Dengue virus detection in Lao PDR and Colombia: Comparative evaluation of PCR tests.

OBJECTIVES: Dengue virus (DENV) detection by polymerase chain reaction (PCR) facilitates diagnosis of dengue fever, which is the most frequent arboviral disease globally. Two studies were performed in countries with high dengue incidence, to assess the diagnostic performance of different PCR techniques. METHODS/RESULTS: Two hundred and seventy-nine acute phase blood samples from febrile patients were analyzed for DENV by the RealStar Dengue RT-PCR kit (Altona Diagnostics) as gold standard in comparison with the Tropical Fever Core multiplex PCR (Fast Track Diagnostics). In total, 102 samples collected in Savannakhet Province (Lao PDR, Southeast Asia) in 2013 and 35 samples from Valledupar (Colombia, South America) tested positive for DENV by RealStar RT-PCR. In comparison, the Tropical Fever Core multiplex PCR detected 65.0% (65/102) and 68.6% (24/35) of these samples as positive for DENV in Savannakhet and Valledupar, respectively. Diagnostic sensitivity of the multiplex PCR strongly correlated with viral load. A subset of DENV PCR-confirmed samples was additionally tested by BNITM in house Dengue Type RT-PCR in comparison with two commercial test kits (RealStar Dengue Type RT-PCR [Altona Diagnostics], Dengue differentiation PCR [Fast Track Diagnostics]). The leading dengue serotype in Savannakhet was DENV-3 (58% [29/50]), while DENV-1 (53.8% [14/26]) was the predominant serotype found in samples collected in Valledupar by BNITM-type PCR. However, three DENV serotypes were circulating in Valledupar and in Savannakhet. In 2015, additional studies found predominantly DENV-4 (71% [12/17]) in Savannakhet. CONCLUSIONS: Both studies emphasized that routine diagnostics in both regions will benefit from an expanded use of highly sensitive pan-dengue PCRs.

Assessment of diagnostic and analytic performance of the SD Bioline Dengue Duo test for dengue virus (DENV) infections in an endemic area (Savannakhet province, Lao People's Democratic Republic).

BACKGROUND: Rapid tests detecting both dengue virus (DENV) NS1 antigen and anti-DENV IgM and IgG antibodies facilitate diagnosis of dengue fever (DF) in resource-poor settings. METHODOLOGY/PRINCIPAL FINDINGS: 92 acute phase serum samples from patients with a PCR-confirmed DENV infection collected in Lao People's Democratic Republic (Lao PDR) in 2013 and 2015 were analyzed with the SD Bioline Dengue Duo test. A subset of 74 samples was additionally tested with the Platelia NS1 antigen test, the Panbio DENV µ-capture ELISA and the Panbio DENV IgG ELISA. IgM seroconversion was assayed using follow-up samples of 35 patients collected in the convalescent phase. 57.6%, 22.8% and 44.6% of acute phase serum samples tested positive in the SD Bioline Dengue Duo NS1, IgM, and IgG test, respectively. Diagnostic sensitivity of the SD Bioline Dengue Duo NS1 test strongly correlated with viral load, decreased rapidly over the acute phase of the disease, and was significantly reduced in presence of high anti-DENV IgG antibody titers resulting from secondary DENV infection. While a good concordance (Cohen's kappa 0.78) was found between the SD Bioline Dengue Duo NS1 test and the Platelia NS1 antigen ELISA, both the SD Bioline Dengue Duo IgM and IgG test displayed a significantly lower sensitivity than the corresponding ELISA tests. CONCLUSIONS/SIGNIFICANCE: The SD Bioline Dengue Duo test is a valuable tool for diagnosis of DENV infections especially when analyzing early acute phase samples with high viral load. Nevertheless, in endemic areas, where secondary flavivirus infections are common, diagnostic sensitivity of the NS1 and IgM test components may be compromised.

Verification and diagnostic evaluation of the RealStar® Middle East respiratory syndrome coronavirus (N gene) reverse transcription-PCR kit 1.0.

Aim: We report the diagnostic evaluation of a confirmatory reverse transcription-PCR (RT-PCR) kit targeting the Middle East respiratory syndrome coronavirus (MERS-CoV) N gene. Material & methods: 33 patient samples from two collections sites in Riyadh, Saudi Arabia, which were pre-characterized via real-time RT-PCR targeting MERS-CoV orf1a and upE, and were tested using the MERS-CoV N gene, as a confirmatory assay. This diagnostic procedure follows a two-step diagnostics scheme, recommended by the WHO. Results: 18/33 samples tested positive, 11/33 tested negative for MERS-CoV RNA and 2/33 showed uncertain results. Conclusion: The results suggest, that the RealStar® MERS-CoV (N gene) RT-PCR kit 1.0 can be considered a suitable and reliable confirmatory assay in combination with the RealStar MERS-CoV RT-PCR kit 1.0 according to the diagnostic scheme recommended by WHO.

Evaluation of RealStar Reverse Transcription-Polymerase Chain Reaction Kits for Filovirus Detection in the Laboratory and Field.

BACKGROUND: Diagnosis of Ebola virus (EBOV) disease (EVD) requires laboratory testing. METHODS: The RealStar Filovirus Screen reverse transcription-polymerase chain reaction (RT-PCR) kit and the derived RealStar Zaire Ebolavirus RT-PCR kit were validated using in vitro transcripts, supernatant of infected cell cultures, and clinical specimens from patients with EVD. RESULTS: The Filovirus Screen kit detected EBOV, Sudan virus, Taï Forest virus, Bundibugyo virus, Reston virus, and Marburg virus and differentiated between the genera Ebolavirus and Marburgvirus The amount of filovirus RNA that could be detected with a probability of 95% ranged from 11 to 67 RNA copies/reaction on a LightCycler 480 II. The Zaire Ebolavirus kit is based on the Filovirus Screen kit but was optimized for detection of EBOV. It has an improved signal-to-noise ratio at low EBOV RNA concentrations and is somewhat more sensitive than the Filovirus kit. Both kits show significantly lower analytical sensitivity on a SmartCycler II. Clinical evaluation revealed that the SmartCycler II, compared with other real-time PCR platforms, decreases the clinical sensitivity of the Filovirus Screen kit to diagnose EVD at an early stage. CONCLUSIONS: The Filovirus Screen kit detects all human-pathogenic filoviruses with good analytical sensitivity if performed on an appropriate real-time PCR platform. High analytical sensitivity is important for early diagnosis of EVD.

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.

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.

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.

JAK2 V617F mutation detection: laboratory comparison of two kits using RFLP and qPCR.

AIM: The JAK2 V617F mutation has been implicated in a variety of diseases mainly related to myeloproliferative disorders including polycythemia vera, essential thrombocythemia, and idiopathic myelofibrosis with an increased demand for testing using molecular techniques. The latter are diversified and all aim to simplify the methods employed for detection. MATERIALS AND METHODS: In this study, two detection kits were compared: one using polymerase chain reaction (PCR)-restriction fragment length polymorphisms (RFLP) (JAK2 Activating Mutation assay; InVivoScribe Technologies, San Diego, CA) and the other using real-time quantitative PCR (JAK2 MutaScreen Kit assay; Ipsogen, Marseilles, France). RESULTS: A total of 80 reactions were compared using the two techniques and the results showed a perfect concordance between the two methods. CONCLUSION: We conclude that both PCR-RFLP and quantitative PCR are extremely useful and sensitive techniques for the detection of the JAK2 V617F mutation with quantitative PCR being more time effective and less expensive.

Distribution of cytokine gene polymorphisms in the general Lebanese population: the first report.

CONTEXT: Cytokines are polypeptide regulatory molecules that play a significant role in inflammatory and regulatory responses of the immune system. Several cytokine gene polymorphisms have been studied to date and have been found to be associated with distorted cytokine production or activity by affecting transcriptional regulation and with vulnerability to a variety of infectious and autoimmune diseases as well as to transplant rejection. RESULTS: We studied 106 healthy Lebanese individuals using polymerase chain reaction/sequence-specific priming technique to detect 22 single-nucleotide polymorphisms within 13 cytokine genes: IL1alpha 889-T/C, IL1beta 511-T/C, IL1beta +3962-T/C, IL1R pst1 1970-T/C, IL1RA mspa1 11100-T/C, IL4Ralpha 1902-G/A, IL12 1188-C/A, IFNgamma 874-A/T, TGFbeta codon 10-C/T, TGF-beta codon 25-G/C, TNFalpha 308-A/G, TNFalpha 238-A/G, IL2 166-G/T, IL2 330-T/G, IL4 1098-T/G, IL4 590-T/C, IL4 33-T/C, IL6 174-C/G, IL6 nt565-G/A, IL10 1082-G/A, IL10 819-C/T, and IL10 592-A/C. We compared our results to those reported in other populations with similarities observed between the Lebanese and the Italian populations. CONCLUSION: The study of different cytokine polymorphisms will aid in understanding the susceptibility of populations to various diseases, and this is the first report from the Lebanese community.