Samwel Ogado

Ceasar Odiwuor

Alfarazdeg Ageed Saad

Simonne De Doncker

Ilse Maes

Thierry Laurent

Sayda El Safi

Margaret Mbuchi

Philippe Büscher

J.-C Dujardin

Gert Van Der Auwera

Jean-Claude Dujardin

Universal PCR assays for the differential detection of all Old World species

Keywords: Old World leishmaniasis, differential diagnosis, Leishmania donovani, Leishmania infantum, Leishmania tropica, Leishmania aethiopica, Leishmania major

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INTRODUCTION

Leishmaniasis describes a spectrum of tropical and sub-tropical diseases, which range from self-healing localized cutaneous lesions to aggravated visceral disease with multi-organ involvement, often fatal if left untreated. The diseases are endemic in 88 countries where an estimated 12 million people are infected, while 350 million being at risk [START_REF] Desjeux | The increase in risk factors for leishmaniasis worldwide[END_REF][START_REF]The world health report 2002: Reducing risks, promoting healthy life[END_REF]. The etiological agents are parasites of the kinetoplastid genus Leishmania, in which two subgenera and some 20 medically important species are currently recognized [START_REF] Fraga | Phylogeny of Leishmania species based on the heat-shock protein 70 gene[END_REF]. Leishmania distribution is dichotomized into the Old (East-Africa, the Mediterranean region and the Indian subcontinent) and the New (Central and South-America) World groups. Five species are endemic in the Old World: L. major, L. aethiopica, L. tropica, L. donovani, and L. infantum, whereby the latter two comprise the so-called L. donovani complex. The distribution of these species is highly dynamic and is expected to be increasingly modulated by global climate changes and associated demographic developments, making species identification on the basis of geographical origin unreliable [START_REF] Ashford | Ecology and epidemiology: Old World[END_REF].

Characterization of Leishmania species is important for clinical case management and epidemiological monitoring of the parasite spread [START_REF] Reithinger | Molecular diagnosis of leishmaniasis: current status and future applications[END_REF][START_REF] Palumbo | Current treatment for cutaneous leishmaniasis: a review[END_REF][START_REF] Shani-Adir | Leishmania tropica in northern Israel: a clinical overview of an emerging focus[END_REF]. Cutaneous leishmaniasis in the Old World can be caused by any of the four following species, each with different disease progression characteristics: L. infantum can also visceralize and cause the lethal visceral leishmaniasis; L. major is often self-curing [START_REF] Morizot | Healing of Old World cutaneous leishmaniasis in travelers treated with fluconazole: drug effect or spontaneous evolution?[END_REF]; L. aethiopica and L. tropica are recalcitrant to treatment [START_REF] Modabber | Consultative meeting to develop a strategy for treatment of cutaneous leishmaniasis[END_REF][START_REF] Hadighi | Unresponsiveness to Glucantime treatment in Iranian cutaneous leishmaniasis due to drugresistant Leishmania tropica parasites[END_REF]. Visceral leishmaniasis caused by L. donovani in East-Africa often does not respond to first line therapy [START_REF] Croft | Drug resistance in leishmaniasis[END_REF],

and is frequently associated with a secondary pathology called post-kala azar dermal leishmaniasis [START_REF] Zijlstra | Post-kala-azar dermal leishmaniasis[END_REF]. As species cannot be identified on the basis of morphology and clinical symptoms alone [START_REF] Molyneux | Morphology, ultrastructure and life cycles[END_REF][START_REF] Weiss | DNA probes and PCR for diagnosis of parasitic infections[END_REF], other techniques are needed.

The current gold standard for species discrimination is multi-locus enzyme electrophoresis [START_REF] Rioux | Taxonomy of Leishmania. Use of isoenzymes. Suggestions for a new classification[END_REF], a cumbersome technique requiring mass parasite cultivation. Molecular methods based upon PCR amplification of a Leishmania DNA fragment have replaced this technique for practical purposes. Many such PCR assays have been reported in literature (reviewed briefly in [START_REF] Laurent | Identification of Old World Leishmania spp. by specific polymerase chain reaction amplification of cysteine proteinase B genes and rapid dipstick detection[END_REF] for the Old World), but they mostly suffer from a lack of validation on all species, fail testing a geographically representative panel of isolates to account for intra-species variability, and are often hampered by limited sensitivity. In addition, several assays require post-PCR handlings not always available in endemic regions, such as sequencing, restriction digests or melting curve analysis, required to reveal polymorphisms within the amplified fragment.

The internal transcribed spacer 1 of the ribosomal DNA repeat unit (rDNA-ITS1) has previously been exploited for Old World species discrimination [START_REF] Schönian | Molecular epidemiology and population genetics in Leishmania[END_REF] using restriction fragment length polymorphisms [START_REF] Schönian | PCR diagnosis and characterization of Leishmania in local and imported clinical samples[END_REF][START_REF] Cupolillo | Intergenic region typing (IRT): a rapid molecular approach to the characterization and evolution of Leishmania[END_REF][START_REF] Bensoussan | Comparison of PCR assays for diagnosis of cutaneous leishmaniasis[END_REF], reverse hybridization assays [START_REF] Nasereddin | Molecular diagnosis of Old World cutaneous leishmaniasis and species identification by use of a reverse line blot hybridization assay[END_REF], and melting curve analysis [START_REF] Talmi-Frank | Detection and identification of Old World Leishmania by high resolution melt analysis[END_REF]. There are an estimated 20 to 200 identical copies in the Leishmania genome, making it a good target for analyzing low parasite amounts. In this study we describe four species-identification PCRs based on rDNA-ITS1, for discrimination of L. aethiopica, L. tropica, L. major and the L. donovani complex.

We also determined the sequence of this region from 24 isolates or strains.

MATERIALS AND METHODS

Reference strains

DNAs from reference strains and isolates were obtained from cultured parasites, and were either grown and extracted at the Institute of Tropical Medicine Antwerp (ITMA, Antwerp, Belgium), or kindly donated by several institutes acknowledged at the end of this manuscript. Concentration of the parasite DNA was measured using the Nanodrop ND-1000 Spectrophotometer (Thermo Scientific, Wilmington, Delaware, USA). Isolates were characterized by rDNA-ITS1 sequencing, or a discriminative PCR targeting cysteine proteinase B [START_REF] Laurent | Identification of Old World Leishmania spp. by specific polymerase chain reaction amplification of cysteine proteinase B genes and rapid dipstick detection[END_REF]. For sequencing, PCR amplicons (Fig. 1) were generated with primers rDNA-10F (5' CAATACAGGTGATCGGACAGG 3') and rDNA-14R

(5' CACGGGGATGACACAATAGAG 3'), and this was followed by direct sequencing without cloning. Table 1 lists the origin of all samples used during the evaluation and validation steps, and the accession numbers of the determined rDNA-ITS1 sequences. The definitions of L. infantum and L. donovani are as in [START_REF] Lukeš | Evolutionary and geographical history of the Leishmania donovani complex with a revision of current taxonomy[END_REF].

Primers and internal controls.

Species-specific PCR primers were designed on the basis of an alignment of rDNA-ITS1 sequences newly determined (Table 1) and downloaded from the EBI sequence data base -5 -(www.ebi.ac.uk). Several primer combinations per species were tested, and a final set (Fig. 1, Table 2) was selected for extensive optimization, evaluation, and validation experiments. For each of the 4 species-specific PCRs, internal positive control templates were developed (Figs. 2,3). These internal controls are added to the respective PCRs, and upon successful amplification they show a product of about 100 bp longer than the Leishmania species-specific amplicon. Such internal control allows to check for correct PCR mix set-up, and sample inhibition. To obtain these internal controls, phage lambda DNA fragments were amplified with composite cloning primer pairs, each consisting of a 3' lambda-specific segment with a Leishmania species-specific 5' extension matching the PCR primers in Table 2. The amplified phage lambda sequences were selected to have a base composition comparable to the respective Leishmania targets. The phage lambda amplicons, flanked by the Leishmania primer annealing sites, were cloned in vector pCR4-TOPO using a TOPO TA cloning kit (Invitrogen, Carlsbad, CA, USA), and verified by sequencing (Fig. 2).

Species-specific PCRs

Five PCRs were used in this study, which are listed in Table 2 and depicted schematically in 2, and HotStarTaq Plus DNA polymerase (Qiagen) was used in each case. PCR LT2 was not used in the evaluation and validation experiments. Primers were ordered from Sigma-Aldrich (Bornem, Belgium), dNTPs from Eurogentec (Seraing, Belgium), and dUTPs from Roche (Vilvoorde, Belgium). Cycling conditions for all PCRs were identical: 5 min at 95°C; followed by 40 cycles of 30 sec 95°C, 40 sec 55°C, and 40 sec 72°C; and finally 5 min at 72°C. All PCRs were performed in a BioMetra T3000 thermocycler (BioMetra, Göttingen, Germany). Products were analyzed on a 2% or 2.5% agarose gel stained with ethidium bromide, and scored positive when an amplicon of the expected size (Table 2) was observed.

Optimization and Evaluation

Following development of the PCR protocols, the detection limit of the PCRs was assessed by using 10, 1, 0.1, and 0.01 pg DNA from 5 different parasite isolates or strains from each species. This was repeated with the addition of DNA extracted from 9 µl naive human blood in each PCR, for which 2.5 µl of 180 µl blood extract eluted in 50 µl was added to the reaction. The discriminatory power for identifying the correct species was assessed using 100 pg of the DNAs listed in Table 1.

The optimal concentration of the recombinant internal control plasmids was determined as the lowest concentration that consistently amplified in repeated experiments, as tested at least in triplicate in a 10 fold dilution series, followed by further refining by using 4 intermediate concentrations. Sensitivity of the internal control amplification to PCR inhibitors was tested by adding 0.01, 0.1, and 1% of SDS (sodium dodecyl sulphate) to the reaction.

Validation and implementation

For the purpose of validating the final PCR protocols, a blinded test panel was compiled, including the strains listed in Table 1. DNA from each strain was included twice in the panel: once at an amount of 100 pg per PCR, and once at an amount of 0.2 pg including a DNA equivalent of 1 µl naive human blood per PCR. In addition, the panel contained 3 negative controls and 3 naïve blood extracted DNAs as quality check for contamination and cross-reactivity. This makes a total of 90 samples, which were supplemented by 6 negative controls by the person performing the test. Positive results were considered valid if no contamination was observed in these 6 added negative controls, and an amplicon of the expected Leishmania size (Table 2) was obtained. Negative PCR results were valid if no product of the expected size was observed, and the internal control amplicon (Table 2, Fig. 2) was amplified (Fig. 3). In case one of these conditions was not met, the PCR was repeated. All PCR mixes were prepared in bulk, containing all reagents except polymerase and Leishmania template. They were stored at -20°C and when needed an aliquot was taken to which polymerase and template were added. For the purpose of implementation in an endemic reference lab, the tests were transferred to the Kenya Medical Research Institute (KEMRI, Nairobi, Kenya), where PCR mixes were produced locally. The tests in KEMRI were performed by a different person as those at ITMA, but using the same blinded test panel DNA.

Clinical samples

We tested our assays on various clinical samples, containing different species of Leishmania and other pathogens. L. donovani samples were obtained from blood, bone marrow, and lymph node of visceral leishmaniasis patients from Sudan. L. infantum samples were obtained from bone marrow of Italian visceral leishmaniasis patients, and spleen taken from Portuguese dogs suffering of canine leishmaniasis. L. major and L. tropica samples were obtained from skin lesions of cutaneous leishmaniasis patients in Tunisia and Kenya. In addition, our assays were performed on blood from patients suffering Plasmodium falciparum malaria (Uganda) and human African trypanosomiasis (Democratic Republic of Congo), and on sputum from tuberculosis patients (Democratic Republic of Congo). DNA from these samples was extracted using various methods, and the labs acknowledged at the end of this manuscript contributed to the test set.

RESULTS

Sequences

The determined rDNA-ITS1 sequences were submitted to the EBI sequence data base, and were assigned accession numbers listed in Table 1.

Optimization and evaluation

Internal control templates and amplicons were successfully obtained and are depicted in Figs. 2 and3. Their amplification was inhibited completely by 0.01% SDS, indicating their ability to detect PCR inhibitors. All PCRs showed the potential to amplify as little as 0.1 pg of DNA, which is about half a parasite's genome. This detection limit was unaffected by the presence of DNA isolated from human blood, nor by the addition of the internal control template in its proper limiting concentration.

For some DNA samples the detection limit was 1pg, for others it was possible to amplify 0.01pg, variations probably caused by DNA degradation. No cross-reactivity of any specific PCR to other -8 -species was observed during the evaluation phase, except for 1 out of 10 L. aethiopica DNAs that reacted in the LM PCR specific for L. major.

Validation

Table 3 lists the results of a blinded panel validation, containing at least 10 isolated DNAs of each species or species complex. Results obtained at ITMA are given, as well as those obtained at KEMRI when discordant. In total 6 assays were included in the validation, both using 100 or 0.2 pg parasite DNA, the latter spiked with naïve human blood DNA. The 6 assays comprise the 4 speciesspecific assays LA, LDI, LM, and LT1 (Table 2) evaluated separately, but also as a combined tool (5 th assay). In the separate evaluation, only the results of one PCR at the time are taken into account (positive or negative), while in the combined assay results from the 4 separate PCRs are considered simultaneously. In the latter case, a species will be assigned only if 1 out of 4 PCRs scores positive, and is listed as undetermined if none or more than 1 PCR shows a positive result. The reason for evaluating the PCRs separately is that in reality it is unlikely that all 4 PCRs are performed on all samples, and more often than not a PCR will be used to confirm only the expected species. Finally, PCR LT2 makes up the 6 th assay, which was performed following validation of the other assays because of a poor sensitivity of LT1.

Implementation

As a proof of principle to assess the performance when using the developed PCRs in another lab, the species-specific PCRs were implemented and validated at KEMRI, a reference lab in Kenya, where leishmaniasis is endemic. Even though the same products and type of thermocycler were used, there was a problem in the amplification of the internal control amplicons, as well as with a slight contamination in the L. donovani complex PCR. This contamination showed a very faint PCR product and was visible in one out of the 6 user-added negative controls but in none of the negative controls of the blinded test panel. When ignoring these faint amplicons, and the fact that in many negative PCRs no internal control amplicon was amplified, the results were almost identical to those obtained at ITMA (Table 3). PCR LT2 (Table 2) was not tested in KEMRI.

Clinical sample analysis

Fig. 4 depicts the results of 4 species-specific PCRs in clinical samples. The species could successfully be identified in the various Leishmania samples, as only one of the 4 PCRs showed a Leishmania product of the correct size (Table 2). In all other cases, only the internal control amplicon amplified. In LT2, occasionally a weak PCR product is seen in samples not containing L. tropica, but this product is slightly smaller than 100 bp, and clearly below the molecular weight of the product seen in the actual L. tropica samples. Equally so, a very weak amplicon is observed from the LA PCR in the left skin lesion scrapping L. tropica sample, but this is much fainter than the L. tropica amplicon. No products were amplified from patients suffering tuberculosis, sleeping sickness, or malaria.

DISCUSSION

In this study we present 4 ITS1-rDNA PCRs that can faithfully distinguish the Old World Leishmania species L. aethiopica, L. major, L. tropica, and the L. donovani species complex comprised of L. donovani and L. infantum. Because the number of sequences available for this region was limited for certain species, we added additional ones to the public repositories (Table 1).

Compared to other assays devoid of post-amplification steps other than agarose gel analysis (reviewed in [START_REF] Laurent | Identification of Old World Leishmania spp. by specific polymerase chain reaction amplification of cysteine proteinase B genes and rapid dipstick detection[END_REF]), our approach has several advantages. (1) First, our assay is universally applicable in the Old World. Intra-and inter-species variability was validated by testing all species from diverse geographical origins (Table 1). It does not allow to differentiate L. infantum from other members of the L. donovani complex, for which alternatives are available [START_REF] Laurent | Identification of Old World Leishmania spp. by specific polymerase chain reaction amplification of cysteine proteinase B genes and rapid dipstick detection[END_REF][START_REF] Botilde | Comparison of molecular markers for strain typing of Leishmania infantum[END_REF][START_REF] Hide | Species-specific PCR assay for L. infantum / L. donovani discrimination[END_REF][START_REF] Haralambous | Development of a molecular assay specific for the Leishmania donovani complex that discriminates L. donovani / Leishmania infantum zymodemes: a useful tool for typing MON-1[END_REF]. (2) Second, the detection limit is sufficient to analyze clinical samples, as between 5 and 1/20 parasite genomes are amplifiable, and we showed amplification in actual clinical samples. (3) Third, our assays include dUTP, which allows to counter the effect of amplicon contamination using the dUTP/UNG system [27]. This can be particularly important in low resource settings, where basic precautions to avoid carry-over contamination are often not easily adhered to, leading to false positive results. (4) Fourth, all 4 assays use the exact same thermal amplification conditions, which allows parallel analysis. As a plus, PCR mixes containing all components except Leishmania template and enzyme can be stored at -20°C, facilitating batch quality control and saving time. (5) Fifth, internal controls allow checking for consistent amplification across different runs, and PCR sample inhibition [START_REF] Pham | Use of lambda phage DNA as a hybrid internal control in a PCR-enzyme immunoassay to detect Chlamydia pneumoniae[END_REF]. Failure to amplify this control in a negative Leishmania sample points to the presence of inhibitors or an erroneously prepared PCR mix, in which case no conclusions must be drawn from the PCR. In a positive sample, it might simply indicate the Leishmania template outcompetes the control for primer annealing. (6)

Finally, as illustrated in Fig. 1, our assays can be used as a nested PCR in case of insufficient sensitivity, whereby the Leishmania-specific primers LITSR and L5.8S [START_REF] Tai | Genetic heterogeneity of ribosomal internal transcribed spacer in clinical samples of Leishmania donovani spotted on filter paper as revealed by single-strand conformation polymorphisms and sequencing[END_REF] generate the outer PCR amplicons. In such case, all 4 specific PCRs must be run in parallel in the second PCR round to ensure specificity.

As can be seen in Table 3, when taking results from the validation PCRs LA, LDI, LM, LT1, and the combined assay, at the 100 pg level the species discrimination and identification performance is 100%, except for L. aethiopica isolate MHOM/ET/67/L86 which cross-reacted in the LT1 PCR. Species identification of this isolate was however based on its unique zymodeme LON33 [START_REF] Blancq | Leishmania in the Old World: 3. The distribution of L. aethiopica zymodemes[END_REF], and the rDNA-ITS1 region (accession FN677356) clearly showed an L. tropica sequence, explaining the positive amplification in the L. tropica PCR. Nevertheless, a limiting amount of L. aethiopica rDNA-ITS1 template is present in the DNA sample, as evidenced by the additional positive L. aethiopica PCR only at the 100 pg level, pointing to a hybrid strain or a mixed culture. The other PCRs showed no cross-reaction at the 0.2 pg level, but some isolates could not be identified. This was the case especially for L. tropica, where only 4 out of 10 DNAs could be typed. Given the fact that amplification failure may be caused by DNA degradation or mismatched primers, it is not surprising that some are not detected at the 0.2 pg level, corresponding to one parasite only. As the L. tropica PCR however identified merely 4 out of 10 L. tropica DNAs when supplied with 0.2 pg, we used an alternative PCR (LT2 in Table 2) including an outer Leishmania-specific DNA primer pair (Fig. 1, [START_REF] Tai | Genetic heterogeneity of ribosomal internal transcribed spacer in clinical samples of Leishmania donovani spotted on filter paper as revealed by single-strand conformation polymorphisms and sequencing[END_REF]) to boost the reaction. This allowed an additional 3 L. tropica to be identified from 0.2 pg, but resulted in an extra L. aethiopica DNA being detected (Table 3). LT2 must therefore be used only in parallel with PCRs LA, LDI, and LM.

-11 -Upon evaluating the blinded test panel in an endemic reference center in Kenya, two problems were identified: the internal control amplicon did not amplify consistently, and a slight L. donovani contamination was observed in one out of six negative controls. Despite using the same products in the PCR mix, this illustrates the necessity to re-optimize the internal control template concentration during implementation. This becomes even more important when changing PCR reagents, such as the type of Taq DNA polymerase. Equally so, depending on the geographical region and type of clinical samples analyzed, one should be aware of potential cross-reactivity against species other than Leishmania. And even though our analysis did not identify cross-reactions against 3 other pathogens, such problems are region and sample dependent, and a validation should be part of any implementation process. In this respect we also highlight that our assays are to be used in second line for Leishmania species identification, only after performing diagnosticgenerally more sensitive assays for parasite detection. During implementation, it is highly recommended to run all 4 PCRs in parallel to assess the performance of each individual PCR by comparing to the combined assay, as was done in Table 3.

Once validated, PCR mixes without DNA polymerase can be prepared in bulk and aliquoted, which allows quality control and saves time. When functioning properly, there is no need to use all 4

PCRs for analyzing each sample (combined assay in Table 3), and one could suffice by using an appropriate selection depending on the exact purpose of the study, and species expected to occur: in endemic settings this would typically be the sympatric species, in travel clinics all species can be found. At most 100 pg Leishmania DNA should be used in the assays, as cross-reactivity for higher concentrations has not been checked for. In clinical samples it is unlikely that a higher amount of Leishmania DNA is present, but if uncertain it is advisable to run all 4 PCRs in parallel to increase the chance of finding unexpected cross-reactions.

Our assays were able to identify the infecting Leishmania species in various clinical samples (Fig. 4), and did not show cross-reaction against Mycobacterium tuberculosis, Plasmodium falciparum, or Trypanosoma brucei. These tests were performed as a proof-of-principle only, as in particular real-life settings the diagnostic specificity and sensitivity must be determined using -12 -standardized sampling methods, and accounting for the Leishmania species and other pathogens circulating in the region.

Our study presents the first step towards a standardized Old World Leishmania species typing assay. It focuses on validation in cultured samples, with emphasis on implementation of the PCRs in other settings, as a starting point for the use in clinical and epidemiological studies for which the proof-of-principle was delivered. Validation of the PCRs and potential unexpected reactions are essential topics to cover before starting any analysis. We are currently in the process of using the here reported PCRs in endemic reference labs on an extended set of clinical samples, and anticipate that the here described versatile PCRs can contribute to adequate management and follow-up of Old World leishmaniasis. b These amplicon sizes can deviate with a few nucleotides depending on the exact strain or isolate.

c Internal control constructs are depicted in Fig. 2, and the amount used in each PCR is given between brackets. The concentration of the internal control template was estimated spectrophotometrically using the Nanodrop ND-1000 Spectrophotometer (Thermo Scientific, Wilmington, Delaware, USA).

d This is the final concentration used in the PCR, inclusive of the 1.5 mM already present in the Coralload PCR buffer.

e Primer annealing positions are depicted schematically in Fig. 1.

-21 - The geographic origin of the isolates and strains is listed in Table 1.

c See Table 2. For each PCR assay, the number of positive PCRs per species or negative sample type is given.

d When there was no agreement between the numbers for the ITMA and KEMRI validation, the KEMRI result is given between brackets.

e For the combined assay, the number of correctly and wrongly identified isolates from each species is listed, as determined from a parallel evaluation of LA, LDI, LM, and LT1 PCRs.

f

The LT2 PCR was not tested in KEMRI, and was not taken into account in the combined assay. 2, and the annealing position of the 5' base is indicated, also delineating the start and end of each amplicon. As the annealing position of all primers is given relative to L. major, fragment sizes calculated from these do not correspond to those indicated in Table 2, except for L. major itself.

Fig. 2.

Overview of the 4 cloned PCR products used as internal controls (Table 2). These PCR products were cloned in the pCR4-TOPO vector (tools.invitrogen.com/content/sfs/vectors/pcr4topo_map.pdf), from which the cloning site sequence is shown below. Amplicons were inserted using the T:A overhang strategy, and the orientation in the obtained plasmids is as shown in the figure. The internal phage lambda fragments are described in the grey boxes, with positions referring to accession J02459 (www.ebi.ac.uk, accession number J02459, release 102). The regions corresponding to the Leishmania species-specific primer sequences (Table 2) are indicated in the outlined boxes, with the arrows showing the 5'-3' orientation. Leon-Rot, Germany) is depicted on the left (M), with indicated fragments of 100 (smallest fragment), 500 (brightest fragment), and 1000 (largest fragment) nucleotides. Expected sizes are shown in Table 2. and Leishmania fragments is as in Table 2 and Fig. 3, and is respectively indicated by white and black arrows on the right.

Fig. 1 .

 1 Fig. 1. The total reaction volume was 25 µl of 1x Coralload PCR buffer (Qiagen, Venlo, The

a

  Only reagents differing between the PCRs are listed. Common reagents in all PCRs (buffer, BSA, dNTPs, dUTP) are described in Materials and Methods.

a

  Negatives are TE in the 100 pg panel, naïve human blood in the 0.2 pg panel. b

Fig. 1 .

 1 Fig. 1. Position of the sequenced and species-specific PCR fragments relative to the first nucleotide of

Fig. 3 .

 3 Fig. 3. Agarose gel image of the Leishmania species-specific PCR products (L), and the same

Fig. 4 .

 4 Fig. 4. Results from 4 species-specific PCRs on clinical samples. The PCRs are indicated on the left,

Table 2 :

 2 Overview of species-specific PCRs a

Table 3 :

 3 Overview of blinded panel validation result

	Sample type or Leishmania species
	L. donovani
	L. aethiopica	L. major	L. tropica	Negative a
	complex
	Total number of samples b

ACKNOWLEDGEMENTS

We thank Gabi Schönian (Institut für Mikrobiologie und Hygiene, Berlin, Germany), Henk Schallig and Gerard Schoone (Koninklijk Instituut voor de Tropen, Amsterdam, The Netherlands), and Isabel Mauricio (London School of Tropical Medicine and Hygiene, London, United Kingdom) for contributing DNA from reference strains used in the study. Clinical samples were obtained from (1) Lenea Campino (Institute of Hygiene and Tropical Medicine, Lisbon, Portugal); (2) Luigi Gradoni and Marina Grammicia (Istituto Superiore di Sanità, Rome, Italy); (3) Hechmi Louzir and Afif Ben Salah (Institut Pasteur de Tunis, Tunis, Tunisia); (4) Chantal Van Overmeir, Umberto d'Alessandro, Pim De Rijk, Leen Rigouts, Françoise Portaels, Stijn Deborggraeve, and Philippe Büscher (Institute of Tropical Medicine Antwerp, Antwerp, Belgium). This study was financed by the commission of the European Community's Sixth Framework Program, priority INCO-DEV, project TRYLEIDIAG, contract 015379. SOCO and GVDA are supported by the third framework program of the Belgian Development Cooperation with ITMA.

The authors declare that they have no conflict of interest.

a Where possible, different parasite strains were used for the evaluation and validation.

b Accession numbers are listed according to species and origin, but are not always determined from the strains or isolates used for the evaluation and validation.

c Species of the L. donovani complex (L. donovani and L. infantum) are defined as in [START_REF] Lukeš | Evolutionary and geographical history of the Leishmania donovani complex with a revision of current taxonomy[END_REF].

g

The L. aethiopica isolate MHOM/ET/67/L86 was identified as L. tropica.


E F W Courrech Staal

Mwjm W J M Wouters

H Boot

Raem A E M Tollenaar

J W Van Sandick

Efw Courrech Staal

Courrech Efw
email: e.courrech@nki.nl

Staal

Quality-of-care indicators for oesophageal cancer surgery

Keywords: Oesophageal Neoplasms, Oesophagectomy, Quality of care, Surgery

Background

Quality-of-care indicators are measurable elements of practice performance that can assess the (change in) quality of the care provided. To date, the literature on quality-of-care indicators for oesophageal cancer surgery has not been reviewed.

Methods

We performed a review of the literature on quality-of-care indicators for oesophageal cancer surgery. The indicators were classified by their nature of care provision (structural, process, or outcome).

Results

One hundred thirty articles were included. For structural measures, most evidence was found for the inverse relationship between hospital or surgeon volume and postoperative mortality.

Few articles described the required infrastructural and organisational elements for oesophageal cancer surgery. Regarding process measures, the most common indicators were determinants of patient selection for surgery. Other process indicators with considerable evidence were found (e.g., multidisciplinary team management), though the number of studies was small. For outcome indicators, the level of evidence for pathological outcome measures was strong. Data on postoperative complications as outcome indicators varied widely.

Conclusion

Since there is considerable variation in the evaluation of quality of care, the uniform use of well-defined quality-of-care indicators to measure and document practice performance holds the promise of improving outcome in patients who undergo oesophageal cancer surgery.

Introduction

Quality assurance in the treatment of cancer is gaining importance since many studies have shown variation in outcome between different providers. In Europe, quality assurance programmes have been introduced in the field of radiotherapy as well as for medical oncology, however, surgical quality control has received less attention 1;2 . Only recently, the European Society for Surgical Oncology (ESSO) has started an international audit program for rectal cancer treatment 3;4 . Few attempts have been made to spread the merits of quality assurance programmes to other tumour types.

Evidence-based guidelines that have been developed for a large variety of cancer treatments worldwide are seldom accompanied by well-defined standards for the evaluation of the quality of surgical care. Donabedian has conceptualized the evaluation of patient care in terms of structure, process, and outcome measures 5 .

Oesophageal cancer ranks sixth on the list of cancer mortality worldwide 6 . In 2008, the incidence in the Netherlands was around 1850 patients per year [START_REF] Patti | A hospital's annual rate of esophagectomy influences the operative mortality rate[END_REF] . It has been recommended to concentrate the surgical treatment of oesophageal cancer in high-volume. The effectiveness of such measures in raising the whole level of care has been questioned 8 .

Preferably, the concentration of oesophageal cancer treatment is accompanied by a national quality assurance program, evaluating the different dimensions of quality of care in all hospitals taking care of these patients [START_REF] Leigh | Surgical specialty, surgical unit volume and mortality after oesophageal cancer surgery[END_REF] . A practical definition of quality of care would be the degree to which health services achieve a level of care deemed adequate by evidencebased quality measures [START_REF] Kuo | Impact of hospital volume on clinical and economic outcomes for esophagectomy[END_REF] .

We have performed a review of the literature to identify evidence-based standards for highlevel quality of care for oesophageal cancer patients who are candidates for surgical therapy. We used the Donabedian quality-of-care model to categorize the identified standards. Furthermore, we aimed to construct a minimum dataset of evidence-based quality-of-care indicators for future registration and benchmarking.

Materials and Methods

Search strategy

A search of the literature on PubMed was performed to find articles published between January 1990 and October 2009 on quality of care in the surgical treatment of oesophageal cancer. Three Medical Subject Heading (MeSH) terms were used: 'oesophageal neoplasms', 'surgery' and 'oesophagectomy'. Studies describing aspects of quality of care were searched by combining these three MeSH terms with the following keywords: 'benchmarking', 'health care', 'hospital mortality', 'hospitals', 'medical audit', 'outcome and process assessment (health care)', 'postoperative complications', 'quality assurance', 'quality indicators', 'referral and consultation'. Articles were selected on the basis of their relevance using pre-defined inand exclusion criteria (Figure 1). Many studies were excluded because they were noncomparative studies. Only original articles were considered for inclusion. Selection was performed independently by two investigators (ECS and MW). A third reviewer (JvS) was consulted in case of disagreement. One hundred thirty articles were included.

Classification of studies

Articles were categorized according to the primary subject of the study: structural, process or outcome measures. Structural components of care are characteristics of the provider, reflecting the setting in which care is delivered (e.g. staff expertise). Process components of care refer to the interactions between the provider (i.e., physician) and the patient (e.g., staging process). Outcome characteristics are measurable short-term outcomes affecting the final outcome of patients (e.g., radicality of resection).

Level of evidence

Of 130 included articles, 13 were randomised controlled trials, 26 were prospective (cohort) studies and 91 were retrospective studies. Because of the comparable level of evidence for most of these included studies, we classified results of evidence according to a scoring system used in a previous article by Lagarde et al:: none (no evidence), minor (only evidence from univariate analysis), considerable (evidence from uni-and multivariate analysis), strong (evidence from several multivariate analyses or evidence from univariate analysis in at least ten articles) [START_REF] Kazui | An attempt to analyze the relation between hospital surgical volume and clinical outcome[END_REF] .

Only characteristics with strong evidence or those with considerable evidence based on at least three articles were entered into a minimum dataset of 'evidence-based' quality-of-care indicators for oesophageal cancer surgery.

Structural measures

Structural variables with corresponding articles are listed in Table 1.

Volume

A wide variation in definitions of hospital volume was found in the various studies on hospital volume and mortality rates (e.g., the definition of high-volume ranged from 6 to 40 resections per year) . Besides the clear association between high volume and low mortality rates, also lower complication rates and shorter admission times have been found in high-volume hospitals as compared to low-volume hospitals 1;2;7;8;13;17;26;27 . Consequently, treatment in highvolume hospitals has been associated with a decrease in hospital charges 1;7;10;12;24 . Other clinical endpoints, such as long-term survival and quality of life, have been studied less often Two out of three studies 18;28;29 could not demonstrate a survival benefit in high-volume hospitals 18;28 and one other study did not find an improved quality of life 30 .

As in studies on the influence of hospital volume, higher annual case volume per surgeon has been associated with lower postoperative mortality rates 6;15;18;31-37 .

Specialization

Volume is related to, but does not equal specialization. Three studies have reported on the influence of subspecialty training of the surgeon on outcome (Table 1) 9;38;39 . In two studies, a lower postoperative mortality rate has been found in patients operated on by cardiothoracic surgeons as compared to that in patients operated on by a general surgeon 9;39 . The third study reported no significant differences in outcome between general and thoracic surgeons [START_REF] Smith | Outcomes of esophagectomy according to surgeon's training: general vs. thoracic[END_REF] . To date, no comparisons between dedicated upper gastrointestinal and general surgical oncologists have been made.

Organization

Few studies specifically addressed the impact of organization of care on the outcome of surgically treated oesophageal cancer patients. In one study, daily rounds by an ICU physician were associated with shorter lengths of stay, lower hospital cost, and less postoperative complications after oesophageal resection, but not with a lower in-hospital mortality rate [START_REF] Dimick | Intensive care unit physician staffing is associated with decreased length of stay, hospital cost, and complications after esophageal resection[END_REF] . In an other large study, a comparison between 225 patients with a night-time nurse-to-patient ratio of 1:1 or 1:2 and 128 patients with a night-time nurse-to-patient ratio of 1:3 or more was made41. Patients in the second group had an increased risk of postoperative complications after oesophageal resection which was associated with an increased use of resources.

Centralization

Following the relationship between volume/specialization and outcome, one may expect that centralization of oesophagectomies at high-volume providers improves outcome. Few studies have actually demonstrated this 1;42;43 . Recently, we have shown a dramatic improvement in mortality and survival after centralization of oesophageal cancer surgery in a region of the Netherlands 9 . This centralization process was accompanied by feedback of detailed clinical data to individual hospitals and surgeons (surgical audit).

Process measures

Process variables with corresponding articles are listed in Table 2.

Patient selection

In several studies, the influence of old age was investigated [START_REF] Alexiou | Surgery for esophageal cancer in elderly patients: the view from Nottingham[END_REF][START_REF] Alibakhshi | The effect of age on the outcome of esophageal cancer surgery[END_REF][START_REF] Ellis | Cancer of the esophagus and cardia: does age influence treatment selection and surgical outcomes?[END_REF][START_REF] Fontes | Is age a risk factor for esophagectomy?[END_REF][START_REF] Johansson | Clinical outcome and long-term survival rates after esophagectomy are not determined by age over 70 years[END_REF][START_REF] Ma | Clinicopathologic characteristics of esophagectomy for esophageal carcinoma in elderly patients[END_REF][START_REF] Rice | Preoperative chemoradiotherapy prior to esophagectomy in elderly patients is not associated with increased morbidity[END_REF][START_REF] Ruol | Results of esophagectomy for esophageal cancer in elderly patients: age has little influence on outcome and survival[END_REF][START_REF] Fang | Three-field lymph node dissection for esophageal cancer in elderly patients over 70 years of age[END_REF][START_REF] Morita | Esophagectomy in patients 80 years of age and older with carcinoma of the thoracic esophagus[END_REF][START_REF] Sabel | Esophageal resection for carcinoma in patients older than 70 years[END_REF][START_REF] Moskovitz | Mortality increases for octogenarians undergoing esophagogastrectomy for esophageal cancer[END_REF][START_REF] Takeno | Esophagectomy in patients aged over 80 years with esophageal cancer[END_REF] . In a minority of studies, older age was associated with an unfavourable postoperative outcome 44;49;52;53;55;56. The use of a risk score may be a quality indicator. Bartels et al evaluated a risk scoring model in a prospective setting and found a marked reduction in post-operative deaths due to better patient selection [START_REF] Bartels | Preoperative risk analysis and postoperative mortality of oesophagectomy for resectable oesophageal cancer[END_REF] .

The relationship between the preoperative nutritional status and the outcome of surgery in patients with oesophageal carcinoma has shown conflicting results [START_REF] Hop | Nutritional status as a risk factor in esophageal surgery[END_REF][START_REF] Mullen | Impact of body mass index on perioperative outcomes in patients undergoing major intra-abdominal cancer surgery[END_REF][START_REF] Kunisaki | Immunonutrition risk factors of respiratory complications after esophagectomy[END_REF][START_REF] Kilic | Impact of obesity on perioperative outcomes of minimally invasive esophagectomy[END_REF] . In one study, underweight patients who underwent major intra-abdominal surgery, e.g., oesophagectomy, had a five-fold increased risk of postoperative mortality [START_REF] Mullen | Impact of body mass index on perioperative outcomes in patients undergoing major intra-abdominal cancer surgery[END_REF] . Though, other studies including exclusively oesophageal cancer patients have not confirmed this 58;60 . In four studies, complication rates for obese patients equalled those for non-obese patients [START_REF] Hop | Nutritional status as a risk factor in esophageal surgery[END_REF][START_REF] Mullen | Impact of body mass index on perioperative outcomes in patients undergoing major intra-abdominal cancer surgery[END_REF][START_REF] Kunisaki | Immunonutrition risk factors of respiratory complications after esophagectomy[END_REF][START_REF] Kilic | Impact of obesity on perioperative outcomes of minimally invasive esophagectomy[END_REF] .

The role of race and socio-economic status on patient selection has received some attention [START_REF] Dominitz | Race, treatment, and survival of veterans with cancer of the distal esophagus and gastric cardia[END_REF][START_REF] Silverstein | Access to care and stage at diagnosis for patients with lung cancer and esophageal cancer: analysis of the Savannah River Region Information System cancer registry data[END_REF][START_REF] Steyerberg | Racial differences in surgical evaluation, treatment, and outcome of locoregional esophageal cancer: a populationbased analysis of elderly patients[END_REF][START_REF] Van Vliet | The role of socio-economic status in the decision making on diagnosis and treatment of oesophageal cancer in The Netherlands[END_REF] . In the United States, African-American patients with oesophageal cancer were less likely to undergo surgical resection compared to Caucasian patients [START_REF] Dominitz | Race, treatment, and survival of veterans with cancer of the distal esophagus and gastric cardia[END_REF][START_REF] Silverstein | Access to care and stage at diagnosis for patients with lung cancer and esophageal cancer: analysis of the Savannah River Region Information System cancer registry data[END_REF][START_REF] Steyerberg | Racial differences in surgical evaluation, treatment, and outcome of locoregional esophageal cancer: a populationbased analysis of elderly patients[END_REF] . In the Netherlands, low socio-economic status proved to be associated with a lower chance of resection [START_REF] Van Vliet | The role of socio-economic status in the decision making on diagnosis and treatment of oesophageal cancer in The Netherlands[END_REF] . These disparities are not fully explained by differences in medical factors [START_REF] Steyerberg | Racial differences in surgical evaluation, treatment, and outcome of locoregional esophageal cancer: a populationbased analysis of elderly patients[END_REF] . It has been suggested that patients' but possibly also physicians' preferences might differ among different socio-economic groups of patients [START_REF] Van Vliet | The role of socio-economic status in the decision making on diagnosis and treatment of oesophageal cancer in The Netherlands[END_REF] .

In one study, there was a relationship between better pretreatment quality of life and lower postoperative mortality [START_REF] Healy | Health-related quality of life assessment at presentation may predict complications and early relapse in patients with localized cancer of the esophagus[END_REF] . Especially reduced physical function as an aspect of pretreatment quality of life was predictive of lower survival in several studies [START_REF] Blazeby | The prognostic value of quality of life scores during treatment for oesophageal cancer[END_REF][START_REF] Fang | Quality of life as a survival predictor for esophageal squamous cell carcinoma treated with radiotherapy[END_REF][START_REF] Chau | Multivariate prognostic factor analysis in locally advanced and metastatic esophago-gastric cancer--pooled analysis from three multicenter, randomized, controlled trials using individual patient data[END_REF][START_REF] Mckernan | The relationship between quality of life (EORTC QLQ-C30) and survival in patients with gastrooesophageal cancer[END_REF] .

Staging

For high-risk surgical procedures, it is important to select only those patients who can be cured. For computed tomography (CT) examination, the level of experience of the radiologist appeared to influence the detection of metastases in patients with oesophageal cancer [START_REF] Van Vliet | Radiologist experience and CT examination quality determine metastasis detection in patients with esophageal or gastric cardia cancer[END_REF] .

Fluorodeoxyglucose positron emission tomography (FDG-PET) has shown its incremental value with the identification of 5 to 17% additional patients with metastases [START_REF] Flamen | Utility of positron emission tomography for the staging of patients with potentially operable esophageal carcinoma[END_REF][START_REF] Imdahl | Impact of FDG-PET for staging of oesophageal cancer[END_REF][START_REF] Kato | The incremental effect of positron emission tomography on diagnostic accuracy in the initial staging of esophageal carcinoma[END_REF][START_REF] Lowe | Comparison of positron emission tomography, computed tomography, and endoscopic ultrasound in the initial staging of patients with esophageal cancer[END_REF][START_REF] Meyers | The utility of positron emission tomography in staging of potentially operable carcinoma of the thoracic esophagus: results of the American College of Surgeons Oncology Group Z0060 trial[END_REF][START_REF] Rankin | Computed tomography and positron emission tomography in the pre-operative staging of oesophageal carcinoma[END_REF][START_REF] Salahudeen | Impact of the introduction of integrated PET-CT into the preoperative staging pathway of patients with potentially operable oesophageal carcinoma[END_REF][START_REF] Wallace | An analysis of multiple staging management strategies for carcinoma of the esophagus: computed tomography, endoscopic ultrasound, positron emission tomography, and thoracoscopy/laparoscopy[END_REF] . Van Vliet et al studied the results of endoscopic ultrasonography (EUS) performed in low-volume EUScentres and found unfavourable results in comparison with those in high-volume EUScentres [START_REF] Van Vliet | Staging of esophageal carcinoma in a low-volume EUS center compared with reported results from high-volume centers[END_REF] .

Treatment choices

Multimodality treatment

Several studies have compared the use of neoadjuvant chemoradiation with surgery alone for patients with oesophageal cancer [START_REF] Nygaard | Preoperative radiotherapy prolongs survival in operable esophageal carcinoma: a randomized, multicenter study of pre-operative radiotherapy and chemotherapy. The second Scandinavian trial in esophageal cancer[END_REF][START_REF] Prise | A randomized study of chemotherapy, radiation therapy, and surgery versus surgery for localized squamous cell carcinoma of the esophagus[END_REF][START_REF] Apinop | A prospective study of combined therapy in esophageal cancer[END_REF][START_REF] Bosset | Chemoradiotherapy followed by surgery compared with surgery alone in squamouscell cancer of the esophagus[END_REF][START_REF] Kelley | Neoadjuvant chemoradiotherapy is not associated with a higher complication rate vs. surgery alone in patients undergoing esophagectomy[END_REF][START_REF] Lin | Induction therapy does not increase surgical morbidity after esophagectomy for cancer[END_REF][START_REF] Zhang | Neoadjuvant chemoradiotherapy for esophageal carcinoma[END_REF][START_REF] Burmeister | Surgery alone versus chemoradiotherapy followed by surgery for resectable cancer of the oesophagus: a randomised controlled phase III trial[END_REF][START_REF] Berger | Morbidity and mortality are not increased after induction chemoradiotherapy followed by esophagectomy in patients with esophageal cancer[END_REF][START_REF] Kesler | A retrospective analysis of locally advanced esophageal cancer patients treated with neoadjuvant chemoradiation therapy followed by surgery or surgery alone[END_REF][START_REF] Reynolds | Neoadjuvant chemoradiation may increase the risk of respiratory complications and sepsis after transthoracic esophagectomy[END_REF][START_REF] Mariette | Impact of preoperative radiochemotherapy on postoperative course and survival in patients with locally advanced squamous cell oesophageal carcinoma[END_REF][START_REF] Ruol | Effects of neoadjuvant therapy on perioperative morbidity in elderly patients undergoing esophagectomy for esophageal cancer[END_REF][START_REF] Walsh | A comparison of multimodal therapy and surgery for esophageal adenocarcinoma[END_REF][START_REF] Urba | Randomized trial of preoperative chemoradiation versus surgery alone in patients with locoregional esophageal carcinoma[END_REF][START_REF] Lee | A single institutional phase III trial of preoperative chemotherapy with hyperfractionation radiotherapy plus surgery versus surgery alone for resectable esophageal squamous cell carcinoma[END_REF][START_REF] Berger | Complete response to neoadjuvant chemoradiotherapy in esophageal carcinoma is associated with significantly improved survival[END_REF] . Although a negative effect of neoadjuvant treatment on postoperative morbidity [START_REF] Reynolds | Neoadjuvant chemoradiation may increase the risk of respiratory complications and sepsis after transthoracic esophagectomy[END_REF] and mortality 84 was found in two separate studies, most studies could not demonstrate these differences 81-83;85-87,89;92-94 . Regarding overall survival, there was a benefit of neoadjuvant chemoradiation in two out of 11 studies 92;94 .

Multidisciplinary team

It is generally believed that a multidisciplinary approach in cancer treatment results in the best achievable outcomes. The added value is hardly measurable. Nevertheless, in a study by Stephens et al, the selection, staging and treatment of patients eligible for oesophagectomy by a multidisciplinary team resulted in a better survival as compared to the survival of patients treated by surgeons alone [START_REF] Stephens | Multidisciplinary team management is associated with improved outcomes after surgery for esophageal cancer[END_REF] .

Surgical approach

Four randomized trials have compared the outcome of a limited transhiatal approach versus an extended transthoracic approach [START_REF] Chu | A prospective randomized comparison of transhiatal and transthoracic resection for lower-third esophageal carcinoma[END_REF][100][101][102] . Differences in postoperative morbidity were in favour of a limited transhiatal approach, and one trial showed a trend towards better survival in patients operated via an extended transthoracic approach 101 . Complete 5-year survival data suggested that patients with a tumour in the distal oesophagus had benefitted from an extended transthoracic resection 103 .

Peri-operative care

Watson et al showed that respiratory complications decreased from 30 to 13% and death due to these complications from 5 to 0%, after introducing the routine use of thoracic epidural analgesia (TEA) 104 . In an other series, TEA lowered the anastomotic leakage rate 105 . The authors suggested a causal relationship between hypoxemia and hypotension due to respiratory hypofunction in patients undergoing oesophagectomy without the use of TEA. It has also been shown that the use of TEA enables early discharge of patients after oesophagectomy 106 .

Pathology reporting

Histopathological assessment of the resection specimen plays an important role in patient management, in confirming whether complete excision has been achieved and in providing essential information on pathological tumour-node-metastasis (TNM) staging. The need to improve the quality of pathology reporting in oesophageal cancer management has been recognised 107;108 .

Outcome measures

Outcome variables with corresponding articles are listed in Table 3.

ICU stay

Length of ICU stay did not influence patients' survival and long-term quality of life 109 .

Postoperative complications

The occurrence of postoperative complications after oesophageal cancer surgery has not only been associated with higher postoperative mortality rates and increased use of resources, but also with worse survival 27;110-119 .

Radicality of resection

Multiple studies 120-126 have shown the independent prognostic value of a microscopically radical (R0) resection [120][121][122][123][124][125][126][127] .

Number of resected lymph nodes

The number of identified lymph nodes is an independent predictor of survival after oesophagectomy for cancer [128][129][130] . According to Peyre et al, a minimum of 23 lymph nodes should be resected 130 .

'Evidence-based' quality indicators for oesophageal cancer surgery

A minimum dataset of 'evidence-based' quality-of-care indicators for the surgical treatment of oesophageal cancer was created based on the identified standards with strong evidence or those with considerable evidence in at least three articles. This dataset is presented in Table 4.

Discussion

This has been the first review of the literature to identify evidence-based standards for highlevel quality of care for oesophageal cancer patients who are candidates for surgery. Results

show that (1) there is strong evidence that both hospital and surgeon volume are important determinants for postoperative mortality, (2) other structural measures, e.g., infrastructure and organization of oesophageal cancer surgery, have been less frequently investigated, (3) the most commonly reported process measures were determinants of patient selection for surgery (e.g., patients' age), ( 4) other process indicators with considerable evidence were found (e.g., multidisciplinary team management), though the number of studies was small, and ( 5) the level of evidence for pathological outcome measures was high.

Structural measures

A plethora of studies concerning the volume-outcome relationship for oesophageal cancer surgery was found. High-volume and specialized care were mostly related to a decreased postoperative mortality, and, in a lesser extent, to lower postoperative morbidity, shorter hospital stay, better survival and lower costs. Volume is only a surrogate for high-level processes of care and does not reveal the mechanisms behind the better outcomes. There is evidence that centralization of oesophageal cancer resections leads to substantial improvements in outcome. Such efforts have been accompanied by continuous measurement and feedback of process and outcome indicators to individual surgeons and their referring colleagues. Only then, improvements in outcome are to be expected [START_REF] Leigh | Surgical specialty, surgical unit volume and mortality after oesophageal cancer surgery[END_REF] . Data on infrastructural or organizational characteristics that lead to success or failure, were very limited (e.g., ICU staffing) or absent (e.g., ICU level).

Process measures

Evidence was found that teams using a risk score for the selection of surgical patients can decrease their postoperative mortality rates [START_REF] Dimick | Hospital volume is related to clinical and economic outcomes of esophageal resection in Maryland[END_REF] . Several risk-prediction models have been proposed for this purpose, such as the Physiologic and Operative Severity Score for the enumeration of Mortality and morbidity (O-POSSUM) [START_REF] Dimick | Surgical volume and quality of care for esophageal resection: do high-volume hospitals have fewer complications?[END_REF] . The O-POSSUM has only been studied retrospectively showing a two-or three-fold overprediction of in-hospital mortality [START_REF] Allareddy | Specificity of procedure volume and in-hospital mortality association[END_REF][START_REF] Bachmann | Cohort study in South and West England of the influence of specialization on the management and outcome of patients with oesophageal and gastric cancers[END_REF][START_REF] Birkmeyer | Potential benefits of the new Leapfrog standards: effect of process and outcomes measures[END_REF] .

Steyerberg et al. developed a more simple risk score, that also included the excess risk on postoperative mortality introduced by operations performed in low-volume hospitals 17 . Again, this risk score could not be validated by others 18;19 .

Age, nutritional or socio-economic status should not be used as selection criteria for oesophageal cancer surgery. On the other hand, in assessing resection rates or surgical outcome, these factors are to be included as case-mix variables. Preoperative feeding to prevent further deterioration of the nutritional state of patients presenting with obstructive symptoms and weight loss could be a valid quality indicator, but the level of evidence for a better outcome is low 20 .

We found evidence -although limited-that volume and experience play a role in the staging process of patients with oesophageal cancer 21;22 . It would be better to assess the whole staging process by calculating the percentage of patients in whom a futile operation has been performed due to inadequate staging.

Neoadjuvant chemoradiation followed by surgery was associated with similar postoperative mortality rates as surgery alone in all studies, and survival rates improved in two studies 23;24 .

If neoadjuvant chemoradiation becomes standard of care, there is a need to formulate quality indicators for its use (e.g., toxicity criteria).

Diagnosis and treatment of oesophageal cancer patients by a dedicated multidisciplinary team could be an important quality indicator, but this is supported by only one paper 25 . Not only the expertise of the surgeon, but also that of the radiologist, anaesthesiologist, ICUphysician and nurses contribute to the outcome of surgery.

There is considerable evidence that the transhiatal approach leads to a reduced postoperative morbidity rate [26][27][28][29] . Presumed that the transthoracic approach with an extended lymphadenectomy is technically more challenging, one could propose that the performance of transthoracic oesophagectomies in patients with a tumour located in the distal oesophagus should be regarded as a quality indicator.

No studies were found on other process indicators such as waiting times or psychological guidance during treatment.

Outcome measures

Outcome measures, like postoperative complication rates, tumour negative surgical margins, and number of retrieved lymph nodes are plausible measures of quality of care for physicians and their patients.

Further investigation is warranted to look for additional valid outcome parameters of quality of care (e.g., hospital readmission rates, pain scores, number of anastomotic dilatations).

Limitations

A major limitation to this review is that there is no MESH term for "quality of care". We have tried to give an overview of the available literature on evidence-based determinants for highleverage quality of care, but our review may have been biased by the choice of our search terms. Secondly, case-mix plays an important role in evaluating differences in outcome after oesophageal cancer surgery. Consequently, for many studies, with a lack of information on patients' co-morbidity, tumour stage distribution, and patient selection criteria, the conclusions are debatable.

Future directions

The uniform use of well-defined quality-of-care indicators to measure and document practice performance holds the promise of improving outcomes in patients who undergo surgical treatment for oesophageal cancer. Recently, another evidence-based review of oesophageal cancer surgery was published 30 . In this review, non-surgical issues were not addressed. The present review places oesophageal cancer surgery in a broader perspective. Improving the level of care for surgical oesophageal cancer patients is a team effort from diagnosis, staging and risk assessment to follow-up and management of late sequelae of treatment. Ideally, each step is monitored by a set of measurable elements which reflect the quality of care.

Several projects have been started in which quality indicators are to be developed, not only based on evidence from the literature, but also on consensus of experts in the field. In Denmark, the Danish National Indicator Project has shown that continuous performance and outcome measurement, using clinical indicators is possible and fruitful in terms of quality improvement 31 . Recently, Bilimoria et al presented an extensive set of quality indicators for pancreatic cancer 32 . After reviewing the literature for potential quality indicators, a Delphi method was used to develop quality indicators consulting several expert panels. In our opinion, a similar procedure should be pursued for oesophageal cancer care. To benchmark the outcome of consensus-based quality indicators, multi-centred data-collection, dataanalysis and feedback of individual data is essential to provide physicians with actionable information about their quality of care.

( 9 )

 9 Wouters MW, Karim-Kos HE, Le Cessie S, Wijnhoven BP, Stassen LP, Steup WH et al. Centralization of esophageal cancer surgery: does it improve clinical outcome? Ann Surg Oncol 2009; 16(7):1789-1798. (10) Leonardi MJ, McGory ML, Ko CY. Quality of care issues in colorectal cancer. Clin Cancer Res 2007; 13(22 Pt 2):6897s-6902s. (11) Lagarde SM, Ten Kate FJ, Reitsma JB, Busch OR, van Lanschot JJ. Prognostic factors in adenocarcinoma of the esophagus or gastroesophageal junction. J Clin Oncol 2006; 24(26):4347-4355. (12) Bartels H, Stein HJ, Siewert JR. Preoperative risk analysis and postoperative mortality of oesophagectomy for resectable oesophageal cancer. Br J Surg 1998; 85(6):840-844.(13) Tekkis PP, McCulloch P, Poloniecki JD, Prytherch DR, Kessaris N, Steger AC. Riskadjusted prediction of operative mortality in oesophagogastric surgery with O-POSSUM. Br J Surg 2004; 91(3):288-295. (14) Lagarde SM, Maris AK, de Castro SM, Busch OR, Obertop H, van Lanschot JJ. Evaluation of O-POSSUM in predicting in-hospital mortality after resection for oesophageal cancer. Br J Surg 2007; 94(12):1521-1526. (15) Nagabhushan JS, Srinath S, Weir F, Angerson WJ, Sugden BA, Morran CG. Comparison of P-POSSUM and O-POSSUM in predicting mortality after oesophagogastric resections. Postgrad Med J 2007; 83(979):355-358. (16) Lai F, Kwan TL, Yuen WC, Wai A, Siu YC, Shung E. Evaluation of various POSSUM models for predicting mortality in patients undergoing elective oesophagectomy for carcinoma. Br J Surg 2007; 94(9):1172-1178.

Acknowledgements

The authors thank Professor Bin BR Kroon for his valuable comments.

Conflict of interest statement

None


Lucien Wald

Philippe Blanc

Bella Espinar

Benoît Gschwind

Mireille Lefèvre

Lionel Ménard

Fabien Wald

Carsten Hoyer-Klick

Marion Schroedter Homscheidt

Claire Thomas

Marion Schroedter-Homscheidt

Early achievements towards an automatic assessment of the uncertainty in solar irradiation using web services

des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Introduction

The solar radiation reaching the ground level on horizontal surfaces is of paramount importance in many applications, from climate to health (ESRA, 2000). The amount of power available on a surface of 1 m² integrated over the whole spectrum of the solar radiation is called surface solar irradiance (SSI). SSI is expressed in W/m². The SSI is an Essential Climate Variable as designed by the Global Climate Observing System in August 2010 [START_REF] Gcos | Global Climate Observing System (GCOS) Essential Climate Variables[END_REF], meaning that it is a parameter of key importance for understanding and monitoring the global climate system. Meteorological networks measure the density of energy received on a horizontal plane at ground level during a certain period, e.g. 15 min, an hour or a day. This is the 15-min, hourly or daily irradiation, also called 15-min, hourly or daily solar exposure, and expressed in MJ/m² or J/cm². The hourly mean of SSI is derived from the hourly irradiation by dividing by the number of seconds in 1 h, i.e. 3600 s. By convention, the daily mean of SSI is computed from the daily irradiation by dividing it by the number of seconds in 24 h, i.e., 86400 s. Accurate assessments of irradiation can now be drawn from satellite data (Blanc et al., 2011a). Stations at ground level measuring irradiation on the long-term (i.e. more than 10 years) are rare and satellites are an accurate way to complement or supplement them. There are several advantages in satellite-derived assessments of irradiation. They cover large areas, such as for example Europe, Africa and the Atlantic Ocean at one glance. Fig. 1 shows an example of the synoptic view offered by the Meteosat series of geostationary satellites. Such images clearly depict clouds and more generally the optical state of the atmosphere. These satellites take images every 15 min. It means that we have an assessment of the 15-min irradiation received by a pixel every 15 min, where a pixel corresponds to a ground surface of 3x3 km² in the subsatellite (or nadir) point. This makes a wealth of information that cannot be supplied by ground-based meteorological networks. Several initiatives were launched in order to create databases of irradiation by processing satellite images [START_REF] Cros | Survey of the main databases providing solar radiation data at ground level[END_REF][START_REF] Wald | Available databases, products and services[END_REF]. Among them, is the SOLEMI project initiated by the German Aerospace Center DLR [START_REF] Meyer | Solar energy mining: High-resolution Meteosat-based service for solar radiation products for Europe, Africa and Asia[END_REF]. Another initiative is the HelioClim project launched by MINES ParisTech in 1997, to increase knowledge on irradiation and to offer irradiation values for any site and any instant over Europe, Africa and the Atlantic Ocean and long period of time, to a wide audience [START_REF] Rigollier | The HelioClim Project: From satellite Images to solar radiation maps[END_REF]Blanc et al., 2011a). The HelioClim-1 database offers daily irradiation for the period 1985-2005. It has been created from archives of images of the Meteosat First Generation satellites. The HelioClim-3 database represents a step forward regarding spatial and time resolution, improving assessment of uncertainty on SSI, and reducing time needed to access recent data. It exploits the enhanced capabilities of the series of satellites Meteosat Second Generation to deliver values of irradiation every 15 min with a spatial resolution of 3 km at nadir. To achieve this, MINES Pa-risTech has set up a routine operation for converting Meteosat data into 15-min irradiation on a nearreal time basis. The SoDa Service (www.soda-is.com) was selected as the means to disseminate the HelioClim databases in order to benefit from the notoriety of this service. SoDa was initially a European project for the integration and exploitation of networked solar radiation databases [START_REF] Wald | SoDa: a project for the integration and exploitation of networked solar radiation databases[END_REF]. It was then established as an operational service in 2003 [START_REF] Gschwind | Three years of experience with the SoDa web service delivering solar radiation information: lessons learned and perspectives[END_REF][START_REF] Gschwind | Converting a successful research project into a sustainable service: the case of the SoDa Web service[END_REF] and is now widely used by communities interested in solar radiation: more than 50 000 users in 2010. Data can be retrieved by users using a standard Internet browser. The high number of requests made to the HelioClim databases, approx. 2 millions in 2010, indicates clearly the high level of interest in irradiation data; the major demand comes from the energy sector.

A challenge: providing assessment of uncertainty

Concern about uncertainty is growing in environment. This concern includes radiation data as solar radiation plays a major role in environment. For example, errors in solar radiation may impact assessment of terrestrial or oceanic biomass. In another example, if erroneous data are used, wrong performances will be obtained in assessing the yield of a solar-powered system, leading to wrong decision in investment or energy policies as demonstrated in the ENVISOLAR project [START_REF] Schroedter-Homscheidt | The ESA-ENVISOLAR project: experience on the commercial use of Earth observation based solar surface irradiance measurements for energy business purposes[END_REF]. Energy producers are aware of this in the last years and have pushed data providers to make efforts to assess and document uncertainty in satellite-derived irradiation. Supplying irradiation data together with associated uncertainty is a new challenge for data providers. Methods converting satellite data are fairly new, say 30 years, and till 2008, no common benchmark was available permitting to compare outcomes of these methods. A number of comparisons were made between results of methods and measurements by ground stations (Abdel [START_REF] Wahab | Assessing surface solar irradiance in Northern Africa desert climate and its long-term variations from Meteosat images[END_REF][START_REF] Blanc | Validating Meteosat-derived surface solar irradiance in Mozambique[END_REF][START_REF] Cano | A method for the determination of the global solar radiation from meteorological satellites data[END_REF][START_REF] Diabaté | An attempt to estimate the diffuse component of solar radiation on horizontal plane from satellite images[END_REF][START_REF] Ineichen | Satellite Application Facilities irradiance products: hourly time step comparison and validation over Europe[END_REF][START_REF] Lefèvre | Using reduced data sets ISCCP-B2 from the Meteosat satellites to assess surface solar irradiance[END_REF][START_REF] Moradi | Evaluation of the Heliosat-II method using daily irradiation data for four stations in Iran[END_REF][START_REF] Njomo | Solar irradiation retrieval in Cameroon from Meteosat satellite imagery using the Heliosat_2 method[END_REF][START_REF] Perez | Comparing satellite remote sensing and ground network measurements for the production of site/time specific irradiance data[END_REF][START_REF] Perez | A new operational model for satellite-derived irradiances: Description and validation[END_REF][START_REF] Raschke | Estimates of global radiation at the ground from the reduced data sets of the International Satellite Cloud Climatology Project[END_REF][START_REF] Rigollier | The method Heliosat-2 for deriving shortwave solar radiation from satellite images[END_REF][START_REF] Rossow | Calculation of surface and top of atmosphere radiative fluxes from physical quantities based on ISCCP data sets.2. Validation and first results[END_REF][START_REF] Wang | Surface solar irradiance from SCIAMACHY measurements: algorithm and validation[END_REF]. It is not easy to compare the results of these comparisons one to each other. A first challenge lies in the quantities used to quantify the uncertainty. There are a number of differences in spatial and temporal resolutions which induce differences in results (Blanc et al., 2011a;[START_REF] England | A study of the errors due to temporal sampling of the earth's radiation budget[END_REF]Pinker/Lazslo, 1991;[START_REF] Zelenka | Effective accuracy of satellite-derived hourly irradiances[END_REF]. The lack of ground-based measuring stations offering long time series of reliable data is another difficulty. It is fairly easy to perform comparisons in, e.g. France or Germany, where a large number of good quality measuring stations are operated; it is much more difficult to find good measurements in many other countries, and even impossible over oceans. The credibility of the uncertainty assessment increases as the number of stations used for its computation increases. Thus, the value of uncertainty assessment made with a limited number of stations is questionable, especially in areas far from these stations. Finally, on pure technical consideration, how uncertainty should be presented and delivered to users should be dealt with.

The authors are tackling these issues in a long standing effort. They are working together aiming at creating and operating an automatic procedure for the assessment of the uncertainty and the delivery of this information to users. The efforts have begun in 2004 during the Envisolar project co-funded by the European Space Agency [START_REF] Schroedter-Homscheidt | The ESA-ENVISOLAR project: experience on the commercial use of Earth observation based solar surface irradiance measurements for energy business purposes[END_REF]. They benefit from other international projects such as the MESOR, MACC and ENDORSE projects co-funded by the European Commission, the Task SHC#36 of the International Energy Agency (IEA) [START_REF] Stackhouse | Towards designing an integrated Earth observation system for the provision of solar energy resource and assessment[END_REF], and the GEOSS (Global Earth Observation System of Systems).

The overall objective can be seen as composed of three specific objectives: 1) assessing the uncertainty of the satellite-derived irradiation compared to a reference; 2) as the uncertainty depends on atmospheric conditions, devising a model that can predict uncertainty at any location and any time; 3) delivering information to users.

Defining a reference and a protocol for uncertainty assessment

The first specific objective is to assess the uncertainty of the satellite-derived irradiation compared to a reference. Discussions were held in the working groups of the IEA SHC#36 and MESOR projects. It was established that the reference should be made of an ad-hoc series of ground-based measurements of recognised and checked quality. The typical accuracy of irradiation measured in the global meteorological network is 3% to 5% in terms of root mean square error, provided that irradiation data has passed some quality check procedures. Therefore, under these conditions, the ground measurements can be seen as an accurate reference against which one may compare the irradiation derived from satellite.

The comparison is made by computing the difference between the two sets of measurements and analysing statistical quantities, such as mean bias error, standard-deviation of error, root-mean square error (RMSE), and correlation coefficient. The choice of these quantities results from consultation of practitioners in ENVISOLAR and MESOR projects [START_REF] Hoyer-Klick | Management and exploitation of solar resource knowledge[END_REF] and is dictated by the knowledge of these practitioners about uncertainty and statistical quantities. These quantities are actually those recommended by the [START_REF] Iso | Guide to the Expression of Uncertainty in Measurement[END_REF]. Other quantities were elaborated relating to agreement between the statistical distributions such as Kolmogorov-Smirnov test integral (KSI) proposed by [START_REF] Espinar | Analysis of different comparison parameters applied to solar radiation data from satellite and German radiometric stations[END_REF]; there are not adopted presently because of the lack of understanding by practitioners.

The protocol for uncertainty assessment is as follows. Firstly, select good quality data from groundbased stations with ad-hoc quality control procedures. Then, perform the comparison between these data and coincident satellite-derived irradiation. Compute and provide the mean irradiation in groundbased observations, number of observations, correlation coefficient, mean bias error, standarddeviation of error, RMSE, and the relative value, expressed in per cent, of the last three quantities to the mean irradiation. Drawing graphs, such as correlograms or scatterplots (fig. 2), is recommended. Optionally, these operations should be performed for irradiation as well as for the clearness index, which is the ratio of the irradiation observed at ground level to the irradiation observed at the top of the atmosphere. Another option is to apply the protocol for selected cases, such as classes of solar zenithal angles or cloud coverage (e.g. clear-sky or overcast conditions). A third option is to perform the above-described operations for the different time scales (e.g. hourly, daily, monthly or yearly time scales) available within the data sets depending on the finer time scale, data missing and the duration of coincident periods.

The result of this benchmarking protocol describes the uncertainty observed for the few selected measuring stations during a limited period of time. This protocol permits to qualify in a similar manner, similar products from different suppliers and is a useful tool to select the most appropriate product for a specific application.

Several limitations exist that make the assessment of the quality of retrieved irradiances a difficult task. [START_REF] Blanc | Validating Meteosat-derived surface solar irradiance in Mozambique[END_REF] discuss the quality of the ground measurements that can be obtained from the World Radiation Data Center (WRDC) and their spatial and temporal scarcity. A severe limitation is due to the large differences in principles of measurements. Single point and temporally integrated data (ground measurements) are compared to spatially integrated and instantaneous data (satellite estimates). An assumption of ergodicity (e.g. here equivalence between the temporal and spatial averages) is usually made. This assumption is correct only if the field is spatially homogeneous at the considered time scale over an area much larger than a pixel. This is generally false when a significant physiographic feature is present. Other local effects such as reflections on the surrounding slopes or the shadows of clouds may add to the difficulty in comparison. Accordingly, an intrinsic discrepancy is expected because of the natural variability of irradiation in space. [START_REF] Zelenka | Effective accuracy of satellite-derived hourly irradiances[END_REF] have observed the local variability of the solar radiation using measurements made by well-calibrated ground stations close to each other. They demonstrate that this variability cannot be ignored. Expressing it as the ratio of the variance relative to the mean value over the area, they found typical variability of 17 % in hourly irradiation for an area of 10 km in radius. This means that within a 10 x 10 km² area, irradiation measured by a series of similar inter-calibrated sensors would exhibit the same mean value but would differ from hour-to-hour, with a relative variance equal to 17 %. Therefore, observing a difference hour-to-hour of 17 % between a single pyranometer located in a pixel cannot mean that the satellite-derived irradiation is of bad quality. These authors established that a substantial part of the difference between satellite-derived data and ground-based measurements comes from three different sources. One is the natural variability of the irradiation as discussed above. The second one is the error in measurements, which may amount from 3 % to 5 %. Finally, the hypotheses of ergodicity and spatial homogeneity of the irradiation may induce an additional error of 3 % of the mean irradiation.

Predicting uncertainty at any location and any time

The application of the protocol to several cases and databases in the MESOR and IEA SHC#36 projects, among others, reveals that the uncertainty depends on atmospheric conditions and other environmental variables. Uncertainty cannot be described by a set of quantities that are constant in time and space, and that could be derived from one or more benchmarks. Consequently, a model should be devised that can predict uncertainty at any location and any time. This is the second specific objective. Previous benchmarks reveal that the most important variables explaining the uncertainty are sun zenithal angle, clearness index, and irradiation itself. All these variables can be known in a deterministic way or already estimated and therefore can be used as inputs to a model predicting uncertainty. In case of daily irradiation, there are only two variables: clearness index, and irradiation itself.

To our knowledge, there is no such published model. MINES ParisTech made a first attempt in 2006 for the HelioClim-1 database for daily irradiation. Based on results of multiple applications of the benchmarking protocol, we established an empirical analytical model.

Let Kc be the clear-sky index. It is the ratio of the irradiation I at ground level to the irradiation Ic that should be observed under a clear sky for this day and location. The model ESRA [START_REF] Rigollier | On the clear sky model of the 4th European Solar Radiation Atlas with respect to the Heliosat method[END_REF] is one of the models that can be used to predict Ic. Then, we define a systematic error (0.05) and a random part in Kc:

α = 0.05 + 0.04 / √ ndays
where ndays is the number of days used to compute the daily irradiation. In case of monthly means of daily irradiation, e.g., ndays will be equal to 30.

We define r as the number of hourly values used to compute the daily irradiation divided by the number of possible hours in the duration of this particular day. The error in Kc is: [1.54 (0.5 -r)

errorKc = α exp[ ln(2) (1 -Kc)] if r > 0.5 errorKc = α exp[ ln(2) (1 -Kc)] exp

] otherwise

The error in daily irradiation is then: error = Ic errorKc The outcome error of this model was considered as equivalent to the RMSE. It was delivered together with the daily irradiation to the users on the SoDa Service. It helps creating awareness on uncertainty among users and helps researchers to gain expertise in this domain.

Though the approach is the same, we have elaborated a more precise model for the HelioClim-3 database taking into account comments from users. In particular, we have decided to deliver lower and upper bounds in irradiation, such that there is a 68% chance that the actual value is comprised between them.

We have selected 29 stations offering time series of hourly irradiation of good quality during the period 2004-2005. These stations are mostly located in Europe with one in South Africa, one in South Algeria and one in Israel. By using the protocol, we have estimated the differences for all stations merged together. Prior to this, the HelioClim-3 estimates were corrected in elevation using the model of Abdel [START_REF] Wahab | Assessing surface solar irradiance in Northern Africa desert climate and its long-term variations from Meteosat images[END_REF] to adjust to the elevation above sea level of the ground stations.

In case of daily irradiation, we have created classes in clearness index, and HelioClim-3 daily mean of irradiance. For each class, we have computed bias (mean value of the difference), standarddeviation, and RMSE. All these quantities were expressed relatively to the HelioClim-3 mean irradiance for this class.

Thus, we obtained abaci whose inputs are clearness index, and HelioClim-3 daily mean of irradiance. There are 6 bins in daily mean of irradiance, in W/m²: [0,100,200,300,400,500,600], and 8 bins in clearness index: [0, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 1.1]. Each class contain relative bias: bias, standard-deviation: std, and RMSE: rmse. The computation of the lower and upper bounds Elow and Eup for the daily mean of irradiance E is as follows:

Elow / E = 1 + bias -std / √ ndays Eup / E = 1 + bias + std / √ nday if (Elow / E) > 0.9, or (Eup / E) < 1.1, then Elow / E = 1 + bias -RMSE / √ ndays Eup / E = 1 + bias + RMSE / √ ndays
The case of irradiation for period less than a day, e.g., hour, 30 min, or 15 min is fairly similar, except that we compute abaci whose inputs are sun zenithal angle, clearness index, and HelioClim-3 hourly (or 30-min, or 15-min) mean of irradiance. There are 5 bins in solar zenithal angle, in degree: [0,30,40,50,60,90], 6 bins in hourly mean of irradiance, in W/m²: [0,200,400,600,800,1000,1400], and 8 bins in clearness index: [0, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 1.1]. Each class contain relative bias, standard-deviation and RMSE.

Let nmin denotes the number of minutes in the period of integration. nmin is equal to 60 for hourly irradiation, and 15 for 15-min irradiation. The computation of the lower and upper bounds Elow and Eup for the hourly (or 30-min, or 15-min) mean of irradiance E is as follows:

Elow / E = max(0, 1 + bias -std * √ (60/nmin))

Eup / E = 1 + bias + std * √ (60/nmin) if [(Elow + Eup)/2 -E)] > 0.8 (Eup -Elow), then Elow / E = 1 + bias -RMSE * √ (60/nmin) Eup / E = 1 + bias + RMSE * √ (60/nmin)
These two quantities are delivered routinely together with the irradiation to the users on the SoDa Service. It brings benefit to companies monitoring the electricity produced by solar-powered systems, since after transformation by a model simulating the system of energy conversion it provides a corresponding plausible range of energy to be produced. Any outlier may mean a problem in the system. The model has been validated by further benchmarks, using other stations in other areas. It reveals to be quite robust though it has been noted that it overestimates the uncertainty in several cases.

Delivering information to users

The third objective is to deliver information to users. The easiest way to do so is to present the uncertainty values on the same Web page containing the irradiation. This is what was made at the earliest stages of our project in the SoDa Service in 2006. Much more can be done today, considering the needs of users to have their computers interacting with the computers providing access to data and the current capabilities in interoperability. We do not discuss here the quantities to be provided that relate to uncertainty or their format. As written before, choice of quantities was made as a function of the knowledge and expectations of practitioners.

We see the delivery of information on uncertainty as a series of challenges, dealing on the one hand with the description of uncertainty and its coding, and on the other hand, with Web services capable of computing uncertainty, thus implementing the workflow by their composition [START_REF] Gschwind | Test of several approaches for the composition of web services in meteorology[END_REF].

As irradiation data are already delivered through the SoDa Service and the IEA SHC#36 / MESOR portal, it is natural to use these dissemination means. The uncertainty quantities are embedded in the flow containing irradiation.

Several services providing irradiation and uncertainty were implemented in the SoDa Service using the proprietary SoDa XML presented at EnviroInfo in 2002 [START_REF] Wald | SoDa: a project for the integration and exploitation of networked solar radiation databases[END_REF]. In this case, there is no specific metadata devoted to the description of uncertainty. The SoDa XML has the advantage of being simple, thus allowing rapid development but is limited regarding interoperability and does not obey current standards promoted by GEOSS [START_REF] Percivall | Geo-processing in cyberinfrastructure: making the web an easy to use geospatial computational platform[END_REF][START_REF] Robinson | GEOSS architecture implementation pilot-II: Integrating technologies and expertise to build GEOSS[END_REF].

More advanced services providing the same information were implemented using WSDL; a revision of the XML schema "SolarResourceKnowledge-2.1.xsd" (Gschwind et al., 2007a) was made to include uncertainty quantities in the product delivered to users. For each instant of observation, e.g., every hour, an irradiation is provided together with the uncertainty attached to this irradiance, as well as a reliability code. The uncertainty can be expressed as a single value, e.g., standard-deviation, or as lower and upper bounds as seen above. These bounds are such that there is a 68% chance that the actual value is comprised between lower and upper bounds. The meaning of the reliability code depends on the type of products. This code denotes whether the irradiation results from an interpolation in time of two adjacent values (for example, the case of a gap during a day), or provides the number of valid 15-min values used to compute a daily average, or the number of valid daily values used to compute a monthly average. This is fully described in the exploitation metadata conveyed by the product. This schema is presently used to disseminate HelioClim and SOLEMI data through the MESOR portal.

Though operational, all these services must be considered as prototypes. They do not permit to achieve the third specific objective "delivering information to users" and further work is needed.

One of our goals is to create Web services permitting practitioners to upload their legacy data, or use archived data measured by ground-based stations in order to perform the protocol. In this way, we expect to increase the number of tests of performances made on the HelioClim and SOLEMI databases by users, therefore increasing the knowledge on the merits of these databases and the sharing of this knowledge. Another series of Web services should be developed implementing the model predicting the accuracy. Actually, this implementation will be part of the development of the Web services providing the irradiation from HelioClim or SOLEMI databases.

Conclusions and perspectives

We have presented here early achievements reached in this long-standing effort for the assessment of uncertainty of solar irradiation data estimated by the processing of satellite images and other Earth Observation data.

International efforts have resulted in a protocol for benchmarking such irradiation data. This was the first objective to attain. The reference is composed of good quality measurements made at ground level by qualified measuring stations. Quantities describing uncertainty are quite standard: bias, standard-deviation, RMSE. Similar irradiation data from different suppliers can be qualified in a similar manner by this protocol. It is a useful tool for users to select the most appropriate database for a specific application.

It is impossible to describe the uncertainty in solar irradiation with constant values in time nor in space, whether absolute or relative. The uncertainty is a function of atmospheric conditions and the irradiation itself, thus is changing rapidly. This communication has presented two models elaborated for the HelioClim databases that predict the uncertainty for any site and any instant. These models have proved to be robust. We may consider that we have reached the second specific objective. However, efforts should be made to better estimate the uncertainty. We believe that this objective can only be reached by improving the method estimating the irradiation from satellite images.

Delivering information on uncertainty to users is our current challenge. Interoperability is a key element in our strategy. Web services will be chained in order to obtain value-added Web services implementing the benchmarking and the uncertainty model. Interoperability is also a means to exploit new or more accurate services, replacing older ones. The Web services will be deployed in the Energy Community Portal (www.webservice-energy.org). The GEOSS-compliant CSW (catalogue service for the web) provided by the EC-funded project EnerGEO (energeo.researchstudio.at) will enable these services to be searched and discovered.

These activities are taking place under the GEOSS umbrella, thus contributing to the Architecture and Data Committee and to Community of Practice "energy". A scenario will be developed in Phase-4 of the GEOSS Architecture Implementation Pilot (AIP-4) exploiting several services where data flow conveys uncertainty information. This research partly contributes to EC-co-funded projects MACC (2009[START_REF] Gcos | Global Climate Observing System (GCOS) Essential Climate Variables[END_REF]( ) and ENDORSE (2011( -2013) ) and to IEA activities in SHC#46 (2011SHC#46 ( -2016)). Cooperation with other projects such as the QA4EO project or EC-co-funded project UncertWeb (2010-2012) is a perspective.

Figure 1

 1 Figure 1 Example of an image taken by the satellite Meteosat on 11 November 2003, at 1100 UTC. Reflectance increases from black to white. Clouds are clearly visible. Copyright Eumetsat, 2003

Figure 2

 2 Figure 2 Example of a correlogram (or scatterplot) between hourly irradiation measured by the meteorological station of Nice (France) and that derived from satellite images (HC-3).

Acknowledgements

The research leading to these results has partly received funding from the European Union's Seventh Framework Programme (FP7/2007-2013) under Grant Agreements no 218793 (MACC project) and no. 262892 (ENDORSE project).