Development and Evaluation of a Novel Fast Broad-Range ITS/LSU DNA PCR and Sequencing Assay (FBR-PCR/S) for Rapid Diagnosis of Invasive Fungal Diseases: Multi-Year Experience in a Large Canadian Healthcare Zone and a Literature Review

This study evaluated the performance of a novel fast broad range PCR and sequencing (FBR-PCR/S) assay for the improved diagnosis of invasive fungal disease (IFD) in high-risk patients in a large Canadian healthcare region. Methods A total of 114 clinical specimens (CS) including bronchoalveolar lavages (BALs) were prospectively tested from 107 patients over a 2-year period. Contrived BALs (n=33) inoculated with known fungi pathogens were also tested to increase diversity. Patient characteristics, fungal stain and culture results were collected from the laboratory information system. Dual-priming oligonucleotide (DPO) primers targeted to the ITS (~350 bp) and LSU (~550 bp) gene regions were used to perform FBR-PCR/S assays on extracted BALs/CS. The performance of the molecular test was evaluated against results of fungal stains and culture, and where available, histopathology, and clinical review for the presence of IFD. FBR-PCR/S assays results were reported in ~8h compared to fungal cultures that took between 4 to 6 weeks. 16S basic (Molzym, Bremen, Germany) kit reagents. The 30 µL reaction contained 7 µL of template DNA and nal concentration of 0.3 µM ITS3DPO_F3 forward primer and 0.2 µM each of ITS4DPO_R5 and LSUDPO3_R reverse primer. FBR-PCR was performed on a Veriti thermocycler (Life Technologies, Carlsbad, CA) under the following cycling conditions: 5 min. initial denaturation at 95 o C, followed by 35 cycles of 94 o C for 10 s, 54 o C for 15 s and 72 0 C for 25 s, with a nal extension of 72 o C for 5 min. PCR product was electrophoresed on a 1.5% agarose gel containing SYBRsafe (Life Technologies). During the PCR reaction, the ITS3DPO_F3/ITS4DPO-R5 F/R primer pair amplify a ~350 bp ITS amplicon, whereas the ITS3DPO_F3/LSUDPO_3R F/R primer pair amplify the ITS region (350 bp) plus ~500-600 bp of the LSU region. The ~900 bp amplicon therefore represented a combined ITS/LSU fragment. Agarose gel electrophoresis conrmed the amplication of fungal DNA: PCR products displaying a band in the expected ~ 350 bp region for ITS and ~900 bp region for ITS/LSU were then puried by Exo-SAP-it (Affymetrix, Santa Clara, CA). results, which may not provide a denitive diagnosis. Our study is the only one performed in a large Canadian healthcare region that has evaluated FBR-PCR/S directly from samples for the rapid detection of suspected IFD. Our unique fungal PCR based on dual target (ITS and LSU) detection using DPO primers and fast protocols allows equivalent or improved diagnostic performance previously reported from other centers who have developed molecular detection assays. Use of a DPO primer approach increased specicity compared to previously reported fungal PCR assays.


Abstract Background
This study evaluated the performance of a novel fast broad range PCR and sequencing (FBR-PCR/S) assay for the improved diagnosis of invasive fungal disease (IFD) in high-risk patients in a large Canadian healthcare region.

Methods
A total of 114 clinical specimens (CS) including bronchoalveolar lavages (BALs) were prospectively tested from 107 patients over a 2-year period. Contrived BALs (n=33) inoculated with known fungi pathogens were also tested to increase diversity. Patient characteristics, fungal stain and culture results were collected from the laboratory information system. Dual-priming oligonucleotide (DPO) primers targeted to the ITS (~350 bp) and LSU (~550 bp) gene regions were used to perform FBR-PCR/S assays on extracted BALs/CS. The performance of the molecular test was evaluated against results of fungal stains and culture, and where available, histopathology, and clinical review for the presence of IFD.

Conclusions
Rapid molecular testing compared to culture has equivalent diagnostic e ciency but improves clinical utility by reporting a rapid species-level identi cation the same dayshift (~8h).

Background
Invasive fungal disease (IFD) has increased signi cantly in the last few decades due to the expansion of patients with acquired immunosuppression (1)(2)(3). IFD results in increased morbidity and mortality and higher healthcare costs (4)(5)(6)(7)(8)(9). Delayed diagnosis is associated with poor clinical outcomes because appropriate treatment measures are not promptly started (1,(10)(11)(12). However, IFD is often di cult to diagnose because clinical and radiographic ndings are non-speci c and traditional microbiological methods such as fungal culture have low sensitivity ranging from 30-60% (13)(14)(15). Too frequently, tissues or body uids are harvested by invasive procedures, and all or most of the specimen is sent to pathology for formalin xation with limited or no tissue being sent for cultures (13). This erroneous clinical practice negates making a microbiological diagnosis of IFD and impedes the ability to properly treat the infection based on a genus-and -species pathogen identi cation. Fresh tissue biopsies are often required to con rm the presence of IFD but specimen processing and homogenization break-up hyphae lowering the recovery of viable microorganisms from cultures. Fungi can also not be accurately identi ed from stained histopathology or cytology sections, and the lack of concordance between pathology and culture results is well recognized (15)(16)(17). Identi cation of the causative pathogen is important for targeting antifungal therapy as well as determining prognosis. Culture-independent microbiology assays for reliable detection and identi cation of a wide-range of fungal pathogens are urgently needed to improve clinical outcomes of IFD.
Broad-range sequencing of fungal DNA has been used as an alternate approach for diagnosing IFD in high-risk patients where delayed appropriate therapy may cause worse outcomes(18-20). Because our regional clinical microbiology laboratory provides services to a wide variety of immunosuppressed patients (e.g., hematological malignancy, HSCT, SOT, diabetes, solid-tumour malignancy, HIV/AIDS, and immunotherapy), it was important to implement broad-range PCR/sequencing to enable rapid fungal identi cation to ensure appropriate therapy because culture take between 4-6 weeks to complete. Prior reports using conventional primers have shown broad range fungal PCR and sequencing targeted to one or more regions of the fungal multicopy ribosomal RNA (rRNA) such as 18S rRNA, D1and D2 regions of 28s rRNA, 5.8S rRNA, and internal transcribed spacers 1 and 2 (ITS1 and ITS2) allowed variable detection and identi cation of fungi in clinical specimens (20)(21)(22)(23)(24)(25). Additionally, clinical specimens routinely tested by fungal culture may be highly contaminated by commensal ora (15), and their human DNA may cross-react in broad-range assays(26). We developed and evaluated a novel fast broad-range dual target DNA PCR and sequencing assay using unique dual-priming oligonucleotide (DPO) primers to try and increase assay speci city while decreasing the time to reported results(26). Our clinical microbiology laboratory previously developed and implemented broad-range PCR for bacterial 16S ribosomal DNA and sequencing using 16S rDNA DPO primers that allows robust sensitivity while improving speci city due to elimination of cross-reactivity with human material (27). The novel FBR-PCR/S assay described herein uses similar fast protocols, DPO primers and procedures to integrate work ow e ciency for technologists performing both the bacterial and fungal broadrange PCR/cycle sequencing assays within a standard ~8 h dayshift, which allows same day reporting of results from either or both assays.
In this multi-year study, we evaluated the performance of our novel FBR-PCR/S assay by comparing it with the results of fungal stains, culture, and conventional broad-range ITS PCR using bronchoalveolar lavages (BALs) and a variety of other clinical specimens in patients with and without suspected IFD. A literature review was also done to compare our result to previously published studies of other laboratory-developed assays, and recently published evaluations of commercial PCR tests.

Study setting and patients
The Calgary Zone, Alberta Health Services (AHS), is one of the largest integrated healthcare jurisdictions in Canada, which provides care to an urban and rural population of ~1.5 million people. Our novel fast fungal BR-PCR/S assay was developed and pre-clinically validated using retrospectively collected and saved clinical samples from patients suspected to have IFD that had either had positive fungal cultures for a wide variety of yeasts and molds or had been inoculated with known fungal isolates.
Patients with and without suspected non-invasive and IFD were then prospectively enrolled over a two-year period (2016-18) based on combined concern of the consulting Infectious Diseases physician for IFD, and the results of fungal stains and culture. Cases were categorized as having con rmed IFD, probable IFD or no fungal disease in the context of clinical review and microbiological work-up (microscopy, culture, and PCR/sequencing). Data were obtained by medical microbiologists (MG and JC) and an infectious diseases specialist (DLC). Data were assessed against previously published clinical and laboratory criteria to inform an expert diagnostic decision(28).

Laboratory setting and specimens
Microbiology testing was performed by the Clinical Section of Microbiology, Calgary Laboratory Services (CLS; Alberta Prevision Laboratories). APL-CLS is a large regional centralized laboratory that performs diagnostic testing for the entire Calgary Zone, including all ambulatory, hospitalized and long-term care patients. Our laboratory also acts as the primary Mycology testing laboratory for Southern Alberta including major rural cities and townships representing a population of ~600K.

Page 4/20
A total of 114 enrolled clinical specimens were categorized as sterile tissues, non-sterile tissues, and sterile body uids (Table 1).
A variety of specimens were enrolled including those from patients with and without suspected IFD that had negative or positive fungal stain/culture results, or a positive stain but negative culture result. Bronchoalveolar lavages (BALs) are collected by experienced pulmonary medicine, critical care, and thoracic surgery specialists according to a standardized regional protocol that sets out the amount of uid, and collection procedures to be used. All other sterile uid and tissue specimens were collected by appropriate sterile techniques, and promptly transported to the microbiology laboratory within 2 h after collection. A Microbiologist (DLC/TG/MG) approved their quality before enrollment. Study specimens were stored at -80 to -86 o C and batched for DNA extraction.

Fungal stain and culture
Clinical specimens were analyzed by microscopy and culture methods for the presence of fungi. Calco uor-White (CW) stain (i.e., CW-Evans blue reagent) with 10% potassium hydroxide was performed on tissue homogenates and sterile body uids. CWstained slides were air-dried then xed with methanol before reading within 24 h of preparation under uorescent microscopy.
Tissue biopsies obtained via open resection were minced for culture. Specimens were inoculated onto general mycology media including inhibitory mold agar (IMA) (Oxoid # MP0950), brain heart infusion agar (BHI) with 5% sheep blood (Oxoid #MP0234) and BHI with antibiotics (BHIA) (i.e., chloramphenicol, gentamicin and cycloheximide) (Oxoid #MP0237) and incubated in 0 2 at 30 o C. In addition, BALs and bronchial wash specimens were inoculated to buffered charcoal yeast extract (BCYE) agar (Oxoid #MP1201). Cultures were assessed for growth daily for the rst 5 days and then biweekly for up to 42 days. Positive cultures were identi ed using various methods including macroscopic colony morphology and microscopic examination, VITEK 2 Yeast ID card (bioMérieux, Laval, Quebec), matrix-assisted laser desorption/ionization-time-of-ight mass spectrometry (MALDI-TOF MS) (bioMérieux, Laval, Quebec), and separate PCR/sequencing analysis at the PLNA reference laboratory using the commercial MicroSEQ™ D2 rDNA Fungal PCR and Sequencing Kit (Applied Biosystems, Thermo Fisher Scienti c).
Some specimens only had a bacterial work-up (Gram stain, culture under aerobic and anaerobic environmental conditions) because fungal culture was not initially ordered by the physician but subsequently one or more yeast/molds grew. An FBR-PCR/S assay was subsequently done to con rm the presence of each recovered yeast/mold species.

Molecular Methods
A clinical isolate of Saccharomyces cerevisiae positive in the FBR-PCR/S assay for both ITS2 and LSU targets was used as the positive control throughout all DNA extraction and FBR-PCR/S assay procedures. The negative extraction control (i.e., extraction reagents only; NEC) was processed and extracted alongside all clinical samples including those inoculated with known fungal isolates. NEC negative control was used throughout all FBR-PCR/S assay procedures.
DNA Extraction a. Fungal Isolates: Fungal isolates obtained from the reference laboratory were extracted in TE buffer using glass beads and bead beating. The DNA concentration of the fungal nucleic acid extract was determined using a Nanodrop spectrophotometer (Thermo-Fisher Scienti c, Mississauga, Ont.). A total of 500 ng DNA was eluted into 100 µL of TE buffer giving a nal template concentration of 5 ng/µL. This amount of fungal DNA was required for reliable detection in the FBR-PCR/S assay. Contrived specimens (n=33) were prepared by adding 500 ng reference isolate DNA to 400 µL of spent BAL uid about to be discarded after completion of clinical testing. The contrived specimens containing 'inoculated' reference isolate DNA were then extracted using the QIAmp UCP Pathogen Mini Kit (Canada-QIAGEN, Toronto, CA). Fungal PCR/sequencing testing was then performed according to the method outlined below. Based on testing of these BALs with a known amount of fungal inoculum, the FBR-PCR/S assay had a limit of detection of ~35 ng of DNA or 1,000 copies/mL. b. Clinical Specimens: Clinical tissue and uid specimens were extracted using QIAmp UCP pathogen Mini Kit (QIAGEN).
Tissues had an extended Proteinase K incubation time, otherwise both tissue and uid protocols were the same. Brie y, a representative tissue specimen of 2-4 mm 3 was nely minced with sterile scalpel and transferred to a sterile 1.5 mL microcentrifuge tube, re-suspended in 400 µL Buffer ATL and 40 µL kit-supplied Proteinase K. Tissue specimens were then brie y vortexed and incubated at 56 o C in a 1000 rpm Eppendorf thermomixer for a minimum of 1 h until the tissue was digested. A minimum 400 µL aliquot of each sterile uid specimen was placed into a sterile 1.5 mL microcentrifuge tube and centrifuged, supernatant discarded, and cell pellet re-suspended in Buffer ATL, Proteinase K. Sterile uid specimens were then brie y vortexed and incubated at 56 o C in a 1000 rpm Eppendorf thermomixer for a minimum of 10 min. DNA in the proteolytic digests were further puri ed according to the manufacturer's instructions. DNA was eluted from tissues and uids in 150 µL and 100 µL of Buffer AVE, respectively. DNA was stored at -20 o C until use. During the PCR reaction, the ITS3DPO_F3/ITS4DPO-R5 F/R primer pair amplify a ~350 bp ITS amplicon, whereas the ITS3DPO_F3/LSUDPO_3R F/R primer pair amplify the ITS region (350 bp) plus ~500-600 bp of the LSU region. The ~900 bp amplicon therefore represented a combined ITS/LSU fragment. Agarose gel electrophoresis con rmed the ampli cation of fungal DNA: PCR products displaying a band in the expected ~ 350 bp region for ITS and ~900 bp region for ITS/LSU were then puri ed A 2 X 2 contingency table was used to calculate the sensitivity, speci city, positive and negative predictive values were calculated using the con rmed or possible clinical diagnosis of IFD based on expert clinical review of each case against internationally recognized diagnostic criteria(28). Performance was calculated using both FBR-PCR/S and fungal culture as the "gold standard" method, because the later is a broad ampli er of both pathogenic, commensal, and contaminating ora and may not be an accurate re ection of the clinical relevance of recovered isolates. Invalid FBR-PCR/S results were de ned as a weakly positive electrophoresis band in either of the LSU/LSU fungal targets with no quality sequence subsequently obtained. A true positive FBR-PCR/S result was one where fungal culture and the molecular assay detected the same microorganism(s) in a patient with suspected, possible, or con rmed IFD. A false-negative molecular result was one where a yeast/fungus was recovered in culture that was not detected by the molecular assay. A false-positive molecular result was one where the FBR-PCR/S assay detected a fungus, there was no growth on fungal culture, and the patient had no evidence of infection. If there was not complete agreement between the fungal culture and molecular assay, results were considered discordant. Resolution of discordant results occurred by repeat FBR-PCR/S testing, repeat testing using conventional ITS primers, and clinical review.
A total of 114 clinical specimens were tested from these patients including 39 (34.2%) BALs and 75 (65.8%) other types of sterile uids and tissues; 7 patients had ≥2 specimens tested (Table 1). BALs and other pulmonary specimens (lung/bronchial/pleural aspirates or uids) (n=51, 44.7%) were the most tested sterile uids. A wide range of different tissue types were tested representing the disseminated nature of IFD. A total of 55 (48.2%) specimens had yeast/fungi recovered from fungal cultures. Twenty (17.5%) specimens only had bacterial cultures done because yeast/fungal culture was not ordered -most of these specimens (n=16, 80%) had negative Gram and CW stains and bacterial cultures, but 1 BAL and 2 abdominal uid specimens grew Candida albicans (despite negative CW), 1 abdominal uid showed yeast in the Gram stain and grew C. albicans, and 1 sinus aspirate grew Aspergillus fumigatus.    (Table 2B). BALs were prone to contamination from patient's airway colonization with Candida spp. and/or Aspergillus spp., which gave initial discrepant results, but most were resolved in favour of the FBR-PCR/S result after repeat testing and clinical review (Table 2A).
FBR-PCR/S analysis made a critical difference to patient management and clinical outcome in 4 unusual cases where fungal cultures were negative (Table 2A). Brain, cheek, and parotid tissue (Specimens 11 to 13) were harvested in the operating room from a critically ill 25 yo diabetic female with rapidly progressive severe necrotizing left facial and sinus infection where sequential specimens remained culture negative but broad-based aseptate hyphae were seen on Grocott's stains in histopathology sections (30). Rapid PCR diagnosis of rhino cerebral Mucormycosis dure to Rhizopus oryzae allowed optimal treatment and management. For Specimen 15, FBR-PCR/S results allowed appropriate management of this patient's intraabdominal abscesses and institution of anti-fungal therapy with cessation of broad-spectrum antibacterial agents. Specimen 18 was harvested under ultrasound guidance in a 49 yo female with a new onset acute myelogenous leukemia and large hepatosplenic lesions thought to be due to candidiasis that were fungal/bacterial culture negative. Diagnosis of hepatosplenic Mucormycosis due to Rhizomucor pusillus enabled immediate appropriate anti-fungal management and drainage, and the patient proceeded to allogenic stem cell bone marrow transplant. Although specimen 19 fungal cultures eventually grew scant amounts of Histoplasma capsulatum after almost 6 weeks of incubation, FBR-PCR/S testing allowed for rapid con rmation of Histoplasmosis, which was also consistent with histopathology sections showing yeast with broad-based budding on Grocott's and PAS stains.

Molecular Assay Performance
The performance of the molecular assay compared to fungal culture is shown in Table 3A for BALs, and Table 3B for other clinical non-BAL specimens. For BALS, the FBR-PCR/S assay had sensitivity, speci city, positive predictive value (PPV) and negative predictive value (NPV) of 88.5%, 100%, 100% and 61.1% compared to culture. Fungal culture had sensitivity, speci city, positive predictive value (PPV) and negative predictive value (NPV) of 100%, 61.1%, 88.5% and 100% compared to the molecular assay for BALs. Overall, fungal culture and the molecular assay had similar diagnostic e ciency (90.2%) for BAL specimens (Table 3A).
For other clinical specimens, the FBR-PCR/S assay and fungal culture had similar performance with sensitivity, speci city, positive predictive value (PPV) and negative predictive value (NPV) of 66.7%, 87.0%, 66.7% and 87.0% compared to culture. Overall, fungal culture and the molecular assay had similar diagnostic e ciency (81.3%) for other types of clinical specimens (Table 3B). Table 3C shows that fungal culture and the molecular detection had similar performance compared to whether the clinical specimen showed fungal elements on CW stain and microscopic examination. Performance of their fungal PCR assay was better in the targeted IFD group [sensitivity (96.6%) and speci city (98.25%)] than in patients suspected of IFD [sensitivity (62.8%) and speci city (71.3%)] (22). Ala-Houhala and colleagues (2017/Finland)(21) used a dual target ITS fungal PCR to test 37 tissue and sterile uid specimens from 279 patients and found a sensitivity, speci city, PPV and NPV of 60.5%, 91.7%, 54.2% and 93.4% respectively. Stempak and colleagues (2019/USA) (31) also showed that fungal PCR testing had equivalent performance on analyses of 65 sterile uid and tissue samples selected based on having all reference methods done (i.e., stains, DNA probes, culture, histopathology). This group did not recommend the routine use of fungal PCR however, because no IFD cases were found that were not diagnoses by the reference methods, and the referred out molecular assay had a prohibitive cost.
Fungal PCR had an excellent performance compared to culture in microscopy positive specimens, and an equivalent performance in microscopy negative specimens. In fact, most patients with IFD were diagnosed by fungal PCR analysis of microscopy negative specimens so testing should be done in non-selected patients without overt immunosuppression. Rampini and colleagues (2016/Switzerland) (19) have also demonstrated similar e cacy of their fungal ITS PCR compared to conventional fungal culture for diagnosing fungal infections in non-immunocompromised patients. They evaluated 251 clinical specimens using both the fungal ITS PCR compared to fungal culture and demonstrated a high concordance of 89.6% and equivalent analytical performance with a sensitivity, speci city, PPV and NPV of 87.7%, 90.3%, 76% and 95.5% respectively (19).

Discussion
Accurate diagnostic of IFD is critical to prompt, appropriate anti-fungal and surgical management to achieve the best clinical outcome for these serious infections. Although fungal culture remains the "gold standard" diagnostic method available in most clinical microbiology laboratories, this study and others demonstrate that molecular testing has an equivalent, and in some cases improved performance for direct specimen analyses (19)(20)(21)(22)(23). However, a major clinical advantage of our fast PCR protocol, is provision of a result in a day (i.e., a single 8-h daytime shift) compared to culture that can take 4 to 6 weeks (13,14). Without a rapid laboratory con rmation of IFD, clinicians must rely on clinical review and histopathology results, which may not provide a de nitive diagnosis.
Our study is the only one performed in a large Canadian healthcare region that has evaluated FBR-PCR/S directly from samples for the rapid detection of suspected IFD. Our unique fungal PCR based on dual target (ITS and LSU) detection using DPO primers and fast protocols allows equivalent or improved diagnostic performance previously reported from other centers who have developed molecular detection assays. Use of a DPO primer approach increased speci city compared to previously reported fungal PCR assays.
However, fungal PCR analysis must be interpreted against the pre-test likelihood of IFD because speci c sample types may have a high rate of fungal contamination as shown by the initial rate of discordant results in this study. Most of these samples, not surprisingly, were bronchoalveolar lavage or other pulmonary samples where harvesting may lead to contamination from commensal fungi in the patient's airway, particularly Candida spp. Penicillium spp. and Aspergillus spp. This has also been reported by other investigators who used panfungal PCR to detect and identify fungi in BAL uids from immunocompromised patients (15,32). Polymicrobial yeast/fungal infections are also di cult to diagnose using PCR because mixed sequencing results may not be interpretable. Culture is a broad ampli er of both pathogens and contaminants as demonstrated in our study where approximately one-third of clinical BALs grew more than one type of fungi, but PCR was only positive for one of them, and the others were often deemed on resolution as contaminants. Even in cases where a single fungus such as Aspergillus spp. is detected in a BAL, clinical correlation must be done to determine whether either the conventional or molecular test result is relevant.
Our study had several limitations including the small number of specimen types and sources enrolled. Because most clinical specimens recover Candida spp. and Aspergillus spp. as the most commonly isolated yeast/fungi, we used mock BAL specimens to broaden the evaluation of the fungal PCR to detect other important yeast/fungal pathogens. Formal chart reviews of each enrolled patient were not done, which may have provided a more detailed clinical assessment of the presence or absence of IFD.
Broad-range FBR-PCR/S analyses targeted to high-risk patients and non-selected non-immunocompromised patients allows for rapid diagnosis of IFD (i.e., 24 h using a fast PCR protocol), and may identify rare types of fungal disease in critically ill patients whose work-up by fungal reference methods is negative. Microscopic examination does not help the clinical laboratory select samples for molecular analyses because most cases of IFD have negative results but culture and/or fungal PCR show infection.
Laboratories using FBR-PCR/S for diagnosis must consult the physician and correlate discordant results against the patient's likelihood of IFD. Interpretation of molecular assay results must be done with the recognition that speci c types of clinical specimens harvested from "sterile" sites may be contaminated with normal commensal fungi. However, use of a DPO primer approach for fungal PCR assay development increases speci city and decreases detection of contaminants.

Conclusions
Rapid FBR-PCR/S testing has equivalent diagnostic e ciency compared to fungal culture with improved speci city, but our novel assay improves clinical utility by reporting a rapid species-level identi cation the same dayshift (~8h).

Declarations
Ethics approval, guidelines, and consent to participate The study was approved, and a waiver of informed consent was granted by the Conjoint Health Ethics Research Board (CHREB), Alberta Health Services, and the University of Calgary (Ethics ID: E-23969). All methods were carried out in accordance with relevant guidelines and regulations.

Consent for publication
Not applicable

Availability of data and materials
The data that support the ndings of this study are available from Alberta Health Services (AHS), Alberta Precision Laboratories (APL) (formerly CLS) but restrictions apply to the availability of these data, which were used under the ethics agreement for the current study, and so are not publicly available. Data are however available from the author upon reasonable request and with permission of AHS/APL.