BACKGROUND
hepatitis a virus  is a small, non-enveloped hepatotropic virus classified into the hepatovirus genus within the picornaviridae family.
its genome consists of an approximately  <dig>  kilobase positive single-strand rna comprising a 5’ untranslated region , a single open reading frame  that encodes both structural and non-structural proteins, and a 3’ utr with a short poly tail.
there is only one serotype of hav.
genomic characterization of hav has been carried out mainly by sequencing of strains from different geographic regions of the world.
firstly, using a short fragment of the vp1/2a junction region, strains were classified in to seven genotypes on the basis of >15% nucleotide variation between isolates, and the subgenotypes with > <dig> % to <15% nucleotide variation  <cit> .
then, the complete genomic data indicated that genotypes ii and vii should be considered a single genotype, based upon the complete vp <dig> sequence .
so, by sequencing of the vp1/2a junction and the vp <dig> gene, three genotypes  divided in two subtypes  have been described for humans and three others  for primates  <cit> .
hav infection is the leading worldwide cause of acute viral hepatitis  <cit> .
there are an annual estimated of  <dig>  million cases of hepatitis a worldwide  <cit> .
optimal use of vaccination can significantly reduce the hepatitis a disease burden and the world health organization position on hepatitis a vaccines depend on the level of endemicity in countries.
in highly endemic countries, large-scale vaccination programmes are not recommended.
in countries of intermediate endemicity, large-scale childhood vaccination may be considered as a supplement to health education and improved sanitation.
and in regions of low endemicity, vaccination against hepatitis a is indicated for individuals with increased risk of contracting the infection such as travelers to areas of intermediate or high endemicity  <cit> .
hav’s geographical distribution is dependent on socioeconomic development and sanitation levels.
in areas with high and very high endemicity , where infections are mostly asymptomatic and epidemics are rare, 50% seroprevalence is reached between the ages of  <dig> and  <dig>  <cit> .
in areas with moderate endemicity , 50% seroprevalence is reached between the ages of  <dig> and  <dig> and epidemics can occur within the general population.
in areas with low endemicity , most of the population is still susceptible to hav, particularly people over 50 years old, and the risk of fulminant hepatitis is higher.
hav is transmitted mainly by the fecal-oral route, either by person-to-person contact or by ingestion of contaminated water and food, particularly shellfish, soft fruits and raw vegetables  <cit> .
hav is stable in the environment and is particularly resistant to disinfectants, heating, pressure and low ph  <cit> .
contamination may occur during growth in the field as well as during processing, storage, distribution or final preparation.
in developed countries, low incidence and low vaccine coverage have led to a high proportion of susceptible individuals, which creates a potential for expanded hepatitis a outbreaks when contaminated products are widely distributed  <cit> .
the development of sensitive, reliable techniques for the detection of hav in food and water samples contributes to the safety of these products  <cit> .
however, detection of hav on the basis of its infectivity is complicated by the absence of a reliable cell culture method and the low contamination levels of food samples.
hav detection is currently based on nucleic acid testing methods.
the international organization for standardization/technical specification   <dig> standard was published in the first half of  <dig> and will be published as iso standard methods after validation.
these protocols target the 5’utr which shows the lowest diversity across hav genotypes  <cit> .
currently, hav genotyping relies on amplification, sequencing and phylogenetic analysis of a portion of the viral genome.
however, these techniques are time-consuming and may lack sensitivity, particularly with food samples, where the level of contamination by enteric viruses is often very low.
alternative approaches for hav genotyping in complex samples  may help to better manage the risk.
indeed, although genotypes i and iii are the most frequently reported worldwide, hav genotypes and hav strains differ in their geographic distribution  <cit> ; strain genotyping can thus give clues to understanding food contamination routes.
currently, very few studies describe alternatives to sequencing for hav genotyping.
in recent years single-nucleotide polymorphism  genotyping has become an area of intense investigation and a valuable tool for diagnosing various pathologies.
various methods for snp detection have been reported including real-time pcr performed with primers and a probe spanning the snp site  <cit> .
the aim of this study was to develop a new approach for the subgenotyping of human hav based on six simplex snp genotyping rt-qpcr assays and to apply this approach to human clinical samples.
RESULTS
design of hav subtype rt-qpcr assays
the hav subtype rt-qpcr assays were designed to give subtype-specific amplification on the basis of snp differentiating the targeted subtype from the others.
in other words, at the snp position, the same nucleotide was found for all the subtypes except for the subtype of targeted hav.
consequently, different regions of hav genome were chosen given their subtype specificity and the absence of major nonspecific homologies on blast analysis.
moreover, degenerated bases were used to detect genetic variation within a given subtype .table  <dig>
genbank accession numbers for hav isolates
genotype
reference strain
genbank accession number
nucleic acid sequences were used to design primers and probes sets.
sequences of primers and probes in the direction 5’-3’
genotype
reference strain
sequence
position
the specific genotype snp is in bold.
probes are fam-bhq except hav 5’utr which is fam-mgb.
*: costafreda et al.
<cit> .
hav genome regions targeted for genotyping.
the different genomic regions used to identify each hav genotype are represented below the hav genome scheme.
sensitivity of subtype-specific rt-qpcr assays
the sensitivity of the simplex subtype–specific rt-qpcr assays was evaluated with serial 10-fold dilutions of in vitro transcribed rna for hav iia, iib, iiia and iiib and genomic rna for hav ia and ib.
from  <dig> ×  <dig> to  <dig> genome copies/assay for ia, ib, iia, iib, and iiib and from  <dig> ×  <dig> to  <dig> ×  <dig> genome copies/assay for iiia were tested.
as shown on table  <dig>  mean rt-qpcr efficiency, derived from the slope parameters, ranged from  <dig> % for iiia to 109% for ib.
r <dig> values were ≥ <dig> .
the limit of detection  obtained for ia, ib, iia and iiib was  <dig> genome copies/assay, whereas the lod of iib was  <dig> genome copies/assay and the lod of iiia was  <dig> genome copies/assay.
the lod of the consensus rt-qpcr assay  <cit>  was in the same range as that of the subtyping rt-qpcr assays at  <dig> genome copies/assay.table  <dig>
characteristics of rt-qpcr standard curves
genome copies/rt-qpcr assay
mean ct values +/− sd
hav ia
hav ib
hav iia
hav iib
hav iiia
hav iiib
hav 5'utr
<dig> x 107
<dig> x 106
<dig> x 105
<dig> x 104
<dig> x 103
<dig>  ±  <dig>
<dig> x 102
<dig>  ±  <dig>
<dig>  ±  <dig>
<dig>  ±  <dig>
<dig>  ±  <dig>
<dig>  ±  <dig>
<dig> x 101
<dig>  ±  <dig>
<dig> x 100
r2
parameters of rt-qpcr amplification curves obtained for hav detection by the rt-qpcr reference method and hav subgenotyping by rt-qpcr assays.
the limit of detection  has been defined as the lowest amount of hav detected in the three experiments and is shown in bold.
nd: not detected.
/ : not analyzed.
specificity of subtype-specific rt-qpcr assays
the specificity of the rt-qpcr assays was assessed by testing hav rna of each subtype at a concentration of  <dig> ×  <dig> genome copies/assay with all the subtype-specific rt-qpcr assays.
as shown on table  <dig>  detection of the specific target was observed for all assays with ct values comprised between  <dig> and  <dig>  consistent with assay sensitivity.
all but one were entirely specific.
the iia-specific assay occasionally allowed amplification of the iib target.
however, this non-specific iib amplification was not observed when as much as  <dig> ×  <dig> genome copies/assay of iib rna was tested in the presence of a low concentration of the specific iia target  .table  <dig>
specificity of subgenotyping rt-qpcr assays
set primers/probe
hav ia
hav ib
hav iia
hav iib
hav iiia
hav iiib
six subtyping rt-qpcr assays were tested with  <dig> x  <dig> genome copies/assay for all subtypes of hav in duplicate in three different experiments.
results are expressed as means cycle threshold  values ± standard deviations .
the number of positive ct values is given in parentheses.
fecal and serum samples analysis
human clinical fecal and serum samples were genotyped by sequencing the vp1/2a region, as described  <cit>  and provided by the nrc.
then, they were tested with the consensus rt-qpcr assay  <cit>  and with all the subtype-specific rt-qpcr assays separately .table  <dig>
stool samples analysis
stool
age/sex
transaminases iu/ml
travel
genotype by sequencing
hav genome copies/g of stool
difference of quantification between 5’utr and subtype rt-qpcr assays
difference of quantification between ia and iia subtype rt-qpcr assays
5’utr
ia
ib
iia
iib
iiia
iiib
each sample was tested with the reference rt-qpcr assay targeting the 5’utr of hav and the  <dig> genotype-specific rt-qpcr assays.
the subtyping results were compared with those obtained with sequencing by the nrc.
concentrations are given in genome copies per gram of stool.
nc = not communicated.
the difference of quantification between 5’utr and subtype rt-qpcr assays is calculated by the formula: ).
the difference of quantification between ia and iia subtypes by rt-qpcr assays is calculated by the formula: ).
serum samples analysis
serum
age/sex
transaminases iu/ml
travel
genotype by sequencing
hav genome copies/μl of serum
difference of quantification between 5’utr and subtype assays
difference of quantification between ia and ib subtype assays
5’utr
ia
ib
iia
iib
iiia
iiib
each sample was tested with the reference rt-qpcr assay targeting the 5’utr of hav and the  <dig> genotype-specific assays.
the subtyping results were compared with those obtained with sequencing by the nrc.
concentrations are given in genome copies per μl of serum.
nc = not communicated.
the difference of quantification between 5’utr and subtype rt-qpcr assays is calculated by the formula: ).
the difference of quantification between ia and ib subtypes by rt-qpcr assays is calculated by the formula: ).
four of the five stool samples and  <dig> out of the  <dig> sera were detected by a single subtype-specific assay that provided a subtype result consistent with vp1/2a sequencing.
the consensus and specific rt-qpcr assays gave similar results with differences of quantification that did not exceed  <dig>  log <dig> .
in stool sample  <dig> provided as a ia subtype by the nrc, subtype-specific assays detected both ia and iia rna, with a iia concentration  <dig>  log <dig> lower than that of the ia subtype .
similarly, in the  <dig> sera provided as the ia subtype by the nrc, subtype-specific assays detected both ia and ib rna, with ib concentrations  <dig>  to  <dig> log <dig> lower than ia.
a single discrepant result was observed for serum sample  <dig> provided as a ib subtype by vp1/ 2a region sequencing and identified as a ia subtype by the subtype-specific rt-qpcr assays .
in conclusion, the subgenotyping rt-qpcr assays allowed detecting 100%  of the clinical samples for the presence of hav.
in total,  <dig> %  of the clinical samples were found to correlate with the genotyping by sequencing the vp1/2a region.
furthermore, positivity for more than one genotype identified by sequencing appeared in  <dig> %  of the clinical samples and a subtype discrepancy in  <dig> %  of the clinical samples.
discussion
although hav has been shown to possess a single conserved antigenic neutralization site  <cit>  leading to a single serotype, hav strains isolated from different parts of the world have been classified into six genotypes , of which genotype i, ii, and iii can infect humans.
genotype i is the most prevalent worldwide, and subtype ia is more common than ib.
the other human genotypes are infrequent.
in areas of low endemicity such as the united states and western europe, ia dominates but all genotypes and subtypes have been reported  <cit> .
genotype ii isolates were originally identified in france in  <dig> and sierra leone in  <dig>  <cit>  and appear to be limited to west africa  <cit> .
genotype iii has been reported in many parts of the world  <cit>  but is prevalent in the indian subcontinent.
an increase in genotype iiia infections has been reported in korea, russia, estonia and in japan.
moreover, iiia and iiib co-circulate broadly with ia and ib strains  <cit> .
phylogenetic analysis is useful to trace back the geographical origin of a given strain and for tracking transmissions of hav.
accurate typing of hav from food samples could thus be helpful for transmission investigations.
however, hav typing from food samples by a classical sequencing approach is often impaired by the low contamination levels, and does not give access to potential contamination by several strains.
indeed, implicated items  in foodborne outbreaks can harbour a heterogeneous hav population that reflects the diversity of the viral strains circulating at the geographic location of item contamination  <cit> .
two commercial quantitative hav rt-qpcr assays have been described.
the detection limit was  <dig> tcid <dig> /ml for the roche kit and  <dig> tcid50/ml for the artus kit.
both kits have been found suitable for detection and quantification of hav but only the roche kit allowed the differentiation between genotype ia and ib after melting curve analysis  <cit> .
the present study introduces six rt-qpcr-based assays for specific molecular genotyping of hepatitis a virus.
to our knowledge, this is the first time that hav subtyping has been achieved by specific qpcr probes.
this subtype identification method can be implemented in diagnostic and research laboratories, avoiding post-pcr analysis and avoiding the problem of low viral loads in food samples.
all subtype assays were found suitable for quantification measurement for comparison with the data obtained with the reference rt-qpcr assay .
the minimal variations  observed for the quantification were potentially due to the differences in amplification efficiencies and calibration curves used.
most of the samples were correctly identified with regard to the genotype provided by vp1/2a sequencing.
in  <dig> samples , the specific rt-qpcr assay identified a major ia strain, the same one determined by vp1/2a sequencing, also in addition to a second subtype, present in a lower concentration.
the conventional genotyping used as a reference assay is a “golden standard assay”.
the design of hav subgenotyping rt-qpcr assays was based on snp in the probe associated with degenerated bases in the primers to enhance the specificity.
nevertheless, cross-reactivity could be only definitively excluded with the entire genome sequencing for the tested samples.
however, co-circulation of the subgenotypes ia and iiia has been reported in india  <cit>  and of ia, ib and iiia in korea  <cit> .
co-circulation of the subgenotypes ia and ib in south africa, south america, europe and the us and the existence of recombination events between subgenotypes have also been observed  <cit> .
indeed, hav exploits all known mechanisms of genetic variation to ensure its survival, including mutation and recombination  <cit> .
hav recombination was originally reported in cell culture  <cit> .
its extent in nature was appreciated only recently  <cit>  and it appears that recombination occurs along the entire length of the genome  <cit> .
the present finding from the stool sample of a patient who had not traveled abroad may be due to a co-infection by ia and iia subtypes.
indeed, hav ia is the dominant strain in france but iia strains have been isolated among french travelers returning from africa as well as from autochthonous cases  <cit> .
a co-infection rather than an event of recombination is suggested because of the huge difference in the concentration of the subtypes.
regarding these two signals, although non-specific amplification due to a very high viral load cannot be excluded, it should be noted that no iia amplification was detected from any of the  <dig> hav ia serum samples.
the discovery of a major ia signal, combined with a 10- to 100-fold lower ib signal in  <dig> sera from patients having traveled abroad  may suggest either an event of recombination or, more likely, a co-infection.
for these samples, the genome copy numbers determined by the 5’-utr assay was not the sum of those determined by subgenotyping rt-qpcr assays together which can be explained by the lack of accurate quantification or by cross reactivity.
as conventional sanger sequencing does not allow accurate identification of multiple species within a sample, the hypotheses could be investigated by cloning and sequencing or by next generation sequencing.
a single sample from a patient contaminated in morocco provided a discrepant result by specific rt-qpcr and sequencing; this sample may correspond to an ia/ib recombinant in the p <dig> region of the hav genome since ia-specific amplification targets the vp <dig> region  and sequencing targets the vp1/2a region .
the sequencing of this sample was attempted but has been unsuccessful may be because of the low viral load.
CONCLUSIONS
it was concluded that the rt-qpcr assays developed in this study are suitable tools for quantification of hav and subtype identification.
they need to be validated by testing a larger number of clinical, environmental and food samples.
conventional genotyping used as a reference assay is a “golden standard assay”, and the rt-qpcr assays described here could be recommended as an additional test to the conventional genotyping and for use in cases of failure of the conventional typing method.
they may be particularly useful for accurately tracing hav in samples with low-level contamination such as food matrices, but also can provide easy identification of a co-infection in human samples.

 hepatitis a virus is a small, non-enveloped hepatotropic virus classified into the hepatovirus genus within the picornaviridae family. its genome consists of an approximately kilobase positive single-strand rna comprising a 5’ untranslated region, a single open reading frame that encodes both structural and non-structural proteins, and a 3’ utr with a short poly tail. there is only one serotype of hav. genomic characterization of hav has been carried out mainly by sequencing of strains from different geographic regions of the world. firstly, using a short fragment of the vp1/2a junction region, strains were classified in to seven genotypes on the basis of >15% nucleotide variation between isolates, and the subgenotypes with > % to <15% nucleotide variation. then, the complete genomic data indicated that genotypes ii and vii should be considered a single genotype, based upon the complete vp sequence. so, by sequencing of the vp1/2a junction and the vp gene, three genotypes divided in two subtypes have been described for humans and three others for primates. hav infection is the leading worldwide cause of acute viral hepatitis. there are an annual estimated of million cases of hepatitis a worldwide. optimal use of vaccination can significantly reduce the hepatitis a disease burden and the world health organization position on hepatitis a vaccines depend on the level of endemicity in countries. in highly endemic countries, large-scale vaccination programmes are not recommended. in countries of intermediate endemicity, large-scale childhood vaccination may be considered as a supplement to health education and improved sanitation. and in regions of low endemicity, vaccination against hepatitis a is indicated for individuals with increased risk of contracting the infection such as travelers to areas of intermediate or high endemicity. hav’s geographical distribution is dependent on socioeconomic development and sanitation levels. in areas with high and very high endemicity, where infections are mostly asymptomatic and epidemics are rare, 50% seroprevalence is reached between the ages of and. in areas with moderate endemicity, 50% seroprevalence is reached between the ages of and and epidemics can occur within the general population. in areas with low endemicity, most of the population is still susceptible to hav, particularly people over 50 years old, and the risk of fulminant hepatitis is higher. hav is transmitted mainly by the fecal-oral route, either by person-to-person contact or by ingestion of contaminated water and food, particularly shellfish, soft fruits and raw vegetables. hav is stable in the environment and is particularly resistant to disinfectants, heating, pressure and low ph. contamination may occur during growth in the field as well as during processing, storage, distribution or final preparation. in developed countries, low incidence and low vaccine coverage have led to a high proportion of susceptible individuals, which creates a potential for expanded hepatitis a outbreaks when contaminated products are widely distributed. the development of sensitive, reliable techniques for the detection of hav in food and water samples contributes to the safety of these products. however, detection of hav on the basis of its infectivity is complicated by the absence of a reliable cell culture method and the low contamination levels of food samples. hav detection is currently based on nucleic acid testing methods. the international organization for standardization/technical specification standard was published in the first half of and will be published as iso standard methods after validation. these protocols target the 5’utr which shows the lowest diversity across hav genotypes. currently, hav genotyping relies on amplification, sequencing and phylogenetic analysis of a portion of the viral genome. however, these techniques are time-consuming and may lack sensitivity, particularly with food samples, where the level of contamination by enteric viruses is often very low. alternative approaches for hav genotyping in complex samples may help to better manage the risk. indeed, although genotypes i and iii are the most frequently reported worldwide, hav genotypes and hav strains differ in their geographic distribution; strain genotyping can thus give clues to understanding food contamination routes. currently, very few studies describe alternatives to sequencing for hav genotyping. in recent years single-nucleotide polymorphism genotyping has become an area of intense investigation and a valuable tool for diagnosing various pathologies. various methods for snp detection have been reported including real-time pcr performed with primers and a probe spanning the snp site. the aim of this study was to develop a new approach for the subgenotyping of human hav based on six simplex snp genotyping rt-qpcr assays and to apply this approach to human clinical samples.  design of hav subtype rt-qpcr assays the hav subtype rt-qpcr assays were designed to give subtype-specific amplification on the basis of snp differentiating the targeted subtype from the others. in other words, at the snp position, the same nucleotide was found for all the subtypes except for the subtype of targeted hav. consequently, different regions of hav genome were chosen given their subtype specificity and the absence of major nonspecific homologies on blast analysis. moreover, degenerated bases were used to detect genetic variation within a given subtype.table genbank accession numbers for hav isolates genotype reference strain genbank accession number nucleic acid sequences were used to design primers and probes sets. sequences of primers and probes in the direction 5’-3’ genotype reference strain sequence position the specific genotype snp is in bold. probes are fam-bhq except hav 5’utr which is fam-mgb. *: costafreda et al.. hav genome regions targeted for genotyping. the different genomic regions used to identify each hav genotype are represented below the hav genome scheme. sensitivity of subtype-specific rt-qpcr assays the sensitivity of the simplex subtype–specific rt-qpcr assays was evaluated with serial 10-fold dilutions of in vitro transcribed rna for hav iia, iib, iiia and iiib and genomic rna for hav ia and ib. from × to genome copies/assay for ia, ib, iia, iib, and iiib and from × to × genome copies/assay for iiia were tested. as shown on table  mean rt-qpcr efficiency, derived from the slope parameters, ranged from % for iiia to 109% for ib. r values were ≥. the limit of detection obtained for ia, ib, iia and iiib was genome copies/assay, whereas the lod of iib was genome copies/assay and the lod of iiia was genome copies/assay. the lod of the consensus rt-qpcr assay was in the same range as that of the subtyping rt-qpcr assays at genome copies/assay.table characteristics of rt-qpcr standard curves genome copies/rt-qpcr assay mean ct values +/− sd hav ia hav ib hav iia hav iib hav iiia hav iiib hav 5'utr x 107 x 106 x 105 x 104 x 103  ±  x 102  ±   ±   ±   ±   ±  x 101  ±  x 100 r2 parameters of rt-qpcr amplification curves obtained for hav detection by the rt-qpcr reference method and hav subgenotyping by rt-qpcr assays. the limit of detection has been defined as the lowest amount of hav detected in the three experiments and is shown in bold. nd: not detected. /: not analyzed. specificity of subtype-specific rt-qpcr assays the specificity of the rt-qpcr assays was assessed by testing hav rna of each subtype at a concentration of × genome copies/assay with all the subtype-specific rt-qpcr assays. as shown on table  detection of the specific target was observed for all assays with ct values comprised between and consistent with assay sensitivity. all but one were entirely specific. the iia-specific assay occasionally allowed amplification of the iib target. however, this non-specific iib amplification was not observed when as much as × genome copies/assay of iib rna was tested in the presence of a low concentration of the specific iia target.table specificity of subgenotyping rt-qpcr assays set primers/probe hav ia hav ib hav iia hav iib hav iiia hav iiib six subtyping rt-qpcr assays were tested with x genome copies/assay for all subtypes of hav in duplicate in three different experiments. results are expressed as means cycle threshold values ± standard deviations. the number of positive ct values is given in parentheses. fecal and serum samples analysis human clinical fecal and serum samples were genotyped by sequencing the vp1/2a region, as described and provided by the nrc. then, they were tested with the consensus rt-qpcr assay and with all the subtype-specific rt-qpcr assays separately.table stool samples analysis stool age/sex transaminases iu/ml travel genotype by sequencing hav genome copies/g of stool difference of quantification between 5’utr and subtype rt-qpcr assays difference of quantification between ia and iia subtype rt-qpcr assays 5’utr ia ib iia iib iiia iiib each sample was tested with the reference rt-qpcr assay targeting the 5’utr of hav and the genotype-specific rt-qpcr assays. the subtyping results were compared with those obtained with sequencing by the nrc. concentrations are given in genome copies per gram of stool. nc = not communicated. the difference of quantification between 5’utr and subtype rt-qpcr assays is calculated by the formula: ). the difference of quantification between ia and iia subtypes by rt-qpcr assays is calculated by the formula: ). serum samples analysis serum age/sex transaminases iu/ml travel genotype by sequencing hav genome copies/μl of serum difference of quantification between 5’utr and subtype assays difference of quantification between ia and ib subtype assays 5’utr ia ib iia iib iiia iiib each sample was tested with the reference rt-qpcr assay targeting the 5’utr of hav and the genotype-specific assays. the subtyping results were compared with those obtained with sequencing by the nrc. concentrations are given in genome copies per μl of serum. nc = not communicated. the difference of quantification between 5’utr and subtype rt-qpcr assays is calculated by the formula: ). the difference of quantification between ia and ib subtypes by rt-qpcr assays is calculated by the formula: ). four of the five stool samples and out of the sera were detected by a single subtype-specific assay that provided a subtype result consistent with vp1/2a sequencing. the consensus and specific rt-qpcr assays gave similar results with differences of quantification that did not exceed log. in stool sample provided as a ia subtype by the nrc, subtype-specific assays detected both ia and iia rna, with a iia concentration log lower than that of the ia subtype. similarly, in the sera provided as the ia subtype by the nrc, subtype-specific assays detected both ia and ib rna, with ib concentrations to log lower than ia. a single discrepant result was observed for serum sample provided as a ib subtype by vp1/ 2a region sequencing and identified as a ia subtype by the subtype-specific rt-qpcr assays. in conclusion, the subgenotyping rt-qpcr assays allowed detecting 100% of the clinical samples for the presence of hav. in total, % of the clinical samples were found to correlate with the genotyping by sequencing the vp1/2a region. furthermore, positivity for more than one genotype identified by sequencing appeared in % of the clinical samples and a subtype discrepancy in % of the clinical samples. discussion although hav has been shown to possess a single conserved antigenic neutralization site leading to a single serotype, hav strains isolated from different parts of the world have been classified into six genotypes, of which genotype i, ii, and iii can infect humans. genotype i is the most prevalent worldwide, and subtype ia is more common than ib. the other human genotypes are infrequent. in areas of low endemicity such as the united states and western europe, ia dominates but all genotypes and subtypes have been reported. genotype ii isolates were originally identified in france in and sierra leone in and appear to be limited to west africa. genotype iii has been reported in many parts of the world but is prevalent in the indian subcontinent. an increase in genotype iiia infections has been reported in korea, russia, estonia and in japan. moreover, iiia and iiib co-circulate broadly with ia and ib strains. phylogenetic analysis is useful to trace back the geographical origin of a given strain and for tracking transmissions of hav. accurate typing of hav from food samples could thus be helpful for transmission investigations. however, hav typing from food samples by a classical sequencing approach is often impaired by the low contamination levels, and does not give access to potential contamination by several strains. indeed, implicated items in foodborne outbreaks can harbour a heterogeneous hav population that reflects the diversity of the viral strains circulating at the geographic location of item contamination. two commercial quantitative hav rt-qpcr assays have been described. the detection limit was tcid /ml for the roche kit and tcid50/ml for the artus kit. both kits have been found suitable for detection and quantification of hav but only the roche kit allowed the differentiation between genotype ia and ib after melting curve analysis. the present study introduces six rt-qpcr-based assays for specific molecular genotyping of hepatitis a virus. to our knowledge, this is the first time that hav subtyping has been achieved by specific qpcr probes. this subtype identification method can be implemented in diagnostic and research laboratories, avoiding post-pcr analysis and avoiding the problem of low viral loads in food samples. all subtype assays were found suitable for quantification measurement for comparison with the data obtained with the reference rt-qpcr assay. the minimal variations observed for the quantification were potentially due to the differences in amplification efficiencies and calibration curves used. most of the samples were correctly identified with regard to the genotype provided by vp1/2a sequencing. in samples, the specific rt-qpcr assay identified a major ia strain, the same one determined by vp1/2a sequencing, also in addition to a second subtype, present in a lower concentration. the conventional genotyping used as a reference assay is a “golden standard assay”. the design of hav subgenotyping rt-qpcr assays was based on snp in the probe associated with degenerated bases in the primers to enhance the specificity. nevertheless, cross-reactivity could be only definitively excluded with the entire genome sequencing for the tested samples. however, co-circulation of the subgenotypes ia and iiia has been reported in india and of ia, ib and iiia in korea. co-circulation of the subgenotypes ia and ib in south africa, south america, europe and the us and the existence of recombination events between subgenotypes have also been observed. indeed, hav exploits all known mechanisms of genetic variation to ensure its survival, including mutation and recombination. hav recombination was originally reported in cell culture. its extent in nature was appreciated only recently and it appears that recombination occurs along the entire length of the genome. the present finding from the stool sample of a patient who had not traveled abroad may be due to a co-infection by ia and iia subtypes. indeed, hav ia is the dominant strain in france but iia strains have been isolated among french travelers returning from africa as well as from autochthonous cases. a co-infection rather than an event of recombination is suggested because of the huge difference in the concentration of the subtypes. regarding these two signals, although non-specific amplification due to a very high viral load cannot be excluded, it should be noted that no iia amplification was detected from any of the hav ia serum samples. the discovery of a major ia signal, combined with a 10- to 100-fold lower ib signal in sera from patients having traveled abroad may suggest either an event of recombination or, more likely, a co-infection. for these samples, the genome copy numbers determined by the 5’-utr assay was not the sum of those determined by subgenotyping rt-qpcr assays together which can be explained by the lack of accurate quantification or by cross reactivity. as conventional sanger sequencing does not allow accurate identification of multiple species within a sample, the hypotheses could be investigated by cloning and sequencing or by next generation sequencing. a single sample from a patient contaminated in morocco provided a discrepant result by specific rt-qpcr and sequencing; this sample may correspond to an ia/ib recombinant in the p region of the hav genome since ia-specific amplification targets the vp region and sequencing targets the vp1/2a region. the sequencing of this sample was attempted but has been unsuccessful may be because of the low viral load.  it was concluded that the rt-qpcr assays developed in this study are suitable tools for quantification of hav and subtype identification. they need to be validated by testing a larger number of clinical, environmental and food samples. conventional genotyping used as a reference assay is a “golden standard assay”, and the rt-qpcr assays described here could be recommended as an additional test to the conventional genotyping and for use in cases of failure of the conventional typing method. they may be particularly useful for accurately tracing hav in samples with low-level contamination such as food matrices, but also can provide easy identification of a co-infection in human samples. 
six subtype-specific rt-qpcr assays were developed for human hav. in food microbiology, this approach is complicated by the low contamination levels of food samples. to date, real-time reverse-transcription pcr has been one of the most promising detection methods due to its sensitivity, specificity and ability to deliver quantitative data in food samples, but it does not provide hav subtyping information. subtyping results concordant with sequencing analysis were obtained from 34/ clinical samples. these rt-qpcr assays may be particularly useful for accurately tracing hav in low-level contaminated samples such as food matrices but also to allow co-infection identification in human samples. hepatitis a virusrt-qpcr assaysgenotypingissue-copyright-statement© the author 2014 hav genotypes and subtypes differ in their geographic distribution and the incidence of hav infection varies considerably among countries, and is particularly high in areas with poor sanitation and hygiene. phylogenetic analyses are traditionally used in clinical microbiology for tracing the geographic origin of hav strains.
 the hepatitis a virus is the most frequent cause of viral hepatitis worldwide and is recognized as one of the most widespread foodborne pathogens. hav genotypes and subtypes differ in their geographic distribution and the incidence of hav infection varies considerably among countries, and is particularly high in areas with poor sanitation and hygiene. phylogenetic analyses are traditionally used in clinical microbiology for tracing the geographic origin of hav strains. in food microbiology, this approach is complicated by the low contamination levels of food samples. to date, real-time reverse-transcription pcr has been one of the most promising detection methods due to its sensitivity, specificity and ability to deliver quantitative data in food samples, but it does not provide hav subtyping information.  six subtype-specific rt-qpcr assays were developed for human hav. the limit of detection of hav was genome copies/assay for subtype iib, genome copies assay for ia, ib, iia and iiib and genome copies/assay for iiia. the specificity of the assays was evaluated by testing reference isolates and in vitro hav rna transcripts. no significant cross reactivity was observed. subtyping results concordant with sequencing analysis were obtained from 34/ clinical samples. co-infection with a minor strain of a different subtype was suggested in cases and a recombinant event in one case.  these rt-qpcr assays may be particularly useful for accurately tracing hav in low-level contaminated samples such as food matrices but also to allow co-infection identification in human samples. keywords hepatitis a virusrt-qpcr assaysgenotypingissue-copyright-statement© the author 2014 