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Fulltext A multiplexed RT-PCR assay for nanopore whole genome sequencing of Tilapia lake virus (TiLV)

Delamare-Deboutteville J, Meemetta W, Pimsannil K, Sangpo P, Gan HM, Mohan CV, et al.

Sci Rep, 2023 Nov 20;13(1):20276.
PMID: 37985860 DOI: 10.1038/s41598-023-47425-w

Tilapia lake virus (TiLV) is a highly contagious viral pathogen that affects tilapia, a globally significant and affordable source of fish protein. To prevent the introduction and spread of TiLV and its impact, there is an urgent need for increased surveillance, improved biosecurity measures, and continuous development of effective diagnostic and rapid sequencing methods. In this study, we have developed a multiplexed RT-PCR assay that can amplify all ten complete genomic segments of TiLV from various sources of isolation. The amplicons generated using this approach were immediately subjected to real-time sequencing on the Nanopore system. By using this approach, we have recovered and assembled 10 TiLV genomes from total RNA extracted from naturally TiLV-infected tilapia fish, concentrated tilapia rearing water, and cell culture. Our phylogenetic analysis, consisting of more than 36 TiLV genomes from both newly sequenced and publicly available TiLV genomes, provides new insights into the high genetic diversity of TiLV. This work is an essential steppingstone towards integrating rapid and real-time Nanopore-based amplicon sequencing into routine genomic surveillance of TiLV, as well as future vaccine development.

Matched MeSH terms: Reverse Transcriptase Polymerase Chain Reaction
Performance evaluation of two multiplex qualitative RT-PCR assays for detection of respiratory infection in paediatric population

Ali U, Zainal M, Zainol Z, Tai CW, Tang SF, Lee PC, et al.

Malays J Pathol, 2023 Aug;45(2):215-227.
PMID: 37658531

INTRODUCTION: Acute respiratory infection (ARI) contributes to significant mortality and morbidity worldwide and is usually caused by a wide range of respiratory pathogens. This study aims to describe the performance of QIAstat-Dx® Respiratory Panel V2 (RP) and RespiFinder® 2SMART assays for respiratory pathogens detection.
MATERIALS AND METHODS: A total of 110 nasopharyngeal swabs (NPS) were collected from children aged one month to 12 years old who were admitted with ARI in UKMMC during a one-year period. The two qPCR assays were conducted in parallel.
RESULTS: Ninety-seven samples (88.2%) were positive by QIAstat-Dx RP and 86 (78.2%) by RespiFinder assay. The overall agreement on both assays was substantial (kappa value: 0.769) with excellent concordance rate of 96.95%. Using both assays, hRV/EV, INF A/H1N1 and RSV were the most common pathogens detected. Influenza A/H1N1 infection was significantly seen higher in older children (age group > 60 months old) (53.3%, p-value < 0.05). Meanwhile, RSV and hRV/EV infection were seen among below one-year-old children. Co-infections by two to four pathogens were detected in 17 (17.5%) samples by QIAstat-Dx RP and 12 (14%) samples by RespiFinder, mainly involving hRV/EV. Bacterial detection was observed only in 5 (4.5%) and 6 (5.4%) samples by QIAstat-Dx RP and RespiFinder, respectively, with Mycoplasma pneumoniae the most common detected.
CONCLUSION: The overall performance of the two qPCR assays was comparable and showed excellent agreement. Both detected various clinically important respiratory pathogens in a single test with simultaneous multiple infection detection. The use of qPCR as a routine diagnostic test can improve diagnosis and management.

Matched MeSH terms: Reverse Transcriptase Polymerase Chain Reaction
A Revised Diagnostic Quantitative RT-PCR for the Detection of Nipah Virus Infection

Patel K, Klena J, Lo MK

Methods Mol Biol, 2023;2682:25-31.
PMID: 37610571 DOI: 10.1007/978-1-0716-3283-3_2

From its discovery in Malaysia in the late 1990s, the spillover of the Nipah virus from its pteropid reservoir into the human population has resulted in sporadic outbreaks of fatal encephalitis and respiratory disease. In this chapter, we revise a previously described quantitative reverse transcription polymerase chain reaction method, which now utilizes degenerate nucleotides at certain positions in the probe and the reverse primer to accommodate the sequence heterogeneity observed within the Nipah henipavirus species.

Matched MeSH terms: Reverse Transcriptase Polymerase Chain Reaction
Development of a TaqMan minor groove binding probe-based quantitative reverse transcription polymerase chain reaction for the detection and quantification of Zika virus

Chin KL, Teoh BT, Sam SS, Loong SK, Tan KK, Azizan NS, et al.

Trop Biomed, 2022 Dec 01;39(4):518-523.
PMID: 36602210 DOI: 10.47665/tb.39.4.005

Zika virus (ZIKV) infection has emerged as a global health concern following epidemic outbreaks of severe neurological disorders reported in Pacific and Americas since 2016. Therefore, a rapid, sensitive and specific diagnostic test for ZIKV infection is critical for the appropriate patient management and the control of disease spread. A TaqMan minor groove binding (MGB) probe-based quantitative reverse transcription-polymerase chain reaction (qRT-PCR) assay was developed based on the conserved sequence regions of 463 ZIKV NS2B genes. The designed ZIKV qRT-PCR assay was evaluated for its detection limit, strain coverage and cross-reactivity. We further assessed the clinical applicability of qRT-PCR assay for ZIKV RNA detection using a total 18 simulated clinical specimens. The detection limit of the qRT-PCR assay was 11.276 ZIKV RNA copies at the 95% probability level (probit analysis, p<= 0.05). Both Asian and African ZIKV strains were detected by the qRT-PCR assay without cross-reacting with DENV-1, DENV-2, DENV-3, DENV-4, CHIKV, JEV, LGTV, GETV and SINV. The qRT-PCR assay demonstrated a perfect agreement (k = 1.000, P < 0.001) with the reference assay; the sensitivity and specificity of the qRT-PCR assay were 100% (95% CI= 79.6-100) and 100% (95% CI= 43.9-100) respectively. The qRT-PCR assay developed in this study is a useful diagnostic tool for the broad coverage detection and quantification of both the Asian and African ZIKV strains.

Matched MeSH terms: Reverse Transcriptase Polymerase Chain Reaction
Fulltext SARS-CoV-2 multiplex RT-PCR to detect variants of concern (VOCs) in Malaysia, between January to May 2021

Fu JYL, Chong YM, Sam IC, Chan YF

J Virol Methods, 2022 Mar;301:114462.
PMID: 35026305 DOI: 10.1016/j.jviromet.2022.114462

Emerging SARS-CoV-2 variants of concern (VOC) have been associated with enhanced transmissibility and immune escape. Next-generation sequencing (NGS) of the whole genome is the gold standard for variant identification for surveillance but is time-consuming and costly. Rapid and cost-effective assays that detect SARS-CoV-2 variants are needed. We evaluated Allplex SARS-CoV-2 Master Assay and Variants I Assay to detect HV69/70 deletion, Y144 deletion, E484K, N501Y, and P681H spike mutations in 248 positive samples collected in Kuala Lumpur, Malaysia, between January and May 2021. Spike variants were detected in 78/248 (31.5 %), comprising 60 VOC B.1.351 (beta) and 18 B.1.1.7 (alpha). With NGS as reference for 115 samples, the sensitivity for detecting the spike mutations was 98.7 % with the Master Assay and 100 % with the Variants I Assay. The emergence of beta variants correlated with increasing COVID-19 infections in Malaysia. The prevalence of alpha VOC and lineage B.1.466.2 was low. These assays detect mutations present in alpha, beta and gamma VOCs. Of the VOCs which have subsequently emerged, the assays should detect omicron (B.1.1.529) but not B.1.617.2 (delta). In conclusion, spike variant PCR assays can be used to rapidly monitor selected SARS-CoV-2 VOCs in resource-limited settings, but require updates as new variants emerge.

Matched MeSH terms: Reverse Transcriptase Polymerase Chain Reaction
Fulltext [Investigation of Group A Rotavirus G10, G12 Genotypes Emerging in Patients with Acute Gastroenteritis in a Tertiary Care Hospital]

Kahraman H, Altay Koçak A, Albakkour K, Muftah H, Dalgıç B, Çağlar K, et al.

Mikrobiyol Bul, 2021 Oct;55(4):553-567.
PMID: 34666655 DOI: 10.5578/mb.20219707

Rotaviruses are the most common cause of viral gastroenteritis with the highest mortality and morbidity rates in children aged 0-5 years. The aim of this study was to determine the frequency of rotavirus infection in patients whose stool samples were sent to microbiology laboratory to investigate the etiology of diarrhea, to investigate the rotavirus genotypes that are common in our region and G10, G12 genotypes that have recently become common in the world. Fecal samples of 476 patients aged between 0-92 years who applied between November 2016 and February 2018 were studied via immunochromatographic rapid test and enzyme-linked immunosorbent assay (ELISA) methods. ELISA positive samples were studied by nested reverse transcriptase chain reaction (RT-PCR) and genotyped by agarose gel electrophoresis. Rotavirus was found positive in 18.3% and 17% of stool samples by immunochromatographic test and ELISA, respectively. All ELISA positive samples were also detected as positive by RT-PCR. 18.5% of female patients and 15.7% of male patients were found to be positive and rotavirus positivity was not statistically significant between genders. The frequency of rotavirus in different age groups was 23.5% (6-12 years), 17.3% (13-24 months) and 16% (25-36 months). It was determined that rotavirus cases were most common in the spring. G1, G2, G3, G4, G9, G10, and G12 were detected in 37%, 7.4%, 16.1%, 6.2%, 9.9%, 2.5%, 26% of the samples, respectively. G12 was the most common genotype after G1. The most common G and P genotype combination was G1P[8] (17.2%). This was followed by G12P[8] (11.11%) and G3P[8] (11.11%). P[8] (53%) was found to be the dominant P genotype. In this study, it was observed that rotavirus, which is the cause of childhood diarrhea, can also be encountered in advanced ages and even new genotypes that infect humans worldwide may also be the causative agents. Therefore, we concluded that it is important to investigate new genotypes such as G10 and G12 in molecular epidemiological studies.

Matched MeSH terms: Reverse Transcriptase Polymerase Chain Reaction
Fulltext Comparative evaluation of osteogenic differentiation potential of stem cells derived from dental pulp and exfoliated deciduous teeth cultured over granular hydroxyapatite based scaffold

Hagar MN, Yazid F, Luchman NA, Ariffin SHZ, Wahab RMA

BMC Oral Health, 2021 May 15;21(1):263.
PMID: 33992115 DOI: 10.1186/s12903-021-01621-0

BACKGROUND: Mesenchymal stem cells isolated from the dental pulp of primary and permanent teeth can be differentiated into different cell types including osteoblasts. This study was conducted to compare the morphology and osteogenic potential of stem cells from exfoliated deciduous teeth (SHED) and dental pulp stem cells (DPSC) in granular hydroxyapatite scaffold (gHA). Preosteoblast cells (MC3T3-E1) were used as a control group.
METHODOLOGY: The expression of stemness markers for DPSC and SHED was evaluated using reverse transcriptase-polymerase chain reaction (RT-PCR). Alkaline phosphatase assay was used to compare the osteoblastic differentiation of these cells (2D culture). Then, cells were seeded on the scaffold and incubated for 21 days. Morphology assessment using field emission scanning electron microscopy (FESEM) was done while osteogenic differentiation was detected using ALP assay (3D culture).
RESULTS: The morphology of cells was mononucleated, fibroblast-like shaped cells with extended cytoplasmic projection. In RT-PCR study, DPSC and SHED expressed GAPDH, CD73, CD105, and CD146 while negatively expressed CD11b, CD34 and CD45. FESEM results showed that by day 21, dental stem cells have a round like morphology which is the morphology of osteoblast as compared to day 7. The osteogenic potential using ALP assay was significantly increased (p

Matched MeSH terms: Reverse Transcriptase Polymerase Chain Reaction
Polymerase chain reaction cycle threshold value as prerequisite for reporting of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) detection

Awang H, Hamzah FH, Ahmad MH, Mahmood MF, Wahab A, Embong K, et al.

Infect Dis (Lond), 2021 05;53(5):390-392.
PMID: 33512265 DOI: 10.1080/23744235.2021.1876913
Matched MeSH terms: Reverse Transcriptase Polymerase Chain Reaction
Kawasaki disease following coronavirus disease 2019 with prolonged fecal viral shedding

Uda K, Okita K, Soneda K, Taniguchi K, Horikoshi Y

Pediatr Int, 2021 05;63(5):597-599.
PMID: 33278321 DOI: 10.1111/ped.14452
Matched MeSH terms: Reverse Transcriptase Polymerase Chain Reaction/methods
COVID-19 testing and diagnosis: A comparison of current approaches

Wong RSY

Malays J Pathol, 2021 Apr;43(1):3-8.
PMID: 33903299

The severe acute respiratory syndrome coronavirus 2 is a novel coronavirus that causes the coronavirus disease 2019 (COVID-19). COVID-19 has been declared a pandemic by the World Health Organisation since March 2020. To date, the number of confirmed COVID-19 cases has exceeded 47 million and more than 1.2 million people have lost their lives to the disease. The disease is spreading at an exponential rate with no signs of slowing down. COVID-19 testing and early diagnosis play a crucial role in not just patient management, but also the prevention of the further spread of the disease. Various diagnostic approaches have been applied to detect SARS-CoV-2 infection. This article will critically review these diagnostic approaches and compare each with the gold-standard, which is viral RNA detection using reverse transcriptase-polymerase chain reaction (RT-PCR).

Matched MeSH terms: Reverse Transcriptase Polymerase Chain Reaction/methods
Fulltext Pre-surgical COVID-19 swabs in surgical patients: An institutional experience in a middle income country

Xavier RG, Roslani AC, Draman Yusof MR, Ng DS, Govindaraju R, Singh S, et al.

Asian J Surg, 2021 03;44(3):560-561.
PMID: 33627224 DOI: 10.1016/j.asjsur.2020.11.028
Matched MeSH terms: Reverse Transcriptase Polymerase Chain Reaction
Fulltext Polycaprolactone-Based Scaffolds Facilitates Osteogenic Differentiation of Human Adipose-Derived Stem Cells in a Co-Culture System

Rozila I, Azari P, Munirah S, Safwani WKZW, Pingguan-Murphy B, Chua KH

Polymers (Basel), 2021 Feb 17;13(4).
PMID: 33671175 DOI: 10.3390/polym13040597

(1) Background: Stem cells in combination with scaffolds and bioactive molecules have made significant contributions to the regeneration of damaged bone tissues. A co-culture system can be effective in enhancing the proliferation rate and osteogenic differentiation of the stem cells. Hence, the aim of this study was to investigate the osteogenic differentiation of human adipose derived stem cells when co-cultured with human osteoblasts and seeded on polycaprolactone (PCL):hydroxyapatite (HA) scaffold; (2) Methods: Human adipose-derived stem cells (ASC) and human osteoblasts (HOB) were seeded in three different ratios of 1:2, 1:2 and 2:1 in the PCL-HA scaffolds. The osteogenic differentiation ability was evaluated based on cell morphology, proliferation rate, alkaline phosphatase (ALP) activity, calcium deposition and osteogenic genes expression levels using quantitative RT-PCR; (3) Results: The co-cultured of ASC/HOB in ratio 2:1 seeded on the PCL-HA scaffolds showed the most positive osteogenic differentiation as compared to other groups, which resulted in higher ALP activity, calcium deposition and osteogenic genes expression, particularly Runx, ALP and BSP. These genes indicate that the co-cultured ASC/HOB seeded on PCL-HA was at the early stage of osteogenic development; (4) Conclusions: The combination of co-culture system (ASC/HOB) and PCL-HA scaffolds promote osteogenic differentiation and early bone formation.

Matched MeSH terms: Reverse Transcriptase Polymerase Chain Reaction
Fulltext Molecular detection of feline leukemia virus in clinically ill cats in Klang Valley, Malaysia

Mummoorthy K, Yasmin AR, Arshad SS, Omar AR, Nur-Fazila SH, Anand P, et al.

Vet World, 2021 Feb;14(2):405-409.
PMID: 33776305 DOI: 10.14202/vetworld.2021.405-409

Background and Aim: Feline leukemia virus (FeLV) is classified as Retroviridae gammaretrovirus. FeLV occurs worldwide, including Malaysia. Thus far, only one decade-old study on molecular characterization of Malaysian FeLV isolates exists, which resulted in a scarcity of updated information of current FeLV isolates circulating in Malaysia. This study was conducted to determine the status of FeLV in clinically ill cats and to study the molecular characterization and phylogenetic relatedness of the current isolates.
Materials and Methods: Convenience sampling was performed in 20 cats from the Gasing Veterinary Hospital in Selangor. Plasma and saliva samples were collected from 15 clinically ill cats and 5 healthy cats subjected to one-step reverse transcription-polymerase chain reaction with primers targeting a highly conserved gene of U3-LTR-gag.
Results: Two clinically ill cats' plasma and saliva samples tested positive for FeLV RNA. Partial nucleotide sequencing and phylogenetic analysis revealed that the current isolates were 94-99% homologous to the previous Malaysian and Japanese FeLV isolates.
Conclusion: Current FeLV isolates from this study displayed higher similarity with the previous Malaysian isolates, signifying that a similar FeLV strain circulated among the cat population in Selangor.

Matched MeSH terms: Reverse Transcriptase Polymerase Chain Reaction
Fulltext Nucleic Acid-Based Diagnostic Tests for the Detection SARS-CoV-2: An Update

Yu CY, Chan KG, Yean CY, Ang GY

Diagnostics (Basel), 2021 Jan 01;11(1).
PMID: 33401392 DOI: 10.3390/diagnostics11010053

The coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) began as a cluster of pneumonia cases in Wuhan, China before spreading to over 200 countries and territories on six continents in less than six months. Despite rigorous global containment and quarantine efforts to limit the transmission of the virus, COVID-19 cases and deaths have continued to increase, leaving devastating impacts on the lives of many with far-reaching effects on the global society, economy and healthcare system. With over 43 million cases and 1.1 million deaths recorded worldwide, accurate and rapid diagnosis continues to be a cornerstone of pandemic control. In this review, we aim to present an objective overview of the latest nucleic acid-based diagnostic tests for the detection of SARS-CoV-2 that have been authorized by the Food and Drug Administration (FDA) under emergency use authorization (EUA) as of 31 October 2020. We systematically summarize and compare the principles, technologies, protocols and performance characteristics of amplification- and sequencing-based tests that have become alternatives to the CDC 2019-nCoV Real-Time RT-PCR Diagnostic Panel. We highlight the notable features of the tests including authorized settings, along with the advantages and disadvantages of the tests. We conclude with a brief discussion on the current challenges and future perspectives of COVID-19 diagnostics.

Matched MeSH terms: Reverse Transcriptase Polymerase Chain Reaction
Genetic Diversity of Recent Infectious Bursal Disease Viruses Isolated From Vaccinated Poultry Flocks in Malaysia

Aliyu HB, Hair-Bejo M, Omar AR, Ideris A

Front Vet Sci, 2021;8:643976.
PMID: 33959650 DOI: 10.3389/fvets.2021.643976

Vaccination is an essential component in controlling infectious bursal disease (IBD), however, there is a lack of information on the genetic characteristics of a recent infectious bursal disease virus (IBDV) that was isolated from IBD vaccinated commercial flocks in Malaysia. The present study investigated 11 IBDV isolates that were isolated from commercial poultry farms. The isolates were detected using reverse transcription-polymerase chain reaction (RT-PCR) targeting the hypervariable region (HVR) of VP2. Based on the HVR sequences, five isolates (IBS536/2017, IBS624/2017, UPM766/2018, UPM1056/2018, and UPM1432/2019) were selected for whole-genome sequencing using the MiSeq platform. The nucleotide and amino acid (aa) sequences were compared with the previously characterized IBDV strains. Deduced aa sequences of VP2HVR revealed seven isolates with 94-99% aa identity to very virulent strains (genogroup 3), two isolates with 97-100% aa identity to variant strains (genogroup 2), and two strains with 100% identity to the vaccine strain (genogroup 1) of IBDV. The phylogenetic analysis also showed that the isolates formed clusters with the respective genogroups. The characteristic motifs 222T, 249K, 286I, and 318D are typical of the variant strain and were observed for UPM1219/2019 and UPM1432/2019. In comparison, very virulent residues such as 222A, 249Q, 286T, and 318G were found for the vvIBDV, except for the UPM1056/2018 strain with a A222T substitution. In addition, the isolate has aa substitutions such as D213N, G254D, S315T, S317R, and A321E that are not commonly found in previously reported vvIBDV strains. Unlike the other vvIBDVs characterized in this study, UPM766/2018 lacks the MLSL aa residues in VP5. The aa tripeptides 145/146/147 (TDN) of VP1 were conserved for the vvIBDV, while a different motif, NED, was observed for the Malaysian variant strain. The phylogenetic tree showed that the IBDV variant clustered with the American and Chinese variant viruses and are highly comparable to the novel Chinese variants, with 99.9% identity. Based on the sequences and phylogenetic analyses, this is the first identification of an IBDV variant being reported in Malaysia. Further research is required to determine the pathogenicity of the IBDV variant and the protective efficacy of the current IBD vaccines being used against the virus.

Matched MeSH terms: Reverse Transcriptase Polymerase Chain Reaction
Fulltext A reverse transcription loop-mediated isothermal amplification for broad coverage detection of Asian and African Zika virus lineages

Teoh BT, Chin KL, Samsudin NI, Loong SK, Sam SS, Tan KK, et al.

BMC Infect Dis, 2020 Dec 11;20(1):947.
PMID: 33308203 DOI: 10.1186/s12879-020-05585-4

BACKGROUND: Early detection of Zika virus (ZIKV) infection during the viremia and viruria facilitates proper patient management and mosquito control measurement to prevent disease spread. Therefore, a cost-effective nucleic acid detection method for the diagnosis of ZIKV infection, especially in resource-deficient settings, is highly required.
METHODS: In the present study, a single-tube reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay was developed for the detection of both the Asian and African-lineage ZIKV. The detection limit, strain coverage and cross-reactivity of the ZIKV RT-LAMP assay was evaluated. The sensitivity and specificity of the RT-LAMP were also evaluated using a total of 24 simulated clinical samples. The ZIKV quantitative reverse transcription-polymerase chain reaction (qRT-PCR) assay was used as the reference assay.
RESULTS: The detection limit of the RT-LAMP assay was 3.73 ZIKV RNA copies (probit analysis, P ≤ 0.05). The RT-LAMP assay detected the ZIKV genomes of both the Asian and African lineages without cross-reacting with other arthropod-borne viruses. The sensitivity and specificity of the RT-LAMP assay were 90% (95% CI = 59.6-98.2) and 100% (95% CI = 78.5-100.0), respectively. The RT-LAMP assay detected ZIKV genome in 9 of 24 (37.5%) of the simulated clinical samples compared to 10 of 24 (41.7%) by qRT-PCR assay with a high level of concordance (κ = 0.913, P

Matched MeSH terms: Reverse Transcriptase Polymerase Chain Reaction/methods
Molecular evidence and hematological alterations associated with the occurrence of coronavirus in domestic dogs in Pakistan

Sulehria MU, Ahmad SS, Ijaz M, Mushtaq MH, Khan AY, Ghaffar A

Trop Biomed, 2020 Dec 01;37(4):963-972.
PMID: 33612749 DOI: 10.47665/tb.37.4.963

Canine Enteric Coronavirus (CCoV) is one of the major enteric pathogen affecting dogs. This study aims to investigate the molecular prevalence, phylogenetic analysis, associated risk factors, and haemato-biochemical alterations in Canine Coronavirus in dogs in district Lahore, Pakistan. 450 fecal samples were collected from symptomatic dogs originating from various pet-clinics and kennels during 2018-2019. Samples were initially analyzed by sandwich lateral flow immunochromatographic assay and then further processed by RT-PCR (reverse transcriptase polymerase chain reaction) targeting the M gene followed by sequencing. RT-PCR based positive (n=20) and negative (n=20) dogs were samples for their blood for the haemato-biochemical analysis. A questionnaire was used to collect data from pet owners, in order to analyze the data for risk factors analysis by chi square test on SPSS. The prevalence of CCoV was 35.1%, and 23.8 % through Sandwich lateral flow immunochromatographic and RT-PCR respectively. Various risk factors like breed, age, sex, vomiting, diarrhea, sample source, body size, cohabitation with other animals, living environment, food, deworming history, contact with other animals or birds feces, and season were significantly associated with CCoV. The CCoV identified in Pakistan were 98% similar with the isolates from China (KT 192675, 1), South Korea (HM 130573, 1), Brazil (GU 300134, 1), Colombia (MH 717721, 1), United Kingdom (JX 082356, 1) and Tunisia (KX156806). Haematobiochemical alterations in CCoV affected dogs revealed anaemia, leucopenia, lymphopenia, neutrophilia, and decreased packed cell volume, and a significant increase in alkaline phosphate and alanine transaminase. It is concluded that infection with canine coronavirus appears widespread among dog populations in district Lahore, Pakistan. This study is the first report regarding the molecular detection and sequence analysis of CCoV in Pakistan.

Matched MeSH terms: Reverse Transcriptase Polymerase Chain Reaction/veterinary
Clinical implications of conventional cytogenetics, fluorescence in situ hybridization (FISH) and molecular testing in chronic myeloid leukaemia patients in the tyrosine kinase inhibitor era - A review

Ankathil R, Ismail SM, Mohd Yunus N, Sulong S, Husin A, Abdullah AD, et al.

Malays J Pathol, 2020 Dec;42(3):307-321.
PMID: 33361712

Chronic myeloid leukaemia (CML) provides an illustrative disease model for both molecular pathogenesis of cancer and rational drug therapy. Imatinib mesylate (IM), a BCR-ABL1 targeted tyrosine kinase inhibitor (TKI) drug, is the first line gold standard drug for CML treatment. Conventional cytogenetic analysis (CCA) can identify the standard and variant Philadelphia (Ph) chromosome, and any additional complex chromosome abnormalities at diagnosis as well as during treatment course. Fluorescence in situ hybridization (FISH) is especially important for cells of CML patients with inadequate or inferior quality metaphases or those with variant Ph translocations. CCA in conjunction with FISH can serve as powerful tools in all phases of CML including the diagnosis, prognosis, risk stratification and monitoring of cytogenetic responses to treatment. Molecular techniques such as reverse transcriptase-polymerase chain reaction (RT-PCR) is used for the detection of BCR-ABL1 transcripts at diagnosis whereas quantitative reverse transcriptase-polymerase chain reaction (qRTPCR) is used at the time of diagnosis as well as during TKI therapy for the quantitation of BCR-ABL1 transcripts to evaluate the molecular response and minimal residual disease (MRD). Despite the excellent treatment results obtained after the introduction of TKI drugs, especially Imatinib mesylate (IM), resistance to TKIs develops in approximately 35% - 40% of CML patients on TKI therapy. Since point mutations in BCR-ABL1 are a common cause of IM resistance, mutation analysis is important in IM resistant patients. Mutations are reliably detected by nested PCR amplification of the translocated ABL1 kinase domain followed by direct sequencing of the entire amplified kinase domain. The objective of this review is to highlight the importance of regular and timely CCA, FISH analysis and molecular testing in the diagnosis, prognosis, assessment of therapeutic efficacy, evaluation of MRD and in the detection of BCR-ABL1 kinase mutations which cause therapeutic resistance in adult CML patients.

Matched MeSH terms: Reverse Transcriptase Polymerase Chain Reaction/methods*
Fulltext Evidence of West Nile virus infection in migratory and resident wild birds in west coast of peninsular Malaysia

Ain-Najwa MY, Yasmin AR, Omar AR, Arshad SS, Abu J, Mohammed HO, et al.

One Health, 2020 Dec;10:100134.
PMID: 32405525 DOI: 10.1016/j.onehlt.2020.100134

West Nile virus (WNV) is a zoonotic mosquito-borne flavivirus that is harbored and amplified by wild birds via the enzootic transmission cycle. Wide range of hosts are found to be susceptible to WNV infection including mammals, amphibians and reptiles across the world. Several studies have demonstrated that WNV was present in the Malaysian Orang Asli and captive birds. However, no data are available on the WNV prevalence in wild birds found in Malaysia. Therefore this study was conducted to determine the serological and molecular prevalence of WNV in wild birds in selected areas in the West Coast of Peninsular Malaysia. Two types of wild birds were screened, namely migratory and resident birds in order to explore any possibility of WNV transmission from the migratory birds to the resident birds. Thus, a cross-sectional study was conducted at the migratory birds sanctuary located in Kuala Gula, Perak and Kapar, Selangor by catching 163 migratory birds, and 97 resident birds from Kuala Gula and Parit Buntar, Perak at different time between 2016 and 2017 (Total, n = 260). Blood and oropharyngeal swabs were collected for serological and molecular analysis, respectively. Serum were screened for WNV antibodies using a commercial competitive ELISA (c-ELISA) (ID Screen® West Nile Competition Multi-species ELISA, ID VET, Montpellier, France) and cross-reactivity towards Japanese Encephalitis virus (JEV) was also carried out using the JEV-double antigen sandwich (DAS) ELISA. Oropharyngeal swabs were subjected to one-step RT-PCR to detect WNV RNA, in which positive reactions were subsequently sequenced. WNV seropositive rate of 18.71% (29/155) at 95% CI (0.131 to 0.260) and molecular prevalence of 15.2% (16/105) at 95% CI (0.092 to 0.239) were demonstrated in migratory and resident wild birds found in West Coast Malaysia. Phylogenetic analyses of the 16 WNV isolates found in this study revealed that the local strains have 99% similarity to the strains from South Africa and were clustered under lineage 2. Evidence of WNV infection in resident and migratory birds were demonstrated in this study. As a summary, intervention between migratory birds, resident birds and mosquitoes might cause the introduction and maintenance of WNV in Malaysia, however the assumption could be further proven by studying the infection dynamics in the mosquitoes present in the studied areas.

Matched MeSH terms: Reverse Transcriptase Polymerase Chain Reaction
Fulltext Sequence and Phylogenetic Analysis of the First Complete Genome of Ricetungro spherical virus in Malaysia

Kannan M, Mohamad Saad M, Zainal Z, Kassim H, Ismail I, Talip N, et al.

Iran J Biotechnol, 2020 Oct;18(4):e2566.
PMID: 34056024 DOI: 10.30498/IJB.2020.2566

Background: Rice tungro disease (RTD) is a viral disease mainly affecting rice in Asia. RTD caused by Rice tungro bacilliform virus and Rice tungro spherical virus. To date, there are only 5 RTSV isolates have been reported.
Objectives: In this study, we aimed to report the complete nucleotide sequence of Malaysian isolate of Rice tungro spherical virus Seberang Perai (RTSV-SP) for the first time. RTSV-SP was characterized and its evolutionary relationship with previously reported Indian and Philippines isolates were elucidated.
Materials and Methods: RTSV-SP isolate was isolated from a recent outbreak in a paddy field in Seberang Perai zone of Malaysia. Its complete genome was amplified by RT-PCR, cloned and sequenced.
Results: Sequence analysis indicated that the genome of RTSV-SP consisted of 12,173 nucleotides (nt). Comparative analysis of 6 complete genome sequences using Clustal Omega showed that Seberang Perai isolate shared the highest nucleotide identity (96.04%) with Philippine-A isolate, except that the sORF-2 of RTSV-SP is shorter than RTSV Philippine-A by 27 amino acid residues. RTSV-SP found to cluster in Southeast Asia (SEA) group based on the whole genome sequence phylogenetic analysis using MEGA X software.
Conclusions: Phylogenetic classification of RTSV isolates based on the complete nucleotide sequences showed more distinctive clustering pattern with the addition of RTSV-SP whole genome to the available isolates. Present study described the isolation and molecular characterization of RTSV-SP.

Matched MeSH terms: Reverse Transcriptase Polymerase Chain Reaction

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