Advances in Molecular Technologies and Platforms for the Diagnosis of Infectious Diseases

2013 ◽  
Vol 810 ◽  
pp. 77-125
Author(s):  
M. Rubayet Hasan
Keyword(s):  
Nucleic Acid ◽  
Infectious Diseases ◽  
Potential Method ◽  
Diagnostic Methods ◽  
Control Measures ◽  
Nucleic Acid Amplification ◽  
Microbial Pathogens ◽  
Immunological Methods ◽  
Clinical Samples ◽  
Single Test

nfectious microbial pathogens constitute the largest cause of morbidity and mortality worldwide. Early diagnosis and rapid infection control measures can lead to improved outcomes, earlier discharges and reduced nosocomial infections. Conventional diagnostic methods for infectious diseases such as microscopy, culture, and immunological methods, in most cases, are not universally applicable, less sensitive and could take from days to months to complete depending on the pathogen. Molecular assays based on nucleic acids such as polymerase chain reaction (PCR) have improved the sensitivity, specificity and turn-around time in diagnostic microbiology laboratories. These tests are particularly important to detect very low levels of pathogens in clinical samples, and for organisms that have long half-lives or are non-culturable. However, individual molecular tests are available for only a limited number of the more common infectious agents. Moreover, infectious disease events arising from novel pathogens or genetic variants have significantly increased, recently, for which, routine diagnostic methods are not yet available. Therefore, molecular methods and technologies capable of detecting multiple pathogens in a single test have become available over the last few years. Although, these methods are based on the conventional nucleic acid amplification and hybridization chemistry, enhanced multiplexing capability has been achieved through innovations in nucleic acid labeling techniques, and post-amplification analytic methods and instrumentation. The availability of these test kits brought a new level of convenience to the physicians ordering practices, and to the laboratory personnel, as they require very little hands on time. However, these tests are yet unaffordable to many laboratories, and in many cases, the sensitivity is poor compared to that of single-target, real-time PCR assays. Looking into the future, the revolutionary, next generation sequencing (NGS) technology is now being considered as a potential method for rapid identification of hundreds of pathogens, in an unbiased manner, with a single test that could significantly benefit patients who are critically ill with undiagnosed disease.

scholarly journals Advanced CRISPR-Cas Effector Enzyme-Based Diagnostics for Infectious Diseases, Including COVID-19

Life ◽  
2021 ◽  
Vol 11 (12) ◽  
pp. 1356
Author(s):  
Sangha Kwon ◽  
Ha Youn Shin
Keyword(s):  
Nucleic Acid ◽  
Infectious Diseases ◽  
Pathogen Detection ◽  
Diagnostic Techniques ◽  
Diagnostic Methods ◽  
Nucleic Acid Amplification ◽  
Detection Methods ◽  
Molecular Diagnostic ◽  
Nucleic Acid Detection ◽  
Time Accuracy

Rapid and precise diagnostic tests can prevent the spread of diseases, including worldwide pandemics. Current commonly used diagnostic methods include nucleic-acid-amplification-based detection methods and immunoassays. These techniques, however, have several drawbacks in diagnosis time, accuracy, and cost. Nucleic acid amplification methods are sensitive but time-consuming, whereas immunoassays are more rapid but relatively insensitive. Recently developed CRISPR-based nucleic acid detection methods have been found to compensate for these limitations. In particular, the unique collateral enzymatic activities of Cas12 and Cas13 have dramatically reduced the diagnosis times and costs, while improving diagnostic accuracy and sensitivity. This review provides a comprehensive description of the distinct enzymatic features of Cas12 and Cas13 and their applications in the development of molecular diagnostic platforms for pathogen detection. Moreover, it describes the current utilization of CRISPR-Cas-based diagnostic techniques to identify SARS-CoV-2 infection, as well as recent progress in the development of CRISPR-Cas-based detection strategies for various infectious diseases. These findings provide insights into designing effective molecular diagnostic platforms for potential pandemics.

scholarly journals Comparative performance and cycle threshold values of 10 nucleic acid amplification tests for SARS-CoV-2 on clinical samples

PLoS ONE ◽  
2021 ◽  
Vol 16 (6) ◽  
pp. e0252757
Author(s):  
Miyuki Mizoguchi ◽  
Sohei Harada ◽  
Koh Okamoto ◽  
Yoshimi Higurashi ◽  
Mahoko Ikeda ◽  
...  
Keyword(s):  
Nucleic Acid ◽  
Diagnostic Performance ◽  
Nucleic Acid Amplification ◽  
Clinical Samples ◽  
Nucleic Acid Amplification Tests ◽  
Cycle Threshold ◽  
E Gene ◽  
Viral Copy Number ◽  
Gene Test ◽  
Positive Results

Background A number of nucleic acid amplification tests (NAATs) for SARS-CoV-2 with different reagents have been approved for clinical use in Japan. These include research kits approved under emergency use authorization through simplified process to stabilize the supply of the reagents. Although these research kits have been increasingly used in clinical practice, limited data is available for the diagnostic performance in clinical settings. Methods We compared sensitivity, specificity, and cycle threshold (Ct) values obtained by NAATs using 10 kits approved in Japan including eight kits those receiving emergency use authorization using 69 frozen-stored clinical samples including 23 positive samples with various Ct values and 46 negative samples. Results Viral copy number of the frozen-stored samples determined with LightMix E-gene test ranged from 0.6 to 84521.1 copies/μL. While no false-positive results were obtained by any of these tests (specificity: 100% [95% CI, 88.9%-100%]), sensitivity of the nine tests ranged from 68.2% [95% CI, 45.1%-86.1%] to 95.5% [95% CI, 77.2%-99.9%] using LightMix E-gene test as the gold standard. All tests showed positive results for all samples with ≥100 copies/μL. Significant difference of Ct values even among tests amplifying the same genetic region (N1-CDC, N2) was also observed. Conclusion Difference in the diagnostic performance was observed among NAATs approved in Japan. Regarding diagnostic kits for emerging infectious diseases, a system is needed to ensure both rapidity of reagent supply and accuracy of diagnosis. Ct values, which are sometimes regarded as a marker of infectivity, are not interchangeable when obtained by different assays.

Amplification-Free DNA Detection Using a Microtip-Sensor Decorated With LNA Probes for Rapid TB Screening

2011 ◽  
Author(s):  
Shinnosuke Inoue ◽  
Woon-Hong Yeo ◽  
Jong-Hoon Kim ◽  
Jae-Hyun Chung ◽  
Kyong-Hoon Lee ◽  
...  
Keyword(s):  
Nucleic Acid ◽  
Staphylococcus Epidermidis ◽  
Genomic Dna ◽  
Bacterial Culture ◽  
Low Cost ◽  
Surrogate Marker ◽  
High Sensitivity ◽  
Diagnostic Methods ◽  
Nucleic Acid Amplification ◽  
Highly Sensitive

Tuberculosis (TB) is an epidemic affecting one-third of the world’s population, mostly in developing and low-resource settings. People having active pulmonary TB are considered highly infectious; therefore, it is critical to identify and treat these patients rapidly before spreading to others. However, the most reliable TB diagnostic methods of bacterial culture or nucleic acid amplification are time-consuming and expensive. The challenge of TB diagnosis lies in highly sensitive and specific screening with low cost. Here, we present an LNA-modified microtip-sensor, which is capable of selectively detecting low-abundance DNA from bacteria. When genomic DNA of Bacillus Calmette-Gue´rin (BCG, a surrogate marker of Mycobacterium bovis), and genomic DNA of Staphylococcus epidermidis (S. epi) are used, the microtip-sensor yields the detection limit of 1,000 copies/mL within 20 minutes. The high sensitivity and specificity approaching nucleic acid amplification methods can potentially overcome the current challenges for rapid TB screening.

scholarly journals CURRENT STATE OF THE PROBLEM OF THE LABORATORY DIAGNOSTICS OF UROGENITAL TRICHOMONIASIS

2013 ◽  
Vol 62 (1) ◽  
pp. 32-41
Author(s):  
Aleksey Nikolayevich Grigoryev
Keyword(s):  
Nucleic Acid ◽  
Reproductive Tract ◽  
Review Literature ◽  
Nucleic Acid Amplification ◽  
Immunological Methods ◽  
Laboratory Diagnostics ◽  
Actual Problem ◽  
Culture Techniques ◽  
Current State ◽  
Wet Mount

The laboratory diagnostics of urogenital trichomoniasis is an actual problem of modern microbiology of infections of the reproductive tract. In the review, literature data on methods of microbiological diagnostics of trichomoniasis are presented, which include microscopy of wet mount and stained preparations, culture techniques, immunological methods and nucleic acid amplification tests.

scholarly journals Comparative Evaluation of Six Molecular Assays based on RT-PCR and Cross Primer Isothermal Amplification for SARS-CoV-2 RNA Detection

2021 ◽  
Author(s):  
Shuang Wu ◽  
Xiaolu Shi ◽  
Qiongcheng Chen ◽  
Yixiang Jiang ◽  
Le Zuo ◽  
...  
Keyword(s):  
Nucleic Acid ◽  
Comparative Study ◽  
Reverse Transcriptase ◽  
Real Time ◽  
Isothermal Amplification ◽  
Nucleic Acid Amplification ◽  
Clinical Samples ◽  
Predictive Values ◽  
Health Crisis ◽  
Sensitivity Specificity

Abstract Background: SARS-CoV-2 is a newly emerged coronavirus, causing the coronavirus disease 2019 (COVID-19) outbreak in December, 2019. As drugs and vaccines of COVID-19 remain in development, accurate virus detection plays a crucial role in the current public health crisis. Quantitative real-time reverse transcriptase-polymerase chain reaction (RT-qPCR) kits have been reliably used for detection of SARS-CoV-2 RNA since the beginning of the COVID-19 outbreak, whereas isothermal nucleic acid amplification-based point-of-care automated kits have also been considered as a simpler and rapid alternative. However, as these kits have only been developed and applied clinically within a short timeframe, their clinical performance has not been adequately evaluated to date. We describe a comparative study between a newly developed cross-priming isothermal amplification (CPA) kit (Kit A) and five RT-qPCR kits (Kits B–F) to evaluate their sensitivity, specificity, predictive values and accuracy. Methods: Fifty-two clinical samples were used including throat swabs (n=30), nasal swabs (n=7), nasopharyngeal swabs (n=7) and sputum specimens (n=8), comprising confirmed (n=26) and negative cases (n=26). SARS-CoV-2 detection was simultaneously performed on each sample using six nucleic acid amplification kits. The sensitivity, specificity, positive/negative predictive values (PPV/NPV) and the accuracy for each kit were assessed using clinical manifestation and molecular diagnoses as the reference standard. Reproducibility for RT-qPCR kits was evaluated in triplicate by three different operators using a SARS-CoV-2 RNA-positive sample. On the basis of the six kits’ evaluation results, CPA kit (Kit A) and two RT-qPCR Kits (Kit B and F) were applied to the SARS-CoV-2 detection in close-contacts of COVID-19 patients. Results: For Kit A, the sensitivity, specificity, PPV/NPV and accuracy were 100%. Among the five RT-qPCR kits, Kits B, C and F had good agreement with the clinical diagnostic reports (Kappa≥0.75); Kits D and E were less congruent (0.4≤Kappa<0.75). Differences between all kits were statistically significant (P<0.001). The reproducibility of RT-qPCR kits was determined using a coefficients of variation (CV) between 0.95% and 2.57%, indicating good reproducibility. Conclusions: This is the first comparative study to evaluate CPA and RT-qPCR kits’ specificity and sensitivity for SARS-CoV-2 detection, and could serve as a reference for clinical laboratories, thus informing testing protocols amid the rapidly progressing COVID-19 pandemic. Keywords: SARS-CoV-2; COVID-19; nucleic acid detection; real-time reverse transcriptase PCR (RT-qPCR); cross-priming isothermal amplification (CPA)

scholarly journals Mycobacteriosis and Tuberculosis: Laboratory Diagnosis

2018 ◽  
Vol 12 (1) ◽  
pp. 41-58 ◽  
Author(s):  
Davood Azadi ◽  
Tahereh Motallebirad ◽  
Kazem Ghaffari ◽  
Hasan Shojaei
Keyword(s):  
Nucleic Acid ◽  
Case Reports ◽  
Diagnostic Algorithm ◽  
Laboratory Diagnosis ◽  
Etiologic Agent ◽  
Control Measures ◽  
Nucleic Acid Amplification ◽  
Nucleic Acid Hybridization ◽  
Original Research ◽  
Mycobacterial Infections

Background:Tuberculosis is one of the most important infectious diseases that has claimed its victims throughout much of known human history. With Koch's discovery of the tubercle bacillus as the etiologic agent of the disease, his sanitary and hygienic measures, which were based on his discovery and the development of a vaccine against tuberculosis by Albert Calmette and Camille Guérin in 1921, an attenuatedMycobacterium bovisstrain, bacilli Calmette-Guérin (BCG), and the discovery of the first antibiotic against tuberculosis, streptomycin by Selman Waksman in 1943, soon led to the opinion that appropriate control measures had become available for tuberculosis and it had been assumed that the disease could ultimately be eradicated.The emergence of resistant strains of this bacteria and widespread distribution of the disease in the world, and the emergence of the AIDS epidemic destroyed any possibility of global control of tuberculosis in the foreseeable future.Objectives:The purpose of this review is to highlight the current scientific literature on mycobacterial infections and provide an overview on the laboratory diagnosis of tuberculosis and non-tuberculosis infections based on conventional phenotypic and modern molecular assays.Method:In this study, a number of 65 papers comprising 20 reviews, 9 case reports, and 36 original research in association with mycobacteriosis and the laboratory diagnosis of mycobacterial infections, were reviewed.Results:Based on our analysis on the published documents methods applied for the laboratory diagnosis of tuberculosis are continually assessed and developed in order to achieve more rapid, less expensive, and accurate results. Acid-fast staining and culture for mycobacteria remain at the core of any diagnostic algorithm with the sensitivity of 20-70% and specificity of 95-98% for AFB microscopy and the sensitivity of 95% and the specificity of 98% for culture based diagnosis. Following growth in culture, molecular tests such as nucleic acid hybridization probes and DNA sequencing may be used for definitive species identification. Nucleic acid amplification methods provide the means for direct detection ofMycobacterium tuberculosisin respiratory specimens without the prerequisite to isolate or culture the organism, leading to more rapid diagnosis and better patient care.Conclusion:As the researchers in a developing country, we strongly believe that despite significant advances in laboratory capacity, in many countries reliable confirmation of suspected mycobacterial diseases is hindered by a lack of knowledge on proper standardized methods, sufficient funds, suitably trained staff and laboratory supplies.

Diagnosing Emerging and Reemerging Infectious Diseases: The Pivotal Role of the Pathologist

2011 ◽  
Vol 135 (1) ◽  
pp. 83-91 ◽  
Author(s):  
Juan P. Olano ◽  
David H. Walker
Keyword(s):  
Nucleic Acid ◽  
Infectious Diseases ◽  
Molecular Diagnostics ◽  
Clinical Setting ◽  
Driving Forces ◽  
Molecular Techniques ◽  
Nucleic Acid Amplification ◽  
Pivotal Role ◽  
Laboratory Diagnostics ◽  
Molecular Tools

Abstract Context—Molecular diagnostics continues to evolve very rapidly, and its impact in the diagnosis of infectious diseases is undeniable. Molecular tools have played a pivotal role in discovering and characterizing several emerging infectious agents and have now become the gold standard for the diagnosis of infectious diseases caused by fastidious or uncultivable agents. Multiple challenges still remain for the widespread use of cost-effective, validated, and commercially available molecular tools. Automated instruments capable of sample processing and multiplex nucleic acid amplification and postamplification analysis have already been approved by the US Food and Drug Administration (FDA) for use in the clinical setting. Nanobiotechnology is beginning to impact laboratory diagnostics in the clinical setting. Objective—To address current nucleic acid techniques used in the clinical laboratory for diagnosis of infectious diseases. FDA-approved tests are listed, as well as molecular techniques (amplification and postamplification analysis). A comprehensive list of emerging pathogens during the last 4 decades is also presented. Biosurveillance systems are discussed in the context of molecular tools. The rapidly evolving field of nanobiotechnology is briefly addressed. Data Sources—Original publications, major reviews, and book chapters were used to present a comprehensive, yet short, review of molecular diagnostics in infectious diseases. Conclusions—We will continue to witness an exponential growth of molecular techniques used for the initial diagnosis of infectious diseases. Molecular tools will also continue to have an impact on disease prognosis and response to therapeutic interventions. Automation, multiplexing, and miniaturization will continue to be driving forces in the development of new instruments.

Empfehlungen zur qualitätsgesicherten Durchführung von Nukleinsäure-Amplifikations - Testungen (NAT) von Blutprodukten. Quality assurance in nucleic acid amplification testing (NAT) of blood products

2005 ◽  
Vol 29 (2) ◽  
Author(s):  
Willi K. Roth
Keyword(s):  
Quality Control ◽  
Nucleic Acid ◽  
Sample Preparation ◽  
Nucleic Acids ◽  
Internal Control ◽  
Control Measures ◽  
Nucleic Acid Amplification ◽  
Blood Products ◽  
Whole Process ◽  
And Storage

AbstractEuropean manufacturers of plasma products and German blood transfusion services were the first to introduce nucleic acid amplification testing (NAT) of blood products in the mid-1990s. Their primary goal was to increase the safety of blood by closing as far as possible the diagnostic window, which exists after the onset of viral infection until the appearance of the first detectable antibodies. Sample preparation, transport and storage are crucial steps in a quality-controlled PCR. Sensitivity and contamination rates highly depend on the sample preparation and storage techniques. Anticoagulants must be selected carefully because some may inhibit the PCR. Dilution of samples by pooling needs to be considered and should be compensated for by subsequent virus enrichment procedures, e.g. centrifugation. The whole process of sample preparation, pooling and virus enrichment must be validated and quality control measures must be implemented. Reagents for the extraction of viral nucleic acids should not pose any risk to the laboratory staff. Nevertheless, the reagents should be highly efficient in liberating viral nucleic acids at high yield and purity for the following amplification reactions. At this critical stage, quality control measures should guarantee an efficient extraction process and contain potential sources of contaminations. Several methods are available for the amplification of nucleic acids. PCR is the most common, especially in in-house assays. The amplification of nucleic acids should be performed as far as possible in a closed system, which may be guaranteed best by real-time PCR approaches. Reaction tubes need never be opened during the amplification because detection can be performed through the closed tube. Amplicons that could contaminate the following PCR reactions will not be released. It is of great importance to blood transfusion services to guarantee that negative results un-equivocally indicate virus negative blood donations. Therefore, internal control sequences should be implemented in each individual PCR reaction in order to monitor that the individual PCR has worked correctly. Besides internal control sequences, external negative and positive controls should be implemented in each PCR run to demonstrate false positive reactions as well as to monitor pre-PCR processes like virus enrichment and extraction. The whole process needs to be validated according to the criteria set in national guidelines or by national authorities. External quality assessment programs are highly recommended.

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