Mycobacteriosis and Tuberculosis: Laboratory Diagnosis

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.

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Laboratory Diagnosis of Zika Virus Infection

Archives of Pathology & Laboratory Medicine ◽

10.5858/arpa.2016-0406-sa ◽

2016 ◽

Vol 141 (1) ◽

pp. 60-67 ◽

Cited By ~ 63

Author(s):

Marie Louise Landry ◽

Kirsten St. George

Keyword(s):

Public Health ◽

Nucleic Acid ◽

Virus Infection ◽

Zika Virus ◽

Laboratory Diagnosis ◽

Government Support ◽

Nucleic Acid Amplification ◽

Diagnostic Tools ◽

Nucleic Acid Amplification Tests ◽

Zika Virus Infection

Context.—The rapid and accurate diagnosis of Zika virus infection is an international priority. Objective.—To review current recommendations, methods, limitations, and priorities for Zika virus testing. Data Sources.—Sources include published literature, public health recommendations, laboratory procedures, and testing experience. Conclusions.—Until recently, the laboratory diagnosis of Zika infection was confined to public health or research laboratories that prepared their own reagents, and test capacity has been limited. Furthermore, Zika cross-reacts serologically with other flaviviruses, such as dengue, West Nile, and yellow fever. Current or past infection, or even vaccination with another flavivirus, will often cause false-positive or uninterpretable Zika serology results. Detection of viral RNA during acute infection using nucleic acid amplification tests provides more specific results, and a number of commercial nucleic acid amplification tests have received emergency use authorization. In addition to serum, testing of whole blood and urine is recommended because of the higher vial loads and longer duration of shedding. However, nucleic acid amplification testing has limited utility because many patients are asymptomatic or present for testing after the brief period of Zika shedding has passed. Thus, the greatest need and most difficult challenge is development of accurate antibody tests for the diagnosis of recent Zika infection. Research is urgently needed to identify Zika virus epitopes that do not cross-react with other flavivirus antigens. New information is emerging at a rapid pace and, with ongoing public-private and international collaborations and government support, it is hoped that rapid progress will be made in developing robust and widely applicable diagnostic tools.

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Expanding detection windows for discriminating single nucleotide variants using rationally designed DNA equalizer probes

Nature Communications ◽

10.1038/s41467-020-19269-9 ◽

2020 ◽

Vol 11 (1) ◽

Author(s):

Guan A. Wang ◽

Xiaoyu Xie ◽

Hayam Mansour ◽

Fangfang Chen ◽

Gabriela Matamoros ◽

...

Keyword(s):

Nucleic Acid ◽

Rural Areas ◽

Simultaneous Detection ◽

Dna Nanotechnology ◽

Quantitative Relationship ◽

Nucleic Acid Amplification ◽

Nucleic Acid Hybridization ◽

Single Nucleotide Variants ◽

Single Nucleotide ◽

Hybridization Probes

Abstract Combining experimental and simulation strategies to facilitate the design and operation of nucleic acid hybridization probes are highly important to both fundamental DNA nanotechnology and diverse biological/biomedical applications. Herein, we introduce a DNA equalizer gate (DEG) approach, a class of simulation-guided nucleic acid hybridization probes that drastically expand detection windows for discriminating single nucleotide variants in double-stranded DNA (dsDNA) via the user-definable transformation of the quantitative relationship between the detection signal and target concentrations. A thermodynamic-driven theoretical model was also developed, which quantitatively simulates and predicts the performance of DEG. The effectiveness of DEG for expanding detection windows and improving sequence selectivity was demonstrated both in silico and experimentally. As DEG acts directly on dsDNA, it is readily adaptable to nucleic acid amplification techniques, such as polymerase chain reaction (PCR). The practical usefulness of DEG was demonstrated through the simultaneous detection of infections and the screening of drug-resistance in clinical parasitic worm samples collected from rural areas of Honduras.

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

LaboratoriumsMedizin ◽

10.1515/jlm.2005.016 ◽

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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Development of a Portable SPR Sensor for Nucleic Acid Detection

Micromachines ◽

10.3390/mi11050526 ◽

2020 ◽

Vol 11 (5) ◽

pp. 526 ◽

Cited By ~ 1

Author(s):

Yafeng Huang ◽

Lulu Zhang ◽

Hao Zhang ◽

Yichen Li ◽

Luyao Liu ◽

...

Keyword(s):

Nucleic Acid ◽

Incident Angle ◽

Incident Light ◽

High Sensitivity ◽

Nucleic Acid Amplification ◽

Nucleic Acid Hybridization ◽

Nucleic Acid Detection ◽

Scanning System ◽

Label Free ◽

Spr Sensor

Nucleic acid detection is of great significance in clinical diagnosis, environmental monitoring and food safety. Compared with the traditional nucleic acid amplification detection method, surface plasmon resonance (SPR) sensing technology has the advantages of being label-free, having simple operation, and providing real-time detection. However, the angle scanning system in many SPR angle modulation detection applications usually requires a high-resolution stepper motor and complex mechanical structure to adjust the angle. In this paper, a portable multi-angle scanning SPR sensor was designed. The sensor only uses one stepping motor to rotate a belt, and the belt pulls the mechanical linkages of incident light and reflected light to move in opposite directions for achieving the SPR angle scanning mode that keeps the incident angle and reflected angle equal. The sensor has an angle scanning accuracy of 0.002°, response sensitivity of 3.72 × 10−6 RIU (refractive index unit), and an angle scanning range of 30°–74°. The overall size of the system is only 480 mm × 150 mm × 180 mm. The portable SPR sensor was used to detect nucleic acid hybridization on a gold film chip modified with bovine serum albumin (BSA). The result revealed that the sensor had high sensitivity and fast response, and could successfully accomplish the hybridization detection of target DNA solution of 0.01 μmol/mL.

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The predictive value of quantitative nucleic acid amplification detection ofClostridium difficiletoxin gene for faecal sample toxin status and patient outcome

10.1101/436451 ◽

2018 ◽

Author(s):

Kerrie A Davies ◽

Tim Planche ◽

Mark H Wilcox

Keyword(s):

Nucleic Acid ◽

Predictive Value ◽

Severe Infection ◽

Laboratory Diagnosis ◽

Nucleic Acid Amplification ◽

Toxin Gene ◽

Diagnostic Study ◽

Increased Risk ◽

Risk Patients ◽

Using Data

AbstractBackgroundLaboratory diagnosis ofClostridium difficileinfection (CDI) remains unsettled, despite updated guidelines. We investigated the potential utility of quantitative data from a nucleic acid amplification test (NAAT) forC. difficiletoxin gene (tg) for patient management.MethodsUsing data from the largest everC. difficilediagnostic study (8853 diarrhoeal samples from 7335 patients), we determined the predicative value of C. difficile tgNAAT (Cepheid Xpert C.diff) low cycle threshold (CT) value for patient toxin positive status, CDI severity, mortality and CDI recurrence. Reference methods for CDI diagnosis were cytotoxicity assay (CTA) and cytotoxigenic culture (CTC).ResultsOf 1281 tgNAAT positive faecal samples, 713 and 917 were CTA and CTC positive, respectively. The median tgNAAT CT for patients who died was 25.5 vs 27.5 for survivors (p = 0.021); for toxin-positivity was 24.9 vs 31.6 for toxin-negative samples (p<0.001) and for patients with a recurrence episode was 25.6 vs 27.3 for those who did not have a recurrent episode (p = 0.111). Following optimal cut-off determination, low CT was defined as ≤25 and was significantly associated with a toxin-positive result (P<0.001, positive predictive value 83.9%), presence of PCR-ribotype 027 (P=0.025), and mortality (P=0.032). Recurrence was not associated with low CT (p 0.111).ConclusionsLow tgNAAT CT could indicate CTA positive patients, have more severe infection, increased risk of mortality and possibly recurrence. Although, the limited specificity of tgNAAT means it cannot be used as a standalone test, it could augment a more timely diagnosis, and optimise management of these at-risk patients.

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Ability of the Digene Hybrid Capture II Test To Identify Chlamydia trachomatis and Neisseria gonorrhoeae in Cervical Specimens

Journal of Clinical Microbiology ◽

10.1128/jcm.37.11.3668-3671.1999 ◽

1999 ◽

Vol 37 (11) ◽

pp. 3668-3671 ◽

Cited By ~ 33

Author(s):

Julius Schachter ◽

Edward W. Hook ◽

William M. McCormack ◽

Thomas C. Quinn ◽

Max Chernesky ◽

...

Keyword(s):

Nucleic Acid ◽

Chlamydia Trachomatis ◽

Neisseria Gonorrhoeae ◽

Fluorescent Antibody ◽

Simultaneous Detection ◽

Nucleic Acid Amplification ◽

Nucleic Acid Hybridization ◽

Attractive Alternative ◽

Single Specimen ◽

Hybrid Capture

The Digene Hybrid Capture II (HCII CT/GC) test is a combination test designed to detect Chlamydia trachomatis andNeisseria gonorrhoeae in a single specimen. It is a nucleic acid hybridization test which uses signal amplification to increase sensitivity. We compared its performance to that of culture on cervical specimens from 1,370 women. Direct fluorescent-antibody assay was used to resolve discrepant results for C. trachomatis. Samples were collected with a proprietary cervical brush or with endocervical swabs. The HCII CT/GC test proved to be sensitive and specific in detecting these organisms. Compared to N. gonorrhoeaeculture, it had a sensitivity of 93% (87/94) and a specificity of 98.5% (1,244/1,263). Compared to C. trachomatis culture, the sensitivity was 97.7% (129/132) and specificity was 98.2% (1,216/1,238). Testing of some specimens with discrepant results by PCR suggested that the test would actually prove to be even more specific if it were compared to a nucleic acid amplification test (NAAT). The sensitivity of C. trachomatis culture was somewhat less, at 88.6% (117/132). The endocervical brush appeared to be better than Dacron swabs for collecting specimens. The HCII CT/GC test offers an attractive format that allows simultaneous detection of C. trachomatis and N. gonorrhoeae with a single specimen. An initial positive result is followed by repeat tests with probes to identify chlamydiae or gonococci. This test is more sensitive than C. trachomatis culture and is at least as sensitive as culture for gonococci. It deserves further evaluation and comparison with NAATs and may well offer an attractive alternative for diagnosis and screening of these infections.

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Use of serology immunoassays for predicting SARS-CoV-2 infection: a serology-based diagnostic algorithm

10.1101/2021.10.23.21265429 ◽

2021 ◽

Author(s):

Alejandro Lazo-Langner ◽

Benjamin Chin-Yee ◽

Jaryd Tong ◽

Lori Lowes ◽

Benjamin D. Hedley ◽

...

Keyword(s):

Validation Cohort ◽

Diagnostic Algorithm ◽

Roc Curves ◽

Control Measures ◽

Nucleic Acid Amplification ◽

Diagnostic Strategy ◽

Roc Curve Analysis ◽

Potential Cost ◽

Derivation Cohort ◽

Infection Control Measures

Background. Detection of viral RNA by nucleic acid amplification testing (NAAT) remains the gold standard for diagnosis of SARS-CoV-2 infection but is limited by high cost and other factors. Whether serology-based assays can be effectively incorporated into a diagnostic algorithm remains to be determined. Herein we describe the development of a serology-based testing algorithm for SARS-CoV-2 infection. Patients and Methods. Between July 2020 and February 2021, we included symptomatic unvaccinated patients evaluated in the Emergency Department of our institution for suspected SARS-CoV-2. All patients had testing by real-time Reverse Transcription Polymerase Chain Reaction. The performance characteristics of five commercial enzymatic serology assays testing for different antibody isotypes were evaluated in a derivation cohort and the assay with the best performance was further tested on a validation cohort. Optimal cut-off points were determined using receiver operating characteristic (ROC) curves and further tested using logistic regression. Results. The derivation and validations cohorts included 72 and 319 patients, respectively. Based on its initial performance, the Elecsys Anti-SARS-CoV-2 assay (Roche Diagnostics) was further tested in the validation cohort. Using ROC curve analysis, we estimated the diagnostic performance for different cut-off points assuming a prevalence of positive tests of 5%. At any given cut-off point the NPV was over 97%. Discussion. This study suggests that an initial diagnostic strategy using the Elecsys Anti-SARS-CoV-2 serology test in symptomatic unvaccinated patients could help to rule out an acute SARS-CoV2 infection and potentially lead to appropriately tailored infection control measures or rational guidance for further testing with a potential cost reduction and increased availability

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