Scaling national and international improvement in virtual gene panel curation via a collaborative approach to discordance resolution.

Clinical validity assessments of gene-disease associations underpin analysis and reporting in diagnostic genomics, and yet wide variability exists in practice, particularly in use of these assessments for virtual gene panel design and maintenance. Harmonization efforts are hampered by the lack of agreed terminology, agreed gene curation standards, and platforms that can be used to identify and resolve discrepancies at scale. We undertook a systematic comparison of the content of 80 virtual gene panels used in two healthcare systems by multiple diagnostic providers in the United Kingdom and Australia. The process was enabled by a shared curation platform, PanelApp, and resulted in the identification and review of 2,144 discordant gene ratings, demonstrating the utility of sharing structured gene-disease validity assessments and collaborative discordance resolution in establishing national and international consensus.

Analysis of primary central nervous system large B-cell lymphoma in the era of high-grade B-cell lymphoma: Detection of two cases with MYC and BCL6 rearrangements in a cohort of 12 cases.

High-grade diffuse large B-cell lymphoma (HG-DLBCL) refers to DLBCL with MYC and BCL2 and/or BCL6 rearrangements (double-hit or triple-hit DLBCL) that exhibits poor prognosis. Double-expressor DLBCL (c-myc+/bcl-2+) has intermediate prognosis when compared to HG-DLBCL. Primary central nervous system lymphoma (PCNSL) has distinct pathophysiology (frequent non-germinal center-like subtype and double-expressor) and has worse prognosis than systemic DLBCL. By fluorescence in situ hybridization (FISH), 25-30% of PCNSLs harbor BCL6 abnormalities with rare alterations in MYC, BCL2, double-hit or triple-hit events. We describe the clinicopathologic features and status of MYC, BCL2 and BCL6 in 12 PCNSLs (7 women, 5 men; median age 63 years; range: 28-79). Six cases showed focal starry-sky pattern. Immunohistochemically, all (100%) were of non-germinal center-like subtype, and 8/10 (80%) cases were double-expressors. Ki-67 ranged from 70 to 100%. FISH was positive in 9/12 (75%) cases: 4 (33%) harbored a BCL6 rearrangement, 3 (25%) had a gain of BCL2, 2 (17%) cases each had a gain of BCL6 and gain of IGH, and gain of MYC and deletion of MYC were observed in 1 case each (8%). Two (16%) cases were MYC/BCL6 double-hit PCNSLs. No MYC/BCL2 or triple-hit cases were identified. Eleven (92%) patients received chemotherapy and one also received whole brain radiation. The median time of follow-up was 4.4 months (range, 0.3-40.3). Seven (58%) patients are alive, 4 (33%) have died, and 1 (8%) had no follow-up. Five alive patients are in remission, including one MYC/BCL6 double-hit PCNSL. Our results add two new cases of rare double-hit PCNSL to the literature.

An examination of radon awareness, risk communication, and radon risk reduction in a Hispanic community.

Purpose: Radiation risk communication is a critical component of radiation protection and the public's understanding of radiation risks and benefits. Risk communication becomes even more complicated when considering cultural and language differences. In the US, many diverse communities face risk communication challenges. We obtained radon testing data to evaluate patterns of radon testing in Allentown, the third largest city in Pennsylvania. Radon exposure is the second leading cause of lung cancer after smoking and is associated with over 21,000 lung cancer deaths in the US annually. It is estimated that 1 in every 15 homes in the US has elevated radon levels above the recommended action level set by the US Environmental Protection Agency. Allentown has some of the highest reported levels of indoor radon in the country, yet only a small portion of the population has tested their homes. This is true particularly among self-identified Hispanics, who make up nearly half of the city's population. This study seeks to (1) characterize the difference in testing rates between self-identified Hispanics and non-Hispanics in Allentown, (2) quantify the level of radon awareness and knowledge, (3) identify potential obstacles to radon testing among the Allentown population that self-identifies as Hispanic, and (4) determine whether more effective risk communication is needed.Method: Radon test results in Allentown were analyzed to better understand the nature of radon testing. To evaluate radon awareness and knowledge, a cross-sectional study was conducted using a face-to-face survey. This data was informative in assessing testing and mitigation practices, ethnicity, income level, age, education level, homeowner status, zip code and primary language.Results: Ethnicity was an independent predictor of radon awareness and knowledge. Statistically significant differences were found between the number of self-identified Hispanics (39%) and non-Hispanics (84%) who indicated that they had ever heard of radon; 13% of Hispanics and 49% of non-Hispanics knew that they lived in an area with typically high radon levels. There was a statistically significant association between self-reported ethnicity and radon testing with non-Hispanics (43%) more likely to test their homes for radon than Hispanics (32%).Conclusion: Individual and community understanding of the risks of exposure to radiation sources such as radon is dependent upon communication that informs and spurs appropriate action. This study demonstrates the need for culturally appropriate radon risk communication strategies targeted to a Hispanic population. Successful communication will raise awareness and knowledge that can lead to better public health protection.

Short-Duration Rapid Chilling of Mastectomy Specimens Does Not Interfere With Breast Cancer Biomarker and Molecular Testing.

CONTEXT.­: A thorough gross examination of specimens for breast cancer requires the tissue to be very thinly sectioned, which is often difficult in large mastectomy samples. We have implemented rapid chilling of mastectomy specimens before formalin fixation. OBJECTIVE.­: To evaluate the effects of rapid chilling of breast tissue on subsequent biomarker and molecular testing. DESIGN.­: Mastectomy specimens were chilled at -80°C for 20 minutes to facilitate uniform sectioning of tissue at 4-mm intervals and enhance proper fixation and identification of small lesions. The integrity of chilled tissue for ancillary and molecular testing was assessed. We identified patients who were diagnosed with breast cancer on biopsy at outside institutions and subsequently underwent mastectomy at our institution during 2010-2014. We compared the results of biomarker testing performed on biopsy tissue with those performed on mastectomy tissue. The quantity and quality of DNA extracted from formalin-fixed, paraffin-embedded (FFPE) mastectomy tissue with invasive carcinoma were assessed by using spectrophotometry and polymerase chain reaction. All Oncotype DX reports from 2011-2014 were reviewed to identify any documented evidence of assay interference caused by rapid chilling of tissue. RESULTS.­: We found essentially 100% concordances in estrogen receptor, progesterone receptor, and human epidermal growth factor receptor 2 gene ERBB2 (HER2/neu) studies. Extracted tumor DNA showed suitable purity and concentration that produced amplified fragments of 300 to 400 base pair lengths by polymerase chain reaction of FFPE tissue. No documented assay interferences were found in the Oncotype DX reports. CONCLUSIONS.­: Short-duration rapid chilling of mastectomy tissue improves gross examination, optimally preserves DNA, allows for molecular testing, and does not interfere with biomarker assessment.

ALK Rearrangements Are Infrequent in Cellular Blue Nevus and Deep Penetrating Nevus.

Recent studies have identified kinase fusions in Spitzoid melanocytic neoplasms, and approximately 10% of Spitzoid neoplasms harbor anaplastic lymphoma kinase (ALK) rearrangements and corresponding ALK immunoreactivity. Deep penetrating nevi (DPN), a subset of melanocytic neoplasms, have histologic and immunohistochemical overlap that have historically supported classification of DPN with blue/cellular blue nevi (CBN). However, HRAS mutations have rarely been detected in DPN, thereby also linking them to Spitz nevi. The purpose of this study was to see if DPN or CBN possess ALK rearrangements, thereby providing more evidence that these melanocytic lesions may be pathogenetically related to Spitzoid neoplasms. Using ALK immunohistochemistry as a surrogate for ALK rearrangement, the authors examined 26 DPN, 30 CBN, and 4 conventional blue nevi. ALK immunoreactive cases underwent fluorescent in situ hybridization to investigate for the presence of ALK gene rearrangement. Patchy and focal ALK immunostaining was found in only 1 case of DPN (1/26, 3.8%). Seven cases of CBN (7/30; 23%) showed ALK immunostaining (6 focal/patchy, 1 strong and diffuse). Fluorescent in situ hybridization using ALK break-apart probes showed various degrees of gain of 2p23 and rare ALK break-apart signals. Four CBN showed ALK rearrangement in 2%-4% of cells. Two cases of CBN showed gain of 2p23 in 10%-20% of cells. In our study, ALK rearrangements are uncommon in both CBN and DPN, making ALK an unlikely driver in tumorigenesis and classification of these melanocytic variants. However, our study did identify ALK molecular changes and immunohistochemical staining patterns that have not been previously described in CBN or DPN.

Ex vivo-expanded natural killer cells demonstrate robust proliferation in vivo in high-risk relapsed multiple myeloma patients.

Highly activated/expanded natural killer (NK) cells can be generated by stimulation with the human leukocyte antigen-deficient cell line K562, genetically modified to express 41BB-ligand and membrane-bound interleukin (IL)15. We tested the safety, persistence, and activity of expanded NK cells generated from myeloma patients (auto-NK) or haploidentical family donors (allo-NK) in heavily pretreated patients with high-risk relapsing myeloma. The preparative regimen comprised bortezomib only or bortezomib and immunosuppression with cyclophosphamide, dexamethasone, and fludarabine. NK cells were shipped overnight either cryopreserved or fresh. In 8 patients, up to 1×108 NK cells/kg were infused on day 0 and followed by daily administrations of IL2. Significant in vivo expansion was observed only in the 5 patients receiving fresh products, peaking at or near day 7, with the highest NK-cell counts in 2 subjects who received cells produced in a high concentration of IL2 (500 U/mL). Seven days after infusion, donor NK cells comprised >90% of circulating leukocytes in fresh allo-NK cell recipients, and cytolytic activity against allogeneic myeloma targets was retained in vitro. Among the 7 evaluable patients, there were no serious adverse events that could be related to NK-cell infusion. One patient had a partial response and in another the tempo of disease progression decreased; neither patient required further therapy for 6 months. In the 5 remaining patients, disease progression was not affected by NK-cell infusion. In conclusion, infusion of large numbers of expanded NK cells was feasible and safe; infusing fresh cells was critical to their expansion in vivo.

BB0238, a presumed tetratricopeptide repeat-containing protein, is required during Borrelia burgdorferi mammalian infection.

The Lyme disease spirochete, Borrelia burgdorferi, occupies both a tick vector and mammalian host in nature. Considering the unique enzootic life cycle of B. burgdorferi, it is not surprising that a large proportion of its genome is composed of hypothetical proteins not found in other bacterial pathogens. bb0238 encodes a conserved hypothetical protein of unknown function that is predicted to contain a tetratricopeptide repeat (TPR) domain, a structural motif responsible for mediating protein-protein interactions. To evaluate the role of bb0238 during mammalian infection, a bb0238-deficient mutant was constructed. The bb0238 mutant was attenuated in mice infected via needle inoculation, and complementation of bb0238 expression restored infectivity to wild-type levels. bb0238 expression does not change in response to varying culture conditions, and thus, it appears to be constitutively expressed under in vitro conditions. bb0238 is expressed in murine tissues during infection, though there was no significant change in expression levels among different tissue types. Localization studies indicate that BB0238 is associated with the inner membrane of the spirochete and is therefore unlikely to promote interaction with host ligands during infection. B. burgdorferi clones containing point mutations in conserved residues of the putative TPR motif of BB0238 demonstrated attenuation in mice that was comparable to that in the bb0238 deletion mutant, suggesting that BB0238 may contain a functional TPR domain.

Chitosan coating to enhance the therapeutic efficacy of calcium sulfate-based antibiotic therapy in the treatment of chronic osteomyelitis.

We demonstrate that coating calcium sulfate with deacetylated chitosan enhances the elution profile of daptomycin by prolonging the period during which high concentrations of antibiotic are released. Coatings reduced initial bolus release of daptomycin by a factor of 10 to approximately 1000 µg/ml, and levels remained above 100 µg/ml for up to 10 days. Chitosan-coated and uncoated calcium sulfate implants with and without 15% daptomycin were evaluated in an experimental model of staphylococcal osteomyelitis through bacteriology scores, radiology, histopathology, and Gram staining. Significant reduction in bacteriology scores was observed for implants containing daptomycin and coated with chitosan compared with all the other groups. We confirm that the use of chitosan-coated calcium sulfate beads for local antibiotic delivery can be correlated with an improved therapeutic outcome following surgical debridement in the treatment of chronic osteomyelitis.

Influenza a outbreak in an ambulatory stem cell transplant center.

BACKGROUND: In the era of cost-consciousness regarding healthcare , provision of medical services in an outpatient setting has become increasingly attractive. We report an influenza outbreak in an ambulatory stem cell transplant center in 2013 that highlights unique identification and infection control challenges in this setting. METHODS: Nasopharyngeal swabs were performed on patients with suspected influenza-like illnesses (ILI), defined by subjective fever or measured temperature of ≥37.7°C (≥100°F) with cough or sore throat during July 25, 2013 through August 7, 2013. In addition, testing was triggered by an elevated C-reactive protein (CRP). Specimens were analyzed by using eSensor Respiratory Viral Panel. Clinical and epidemiologic information was collected in real time, and frequencies were calculated on demographics, baseline clinical parameters, treatment methods, comorbidities, and symptoms of affected persons. RESULTS: Thirty-one patients had influenza A (H3N2) infection during July 25, 2013 through August 7, 2013. Only 7 patients (23%) met the Centers for Disease Control and Prevention and Council of State and Territorial Epidemiologists ILI case definition. Twenty-five patients (81%) had received ≥1 transplant, with 13 (42%) having occurred within 1 year before the outbreak. Twenty-five patients (81%) had received B-cell active chemotherapy <60 days before influenza diagnosis, 6 (19%) were neutropenic, and 25 (81%) lymphopenic. Among clinical and laboratory markers analyzed, abnormal CRP was the most sensitive screening tool for influenza. Twelve (39%) patients were hospitalized (median stay, 10 days; range, 2-20). No deaths occurred. CONCLUSIONS: Immunocompromised hosts with influenza have atypical presentations. Existing surveillance case definitions might be insufficient to reliably identify influenza outbreaks in such patients.

Use of xylitol to enhance the therapeutic efficacy of polymethylmethacrylate-based antibiotic therapy in treatment of chronic osteomyelitis.

Using a rabbit model of postsurgical osteomyelitis, we demonstrate that incorporation of xylitol into polymethylmethacrylate (PMMA) bone cement enhances the elution of daptomycin under in vivo conditions. We also demonstrate that this can be correlated with an improved therapeutic outcome in the treatment of a chronic bone infection following surgical debridement.

Evaluation of dynamic [18F]-FDG-PET imaging for the detection of acute post-surgical bone infection.

Diagnosing bone infection in its acute early stage is of utmost clinical importance as the failure to do so results in a therapeutically recalcitrant chronic infection that can only be resolved with extensive surgical intervention, the end result often being a structurally unstable defect requiring reconstructive procedures. [(18)F]-FDG-PET has been extensively investigated for this purpose, but the results have been mixed in that, while highly sensitive, its specificity with respect to distinguishing between acute infection and sterile inflammatory processes, including normal recuperative post-surgical healing, is limited. This study investigated the possibility that alternative means of acquiring and analyzing FDG-PET data could be used to overcome this lack of specificity without an unacceptable loss of sensitivity. This was done in the context of an experimental rabbit model of post-surgical osteomyelitis with the objective of distinguishing between acute infection and sterile post-surgical inflammation. Imaging was done 7 and 14 days after surgery with continuous data acquisition for a 90-minute period after administration of tracer. Results were evaluated based on both single and dual time point data analysis. The results suggest that the diagnostic utility of FDG-PET is likely limited to well-defined clinical circumstances. We conclude that, in the complicated clinical context of acute post-surgical or post-traumatic infection, the diagnostic utility accuracy of FDG-PET is severely limited based on its focus on the increased glucose utilization that is generally characteristic of inflammatory processes.

Highly activated and expanded natural killer cells for multiple myeloma immunotherapy.

BACKGROUND: Patients with gene expression profiling-defined high-risk myeloma in relapse have poor outcomes with current therapies. We tested whether natural killer cells expanded by co-culture with K562 cells transfected with 41BBL and membrane-bound interleukin-15 could kill myeloma cells with a high-risk gene expression profile in vitro and in a unique model which recapitulates human myeloma. DESIGN AND METHODS: OPM2 and high-risk primary myeloma tumors were grown in human fetal bone implanted into non-obese diabetic severe combined immunodeficiency mice with a deficient interleukin-2 receptor gamma chain. These mice are devoid of endogenous natural killer and T-cell activity and were used to determine whether adoptively transferred expanded natural killer cells could inhibit myeloma growth and myeloma-associated bone destruction. RESULTS: Natural killer cells from healthy donors and myeloma patients expanded a median of 804- and 351-fold, respectively, without significant T-cell expansion. Expanded natural killer cells killed both allogeneic and autologous primary myeloma cells avidly via a perforin-mediated mechanism in which the activating receptor NKG2D, natural cytotoxicity receptors, and DNAX-accessory molecule-1 played a central role. Adoptive transfer of expanded natural killer cells inhibited the growth of established OPM2 and high-risk primary myeloma tumors grown in the murine model. The transferred, expanded natural killer cells proliferated in vivo in an interleukin-2 dose-dependent fashion, persisted up to 4 weeks, were readily detectable in the human bone, inhibited myeloma growth and protected bone from myeloma-induced osteolysis. CONCLUSIONS: These studies provide the rationale for testing expanded natural killer cells in humans.

A phase II trial of single agent bevacizumab in patients with relapsed, aggressive non-Hodgkin lymphoma: Southwest oncology group study S0108.

This is the first report of the Southwest oncology group phase II trial of single agent bevacizumab in patients with relapsed, aggressive non-Hodgkin lymphoma (NHL). Fifty-two patients in first or second relapse with diffuse large B-cell or mantle cell lymphoma were enrolled. Patients were treated with bevacizumab at 10 mg/kg every 2 weeks. Therapy was well tolerated with no unexpected toxicities observed. Six-month progression-free survival (PFS) was 16% with a response rate of 2% and median duration of response or stable disease of 5.2 months (range 3.5-72.7). Vascular endothelial growth factor A (VEGF) and VEGF receptor expression was observed in 70% and 65% of specimens, respectively. In an exploratory subgroup analysis, baseline urine VEGF and plasma vascular cell adhesion molecule-1 (VCAM) levels correlated with survival. Prolonged PFS in several patients as well as biomarker studies suggest the VEGF pathway plays an important role in aggressive NHL. Clinical trials combining active chemotherapy regimens with VEGF targeted agents are currently in progress.

Frequent engagement of the classical and alternative NF-kappaB pathways by diverse genetic abnormalities in multiple myeloma.

Mechanisms of constitutive NF-kappaB signaling in multiple myeloma are unknown. An inhibitor of IkappaB kinase beta (IKKbeta) targeting the classical NF-kappaB pathway was lethal to many myeloma cell lines. Several cell lines had elevated expression of NIK due to genomic alterations or protein stabilization, while others had inactivating mutations of TRAF3; both kinds of abnormality triggered the classical and alternative NF-kappaB pathways. A majority of primary myeloma patient samples and cell lines had elevated NF-kappaB target gene expression, often associated with genetic or epigenetic alteration of NIK, TRAF3, CYLD, BIRC2/BIRC3, CD40, NFKB1, or NFKB2. These data demonstrate that addiction to the NF-kappaB pathway is frequent in myeloma and suggest that IKKbeta inhibitors hold promise for the treatment of this disease.

External quality assessment of a DNA sequence-based scheme for epidemiological typing of Legionella pneumophila by an international network of laboratories.

We report the results of an international external quality assessment (EQA) program to assess the performance of laboratories in genotyping Legionella pneumophila isolates using the standard European Working Group for Legionella Infections sequence-based typing protocol. Three coded distributions of L. pneumophila isolates were sent to laboratories in 12, 14, and 20 countries, respectively. The data were returned by 11 of 16, 18 of 19, and 27 of 29 centers, respectively. Incomplete submission of data resulted in exclusion from certain aspects of the analyses. The number of centers achieving 100% score, for all loci tested, rose successively from 50% (5 of 10) for the first EQA distribution, to 56% (9 of 16) for the second EQA distribution, to 76% (19 of 25) for the third EQA distribution. A number of additional centers made only a few errors (one to three) in each distribution. Sequence data from the first two distributions were collected in flat text file format and using specially developed software, the sequence quality tool (SQT), in the third distribution. The SQT allows users to upload trace files in standard file formats, automates basecalling using phred and phrap software, contig assembly, trimming, and matching against a reference library. The program described here allow users an independent measure of sequence quality, and such schemes are vital in order to identify strengths and weakness in centers responsible for the generation of genotyping data in legionella outbreak investigation. The present study demonstrates that DNA sequence data can be highly reproducible but, when independently assessed, in practice frequently falls short of this goal. However, experience and training in the methodology results in increased performance.

The anti-angiogenesis agent, AG-013736, has minimal activity in elderly patients with poor prognosis acute myeloid leukemia (AML) or myelodysplastic syndrome (MDS).

AG-013736 is an oral anti-angiogenesis agent with activity against a variety of receptor tyrosine kinases, including VEGFR-1, VEGFR-2, VEGFR-3, c-kit, and PDGFR-beta. A phase 2 study was conducted in patients with poor prognosis AML or MDS. Twelve patients (six AML; six MDS) were treated with AG-013736 at a dose of 10mg orally daily for a median of 56 days (range, 1-248 days). Median age was 80 years (range, 58-88 years). Grade 3 or 4 drug-related toxicities included hypertension (42%), mucositis (8%) and deep venous thrombosis (8%). No objective responses occurred; two patients with MDS had stable disease for 8.3 and 6.2 months, respectively. Bone marrow expression of VEGFR-1 and VEGFR-2 was observed in 11% and 0% of patients, respectively. Sustained decreases in soluble VEGFR-2 plasma levels with concomitant elevation in plasma VEGF and placental growth factor levels were obtained during the course of therapy with AG-013736. AG-01736 had minimal biologic or clinical activity in this elderly patient population.

Lenalidomide: targeted anemia therapy for myelodysplastic syndromes.

Lenalidomide, an IMiD drug (a novel type of immunomodulating drug) was recently approved by the US Food and Drug Administration for the treatment of transfusion-dependent anemia in patients with myelodysplastic syndromes (MDS) and interstitial deletions of chromosome 5q [del(5q)]. This review examines the clinical experience from the MDS-001 and MDS-003 clinical trials that led to this approval, the results of biological correlates supporting the targets of drug action, and the results from a non-del(5q) multicenter study (MDS-002). Lenalidomide treatment resulted in both erythroid and cytogenetic responses in the majority of patients with del(5q), accompanied by reductions in inflammatory cytokine generation and marrow microvessel density and improvement in primitive hematopoietic progenitor recovery. Central pathology review showed that resolution of cytologic dysplasia was common in patients with del(5q) but was infrequent in erythroid-responding patients without the chromosome 5 deletion. These findings indicate that lenalidomide promotes erythropoiesis in lower-risk MDS, with two apparently distinct mechanisms of action: suppression of the ineffective del(5q) clone and promotion of effective erythropoiesis in non-del(5q) MDS progenitors. These studies identified lenalidomide as a highly active erythropoietic- and cytogenetic-remitting agent in lower-risk MDS patients who otherwise would not be expected to benefit from recombinant erythropoietin therapy. The most common adverse reactions include dose-dependent neutropenia and thrombocytopenia that are more pronounced in patients with del(5q) in whom early suppression of the clone is expected.

ER-associated protein degradation is a common mechanism underpinning numerous monogenic diseases including Robinow syndrome.

Correct folding of nascent polypeptide chains within the ER is critical for function, assembly into multi-subunit complexes and trafficking through the exocytic pathway for secretory and cell surface proteins. This process is rather inefficient, and a substantial proportion of nascent polypeptides is rejected by an ER quality control system and targeted for degradation. In some cases, only a minor fraction of nascent chains is correctly folded, and the smallest alteration to polypeptide primary structure (i.e. point mutation) can result in the complete loss of function with inherent pathological consequences; cystic fibrosis and emphysema result from such mutations. We have taken a bioinformatic approach to parse a large database of known disease susceptibility genes for candidates whose disease-associated alleles are likely prone to misfolding in the ER. Surprisingly, we find that proteins with ER-targeting signals are over represented in this database when compared with all predicted proteins in the human genome (45 versus 30%). We selected a subgroup of proteins that were positive for both an ER-targeting signal and a membrane-anchoring domain and thereby identified several ER-associated degradation diseases candidates. To determine whether our analysis had identified new ER-degradation substrates, we established that ER retention is indeed the mechanism underlying Robinow syndrome (RRS), one of the identified candidates. Specifically, mutant alleles of ROR2 that are associated with RRS are retained within the ER, whereas wild-type and non-pathogenic alleles are exported to the plasma membrane. These data both uncover a major pathogenic factor for RRS and indicate that misfolding of secretory proteins is likely to significantly contribute to human disease and morbidity.