scholarly journals Role of Laboratory Medicine in SARS-CoV-2 Diagnostics. Lessons Learned from a Pandemic

Healthcare ◽  
2021 ◽  
Vol 9 (7) ◽  
pp. 915
Author(s):  
Irena Duś-Ilnicka ◽  
Aleksander Szymczak ◽  
Małgorzata Małodobra-Mazur ◽  
Miron Tokarski
Keyword(s):  
Molecular Biology ◽  
Real Time ◽  
Real Time Pcr ◽  
Laboratory Medicine ◽  
Lessons Learned ◽  
Diagnostic Methods ◽  
Medical Community ◽  
Medical Diagnostic ◽  
Novel Coronavirus ◽  
Available Information

Since the 2019 novel coronavirus outbreak began in Wuhan, China, diagnostic methods in the field of molecular biology have been developing faster than ever under the vigilant eye of world’s research community. Unfortunately, the medical community was not prepared for testing such large volumes or ranges of biological materials, whether blood samples for antibody immunological testing, or salivary/swab samples for real-time PCR. For this reason, many medical diagnostic laboratories have made the switch to working in the field of molecular biology, and research undertaken to speed up the flow of samples through laboratory. The aim of this narrative review is to evaluate the current literature on laboratory techniques for the diagnosis of SARS-CoV-2 infection available on pubmed.gov, Google Scholar, and according to the writers’ knowledge and experience of the laboratory medicine. It assesses the available information in the field of molecular biology by comparing real-time PCR, LAMP technique, RNA sequencing, and immunological diagnostics, and examines the newest techniques along with their limitations for use in SARS-CoV-2 diagnostics.

DEVELOPMENT OF MULTIPLEX REAL-TIME PCR ASSAY FOR SIX PATHOGENS: RAPID TOOL FOR DIAGNOSIS OF BACTERIAL MENINGITIS

2019 ◽  
pp. 60-66
Author(s):  
Viet Quynh Tram Ngo ◽  
Thi Ti Na Nguyen ◽  
Hoang Bach Nguyen ◽  
Thi Tuyet Ngoc Tran ◽  
Thi Nam Lien Nguyen ◽  
...  
Keyword(s):  
Bacterial Meningitis ◽  
Real Time ◽  
Real Time Pcr ◽  
Diagnostic Methods ◽  
Type B ◽  
E Coli ◽  
Pcr Assays ◽  
Set Up ◽  
Etiological Agents ◽  
Suis Serotype

Introduction: Bacterial meningitis is an acute central nervous infection with high mortality or permanent neurological sequelae if remained undiagnosed. However, traditional diagnostic methods for bacterial meningitis pose challenge in prompt and precise identification of causative agents. Aims: The present study will therefore aim to set up in-house PCR assays for diagnosis of six pathogens causing the disease including H. influenzae type b, S. pneumoniae, N. meningitidis, S. suis serotype 2, E. coli and S. aureus. Methods: inhouse PCR assays for detecting six above-mentioned bacteria were optimized after specific pairs of primers and probes collected from the reliable literature resources and then were performed for cerebrospinal fluid (CSF) samples from patients with suspected meningitis in Hue Hospitals. Results: The set of four PCR assays was developed including a multiplex real-time PCR for S. suis serotype 2, H. influenzae type b and N. meningitides; three monoplex real-time PCRs for E. coli, S. aureus and S. pneumoniae. Application of the in-house PCRs for 116 CSF samples, the results indicated that 48 (39.7%) cases were positive with S. suis serotype 2; one case was positive with H. influenzae type b; 4 cases were positive with E. coli; pneumococcal meningitis were 19 (16.4%) cases, meningitis with S. aureus and N. meningitidis were not observed in any CSF samples in this study. Conclusion: our in-house real-time PCR assays are rapid, sensitive and specific tools for routine diagnosis to detect six mentioned above meningitis etiological agents. Key words: Bacterial meningitis, etiological agents, multiplex real-time PCR

scholarly journals Cryptosporidium spp. during chemotherapy: a cross-sectional study of 94 patients with malignant solid tumor

2021 ◽  
Vol 41 (5) ◽  
pp. 293-298
Author(s):  
Mehmet Karabey ◽  
Hüseyin Can ◽  
Tülay Öncü Öner ◽  
Mert Döşkaya ◽  
Sedef Erkunt Alak ◽  
...  
Keyword(s):  
Sample Size ◽  
Real Time ◽  
Solid Tumors ◽  
Real Time Pcr ◽  
Tertiary Care ◽  
Diagnostic Methods ◽  
Cross Sectional ◽  
Cryptosporidium Spp ◽  
Stool Samples ◽  
Pcr Targeting

BACKGROUND: Cryptosporidium spp . is a protozoan parasite that infects many vertebrate animals, including humans. Since Cryptosporidium spp . can cause chronic life-threatening diarrhea and severe malabsorption in immunocompromised patients, we investigated the prevalence of this parasite among patients undergoing chemotherapy for malignant solid tumors. OBJECTIVE: Investigate the prevalence of Cryptosporidium spp . in stool samples. DESIGN: Cross-sectional. SETTING: Tertiary care. PATIENTS AND METHODS: Stool samples were collected from adult patients with malignant solid tumors receiving chemotherapy and diarrhea. Cryptosporidium spp . prevalence was determined using Ziehl–Neelsen staining, ELISA, and real-time PCR targeting of the COWP gene. MAIN OUTCOME MEASURE: The prevalence of Cryptosporidium spp . in patients undergoing chemotherapy for malignant solid tumors. SAMPLE SIZE: 94 RESULTS: The prevalence was 2.1% (2/94), 5.3% (5/94), and 5.3% (5/94) as detected by Ziehl–Neelsen staining, real-time PCR and ELISA, respectively. The prevalence reached 8.5% (8/94) using all results obtained from the three methods. Among eight positive stool samples, four were positive by at least two different methods (Ziehl–Neelsen staining-ELISA or ELISA-real-time PCR) whereas the remaining four were positive by either ELISA or real-time PCR. CONCLUSION: These findings show the risk of cryptosporidiosis in cancer patients and the necessity to use at least two diagnostic methods during the diagnosis of cryptosporidiosis to reach more accurate and trustworthy results. LIMITATIONS: Further studies with a larger sample size are recommended. CONFLICT OF INTEREST: None.

scholarly journals Comparison of Loop-Mediated Isothermal Amplification and Real-Time PCR Assays for Detection ofStrongyloidesLarvae in Different Specimen Matrices

2019 ◽  
Vol 57 (4) ◽  
Author(s):  
Matthew R. Watts ◽  
Rady Kim ◽  
Vishal Ahuja ◽  
Gemma J. Robertson ◽  
Yasmin Sultana ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Chronic Infection ◽  
Lamp Assay ◽  
Isothermal Amplification ◽  
Diagnostic Methods ◽  
Percentage Agreement ◽  
Content Type ◽  
Loop Mediated Isothermal Amplification ◽  
Stool Specimens

ABSTRACTStrongyloides stercoraliscan cause disease that ranges from asymptomatic chronic infection to fatal hyperinfection. Diagnosis from stool can be challenging because the most sensitive conventional tests require live larvae to be effective and there can be low larval output in chronic infection. Nucleic acid amplification tests (NAAT) have been developed to complement existing diagnostic methods. We compared a recently developed loop-mediated isothermal amplification (LAMP) assay with a real-time PCR that has previously been validated with larval microscopy. The limits of detection—quantified using serial dilutions of DNA extracts from singleStrongyloides rattithird-stage (L3) larvae spiked into approximately 250 µl of 5 differentS. stercoralis-negative stool specimens—were 10−3(1/5 replicates) and 10−2(1/5 replicates) dilutions for PCR and LAMP, respectively. PCR was positive for 4/5 replicates at 10−2. LAMP was compared to PCR using extracts from 396 stool specimens collected in Bangladesh and Australia, of which 53 were positive and 343 were negative by PCR. The positive percentage agreement of LAMP was 77.3% (95% score confidence interval [CI], 64.5 to 86.6). The negative percentage agreement was 100% (95% CI, 98.9 to 100). In a preliminary investigation, PCR and LAMP assays were positive using DNA extracted from serum (PCR, 3/16 extracts; LAMP, 2/16 extracts) and bronchoalveolar lavage fluid (PCR and LAMP, 2/2 extracts), demonstrating proof of concept. Compared to PCR, the lower number of positive results using the LAMP assay may have been due to reaction inhibitors and DNA degradation, and strategies to improve the LAMP assay are discussed.

scholarly journals Evaluation of Two DNA Extraction Methods for Detection of Strongyloides stercoralis Infection

2018 ◽  
Vol 56 (4) ◽  
Author(s):  
Beatrice Barda ◽  
Rahel Wampfler ◽  
Somphou Sayasone ◽  
Khampheng Phongluxa ◽  
Syda Xayavong ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Strongyloides Stercoralis ◽  
Extraction Methods ◽  
Molecular Techniques ◽  
Diagnostic Methods ◽  
Content Type ◽  
Stool Samples ◽  
Dna Extraction Methods ◽  
Baermann Method

ABSTRACT Strongyloides stercoralis is present worldwide, but its prevalence is still uncertain, mainly due to the lack of sensitivity of diagnostic methods. Molecular techniques are under development, but a standardized protocol is still unavailable. We compared the sensitivity of real-time PCR, using two extraction protocols, with that of the Baermann technique. Samples were collected in the framework of the baseline screening of a randomized clinical trial evaluating moxidectin against S. stercoralis in Lao People's Democratic Republic. Two stool samples from each participant were processed by the Baermann method, and one subsample was processed by PCR. DNA was extracted using the QIAamp DNA stool minikit based on the standard protocol for the QIAamp DNA minikit (QIA) and using a modification of the QIA procedure (POL). Subsequently, all extracted samples were analyzed by real-time PCR. Overall, 95 samples were analyzed by the three diagnostic methods. Sixty-nine (72.6%) samples were positive according to the Baermann method, 25 (26.3%) by the QIA method, and 62 (65.3%) by the POL method. The sensitivities were 86% (95% confidence interval [CI], 76.7 to 92.9), 31.0% (95% CI, 21.3 to 42.6), and 78.0% (95% CI, 66.8 to 86.1) for the Baermann, QIA, and POL methods, respectively. The sensitivities calculated for each day of the Baermann method separately were 60% (48.4 to 70.8%) and 64% (52.2 to 74.2%) for days 1 and 2, respectively. In conclusion, the POL method revealed a good performance and was comparable to the Baermann test performed on two stool samples and superior to the Baermann method performed on one stool sample. Additional studies are needed to standardize a PCR protocol for S. stercoralis diagnosis.

Roles of the multiplex real-time PCR assay and β-D-glucan in a high-risk population for intra-abdominal candidiasis (IAC)

2019 ◽  
Vol 58 (6) ◽  
pp. 789-796 ◽  
Author(s):  
J Fortún ◽  
M J Buitrago ◽  
F Gioia ◽  
E Gómez-Gª de la Pedrosa ◽  
M E Alvarez ◽  
...  
Keyword(s):  
High Risk ◽  
Real Time ◽  
Real Time Pcr ◽  
Volume Overload ◽  
Diagnostic Methods ◽  
Practice Volume ◽  
High Risk Population ◽  
Pcr Assay ◽  
European Consensus ◽  
Abdominal Candidiasis

Abstract Multiplex quantitative real-time PCR (MRT-PCR) using blood can improve the diagnosis of intra-abdominal candidiasis (IAC). We prospectively studied 39 patients with suspected IAC in the absence of previous antifungal therapy. Blood cultures, MRT-PCR, and β-D-glucan (BDG) in serum were performed in all patients. IAC was defined according to the 2013 European Consensus criteria. For MRT-PCR, the probes targeted the ITS1 or ITS2 regions of ribosomal DNA. Candidaemia was confirmed only in four patients (10%), and IAC criteria were present in 17 patients (43.6%). The sensitivity of MRT-PCR was 25% but increased to 63.6% (P = .06) in plasma obtained prior to volume overload and transfusion; specificity was above 85% in all cases. BDG performance was improved using a cutoff > 260 pg/ml, and improvement was not observed in samples obtained before transfusion. In this cohort of high risk of IAC and low rate of bloodstream infection, the performance of non-culture-based methods (MRT-PCR or BDG) was moderate but may be a complementary tool given the limitations of diagnostic methods available in clinical practice. Volume overload requirements, in combination with other factors, decrease the accuracy of MRT-PCR in patients with IAC.

scholarly journals Evaluation of real-time PCR and conventional diagnostic methods for the detection of Clostridium difficile-associated diarrhoea in a prospective multicentre study

2007 ◽  
Vol 56 (1) ◽  
pp. 36-42 ◽  
Author(s):  
Renate J. van den Berg ◽  
Norbert Vaessen ◽  
Hubert P. Endtz ◽  
Tanja Schülin ◽  
Eric R. van der Vorm ◽  
...  
Keyword(s):  
Clostridium Difficile ◽  
Multicentre Study ◽  
Real Time ◽  
Real Time Pcr ◽  
Cytotoxicity Assay ◽  
Diagnostic Methods ◽  
Cell Cytotoxicity ◽  
Predictive Values ◽  
Prospective Multicentre Study ◽  
Faecal Samples

In this prospective multicentre study, an enzyme-linked fluorescent assay (VIDAS CDA2; bioMérieux), an enzyme-linked assay [Premier Toxins A and B (PTAB); Meridian] and an in-house real-time PCR amplifying the tcdB gene were compared with the cell cytotoxicity assay used as the ‘gold standard’ for diagnosis of Clostridium difficile-associated diarrhoea (CDAD). Faecal samples from patients with a request for C. difficile diagnosis and samples from patients with diarrhoea hospitalized for at least 72 h were collected for 3 consecutive months from four university medical centres in The Netherlands. In total, 547 faecal samples were obtained from 450 patients. Of 540 samples available for all of the assays, 84 (15.6 %) showed a positive result in one or more assays. The cell cytotoxicity assay was positive in 31 samples (5.7 %) from 28 patients. A diagnosis of CDAD was not considered by the physician in 5 (23.8 %) of 21 patients with CDAD who were hospitalized for at least 72 h. Compared with the cell cytotoxicity assay, the sensitivity of VIDAS, PTAB and PCR was 83.9, 96.8 and 87.1 %, respectively. The specificity of VIDAS, PTAB and PCR was 97.1, 94.3 and 96.5 %, respectively. The positive and negative predictive values for VIDAS, PTAB and PCR were 63.4 and 99.0 %, 50.9 and 99.8 %, and 60.0 and 99.2 %, respectively. Of 61 samples that were positive in one, two or three of the assays, 56 were available for discordance analysis. Discordance analysis was performed by culture of toxinogenic strains. The concordance of VIDAS, PTAB and PCR with culture was 53.6 % (30/56), 55.4 % (31/56) and 71.4 % (40/56), respectively. It was concluded that real-time PCR had the highest concordance with toxinogenic culture and is therefore the preferred method for diagnosing CDAD in faecal samples. It was also concluded that diagnosis of patients with diarrhoea who have been hospitalized for more than 72 h should focus mainly on the detection of C. difficile, irrespective of the physician's request.

scholarly journals Confirmation of multi-parallel quantitative real-time PCR as the gold standard for detecting soil-transmitted helminths in stool

2021 ◽  
Author(s):  
Marina Papaiakovou ◽  
Nils Pilotte ◽  
Julia Dunn ◽  
David TJ Littlewood ◽  
Rubén O Cimino ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Gold Standard ◽  
Low Cost ◽  
Strongyloides Stercoralis ◽  
Diagnostic Methods ◽  
Quantitative Real Time Pcr ◽  
Soil Transmitted Helminths ◽  
Stool Samples ◽  
Kappa Agreement

AbstractDue to its simplicity and cost-effectiveness, microscopy has seen extensive field-use as the diagnostic standard for the detection of soil-transmitted helminths (STH) in stool samples. However, the sensitivity of microscopy-based detection is inadequate in reduced-transmission settings where worm burden is oftentimes low. Equally problematic, eggs of closely related species oftentimes have indistinguishable morphologies, leading to species misidentification. In light of these shortcomings, the purpose of this study was to demonstrate multi-parallel quantitative real-time PCR (qPCR) as the new “gold standard” for STH detection. Accordingly, stool samples from non-endemic participants were spiked with limited numbers of eggs or larvae (1 to 40) of five different species of STH. DNA extracts were tested using two unique multi-parallel real-time PCR-based diagnostic methods. These methods employed different target sequences (ribosomal internal transcribed spacer, or highly repetitive non-coding regions), to evaluate the detection of DNA from as little as one egg per sample. There was a statistically significant kendall correlation between egg/larvae counts and qPCR from both methods for Trichuris trichiura (0.86 and 0.872 for NHM and Baylor assays) and a strong correlation (0.602 and 0.631 for NHM and Baylor assays, respectively) for Ascaris lumbricoides. Less strong but still significant was the Kendall Tau-b value for A. duodenale (0.408 for both) and for S. stercoralis (0.483 and 0.653, respectively). In addition, using field stool samples from rural Argentina both assays had fair to moderate kappa agreement (0.329-0.454), except for Strongyloides stercoralis (0.121) that both assays had slight agreement. In spite of the small cohort of samples, both qPCR assays, targeting of two independent genomic regions, provided reproducible results and we believe that, low cost multi-parallel quantitative real-time PCR-based diagnostics should supplant microscopy as the new gold standard for stool-based detection of soil transmitted helminths in public-health and community settings.

scholarly journals Identification of Mycoses in Developing Countries

2019 ◽  
Vol 5 (4) ◽  
pp. 90 ◽  
Author(s):  
Amir Arastehfar ◽  
Brian L. Wickes ◽  
Macit Ilkit ◽  
David H. Pincus ◽  
Farnaz Daneshnia ◽  
...  
Keyword(s):  
Developing Countries ◽  
Real Time ◽  
Real Time Pcr ◽  
Fungal Infections ◽  
Point Of Care ◽  
Fungal Species ◽  
Lateral Flow ◽  
Lateral Flow Assay ◽  
Diagnostic Methods ◽  
Diagnostic Tools

Extensive advances in technology offer a vast variety of diagnostic methods that save time and costs, but identification of fungal species causing human infections remains challenging in developing countries. Since the echinocandins, antifungals widely used to treat invasive mycoses, are still unavailable in developing countries where a considerable number of problematic fungal species are present, rapid and reliable identification is of paramount importance. Unaffordability, large footprints, lack of skilled personnel, and high costs associated with maintenance and infrastructure are the main factors precluding the establishment of high-precision technologies that can replace inexpensive yet time-consuming and inaccurate phenotypic methods. In addition, point-of-care lateral flow assay tests are available for the diagnosis of Aspergillus and Cryptococcus and are highly relevant for developing countries. An Aspergillus galactomannan lateral flow assay is also now available. Real-time PCR remains difficult to standardize and is not widespread in countries with limited resources. Isothermal and conventional PCR-based amplification assays may be alternative solutions. The combination of real-time PCR and serological assays can significantly increase diagnostic efficiency. However, this approach is too expensive for medical institutions in developing countries. Further advances in next-generation sequencing and other innovative technologies such as clustered regularly interspaced short palindromic repeats (CRISPR)-based diagnostic tools may lead to efficient, alternate methods that can be used in point-of-care assays, which may supplement or replace some of the current technologies and improve the diagnostics of fungal infections in developing countries.

scholarly journals Development of Primers and Probes for Genus and Species Specific Detection of ‘Candidatus Liberibacter Species’ by Real-Time PCR

Plant Disease ◽  
2013 ◽  
Vol 97 (9) ◽  
pp. 1235-1243 ◽  
Author(s):  
G. Ananthakrishnan ◽  
N. Choudhary ◽  
Avijit Roy ◽  
V. G. Sengoda ◽  
E. Postnikova ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Diagnostic Methods ◽  
Rrna Gene ◽  
Single Pair ◽  
Degenerate Primers ◽  
Specific Primers ◽  
Genus Level ◽  
Candidatus Liberibacter ◽  
Species Specific

Huanglongbing (HLB), also known as citrus greening, is currently the most devastating disease impacting citrus production. The disease is associated with three different ‘Candidatus Liberibacter species’, ‘Ca. Liberibacter asiaticus’, ‘Ca. Liberibacter americanus’, and ‘Ca. Liberibacter africanus’, which induce similar and overlapping symptoms. When HLB-symptomatic trees are tested, one of the Candidatus Liberibacters is normally detected by conventional or real-time PCR (qPCR). The most widely used assays use primers and probes based on the 16S ribosomal RNA (rRNA) gene. The 16S rRNA-based assays to detect the three species are species-specific and must be performed sequentially. We describe a single assay that detected all species of ‘Ca. Liberibacter’ at the genus level, providing increased convenience. Recent molecular analyses of ‘Ca. Liberibacter species’ and other bacteria suggest that the rpoB gene (encoding the β-subunit of RNA polymerase) provides an alternative target for bacterial identification. We report here the design of a single pair of degenerate primers and a hybridization probe corresponding to the rpoB region and their application for the detection of all three citrus ‘Ca. Liberibacter species’, enabling detection of ‘Ca. Liberibacter’ at the genus level. In addition, species-specific primers and probes based on the rplJ/rplK genes were designed and used for detection at the species level in a multiplexed format. Both the genus- and species-specific assays were validated in both SYBR Green I and TaqMan formats, and with both plant and insect extracts that contained the pathogen. These one-step qPCR diagnostic methods are useful for the detection of all species of Liberibacter infecting citrus. In addition, the degenerate genus-specific primers and probe successfully detected ‘Ca. Liberibacter solanacearum’, a psyllid-transmitted pathogen associated with disease in tomato, carrot, and potato.

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