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Fulltext Diagnosis and potential treatments for acute hepatopancreatic necrosis disease (AHPND): a review

Santos HM, Tsai CY, Maquiling KRA, Tayo LL, Mariatulqabtiah AR, Lee CW, et al.

Aquac Int, 2020;28(1):169-185.
PMID: 32834683 DOI: 10.1007/s10499-019-00451-w

Acute hepatopancreatic necrosis disease (AHPND) or formerly known as early mortality syndrome (EMS) is an emerging disease that has caused significant economic losses to the aquaculture industry. The primary causative agent of AHPND is Vibrio parahaemolyticus, a Gram-negative rod-shaped bacterium that has gained plasmids encoding the fatal binary toxins Pir A/Pir B that cause rapid death of the infected shrimp. In this review, the current research studies and information about AHPND in shrimps have been presented. Molecular diagnostic tools and potential treatments regarding AHPND were also included. This review also includes relevant findings which may serve as guidelines that can help for further investigation and studies on AHPND or other shrimp diseases.

Matched MeSH terms: Pathology, Molecular
Fulltext Evaluation of in silico predictors on short nucleotide variants in HBA1, HBA2, and HBB associated with haemoglobinopathies

Tamana S, Xenophontos M, Minaidou A, Stephanou C, Harteveld CL, Bento C, et al.

Elife, 2022 Dec 01;11.
PMID: 36453528 DOI: 10.7554/eLife.79713

Haemoglobinopathies are the commonest monogenic diseases worldwide and are caused by variants in the globin gene clusters. With over 2400 variants detected to date, their interpretation using the American College of Medical Genetics and Genomics (ACMG)/Association for Molecular Pathology (AMP) guidelines is challenging and computational evidence can provide valuable input about their functional annotation. While many in silico predictors have already been developed, their performance varies for different genes and diseases. In this study, we evaluate 31 in silico predictors using a dataset of 1627 variants in HBA1, HBA2, and HBB. By varying the decision threshold for each tool, we analyse their performance (a) as binary classifiers of pathogenicity and (b) by using different non-overlapping pathogenic and benign thresholds for their optimal use in the ACMG/AMP framework. Our results show that CADD, Eigen-PC, and REVEL are the overall top performers, with the former reaching moderate strength level for pathogenic prediction. Eigen-PC and REVEL achieve the highest accuracies for missense variants, while CADD is also a reliable predictor of non-missense variants. Moreover, SpliceAI is the top performing splicing predictor, reaching strong level of evidence, while GERP++ and phyloP are the most accurate conservation tools. This study provides evidence about the optimal use of computational tools in globin gene clusters under the ACMG/AMP framework.

Matched MeSH terms: Pathology, Molecular
Fulltext Molecular pathology quality control in Southeast Asia: Results of a multiregional quality assurance study from MASTER KEY Asia

Okuma HS, Yoshida H, Kobayashi Y, Arakaki M, Mizoguchi C, Inagaki L, et al.

Cancer Sci, 2023 Jun;114(6):2664-2673.
PMID: 36919757 DOI: 10.1111/cas.15790

Tissue specimen quality assurance is a major issue of precision medicine for rare cancers. However, the laboratory standards and quality of pathological specimens prepared in Asian hospitals remain unknown. To understand the methods in Southeast Asian oncology hospitals and to clarify how pre-analytics affect the quality of formalin-fixed paraffin-embedded (FFPE) specimens, a questionnaire surveying pre-analytical procedures (Part I) was administered, quality assessment of immunohistochemistry (IHC) staining and DNA/RNA extracted from the representative FFPE specimens from each hospital (Part II) was conducted, and the quality of DNA/RNA extracted from FFPE of rare-cancer patients for genomic sequencing (Part III) was examined. Quality measurements for DNA/RNA included ΔΔCt, DV200, and cDNA yield. Six major cancer hospitals from Malaysia, Philippines, and Vietnam participated. One hospital showed unacceptable quality for the DNA/RNA assessment, but improved by revising laboratory procedures. Only 57% (n = 73) of the 128 rare-cancer patients' specimens met both DNA and RNA quality criteria for next-generation sequencing. Median DV200 was 80.7% and 64.3% for qualified and failed RNA, respectively. Median ΔΔCt was 1.25 for qualified and 4.89 for failed DNA. Longer storage period was significantly associated with poor DNA (fail to qualify ratio = 1579:321 days, p

Matched MeSH terms: Pathology, Molecular*
Fulltext Adapting the ACMG/AMP variant classification framework: A perspective from the ClinGen Hemoglobinopathy Variant Curation Expert Panel

Kountouris P, Stephanou C, Lederer CW, Traeger-Synodinos J, Bento C, Harteveld CL, et al.

Hum Mutat, 2022 Aug;43(8):1089-1096.
PMID: 34510646 DOI: 10.1002/humu.24280

Accurate and consistent interpretation of sequence variants is integral to the delivery of safe and reliable diagnostic genetic services. To standardize the interpretation process, in 2015, the American College of Medical Genetics and Genomics (ACMG) and the Association for Molecular Pathology (AMP) published a joint guideline based on a set of shared standards for the classification of variants in Mendelian diseases. The generality of these standards and their subjective interpretation between laboratories has prompted efforts to reduce discordance of variant classifications, with a focus on the expert specification of the ACMG/AMP guidelines for individual genes or diseases. Herein, we describe our experience as a ClinGen Variant Curation Expert Panel to adapt the ACMG/AMP criteria for the classification of variants in three globin genes (HBB, HBA2, and HBA1) related to recessively inherited hemoglobinopathies, including five evidence categories, as use cases demonstrating the process of specification and the underlying rationale.

Matched MeSH terms: Pathology, Molecular
Detection of β-globin Gene Mutations Among β-thalassaemia Carriers and Patients in Malaysia: Application of Multiplex Amplification Refractory Mutation System-Polymerase Chain Reaction

Hassan S, Ahmad R, Zakaria Z, Zulkafli Z, Abdullah WZ

Malays J Med Sci, 2013 Jan;20(1):13-20.
PMID: 23613656

β-thalassaemia is one of the most common single-gene disorders worldwide. Each ethnic population has its own common mutations, accounting for the majority of cases, with a small number of mutations for the rarer alleles. Due to the heterogeneity of β-thalassaemia and the multi-ethnicity of Malaysians, molecular diagnostics may be expensive and time consuming.

Matched MeSH terms: Pathology, Molecular
Molecular diagnosis using multi drug delivery network and stability

Jalil MA, Innate K, Suwanpayak N, Yupapin PP, Ali J

Artif Cells Blood Substit Immobil Biotechnol, 2011 Dec;39(6):357-65.
PMID: 21999106 DOI: 10.3109/10731199.2011.618134

By using a pair of tweezers to generate the intense optical vortices within the PANDA ring resonator, the required molecules (drug volumes) can be trapped and moved dynamically within the molecular bus networks, in which the required diagnosis or drug delivery targets can be performed within the network. The advantage of the proposed system is that the proposed diagnostic method can perform within the tiny system (thin film device or circuit), which can be available for a human embedded device for diagnostic use. The channel spacing of the trapped volumes (molecules) within the bus molecular networks can be provided.

Matched MeSH terms: Pathology, Molecular/instrumentation; Pathology, Molecular/methods*
Current Perspectives on Therapies, Including Drug Delivery Systems, for Managing Glioblastoma Multiforme

Bhaskaran M, Devegowda VG, Gupta VK, Shivachar A, Bhosale RR, Arunachalam M, et al.

ACS Chem Neurosci, 2020 10 07;11(19):2962-2977.
PMID: 32945654 DOI: 10.1021/acschemneuro.0c00555

Glioblastoma multiforme (GBM), a standout among the most dangerous class of central nervous system (CNS) cancer, is most common and is an aggressive malignant brain tumor in adults. In spite of developments in modality therapy, it remains mostly incurable. Consequently, the need for novel systems, strategies, or therapeutic approaches for enhancing the assortment of active agents meant for GBM becomes an important criterion. Currently, cancer research focuses mainly on improving the treatment of GBM via diverse novel drug delivery systems. The treatment options at diagnosis are multimodal and include radiation therapy. Moreover, significant advances in understanding the molecular pathology of GBM and associated cell signaling pathways have opened opportunities for new therapies. Innovative treatment such as immunotherapy also gives hope for enhanced survival. The objective of this work was to collect and report the recent research findings to manage GBM. The present review includes existing novel drug delivery systems and therapies intended for managing GBM. Reported novel drug delivery systems and diverse therapies seem to be precise, secure, and relatively effective, which could lead to a new track for the obliteration of GBM.

Matched MeSH terms: Pathology, Molecular
Analysis of model-based and model-free CEST effect quantification methods for different medical applications

Foo LS, Yap WS, Hum YC, Manan HA, Tee YK

J Magn Reson, 2020 01;310:106648.
PMID: 31760147 DOI: 10.1016/j.jmr.2019.106648

Chemical exchange saturation transfer (CEST) magnetic resonance imaging (MRI) holds great potential to provide new metabolic information for clinical applications such as tumor, stroke and Parkinson's Disease diagnosis. Many active research and developments have been conducted to translate this emerging MRI technique for routine clinical applications. In general, there are two CEST quantification techniques: (i) model-free and (ii) model-based techniques. The reliability of these quantification techniques depends heavily on the experimental conditions and quality of the collected data. Errors such as noise may lead to misleading quantification results and thus inaccurate diagnosis when CEST imaging becomes a standard or routine imaging scan in the future. This paper investigates the accuracy and robustness of these quantification techniques under different signal-to-noise (SNR) levels and magnetic field strengths. The quantified CEST effect before and after adding random Gaussian White Noise using model-free and model-based quantification techniques were compared. It was found that the model-free technique consistently yielded larger average percentage error across all tested parameters compared to its model-based counterpart, and that the model-based technique could withstand SNR of about 3 times lower than the model-free technique. When applied on noisy brain tumor, ischemic stroke, and Parkinson's Disease clinical data, the model-free technique failed to produce significant differences between normal and abnormal tissue whereas the model-based technique consistently generated significant differences. Although the model-free technique was less accurate and robust, its simplicity and thus speed would still make it a good approximate when the SNR was high (>50) or when the CEST effect was large and well-defined. For more accurate CEST quantification, model-based techniques should be considered. When SNR was low (<50) and the CEST effect was small such as those acquired from clinical field strength scanners, which are generally 3T and below, model-based techniques should be considered over model-free counterpart to maintain an average percentage error of less than 44% even under very noisy condition as tested in this work.

Matched MeSH terms: Pathology, Molecular/instrumentation*
Development of multiplex loop mediated isothermal amplification (m-LAMP) label-based gold nanoparticles lateral flow dipstick biosensor for detection of pathogenic Leptospira

Nurul Najian AB, Engku Nur Syafirah EA, Ismail N, Mohamed M, Yean CY

Anal Chim Acta, 2016 Jan 15;903:142-8.
PMID: 26709307 DOI: 10.1016/j.aca.2015.11.015

In recent years extensive numbers of molecular diagnostic methods have been developed to meet the need of point-of-care devices. Efforts have been made towards producing rapid, simple and inexpensive DNA tests, especially in the diagnostics field. We report on the development of a label-based lateral flow dipstick for the rapid and simple detection of multiplex loop-mediated isothermal amplification (m-LAMP) amplicons. A label-based m-LAMP lateral flow dipstick assay was developed for the simultaneous detection of target DNA template and a LAMP internal control. This biosensor operates through a label based system, in which probe-hybridization and the additional incubation step are eliminated. We demonstrated this m-LAMP assay by detecting pathogenic Leptospira, which causes the re-emerging disease Leptospirosis. The lateral flow dipstick was developed to detect of three targets, the LAMP target amplicon, the LAMP internal control amplicon and a chromatography control. Three lines appeared on the dipstick, indicating positive results for all representative pathogenic Leptospira species, whereas two lines appeared, indicating negative results, for other bacterial species. The specificity of this biosensor assay was 100% when it was tested with 13 representative pathogenic Leptospira species, 2 intermediate Leptospira species, 1 non-pathogenic Leptospira species and 28 other bacteria species. This study found that this DNA biosensor was able to detect DNA at concentrations as low as 3.95 × 10(-1) genomic equivalent ml(-1). An integrated m-LAMP and label-based lateral flow dipstick was successfully developed, promising simple and rapid visual detection in clinical diagnostics and serving as a point-of-care device.

Matched MeSH terms: Pathology, Molecular
Global systematic review of primary immunodeficiency registries

Abolhassani H, Azizi G, Sharifi L, Yazdani R, Mohsenzadegan M, Delavari S, et al.

Expert Rev Clin Immunol, 2020 07;16(7):717-732.
PMID: 32720819 DOI: 10.1080/1744666X.2020.1801422

INTRODUCTION: During the last 4 decades, registration of patients with primary immunodeficiencies (PID) has played an essential role in different aspects of these diseases worldwide including epidemiological indexes, policymaking, quality controls of care/life, facilitation of genetic studies and clinical trials as well as improving our understanding about the natural history of the disease and the immune system function. However, due to the limitation of sustainable resources supporting these registries, inconsistency in diagnostic criteria and lack of molecular diagnosis as well as difficulties in the documentation and designing any universal platform, the global perspective of these diseases remains unclear.
AREAS COVERED: Published and unpublished studies from January 1981 to June 2020 were systematically reviewed on PubMed, Web of Science and Scopus. Additionally, the reference list of all studies was hand-searched for additional studies. This effort identified a total of 104614 registered patients and suggests identification of at least 10590 additional PID patients, mainly from countries located in Asia and Africa. Molecular defects in genes known to cause PID were identified and reported in 13852 (13.2% of all registered) patients.
EXPERT OPINION: Although these data suggest some progress in the identification and documentation of PID patients worldwide, achieving the basic requirement for the global PID burden estimation and registration of undiagnosed patients will require more reinforcement of the progress, involving both improved diagnostic facilities and neonatal screening.

Matched MeSH terms: Pathology, Molecular
Enhanced production of short-peptide tagged Ss3a recombinant protein as a novel potential biomarker for strongyloidiasis

Mohd-Hassan NH, Noordin R, Arifin N

Trop Biomed, 2020 Sep 01;37(3):578-586.
PMID: 33612773 DOI: 10.47665/tb.37.3.578

Strongyloidiasis is a mysterious yet important parasitic disease that is hard to diagnose. While microscopic examination remains a "controversial" gold standard method, improved diagnosis is achieved through confirmatory assays with serological and/or molecular diagnostic approaches. In the current serodiagnosis of strongyloidiasis, recombinant proteins have been adopted in place of the use of native parasite antigens, although the availability of diagnostically potential proteins are still limited. Here, we introduce a novel Strongyloides recombinant protein that is uniquely attached to two different short peptide tags as a potential diagnostic biomarker for serodiagnosis of strongyloidiasis, namely lysine (7K) and aspartic acid (7D). The work presented focus on improving the yield and purity of the previously unexpressed recombinant protein. Preliminary diagnostic evaluation of the recombinant favors Ss3a7K protein owing to its higher antigenicity performance with 80% sensitivity and 100% specificity, respectively.

Matched MeSH terms: Pathology, Molecular
Fulltext A Thermostabilized, One-Step PCR Assay for Simultaneous Detection of Klebsiella pneumoniae and Haemophilus influenzae

Khazani NA, Noor NZ, Yean Yean C, Hasan H, Suraiya S, Mohamad S

J Trop Med, 2017;2017:7210849.
PMID: 28386286 DOI: 10.1155/2017/7210849

Klebsiella pneumoniae and Haemophilus influenzae are two common pathogens associated with respiratory tract infections. The identification of these pathogens using conventional molecular diagnostic tests requires trained personnel, cold-chain transportation, and storage-dependance, which does not render them user-friendly. The aim of this study was to develop a thermostabilized, cold-chain-free, one-step multiplex PCR for simultaneous detection of K. pneumoniae and H. influenzae. The multiplex PCR assay was designed to amplify the php gene of K. pneumoniae (202 bp) and p6 gene of H. influenzae (582 bp). In addition, the specific primer to amplify glm gene of Helicobacter pylori (105 bp) was included as an internal amplification control. Subsequently, the designed primers and all PCR reagents were thermostabilized by lyophilization. The stability of the thermostabilized PCR was evaluated using the Q(10) method. The sensitivity and specificity of performances for thermostabilized PCR were evaluated using 127 clinical isolates and were found to be 100% sensitive and specific. The thermostabilized PCR mix was found to be stable for 30 days and the Q10 accelerated stability was found to be 3.02 months. A cold-chain-free, PCR assay for easy, rapid, and simultaneous detection of K. pneumoniae and H. influenzae was successfully developed in this study.

Matched MeSH terms: Pathology, Molecular
Fulltext Cri-du-chat syndrome: application of array cgh in diagnostic evaluation

Juriza, I., Sharifah Azween, S.O., Azli, I., Zarina, A.L., Mohd Fadly, M.A., Zubaidah, Z., et al.

Medicine & Health, 2010;5(2):108-113.
MyJurnal

The human genome contains many submicroscopic copy number variations which includes deletions, duplications and insertions. Although conventional karyotyping remains an important diagnostic tool in evaluating a dysmorphic patient with mental retardation, molecular diagnostic technology such as array comparative genomic hybridization (aCGH) has proven to be sensitive and reliable in detecting these submicroscopic anomalies. A 3 month-old infant with dysmorphic facies, microcephaly and global developmental delay was referred for genetic evaluation. Preliminary karyotyping which was confounded by the quality of metaphase spread was normal; however, aCGH detected a 30.6Mb deletion from 5p15.33-p13.3. This case illustrates the usefulness of aCGH as an adjunctive investigative tool for detecting chromosomal imbalances.

Matched MeSH terms: Pathology, Molecular
Fulltext Beta thalassaemia mutations in Malays: a simplified cost-effective strategy to identify the mutations

George, E., Teh, L.K., Rosli, R., Lai, M.I., Tan, J.A.M.A.

Malaysian Journal of Medicine and Health Sciences, 2012;8(1):45-53.
MyJurnal

Beta (β)- thalassaemia is a public health problem in Malaysia. The carrier rate is estimated to be 4.5% by micro-mapping studies particularly among Malays who comprise 53.5% of the population in Malaysia. The common diagnostic method in Malaysia for mutation detection is by amplification refractory mutation system (ARMS). It allows single mutation detection in each reaction but is labour intensive and time consuming when many mutations need to be identified. The purpose of this study was to develop a diagnostic tool for effective mutation detection of beta thalassaemia in Malay patients and compare its efficacy with ARMS-PCR, the current method in use. Methods: Reverse dot blot hybridization (RDBH) technique was incorporated in the development of two strip assays [RDBH-Strip M(6) and RDBH-Strip C(6)] to identify common beta thalassaemia mutations in the Malays. The panels of selected mutations were based on the mutation frequencies in Malaysia reported in previous studies. RDBH-Strip M(6) was applied as step 1 and RDBH-Strip C(6) was applied as step 2 for unidentified mutations. The strips were validated with the gold standard method, ARMS- PCR. Results: One hundred and thirty seven Malay patients with 274 alleles were studied. In Step 1 mutation detection, 238 alleles (86.9%) were identified in 119 of patients by RDBH-Strip M(6). Step 2 resulted in a further detection of 20 alleles in another 10 patients by RDBH-Strip C(6). The combination of both strips resulted in the identification of 258 alleles in 129 (94.6%) of 137 Malay patients. The strip assays were 100% sensitive and specific when compared with ARMS-PCR method. Conclusion: Two strip assays utilising the RDBH technique were developed to identify common β-thalassaemia mutations in Malays. The RDBH Strip M(6) identified 86.9% of the mutations and the RDBH-Strip C (6) detected further 7.3% alleles. This two step strategy was found to be rapid and cost effective for the direct diagnosis of β-thalassaemia mutations in the Malays. The remaining unidentified mutations would require DNA sequencing. It can serve as a specific molecular diagnostic tool for effective diagnosis of
β-thalassaemia mutations in this ethnic group.

Matched MeSH terms: Pathology, Molecular
Fulltext Developing new primers for molecular identification of plasmodium spp. recorded in Sabah

Chua, T.H., Stanis, C.S., Song, B.K., Lau, Y.L., Jelip, P., Lau, T.Y.

Borneo Journal of Medical Sciences, 2015;9(1):3-10.
MyJurnal

Malaria is a major public health problem in tropical and subtropical areas, caused by five
species of Plasmodium (P. falciparum, P. vivax, P. malariae, P. ovale andP. knowlesi) and is the leading cause of morbidity and mortality worldwide. We have developed molecular markers for three genes viz, Cytb, dhfr and Msp-1 gene and designed a protocol for rapid molecular diagnostics of the four malaria parasites prevalent in Southeast Asia. The new primers were used on the blood
samples containing Plasmodium parasites by conventional PCR. The result was compared with
the nested PCR of Singh et al. (2004) and the microscopy method. The result shows that the new
set of primers had successfully amplified all four human malaria parasite species. These primers
were 100% sensitive and more specific than microscopy and PCR identification using these
primers was faster than the nested PCR. These alternative primers should provide powerful and
rapid molecular diagnostic method for detecting Plasmodium species as well as providing reliable
data for epidemiology study. These primers have the potential to be combined and used in
multiplex PCR.

Matched MeSH terms: Pathology, Molecular
Development of Molecular Diagnosis by PCR for the Detection of Infection and Gene Expression for Nipah Virus (NiV)

Pasha F, Alatawi A, Amir M, Faridi U

Pak J Biol Sci, 2020 Jan;23(8):1086-1095.
PMID: 32700860 DOI: 10.3923/pjbs.2020.1086.1095

BACKGROUND AND OBJECTIVE: The epidemiology of Nipah virus (NiV) was shortly seen in many Asian countries like Malaysia, Bangladesh and India most recently. Nipah virus also synonym as bat born virus is transmitted primarily by fruit bats. The 2 different strains transmitted are Hendra (highly pathogenic) and Cedar (non-pathogenic). The present study was attempt to develop recombinant protein based reagents for molecular diagnosis of Nipah.
MATERIALS AND METHODS: The different primer sets were developed using bioinformatics software DNASTAR. The E. coli cells were used for recombinant protein expression.
RESULTS: The NiV 'G' region primers were designed and amplified for 1 kb fragment and cloned. The NiV 'G' fragments were sub-cloned in pET-28(+) B and pGEX-5x-1. Recombinant protein thus obtained in soluble form in both the cases was essayed using western blot. The result showed the protein expression yield was more in pET-28(+) B with low stability and vice versa for pGEX-5x-1.
CONCLUSION: The antibodies raised from the protein can be used as diagnostic reagent for detection of NiV. Thus, a new diagnostic technique can be industrialized.

Matched MeSH terms: Pathology, Molecular
Feasibility of the salivary transcriptome as a novel biomarker in determining disease susceptibility

Hidayat MFH, Milne T, Cullinan MP, Seymour GJ

J Periodontal Res, 2018 Jun;53(3):369-377.
PMID: 29280135 DOI: 10.1111/jre.12522

BACKGROUND AND OBJECTIVE: The salivary transcriptome may present as a readily available and non-invasive source of potential biomarkers. The development of chronic periodontitis is determined by individual patient susceptibility; hence, the aim of this study was to determine the potential of the salivary transcriptome as a biomarker of disease susceptibility using chronic periodontitis as an example.
MATERIAL AND METHODS: Using an Oragene® RNA kit, the total RNA was purified from the saliva of 10 patients with chronic periodontitis and 10 patients without chronic periodontitis. The quantity and quality of the total RNA was determined, and a measure of gene expression via cDNA was undertaken using the Affymetrix microarray system. The microarray profiling result was further validated by real-time quantitative polymerase chain reaction.
RESULTS: Spectrophotometric analysis showed the total RNA purified from each participant ranged from 0.92 μg/500 μL to 62.85 μg/500 μL. There was great variability in the quantity of total RNA obtained from the 2 groups in the study with a mean of 10.21 ± 12.71 μg/500 μL for the periodontitis group and 15.97 ± 23.47 μg/500 μL for the control group. Further the RNA purity (based on the A260 /A280 ratio) for the majority of participants (9 periodontitis and 6 controls) were within the acceptable limits for downstream analysis (2.0 ± 0.1). The study samples, showed 2 distinct bands at 23S (3800 bp) and 16S (1500 bp) characteristic of bacterial rRNA. Preliminary microarray analysis was performed for 4 samples (P2, P6, H5 and H9). The percentage of genes present in each of the 4 samples was not consistent with about 1.8%-18.7% of genes being detected. Quantitative real-time polymerase chain reaction confirmed that the total RNA purified from each sample was mainly bacterial RNA (Uni 16S) with minimal human mRNA.
CONCLUSION: This study showed that minimal amounts of human RNA were able to be isolated from the saliva of patients with periodontitis as well as controls. Further work is required to enhance the extraction process of human mRNA from saliva if the salivary transcriptome is to be used in determining individual patient susceptibility.

Matched MeSH terms: Pathology, Molecular/methods*
Fulltext Development of conventional and real-time multiplex PCR assays for the detection of nosocomial pathogens

Anbazhagan D, Mui WS, Mansor M, Yan GO, Yusof MY, Sekaran SD

Braz J Microbiol, 2011 Apr;42(2):448-58.
PMID: 24031653 DOI: 10.1590/S1517-83822011000200006

Nosocomial infections are major clinical threats to hospitalised patients and represent an important source of morbidity and mortality. It is necessary to develop rapid detection assays of nosocomial pathogens for better prognosis and initiation of antimicrobial therapy in patients. In this study, we present the development of molecular methods for the detection of six common nosocomial pathogens including Escherichia coli, Staphylococcus aureus, Streptococcus pneumoniae, Klebsiella pneumoniae, Pseudomonas aeruginosa and Acinetobacter spp. Conventional multiplex PCR and SYBR Green based real time PCR assays were performed using genus and species specific primers. Blind testing with 300 clinical samples was also carried out. The two assays were found to be sensitive and specific. Eubacterial PCR assay exhibited positive results for 46 clinical isolates from which 43 samples were detected by real time PCR assay. The sensitivity of the assay is about 93.7% in blind test isolates. The PCR results were reconfirmed using the conventional culture method. This assay has the potential to be a rapid, accurate and highly sensitive molecular diagnostic tool for simultaneous detection of Escherichia coli, Staphylococcus aureus, Streptococcus pneumoniae, Klebsiella pneumoniae, Pseudomonas aeruginosa and Acinetobacter spp. This assay has the potential to detect nosocomial pathogens within 5 to 6 hours, helping to initiate infection control measures and appropriate treatment in paediatric and elderly (old aged) patients, pre-and post surgery patients and organ transplant patients and thus reduces their hospitalization duration.

Matched MeSH terms: Pathology, Molecular
Fulltext Advances in the Diagnosis of Foot-and-Mouth Disease

Wong CL, Yong CY, Ong HK, Ho KL, Tan WS

Front Vet Sci, 2020;7:477.
PMID: 32974392 DOI: 10.3389/fvets.2020.00477

Foot-and-mouth disease (FMD) is a devastating livestock disease caused by foot-and-mouth disease virus (FMDV). Outbreaks of this disease in a country always result in conspicuous economic losses to livestock industry and subsequently lead to serious socioeconomic damages due to the immediate imposition of trade embargo. Rapid and accurate diagnoses are imperative to control this infectious virus. In the current review, enzyme-linked immunosorbent assay (ELISA)-based methods used in FMD diagnosis are extensively reviewed, particularly the sandwich, liquid-phase blocking, and solid-phase competition ELISA. The differentiation of infected animals from vaccinated animals using ELISA-based methods is also highlighted, in which the role of 3ABC polyprotein as a marker is reviewed intensively. Recently, more studies are focusing on the molecular diagnostic methods, which detect the viral nucleic acids based on reverse transcription-polymerase chain reaction (RT-PCR) and RT-loop-mediated isothermal amplification (RT-LAMP). These methods are generally more sensitive because of their ability to amplify a minute amount of the viral nucleic acids. In this digital era, the RT-PCR and RT-LAMP are progressing toward the mobile versions, aiming for on-site FMDV diagnosis. Apart from RT-PCR and RT-LAMP, another diagnostic assay specifically designed for on-site diagnosis is the lateral flow immunochromatographic test strips. These test strips have some distinct advantages over other diagnostic methods, whereby the assay often does not require the aid of an external device, which greatly lowers the cost per test. In addition, the on-site diagnostic test can be easily performed by untrained personnel including farmers, and the results can be obtained in a few minutes. Lastly, the use of FMDV diagnostic assays for progressive control of the disease is also discussed critically.

Matched MeSH terms: Pathology, Molecular
Panspecies molecular assays detect viral pathogens missed by real-time PCR/reverse-transcriptase PCR among pneumonia patients, Sarawak, Malaysia

Fieldhouse JK, Bailey ES, Toh TH, Hii KC, Mallinson KA, Ting J, et al.

Trop Dis Travel Med Vaccines, 2020;6:13.
PMID: 32817802 DOI: 10.1186/s40794-020-00114-2

Background: In a year-long pneumonia etiology study conducted June 2017 to May 2018 in Sarawak, Malaysia, 599 patients' nasopharyngeal swab specimens were studied with real-time polymerase chain reaction (rPCR)/ reverse-transcription (rRT-PCR) assays for respiratory pathogens known to contribute to the high burden of lower respiratory tract infections. The study team sought to compare real-time assay results with panspecies conventional molecular diagnostics to compare sensitivities and learn if novel viruses had been missed.
Methods: Specimens were studied for evidence of adenovirus (AdV), enterovirus (EV) and coronavirus (CoV) with panspecies gel-based nested PCR/RT-PCR assays. Gene sequences of specimens positive by panspecies assays were sequenced and studied with the NCBI Basic Local Alignment Search Tool software.
Results: There was considerable discordance between real-time and conventional molecular methods. The real-time AdV assay found a positivity of 10.4%; however, the AdV panspecies assay detected a positivity of 12.4% and the conventional AdV-Hexon assay detected a positivity of 19.6%. The CoV and EV panspecies assays similarly detected more positive specimens than the real-time assays, with a positivity of 7.8% by the CoV panspecies assay versus 4.2% by rRT-PCR, and 8.0% by the EV panspecies assay versus 1.0% by rRT-PCR. We were not able to ascertain virus viability in this setting. While most discordance was likely due to assay sensitivity for previously described human viruses, two novel, possible zoonotic AdV were detected.
Conclusions: The observed differences in the two modes of amplification suggest that where a problem with sensitivity is suspected, real-time assay results might be supplemented with panspecies conventional PCR/RT-PCR assays.

Matched MeSH terms: Pathology, Molecular

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