Normandy with a switch from the 343 A group to the B group being noted during the winter of 2003-04. Finally, molecular tools 344 developed for the M gene appear to be useful for diagnosis and molecular analysis, since the 345 four subgroups are detected and can be discriminated between in a significant manner.

W 4 7 W 4 8 W 4 9 W 5 0 W 5 1 W 5 2 W 5 3 / 1 W 2 W 3 W 4 W 5 W 6 W 7 W 8 W 9 W 1 0 W 1 1 W 1 2 W 1 3

Weeks

W 4 7 W 4 8 W 4 9 W 5 0 W 5 1 W 5 2 W 5 3 / 1 W 2 W 3 W 4 W 5 W 6 W 7 W 8 W 9 W 1 0 W 1 1 W 1 2 W 1 3

  Phylogenic evolution of hMPV strains detected during the outbreaks circulating in Normandy for three consecutive winter to spring periods. (a) Prominence of hMPV group A and B during each winter; (b) Strain repartition between subgroup A1, A2, B1 and B2 during each winter (c) hMPV sequence analysis for each outbreak. 2x1mm (600 x 600 DPI)

  infection (LRI) with a final diagnosis of bronchiolitis. During the winter of 2003-04, all viral 39 clusters (A1, A2, B1 and B2) that circulated in France shifted progressively from the A group 40 to the B group. This study determined the prevalence of hMPV in Normandy, its clinical 41 impact and permitted the analysis of the molecular evolution during the successive outbreaks.

  of 2002 to 2004, other viruses were scrutinized for by a DFA test or by 199 using culture models or molecular tools. Human metapneumovirus was the fourth most 200

  (71.7%), respiratory distress syndrome (30.4%) and otitis (17.4%). Nineteen children (41.3%) 213 presented with an upper respiratory tract infection and 26 children (56.5%) with a lower 214 respiratory tract infection. The final clinical diagnosis was bronchiolitis in 17 cases (37%), 215 broncho-pneumonia in six cases (13%) and acute exacerbation of asthma in three cases 216 (6.6%). Signs of gastroenteritis were present in 17 (30.4%) of hMPV-infected children and 217 rotavirus or adenovirus were present in nine cases. Seven hMPV-infected children (15.2%) 218 required oxygen therapy. 219Epidemics of hMPV infections 220During the winter of 2002-03, the hMPV and hRSV epidemics appeared at the end of 221 November and they peaked almost immediately at three to four weeks later. In 222 contradistinction, during the winters of 2003-04 and 2004-05, the hMPV and hRSV epidemics 223 continued more slowly. Human metapneumovirus seemed to have a 224 constant presence without any obvious peak in activity (Figure1). 225

  number of positive cases may have been underestimated although it was shown 249

Figure 1 :

 1 Figure 1: Weekly comparison of the outbreak of hRSV, influenza viruses A and B (MIA/B) and hMPV 615 in a paediatric population. (a) from November to March, 2002-03; (b) from 2003-04; (c) from 2004-05 616 2002-03 617 (a) 618

Figure 2 :Figure 3 :

 23 Figure 2: Phylogenic analysis performed for the M gene for 107 sequenced hMPV isolates in Normandy 653 during three consecutive winters. The tree was built with Mega 3.1 software using the neighbour joining 654 method. Bootstrap values (1000 replicates) are reported on branch nodes of the tree. Four genotypes are 655 indicated. The strains obtained in Normandy are written as follows: Cae-isolation winter (A strain 656 isolated during the winter 2002-03 is notified 03)-Number of strain-Quantity of sample containing this 657 strain. The Genbank access numbers of the referenced strains are NL00-1(AF371337), NL99-658 1(AY525843), Can98-75(AY297748), Can97-83(AY297749), JPS03-240(AY530095). 659

Table 2 :

 2 Analysis of the epidemiological study in the paediatric patient population during three consecutive winters from November 2002 to March 2005. These data were obtained only in children less than two years old.

	Winter 2002-03 Winter 2003-04* Winter 2004-05

Table 1 :

 1 Virus repartition in positive respiratory samples from children hospitalized during 598 three consecutive winters from November 2002 to March 2005.

	346
	347
	348

Weeks Number of cases in 2003/04

	624					2004-05
	625	(c)
			45
			40
		Number of cases in 2004/05	W 4 6 0 5 10 15 20 25 30 35	r W 4 9 W 5 0 W 5 1 o W 4 8 F W 4 7	W 5 2 W 5 3 / 1	W 2	W 3	W 4	W 5	W 6	W 7	W 8	W 9 W 1 0 W 1 1 W 1 2 W 1 3
	626			P e	Weeks MIA/B RSV hMPV
	627 628			e r
	629 630 631 632 633 634 635 636 637 638 639				R e v i e w
	640
	641
	642
	643
	644
	645
	646
	647
	648
	649
	650					MIA/B RSV hMPV
	622 651
	623 652
						22


Tilak ; Ghosh

Dr Ian David

Lewis

M.D, FRCP. Prof A T R Axon

Dr Simon Everett

FRCP Consultant Gastroenterologist

Review article: methods of measuring gastric acid secretion

Keywords: Basic science < Topics, Diagnostic tests < Topics, Stomach and duodenum < Organ-based, Peptic ulcer disease < Disease-based, Acidity

Background: Gastric acid has an important pathophysiological role in human beings. Numerous methods have been evaluated over the years in an attempt to measure gastric acid and stomach acidity, to study the role of gastric acid in gastrointestinal diseases in humans and to evaluate the effects of acid suppressing drugs. Aim: To review methods which have been used to measure gastric acid and gastric acidity.

Methods: Searches of the electronic databases PUBMED, MEDLINE and EMBASE, were performed with articles restricted to English language and human subjects. References were also identified from the bibliographies of selected articles. Results: Methods for measuring gastric acid include both invasive and non invasive techniques. Invasive tests include the conventional gastric acid aspiration tests, gastric pH measurement techniques and endoscopic methods. Non invasive methods use urinary analysis, breath analysis, serum pepsinogens assay, scintigraphic techniques, impedence tomography and alkaline tide for measurement of gastric acid. Conclusions: Several methods of measuring gastric acid exist. Invasive tube tests are uncomfortable and time consuming whereas most of the noninvasive methods are at best semi quantitative and useful in detecting low or

Introduction

It had been known for a long time that the human stomach secreted an acidic fluid but its exact nature was not clear until the early 19 th century when Prout showed the presence of hydrochloric acid in gastric juice. [START_REF] Prout | On the nature of acid and saline matters usually existing in stomach of animals[END_REF] Beaumont, with his famous experiments on a patient with a permanent gastric fistula, provided more insight into the factors that influence the secretion of gastric acid in his work, Experiments and Observations on the Gastric Juice and the Physiology of Digestion. [START_REF] Beaumont | Experiments and Observations on Gastric-Juice and Physiology of Digestion[END_REF] Since then, a variety of methods of measuring gastric acid both qualitatively and quantitatively have been discovered and tested in different pathological states, adding to our knowledge about gastric acid. Before the discovery of H.pylori it was widely believed that peptic ulcer disease was caused by excess acid secretion but following the recognition of this organism's role in the pathogenesis of peptic ulcer disease and its effect on acid secretion, interest in measuring gastric acid output declined. The widespread use of endoscopy and the invasive nature of reliable quantitative methods of measuring gastric acid have contributed to this trend.

However there still exists a need to measure gastric acid secretion. Gastric acid is believed to be important as a protective mechanism against ingested pathogens. [START_REF] Martinsen | Gastric juice: a barrier against infectious diseases[END_REF][START_REF] Cook | Infective gastroenteritis and its relationship to reduced gastric acidity[END_REF] Low acid output states may predispose to enteric infections e.g. E. coli and V. cholerae, whereas the use of acid suppressant drugs such as proton pump inhibitors have been associated with a higher incidence of gastroenteritis, pneumonia and Clostridium difficile infections. [START_REF] Giannella | Influence of gastric acidity on bacterial and parasitic enteric infections. A perspective[END_REF][START_REF] Sack | Gastric acidity in cholera and noncholera diarrhoea[END_REF][START_REF] Laheij | Risk of community-acquired pneumonia and use of gastric acid-suppressive drugs[END_REF][START_REF] Canani | Therapy with gastric acidity inhibitors increases the risk of acute gastroenteritis and community-acquired pneumonia in children[END_REF][START_REF] Dial | Use of gastric acid-suppressive agents and the risk of community-acquired Clostridium difficile-associated disease[END_REF] High levels of acidity are seen in duodenal ulcers and patients with Zollinger-Ellison syndrome whilst low or absent acid secretion is found in pernicious anaemia , atrophic gastritis and gastric carcinoma. [START_REF] Kwiecien | Gastric analysis with fractional test meals (ethanol, caffeine, and peptone meal), augmented histamine or pentagastrin tests, and gastric pH recording[END_REF] In countries with a high incidence of gastric cancer there have been attempts at identifying patients with extensive gastric atrophy, as this is believed to be a precursor for the intestinal type of gastric cancer. Gastric atrophy results in reduction of parietal cell mass and can cause reduction or absence of detectable gastric acid.

Hence an absent or reduced gastric acid output state (achlorhydria or hypochlorhydria) can be a marker of increased risk of developing gastric cancer. [START_REF] Hitchcock | Screening for gastric cancer[END_REF] Thus, methods to detect reduced gastric acid output, particularly non invasive techniques, have the potential to be used as screening tools to detect patients at risk for this condition. Gastric acid measurement may also be useful in pharmacological trials assessing the acid suppressing capacity of new drugs and in modifying treatment in dyspeptic patients who do not appear to be responding to acid suppressive medications.

Another use of gastric acid measurement is to distinguish between causes of fasting hypergastrinaemia. Under normal physiological conditions the hormone gastrin stimulates gastric acid secretion and the presence of acid in the gastric antrum in turn inhibits gastrin release. Increased levels of gastrin associated with an increased stomach pH can be due to gastric atrophy, use of acid suppressing drugs or after vagotomy. On the other hand Conventional methods of measuring gastric acid secretion involve aspiration of gastric contents through a tube placed in the stomach during stimulation of acid secretion by histamine, pentagastrin or tetragastrin. [START_REF] Kay | Effect of large doses of histamine on gastric secretion of HCI; an augmented histamine test[END_REF][START_REF] Johnston | Use of pentagastrin in a test of gastric acid secretion[END_REF][START_REF] Hansen | The effect of tetragastrin on gastric acid secretion. Reproducibility and comparison with the pentagastrin test and the augmented histamine test[END_REF] However these tests are uncomfortable, time consuming and can cause side effects. As a result several alternative approaches to measuring gastric acid have been evaluated over the years including some novel approaches. This review aims to evaluate these tests along with the established conventional tests.

Methods

We searched the following electronic databases: Pubmed, Medline and Embase with the following keywords: gastric acid measurement, pentagastrin test, histamine test, pepsinogens, intragastric pH, Heidelberg capsule, breath test for gastric acid and alkaline tide. The key words were chosen to maximize the chances of obtaining articles related to the above known methods for gastric acid estimation. The search was confined to articles in the English

Results

For clarity the methods of gastric acid measurement have been divided into two broad groups, invasive and non-invasive, as detailed in table 1.

A) Invasive methods

Conventional aspiration tests for gastric acid measurement.

Conventional methods for measuring gastric acid are performed by aspiration of gastric contents through a nasogastric tube during gastric acid stimulation.

These tests are regarded as the most accurate quantitative tests for measuring gastric acid secretion. The principle underlying these tests is to aspirate gastric acid through a tube placed in the most dependent part of the stomach and determining the acid content by measuring the volume of the gastric juice aspirated and its acidity. Gastric contents can be aspirated either constantly with a vacuum pump or intermittently, manually by a syringe. After aspiration the acidity of the gastric acid can be derived by titration to a pH 7 using an alkaline solution and chemical indicators or pH meters. The position of the gastric tube in the most dependent part of the stomach can be confirmed either radiologically or by a water recovery test. In the water recovery test, the subject drinks 20 mls of water and if 16-20 mls of it can be aspirated via the gastric tube it can be taken as a marker that the tube is in a satisfactory position. [START_REF] Findlay | Comparative evaluation of water recovery test and fluoroscopic screening in positioning a nasogastric tube during gastric secretory studies[END_REF] Findley et al. compared both the above methods and found no significant differences in measurement of acid output using either the radiological method or the water recovery test. [START_REF] Findlay | Comparative evaluation of water recovery test and fluoroscopic screening in positioning a nasogastric tube during gastric secretory studies[END_REF] These tests can measure both basal and stimulated acid secretion. Basal acid output (BAO) represents the output of a fasting, unstimulated stomach. [START_REF] Meeroff | The nomenclature of gastric acid output[END_REF] The levels of BAO can fluctuate diurnally, even hourly and is obtained by aspiration of gastric acid for an hour (i.e. sum of four 15 minute collections) from a fasted stomach. It is thought to reflect the background vagal tone mediated via acetylcholine. Maximal acid output (MAO) and Peak acid output (PAO) on the other hand are measures of stimulated acid secretion. Maximal acid output (MAO) is defined as the total acid secretion for the period of 60 minutes after administration of a gastric stimulant that cannot be increased by higher doses of the stimulant. It corresponds to the sum of the four 15 minute collections of gastric acid after stimulation and correlates to the parietal cell mass. [START_REF] Iijima | A new endoscopic method of gastric acid secretory testing[END_REF][START_REF] Card | The relationship between the acid output of the stomach following "maximal" histamine stimulation and the parietal cell mass[END_REF] Peak acid output (PAO) on the other hand is the highest rate of gastric acid secretion that can be achieved in an individual after injection of a gastric stimulant. [START_REF] Meeroff | The nomenclature of gastric acid output[END_REF] [START_REF]Manual of Gastroenterologic Procedures[END_REF] Initial methods of stimulating gastric acid involved the administration of test meals to subjects, following which stomach contents were aspirated and acid content was calculated by titration. [START_REF] Lawrie | The Measurement of Gastric Acid[END_REF] However, partial neutralization of the acid by the test meal, the practical difficulties of trying to titrate a food filled gastric fluid and the fact that these tests were neither precise nor reproducible led to the development of newer techniques for measuring stimulated gastric acid secretion.

In most techniques the basal acid output is measured first, gastric stimulant is given following which the maximal and peak acid output is obtained. The earlier tests used histamine as the gastric acid stimulant given intravenously or subcutaneously, either as a bolus or infusion. The best known method is the augmented histamine test described by Kay in 1953. [START_REF] Kay | Effect of large doses of histamine on gastric secretion of HCI; an augmented histamine test[END_REF] Histamine in a dose of 40µg/kg was administered subcutaneously and peak acid output could be obtained within one hour. However, histamine had side effects that included nausea, palpitations and hypotension and this necessitated the administration of a histamine 1 receptor antagonist (H 1 antagonist) prior to the histamine injection. An analogue of histamine called Betazole (Histalog) which had a lower incidence of side effects has also been used at a dose of 2mg/kg . [START_REF] Kwiecien | Gastric analysis with fractional test meals (ethanol, caffeine, and peptone meal), augmented histamine or pentagastrin tests, and gastric pH recording[END_REF][START_REF] Ward | Comparison of histalog and histamine as stimulants for maximal gastric secretion in human subjects and in dogs[END_REF][START_REF] Goldenberg | A clinical evaluation of the maximal histalog test[END_REF] However, with the introduction of pentagastrin in the late 1960s , the use of histamine and its analogue declined. Pentagastrin is a derivative of gastrin and is a synthetic polypeptide containing the 5 Carbon-terminal amino acid sequence of gastrin. It has the ability to stimulate gastric acid secretion maximally in the same manner as gastrin but is less expensive, has fewer side effects and can produce a peak acid output in a shorter time compared to histamine. [START_REF] Konturek | Pioglitazone, a specific ligand of the peroxisome proliferator-activated receptor gamma reduces gastric mucosal injury induced by ischaemia/ reperfusion in rat[END_REF] It is believed that its major mechanism of action is to stimulate the release of histamine from the enterochromaffin cells, that in turn stimulates the histamine 2 (H 2 ) receptors on the parietal cells. [START_REF] Schubert | Gastric secretion[END_REF] . Pentagastrin can be administered intravenously, subcutaneously or intramuscularly as a bolus or an infusion. In the method described by Johnston and Jepson an intramuscular dose of 6 micrograms/kg of pentagastrin was used and the peak acid output was obtained during the 20 min period, 10 to 30 minutes following its administration. The pentagastrin test has been found to be reproducible in several studies, though in a relatively small number of individuals. [START_REF] Johnston | Use of pentagastrin in a test of gastric acid secretion[END_REF][START_REF]Pentagastrin as a stimulant of maximal gastric acid response in man. A multicentre pilot study[END_REF] Tetragastrin (Figure 1), a tetra peptide analogue of gastrin, which has similar gastric stimulatory effect as pentagastrin with minimal side effects, has also being used as a gastric stimulant in several studies at a dose of 4 micrograms/kg. [START_REF] Iijima | A new endoscopic method of gastric acid secretory testing[END_REF][START_REF] Koster | Comparative effects of tetragastrin and histamine on acid and intransic factor secretion in man[END_REF] A further gastric stimulant used in gastric acid measurement studies is Gastrin Releasing Peptide (GRP). [START_REF] Harris | Eradication of Helicobacter pylori in patients with duodenal ulcer lowers basal and peak acid outputs to gastrin releasing peptide and pentagastrin[END_REF][START_REF] El-Omar | A substantial proportion of non-ulcer dyspepsia patients have the same abnormality of acid secretion as duodenal ulcer patients[END_REF] GRP is a neuropeptide, which stimulates the G cells in the antrum to secrete gastrin which in turn stimulates gastric acid secretion. This test has the advantage of simultaneously assessing gastrin secretion and gastric acid secretion in an individual and also replicates the changes in hormonal secretion occurring after intake of food i.e. release of gastrin and secretion of inhibitory substances e.g. somatostatin. The gastric acid secretion response to this stimulus has also been found to be reproducible and there have been no major adverse events noted on a significant number of individuals who were subjected to this test. [START_REF] Mccoll | Review article: gastrin releasing peptide and its value in assessing gastric secretory function[END_REF] It has been shown that GRP stimulated gastric acid may be increased six times in patients with duodenal ulcer who harbour H. pylori and about a three times in individuals with non ulcer dyspepsia and H. pylori positivity, compared with healthy volunteers. [START_REF] El-Omar | A substantial proportion of non-ulcer dyspepsia patients have the same abnormality of acid secretion as duodenal ulcer patients[END_REF] The GRP test may even be more sensitive than the pentagastrin stimulation test in assessing acid secretion in patients with duodenal ulcer. [START_REF] Mccoll | Review article: gastrin releasing peptide and its value in assessing gastric secretory function[END_REF] However the aspiration methods have some common shortcomings. They can underestimate the gastric acid as some part of the acid is lost due to gastric emptying, some of the acid is neutralized by the alkaline secretions and part of the hydrogen ions diffuse back into the gastric mucosa. These tests involve removal of gastric juice from the stomach which can itself influence the measurement. They may be difficult to perform in conditions where gastric aspirates are small e.g. in tests involving drugs which suppress gastric acid secretion. In addition the invasive nature of these tests makes them uncomfortable for patients. With the advent of endoscopy the clinical use of these tests has declined and they are nowadays used mainly in experimental studies.

Fractional test meals-Intra gastric titration methods

Fordtran and Walsh developed the intragastric titration method in 1973 for measuring gastric acid secretion. [START_REF] Fordtran | Gastric acid secretion rate and buffer content of the stomach after eating. Results in normal subjects and in patients with duodenal ulcer[END_REF] This method involved ingestion of a meal of pH approximately 5.5 and subsequent aspirations of small volumes of gastric contents at regular time intervals with determination of its pH. In the initial study subjects ingested a solid meal. However subsequent studies used liquid or homogenized mixed meals which were introduced into the stomach by a nasogastric tube. When the pH value of the gastric contents fell below 5.5, alkali (sodium bicarbonate) was added to the stomach in order to keep the pH at 5.5. The amount of alkali infused in the stomach per unit time to maintain the pH at the predetermined level was thus equivalent to the amount of acid produced. [START_REF] Fordtran | Gastric acid secretion rate and buffer content of the stomach after eating. Results in normal subjects and in patients with duodenal ulcer[END_REF] This test suffered from several drawbacks including the fact that maintaining the stomach pH at a level of 5.5 was not physiological and hence did not reflect the normal pattern of pH changes in the stomach after ingestion of food. Maintaining the stomach at this pH also could cause disturbances in the feedback inhibition of gastrin and spuriously high acid levels. Such a high pH could also increase the gastric emptying time, retard protein digestion and decrease the back diffusion of H+ from the stomach lumen into blood leading to erroneous results. [START_REF] Fordtran | Gastric acid secretion rate and buffer content of the stomach after eating. Results in normal subjects and in patients with duodenal ulcer[END_REF] Addition of the alkaline solution constantly to the stomach could also lead to distension of the stomach leading to increased gastric acid secretion. An important technical problem of this test was ensuring an appropriate mixing of the alkali and the gastric contents. If inadequate mixing occurred, alkali which was not neutralised would be then the intragastric titration technique, though the results obtained by the intragastric titration method was 33% higher than the conventional tube test result . [START_REF] Halter | A comparison between intragastric titration and aspiration technique under basal conditions and after food or pentagastrin stimulation[END_REF] However this technique like others is now rarely used as more advanced pH based studies have been developed.

Endoscopic methods to estimate gastric acid secretion

Quantitative methods

A variety of endoscopic techniques have been used to measure gastric acid secretion. They have ranged from measuring the pH of aspirated gastric juice to a visual assessment of the extent of the acid producing mucosa after staining. [START_REF] Iijima | A new endoscopic method of gastric acid secretory testing[END_REF][START_REF] Eisenband | Endoscopic estimate of gastric acid secretion[END_REF][START_REF] Yamaguchi | Precise gastric secretory studies using a new fiber-gastroscope[END_REF][START_REF] Okuda | An Endoscopic Method to Investigate Gastric Acid Secretion[END_REF] Yamaguchi et al. developed a method where gastric juice was aspirated under direct endoscopic visualization every 10 minutes before and after stimulation with tetragastrin during a 2 hour period in 24 subjects. [START_REF] Yamaguchi | Precise gastric secretory studies using a new fiber-gastroscope[END_REF] On comparison with standard tube tests performed on the same subjects the authors found no significant difference in acid secretion detected by the two methods. However the length of the procedure was too long for it to be used widely in clinical practise. Eisenband and Wenger used a shorter technique.

They measured the pH of the gastric content 10-12 minutes after intravenous pentagastrin injection (3µg/kg) in 27 patients. Gastric acid levels were then calculated based on arbitrary estimates of gastric acid volume which was the 14 major drawback of their study. [START_REF] Eisenband | Endoscopic estimate of gastric acid secretion[END_REF] It was interesting to note that though they used arbitrary values for calculating gastric acid levels, when they compared their method with the conventional tube tests, the linear correlation coefficient was 0.99.

Another technique described by Iijama et al. in 1998 measured stimulated acid secretion. [START_REF] Iijima | A new endoscopic method of gastric acid secretory testing[END_REF] Their technique involved sampling gastric secretion endoscopically for a period of 10 minutes, 20 minutes after tetragastrin injection (4 micrograms/kg IM) and comparing it with the conventional tube tests. The volume of the sample collected over the 10 minutes was measured and the hydrogen ion concentration (H + ) in the sample was determined by titration. The acid output for the 10mins could then be calculated by multiplying the volume of the sample with the H + concentration. The test was conducted in 15 individuals of whom 11 were healthy volunteers. There was good correlation between the values obtained by the test and maximal acid output and peak acid output measured by the conventional tube tests (r= 0.90 and 0.92 respectively, Figure 2). The test also showed good reproducibility in 10 of the healthy volunteers. This test had the disadvantage of not being able to measure the basal acid output (BAO).However, the Endoscopic Gastrin Test has subsequently been used successfully in a variety of studies in Japan. [START_REF] Iijima | Changes in gastric acid secretion assayed by endoscopic gastrin test before and after Helicobacter pylori eradication[END_REF][START_REF] Inomata | Preservation of gastric acid secretion may be important for the development of gastroesophageal junction adenocarcinoma in Japanese people, irrespective of the H. pylori infection status[END_REF][START_REF] Abe | The prevalence of Helicobacter pylori infection and the status of gastric acid secretion in patients with Barrett's esophagus in Japan[END_REF]

Qualitative methods

A novel method of assessing gastric acid secretion was described by Okuda et al. in 1965 called the Congo Red Test (CRT). [START_REF] Okuda | An Endoscopic Method to Investigate Gastric Acid Secretion[END_REF] The test was based on the [START_REF] Toth | Modified endoscopic Congo red test: A rapid method to visualize gastric acid secretion[END_REF] In this test, pentagastrin was given intravenously (0.2µg/kg) instead of intramuscularly as was the case with the original method and this considerably shortened the duration of the examination. However these tests not only involve endoscopy but also do not provide quantitative measurements of gastric acid and hence are not widely used.

Intragastric pH measurement

Esophageal pH monitoring by intranasally placed catheter systems have been used for some time. These systems have also been adapted for measurement of gastric pH. Intragastric pH is measured by means of a pH measuring electrode in the stomach inserted by the nasogastric route. Though several types of electrodes are available for pH measurement, the glass or antimony electrodes are mostly used in intragastric pH measurement. They can be connected to a recording device directly, or indirectly by radiotelemetry. [START_REF] Van Herwaarden | 24-h recording of intragastric pH: technical aspects and clinical relevance[END_REF] The positioning of the electrode is vital as different parts of [START_REF] Fackler | Ambulatory gastric pH monitoring: proper probe placement and normal values[END_REF][START_REF] Cilluffo | Reproducibility of ambulatory gastric pH recordings in the corpus and antrum. Effect of food, time, and electrode position[END_REF] The probe can be placed with the help of fluoroscopy or manometrically with the latter method being commonly used (10cms below the upper margin of the Lower Esophageal Sphincter). [START_REF] Hirano | ACG practice guidelines: esophageal reflux testing[END_REF] However this method is invasive, uncomfortable and can lead to restriction in activity and diet of patients. Fass et al. in their study of fifty four patients undergoing 24 hrs esophageal pH studies showed a significant decrease in time spent being active and also the number of meals by the subjects during the test. [START_REF] Fass | Effect of ambulatory 24-hour esophageal pH monitoring on reflux-provoking activities[END_REF] In addition, catheter migration is an associated problem leading to inaccuracies. [START_REF] Chang | A novel placement method of the Bravo wireless pH monitoring capsule for measuring intragastric pH[END_REF] To counteract this, the wireless capsule pH measurement system -BRAVO™ pH monitoring system has been utilized (Figure 3). It can be inserted endoscopically, is better tolerated and can be used for a longer duration i.e. 48hours compared to 24 hrs with the catheter based techniques.

As with the catheter based techniques the BRAVO™ system has been used in measuring intragastric pH. [START_REF] Chang | A novel placement method of the Bravo wireless pH monitoring capsule for measuring intragastric pH[END_REF][START_REF] Banerjee | Bravo capsule pH monitoring[END_REF] One of the early drawbacks was the early dislodgement of the capsule from the stomach wall before the 48 hrs period had elapsed, though techniques have now been developed to fix the capsule in the stomach including a clipping technique developed by Chang et al. [START_REF] Yamaguchi | Placement of the Bravo wireless pH monitoring capsule onto the gastric wall under endoscopic guidance[END_REF][START_REF] Chang | A novel placement method of the Bravo wireless pH monitoring capsule for measuring intragastric pH[END_REF] Intragastric pH measurements have an important role relating to quantifying acid suppression within health and various disease states. However it is most useful in studies assessing the effect of acid suppressing drugs on gastric Stomach acidity measured by the above method can be expressed either as a pH value or as a hydrogen ion (H + ) activity. The H + activity can be calculated from pH values by the formula (pH= -log 10 [H + ]). Studies comparing intragastric pH using intraluminal electrodes and that from aspirated gastric juice have shown a good correlation. However where calculated H+ activity has been compared with the actual H + activity measured form gastric aspirates there have been significant differences. Hence some concerns have been raised in using pH values as a measure of gastric acid. [START_REF] Van Herwaarden | 24-h recording of intragastric pH: technical aspects and clinical relevance[END_REF] It should be borne in mind that gastric pH neither gives a quantitative evaluation of the volume of acid secretion nor does it necessarily correlate to gastric acid secretion as several factors like food, gastric emptying, bicarbonate secretion, dilutional effect of saliva and intestinal secretions can all influence the pH of gastric liquid.

B) Non invasive methods of gastric acid measurement.

Dye tests. One of the earlier non-invasive methods of measuring gastric acid was described by Segal in 1950 using a cation exchange indicator compound. [START_REF] Segal | Determination of gastric acidity without intubation by use of cation exchange indicator compounds[END_REF] This method depended on the fact that free acid (hydrochloric acid in stomach) caused dissociation of an ingested cationic exchange resin compound. The cation would then be absorbed by the intestine and be excreted in the urine where it could be detected. The first exchange resin used was "Diagnex" in which hydrogen ions were replaced by quininium ions that could be measured fluorometrically but required ether-sulphuric acid extraction of urine making it difficult for routine use. To simplify this method, Segal et al. subsequently used azure A dye combined with the cationexchange resin (Diagnex blue). The presence of gastric acid could be detected by direct visual comparison of the colour of the subject's urine with standards. [START_REF] Segal | Tubeless gastric analysis with an azure A ionexchange compound[END_REF] The dye was released at pH 3.2 and below with a maximal release at pH 1.5. Hence patients with Achlorhydria, in whom the pH of the stomach did not decline below 3.2, did not excrete the dye. By observing the change in colour of the test urine 2 hours after ingestion of the dye, participants without free gastric acid could thus be identified. Segal et al.

initially used caffeine benzoate as a gastric stimulant. However it was later found that using betazole hydrochloride as the gastric stimulant showed production of gastric acid in 51% of the patients who were thought to be achlorhydric by using caffeine benzoate. [START_REF] Segal | Gastric hydrochloric acid secretory response to orally administered betazole hydrochloride[END_REF] Hence in later tests, caffeine was replaced by betazole as the gastric stimulant. This method has been tested further in several studies. [START_REF] Bock | Tubeless Gastric Analysis with Azure a and Betazole Hydrochloride[END_REF][START_REF] Correia | Tubeless gastric analysis with azure A and maximal histamine stimulation[END_REF][START_REF] Bolt | A clinical evaluation of tubeless gastric analysis[END_REF] Although this test did not quantitatively measure gastric acid, it was felt that the most useful application would be to screen patients for achlorhydria. [START_REF] Bock | Tubeless Gastric Analysis with Azure a and Betazole Hydrochloride[END_REF] However, the dye test had 2 major disadvantages. Firstly it depended upon the functional integrity of the kidney and secondly patients who tested negative on the test i.e. did not excrete the dye in urine, were not necessarily achlorhydric as some individuals may not secrete gastric acid under basal conditions. With the advent of newer and more reliable methods to detect gastric acid secretion this test is no longer used.

Serum dye tests

Passaro et al in 2002 developed a similar method to detect achlorhydria but measured the indicator in blood and serum. They used quininium resin that liberated free quinine at pH<3.5, which could be measured in blood serum fluorometrically. They found a 95% accuracy rate for detecting hypochlorhydria (induced by ingestion of a proton pump inhibitor). However the study was performed in only 10 individuals and has not been further validated. [START_REF] Passaro | A modification of the quininium resin test for assessing gastric acidity[END_REF]

Gastric acid measurement with a pH sensitive endoradiosonde (Heidelberg capsule)

In the 1960's a non invasive method of measuring gastric acid was described by Noeller et al. using a pH sensitive electroradiosonde, called the Heidelberg capsule. [START_REF] Noller | Proceedings of the IIIrd International Conference on Medical Electronics[END_REF] The capsule contained a battery, a pH sensor and a radio transmitter. The pH sensor comprised an antimony electrode that was in Hence by measuring the transmitted frequency of the capsule in a solution, its pH could be calculated. In this technique, subjects ingested the capsule and a basal pH was calculated. Subsequently a saturated bicarbonate solution was ingested causing a rise in intragastric pH, stimulating gastric acid secretion and then a fall in pH back to its basal level. The time taken for the pH to fall to its basal level was the alkali test time and represented a measure of the gastric acid secreted. This test was compared with the standard tube test and showed acceptable correlation ranging from 0.74 to 0.68. [START_REF] Connell | Assessment of Gastric Function by Ph Telemetering Capsule[END_REF][58] The capsule is still in use but has been technically modified and is smaller in size. [START_REF] Shelton | Eradication of Helicobacter pylori is associated with increased exposure to delavirdine in hypochlorhydric HIV-positive patients[END_REF]

Serum pepsinogens and their use as a marker of gastric acid secretion.

Pepsinogens belong to the aspartic proteinase group of enzymes which act on peptide bonds in proteins. They are converted into the active enzyme pepsin by gastric acid. They act between a pH of 1.5-5.0 and initiate protein digestion. [START_REF] Gritti | Pepsinogens: physiology, pharmacology pathophysiology and exercise[END_REF] Pepsinogens can be divided into two groups; pepsinogen I (PGA or PGI, includes pepsinogens 1-5) and pepsinogen II (PGC or PGII, includes ). These two groups are not only biochemically and immunologically different but are secreted by different parts of the gastrointestinal tract. Pepsinogen I is produced by chief cells and the mucus neck cells of the gastric fundus. Pepsinogen II is secreted in addition by the pyloric glands and Brunner's glands in the duodenum. [START_REF] Samloff | Cellular localization of group I pepsinogens in human gastric mucosa by immunofluorescence[END_REF][START_REF] Samloff | Cellular localization of the group II pepsinogens in human stomach and duodenum by immunofluorescence[END_REF] In healthy people the concentration of pepsinogen levels are thought to be stable in single individuals but can vary from individual to individual depending on their age, height, sex, weight, ethnicity, diet and circadian rhythms. [START_REF] Gritti | Pepsinogens: physiology, pharmacology pathophysiology and exercise[END_REF] Pepsinogen levels vary in conditions affecting the gastric mucosa and they reflect the structural and functional status of the gastric mucosa. [START_REF] Iijima | Serum pepsinogen concentrations as a measure of gastric acid secretion in Helicobacter pylori-negative and -positive Japanese subjects[END_REF] High levels are found where there is active inflammation of the gastric mucosa such as in superficial gastritis, decreased levels however are seen in chronic gastritis and especially in severe atrophic gastritis with intestinal metaplasia and in gastric cancer. [START_REF] Miki | Usefulness of gastric cancer screening using the serum pepsinogen test method[END_REF][START_REF] Kitahara | Accuracy of screening for gastric cancer using serum pepsinogen concentrations[END_REF][START_REF] Miki | The significance of low serum pepsinogen levels to detect stomach cancer associated with extensive chronic gastritis in Japanese subjects[END_REF][START_REF] Miki | Clinical application of serum pepsinogen I and II levels for mass screening to detect gastric cancer[END_REF] Studies have shown that levels of PGI and PGI/II ratio can be correlated to gastric acid output. [START_REF] Iijima | Serum pepsinogen concentrations as a measure of gastric acid secretion in Helicobacter pylori-negative and -positive Japanese subjects[END_REF] This correlation between gastric acid and pepsinogens depends upon the H. pylori status of an individual. Iijama et al. in his study found that in patients who were H. pylori positive, there was a reasonable correlation between gastric acid secretion with PGI/II ratio and serum PGI levels (r=0.40 and 0.53) However in H.pylori negative subjects, serum PGI levels were found to have a better correlation with acid secretion (r=0.57) and the PGI/II ratio showed no correlation (Figure 4). The main use, however, of measuring pepsinogens in clinical practice is to detect subjects with significant gastric atrophy who are at risk of developing gastric cancer. Gastric atrophy with intestinal metaplasia is believed to be a precursor lesion for the intestinal type of gastric cancer. [START_REF] Komoto | Helicobacter pylori infection and gastric neoplasia: Correlations with histological gastritis and tumor histology[END_REF] It is believed that as gastric atrophy progresses, levels of PGI decrease whereas PGII levels remain fairly constant. This leads to a fall in the PGI: PGII ratio and a combination of a low PGI level with a low PGI: PGII ratio is considered to be a marker of gastric atrophy. This finding of a low PGI levels and low PGI: PGII ratio has been correlated with histologically proven gastric atrophy particularly corpus atrophy. [START_REF] Urita | Serum pepsinogens as a predicator of the topography of intestinal metaplasia in patients with atrophic gastritis[END_REF][START_REF] Cao | Screening of atrophic gastritis and gastric cancer by serum pepsinogen, gastrin-17 and Helicobacter pylori immunoglobulin G antibodies[END_REF] Breath tests for estimating gastric acid secretion.

The hydrogen breath test.

Breath analysis as a method of estimation of various metabolic processes has been around for some time. Sack and Stephenson in 1985 described a novel method of estimating gastric acid by orally administering magnesium (gelatine capsules containing finely ground magnesium metal) in different doses ranging from 10-200mg, which reacts with acid in the stomach to produce hydrogen gas, the amount of which was used as a proxy for gastric acid. was measured by gas chromatography of the breath sample. By subtracting the basal hydrogen levels from the total hydrogen levels in exhaled air, the excess hydrogen produced due to action of gastric acid with magnesium could be ascertained, which could be related to the gastric acid secretion. This test was further validated in 1987 in 36 subjects who also underwent the standard intubation test. The gastric acid stimulant used was Betazole. A correlation coefficient of 0.70 was found between the stimulated acid output values and the excess amount of hydrogen exhaled during the test. 9 individuals went on to have a repeat breath test and the test-retest correlation coefficient was 0.83. This test has been suggested as a screening test for hypochlorhydria in view of its simplicity and non-invasive nature. [START_REF] Stephensen | Comparison of noninvasive breath hydrogen test for gastric acid secretion to standard intubation test in adults[END_REF] Humbert et al. in 1997 used a modified version of the Magnesium hydrogen breath test where they used end expiratory breath sampling only and compared it with the pentagastrin test for gastric acid secretion. They studied patients with pernicious anaemia and those with healed duodenal ulcer with normal acid secretion. They measured the peak value of hydrogen in breath and found it to have 95.2% sensitivity and 100 % specificity in detecting achlorhydria. [START_REF] Humbert | Magnesium hydrogen breath test using end expiratory sampling to assess achlorhydria in pernicious anaemia patients[END_REF] As with most non invasive tests it could not detect gastric acid quantitatively and has been suggested solely as a test to detect achlorhydria.

The calcium carbonate breath test

Clough and Axon introduced the Calcium carbonate breath test in 2009 as another method to assess stimulated gastric acid secretion quantitatively by breath analysis. They incorporated [START_REF] Kay | Effect of large doses of histamine on gastric secretion of HCI; an augmented histamine test[END_REF] C, which is a non radioactive isotope of carbon, into calcium carbonate and administered it orally to a healthy volunteer. It was based on the principle that calcium carbonate labelled with the [START_REF] Kay | Effect of large doses of histamine on gastric secretion of HCI; an augmented histamine test[END_REF] C was acted upon by hydrochloric acid in the stomach producing [START_REF] Kay | Effect of large doses of histamine on gastric secretion of HCI; an augmented histamine test[END_REF] C labelled carbon dioxide ( 13 CO 2 ).

Ca [START_REF] Kay | Effect of large doses of histamine on gastric secretion of HCI; an augmented histamine test[END_REF] CO 3 +2HCl→CaCl 2 +H 2 O+ 13 CO 2 ↑

The 13 CO 2 was absorbed by the lungs and eliminated in breath. By measuring the ratio of 13 CO 2 to 12 CO 2 in the breath sample via a mass spectrometer the amount of acid secreted by the stomach was calculated. The test used various regimens including 500mg every 5 mins and 750mg every 10mins of the labelled calcium carbonate. This test has been thought to measure stimulated acid output and could detect an 83% reduction in gastric acid production after acid suppression therapy with proton pump inhibitors. This test has the added advantage of measuring gastric acid secretion quantitatively, which was a major drawback of the other non invasive methods. However this technique was performed on only one volunteer and was not compared with conventional tests, hence necessitating further studies to validate it. [START_REF] Clough | The Calcium Carbonate Breath Test, a noninvasive test of stimulated gastric acid secretion: preliminary communication[END_REF] Measurement of gastric acid using Scintigraphic techniques and applied potential tomography.

Use of 99m Tc-pertechnetate scintigraphy as a method of measuring gastric acid was described in late 1970's. [START_REF] Taylor | A single scan technique for estimating acid output[END_REF][START_REF] Keane | An assessment of dynamic gastric scanning with 99Tcm-pertechnetate[END_REF] Measurement of the accumulated Tc-pertechnetate in the stomach by scintigraphy after intravenous injection of the tracer was thought to be related to gastric acid secretion. However it used radioactive elements and doubts were expressed about its reliability so it has not been widely used.

Applied Potential Tomography (APT) is a technique that measures the resistivity of gastric contents through 16 electrodes placed in a circular manner on the abdomen at the level of the gastric fundus/body. It can build up tomographic images of gastric resistivity. Gastric acid secretion causes gastric resistivity to fall, which can be recorded using this technology and used as a measure of gastric acid secretion. It is non-invasive and non-radioactive.

Baxter et al. used the technique in 20 healthy volunteers and 13 patients with peptic ulcer disease (who also underwent acid measurement by conventional methods). [START_REF] Baxter | Evaluation of applied potential tomography as a new non-invasive gastric secretion test[END_REF] They found a significant correlation (r=0.85, p<0.001) between the basal change in resistivity of gastric contents with basal acid output and the maximum change in resistivity, after gastric stimulation with pentagastrin, with a maximal acid output (r=0.58, p<0.05) in the group with peptic ulcer disease (Figure 5). Sarkar et al. also used the technique in 19 healthy volunteers and compared it to conventional intubation tests. [START_REF] Sarker | Noninvasive assessment of gastric acid secretion in man. Application of electrical impedance tomography (EIT)[END_REF] They found a correlation coefficient of r=0.65 between the two methods for stimulated acid output.

Though it is non invasive and a simple test, it cannot quantitatively estimate gastric acid. Hence it may be useful only as a non invasive method to detect low acid output states.

Alkaline tide in measurement of gastric acid secretion

Alkaline Tide is recognized phenomenon in the physiology of gastric acid secretion. The intracellular pH of parietal cells is regulated by a complex system of ion channels and exchangers. [START_REF] Niv | The alkaline tide phenomenon[END_REF] Secretion of H + ions by the parietal cells into the gastric lumen leads to secretion of bicarbonate (HCO 3 -) ions into the blood stream. The resulting alkalization of blood and urine has been described as the alkaline tide and was found to transiently occur after meals. [START_REF] Rune | The metabolic alkalosis following aspiration of gastric acid secretion[END_REF] This phenomenon has been used as an indirect method of measuring gastric acid secretion. The alkaline tide effect leads to changes in the base excess of blood. After gastric stimulation by histamine or pentagastrin, the base excess in arterialized venous blood can be measured and from this the alkaline tide can be calculated.

Niv et al. studied 12 patients with duodenal ulcer where the response of the alkaline tide to pentagastrin stimulation and its correlation to stimulated gastric acid (measured by gastric aspiration at a separate date) was analysed. They found a good correlation (r=0.86) between the peak acid output ( PAO) and the alkaline tide 45 minutes after pentagastrin injection. [START_REF] Niv | Further applications of blood gas analysis to gastric acidity determination[END_REF] This test can thus be used as an indirect measure of gastric acid secretion.

Conclusion

Over the years a number of invasive and non-invasive ways of measuring gastric acid secretion have been described. However as the conventional

  secretion. Further attempts to explore new methods for measuring gastric acid are therefore warranted.

5

 5 Levels of gastric acid secretion differ in various disease states and their measurement may help in the diagnosis and treatment of these diseases.

  a low stomach pH can be caused by Zollinger-Ellison syndrome, chronic antral H. pylori infection, renal failure and gastric outlet obstruction.[START_REF] Metz | A retrospective study of the usefulness of acid secretory testing[END_REF] Apart from clinical use, the measurement of gastric acid has an important role in clinical and pharmacological research. Studies related to the epidemiology of acid peptic disorders as well as introduction and assessment of new and existing drug therapies for suppression of gastric acid are further areas where gastric acid measurement is still important.

  subjects and from years 1950-2010. Abstracts were then studied and articles which dealt mainly with the methodology and use of the test in studies related to measuring gastric acid in health and disease were chosen and full text versions were obtained. Most of these articles were several decades old and many of the further references were identified from the bibliography of the selected articles.

16

 16

24

 24

  stomach leading to an overestimation of the gastric acid. In spite of these drawbacks, Halter et al. in his study of 16 healthy volunteers showed a good correlation (correlation coefficient-r=0.85) between values of maximal gastric acid output (MAO) obtained by the conventional aspiration tests and

  inspection of acid producing gastric mucosa and relies on a pH dependent colour change. In the test, Congo red dye, which changes colour at pH<3, is sprayed onto gastric mucosa after pentagastrin stimulation (6µg/kg) and the change in colour of the dye from red to dark blue or black is observed. The extent of the area showing the change is assessed semiquantitatively and gives an estimate of the acid secretory ability of the gastric mucosa. This test has been used to help in the diagnosis of early gastric cancer and gastric atrophy related changes. However the original method was time consuming and prolonged endoscopy by 10-20 minutes. The Modified Congo Red Test (MRCT) was described by Tooth et al. in 2003.

  have different pH. Fackler et al. demonstrated that positioning the probe in the fundus was the optimal position for measurement of gastric pH whereas Cilluffo et al. showed in post prandial states pH measurement in the antrum was more dependable.

  low acid output states, particularly during use of acid suppressing drugs, conventional methods to measure gastric acid may be technically difficult due to problems in aspirating low volumes of acid leading to inaccurate results. The intragastric pH measurement method is very useful in these situations (in particular where 2 or more acid suppressing drugs are being compared) to detect the subtle differences in gastric acidity by measuring a difference of intragastric pH rather than actual acid secretion.

  the fluid whose pH was to be measured and an internal silver chloride electrode, the two being separated from each other by a semipermeable membrane. Changes in the pH altered the voltage output of the battery, which in turn altered the frequency of the radio transmitter. The frequency was picked up by antennae worn by the patient connected to an amplifying receiver and a recorder. The test depended on the fact that a known change in pH could cause a change in the transmitted radio frequency.

63

 63

71

 71

Mg+2HCL→MgCl 2 +H 2 ↑

 2 Normal individuals in a fasting state produce a small amount of H 2 in their exhaled air. A basal hydrogen excretion rate in expired air was initially calculated. Participants were then given magnesium orally following administration of a gastric secretory stimulant. The hydrogen concentration

Females 33 Figure 1 :

 331 Figure 1: Structure of Tetragastrin

Figure 2 : 35 Figure 3 :

 2353 Figure 2: Correlation between MAO & PAO estimated by conventional tube

Figure 4 :

 4 Figure 4: Figure showing correlation between serum gastric acid measured by

Figure 5 :Figure 1 :Figure 2 :Figure 3 :Figure 4 :Figure 5 :

 512345 Figure 5: Relationship between gastric resisitivity (APT units) against basal

Table I : Invasive and non invasive methods of measuring gastric acid secretion Invasive methods of measuring gastric acid

 I

	tests are invasive and uncomfortable for patients, their use has declined. The Tables has its own procedural risks. Non invasive methods of measuring gastric acid test. used in day to day clinical practice
	widespread use of endoscopy has led to a further decline in their use in the Congo red test No No Gives a qualitative assay of gastric acid Method In use in Quantitative Comment
	clinical setting. However gastric acid secretion remains an important element (CRT) and output only and also requires endoscopy clinical measure of Table 2
	of gastric physiology in health and disease. The ability to measure it modified CRT. with its associated risks. practice gastric acid Scintigraphy and No No Scintigraphy uses radioactive Gastric acid levels in health and disease (Pentagastrin stimulation test)
	accurately and non invasively would serve a variety of clinical and Method In use in Quantitative Comment (Yes/No) (Yes/No) applied potential materials. Applied potential
	experimental needs. Further attempts to refine existing methods and to routine measure of Dye tests (urinary No longer No These tests were used to detect low tomographic tomography does not give a BAO(mmol/hr) MAO(mmol/hr) PAO(mmol/hr)
	explore new methods for measuring gastric acid are warranted. clinical gastric acid and serum). used acid output states but results were not techniques. quantitative measure of gastric acid. Average(range) Average(range) Average(range)
	F o r F o r practice (Yes/No) Conventional F o r Intragastric pH measurements Yes F o r Heidelberg Still used F o r Alkaline tide. No Normal Individuals F r o Males	secretion (Yes/No) No. It gives a measure No No	Initially technique was developed for esophageal pH studies but now also completely reliable hence no longer used. This technique measures gastric pH Both tests are not in regular clinical use. Useful as a indirect method of
	aspiration tests Test meals. capsule.	P e P e No Yes P e of gastric pH P e but a more techinically P e P e	The aspiration tests are the gold used for intra gastric pH measurements. New system includes the wireless only. It is still available and has been used in clinical settings and estimating gastric acid. Not a quantitative method and mostly an
		supeior	e r e r e r e r e r e r	standard tests against which most other BRAVO™ capsule. The method does not experimental work. experimental technique.
	Augmented histamine test. Pentagastrin & Tetragastrin test Endoscopic tests Serum Pepsinogens.	No Yes version. Yes (mostly as a screening test for gastric	R e v i e w R e v i e w Yes Yes methods of gastric acid measurement are compared. However they are invasive and uncomfortable. Pentagastrin and tetragastrin are presently used as gastric stimulants. R e v i e w give a measure of gastric acid secretion but a measure of gastric acidity. R e v i e w No R R The main use of pepsinogens have e e been mostly in diagnosis of gastric atrophy, hence for screening of v i e v i e stomach cancers. Their role in gastric acid measuremet is limited. w w
	Endoscopic	Yes malignancy) Yes	Used recently in Japan. Short duration
	Gastrin test (EGT). Breath tests	No		Yes (The	and gives a quantitative measurement of The hydrogen breath test does not
	including the			calcium	gastric acid. Has been compared to give us a quantitative measure of
	hydrogen breath			carbonate	standard tube test with good corelation. gastric acid which the calcium
	test and calcium			breath test)	However requires an endoscopy as a carbonate does. However these are
	carbonate breath				part of the method which is invasive and still experimental methods and not
				29 30 31 32


Hana Zelena

Vlasta Zvolankova Vlasta

Jana Zuchnicka

Katerina Liszkova

Professor Anna Papa
email: annap@med.auth.gr

Zelena Hana

Zvolankova Vlasta

Zuchnicka Jana

Liszkova Katerina

Hantavirus infection during a stay in a mountain hut in northern Slovakia

Keywords: Hantavirus, Dobrava/Belgrade virus, Slovakia, Czech Republic Hantavirus, Dobrava/Belgrade virus, Slovakia, Czech Republic

à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

 [START_REF] Avsic-Zupanc | Hemorrhagic fever with renal syndrome in the Dolenjska region of Slovenia -a 10-year survey[END_REF][START_REF] Papa | Hantavirus infections in Greece -an update[END_REF].

Since the first report of hantavirus infections in Breclav, a Czech town in the South

Moravian region [START_REF] Kobzik | Laboratory-confirmed cases of hemorrhagic fever with renal syndrome which occurred in Breclav 1989-1990[END_REF], only a limited number of HFRS cases have been detected in the Czech Republic [START_REF] Heyman | Situation of hantavirus infections and haemorrhagic fever with renal syndrome in European countries as of December 2006[END_REF]. In August 2008, a 15year boy was infected in Ostravice village, close to the borders of the Czech Republic with Slovakia and Poland, and presented a severe form of HFRS; the patient was hospitalized in the intensive care unit and recovered after 23 days in hospital [START_REF] Papa | Genetic detection of Dobrava/Belgrade virus in a Czech patient with Haemorrhagic fever with renal syndrome[END_REF]. Application of molecular methods showed that the causative agent was DOBV (strain Beskydy/08), with sequences clustering with respective ones obtained from tissues of A. flavicollis trapped in Slovakia, Greece and Slovenia [Papa et al., 2009] [START_REF] Papa | Genetic detection of Dobrava/Belgrade virus in a Czech patient with Haemorrhagic fever with renal syndrome[END_REF] (Figure 1).

Case 1.

The owner of the hut, a 29-year old hitherto healthy male (patient CT) resident of Bohumín, a small town in the southeastern part of the Czech Republic, went to the hut one day earlier than the rest of the group in order to clean the place. Thirteen days later (December, 10, day 1), he suddenly presented high fever (39°C) accompanied by headache, diarrhea, vomiting, and lower back and abdominal pain. He received antipyretics according to his physician recommendations. However, his condition deteriorated and on December 15 (day 6), he was admitted to the Department of Internal Diseases in the regional hospital in Bohumín. Three days later, he presented signs of gut and skin bleeding, and the patient developed renal failure with proteinuria, hematuria and hypertension. Hemodialysis was not applied.

The patient presented leucopenia 3.5 x 10 9 /l (normal range 4-10 x 10 9 /l), thrombocytopenia 18 x 10 9 /l (normal range 150-400 x 10 9 /l), and elevated liver enzymes.

Serum urea increased to 21.0 mmol/l (normal range 2.8-7.2 mmol/l), creatinine to 245 µmol/l (normal range 62-115 µmol/l), CRP to 45 mg/l (normal range 0-10 mg/l). He presented high values of D-dimers (maximum of 18.25 mg/l, normal range 0-0.2 mg/l);

Extraction of viral RNA and polymerase chain reactions

Viral RNA was extracted from the earliest available serum samples (taken on day 7 from patient CT and on day 13 from patient SP) using the QIAamp Viral RNA kit (Qiagen, Hilden, Germany) and two RT-nested PCRs were performed using two sets of primers designed to detect hantaviruses associated with rodents of the Murinae subfamily (HTN, DOB, SEO). One primer set (MS120C -MS1170R for the first round and MS364C -MS963R for the second round) was used for the amplification of a 590-bp fragment of the N coding region, and a second primer set (MM1470C -MM2029R for the first round and MM1674C -MM1990R for the second round) was used for the amplification of a 317-bp fragment of partial G1 coding region [START_REF] Papa | Retrospective serological and genetic study of the distribution of hantaviruses in Greece[END_REF]Papa et al., 2009].

Sequencing and phylogenetic analysis

Sequencing of the PCR products was performed in an ABI 3110 genetic analyzer (Applied Biosystems, Foster City, CA). Sequences were aligned by CLUSTAL W, while phylogenetic trees based on S and M segment sequences were constructed by Neighborjoining method using the modules DNAdist, Neighbor and Consense from the PHYLIP software [START_REF] Felsenstein | PHYLIP (Phylogeny Inference Package) version 3.5c. Distributed by the author[END_REF]

RESULTS

IgM and IgG antibodies against HTNV were detected in all serum samples tested, while antibodies against PUUV were either not detected or the titers were low (Table 1).

Serology results suggested that the causative agent was a hantavirus closer antigenically to HTNV. Products of the expected size were obtained by both RT-nested PCRs, while sequencing analysis confirmed that the causative agent was DOBV. The two DOBV sequences (Cadca-CT/08 and Cadca-SP/08) were identical. The closest strain genetically was the Czech strain Beskydy/08, presenting one nucleotide difference in S segment, and no difference in the M segment. In both S and M segment phylogenetic trees, the sequences from both patients and those of the Czech strain (Beskydy/08, Cadca-CT/08, Cadca-SP/08) comprise a distinct subclade in the clade containing DOBV sequences associated with A. flavicollis (Figure 2A and2B). In the S segment, Slovakian strain differs by 8.7% and 8.6% from DOBV strains isolated from A. flavicollis in Greece and Slovenia respectively, and by 9.5% from strain 400Af/98 from East Slovakia, while in the M segment the Slovakian strain differs by 15.5% from the strain 400Af/98, and by 16.7% and 18.6% from strains isolated in Greece and Slovenia respectively.

DISCUSSION

DOBV is highly endemic in the Balkan peninsula, where cases are observed usually in the summer time [START_REF] Avsic-Zupanc | Hemorrhagic fever with renal syndrome in the Dolenjska region of Slovenia -a 10-year survey[END_REF][START_REF] Papa | Hantavirus infections in Greece -an update[END_REF]. In Central and Northern Europe HFRS cases are caused mainly by PUUV [START_REF] Heyman | Hantavirus infections in Europe: from virus carriers to a major public-health problem[END_REF][START_REF] Vapalahti | Hantavirus infections in Europe[END_REF]. However, during the last decade, cases vaused by DOBV have been reported in Slovakia, Germany, Poland and the Czech Republic [START_REF] Klempa | First molecular identification of human Dobrava virus infection in central Europe[END_REF][START_REF] Nowakowska | The first established focus of hantavirus infection in Poland, 2007[END_REF][START_REF] Papa | Genetic detection of Dobrava/Belgrade virus in a Czech patient with Haemorrhagic fever with renal syndrome[END_REF][START_REF] Sibold | Dobrava hantavirus causes hemorrhagic fever with renal syndrome in Central Europe and is carried by two different Apodemus mice species[END_REF]. The patients of the the most important [START_REF] Tsergouli | Cytokine levels in hemorrhagic fever with renal sydrome caused by Dobrava-Belgrade virus[END_REF].

The Slovakian and the Czech sequences cluster with DOBV strains associated with A.

flavicollis, and this is in accordance that this virus causes more severe disease than the other hantaviruses in Europe. Studies in rodents in the area will help the identification of 10 the exact host of these viruses. It has to be mentioned that DOBV spill-over events have been reported in rodents [START_REF] Schlegel | Dobrava-Belgrade virus spillover infections, Germany[END_REF]Weidmann et al., 2005].

A geographical clustering of sequences has been reported among hantaviruses [START_REF] Papa | Retrospective serological and genetic study of the distribution of hantaviruses in Greece[END_REF]Weidmann et al., 2005]. The strains of the present study differ greatly from those detected in East Slovakia, and, more interestingly, in the S segment they are closer genetically to Greek DOBV strains than to East Slovakian ones. . It should be noted that there are high mountains between Cadca region, the place where patients of the present study were infected, and Kosice, the place were strain East Slovakia/400Af/98 was isolated. Full genome sequencing of the strains will reveal whether recombination or reassortment events took place.

The

  were slightly affected. Renal biopsy was suggestive of progressive acute glomerulonephritis, while the thrombocytopenia was considered as immune-associated. As his condition deteriorated rapidly, on day 10 he was transferred to the Intensive Care Unit of the Department of Internal Diseases in the University Hospital in Ostrava. His clinical condition, the renal and liver function, and the laboratory findings returned subsequently to normal and he was discharged from the hospital on day 19, with the diagnosis of hemolytic uremic syndrome. On January 22, 2009, (day 43) during a follow-up examination in a mobile medical facility, a hantavirus infection was suspected.Case 2On December 26, 2008, 28 days after the overnight in the mountain hut, a second member of the group, a 27-year old hitherto healthy male (SP), developed a sudden high fever (40°C) with profuse diarrhoea, vomiting, headache and vertigo. On day 4 the patient collapsed, and he was admitted to the Department of Internal Diseases of the regional hospital in Bohumín. His condition deteriorated rapidly with hypovolemic and haemorrhagic shock leading to multiorgan failure with predominant renal failure and anuria. His condition was complicated by peripheral edema of the lung. The patient had disseminated intravascular coagulation with heavy bleeding from the needle puncture sites, and then, ongoing bleeding into the adrenal and retroperitoneal space, prompting urgent transfer to the Department of Anaesthesiology and Resuscitation in the University Hospital in Ostrava. High levels of procalcitonin (88.6 µg/l, normal range 0-0.5 µg/l) were observed, however, with no evidence of bacterial infection. Hemodialysis and tracheostomy were applied. The patient remained comatose for several weeks. Laboratory findings included leucocytosis 24.4 x 10 9 /l (normal range 4-10 x 10 9 /l), thrombocytopenia 3 x 10 9 /l (normal range 150-400 x 10 9 /l), elevated liver enzymes -ALT 5.97 µkat/l (normal range 0.15-0.75 µkat/l), AST 8.44 µkat/l (normal range 0.15-0.85 µkat/l). Serum urea increased to 20.3 mmol/l (normal range 2.8-7.2 mmol/l), creatinine to 474 µmol/l (normal range 62-115 µmol/l). The maximum value of CRP was 127 mg/l (normal range 0-10 mg/l). He revealed extreme values of D-dimers with the maximum value 24.67 mg/l (normal range 0-0.2 mg/l); other coagulatiaon parameters were also affected -prothrombin time 26 s (normal range 11-16 s), and activated partial thromboplastin time 80.9 s (normal range 25-35 s). The condition of the patient was complicated further by lifethreatening nosocomial infections caused by methicillin-resistant Staphylococcus aureus and Candida spp. He was discharged after 68 days in hospital, with most of the time in the Department of Anaesthesiology and Resuscitation. Serology Serum samples taken on days 7 and 76 from patient CT and on days 13, 43 and 85 from patient SP were tested for IgG and IgM antibodies to Hantaan virus (HTNV) and PUUV using ELISA (PROGEN Biotechnik, Heidelberg, Germany). IgM and IgG serology results are considered positive when the index value (optical density divided by the cut off value) is ≥ 2 and ≥ 1.5 respectively.

  infected in November 2008 in Slovakia, in an area 30 km far from Ostravice village(South-eastern Czech Republic), where an HFRS case was observed in August 2008, suggesting that this area of Central Europe is an endemic focus for HFRS.The occurrence of the cases in winter could be explained by the fact that rodents entered the hut trying to be protected from the cold. Patient CT presented the disease earlier than patient SP (incubation time 13 days for patient CT versus 28 days for patient SP), most probably because CT inhaled the largest quantity of aerosolized excreta, as he was the first who entered the hut, he cleaned the place and he stayed more time there than the rest group. Two other members of the group tested negative for the presence of IgG and IgM antibodies, while the rest refused to be tested. Different factors, including variations in immune response might explain why only patient SP (who stayed the same time as the rest) acquitted the disease. Although the incubation time of patient SP was longer than that of patient CT, his course of the disease was much more severe, accompanied by heavy hemorrhages, and more affected hematological and biochemical parameters.Cytokines and chemokines might be responsible for the different response, which resulted in prolongation of the hospitalization time and further acquisition of nosocomial infections. Preliminary studies have shown that a large number of cytokines and chemokines play a catalytic role in the course and the outcome of HFRS; among them PDGF, RANTES, IP-10, MIP-1b, IL-1ra, IL-9, GM-CSF, IFN-g, eotaxin, and MCP-1 are

Figure 1 .

 1 Figure legends

Figure 2 .

 2 Figure 2. Phylogenetic trees based on a 590-bp fragment of the S RNA segment (A) and a 317-

Table 1 .

 1 Detection of Hantaan virus (HTNV) and Puumala virus (PUUV) reactiveantibodies in patients at different time points using commercial ELISA. N: negative result.

		Day of	Index value in	Index value in
		disease	HTNV ELISA	PUUV ELISA
			IgG	IgM	IgG	IgM
	Patient CT	7	3.21	4.05	N	N
		76	2.41	1.46	N	N
	Patient SP	13	3.27	6.08	1.28	N
		43	3.39	5.63	1.49	N
		85	3.57	1.49	2.14	N

INTRODUCTION

Hantaviruses (genus Hantavirus, family Bunyaviridae) are enveloped, single-stranded RNA viruses with three-segmented genome, causing to humans hemorrhagic fever with renal syndrome (HFRS) in Eurasia and hantavirus pulmonary syndrome (HPS) in the Americas

[START_REF] Jonsson | A global perspective on hantavirus ecology, epidemiology, and disease[END_REF]

. Humans are infected mainly by inhalation of infected rodents' excreta. Five hantavirus genotypes associated with rodents are known to circulate in Europe: Puumala virus (PUUV), Dobrava/Belgrade virus (DOBV), Saarema virus (SAAV), Tula virus (TUUV) and Seoul virus (SEOV). The most pathogenic is DOBV, carried by Apodemus flavicollis, with fatality rate up to 10% [Avsic-Zupanc

John Wiley & SonsJournal of Medical Virology

John Wiley & Sons

Journal of Medical Virology