Comparison between the accuracy of chest computerized tomography vs. reverse transcriptase polymerase chain reaction in a tertiary care center in Lebanon; along with their correlation to mortality, morbidity and symptoms in COVID-19 patients.

Objectives: Chest Computerized Tomography has been widely used in COVID patients' assessment. Hence the question arises as to whether there is any correlation between the Ct value and findings on Chest CT scan or clinical presentation of the patient. We wanted to test the hypothesis of whether low Ct values (≤30) in RT-PCR were associated with a high mortality rate, CT scan findings, or with comorbidities such as immunosuppression and lung disease. Methods: The radiographic records and RT-PCR Ct values of 371 COVID patents diagnosed at the American University of Beirut Medical Center were reviewed. Results: We found out that the sensitivity of chest CT scan compared to RT-PCR, the gold standard, turned out to be 74% (95% CI 69-79%). Specificity, on the other hand was 33% (95% CI 16-55%). The positive predictive value of CT was 94% (95% CI 91-97%) and the negative predictive value was 8% (95% CI 4-16%). low Ct values in RT-PCR were not associated with a higher mortality rate (p-value = 0.416). There was no significant positive association between low Ct value and suspicious CT scan findings (typical and indeterminate for COVID-19), with a p-value of 0.078. There was also no significant association between low Ct value and immunosuppression (p-value = 0.511), or lung disease (p-value =0.06). CT scan findings whether suspicious or not for COVID-19 infection, were not shown to be significantly associated with respiratory symptoms of any kind.No association was found between a history of lung disease, immunosuppression and suspicious CT scan findings for COVID-19. Conclusion: As long as this pandemic exists, nucleic acid testing was and remains the gold standard of COVID-19 diagnosis worldwide and in our community as it has a superior diagnostic accuracy to CT scan and higher sensitivity (94% vs 74%).

Late antibody-mediated rejection in a kidney transplant recipient: COVID 19 induced?

BACKGROUND: Antibody-mediated rejection (AMR) was described in kidney transplant patients after viral infections, such as the cytomegalovirus. Very few cases were recently reported after severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection, probably in the context of lowering of immunosuppressive therapy. To date, no direct immunological link was proved to explain a connection between the coronavirus disease 19 (COVID-19) infection and antibody-mediated rejection (AMR) if it exists. CASE PRESENTATION: Here we try to find this association by presenting the case of a low immunological risk patient who presented, six years post-transplant, with c4d negative antibody mediated rejection due to an anti-HLA-C17 de novo donor specific antibody (DSA) after contracting the coronavirus disease 19. The HLA-Cw17 activated the antibody-dependent cell-mediated cytotoxicity via the KIR2DS1 positive NK cells. DISCUSSION AND CONCLUSIONS: This case report may prove a direct role for COVID-19 infection in AMRs in the kidney transplant recipients, leading us to closely monitor kidney transplant recipients, especially if they have "at-risk" donor antigens.

Microsatellite Instability in Near East Sebaceous Neoplasms: Toward Improved Prediction.

Sebaceous neoplasms (SN) comprise a heterogeneous spectrum of tumors with different biological behaviors. In the Near-East Region (NER), microsatellite instability (MSI) in SN's development, and its correlation with the clinicopathologic features of tumors is not well elucidated. A cohort of 225 SN patients (40 benign SNs and 185 sebaceous carcinomas) from the NER was retrospectively reviewed. Clinical variables and available follow-up information were recorded. MSI proteins (MLH1, MSH2, MSH6, and PMS2) as well as P53, P16, EMA, CD8, and PDL-1 expressions were examined by immunohistochemistry. Detection of human papilloma virus was determined by polymerase chain reaction. Microscopic features such as mitotic count and tumor-infiltrating lymphocytes were documented. A minority of SNs from benign (n=2) or malignant (n=3) tumors in the NER exhibit MSI (2.2%). MSI is exclusively found in patients with extraocular lesions (back, n=5) and presented a poor outcome. Among these, PMS2 protein was mostly lost (average=80%, n=4). SN with MSI exhibited a significant increase in p53 expression, (average=62.10%, P=0.002). There was no significant correlation between MSI status and any of the following: PD-L1, CD8, p16, and human papilloma virus infection. Microscopically, SN with MSI show significantly higher mitotic count, cystic changes and increased tumor-infiltrating lymphocytes. MSI is rarely found in NER's SN. When detected, it is exclusively in extraocular SNs with minimal predicative microscopic features and worse outcome.

Sebaceous neoplasms: prevalence of HPV infection and relation to immunohistochemical surrogate markers.

BACKGROUND: Sebaceous neoplasms (SNs) and carcinomas (SCs) represent rare skin adnexal tumours. OBJECTIVES: To establish the prevalence of HPV in SNs, assess the relationship between HPV, p16 and p53 expression, and further elucidate the carcinogenetic course of SCs. MATERIALS & METHODS: A total of 113 resected SNs (five sebaceous adenomas, 10 sebaceomas and 98 SCs) from the Near-East were reviewed. Clinical information (age, gender, size and anatomical location), microscopic variables, and expression of several immunohistochemical markers (EMA, CK5/6, p63, p40, AR, p16 and p53) were documented. Cases were evaluated by fluorescently labelled PCR for HPV detection, followed by DNA microarray hybridization for subtype detection. RESULTS: HPV infection was detected in 9.4% of SNs: 28.6% sebaceous adenomas (HPV-16 and HPV-66), 9.1% sebaceomas (HPV-18) and 8.1% SCs. High-risk HPV types (HPV-16, -18, -52 and -66) constituted 90.9% of HPV infections. Histologically, HPV-positive SCs showed significantly milder cytologic atypia and patchy cellular necrosis. p16 was expressed in SNs irrespective of HPV status (20.0%, 33.3% and 65.5% of HPV-negative sebaceous adenomas, sebaceomas, and SCs, respectively), and p53 was abnormally expressed in 95.5% of HPV-negative SCs and all HPV-positive SCs. CONCLUSION: HPV infection is significantly present in benign and malignant SNs. HPV-positive SCs exhibit less cytologic atypia and necrosis than HPV-negative cases. p16 is not a surrogate marker of HPV infection in the SN setting. Further elucidation of various carcinogenic mechanisms in SCs will allow clinicians to single out the various populations at risk, optimize possible preventive strategies and develop targeted therapies.

PD-L1 expression in sebaceous carcinomas.

BACKGROUND: Traditional systemic treatments for unresectable, recurrent, and/or advanced sebaceous carcinoma (SC) are ineffective. Tumoral immune microenvironment characterization is essential for considering immune checkpoint inhibitors as a treatment option. METHODS: A total of 173 resected SCs were reviewed. Clinical information, lesion size, and location were collected. Microscopic examination documented histopathologic features and expression of immunohistochemical markers PD-L1 and CD8. PD-L1 percentage was assessed amongst tumor (PD-L1 + Tu) and immune infiltrating cells (PD-L1 + Inf). Each case was attributed a combined positive score (CPS) following Head and Neck squamous cell carcinoma recommendations. PD-L1 expression was evaluated according to clinicopathologic parameters. Human Papilloma Virus presence (HPV) was analyzed using PCR microarray scanning. RESULTS: A therapeutically relevant CPS was seen in 51.4% of cases. Higher PD-L1 + Tu, PD-L1 + Inf, and CPSs were positively associated with greater lesion size and an extraocular location. No association was seen with patient age or gender. 9.2% of SCs showed PD-L1 + Tu ≥ 1, while 52.0% showed PD-L1 + Inf ≥ 1. A higher CD8 + T-lymphocyte density was significantly associated with a higher CPS, PD-L1 + Tu, and PD-L1 + Inf. Tumor-associated T-cell infiltrate's density was higher along tumor periphery. HPV-16, HPV-43, HPV-52, and HPV-66 were detected in 8.4% of SCs. There was no significant association between HPV status, PD-L1 expression, and CPS. A significant number of SCs express PD-L1 at therapeutic levels. Nevertheless, PD-L1 expression shows a higher intertumoral heterogeneity, in extraocular than in biologically distinct periocular cases. CONCLUSION: Our data support the need for large-scale prospective studies evaluating anti-PD-L1 immunotherapy mainly in extraocular SC treatment.

HPV-related oropharyngeal cancer prevalence in a middle eastern population using E6/E7 PCR.

BACKGROUND: Given the paucity of data and widely variable rates that have been reported, the main objective of this study was to examine the prevalence of HPV-positivity in oropharyngeal squamous cell carcinoma (OPSCC) in Middle Eastern patients presenting to one of the region's largest tertiary care centers using polymerase chain reaction (PCR) amplification of the HPV E6/E7 oncogenes, a highly sensitive and specific method of detection. METHODS: Medical charts and archived pathological specimens were obtained for patients diagnosed with biopsy proven oropharyngeal cancer who presented to the American University of Beirut Medical Center between 1972 and 2017. DNA was extracted from paraffin-embedded specimens and tested for 30 high-risk and low-risk papilloma viruses using the PCR-based EUROarray HPV kit (EuroImmun). RESULTS: A total of 57 patients with oropharyngeal cancer were initially identified; only 34 met inclusion/exclusion criteria and were included in the present study. Most patients were males (73.5%) from Lebanon (79.4%). The most common primary tumor site was in the base of tongue (50%), followed by the tonsil (41.2%). The majority of patients (85.3%) tested positive for HPV DNA. CONCLUSION: The prevalence of HPV-positivity amongst Middle Eastern OPSCC patients, specifically those from Lebanon, may be far greater than previously thought. The Lebanese population and other neighboring Middle Eastern countries may require a more vigilant approach towards HPV detection and awareness. On an international level, further research is required to better elucidate non-classical mechanisms of HPV exposure and transmission.

Vitamin D receptor biochemical and genetic profiling and HLA-class II genotyping among Lebanese with multiple sclerosis - A pilot study.

BACKGROUND: Multiple sclerosis (MS) is an autoimmune demyelinating disease affecting mostly young adult females with multifactorial etiology. Recent studies suggested that adequate vitamin D levels may lower the risk of developing MS. OBJECTIVES: Our aim was to explore the relationship between vitamin D receptor (VDR) polymorphism, HLA-DR locus genotype, and serum vitamins D and A levels in the Lebanese population. METHODS: Fifty MS patients were recruited for this study. The control group consisted of 48 healthy and 51 patients with other neurological disorders (non-MS). Biochemical analysis included serum 25 hydroxyvitamin D (25OHD) and vitamin A. Molecular analysis targeted VDR genotypes (ApaI, TaqI and BsmI) and low resolution HLA typing for DRB1 locus. RESULTS: Healthy and non-MS groups had comparable parameters and were combined into one control group. No significant differences were found between MS and control groups for VDR genotypes. The frequency of HLA-DRB1*15 was significantly higher in MS patients (22%) compared to controls (8%) (p=0.018). Odds ratio for MS in the presence of DRB1*15 allele was 3.21 (p=0.018). Cosegregation with A (ApaI) and b (BsmI) alleles did not influence the risk for MS. 25OHD levels were significantly higher in MS patients compared to controls (p=0.002), due to more frequent oral supplementation (p=0.005). Vitamin A levels were comparable between the two groups. When all parameters were included in a logistic regression model adjusted for supplementation, only HLA-DRB1*15 (OR=3.42; p=0.027) contributed significantly to MS risk. CONCLUSION: There was no association between serum vitamin D or A or VDR genotypes and MS. HLA-DRB1*15 was the major factor imposing more than 3 folds greater risk for developing MS among Lebanese.

First report on HLA-DPA1 gene allelic distribution in the general Lebanese population.

AIMS: HLA-DPA1 is an important marker in bone marrow and organ transplantation and a highly emerging screening parameter in histocompatibility laboratories. Being highly polymorphic, it has another significant value in detecting population origins and migrations. This is the first study to assess DPA1 allele frequencies in an Arab population. METHODS: The HLA DPA1 alleles were identified using the One-Lambda assays on a Luminex reverse SSO DNA typing system. Our study included 101 individuals coming from different Lebanese geographical areas representing the different communities and religious sects of the country. RESULTS: We compared the results of this study to 16 different populations and found very interesting similarities and differences between Lebanese people and individuals of European ancestry. CONCLUSION: This study is the first to describe the different allelic frequencies of HLA-DPA1 in the Lebanese population and will serve as a template that can be later used for disease association studies both at the level of the country and internationally.

Comparison of the Artus RotorGene and COBAS Ampliprep/COBAS TaqMan Platforms for the Detection of Cytomegalovirus: Experience of a Tertiary Care Center.

INTRODUCTION: Cytomegalovirus (CMV) is a member of the Herpesviruses family. CMV infection rarely causes serious disease in otherwise healthy individuals, however, infection/reactivation among immunocompromised patients, including those undergoing hematopoietic stem cell transplantation (HSCT), can be critical and is associated with high rates of morbidity and mortality. The detection of CMV in blood using real-time polymerase chain reaction (qPCR) methods is the most sensitive and specific technique providing for a well-determined preemptive treatment cutoff. AIM: This study compares the performance of two new CMV qPCR platforms, COBAS(®) Ampliprep/COBAS(®) TaqMan(®) (Roche Molecular Diagnostics) and Artus RotorGene (QIAGEN). METHODS: A total of 99 patients referred for CMV testing at AUBMC were tested using the Artus CMV RG PCR kit and the COBAS AmpliPrep/COBAS TaqMan CMV kit as per the manufacturers' recommendations. RESULTS: The difference between the two methods was within the allowable error for 97 out of 99 specimens (98%), with a correlation coefficient r = 0.80. CONCLUSION: The Artus CMV RG PCR Kit and the COBAS AmpliPrep/COBAS TaqMan CMV kit are both acceptable assays that can be used for the sensitive detection and quantitation of CMV mainly in peripheral blood specimens.

Invivoscribe BIOMED-2 primer mixes in B-cell immunoglobulin gene rearrangement studies: experience of a molecular diagnostics laboratory in a major tertiary care center.

AIMS: To determine the frequency of positive reactions obtained using the Invivoscribe BIOMED-2 kit for B-cell gene rearrangement studies in leukemias and lymphomas. MATERIALS AND METHODS: We reviewed the gel patterns for 192 samples tested, using the above-mentioned kit and matched the positive signal with the corresponding mix available in the assay kit. RESULTS: 92.2% had immunoglobulin heavy-chain clonality, of which 74% were detected by the IgH VH-FR1+JH primer set, 75.5% by IgH VH-FR2+JH primer set, 65.1% by IgH VH-FR3+JH primer set, 26% by IgH DH+JH primer set, and 2.1% by IgH DH7+JH primer set. In addition, 55.7% had clonality in the kappa light chain, where 33.3% were positive by the IgK Vκ +Jκ primer set and 39.6% by IgK Vκ and INTR+Kde primer sets. Clonality in the lambda light chain of immunoglobulins was detected in 17.7% of specimens tested using the IgL Vλ +Jλ primer set. CONCLUSION: All primer mixes provided by the assay were positive. Thus, the Invivoscribe BIOMED-2 B-cell gene rearrangement kit is very reliable in adequately covering all targets represented by the master mixes. This assay is an integral part of the differential diagnosis of clonal populations of cells. Our report is the first in the literature that describes the full range of coverage of the BIOMED-2 primer mixes provided in this assay.

Implementation of the Cepheid Xpert EV assay for rapid detection of enteroviral meningitis: experience of a tertiary care center and a technical review.

AIMS: Rapid molecular detection of enterovirus in cerebrospinal fluid (CSF) has revolutionized the diagnosis and treatment modalities of patients with meningitis and largely affected cost of unnecessary antibiotic use and length of stay in hospitals. MATERIALS AND METHODS: Between January 2010 and July 2012, we tested 220 CSF samples on the GeneXpert DX real-time polymerase chain reaction system (by Cepheid) using the Xpert EV kit assays. In addition, we reported 14 sample results cross-validated with an external referral laboratory as well as the results of external proficiency testing. RESULTS: Out of all tested samples, 19% were positive for enterovirus. Furthermore, all referred and proficiency testing specimens were concordant with the peer-reviewed and tested samples. CONCLUSION: Implementation of the Cepheid Xpert EV assay for rapid detection of enteroviral meningitis has been successful in our laboratory. This molecular diagnostic test is currently utilized by our staff physicians as a major tool in assessment and management of patients with meningitis.

HLA class I allele frequencies in the Lebanese population.

The highly polymorphic Human Leukocyte Antigen system encompasses different loci that have been studied in transplantation as well as diseases and population associated research. This study is the first and largest of its kind to describe the distribution of HLA-A, -B and -C alleles in Lebanon. Respectively, 1994, 1309 and 1163 Lebanese individuals referred for HLA typing and possible bone marrow/kidney donation were tested for HLA-A, HLA-B and HLA-C alleles using the polymerase chain reaction/Sequence specific priming (PCR-SSP) method. Our data were compared to that of several populations with interesting and common findings shared with the Moroccan, Jordanian, Tunisian, Omani, Korean, Chinese, Japanese, Peruan, Bulgarian, Irish, Polish, Spanish, Swiss, American, African and Brazilian populations. The following data concerning the Lebanese population will help future investigators to study the relation of HLA-A, -B and -C alleles with common diseases in Lebanon and will add to the available international literature. This new data will serve as a major reference report in the region.

Significance of the use of the ViennaLab "Cardiovascular Disease panel" (CVD) Assay as a reflex test for the "Factor V/II/MTHFR Assay".

INTRODUCTION: Trends toward identifying risk factors of thrombotic complications had become essential as an attempt to prevent and decrease the incidence of the complications. Thrombosis has been associated with predisposing factors like mutations in FV, PTH, MTHFR and other genes. AIM: Evaluate whether the CVD StripAssay has an added value in the screening for more thrombophilia risk factors, which may predispose for the development of cardiovascular diseases and other thrombotic clinical conditions. METHODS: We compared the results for 94 patients who were previously tested for Factor V, Factor II and MTHFR gene mutations using the ViennaLab FV-PTH-MTHFR StripAssay, and for whom additional testing for the Cardiovascular Disease panel (CVD StripAssay, ViennaLab) was requested. RESULTS: Using the CVD StripAssay, 66% of patients who had no mutations when tested using the FV-PTH-MTHFR StripAssay or carried a mutation for MTHFR, were found to have additional genes' SNPs or mutations that are highly associated with a risk of thrombosis as per the available international literature. CONCLUSION: This observation is of extreme importance in clinical practice for the introduction of the extended CVD panel into routine molecular diagnostic test menus and highlights the importance of genetic analysis of the implicated genes in the management of patients with a thrombotic episode presentation.

HLA class II allele frequencies in the Lebanese population.

AIMS: Being one of the most polymorphic genetic systems , the Human Leukocyte Antigen system is divided into class I (HLA-A, HLA-B and HLA-C) and class II (HLA-DP, -DQ and -DR). This study is the first and largest of its kind to describe the distribution of HLA-DQB1 and HLA-DRB1 alleles in Lebanon and the region. METHODS: Respectively, 560 and 563 Lebanese individuals referred for HLA typing and possible bone marrow/kidney donation were tested for HLA-DQB1 and HLA-DRB1 alleles using the polymerase chain reaction/sequence specific priming (PCR-SSP) method. RESULTS: Our data were compared to that of several populations with interesting common findings between the Lebanese, Jordanian, Bahraini, Saudi, Kuwaiti, Tunisian, Korean, Japanese, Thai, Irish, Bulgarian and Polish populations. CONCLUSION: These data about the Lebanese population are going to aid future researchers to study the relation of HLA-DQB1 and HLA-DRB1 alleles with major and common diseases in the Lebanese population and will add to the available international literature associated with these loci. In addition it will serve as a reference for the future national bone marrow registry program in our country. We also reviewed the literature for the described association between HLA-DRB1 and -DQB1 loci and different disease entities.