Validation of Whole-Slide Imaging for Histolopathogical Diagnosis: Current State

Rapid advances in informatics and technological improvements have led to the development of high-throughput whole-slide imaging (WSI) scanners able to produce high-quality digital images, which allow achieving a correct diagnosis of the biopsies using virtual viewers. This technology is currently prepared to be introduced in the departments of pathology for routine diagnosis. The aim of this review is to analyze the current evidence regarding the use of WSI in primary or routine diagnosis in the different subspecialties of pathology. An increasing number of studies have shown almost perfect inter- and intraobserver agreement between the diagnoses obtained with WSI and the classical diagnoses based on conventional light microscopy. The only exception seems to be cytology, which still requires some technological development. Although validation studies are needed in some areas of pathology, growing evidence indicates that WSI is a reliable tool for routine diagnosis. Pathologists have a positive perception of the ergonomics of the workstations, the low magnification of WSI and the possibility of making annotations and measurements. WSI can be used from any device and anywhere, thereby providing great opportunities for teleconsultation. New technologies such as the recognition of histopathology patterns using image analysis may facilitate diagnosis and improve the reproducibility among pathologists in the future

Validation of whole-slide imaging in the primary diagnosis of liver biopsies in a University Hospital

BACKGROUND: Experience in the use of whole slide imaging (WSI) for primary diagnosis is limited and there are no comprehensive reports evaluating this technology in liver biopsy specimens. AIMS: To determine the accuracy of interpretation of WSI compared with conventional light microscopy (CLM) in the diagnosis of needle liver biopsies. METHODS: Two experienced liver pathologists blindly analyzed 176 consecutive biopsies from the Pathology Department at the Hospital Clinic of Barcelona. One of the observers performed the initial evaluation with CLM, and the second evaluation with WSI, whereas the second observer performed the first evaluation with WSI and the second with CLM. All slides were digitized in a Ventana iScan HT at 400x and evaluated with the Virtuoso viewer (Roche diagnostics). We used kappa statistics (kappa) for two observations. RESULTS: Intra-observer agreement between WSI and CLM evaluations was almost perfect (96.6%, kappa=0.9; 95% confidence interval [95% CI]: 0.9-1 for observer 1, and 90.3%, kappa=0.9; 95%CI: 0.8-0.9 for observer 2). Both native and transplantation biopsies showed an almost perfect concordance in the diagnosis. CONCLUSION: Diagnosis of needle liver biopsy specimens using WSI is accurate. This technology can reliably be introduced in routine diagnosis

Histological characteristics of HPV-associated and -independent squamous cell carcinomas of the vulva: A study of 1594 cases

There are at least two different etio-pathogenic pathways for the development of vulvar squamous cell carcinoma (VSCC): one associated with infection by human papillomavirus (HPV) and another independent of HPV. We aimed to describe the histological characteristics of HPV-associated and HPV-independent tumors and to determine the best strategy to identify HPV in VSCC. A single paraffin block was available for review from a series of 1594 VSCCs. In all cases HPV DNA detection was analyzed using the SPF10PCR/DEIA/LiPA25 system and p16 immunohistochemistry (IHC). A tumor was considered as unquestionably HPV-associated if both HPV DNA and p16 IHC were positive. A tumor was considered indisputably HPV-independent if both HPV DNA and p16 IHC were negative. Two groups of tumors were classified as non-conclusive: 1) HPV DNA+/p16-; and 2) HPV DNA-/p16+. WHO typing and a thorough histological evaluation were conducted in all cases. 441 tumors were HPV DNA+ with 367 cases (23.0%) being HPV DNA+/p16+. These HPV DNA+/p16+ tumors were more frequently basaloid or warty (49.8%), but 36.5% were of the keratinizing type. 1153 tumors were HPV DNA-, with 1060 cases (66.5%) being HPV DNA-/p16-. These HPV DNA-/p16- tumors were mostly keratinizing (81.2%) but were occasionally basaloid or warty (5.2%). The features of HPV DNA-/p16+ cases (n=93) were similar to those of the HPV-associated VSCC, and HPV DNA+/p16- (n=74) cases had a more diverse profile, although they were more similar to HPV-independent tumors. Several histological characteristics were more frequently associated with HPV-related VSCC (koilocytotic-like change, necrosis, moderate to marked pleomorphism, invasive front in nests; p<0.001), however, none of these characteristics allowed differentiation between HPV-associated and -independent VSCC. In conclusion, histological criteria do not allow differentiation between HPV-associated and -independent VSCC. p16 alone is a clinically easy strategy to determine HPV status in VSCC. This article is protected by copyright. All rights reserved

HPV-independent Precursors Mimicking High-grade Squamous intraepithelial Lesions (HSIL) of the Vulva

Two etiopathogenic types of vulvar squamous cell carcinoma (VSCC) have been described: human papillomavirus (HPV)-associated and HPV-independent. Precursor lesions, frequently identified in the adjacent skin, are also distinct in the 2 types of VSCC: high-grade squamous intraepithelial lesions (HSILs) in HPV-associated VSCC and differentiated vulvar intraepithelial neoplasia (dVIN) or vulvar acanthosis with altered differentiation in HPV-independent VSCC. Although HPV-independent precursors mimicking HSIL have been described in the vulva, their frequency and morphologic spectrum have not been completely characterized. We explored, in a large series of HPV-independent VSSC, the frequency and the histologic features of precursors mimicking HSIL. We included 779 DNA HPV-negative/p16-negative VSCC with at least 1\xE2\x80\x89cm of adjacent skin. We evaluated the histologic and immunohistochemical (p16 and p53) characteristics of the intraepithelial lesions, focusing on precursors mimicking HPV-associated vulvar HSIL. A total of 254 tumors (33%) had adjacent premalignant lesions. Of them, 186 (73%) had dVIN, 22 (9%) had vulvar acanthosis with altered differentiation, and 46 (18%) had lesions that mimicked HSIL. The mean age of the patients with these HSIL-like lesions was 72\xC2\xB115 years. Twenty-six of these HSIL-like lesions had basaloid morphology, 13 warty, and 7 mixed basaloid/warty features. All the HSIL-like precursors were DNA HPV-negative/p16-negative; 74% of them showed p53 abnormal staining and 35% of them had areas of conventional dVIN. In conclusion, about one fifth of the HPV-independent precursors mimic HSIL, showing either basaloid or warty features. Older age and the presence of areas of typical HPV-independent intraepithelial lesions, together with p16 negativity, should raise suspicion of an HPV-independent etiology

Quality of care and maternal mortality in a tertiary-level hospital in Mozambique: a retrospective study of clinicopathological discrepancies

Background: Although an increasing number of pregnant women in resource-limited areas deliver in health-care facilities, maternal mortality remains high in these settings. Inadequate diagnosis and management of common life-threatening conditions is an important determinant of maternal mortality. We analysed the clinicopathological discrepancies in a series of maternal deaths from Mozambique and assessed changes over 10 years in the diagnostic process. We aimed to provide data on clinical diagnostic accuracy to be used for improving quality of care and reducing maternal mortality. Methods: We did a retrospective analysis of clinicopathological discrepancies in 91 maternal deaths occurring from Nov 1, 2013, to March 31, 2015 (17 month-long period), at a tertiary-level hospital in Mozambique, using complete diagnostic autopsies as the gold standard to ascertain cause of death. We estimated the performance of the clinical diagnosis and classified clinicopathological discrepancies as major and minor errors. We compared the findings of this analysis with those of a similar study done in the same setting 10 years earlier. Findings: We identified a clinicopathological discrepancy in 35 (38%) of 91 women. All diagnostic errors observed were classified as major discrepancies. The sensitivity of the clinical diagnosis for puerperal infections was 17% and the positive predictive value was 50%. The sensitivity for non-obstetric infections was 48%. The sensitivity for eclampsia was 100% but the positive predictive value was 33%. Over the 10-year period, the performance of clinical diagnosis did not improve, and worsened for some diagnoses, such as puerperal infection. Interpretation: Decreasing maternal mortality requires improvement of the pre-mortem diagnostic process and avoidance of clinical errors by refining clinical skills and increasing the availability and quality of diagnostic tests. Comparison of post-mortem information with clinical diagnosis will help monitor the reduction of clinical errors and thus improve the quality of care

Limitations to current methods to estimate cause of death: a validation study of a verbal autopsy model

Background: Accurate information on causes of death (CoD) is essential to estimate burden of disease, track global progress, prioritize cost-effective interventions, and inform policies to reduce mortality. In low-income settings, where a significant proportion of deaths take place at home or in poorly-resourced peripheral health facilities, data on CoD often relies on verbal autopsies (VAs). Validations of VAs have been performed against clinical diagnosis, but never before against an acceptable gold standard: the complete diagnostic autopsy (CDA). Methods: We have validated a computer-coded verbal autopsy method -the InterVA- using individual and population metrics to determine CoD against the CDA, in 316 deceased patients of different age groups who died in a tertiary-level hospital in Maputo, Mozambique between 2013 and 2015. Results: We found a low agreement of the model across all age groups at the individual (kappa statistic ranging from -0.030 to 0.232, lowest in stillbirths and highest in adults) and population levels (chance-corrected cause-specific mortality fraction accuracy ranging from -1.00 to 0.62, lowest in stillbirths, highest in children). The sensitivity in identifying infectious diseases was low (0% for tuberculosis, diarrhea, and disseminated infections, 32% for HIV-related infections, 33% for malaria and 36% for pneumonia). Of maternal deaths, 26 were assigned to eclampsia but only four patients actually died of eclampsia. Conclusions: These findings do not lead to building confidence in current estimates of CoD. They also call to the need to implement autopsy methods where they may be feasible, and to improve the quality and performance of current VA techniques

P53 in Penile Squamous Cell Carcinoma : A Pattern-Based Immunohistochemical Framework with Molecular Correlation

Penile squamous cell carcinomas harbouring mutations of TP53 have an increased risk of lymph node metastases and an impaired prognosis, but the mutational analysis of the TP53 gene is not available in many pathology laboratories. Although p53 immunohistochemistry (IHC) has been proposed as an alternative to the molecular analysis, the current method of evaluation of p53 IHC has many inaccuracies. The aim of our study was to determine, in a series of 40 penile tumours, if a recently described pattern-based framework of p53 IHC evaluation correlates better than the classical method with the TP53 mutational status. Our results show that the new method has a very good correlation with TP53 mutations (95% sensitivity; 92% specificity), higher than that of the classical method, and can be considered as a reliable surrogate of the TP53 mutational status. This new framework can help clinicians to better define risk groups and refine treatment strategies. p53 immunohistochemistry (IHC) has been proposed as a surrogate for TP53 mutations in penile squamous cell carcinomas (PSCC). We aimed to evaluate the performance of a pattern-based evaluation of p53 IHC in PSCC. Human papilloma virus (HPV) DNA testing, p16 and p53 IHC, and whole exome sequencing were performed in a series of 40 PSCC. p53 IHC was evaluated following a pattern-based framework and conventional p53 IHC evaluation. Out of 40 PSCC, 12 (30.0%) were HPV-associated, and 28 (70.0%) were HPV-independent. The agreement between the p53 IHC pattern-based evaluation and TP53 mutational status was almost perfect (k = 0.85). The sensitivity and accuracy of the pattern-based framework for identifying TP53 mutations were 95.5% and 92.5%, respectively, which were higher than the values of conventional p53 IHC interpretation (54.5% and 70.0%, respectively), whereas the specificity was the same (88.9%). In conclusions, the pattern-based framework improves the accuracy of detecting TP53 mutations in PSCC compared to the classical p53 IHC evaluation

Unmasking the hidden tuberculosis mortality burden in a large postmortem study in Maputo Central Hospital, Mozambique

Sensitive tools are needed to accurately establish the diagnosis of tuberculosis (TB) at death, especially in low-income countries. The objective of this study was to evaluate the burden of TB in a series of patients who died in a tertiary referral hospital in sub-Saharan Africa using an in-house real time PCR (TB-PCR) and the Xpert MTB/RIF Ultra (Xpert Ultra) assay.Complete diagnostic autopsies were performed in a series of 223 deaths (56.5% being HIV-positive), including 54 children, 57 maternal deaths and 112 other adults occurring at the Maputo Central Hospital, Mozambique. TB-PCR was performed in all lung, cerebrospinal fluid and central nervous system samples in HIV-positive patients. All samples positive for TB-PCR or showing histological findings suggestive of TB were analysed with the Xpert Ultra assay.TB was identified as the cause of death in 31 patients: 3/54 (6%) children, 5/57 (9%) maternal deaths and 23/112 (21%) other adults. The sensitivity of the main clinical diagnosis to detect TB as the cause of death was 19.4% (95% CI: 7.5-37.5) and the specificity was 97.4% (94.0-99.1) compared to autopsy findings. Concomitant TB (TB disease in a patient dying of other causes) was found in 31 additional cases. Xpert Ultra helped to identify 15 cases of concomitant TB. In 18 patients, " - " DNA was identified by TB-PCR and Xpert Ultra in the absence of histological TB lesions. Overall, 62 cases (27.8%) had TB disease at death and 80 (35.9%) had TB findings.The use of highly sensitive, easy to perform molecular tests in complete diagnostic autopsies may contribute to identifying TB cases at death that would have otherwise been missed. Routine use of these tools in certain diagnostic algorithms for hospitalised patients needs to be considered. Clinical diagnosis showed poor sensitivity for the diagnosis of TB at death

High within-host diversity found from direct genotyping on post-mortem tuberculosis specimens in a high-burden setting

Objectives: To characterize the clonal complexity in Mycobacterium tuberculosis (MTB) infections considering factors that help maximize the detection of coexisting strains/variants. Methods: Genotypic analysis by Mycobacterial Interspersed Repetitive-Unit-Variable-Number Tandem-Repeats (MIRU-VNTR) was performed directly on 70 biopsy specimens from two or more different tissues involving 28 tuberculosis cases diagnosed post-mortem in Mozambique, a country with a high tuberculosis burden. Results: Genotypic data from isolates collected from two or more tissues were obtained for 23 of the 28 cases (82.1%), allowing the analysis of within-patient diversity. MIRU-VNTR analysis revealed clonal diversity in ten cases (35.7%). Five cases showed allelic differences in three or more loci, suggesting mixed infection with two different strains. In half of the cases showing within-host diversity, one of the specimens associated with clonal heterogeneity was brain tissue. Conclusions: Direct MTB genotyping from post-mortem tissue samples revealed a frequent within-host Mycobacterium tuberculosis diversity, including mixed and polyclonal infections. Most of this diversity would have been overlooked if only standard analysis of respiratory specimens had been performed

High prevalence and mortality due to Histoplasma capsulatum in the Brazilian Amazon: An autopsy study

Background: Histoplasmosis is acquired by inhalation of spores of the dimorphic fungus Histoplasma spp. Although this pathogen is distributed worldwide, it is more prevalent in the Americas. However, the real burden of histoplasmosis remains undefined in many endemic regions. Methodology: We conducted a series of 61 autopsies to individuals who died in a hospital in the Brazilian Amazon focused on infectious diseases. We performed a detailed histological and microbiological evaluation with genetic characterization of Histoplasma strains with the aim to evaluate the contribution of histoplasmosis to morbidity and mortality. Additionally, we assessed the clinicopathological correlation. Principal findings: Evidence of Histoplasma infection was detected in 21 patients (34%). Eight cases were disseminated infections, all of them occurred in HIV-positive patients. Six cases were localized histoplasmosis, limited to the lungs. In seven patients Histoplasma DNA was detected by PCR in patients with no histological lesions. Histoplasma infection was detected in 38% of HIV-positive patients and was a major contributor to death in 22% of them. Lungs, liver and spleen were affected in all cases of disseminated histoplasmosis. Phylogenetic analysis of the strains suggested a high diversity of Histoplasma species circulating in the Brazilian Amazon. Histoplasmosis was clinically missed in 75% of the disseminated infections. Conclusions: The high incidence of histoplasmosis, the low index of clinical suspicion, and the severity of the disseminated disease highlight the need of proactively implementing sensitive routine screening methods for this pathogen in endemic areas. Antifungal prophylaxis against Histoplasma should be encouraged in the severely immunocompromised HIV patients in these areas. In conclusion, substantial mortality is associated with disseminated histoplasmosis among HIV-positive patients in the Brazilian Amazon