Use of the Human Coronavirus 2012 (MERS) GeneSig kit for MERS-CoV detection.

INTRODUCTION: Mortality due to MERS-CoV infection is common especially among immunocompromised patients. The pathogenesis and the transmission mode of this virus are still not well understood. The name of the virus is derived from the area of its appearance and the genomic sequence that was used in the development of qRT-PCR assays for MERS-CoV detection was retrieved from the first detected case isolate. The employed assays target various regions including the area upstream of the envelope gene (upE) that is used for screening and the open reading frames (ORF) 1a and 1b used for confirmation. AIM: This study assesses the use of a MERS-CoV specific assay for screening of respiratory samples in anticipation of the possible spread of the virus in the region. METHODS: 46 respiratory specimens were tested using the qualitative one-step qRT-PCR GeneSig Human Coronavirus 2012 (MERS) kit (PrimerDesign™). RESULTS: Out of the 46 tested samples, 45 were negative for MERS-CoV and one sample was found MERS-CoV positive. CONCLUSION: The GeneSig Human Coronavirus 2012 (MERS) kit is very useful for the screening of suspected respiratory cases in the Middle East area as well as other regions.

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.

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.

TPOX Triallelic Genotype: An Interesting Pattern to Be Noted in Bone Marrow Transplantation Monitoring.

AIMS: TPOX triallelic genotypic pattern has been described in the setting of forensic and paternity testing but not in bone marrow transplantation (BMT) monitoring for graft engraftment. MATERIALS AND METHODS: A total of 50 cases have been studied using the AmpFLSTR(®) Identifiler™ polymerase chain reaction amplification kit as part of the workup of patients and donors before and after BMT at the American University of Beirut Medical Center. RESULTS: Of the 50 studied cases, 49 showed typical allelic patterns of the variable short tandem repeats detected by the assay; however, one single patient showed a biallelic TPOX genotype in the pre-BMT specimen but a triallelic pattern in the post-BMT sample. CONCLUSION: Triallelic patterns of TPOX should also be considered in the context of BMT monitoring testing where misinterpretation of the allelic pattern can lead to wrong unwanted conclusions related to the graft condition and proper quantification of donor DNA.

KIR genotype distribution among Lebanese patients with Hodgkin's lymphoma.

INTRODUCTION: In addition to their important role in fighting infection, natural killer cells are cytotoxic to cancer cells. Studies demonstrated that some KIR genes were responsible for the reduction of the risk of Hodgkin's lymphoma (HL) while others were associated with an increased risk of HL. AIM: The aim of this study is to assess KIR genotypic distribution in Lebanese patients with Hodgkin's lymphoma. METHODS: KIR genotype was analyzed in 41 HL patients and 120 healthy Lebanese individuals using the KIR Genotyping SSP kit. RESULTS: No significant association between HL and any KIR gene was found. Among HL patients, the AA, AB, and BB genotype frequencies were, respectively, 41.46%, 43.9% and 14.63% with an A:B ratio of 1.73:1. As for the controls, the AA, AB, and BB genotype frequencies were, respectively, 39.17%, 50%, and 10.83% with an A:B ratio of 1.79:1. CONCLUSION: In this first study from the Mediterranean region, KIR genotype does not seem to be associated with Hodgkin's lymphoma. Further clinical and translational research is needed to rule out the protective or predisposing role of KIR genes in this important clinical entity.

Comparison of the performance of the Cepheid Xpert HemosIL Factor II and Factor V and the ViennaLab FV-PTH-MTHFR StripAssay kits for molecular thrombophilia profiling.

AIMS: To compare the performance of two assays used for the detection of mutations/polymorphisms in the Factor V, Factor II, and methylenetetrahydrofolate reductase genes among patients referred for the management of a thrombotic event. MATERIALS AND METHODS: We tested 40 different patient samples using two assays, the ViennaLab FV-PTH-MTHFR StripAssay and the Cepheid Xpert HemosIL. RESULTS: The two assays were 100% concordant in their produced results with no samples failing the testing procedures in both. CONCLUSION: This is the first report to evaluate the performance of the ViennaLab FV-PTH-MTHFR StripAssay and the Cepheid Xpert HemosIL. Both assays can be introduced to the operation of molecular diagnostic laboratories to cover the referrals from different disciplines, especially in tertiary care centers with emergency departments.

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.

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 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.

Natural Killer Cell Immunoglobulin-like Receptor (KIR) genotypes in Follicular Lymphoma patients: results of a pilot study.

AIMS: The Natural Killer Cell Immunoglobulin-like Receptor (KIR) genotype profiling in Follicular Lymphoma has not been reported before in the literature. MATERIALS AND METHODS: DNA extracted from 20 Follicular Lymphoma patients and 62 healthy controls was analyzed for KIR genotyping using a polymerase chain reaction/sequence specific primers technique (PCR/SSP) for the presence of 16 KIR gene and pseudogene loci. RESULTS: The AA, AB, and BB genotype frequencies were, respectively, 20%, 60% and 20% with an A:B ratio of 1:1. KIR 2DL4, KIR 3DL2, KIR 3DL3, and KIR 3DP1*003 were presented in all individuals. No significant difference between patients and controls was detected. CONCLUSION: KIR genotyping profile does not seem to be associated with Follicular Lymphoma. The results presented in this pilot research represent the first international report about this important clinical entity.

Laboratory referral rates of genetic tests for thrombophilia workup in a major referral center.

AIMS: The rate of laboratory referrals for thrombophilia patients' genetic workup was assessed and compared among the medical and surgical specialties and subspecialties at a major tertiary care center in Lebanon. METHODS: DNA extraction was performed using the PEL-FREEZ extraction kit (PEL-FREEZ; DYNAL) and the Factor V, prothrombin, and methylenetetrahydrofolate reductase genotypic profiles were done using the FV-PTH-MTHFR StripAssay kit (ViennaLab) that employs a polymerase chain reaction-reverse hybridization method. A total of 2238 referred cases were analyzed. RESULTS: Around 42.23% of all referred cases turned out to have a thrombosis-associated mutation. Referrals from medical and surgical specialties were almost equal. In the surgical specialty, most referrals came from the department of Obstetrics and Gynecology, while in the medical speciality, most of the workup referrals originated from the Hematology/Oncology physicians. However, low referral rates were reported from the emergency department and family medicine practitioners. CONCLUSION: Genetic testing for thrombophilia workup is gaining more importance among the different medical and surgical specialties and is worth being introduced into the offered test lists of all established molecular diagnostics laboratories.

Frequency of triple mutations involving factor V, prothrombin, and methylenetetrahydrofolate reductase genes among patients referred for molecular thrombophilia workup in a tertiary care center in Lebanon.

AIM: Molecular diagnostics has markedly improved the diagnosis and workup of different clinical conditions including hypercoagulable state or thrombophilia where different genes are involved. In this report, which is the largest report in the medical literature and the first in Lebanon, we describe the prevalence of simultaneous mutations in the three major thrombophilia genes Factor V, Factor II, and methylenetetrahydrofolate reductase. MATERIALS AND METHODS: Using a polymerase chain reaction and reverse hybridization assay for the corresponding mutations identification, 2248 referred cases were analyzed. RESULTS: Only 25 cases were found to be simultaneously positive for the three mutations at a prevalence rate of 1.1%. CONCLUSION: Compared with other populations, this prevalence rate is considered high, possibly the highest, and warrants future clinical studies and follow-up.

JAK2 V617F "indeterminate" results by MutaScreen can be easily resolved using MutaQuant kits.

AIM: Janus kinase 2 (JAK2) V617F mutation testing has revolutionized the classification of myeloproliferative disorders, for which several tests have been introduced for qualitative and quantitative diagnostics including the MutaScreen and MutaQuant kits by IPSOGEN. One interesting technical observation are those values detected by MutaScreen kits, which have typically "indeterminate" meaning at a provided reference strand cutoff point and cannot be classified as either positive or negative for JAK2 V617F mutation. RESULTS: We ran 10 different patients with such a finding using the MutaQuant kit and got a better resolution and interpretation into clear-cut negative or positive cases, which were also clinically followed and confirmed as being nonmyeloproliferative or myeloproliferative entities, respectively. CONCLUSION: We propose that it is important to not consider the indeterminate or "at-the-reference strand" results obtained by MutaScreen as positive but rather perform additional testing using MutaQuant kits or other JAK2 quantitative assays. For laboratories that can afford it and utilize both assays, it may be a better strategy to directly initiate diagnostic testing using the MutaQuant rather than the MutaScreen kit.

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.

Proposed algorithm for the best detection of different bcr-abl gene fusion transcripts in molecular diagnostics laboratories: experience of a major referral center.

AIMS: Detection of bcr-abl transcripts is important both in diagnosis as well as in prognostication and treatment modalities of different types of leukemia, both chronic and acute. However, the techniques employed are variable and different among laboratories. Our aim was to share with other labs a strategy/algorithm that we find highly useful for implementation to best detect all bcr-abl fusion transcripts for proper patient management. METHODS: We have used two techniques for the detection of bcr-abl transcripts, an in-house developed polymerase chain reaction and a real-time quantitative commercial polymerase chain reaction (PCR) kit and tested 849 patients referred for initial screening for bcr-abl. RESULTS: Out of 849 cases, 146 (17.2%) were positive for bcr-abl. Around 92.11% of the total bcr-abl positive cases (N=76) detected by the real-time quantitative technique were also positive by the gel-based PCR assay; however, six cases (around 7.89%) were missed by the real-time assay and detected by the other technique in chronic myelogenous leukemia-proven cases. CONCLUSION: We highly encourage other laboratories to perform testing using a simple and inexpensive gel-based PCR screening assay followed by a real-time quantitative assay for a baseline bcr-abl expression level. This combination will enable laboratories to detect all the reported fusion transcripts in accordance with the clinical presentation of the patient as well as other laboratory tests for the best use of this genetic test in patient management and care.

JAK2 V617F gene mutation in the laboratory work-up of myeloproliferative disorders: experience of a major referral center in Lebanon.

AIMS: JAK2 V617F mutation is gaining more acceptance in laboratory testing as part of the differential diagnosis work-up of myeloproliferative disorders (MPD). This report is the first of its kind from Lebanon that analyzes the distribution of this mutation among a series of referred cases to a major tertiary referral center. METHODS: Real-time polymerase chain reaction using JAK2 V617F MutaScreen assay (IPSOGEN Cancer Profiler) was performed on 229 patients. RESULTS: JAK2 V617F mutation was found to be positive in 100% of polycythemia vera cases, 68.29% of essential thrombocythemia cases, and 55.28% of all MPD cases whereas negative in idiopathic erythrocytosis, reactive thrombocytosis, and other non-MPD cases such as acute chronic myeloid leukemias. CONCLUSION: Our unique study in this sample of Lebanese patients shows extensive similarities of positivity of JAK2 V617F as compared with the international literature and for the same categories of clinical entities. This will constitute a baseline for future clinical studies that would also help determine prognosis of cases based on the absence or presence of this mutation.

The use of a reverse hybridization strip assay for the study of hemochromatosis-associated gene mutations in Lebanon.

AIMS: Hereditary hemochromatosis (HHC) is the most commonly identified autosomal recessive genetic disorder in the Caucasian population and HFE gene mutations are highly concentrated among European populations. This is the first study that screens for HHC-related gene mutations in a healthy Lebanese sample population. METHODS: Using the reverse hybridization Hemochromatosis StripAssay A from ViennaLab, the DNA extracted from a total of 116 healthy volunteers (59 males and 57 females) was analyzed, looking for 18 different mutations in the HFE, ferroportin, and transferrin genes. RESULTS: For the HFE gene, the C282Y mutation was not detected, but the H63D mutation was found with an overall carrier frequency of 25.8% (24.1% heterozygous and 1.7% homozygous). None of the mutations in the transferrin and ferroportin genes was identified. CONCLUSIONS: The Hemochromatosis StripAssay A from ViennaLab provides an easy and reliable technique for simultaneous screening of the different HFE gene mutations. This first study in Lebanon represents a baseline report for further future studies in the field using this easy technique with a reasonable turnaround time for diagnosis. We also note that ferroportin and transferrin gene mutations have not been detected in this population sample and larger clinical studies will be needed to better estimate their prevalence.