Piotr Grabarczyk
email: pgrabarczyk@ihit.waw.pl

Aleksandra Kalińska

Mahmut Kara

Renata Wieczorek

Anna Ejduk

Ewa Sulkowska

Sydonia Golębiowska-Staroszczyk

Michal Matysiak

Sally A Baylis

Ewa Brojer

Sydonia Gołębiowska-Staroszczyk

Michał Matysiak

Identification and characterization of acute infection with Parvovirus B19 genotype 2 in immunocompromised patients in Poland

Keywords: parvovirus B19, variants, genotype 2, immunocompromised patients parvovirus B19, variants, genotype 2, immunocompromised patients

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.

Human parvovirus B19 (B19V) is distributed worldwide and is associated with a wide spectrum of diseases including: erythema infectiosum (Fifth disease) in children, arthropathy in adults; hydrops fetalis, intrauterine death or other complications during pregnancy; aplastic crisis in patients with increased erythropoiesis, anemia in immunocompromised or immunodeficient patients and occasionally hepatitis, vasculitis, myocarditis and others [START_REF] Young | Mechanisms of disease. Parvovirus B19[END_REF][START_REF] De Jong | Parvovirus B19 infection in pregnancy[END_REF].

In immunocompetent individuals, viral load rapidly increases during the first week of infection and may exceed 1 x 10 12 genome equivalents/ml. The humoral immune response plays a crucial role in viral clearance. In the following 7-14 days, viremia declines, B19-specific immunoglobulin M (IgM) appears and may persist for up to 6 months. Specific IgG is detectable by the third week following infection and remains for years. Viral DNA has been shown to persist in the circulation of immunocompetent individuals for several years accompanying the specific B19V IgG.

The virus may also persist in bone marrow, synovial membrane, skin and other tissues [Parsyan and Candotti 2007].

B19V has been grouped into three distinct genotypes [START_REF] Nguyen | Novel human erythrovirus associated with transient aplastic anemia[END_REF][START_REF] Nguyen | Identification and characterization of a second novel human erythrovirus variant, A6[END_REF][START_REF] Servant | Genetic diversity within human erythroviruses: identification of three genotypes[END_REF] and further sub-genotypes [START_REF] Toan | Phylogenetic analysis of human parvovirus B19, indicating two subgroups of genotype 1 in Vietnamese patients[END_REF]Parsyan et al., 2007;Corcoran et al., 2009]. [START_REF] Blümel | Characterization of parvovirus B19 genotype 2 in KU812Ep6 cells[END_REF][START_REF] Baylis | A proficiency testing study to evaluate laboratory performance for the detection of different genotypes of parvovirus B19[END_REF][START_REF] Baylis | Evaluation of different assays for the detection of parvovirus B19 DNA in human plasma[END_REF][START_REF] Baylis | Effect of probe binding mutations in an assay design to detect parvovirus B19: implications for the quantitation of different virus genotype[END_REF][START_REF] Hokynar | Detection and differentiation of human parvovirus variants by commercial quantitative real-time PCR tests[END_REF]]. In the present paper the results of a study on B19V polymorphism in patients with symptomatic B19V infection in Poland and the clinical course of non-genotype 1 infections are presented.

Materials and Methods

Patients and samples

30 patients infected with B19V were included in the study. They were selected from a group of sixty nine B19V DNA positive patients who were identified by testing of clinical samples from patients in hematological, obstetric and gynaecological wards.

B19V antibodies detection and DNA analysis

Testing for B19V-specific antibodies was performed using the recomWell Parvovirus B19 IgG/IgM ELISA (Mikrogen GmbH, Neuried, Germany) according to the manufacturer's instructions.

B19V DNA was studied by two alternative quantitative real time PCR methods, performed using the ABI Prism 7700 (Applied Biosystems, Singapore) or Corbett 3000 (Corbett Research, Sydney, Australia) platforms, respectively [START_REF] Aberham | A quantitative, internally controlled real-time PCR assay for the detection of parvovirus B19 DNA[END_REF][START_REF] Candotti | Identification and characterization of persistent human erythrovirus infection in blood donor samples[END_REF]. Both methods utilize TaqMan hydrolysis probes designed within the VP1 and the NS1 genes, respectively [START_REF] Aberham | A quantitative, internally controlled real-time PCR assay for the detection of parvovirus B19 DNA[END_REF][START_REF] Candotti | Identification and characterization of persistent human erythrovirus infection in blood donor samples[END_REF]. In the second method, additional primers and probes were IU/reaction and 20 IU/reaction for the respective VP1 and NS1 assays. Both methods detect genotypes 1-3; however the VP1-specific method, used until the end of 2006, has since been shown to under quantify genotype 3 [START_REF] Baylis | Effect of probe binding mutations in an assay design to detect parvovirus B19: implications for the quantitation of different virus genotype[END_REF].

Amplification was combined with one of two DNA extraction procedures: QIAamp DNA Blood Mini Kit (Qiagen, Hilden, Germany) or Nuclisens Extractor (Nuclisens automated isolation reagents, BioMerieux, Boxtel, the Netherlands). Both procedures were performed according to the manufacturer's instructions.

Sequencing and phylogenetic analysis

For genotype analysis, a ~1 kb region corresponding to NS1/VP1u was amplified by nested PCR [START_REF] Servant | Genetic diversity within human erythroviruses: identification of three genotypes[END_REF] and both strands were directly sequenced (ABI PRISM 3.1 Big DyeTerminator Cycle Sequencing Ready Reaction Kit, Applied Biosystems, Warrington, UK). Sequences were aligned using CLUSTAL W [START_REF] Chenna | Multiple sequence alignment with the CLUSTAL series of programs[END_REF]] and a topology tree was constructed using MEGA 4.0 software [START_REF] Tamura | MEGA4: Molecular Evolutionary Genetics Analysis (MEGA) Software Version 4.0[END_REF], by using the Jukes-Cantor parameter estimation to generate the nucleotide distance matrix. To evaluate the phylogenetic relevance, bootstrapping analysis with 1,000 replicates was performed by neighbour-joining and the Jukes-Cantor parameter. Erythrovirus sequences from the GenBank were used as reference sequences: genotype 1, the strain Au (M13178) [START_REF] Shade | Nucleotide sequences and genome organization of human parvovirus B19 isolated from the serum of a child during aplastic crisis[END_REF] and two strains from Vietnam (DQ225149 -subtype 1a and DQ357064 -subtype1b, respectively) [START_REF] Toan | Phylogenetic analysis of human parvovirus B19, indicating two subgroups of genotype 1 in Vietnamese patients[END_REF]; genotype 2, the strain A6 (AY044266) [START_REF] Nguyen | Identification and characterization of a second novel human erythrovirus variant, A6[END_REF] and the strain LaLi (AY064475) [START_REF] Hokynar | A new parvovirus genotype persistent in human skin[END_REF]; and the genotype 3a virus termed V9 (AX003421) [START_REF] Nguyen | Novel human erythrovirus associated with transient aplastic anemia[END_REF] and the genotype 3b virus D91.1 (AY083234) [START_REF] Servant | Genetic diversity within human erythroviruses: identification of three genotypes[END_REF].

To avoid contamination with DNA amplicons, precautions, which have been described previously, were used during all steps connected with DNA detection or analysis by PCR [START_REF] Kwok | Avoiding false positives with PCR[END_REF].

Results

The clinical and virological characterization of the patients are presented in the Table 1. The patients were immunocompromised (except for P1-3, P9, P11, P16, P17, P19, P21, P24 and P25), due to underlying illness, immunosuppressive treatment or chemotherapy. The samples studied originated from symptomatic individuals mostly during the acute phase of infection. Out of 28 patients, where B19V serology testing was available, sixteen samples were positive for both anti-B19V IgM and IgG, one for IgM alone, and in the case of 8 immunocompromised patients, anti-B19V was not detectable (Table 1). In 3 further individuals specific IgG was found. B19V DNA loads ranged from 10 2 to 10 11 IU/ml. For 17 of the patients, B19V infection markers were monitored for between 2 and 255 weeks. For all patients, where data for longer than 1 month were available, B19V DNA levels declined, however complete disappearance of the viral nucleic acid from plasma was not observed. Maximum period of observed infection in immunocompetent and immunocompromised patients, was 124 and 255 weeks respectively. In one case, where virus was monitored for 114 weeks, patient (P7) was treated with immunosupressed drugs and despite high doses of intravenous immunoglobulin (IVIG), high levels of B19V viremia and severe anemia were observed.

The majority of B19V strains sequenced were genotype 1. However, of 30 strains sequenced, two (6.7%) genotype 2 viruses were identified and were amongst the 19 for genotype 2. Comparing genotype 1 and genotype 2, diversity ranged between 7% and 8%. Strains P29 and P30 showed 98% nucleotide identity with genotype 2 reference strains (A6 and LaLi) and phylogenetic analysis confirmed the clustering of P29 and P30 with the genotype 2 reference strains (Figure 1). Twenty-seven of the strains grouped with the genotype 1a reference sequence, however one strain (P28) was located between the type 1a and 1b sub-groups. The GenBank accession numbers of the nucleotide sequences determined in the study are FN827057 to FN827097.

For some of the genotype 1 strains, high levels of identity were observed. Sample pairs P1 and P2 and P16, P17 were taken from pregnant women with hydrops fetalis and their fetuses by cordocentesis. In each case, the respective pairs from mother and fetus were identical to each other over the region sequenced. In addition, isolate P18 was identical to P16 and P17. Strain P18 originated from a patient hospitalized in a children's ward, in the same city and during same infection season as P16 and P17. Samples P20 and P21 were identified in a bone marrow donor and recipient, and were again identical to one another. Identical sequences or with single nucleotide changes (99.86-100% identity) were isolated from three patients (P6, P7 and P8) in the same children's ward who, prior to the onset symptoms of B19V infection, had been admitted to hospital at the same time due to hematological disease.

In Figure 2, the course of the two genotype 2 infections in patients P29 and P30 is presented. Patient P29 (Figure 2A), a 29 year-old woman, underwent kidney transplantation in November 2003 and received the graft from 46 year old woman. was treated with intravenous immunoglobulin (IVIG (Sandoglobulin) 24 g /day for 5 days). and the viral load fell (10 2 IU/ml plasma) and fever, rash and joint pain improved and gradual normalization of peripheral blood parameters was observed (7 th day: RBCs 2.35x10 6 /µL, WBCs 1.94x10 3 /µL, Plt: 190x10 3 /µL; 12 th day: RBCs 3.70x10 6 /µL, WBC 3.65x10 3 /µL, Plt 251x10 3 /µL, retikulocytes 4.21%). Both anti-B19V

IgM and IgG were detectable by the 15 th day of observation. By the 30 th day, until the end of the period of serology monitoring, only anti-B19V IgG was observed. Due to planned chemotherapy and hematopoietic stem cell transplantation, IVIG was administered twice more to consolidate treatment. No symptoms were observed, however DNA B19V remained detectable for a total of 85 days until the day of the patient's death caused by a leukemia relapse. Testing of the last available sample revealed 2 x 10 4 IU B19V DNA/ml.

Discussion

In the present study, where patients with clinical manifestation of B19V were studied, two genotypes of B19V were found in Poland. Genotype 1 was the predominant type, with some genotype 2 strains being detected less frequently. To the best of our knowledge, this is the first report of genotype 2 cases in Eastern

Europe. The data presented imply that in Poland genotype 2 could be responsible for a significant proportion of acute symptomatic infections, especially in immunocopromised patients. A similar case of genotype 2 infection has been described in a kidney transplant recipient by [START_REF] Liefeldt | Recurrent high level Parvovirus B19/genotype 2 viremia in a renal transplant recipient analysed by real-time PCR for simultaneous detection of genotype 1 to 3[END_REF]. The patient developed persistent viremia associated with chronic anemia and pure red cell aplasia combined with recurrent high viremic periods [START_REF] Liefeldt | Recurrent high level Parvovirus B19/genotype 2 viremia in a renal transplant recipient analysed by real-time PCR for simultaneous detection of genotype 1 to 3[END_REF]. These nosocomial cases -the two described in present study and the one described by [START_REF] Liefeldt | Recurrent high level Parvovirus B19/genotype 2 viremia in a renal transplant recipient analysed by real-time PCR for simultaneous detection of genotype 1 to 3[END_REF] occurred within a fairly close geographical area (i.e. Poland and variants may be epidemiologically restricted to certain geographical areas. In a previous report, [START_REF] Hübschen | Phylogenetic analysis of human parvovirus B19 sequences from eleven different countries confirms the predominance of genotype 1 and suggest the spread of genotype 3b[END_REF] found two single cases of genotype 3b B19V, but not genotype 2, in rash/fever patients from South-East Europe (Greece and Bulgaria) and suggested spread of genotype 3b from Africa to Europe.

In the study presented, the clinical spectrum associated with genotype 2 was found to be similar to that observed with genotype 1 infection. This was not unexpected, since the study aimed to detect variants in groups of patients with symptoms suggestive of B19V infection.The genotype 2 B19V infection in the kidney transplant recipient did not manifest clinically during pregnancy. However, it is likely to be due to the presence of protective antibodies at the start of pregnancy rather than lack of pathogenic potential in the fetus.

These observations together with problems in genotype 2 detection described previously [START_REF] Hokynar | Detection and differentiation of human parvovirus variants by commercial quantitative real-time PCR tests[END_REF][START_REF] Cohen | Genetic variants of parvovirus B19 identified in the United Kingdom: implications for diagnostic testing[END_REF][START_REF] Baylis | A proficiency testing study to evaluate laboratory performance for the detection of different genotypes of parvovirus B19[END_REF] demonstrate that proper diagnosis, particularly in the case of immunosupressed patients is very important. In addition, the data highlighted in the present study indicate that lack of detectable antibodies to B19V in immunocompromised patient is a frequent phenomenon, emphasizing the importance of B19V DNA analysis in patients [START_REF] Amiot | Spontaneous recovery from severe parvovirus B19 pure red cell aplasia in heart transplant recipient, as demonstrated by marrow culture[END_REF][START_REF] Oeda | Parvovirus B19 infection causing severe peripheral blood thrombocytopenia and persistent viremia[END_REF][START_REF] Liefeldt | Recurrent high level Parvovirus B19/genotype 2 viremia in a renal transplant recipient analysed by real-time PCR for simultaneous detection of genotype 1 to 3[END_REF].

Previously genotype 2 B19V DNA has been detected in 2.5% of plasmaderived clotting factors, more frequently in older concentrates than those manufactured more recently [START_REF] Schneider | Contamination of coagulation factor concentrates with human parvovirus B19 genotype 1 and 2[END_REF]. Genotype 2 B19V has been identified in blood donors and in plasma for fractionation albeit only rarely [START_REF] Blümel | Characterization of parvovirus B19 genotype 2 in KU812Ep6 cells[END_REF][START_REF] Koppelman | Parvovirus B19 genotype 1 and 2 detection with real-time polymerase chain reaction assays[END_REF][START_REF] Baylis | Standardization of nucleic acid amplification technique (NAT)based assays for different genotypes of parvovirus B19: a meeting summary[END_REF]. Genotype 2 strains have been found occasionally in patients in France [START_REF] Servant | Genetic diversity within human erythroviruses: identification of three genotypes[END_REF], England [START_REF] Cohen | Genetic variants of parvovirus B19 identified in the United Kingdom: implications for diagnostic testing[END_REF], Italy [START_REF] Corcioli | Tissue persistance of parvovirus B19 genotypes in asymptomatic persons[END_REF], Germany [START_REF] Liefeldt | Recurrent high level Parvovirus B19/genotype 2 viremia in a renal transplant recipient analysed by real-time PCR for simultaneous detection of genotype 1 to 3[END_REF][START_REF] Schneider | Simultaneous persistence of multiple genome variants of human parvovirus B19[END_REF],

United States [START_REF] Nguyen | Identification and characterization of a second novel human erythrovirus variant, A6[END_REF][START_REF] Wong | Prevalence of parvovirus B19 in liver tissue: no association with fulminant hepatitis or hepatitis-associated aplastic anemia[END_REF], Brazil [START_REF] Sanabani | Sequence variability of human erythroviruses present in bone marrow of Brazil patients with various parvovirus B19-related hematological symptoms[END_REF],

Vietnam [START_REF] Toan | Phylogenetic analysis of human parvovirus B19, indicating two subgroups of genotype 1 in Vietnamese patients[END_REF] and South Africa [START_REF] Corcoran | Genetic Variants of human parvovirus B19 in South Africa: co-circulation of three genotypes and identification of a novel subtype of genotype 1[END_REF] and represents a minority of strains. Although in a significant number of these reports viremia was the result of persistent infections characterized by the presence of a low viral load or DNA B19V detectable in certain tissues from older subjects [START_REF] Schneider | Simultaneous persistence of multiple genome variants of human parvovirus B19[END_REF][START_REF] Sanabani | Sequence variability of human erythroviruses present in bone marrow of Brazil patients with various parvovirus B19-related hematological symptoms[END_REF][START_REF] Toan | Phylogenetic analysis of human parvovirus B19, indicating two subgroups of genotype 1 in Vietnamese patients[END_REF][START_REF] Corcioli | Tissue persistance of parvovirus B19 genotypes in asymptomatic persons[END_REF]. Analysis of the proportion of B19V variants in tissues in relationship to patients' age led to the proposal that genotype 1 and 2 circulated widely and with equal frequency in Northern and Central Europe, from the 1930s until the 1950s. However, genotype 2 appears to have disappeared thereafter, as confirmed by patient sera collected during the 1980s and 1990s [START_REF] Norja | Bioportfolio: lifelong persistance of variant and prototypic erythrovirus DNA genomes in human tissue[END_REF]. [START_REF] Norja | Rapid sequence change and geographical spread of human parvovirus B19: comparison of B19 virus evolution in acute and persistent infection[END_REF] suggested that the infrequent number of cases of genotype 2 infections recorded in Europe may have arisen by the transmission of reactivated virus from persistently infected individuals rather than by the continued low-level circulation of this genotype in the community.

In the case of the kidney transplant recipient described in present paper, no data are available regarding blood transfusion, however transmission of persistent genotype 2 virus from the kidney donor (a 46 year old woman) cannot be excluded as it was described previously by Yango and colleagues [2002]. The leukemia patient infected with genotype 2, had not been transfused for at least for two months prior to the onset of infection and this case may suggest current circulation of the variant virus in the community, and not the effect of reactivation of persistent virus infection from the blood or the graft from the donor.

In present study we observed persistent viremia in both immunocompetent and immunocompromised patients, lasting at least ~2.5 and ~5 years, respectively. Similar observations have been reported previously [START_REF] Tang | Dose-by-dose virological and hematological responses to intravenous immunoglobulin in an immunocompromised patient with persistent parvovirus B19 infection[END_REF]Liefeldt et al.2005;Wąsak-Szulkowska et al. 2007;[START_REF] Dobec | Prolonged parvovirus B19 viremia in spite of neutralizing antibodies after erythema infectiosum in pregnancy[END_REF][START_REF] Lefrère | Persistent B19 infection in immunocompetent individuals: implications for transfusion safety[END_REF]] and the probable causes of such infections have been reviewed by Parsyan and Candotti [2007]. In case of kidney graft recipient infected with genotype 2 B19V it seems that lack of viral clearance was mainly connected with immunosuppressive therapy impairing the humoral immune response. Consequently, no seroconversion was observed. Similar prolonged infection with B19V genotype 2, lasting ~2.5 years, was observed previously in a renal transplant recipient, with levels 4.2 x 10 11 IU/ml of B19V DNA and severe anemia [START_REF] Liefeldt | Recurrent high level Parvovirus B19/genotype 2 viremia in a renal transplant recipient analysed by real-time PCR for simultaneous detection of genotype 1 to 3[END_REF]].

In the present study transmission of certain genotype 1 stains could be identified, not only from mother to fetus, but the same strain was detected in a bone marrow donor and recipient (clinical details were described previously by [START_REF] Wąsak-Szulkowska | Pure red cell aplasia due to parvovirus B19 infection transmitted probably through hematopoietic stem cell transplantation[END_REF]), in patients from a children's hematological ward and even in patients residing in the same region during the same infection season. These results are in agreement with previous reports where data were presented, concerning strains obtained from acute-phase serum samples of patients with erythema infectiosum and transient aplastic crisis, the B19V viruses circulating during the particular outbreak showed high levels of identity up to 100% [START_REF] Erdman | Genetic diversity of human parvovirus B19: sequence analysis of the VP1/VP2 gene from multiple isolates[END_REF][START_REF] Hemauer | Sequence variability among different parvovirus B19 isolates[END_REF]. Since seasonal outbreaks of B19V occur which cause epidemics every few years, there is the potential for rapid changes in the epidemiology of the virus. It is possible that similar to the situation for genotype 1

Figure 2 .

 2 Figure 2. Course of B19V genotype 2 infection in two immunosupressed women: A) renal transplant patient with pure red cell aplasia and B) leukemia patient with fever, rash, joint aches and anemia at the time of B19V diagnosis. Virological markers are indicated on the figure: viral loads (▲-▲) and antibody status. Levels of hemoglobin are also shown (•--•). In panel A, the transplant recipient, the change in the immunosuppressive regime is shown (↓); the time when the patient was pregnant is indicated. In panel B, the leukemia patient, three rounds of IVIG treatment (Sandoglobulin 24g a day for 5 successive days) are shown (↓↓↓↓↓). 173x212mm (96 x 96 DPI)

  Testing was performed between 2004 and 2008 in the Molecular Biology Laboratory at Institute of Haematology and Transfusion Medicine (IHBT). Samples were selected for further analysis based upon the availability of sufficient volumes of plasma. The results of the additional analyses are reported below. Nineteen of the patients studied were immunocompromised. The study was approved by the Institute of Haematology and Transfusion Medicine Ethics Committee, Warsaw, Poland.

  detection of CCR5 in plasma as an internal control. Amplification was carried out using TaqMan Universal PCR Master mix (Roche Branchbur, New Jersey, USA) in volume of 25 µl including 10 µl of template. The 100% detection limit is 5

  10.5%). Variability of a ~700 bp region of the genome (spanning the NS1/VP1u junction) ranged from 0 to 2% for genotype 1 and was 2%

8

  Four months later she developed pure red cell aplasia (peripheral blood cell count (RBC) 2.67x10 6 /µL) had with very low levels of hemoglobin (Hb) and reticulocyte count (7.6g/dL and 0.5%, respectively). The patient had a high viral load (~10 10 IU B19V DNA/ml) and was positive for anti-B19V IgM and IgG. Bone marrow examination revealed depletion of most erythroid elements. Based on the result of B19V testing, a change of immunosupression treatment from tacrolimus to cyclosporin was introduced and viremia fell (10 7 IU B19V DNA/ml) and hematological parameters normalized (Hb >10.2 g/dL and RBCs >3.6x10 6 /µL). During the next few months, B19V DNA levels remained around 10 5 IU/ml and infection was asymptomatic. After 10 weeks of B19V infection the woman became pregnant; ultrasound examination of the fetus failed to reveal any signs of B19V infection. The baby was subsequently delivered at full term and in good health. No episodes of symptomatic reactivation were later observed in the mother. In this case B19V DNA was detected for 255 weeks (5 years). Both anti-B19V IgM and IgG were detected in plasma of the patient throughout the five year period. The prolonged infection and atypical seroconversion was probably the result of the immunosuppressive therapy.In Figure2B, the second case of genotype 2 infection in an 18 year old woman is presented. In January 2008, the patient was diagnosed with T-lymphoblastic leukemia. She was treated according to PALG-4 2002 protocol and later reinduction protocol FLAM, morphology normalized in March 2008. On the 10 th May she was hospitalized due to high fever reaching 40 o C, rash, aching joints and joint edema.Pancytopenia was observed by blood examination: white blood cells (WBCs) 0.8 x10 3 /µL, RBCs 2.77 x10 6 /µL, Hb 9.1 g/dl, and platelets (Plt) 48 x10 3 /µL. Examination of the bone marrow revealed reticulopenia (reticulocytes 0.08%). Viral loads were 4.6x 10 9 IU B19V DNA/ml and both anti-B19V IgM and IgG were detected. The patient

  ) and may favor the hypothesis that the circulation of B19V

Figure1.Figure 2 .

 2 Figure1. Phylogenetic analysis of the B19V strains identified in the

Table 1 .

 1 Clinical and virological characterization of B19V DNA positive samples.

	B19V, epidemic spread of genotype 2 could occur in the future in Eastern Europe or
	in other areas, resulting in reemergence of this B19V variant.
	In conclusion, in the present study two cases of symptomatic genotype 2
	infection in immunocompromised patients by prospective testing. Such cases

John Wiley & SonsJournal of Medical Virology

Acknowledgments

The authors thank Anna Maria Eis-Hübinger for critical reading of the manuscript and helpful discussions.

John Wiley & Sons

Journal of Medical Virology

Conflict of interest

The authors declare no conflict of interest.


E Koorambas
email: elias.koor@gmail.com

On the geometric unification of gravity and dark energy

Keywords: number(s): 11.25.Mj, 95.36+x, 04.30.W Alternative theories of gravity, Dark energy, Gravity waves Contents

In the framework of Finslerian geometry, we propose a geometric unification between traditional gauge treatments of gravity, represented by metric field, and dark energy, which arises as a corresponding gauge potential from the single SU (2) group. Furthermore, we study the perturbation of gravity waves caused by dark energy. This proposition may have far reaching applications in astrophysics and cosmology.

Introduction

Observations of type Ia supernovae and of large-scale structure (LSS), in combination with measurements of the characteristic angular size of fluctuations in the cosmic microwave background (CMB)( [1], [2], [3], [4], [START_REF] Filippenko | Measuring and Modeling the Universe[END_REF], [6], [7], [8], [9], [10]) provide evidence that the expansion of the Universe is accelerating. This acceleration is attributed to the "dark energy", a hypothetical energy with negative pressure ( [1], [2], [3], [4], [START_REF] Filippenko | Measuring and Modeling the Universe[END_REF], [6]). Evidence for the presence of a dark energy is also provided by an independent, albeit more tentative, investigation of the integrated Sachs-Wolfe (ISW) ( [11]). The dark energy may result from Einstein's cosmological constant (which has a phenomenally small value); from evolving scalar fields ( [12]); and from a weakening of gravity in our 3 + 1 dimensions by leaking into the higher dimensions, as required in string theories ( [13]). These explanations may have crucial borader implications on fundamental physics. This has stimulated further efforts to confirm the initial results on dark energy, test possible sources of error, and extend our empirical knowledge of this newly discovered component of the Universe.

The gauge theory of gravity is based on the gauge principle and was suggested immediately after the formulation of the gauge theory ( [14], [15], [16], [17], [18], [19], [20], [21], [22], [23]). In the traditional gauge treatment of gravity, the Lorentz group is localized and the gravitational field is not represented by gauge potential, but by a metric field ( [18], [20]).

In this paper we use the framework of Finslerian geometry ( [24], [START_REF] Matsumoto | Foundation of Finsler Geometry and Special Finsler Space[END_REF], [START_REF] Stavrinos | Deviation of geodesic in the gravitational filed of Finslerian spacetime[END_REF], [START_REF] Stavrinos | Finslerian Metric based on the Gravitensional and electromagnetic Fields[END_REF], [START_REF] Antonelli | The Theory of Sprays and Finsler space with Application in Physics and Biology[END_REF], [START_REF] Beil | The Finsler Geometry and a Unified Field Theory[END_REF]) to propose a geometric unification between traditional gauge treatment of gravity, represented by the metric field g  , and dark energy. Which appears as a corresponding gauge potential B  , arising naturally from the gauge treatment of the single SU (2) group ( [30], [31], [32]).

We demonstrate that the dark energy would result naturally as a geometric effect of Finsler space, rather than being an additional suggestion. Furthermore, we study the dark energy perturbation of gravity wave, and discuss some wider potential applications of this in astrophysics and cosmology.

On the Finslerian geometric unification of gravity and dark energy

In the framework of a Riemannian approach, where two nearby particles are subject to the traditional gravitational field g  (free-falling), the Equation of Deviations of Geodesics (EDG) takes the form:

kk kk D n d x B u u B u ds G ds          (2)
where B  is the dark energy field strength:

,,

B B B

     ; and B  is the dark energy gauge potential arising from the single SU (2) group [30]. Equation (2) thus modifies equation (1) in the general form:

2 2 Dn R n n n ds           (3)
where

DB u B B u ds                
; and  is a constant 3) is reduced to (1), where only the gravitational field is present.   also governs the relative acceleration between two nearby particles in the flat space with

0 a R    .
From the above we conclude than Riemannian geometry does not provide a sufficient framework for the geometric unification between gravity and the vector dark energy. The disadvantage of Riemannian geometry is that the equation of motion of a particle subject to the action of a gravitational and dark energy field doesn't occur physically from the geometry of space-time and it is necessary to be imposed as an independent axiom. Riemannian geometry can be extended through the introduction of the Finsler space [START_REF] Matsumoto | Foundation of Finsler Geometry and Special Finsler Space[END_REF]. The metric function of the Finsler space is given by:

    , F x V g x V V B V        ([29]), (4)
where g  is the Riemannian metric tensor and B  is the dark energy vector potential. The metric f  of Finslerian space [START_REF] Beil | The Finsler Geometry and a Unified Field Theory[END_REF] is given by

22 1 2 F f VV      (5) f g h     , (6)
where g  is the Riemannian metric tensor and h  the metric tensor. The latter is given by

  2 2 sl sl h V g B B B V B r             , (7)
where

1 16 G    , k g x x      , ( 8
) 1 k r g g g x x          ,     1 2 ij ji ij a a a  (9)
A space endowed with the metric tensor ( 6) is called a Randeres space [START_REF] Beil | The Finsler Geometry and a Unified Field Theory[END_REF].

The presence of the dark energy component h  in space-time causes the isotropy of space to break down. The geodesic equation for this space is:

  2 , 0 dx xx ds        (10)     2, x x x x x B x                    (11) From   the connection coefficient    of this space can be derived   
, by analogy with the Berwald connection coefficient [START_REF] Antonelli | The Theory of Sprays and Finsler space with Application in Physics and Biology[END_REF]. By analogy to the Berwald curvature tensor [START_REF] Antonelli | The Theory of Sprays and Finsler space with Application in Physics and Biology[END_REF], we may associate with connection coefficient    a curvature tensor.

i i i hjk hjk hjk RB     , ( 12
)
where

i hjk R
is the Riemannian curvature tensor that came from the metric g  , and i

hjk B is given by

                1 3 1 2 i i m i hjk h jk h m k j h j k i m i i h h m h k j j k j k mi hm j B B B g B B B B u B x g B u B x u u B                   (13)
where

ij i g u   , k g x x      and     2 ij ji ij t t t  . ( 14
)
The equation of geodesic deviation is given by

  2 ,0 i ij j i z H x x z u      , ( 15
)
where i i h k j

hjk xx      , j z is the deviation vector and x  is the tangent vector. As i

hjk   has a part independent of velocity m x , we have the relation

          1 3 0 i m i i h h m h k j j k j k mi hm j u B x g B u B x u u B                (16)
By equation ( 16) equation ( 12) becomes

        1 2 i i i n i i hjk hjk h jk h m k j h j k H x R B B g B B B B     (17)
By equation (17) we derive the action of the system

  i hj mn hjk mn S H x g R B B     (18)
Action (18) yields the field's equations of gravity and dark energy, respectively, as follows:

1 8 2 ij ij ij R g R T   (19)
()

m n n m g B J    (20)
For equations ( 12) and ( 15) we observe that the deviation vector j z has two terms: a pure gravitational deviation, represented by i hjk R in the curvature tensor equation ( 12), which we would observe if there was no dark energy field, and the admixture of gravitational and dark energy deviations, represented by i hjk B in the curvature tensor equation (12). We examine the following cases: (21) where the deviation equation ( 15) becomes a Riemannian one.

For 0 i hjk B  we have 2 2 ( , ) 0 i i i h k hjk z R x x x x z u     
  3 11 // 22 i k l l k l m n m n k m n m n k l H g x B u B u u x B mn          (22)
The last relation shows that dark energy field is incorporated in the geometry of space. The second part i hjk B of the full curvature i hjk H  equation ( 12) describes the dark force that two freely falling particles of masses 1 m and 2 m would exercise on each other. In such a case, the dark force would result naturally as a geometrical effect and it would not be necessary for us to impose it in addition.

i i i h k hjk z B x x x x z u      (23)
In this case the first term of the Randers tensor corresponds to the Lorenz metric. The metric function ( , ) F x x  can, then, be expressed in the form:

( , ) ( ) i i F x x n x x B x x         , 1 16 G    (24)
This metric function is interesting for a possible linear theory caused by the dark energy field.

The dark energy perturbation of gravity wave

As an extension of the theory of gravitational waves described by General Relativity, we introduce a Finslerian metric, representing the Finslerian perturbation of Riemannian metric( [START_REF] Stavrinos | Deviation of Geodesics and Gravitational Waves in Finsler Space[END_REF], [START_REF] Balan | Weak Gravitational Models Based on Beil Metrics[END_REF], [START_REF] Balan | Finslerian (a, b)-Metrics in Weak Gravitational Models[END_REF])

( , ) ( ) ( , ) f x y g x x y      , | | 1   (25)
Where g  is the Riemannian metric tensor and ( , )

xy  
is the Finslerian perturbation to the Riemannian metric tensor. Μetric tensor [START_REF] Matsumoto | Foundation of Finsler Geometry and Special Finsler Space[END_REF] can be called a post Riemannian metric tensor [START_REF] Ikeda | Advanced Studies in Applied Geometry[END_REF].

Here, the Finslerian perturbation of Riemannian metric represents the dark energy perturbation of the gravity wave. This observation invites us to consider a Finslerian manifold, whose metric function contains two masslesss dark energy fields with 4-pontensioal vectors, (1) i B and ( 2 ) i B

, in the following form:

      (1) ( 2 )
, ( , )

i j i ij i i F x V g x V V B B V x V       , (26)
where

/ ii V dx ds  is a 4-velocity of a particle,  is a constant, (2) (1) i i BB  
is the interaction term of two dark energy fields ,  is a constant, and ( , )

xV  is an homogeneous function of 1 st degree, assumed to be scalar in the Finslerian manifold [START_REF] Matsumoto | Foundation of Finsler Geometry and Special Finsler Space[END_REF].

The last term of equation ( 26) corresponds to the gravitational field induced by the interaction between the dark energy fields. It contains the information of the gravitational field caused by the interaction of the dark energy field. This gravitational field affects the motion of every physical object in space-time. Applying equation [START_REF] Filippenko | Measuring and Modeling the Universe[END_REF] to equation ( 26) we obtain the following metric tensor for this field:

ij ij ij f g h   , | | 1   , (27)
where g  is the Riemannian metric tensor corresponding to the gravity wave perturbation ( [START_REF] Stavrinos | Deviation of Geodesics and Gravitational Waves in Finsler Space[END_REF], [START_REF] Balan | Weak Gravitational Models Based on Beil Metrics[END_REF], [START_REF] Balan | Finslerian (a, b)-Metrics in Weak Gravitational Models[END_REF]), and h  describes the interactions between the gravity wave and dark energy, and dark energy to itself.

| | 1 g   ) to Minkowski metric ( 1, 1, 1, 1) n diag       ([

 

2 ' ' 2 ' 22 2 2 ( ) 2 ( ) ( ) ' sl s i j i j l ij s s i j i j ij i l ij ij h V g B B B V B r x g B h VB                              (28)
i i i B B B and 2 1 ' 2 ij ij     . (1) ( 2 )

Conclusion

The equation of deviations of geodesics in Finsler space allows incorporation of dark energy in the geometry of space. We demonstrate that the dark energy would result naturally as a geometric effect and it would not necessary for us to impose it in addition. In a way the geometric unification between gravity and dark energy massless vector field is achieved. We also find that, whenever the dark energy component h  is present in space-time, the isotropy of space breaks down. In the framework of Finsler space, we also predict a dark energy perturbation of gravity waves.

  derivative; and R   is the Riemann tensor.The equation of motion of two nearby particles subjected to the action of the massless vector B-field of dark energy is obtained by introducing the Lorentz term into the geodesics equation(1) and replacing the four-acceleration

  describes the external interaction between two nearby mass particles due to dark energy. For 0  , relation (

  the geodesic deviations are governed by the dark energy and relation(15) is reduced to:



  denotes the partial differentiation with respect to i V ,


Angela Pista
email: angela.pista@insa.min-saude.pt

Nuno Verdasca

Ana Oliveira

Clinical Performance of the CLART Human Papillomavirus 2 Assay Compared with the Hybrid Capture 2 Test

Keywords: HPV, CLART Human Papillomavirus 2, HC2, screening, genotyping

come

95% CI: 64.3-77.5). When all 17 HR-HPV types were considered, CLART showed a clinical sensitivity of 96.9 % (95% CI: 93.8-98.5) and a clinical specificity of 71.9% (95% CI: 64.9-78.0).

In conclusion, the CLART assay is efficient, sensitive, reproducible, and has a similar performance to HC2 for cervical intraepithelial neoplasia grade 2 or worse. Furthermore, this assay has the advantage of detecting and genotyping 35 HPV types by a single test, which can provide additional information on the predictive value of infection with HR-HPV.

Keywords:

HPV; CLART Human Papillomavirus 2; HC2; screening; genotyping

INTRODUCTION

Epidemiological studies have established that certain human papillomavirus (HPV) genotypes are related etiologically to cervical cancer development [START_REF] Koutsky | A cohort study of the risk of cervical intraepithelial neoplasias grade 2 or 3 in relation to papillomavirus infection[END_REF][START_REF] Schiffman | Epidemiologic evidence showing that human papillomavirus infection causes most cervical intraepithelial neoplasia[END_REF][START_REF] Bosch | Prevalence of human papillomavirus in cervical cancer: a worldwide perspective[END_REF]. More than 120 HPV types have been identified, of which approximately 40 can infect the mucosa of the genital tract. According to the oncogenic potential, these HPV types can be classified into high-risk (HR), associated with premalignant lesions and cervical cancer, and low-risk (LR), found mainly in benign lesions [START_REF] Munoz | Epidemiological classification of human papillomavirus types associated with cervical cancer[END_REF][START_REF] Bernard | Classification of papillomaviruses (PVs) based on 189 PV types and proposal of taxonomic amendments[END_REF]. Most infections will clear spontaneously, but persistent infection with HR-HPV is a strong predictor of the development of high-grade cervical intraepithelial neoplasia (CIN) and cancer of the cervix uteri [START_REF] Nobbenhuis | Relation of human papillomavirus status to cervical lesions and consequences for cervical-cancer screening: a prospective study[END_REF][START_REF] Kjaer | Type-specific persistence of high risk human papillomavirus (HPV) as indicator of high grade cervical squamous intraepithelial lesions in young women: population-based prospective follow-up study[END_REF].

In areas where the Pap smear is the primary screening method, the development of premalignant lesions may be attributed to the low reproducibility and sensitivity of the Papanicolau test [START_REF] Renshaw | Measuring sensitivity in gynaecologic cytology: A review[END_REF]. Several studies [START_REF] Bosch | Chapter 1: human papillomavirus and cervical cancer-burden and assessment of causality[END_REF][START_REF] Cuzick | Management of women who test positive for high-risk types of human papillomavirus: The HART study[END_REF][START_REF] Cuzick | Overview of human papillomavirus-based and other novel options for cervical cancer screening in developed and developing countries[END_REF][START_REF] Bulkmans | Human papillomavirus DNA testing for the detection of cervical intraepithelial neoplasias grade 3 and cancer: 5-year follow-up of a randomised controlled implementation trial[END_REF][START_REF] Szarewski | Comparison of predictors for high-grade cervical intraepithelial neoplasia in women with abnormal smears[END_REF] have shown that the combined use of cytology and HPV DNA testing in women above 30 years old can improve the sensitivity and the negative predictive value (NPV) of screening. HPV testing can also provide the reassurance of extended intervals between screenings, and can be costeffective for the detection of high-grade lesions in women with equivocal cytological abnormalities.

Indeed, recent studies [START_REF] Castle | Human papillomavirus type 16 infections and 2-year absolute risk of cervical precancer in women with equivocal or mild cytologic abnormalities[END_REF][START_REF] Lai | Role of human papillomavirus genotype in prognosis of early-stage cervical cancer undergoing primary surgery[END_REF] have shown that a single positive result for either HPV 16 or 18 has a high predictive value for CIN grade 2 or worse (≥CIN2). Several HPV assays with various levels of sensitivity and specificity have been made available recently. However, clinical validation is required before these tests can be used as a "stand-alone" method in cervical cancer screening programs. Tests with significantly higher sensitivity for the detection of HPV DNA than the Hybrid Capture 2 test (HC2) (Qiagen, Hilden, Germany) could detect latent infections that are irrelevant clinically, which may lead to overtreatment of women [START_REF] Snijders | The clinical relevance of human papillomavirus testing: relationship between analytical and clinical sensitivity[END_REF][START_REF] Meijer | Guidelines for human papillomavirus DNA test requirements for primary cervical cancer screening in women 30 years and older[END_REF]. Recently, an assay based on PCR and hybridization, the CLART Human Papillomavirus 2 (CLART) (Genomica, Madrid, Spain) was developed for the detection and genotyping of 35 HPV types (20 HR-HPV and 15 LR-HPV), in single or multiple infections.

The aim of this study was to evaluate the performance of the CLART assay in comparison with the HC2 test in women for whom histological results were available. Further, the clinical performance was evaluated for each histological grade, using a clinical cut-off of CIN grade 2 or worse (≥CIN2). All samples were tested by both the CLART and the HC2 assays. Discrepant results were analysed further using the PapilloCheck HPV genotyping system (Greiner Bio-One, Frickenhausen, Germany).

To assess the reproducibility of the CLART assay, 75 samples were retested randomly twice by two operators.

Each HPV test was carried out independently of each other according to the manufacturer's instructions. DNA was isolated from 1 ml of cellular suspension by using the NucliSENS easyMAG (BioMerieux, Boxtel, The Netherlands) system, as specified in the manufacturer's instructions. Nucleic acids were eluted in a final volume of 100 µl and stored at -20 ºC until use for PCR analysis.

CLART Human Papillomavirus 2

This methodology uses biotinylated primers that amplify a fragment a 450 bp fragment within the HPV L1 region. Co-amplification of an 892 bp region of the CFTR gene and a 1202 bp fragment of a transformed plasmid provides a control to ensure DNA extraction adequacy and PCR efficiency.

Amplicons are detected by hybridization in a low-density microarray containing triplicate DNA probes specific to 35 types (HPV 6,11,16,18,26,31,33,35,39,40,42,43,44,45,51,52,53,54,56,58,59,61,62,66,68,70,71,72,73,81,82,83,84,85,and 89). Semi-quantitative results can be obtained in an automatic reader. The HC2 test is a sandwich capture molecular hybridization assay that uses a signal amplification detection method based on chemiluminescence. Thirteen HR-HPV types (HPV 16,18,31,33,35,39,45,51,52,56,58,59,and 68) can be detected with this test. The resultant DNA:RNA hybrids are captured on a microplate, and the emitted light is measured in a luminometer as relative light units (RLU). Samples were considered as positive if the ratio RLU/cut-off was greater than 1.0 (equivalent to 1.0 pg HPV DNA/ml). All the cut-offs between 1 and 2.5 were retested and all were greater than 1.0 RLU/cut-off (data not shown).

PapilloCheck HPV Genotyping

The PapilloCheck system allows the genotyping of 24 HPV types (HPV 6,11,16,18,31,33,35,39,40,42,43,44/55,45,51,52,53,56,58,59,66,68,70,73 and 82). This assay uses a multiplex PCR that amplify a 350 bp fragment of the E1 gene of HPV. Co-amplification of the human gene ADAT1 is used as an internal control. The hybridization is performed on a microarray chip, which is automatically scanned and analysed using the CheckScanner™ at both 532 nm and 635 nm and the CheckReport software, respectively.

Statistical Analysis

For comparative purposes with the HC2 test, results of the CLART assay were considered as positive if one of the following 13 HR-HPV types (CLART-13 HR-HPV) was present: HPV 16,18,31,33,35,39,45,51,52,56,58,59, and 68. In addition, the evaluation was performed also considering 17 (probably) HR-HPV types (CLART-17 HR-HPV) (HPV 16,18,31,33,35,39,45,51,52,53,56,58,59,66,68,73 and 82) [START_REF] Bernard | Classification of papillomaviruses (PVs) based on 189 PV types and proposal of taxonomic amendments[END_REF].

The sensitivity, specificity, negative predictive value (NPV), and positive predictive value (PPV)

were calculated using 2x2 contingency tables with 95% confidence intervals (95% CI). All P-values were obtained using the Fisher's Exact Test or McNemar Χ 2 for comparison of matched-pair samples.

P-values <0.05 were considered statistically significant. Agreement between assays was assessed by

RESULTS

A total of 425 cervical samples from women aged 25-63 years old (mean age: 34.4±10.0 years; median age: 35 years old) were studied. From these, a subset of 405 samples was available for clinical evaluation of the CLART assay (Table I). Results of HPV DNA assays and clinical diagnosis (based on cytology and biopsy) were shown in Table I. Overall, HR-HPV types were detected in 268 cases (63.1%) with CLART-13 HR-HPV, in 275 cases (64.7%) with CLART-17 HR-HPV, and in 274 (64.5%) with the HC2 test (Table I).

Clinical performance of the two tests for CIN grade 2 or worse (≥CIN2) was very good and comparable, with sensitivities of 96% for CLART-13 HR-HPV and HC2, and 96.9% for CLART-17 HR-HPV. The specificity and NPV of all the tests were also comparable (Table II). No significant statistical differences were found among assays, in terms of sensitivity, specificity and NPV (P=0.000).

Overall, agreement between the CLART and the HC2 was very good (k=0.969, 95% CI: 0.941-0.969; concordance: 98.6% ±1.2 for 13 HR-HPV, and k=0.974, 95% CI: 0.941-0.988; concordance:

98.8% ±1.1 for 17 HR-HPV; P=0.000). Compared with the HC2 test, the CLART-13 HR-HPV assay showed a sensitivity, specificity, PPV, and NPV of 97.8% (95% CI: 96.8-97.8), 100% (95% CI: 98.1-100), 100% (95% CI: 98.9-100), and 96.2% (95% CI: 94.4-96.2), respectively (Table III). For 17 HR-HPV the CLART assay showed a sensitivity, specificity, PPV, and NPV of 99.3% (95% CI: 98.1-99.8), 98% (95% CI: 95.9-98.9), 98.9% (95% CI: 97.7-99.4), and 98.7% (95% CI: 96.5-99.6), respectively (Table III).

Discordant results were observed in 6 samples, which were positive by the HC2 test and negative by the CLART assay for the same 13 HR-HPV types. Considering CLART-17 HR-HPV, 2 samples were negative and 4 were positive (for HPV 53,66,and 82). Further testing by the PapilloCheck revealed a total concordance with the results obtained by CLART-17 HR-HPV (Table IV).

In addition, the CLART assay was evaluated for reproducibility (n=75). The assay showed a concordance of 98.7% (±2.7%) and an agreement of 0.972 (95% CI: 0.919-1) for the samples tested (data not shown).

DISCUSSION

Recent studies [START_REF] Castle | Human papillomavirus type 16 infections and 2-year absolute risk of cervical precancer in women with equivocal or mild cytologic abnormalities[END_REF][START_REF] Lai | Role of human papillomavirus genotype in prognosis of early-stage cervical cancer undergoing primary surgery[END_REF][START_REF] Cuzick | Overview of human papillomavirus-based and other novel options for cervical cancer screening in developed and developing countries[END_REF] have shown that detection of HR-HPV, in particular of HPV 16 and 18, can be an important aspect to consider in cervical cancer screening programs and algorithms. Since most HR-HPV infections can clear spontaneously, a clinically useful HPV test needs to have an optimal clinical sensitivity and specificity in order to effectively detect CIN grade 2 or worse (≥CIN2). Tests with a better sensitivity than that of the HC2 will detect a large number of latent infections that are clinically irrelevant and lead to an overtreatment of women with transient HPV infections [START_REF] Snijders | The clinical relevance of human papillomavirus testing: relationship between analytical and clinical sensitivity[END_REF][START_REF] Meijer | Guidelines for human papillomavirus DNA test requirements for primary cervical cancer screening in women 30 years and older[END_REF].

In this study, the performance of a new commercially available CE-marked genotyping assay (CLART Human Papillomavirus 2) that allows the detection and genotyping of 35 HPV types was compared with the HC2 test for the same 13 HR-HPV types, as well as for 17 HR-HPV types, which includes HPV 53, 66, 73 and 82. Moreover, the clinical performance was evaluated on a subset of 405 samples with histological results.

Both the CLART-13 HR-HPV and the HC2 assays showed an identical clinical performance, with a clinical sensitivity of 96%. For 17 HR-HPV types, the clinical sensitivity of the CLART assay was 96.9%. Regarding the specificity and the NPV, the results of the CLART and the HC2 were also very similar. In large population-based trials, the HC2 test had a specificity of ≥95% [START_REF] Meijer | Guidelines for human papillomavirus DNA test requirements for primary cervical cancer screening in women 30 years and older[END_REF]. In this study, all the assays had a positive rate ranging from 26.4 to 28.7% in the control group (CIN grade 1 or less), which can explain the lower clinical specificity observed (71.9% for CLART-17 HR-HPV, 73.6% for CLART-13 HR-HPV, and 71.4% for the HC2), compared to ≥95% specificity in a screening population [START_REF] Ronco | New technologies for cervical cancer screening working group. Results at recruitment from a randomized controlled trial comparing human papillomavirus testing alone with conventional cytology as the primary cervical cancer screening test[END_REF][START_REF] Meijer | Guidelines for human papillomavirus DNA test requirements for primary cervical cancer screening in women 30 years and older[END_REF]. Similar data were reported by using other tests [START_REF] Halfon | Comparison of the clinical performance of carcinogenic HPV typing of the Linear Array and Papillocheck® HPVscreening assay[END_REF][START_REF] Schopp | Evaluation of the performance of the novel PapilloCheck HPV genotyping test by comparison with two other genotyping systems and the HC2 test[END_REF].

Analytical performance of the CLART assay showed highly comparable outcomes, with very good values of sensitivity, specificity, PPV, NPV and concordance compared with the HC2 test. In addition, the CLART assay reproducibility was very good (k=0.972).

HPV discrepancies between the CLART assay and the HC2 test were found in 6 cases. Analysis of these cases by the PapilloCheck system indicated a total concordance with the results obtained by the CLART assay. As explained elsewhere [START_REF] Snijders | The clinical relevance of human papillomavirus testing: relationship between analytical and clinical sensitivity[END_REF], it is necessary to distinguish between clinical and analytical sensitivity. Whether a method with a higher analytical sensitivity would result in a better performance in terms of clinical sensitivity and specificity is still unknown.

In conclusion, the CLART Human Papillomavirus 2 assay showed an excellent performance, very similar to that of the HC2 test, which has a defined clinically cut-off. The CLART assay is efficient, sensitive, and reproducible, Furthermore, this assay has the advantage of detecting and genotyping 35 HPV types, in single or multiple infections, by a single test, which can be important for the individual risk stratification of women found to be infected persistently by HR-HPV types, as well as for the population studies required for vaccination trials and for monitoring the efficacy of HPV vaccines.

  comprised 425 archived cervical samples from sexually active women, attending at primary Health-Care Clinics of the National Health Service and Gynaecological Outpatient Clinics. Although not a screening population, the advantage was a broad range of outcomes and a high disease rate, which would enable accurate evaluation of sensitivity and specificity in a relative small sample. All cervical samples were collected in ThinPrep PreservCyt medium (Cytyc UK, Crawley, West Sussex, UK) during clinical examination for cytological analyses. The residual liquidbased cytology (LBC) was used for HPV testing. In 405 samples, the final clinical diagnosis was based on histological examination of biopsy samples obtained at colposcopy. As suggested byWentzensen and colleagues [2009] and according to histology, 178 out of the 405 women (44%) were considered to have CIN grade 1 or less (≤CIN1, regarded as controls), while 227 (56%) were diagnosed as CIN grade 2 or worse (≥CIN2, regarded as cases). No patient was sampled solely for the purpose of this research study.

  Cohen's kappa (k) statistics. All analyses were conducted using the SPSS (Statistical Package for the Social Sciences; SPSS, Inc., Chicago, IL) version 16.0 software.

TABLE I .

 I Characteristics of the studied cervical samples

	CYTOLOGICAL RESULTS (N=425)	N (%)
	Normal		63 (14.8)
	ASCUS		111 (26.1)
	LSIL		127 (29.9)
	HSIL		118 (27.8)
	SCC		6 (1.4)
	HISTOLOGICAL RESULTS (N=425)
	Normal		95 (22.4)
	CIN1		63 (14.8)
	CIN2 CIN3 SCC ND	F o r	118 (27.8) 98 (23.1) 8 (1.9) 43 (10.1)
	CLINICAL DIAGNOSIS (cytology + biopsy; N=405) ≤CIN1 178 (44.0) P
	≥CIN2	227 (56.0) e
	ND CLART-13 HR (N=425)		20 e r
	Positive		268 (63.1)
	Negative CLART-17 HR (N=425) Positive Negative HC2 (N=425) Positive Negative		157 (36.9) R e 275 (64.7) 150 (35.3) v i e 274 (64.5) 151 (35.5) w
	ASCUS, atypical squamous cells of undeterminated significance; LSIL, low-grade
	squamous intraepithelial lesion; HSIL, high-grade squamous intraepithelial lesion;
	SCC, squamous cell carcinoma; CIN1, cervical intraepithelial neoplasia grade 1;
	CIN2, cervical intraepithelial neoplasia grade 2; CIN3, cervical intraepithelial
	neoplasia grade 3; ND, no determined.

TABLE II .

 II Clinical performance for ≥CIN2 based in Pap smear and biopsy diagnosis of 405 samples

			Clinical Diagnosis	Sensitivity (%)	Specificity (%)	PPV (%)	NPV (%)
			≤CIN1	≥CIN2	(95% CI)	(95% CI)	(95% CI)	(95% CI)
			(n=178)	(n=227)
		P	47	218
					96.0	73.6	82.2	93.6
	CLART-13 HR	N	131	9
					(92.6-97.9)	(66.7-79.5)	(79.4-82.7)	(93.0-94.2)
		Positivity (%)	26.4	96.0
		P	50	220
					96.9	71.9	81.5	94.8
	CLART-17 HR	N	128	7
					(93.8-98.5)	(64.9-78.0)	(79.9-83.1)	(94.3-95.3)
		Positivity (%)	28.1	96.9
	HC2	P N Positivity (%) F o r 51 127 28.7	218 9 96.0	96.0 (92.6-97.9)	71.4 (64.3-77.5)	81.0 (79.4-82.7)	93.4 (92.8-94.0)
			P
			e
			e r
				R
				e
					v i e
					w

≤CIN1, cervical intraepithelial neoplasia grade 1 or less; ≥CIN2, cervical intraepithelial neoplasia grade 2 or worse; PPV, positive predictive value; NPV, negative predictive value; CI, confidence interval; P, HPV positive; N, HPV negative.

TABLE III .

 III Analytical performance of CLART and HC2 in 425 samples

		HC2	Concordance	k	Sensitivity	Specificity	PPV	NPV
		P	N	% ±SD	(95% CI)	(95% CI)	(95% CI)	(95% CI)	(95% CI)
	P	268	0	98.6	0.969	97.8	100	100	96.2
	CLART-13 HR
	N	6	151	±1.2	(0.941-0.969)	(96.8-97.8)	(98.1-100)	(98.9-100)	(94.4-96.2)
	P	272	3	98.8	0.974	99.3	98.0	98.9	98.7
	CLART-17 HR
	N	2	148	±1.1	(0.941-0.988)	(98.1-99.8)	(95.9-98.9)	(97.7-99.4)	(96.5-99.6)
	PPV, positive predictive value; NPV, negative predictive value; k, agreement; CI, confidence interval; SD, standard deviation; P, HPV positive; N, HPV negative.
		F o r
				P
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TABLE IV .

 IV Discordant results between CLART and HC2

			ABSTRACT
	Persistent infection by high-risk human papillomavirus (HR-HPV) is a cause of cervical cancer. The
	Case	CLART-13 HR-HPV	CLART-17 HR-HPV	HC2	PapilloCheck	Cytology / Biopsy
	use of HPV detection in cervical screening programs may improve the ability to identify women at risk
	1	N	HPV 66	P	HPV 66	CIN1
	of cervical cancer. Therefore, the development of appropriate methods for the detection of HR-HPV is
	2	N	N	P	N	CIN1
	3 essential. The aim of this study was to evaluate the clinical performance of the CLART Human N N P N Normal
	4 Papillomavirus 2 assay (CLART) in comparison with the Hybrid Capture 2 test (HC2), using a clinical N HPV 82 P HPV 82 CIN1
	5 cut-off of cervical intraepithelial neoplasia grade 2 or worse. Discrepant results were analysed further N HPV 53 P HPV 53 ASCUS
	6 by the PapilloCheck HPV genotyping system. N HPV 66	P	HPV 66	ASCUS
	P, HPV positive; N, HPV negative; ASCUS, atypical squamous cells of undeterminated significance; CIN1, cervical intraepithelial neoplasia grade 1. In the 425 studied women, HR-HPV positivity rates were similar by both tests (CLART-13 HR-HPV:
	63.1%; CLART-17 HR-HPV: 64.7%; HC2: 64.5%). Agreement between CLART-13 HR-HPV (k=0.969; F concordance level 98.6%), CLART-17 HR-HPV (k=0.974; concordance level 98.8%) and HC2 were o r very good. When 13 HR-HPV types were considered, the two tests showed a clinical sensitivity of
	96% (95% CI: 92.6-97.9). The clinical specificity of CLART-13 HR-HPV was 73.6% (95% CI: 66.7-P e 79.5) for cervical intraepithelial neoplasia grade 2 or worse, which was comparable to HC2 (71.4%;
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ACKNOWLEDGMENT

We thank Fatima Ribeiro for her help with sample processing and Dr. Alexandra Nunes for critical review of the manuscript. We are grateful to Genomica (Madrid, Spain) for providing the CLART Human Papillomavirus 2 reagents. This research study was sponsored by grants of Fundação Calouste Gulbenkian (Ref. 96507) and Comissão de Fomento de Investigação em Cuidados de Saúde (Ref. 13374). Ethical approval for the study was obtained. Authors declare that they had no conflicts of interest.


Carlo Galli
email: carlo.galli@uniroma1.it

Mahelet Patrizia Gentili

Aweke Tadesse

Aweke Mahelet

Carlo Tadesse

Patrizia Galli

Gentili

Prof Carlo Galli

Hydrogen abstraction from

Keywords: Aminoxyl radicals, hydrogen atom transfer, kinetics, bond energy, stereoelectronic effects Aminoxyl radicals, hydrogen atom transfer, kinetics, bond energy, oxidation reactions, stereoelectronic effects

may come

INTRODUCTION

Removal of hydrogen atom from a substrate is a fundamental event that allows the subsequent interaction with dioxygen and therefore leads to an oxidation outcome. [1] Transfer of hydrogen atom from C-H bonds to free-radical species, and to oxygen-centred radicals in particular, takes place in many instances, [2] including the degradation of chemicals in the troposphere, [START_REF] Warnatz | Combustion Chemistry[END_REF] biochemical reactions, [START_REF] Silverman | The Organic Chemistry of Enzyme-Catalyzed Reactions[END_REF] synthetic organic transformations, [START_REF] Arends | Modern Oxidation Methods[END_REF] but also the pathogenesis of some diseases. [START_REF] Cadenas | [END_REF] Oxygen-centred radicals, such as the aminoxyl radicals (R 2 NO . ) from appropriate hydroxylamine (R 2 NO-H) precursors, participate in many industrial procedures, [START_REF] Arends | Modern Oxidation Methods[END_REF]7,8] and promote the environmental benign aerobic oxidation of a number of substrates by H-abstraction. [START_REF] Galli | Nitroxyl Radicals[END_REF][START_REF] Galli | [END_REF][START_REF] Ishii | Modern Oxidation Methods[END_REF] Phthalimide N-oxyl radical (PINO) provides a prominent case (Fig. 1), and the synthetic value of PINO-induced oxidations is recognised and has prompted studies upon the reactivity features of the aminoxyl radical intermediates. [START_REF] Galli | Nitroxyl Radicals[END_REF][START_REF] Galli | [END_REF][START_REF] Amorati | [END_REF]13] Kinetic surveys have been already performed with PINO or else with its X-aryl-substituted cognates, [13][14][15][16][17] and have been recently complemented by a kinetic investigation with another aminoxyl radical, that is benzotriazole N-oxyl (BTNO), [18] originating from precursor 1-hydroxybenzotriazole (HBT). [19] Another kinetic study had been reported with bis(trifluoromethyl)aminoxyl radical, [20] (CF 3 ) 2 N-O . (dubbed here BTFN). We now compare the previous kinetic data with newly acquired ones concerning the H-abstraction reactivity of 6-trifluoromethyl-benzotriazol-N-oxyl radical (dubbed TFNO) (Fig. 1) towards H-donors. Mechanistic evidence has been collected with this particular aminoxyl radical, and a deeper insight into the H-abstraction reactivity of this class of compounds becomes now possible. In addition, and by resorting to suitable substrates, we gain a quantitative appreciation of the stereoelectronic effects that drive the H-abstraction step by the aminoxyl radicals.

RESULTS AND DISCUSSION

Characterization of the radical TFNO

The generation of an aminoxyl radical from the parent hydroxylamine (>NO-H) is in principle possible by either H-atom abstraction or else by one-electron abstraction followed by deprotonation. [9,10] The resulting >N-O . species can be either persistent or short-living depending on the structure, and characterized by appropriate spectroscopic methods. [START_REF] Galli | Nitroxyl Radicals[END_REF] For example, PINO was generated from precursor N-hydroxyphthalimide (HPI) by oxidation with Pb(OAc) 4 in AcOH solution, and showed an electronic spectrum with maximum of absorbance at 380 nm. [14] It underwent spontaneous decay with a half-life of 7900 s. Analogously, BTNO was generated from precursor HBT by oxidation with cerium(IV) ammonium nitrate (CAN) in MeCN solution; it showed λ max at 474 nm and a spontaneous decay with half-life of 110 s. [19] Additional characterization of these aminoxyl radicals was possible by means of laser flash photolysis, cyclic voltammetry and EPR spectroscopy. [START_REF] Galli | Nitroxyl Radicals[END_REF][START_REF] Galli | [END_REF] The latter technique was the one formerly employed in the kinetic investigation with BTFN (Fig. 1) in Freon. [20] We report here on the first generation of TFNO from the commercially available 6-CF 3 -1hydroxy-benzotriazole (TFBT) precursor. Following the addition of a stoichiometric amount of the monoelectronic oxidant CAN (E° 1.3 V vs NHE) [18] to a 0.8 mM solution of TFBT (curve •) in MeCN at 25 °C, a new and broad absorption band develops in the 350-600 nm range (curve ▼), with λ max at 450 nm and ε 1516 M -1 cm -1 (Fig. 2). The generated species is not stable but decays according to an exponential curve having half-life of 70 s. In agreement with the quantitative oxidation of TFBT by the one-electron oxidant CAN, investigation by cyclic voltammetry (CV) shows a reversible scan for a 2 mM solution of TFBT in MeCN in the presence of pyridine (4 mM) and 0.1 M LiClO 4 (Fig. 3), from which the redox potential (E°) of the >NO . />NO -couple is obtained as 1.06 V vs NHE (normal hydrogen electrode). In buffered (pH 6.9) water solution the E° is 1.16 V vs NHE. These data are in keeping with a reversible one-electron oxidation of the conjugate base of TFBT to TFNO (Scheme 1). [START_REF]A pK a value of 4.0 is given for TFBT: I[END_REF] Because of the limited stability of TFNO, we have not been able to carry out the determination of the energy value of the NO-H bond of TFBT by EPR equilibration methods, [22] but nevertheless have attained an evaluation of that BDE NO-H as 86±1 kcal/mol from resorting to a thermochemical cycle. [23] By and large, all the evidence collected on TFNO is in keeping with the formerly reported characterization of the parent BTNO species from HBT (Table 1). [18] In particular, the BDE NO-H values are almost equal within the errors (see Experimental).

(

-TFBT
TFNO is generated in the spectrophotometric cuvette by quickly mixing a 0.5 mM CAN solution with a 1 mM TFBT solution, both in MeCN at 25 °C. The reading of the initial absorption at 450 nm confirms quantitative generation of TFNO. Fast addition of a solution of the C-H bearing substrate marks the beginning of the kinetic experiment; three-to-five initial concentrations of the substrate are typically investigated in concentration exceeding that of TFNO by 10-to-100 fold. The time-dependent depletion of TFNO at OD 450 is monitored by stopped-flow spectrophotometry because the reaction times are in general in the few-seconds range. Under these pseudo first-order conditions, the H-abstraction rate from the substrate is higher than the spontaneous decay of TFNO in all cases reported in Table 2. The pseudo first-order rate constants k', determined from at least duplicated experiments, are converted into second-order H-abstraction rate constants (k H ) by determining the slope of a k' vs [subst] ° linear plot (Fig. 4). [18] No investigation is possible with either H-donor substrates too reactive for our experimental equipment, or slower-reacting ones whose 'apparent' first-order rate constant matches the k (decay) of TFNO. Typical uncertainty in the kinetic determinations ranges from 2 to 5%. (Table 2)

It is appropriate to state that, although the k H data in Table 2 cover a range exceeding two powers of ten, the corresponding range of BDE values is unfortunately not wide enough, nor the latter data are known with sufficient precision, to warrant a significant correlation of reactivity with bond energy, as we were able to do in the previous investigation with BTNO. [18] Nevertheless, the k H values of Ph 2 CH 2 and Ph 2 CHOH (5.4 and 25 M -1 s -1 ) obtained here with TFNO do scale with the available BDE C-H values (84 and 78 kcal/mol, respectively). [START_REF] Luo | Comprehensive Handbook of Chemical Bond Energies[END_REF] Likewise, the rate constants of [START_REF] Luo | Comprehensive Handbook of Chemical Bond Energies[END_REF] Therefore, a higher HAT reactivity for weaker bonds can be inferred, a result consistent with analogous findings for PINO and BTNO. [14,16,17] Summing it up, the reactivity features of TFNO in the HAT process (Table 2) are akin to those of other aminoxyl radicals, [START_REF] Galli | [END_REF] including the finding of an accelerating effect from electron-donor and the retarding effect from electron-withdrawing substituents on the benzylic substrate. Significant figures for this comparison of reactivity among aminoxyl radicals are selected and given in Table 3, proper allowance being made to the different solvents employed (no major effects upon the rates are however expected in these radical processes [14,15,[START_REF] Koshino | [END_REF] ).

(Table 3)

It is worth to point out that the reactivity of abstraction of benzylic hydrogens by the present aminoxyl radicals is substantially lower than that documented for other oxygen-centred radicals, [10,20,26] such as HO . or t-BuO . , the second-order rate constants of which being in the 10 6 -10 9 range for similar substrates under similar conditions. Because HO . and t-BuO . form much stronger O-H bonds upon HAT (119 and 109 kcal/mol, respectively) [START_REF] Luo | Comprehensive Handbook of Chemical Bond Energies[END_REF] than the aminoxyl radicals (>NO-H ca. 80-89 kcal/mol), [START_REF] Galli | Nitroxyl Radicals[END_REF]17] a fair convergence of thermodynamic and reactivity features emerges: the stronger the O-H bond formed by the abstracting oxygen-radical (RO . ) the higher the H-abstraction reactivity. More on this point, and although the difference in BDE NO-H values among the hydroxylamine-precursors of the aminoxyl radicals of Table 3 is admittedly quite small, some comments about their different reactivity are anyhow possible. In fact, there is no doubt that PINO is more reactive than BTFN, in keeping with the higher energy of the NO-H bond it forms on Habstraction (88 vs 84 kcal/mol). [START_REF] Galli | [END_REF] The BDE NO-H of TFBT and HBT are instead intermediate in value between the two above cases and indeed very close together, [START_REF] Galli | [END_REF]22] so that it is a little puzzling that TFNO comes out three-to-ten fold more reactive than BTNO. However, because the >N-O . radicals are known to be electrophilic, [START_REF] Galli | Nitroxyl Radicals[END_REF][START_REF] Galli | [END_REF]17,[START_REF] Minisci | [END_REF] the presence of the electron-withdrawing CF 3 -substituent in TFNO may confer additional polar stabilisation to the transition state of the H-atom transfer (cf. Scheme 2). It will assist the development of a partial negative charge on the abstracting aminoxyl radical, as mirrored by the favourable effect from the electron-donor substituents (X) on the benzylic alcohol counterpart, where a partial positive charge develops (vide also infra).

Scheme 2. Polar effects upon the transition state of a radical

HAT process by an electrophilic aminoxyl radical. Therefore, the H-abstraction reactivity of the aminoxyl radicals appears to be not only dominated by the enthalpic requirements of the NO-H bond energy in formation, but also by the polar effects sketched in Scheme 2 that may provide electrostatic stabilization to the transition state of the radical HAT route.

Mechanistic insight

Recent studies have supported the capability of the aminoxyl radicals to remove electrons from electron-rich substrates, giving an electron-transfer (ET) route as opposed to the expected HAT pathway (Scheme 3). [START_REF] Galli | [END_REF]28,29] Inspired by the finding that polar effects (Scheme 2) are relevant for TFNO and make it somewhat more reactive than BTNO, i.e., the parent aminoxyl radical for which a clean HAT route had been substantiated, [18,19] we have deemed that a full mechanistic characterization of the reaction of TFNO towards H-donors was required in the event that the competing ET route could take over.

Scheme 3. Competition of ET vs HAT routes with an aminoxyl radical.

a -Hammett correlation. The H-abstraction reactivity of TFNO towards X-substituted benzyl alcohols (from k H data in Table 2; cf. equation 1) correlates with the σ + parameter of the Xsubstituents according to an Hammett-like equation (log k X /k H = ρσ + ), [START_REF] Lowry | Mechanism and Theory in Organic Chemistry[END_REF] and gives a linear plot with ρ = -0.62 (±0.06) (Fig. 5). This is indeed a small slope, as expected for a radical process, [18] and compares well with a ρ value of -0.55 for the reaction of BTNO vs the same substrates in MeCN, [19] as well as with a ρ of -0.41 for PINO whenever generated from HPI by Pb(OAc) 4 in AcOH solution. [14] Additional ρ values (in the -0.5 to -0.7 range) are reported for X-substituted PINO's generated from X-substituted HPIs in MeCN solution. [17] In all these cases better Hammett correlations are obtained vs the σ + parameter of the X-substituents than with the simple σ parameter, in line with the electrophilicity of the aminoxyl radicals. [17,[START_REF] Minisci | [END_REF] Thus, an uniform pattern of selectivity among these structurally similar >N-O . species does emerge. Small ρ values such as these are more compatible with a single-step HAT route rather than with the formal alternative of a concerted proton-electron transfer route (CPET; cf. last structure in Scheme 2). [28,[START_REF] Mayer | [END_REF] For example, in the recently reported oxidation of 4-X-substituted N,N-dimethylanilines by the very BTNO, a much larger ρ value of -3.8 has been obtained, and judged compatible with a two step electron transferproton transfer mechanism. [32] towards a few deuteriated substrates, pertinent data being collected in Table 4.

(Table 4)

The large values of the intermolecular k H /k D ratio, ranging from 6 to 17, support a single-step ratedetermining H-abstraction step, and confirm analogous results (in the 6-to-27 range) obtained for PINO and BTNO. [START_REF] Galli | Nitroxyl Radicals[END_REF]14,15,17,18,[START_REF] Koshino | [END_REF] The contribution from tunnelling is clear, as also documented elsewhere. [14,17] c -Products study for reaction of TFNO with a probe substrate. As described previously, [33] the test substrate 1 presents the distinct advantage of giving rise to two different end-products depending on the operating mechanism of oxidation. In fact, under genuine ET conditions, 1 gives a transient 1 .+ intermediate that cleaves the C α -C β bond of the side-chain, and affords the corresponding aldehyde (2) plus tert-butyl radical (Scheme 4). [34] Scheme 4. Products study for reaction of TFNO with probe substrate 1:

C α -C β (above) vs C α -H (below) bond cleavage.
The cleavage is driven by steric and stereoelectronic factors that make the loss of a tertiary radical easy. [34] Conversely, under bona fide radical HAT conditions, 1 undergoes cleavage of the C α -H benzylic bond, and produces the corresponding ketone 3 exclusively. [33,34] This clear-cut behaviour makes 1 a significant probe, capable of differentiating the mechanism of an oxidation reaction from product analysis.

Upon oxidation of 1 with TFNO in MeCN (cf. Experimental) a 4% yield of ketone 3 is obtained with no traces of the aldehyde 2, the rest of mass balance being the unreacted 1. Under similar conditions, BTNO gave a strictly comparable result. [19,33] Thus, the HAT pathway is utterly confirmed for both these aminoxyl radicals, and any competition by an ET route (cf. Scheme 3) can be discarded.

Stereoelectronic effects

In line with the concept of the anomeric effect, [35,[START_REF] Kirby | The Anomeric Effect and Related Stereoelectronic Effects at Oxygen[END_REF] Griller et al. [START_REF] Griller | [END_REF] have convincingly demonstrated that conjugation from the lone-pair of a heteroatom weakens an adjacent C-H bond and stabilizes the intervening C-radical generated by H-abstraction. The contribution from this weakening effect upon the energy of the adjacent C-H bond is the greater the more co-linear are C-H bond and lonepair. [START_REF] Griller | [END_REF]38] Fig. 6 shows the case of an amine where the effect reportedly accounts for a 7-9 kcal/mol weakening of the adjacent C-H bond. A computational study similarly attains a 5 kcal/mol weakening of the α C-H bond for conjugation from an oxygen lone-pair. [39] Figure 6. Stereoelectronic interaction between the lone-pair of an amine and an adjacent C-H bond. Reprinted with permission from Griller et al. (ref. 37). Copyright (1981) American Chemical Society

For torsion angles (θ) wider than 30°, the relevance of the effect fades away drastically. [START_REF] Griller | [END_REF] Our finding of an appreciable rate increase when passing from Ph 2 CH 2 to Ph 2 CHOH (Table 2) is likely due to the profitable stereoelectronic interaction from the oxygen lone-pair of the alcohol with the scissile α C-H bond, an interaction that clearly lacks in the hydrocarbon counterpart, thereby confirming the analogous finding in the kinetic study with BTNO. [18] By exploiting the higher reactivity of TFNO than BTNO in HAT reactions, we have been able

to extend the previous kinetic investigation on the stereoelectronic effects [40,41] to additional and significant substrates. Table 5 gives the results for H-abstraction by TFNO, the investigated substrates being organised pairwise with their experimental k H and BDE C-H values (whenever available). [START_REF] Luo | Comprehensive Handbook of Chemical Bond Energies[END_REF] (Table 5)

A few comments are in order:

-Abstraction of hydrogen from a C-H bond adjacent to an oxygen atom is always faster than from the parent hydrocarbon (entries 1-3), [42] as mirrored by the corresponding BDE C-H data, [START_REF] Luo | Comprehensive Handbook of Chemical Bond Energies[END_REF] owing to the favourable stereoelectronic interaction between oxygen lone-pair and C-H bond (cf. Fig. 6);

-whenever the availability of the adjacent oxygen lone-pair is lessened by resonance with an electron withdrawing group, such as a carbonyl (Fig. 7), the stereoelectronic assistance in Fig. 6 is reduced (entries 4-6); [43] Figure 7. Reduced availability of the oxygen lone-pair owing to conjugation with an adjacent group.

-the stereoelectronic effect from the lone-pair of nitrogen is somewhat stronger than from oxygen (entry 7). [START_REF] Griller | [END_REF][38][39] -clearly, the stereoelectronic effect is particularly appreciable here because the >N-O . species is an electrophilic radical, [START_REF] Galli | Nitroxyl Radicals[END_REF][START_REF] Galli | [END_REF][START_REF] Amorati | [END_REF]13,17,[START_REF] Minisci | [END_REF] and therefore susceptible to both bond-polarisation effects (cf. Fig. 6) and polar effects (see Scheme 2) upon reactivity. Besides the stereoelectronic effect pertaining to the lone-pair of an adjacent heteroatom, Habstraction from a benzylic (or allylic) C-H bond is faster whenever cyclic systems are considered, as opposed to open-chain counterparts. [40] This is confirmed when comparing the values of BDE C-H for the cyclic/open pairs in entries 8-10, and supports previous kinetic findings in HAT processes by BTNO or PINO. [START_REF] Galli | [END_REF]40] Clearly, the homolytic cleavage of a benzylic (or allylic) C-H bond is assisted by π-conjugation with an aromatic (or vinylic) moiety that stabilizes the intervening carbon-radical.

Such assistance is fostered in a cyclic compound where co-linearity between the C-H bond being cleaved and the adjacent π-system is enforced by steric restrictions from the ring system (Fig. 8), whereas in the corresponding open-chain compound the appropriate co-linearity would be somewhat disrupted owing to conformational freedom. [40,41] Figure 8. Enforced co-linearity of a benzydrylic radical with the p-orbitals of the adjacent aromatic rings, as due to the planar 5-membered ring interconnecting the two π-systems (the case of entry 8 in Table 5 is shown).

CONCLUSIONS

Rate constants of H-abstraction (k H ) by TFNO from H-donor substrates have been determined

spectroscopically. Hammett correlation of the reactivity of TFNO vs X-substituted benzyl alcohols affords a small value for ρ (i.e., -0.62), compatible with a single-step radical H-atom abstraction from the benzylic position, rather than with a concerted proton-electron transfer route (cf. Scheme 3), [29] that it is expected to provide larger ρ values (as -3.8, [START_REF] Mayer | [END_REF]32] or even larger [44] ). A product study for reaction of TFNO with a probe substrate confirms a HAT route of oxidation. The primary kinetic isotope effect supports the H-abstraction as rate determining. The HAT reactivity has been compared within a series of four aminoxyl radicals and an uniform patter of selectivity (Table 3) does emerge.

In particular, the H-abstraction from H-donor substrates appears to be the faster the stronger the O-H bond (i.e., BDE NO-H ) that the >N-O . species gives rise to in the HAT step. In this respect, the slightly higher reactivity of TFNO than the parent BTNO species is at odds with the comparable values of their BDE NO-H and ascribed to the electron-withdrawing CF 3 -substituent in TFNO, that confers additional polar stabilisation to the transition state of the radical H-atom transfer (Scheme 2). Finally, and by exploiting the higher reactivity of TFNO than BTNO in HAT reactions, we have been able to substantiate a wider number of exemplary cases of stereoelectronic effects on the HAT reactivity. In the inspected cases the conjugation of a C-H bond with either the lone-pair of an adjacent heteroatom or a π-system weakens the C-H bond and fosters its cleavage by the aminoxyl radical. The efficiency of the interaction is enhanced whenever a proper co-linear conformation is attained (Fig. 6), and this is particularly well met in rigid cyclic substrates (Fig. 8) where the loss of conformational freedom is not severe.

EXPERIMENTAL

General

Commercial TFBT (Aldrich) was used as received; all the substrates were purchases and used without further purification, if not already available in the laboratory. [18] In particular, substrate 1 and products 2 and 3 were available from a previous study. [33] Cerium(IV) ammonium nitrate (CAN; Erba RPE) was oven-dried before use. [18] Reagent grade acetonitrile (Erba RPE) was used as the solvent. Bis α-deuteriated benzyl alcohols (cf. Table 4) were already available in the laboratory or synthesized as described previously [33,45] by reduction of the corresponding carboxylic acids with LiAlD 4 in dry THF. Full 1 H-NMR (samples concentration: 65 mM) and MS characterisation of the bis α-deuteriated benzyl alcohols were as follows.

[ Instrumentation. The kinetic study was carried out with a Hi-Tech SFA-12 stopped flow instrument, interfaced to a HP 8453 diode array spectrophotometers, and having a thermostated cell holder. A thermocouple was employed for reading the temperature in the quartz cuvette. A VARIAN 3400/Star gas chromatograph, fitted with a 20 m × 0.25 mm methyl silicone gum capillary column, was employed in the GC analyses. GC-MS analyses were performed on a HP 5892 GC, equipped with a 12 m × 0.2 mm methyl silicone gum capillary column and coupled to a HP 5972 MSD instrument operating at 70 eV.

Kinetic Procedure. The TFNO species was generated in the cuvette of the stopped-flow by adding a 0.5 mM solution of CAN to a 1.0 mM solution of TFBT, both in MeCN. A broad absorption band developed almost immediately (8 ms) in the 350-600 nm region (λ max at 450 nm, ε 1516 dm 3 mol -1 cm -1 ). The OD 450 reading was not stable, but decayed with a half-life of 70 s. The rate of decay was unaffected by the use of de-aerated MeCN. Rate constants of H-abstraction from H-donor substrates were determined at 25 °C in MeCN, by following the decrease of OD 450 of TFNO. The initial concentration of the substrate was in excess with respect to the TFNO species, and in the 5÷25 mM range, so to enable a pseudo first-order treatment of the kinetic data. Plots of (OD t -OD ∞ ) vs time were well fitted by a first-order exponential over more than three half-lives, and the rate constant (k') reckoned. From a plot of three-to five k' vs [subst] ° data pairs, the second-order rate constant of Habstraction (k H ) was obtained. The intermolecular kinetic isotope effect for bis α-deuteriated-Xsubstituted benzyl alcohols (Table 4) was calculated from the ratio of the k H and k D rate constants with TFNO. Products study with probe substrate (1). As described previously, [18,22,33] 60 mmol of substrate 1, 20 mmol of TFBT and 20 mmol of CAN were let to react at room temperature in 3 mL MeCN. After a 5 h reaction time and conventional workup with ethyl acetate, GC-MS analysis enabled us to detect the formation of ketone 3 and the absence of aldehyde 2; no other products besides unreacted 1 were detected. Quantitative determination of the yield of 3 (i.e., 4%) was carried out by GC with the internal standard method.

Electrochemical Determinations. Cyclic voltammetry at the steady disc electrode (glassy-carbon disc, 1.5 mm in diameter) [46] was carried out with TFBT (2 mM) at 25°C in MeCN in the presence of pyridine (4 mM) and 0.1 M LiClO 4 (Fig. 3). A three-electrode circuit was used with a potentiostat with a positive feedback ohmic-drop compensation and a MPLI Hardware (Vernier Software & Technology) controlled by a program written in C++ language for Windows 95/98. [START_REF] Dr | Mottier for writing and implementing the electrochemical software for us[END_REF] A platinum wire (1 cm 2 ) and a saturated calomel electrode (SCE) as reference were used; the E° value is obtained as the mean of the cathodic and anodic peak values of the reversible scan (Fig. 3), [START_REF] Lund | Organic Electrochemistry[END_REF] and referred to the normal hydrogen electrode (NHE), NHE = SCE + 0.241 V. [START_REF] Ives | Reference Electrodes[END_REF] Scan rates in the 10 mV/s to 10 V/s range were investigated.

BDE Determination.

The BDE NO-H of TFBT has been obtained from the following thermochemical cycle (pertinent data in Table 1, to be converted into kcal/mol), the value of E 2 ° being 54 kcal/mol: [22,23]

R 2 NO-H R 2 NO -+ H + R 2 NO . + H . pK a BDE E°E°1 2 R 2 NO-H
and equation: [START_REF] Galli | Nitroxyl Radicals[END_REF]23]

BDE NO-H = pK a (R 2 NOH) + E 1 ° (R 2 NO . / R 2 NO -) + E 2 ° (H + /H . ) (2)
This calculation has been calibrated in the case of HPI, obtaining a BDE NO-H value of 88 kcal/mol vs the experimental value of 88.3 kcal/mol. [START_REF] Galli | Nitroxyl Radicals[END_REF] This suggested us that a reasonable confidence limit for the calculated values is ± 1 kcal/mol (in Tables 1 and3).

F

(sub A /sub B ) b 1 Ph 2 CHOH k H = 25 M -1 s -1 BDE C-H = 78 kcal/mol Ph 2 CH 2 k H = 5.4 M -1 s -1 BDE C-H = 84 kcal/mol 9 2 H OH k H = 190 M -1 s -1 BDE C-H = 73 kcal/mol H H k H = 24 M -1 s -1 BDE C-H = 82 kcal/mol 16 3 (PhCH 2 ) 2 O k H = 22 M -1 s -1 BDE C-H = ca. 82 kcal/mol PhCH 2 CH 2 Ph k H = 0.88 M -1 s -1 BDE C-H = 87 kcal/mol

Fig. 2 .

 2 Fig. 2. UV-Vis spectrum of TFBT (0.8 mM) in MeCN in the absence of CAN ( ); in the presence of CAN (0.8 mM) (▼) 15 msec after mixing.

Figure 1 .

 1 Figure 1. Aminoxyl radicals (>N-O . ) of interest in this study (full names are given in the text).

Figure 2 .

 2 Figure 2. UV-Vis spectrum of TFBT (0.8 mM) in MeCN in the absence of CAN ( ); in the presence of CAN (0.8 mM) (▼) 15 msec after mixing.

Figure 3 .Scheme 1 .

 31 Figure 3. Cyclic voltammetry in MeCN solution (V vs Ag/AgCl): [TFBT] = 2 mM, [Py] = 4 mM.

Figure 4 .

 4 Figure 4. Pseudo first-order (k') rate constants for H-abstraction from 4-Cl-benzyl alcohol by TFNO (0.5 mM) in MeCN at 25 °C. The slope of the linear plot gives the second-order rate constant k H ; the ratio of the initial concentrations of TFNO and substrate is indicated.

Figure 5 .

 5 Figure 5. Hammett correlation for reaction of TFNO with X-substituted benzyl alcohols in MeCN solution at 25 °C.

  isotope effect determination. The kinetic survey with TFNO has been extended

Figure 2 .

 2 Figure 2. UV-Vis spectrum of TFBT (0.8 mM) in MeCN in the absence of CAN ( ); in the presence of CAN (0.8 mM) (▼) 15 msec after mixing.

Figure 3 .

 3 Figure 3. Cyclic voltammetry in MeCN solution (V vs Ag/AgCl): [TFBT] = 2 mM, [Py] = 4 mM.

Figure 4 .

 4 Figure 4. Pseudo first-order (k') rate constants for H-abstraction from 4-Cl-benzyl alcohol by TFNO (0.5 mM) in MeCN at 25 °C. The slope of the linear plot gives the second-order rate constant k H ; the ratio of the initial concentrations of TFNO and substrate is indicated.

Figure 5 .

 5 Figure 5. Hammett correlation for reaction of TFNO with X-substituted benzyl alcohols in MeCN solution at 25 °C.

Figure 6 .Figure 7 .

 67 Figure 6. Stereoelectronic interaction between the lone-pair of an amine and an adjacent C-H bond. Reprinted with permission from Griller et al. (ref. 37). Copyright (1981) American Chemical Society

Figure 8 .Scheme 1 .Scheme 2 .Scheme 3 .Scheme 4 .Figure 3 .Figure 4 .

 8123434 Figure8. Enforced co-linearity of a benzydrylic radical with the p-orbitals of the adjacent aromatic rings, as due to the planar 5-membered ring interconnecting the two π-systems (the case of entry 8 in Table5is shown).

2 Figure 5 .

 25 Figure 5. Hammett correlation for reaction of TFNO with X-substituted benzyl alcohols in MeCN solution at 25 °C.

Figure 6 .

 6 Figure 6. Stereoelectronic interaction between the lone-pair of an amine and an adjacent C-H bond. Reprinted with permission from Griller et al. (ref. 37). Copyright (1981) American Chemical Society

Figure 7 .Figure 8 .

 78 Figure 7. Reduced availability of the oxygen lone-pair owing to conjugation with an adjacent group.

Scheme 2 .Scheme 3 .

 23 Scheme 2. Polar effects upon the transition state of a radicalHAT process by an electrophilic aminoxyl radical.

Scheme 4 .

 4 Scheme 4. Products study for reaction of TFNO with probe substrate 1: C α -C β (above) vs C α -H (below) bond cleavage.

Table

  )

	The spontaneous decay of TFNO is strongly accelerated in the presence of H-donor substrates
	bearing C-H bonds of suitable energy. Accordingly, it becomes possible to investigate the H-atom
	transfer (HAT) reactivity (k H ) of TFNO from substrates such as benzyl alcohols, alkylarenes or
	allylalkanes (equation 1) by using UV-Vis spectroscopy, and monitoring the disappearance of the
	OD 450 of TFNO under the above described conditions in MeCN solution at 25 °C.
	k H	O 2
	TFNO + ArCH 2 OH → ArCH( . )OH → product (e.g. ArCHO) (1)
	F o r
	P
	e
	e r
	R
	e
	v i e
		w
	Hydrogen-abstraction reactivity by TFNO

  CH 3 vs MeO-C 6 H 4 CH 2 CH 3 vs MeO-C 6 H 4 CHMe 2 are compatible with the corresponding values of BDE C-H (ca. 89, 86 and 84 kcal/mol, respectively).

	abstraction of a primary vs secondary vs tertiary benzylic hydrogen atom from, respectively, MeO-
	C 6 H 4
	F o r
	P
	e
	e r
	R
	e
	v i e
	w

Table 1 .

 1 Comparison of the properties of the >NO-H and >NO . species in the benzotriazole family From ref. 21. b In the presence of pyridine; see also ref. 22. c Calculated from a thermochemical cycle (± 1 kcal/mol), see Experimental and ref. 22. d Spontaneous decay, k (decay) .

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

a

Table 2 .

 2 Second-order rate constants of H-abstraction (k H ) from H-donors (RH) by TFNO at 25 °C in MeCN. Available bond dissociation energy values (BDE C-H ) of RH are given. 3%. b From refs. 18 and 24. c Not available.

	Substrate, RH	k H (M -1 s -1 ) a	BDE C-H (kcal/mol) b
	4-MeO-C 6 H 4 CH 2 OH	54	78-82
	3-MeO-C 6 H 4 CH 2 OH	26	78-82
	4-Me-C 6 H 4 CH 2 OH	40	78-82
	PhCH 2 OH	23	80
	4-Cl-C 6 H 4 CH 2 OH	15	83-85
	4-CF 3 -C 6 H 4 CH 2 OH	7.6	83-85
	4-NO 2 -C 6 H 4 CH 2 OH	7.2	83-85
	(PhCH 2 ) 2 O	22	n.a. c
	4-MeO-C 6 H 4 CH 3	1.8	89-90
	4-MeO-C 6 H 4 CH 2 CH 3	6.2	85-87
	4-MeO-C 6 H 4 CHMe 2	27	83-85
	PhCH(Me)OH	23	n.a. c
	cyclohexene	27	82
	Ph 2 CH 2	5.4	84
	Ph 2 CHOH	25	78
	fluorene	24	82
	PhCH 2 CN	0.4	n.a. c
	a Experimental conditions: [TFNO] 0.5 mM, [RH] 5-to-25 mM in MeCN solution.
	Determinations in triplicate; typical error ±

Table 3 .

 3 Comparison of kinetic data (k H , M -1 s -1 , at 25 °C) of H-abstraction among aminoxyl radicals (structures and names in Fig. 1; n.d. stands for not determined) From ref 24. b From oxidation of HPI with Pb(OAc) 4 at 25 °C, refs. 14,25. c From ref 20, at 25 °C. d From oxidation of HBT with CAN at 25 °C, ref 18. e Experimental value (± 0.5 kcal/mol), refs 9 and 10. f A reasonable confidence limit for our calculated BDE NO-H values is ± 1 kcal/mol, ref 22. g As p-MeO-derivatives.

		PINO, b	BTFN, c	BTNO, d	TFNO
	Substrate, RH	in AcOH	in Freon	in MeCN	in MeCN
	(BDE C-H , in kcal/mol) a	BDE NO-H = 88.3 kcal/mol e	BDE NO-H = 84.0 kcal/mol e	BDE NO-H = 85 kcal/mol f	BDE NO-H = 86 kcal/mol f
	PhCH 2 OH (80)	11	n.d.	1.8	23
	p-MeO-C 6 H 4 CH 2 OH (ca. 79)	45	n.d.	7.8	54
	PhCH 3 (89)	0.62	8.9 10 -3	0.26 g	1.8 g
	PhCH 2 CH 3 (85)	5.4	0.3	0.70 g	6.2 g
	PhCHMe 2 (84)	27	0.3	0.55 g	54 g
	Ph 2 CHOH (78)	58	n.d.	3.2	25
	Ph 2 CH 2 (84)	13	0.48	0.72	5.4
	Ph 3 CH (81)	59	8.8	2.3	n.d.
	Fluorene (82)	40	n.d.	3.8	24
	a

Table 4 .

 4 Kinetic data of either H-or D-abstraction by TFNO from a series of benzylic alcohols, in MeCN at 25 °C

	Substrate	k H or k D (M -1 s -1 )	k H /k D
	4-MeOC 6 H 4 CH 2 OH	54	6.3
	4-MeOC 6 H 4 CD 2 OH	8.6
	PhCH 2 OH	23	16
	PhCD 2 OH	1.4
	4-CF 3 C 6 H 4 CH 2 OH	7.6	17
	4-CF 3 C 6 H 4 CD 2 OH	0.44

Table 5 .

 5 Rate constants of H-abstraction by TFNO in MeCN solution at 25 °C a

	Entry	substrate A	substrate B	k rel

  A rate value too high for our spectrophotometric mixing device (8 msec of dead-time; i.e. k H > 80 M -1 s -1 ). Aminoxyl radicals (>N-O . ) of interest in this study (full names are given in the text).

	Legends
	Figure 1.
	F o r
	P
	e
	e r
	R e BDE C-H = 78 kcal/mol v i e	7.6
	w

a BDE data from ref

24. b

The ratio has been normalised for the number of equivalent H-atoms (i.e., 'intrinsic reactivity' per H-atom). c Rate value comparable to the spontaneous decay of TFNO (i.e., < 0.3 M -1 s -1 ). d Not available. e

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Journal of Physical Organic Chemistry

Acknowledgements

The Authors thank the Italian MIUR for financial support (COFIN and FIRB projects).