Laboratory Diagnostic Tools for Syphilis: Current Status and Future Prospects

With the increasing number of patients infected with syphilis in the past 20 years, early diagnosis and early treatment are essential to decline syphilis prevalence. Owing to its diverse manifestations, which may occur in other infections, the disease often makes clinicians confused. Therefore, a sensitive method for detecting T. pallidum is fundamental for the prompt diagnosis of syphilis. Morphological observation, immunohistochemical assay, rabbit infectivity test, serologic tests, and nucleic acid amplification assays have been applied to the diagnosis of syphilis. Morphological observation, including dark-field microscopy, silver-staining, and direct fluorescent antibody staining for T. pallidum, can be used as a direct detection method for chancre specimens in primary syphilis. Immunohistochemistry is a highly sensitive and specific assay, especially in the lesion biopsies from secondary syphilis. Rabbit infectivity test is considered as a sensitive and reliable method for detecting T. pallidum in clinical samples and used as a historical standard for the diagnosis of syphilis. Serologic tests for syphilis are widely adopted using non-treponemal or treponemal tests by either the traditional or reverse algorithm and remain the gold standard in the diagnosis of syphilis patients. In addition, nucleic acid amplification assay is capable of detecting T. pallidum DNA in the samples from patients with syphilis. Notably, PCR is probably a promising method but remains to be further improved. All of the methods mentioned above play important roles in various stages of syphilis. This review aims to provide a summary of the performance characteristics of detection methods for syphilis.

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Advances in Directly Amplifying Nucleic Acids from Complex Samples

Biosensors ◽

10.3390/bios9040117 ◽

2019 ◽

Vol 9 (4) ◽

pp. 117 ◽

Cited By ~ 4

Author(s):

Faye M. Walker ◽

Kuangwen Hsieh

Keyword(s):

Nucleic Acid ◽

Sample Preparation ◽

Point Of Care ◽

Nucleic Acid Amplification ◽

Current Status ◽

Diagnostic Tools ◽

Complex Samples ◽

Lab On Chip ◽

On Chip ◽

Effective Visualization

Advances in nucleic acid amplification technologies have revolutionized diagnostics for systemic, inherited, and infectious diseases. Current assays and platforms, however, often require lengthy experimental procedures and multiple instruments to remove contaminants and inhibitors from clinically-relevant, complex samples. This requirement of sample preparation has been a bottleneck for using nucleic acid amplification tests (NAATs) at the point of care (POC), though advances in “lab-on-chip” platforms that integrate sample preparation and NAATs have made great strides in this space. Alternatively, direct NAATs—techniques that minimize or even bypass sample preparation—present promising strategies for developing POC diagnostic tools for analyzing real-world samples. In this review, we discuss the current status of direct NAATs. Specifically, we surveyed potential testing systems published from 1989 to 2017, and analyzed their performances in terms of robustness, sensitivity, clinical relevance, and suitability for POC diagnostics. We introduce bubble plots to facilitate our analysis, as bubble plots enable effective visualization of the performances of these direct NAATs. Through our review, we hope to initiate an in-depth examination of direct NAATs and their potential for realizing POC diagnostics, and ultimately transformative technologies that can further enhance healthcare.

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A Current Microbiological Picture of Mycobacterium Isolates from Istanbul, Turkey

Polish Journal of Microbiology ◽

10.33073/pjm-2020-021 ◽

2020 ◽

Vol 69 (2) ◽

pp. 185-191

Author(s):

BILGE SUMBUL ◽

MEHMET ZIYA DOYMAZ

Keyword(s):

Multidrug Resistant ◽

Mycobacterium Abscessus ◽

Diagnostic Methods ◽

Current Status ◽

Clinical Samples ◽

Mycobacterium Fortuitum ◽

Mycobacterium Species ◽

Full Spectrum ◽

Number Of Patients ◽

Lowenstein Jensen

Despite advances in diagnosis and treatment, tuberculosis (TB) continues to be one of the essential health problems throughout the world. Turkey is considered to be endemic for TB. In this study, we analyzed the distribution of Mycobacterium species, compare the diagnostic methods, and susceptibilities to anti-tuberculosis drugs of TB isolates. The aim was to document the current status and to provide a frame of reference for future studies. In this study, 278 Mycobacterium species isolated from 7,480 patients between September 2015 and June 2019 were included. Löwenstein-Jensen medium (LJ) and MGIT 960 were used for the isolation of strains. Susceptibility to 1st-line anti-tuberculosis drugs was determined. Positivity rates in clinical samples were as follows: 1.4% for direct microscopic acid-fast bacilli (AFB) detection, 3.4% for growth on the LJ, and 3.7% for growth on MGIT-960. Two hundred thirty-three isolates were identified as Mycobacterium tuberculosis complex (MTBC) and 45 were non-tuberculous mycobacteria (NTMs). Eleven of the NTMs (24.4%) were Mycobacterium fortuitum group isolates, and eight NTMs (17.7%) were Mycobacterium abscessus complex isolates. A number of patients diagnosed with tuberculosis peaked twice between the ages of 20–31 and 60–71. A hundred and eighty-two MTBC isolates (78.1%) were susceptible to all 1st-line anti-tuberculosis drugs, while 51 isolates (21.9%) were resistant to at least one drug tested. The multidrug-resistant tuberculosis rate was 13.7% among resistant strains and 3% in all strains. The liquid cultures were better for detection of both MTBC and NTMs isolates. The data demonstrate that MTBC continues to be challenge for this country and indicates the need for continued surveillance and full-spectrum services of mycobacteriology laboratory and infectious diseases.

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

PLoS ONE ◽

10.1371/journal.pone.0252757 ◽

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.

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Amplicon-Based Detection and Sequencing of SARS-CoV-2 in Nasopharyngeal Swabs from Patients With COVID-19 and Identification of Deletions in the Viral Genome That Encode Proteins Involved in Interferon Antagonism

Viruses ◽

10.3390/v12101164 ◽

2020 ◽

Vol 12 (10) ◽

pp. 1164

Author(s):

Shona C. Moore ◽

Rebekah Penrice-Randal ◽

Muhannad Alruwaili ◽

Nadine Randle ◽

Stuart Armstrong ◽

...

Keyword(s):

Viral Genome ◽

False Negative ◽

Point Mutations ◽

Read Length ◽

Diagnostic Tools ◽

Clinical Samples ◽

Nucleotide Polymorphisms ◽

Pathogenic Potential ◽

Number Of Patients ◽

Interferon Antagonism

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of coronavirus disease 2019 (COVID-19). Sequencing the viral genome as the outbreak progresses is important, particularly in the identification of emerging isolates with different pathogenic potential and to identify whether nucleotide changes in the genome will impair clinical diagnostic tools such as real-time PCR assays. Although single nucleotide polymorphisms and point mutations occur during the replication of coronaviruses, one of the biggest drivers in genetic change is recombination. This can manifest itself in insertions and/or deletions in the viral genome. Therefore, sequencing strategies that underpin molecular epidemiology and inform virus biology in patients should take these factors into account. A long amplicon/read length-based RT-PCR sequencing approach focused on the Oxford Nanopore MinION/GridION platforms was developed to identify and sequence the SARS-CoV-2 genome in samples from patients with or suspected of COVID-19. The protocol, termed Rapid Sequencing Long Amplicons (RSLAs) used random primers to generate cDNA from RNA purified from a sample from a patient, followed by single or multiplex PCRs to generate longer amplicons of the viral genome. The base protocol was used to identify SARS-CoV-2 in a variety of clinical samples and proved sensitive in identifying viral RNA in samples from patients that had been declared negative using other nucleic acid-based assays (false negative). Sequencing the amplicons revealed that a number of patients had a proportion of viral genomes with deletions.

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Development of a Molecular Method for the Detection of Enteric Viruses in Oysters

Journal of Food Protection ◽

10.4315/0362-028x-58.12.1357 ◽

1995 ◽

Vol 58 (12) ◽

pp. 1357-1362 ◽

Cited By ~ 12

Author(s):

LEE-ANN JAYKUS ◽

RICARDO DE LEON ◽

MARK D. SOBSEY

Keyword(s):

Cell Culture ◽

Nucleic Acid ◽

Hepatitis A Virus ◽

Enteric Virus ◽

Nucleic Acid Amplification ◽

Detection Methods ◽

Ammonium Bromide ◽

Rt Pcr ◽

Initial Inoculum ◽

Ctab Precipitation

Detection of enteric virus contamination of shellfish is limited by current methodology, which is time-consuming, tedious, and lacking in sensitivity due to reliance on cell culture infectivity. Alternative detection methods based on nucleic acid amplification have been hampered by high sample volumes and the presence of enzymatic inhibitors. The goal of this study was to develop methods to purify and concentrate intact virions from oyster extracts to a volume and quality compatible with viral genomic nucleic acid amplification by reverse transcriptase-polymerase chain reaction (RT-PCR). Fifty-gram oyster samples were homogenized and processed by standard adsorption-elution precipitation methodology and then seeded with 105 PFU of poliovirus 1 (PV1) or hepatitis A virus (HAV). Seeded viruses were concentrated by fluorocarbon extraction, polyethylene glycol (PEG) precipitation, chloroform extraction, and cetyltrimethyl ammonium bromide (CTAB) precipitation to a volume of 100 μl with removal of RT-PCR inhibitors. Virus recovery after elution of PEG precipitates was 50% for PVI and IS to 20% for HAV as evaluted by cell culture infectivity. The CTAB precipitation step yielded a concentrated sample which was directly compatible with RT-PCR reactions and capable of detecting about 100 placque=forming units (PFU) of PVl or HAV. When 50-g oyster extracts were seeded and processed by the entire concentration and purification scheme, direct RT-PCR detection of viral genomic RNA was possible at initial inoculum levels of 104 PFU of HAV and 103 PFU of PV1, with recoveries of 1 to 5% of seeded viruses.

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Nucleic acid amplification-based methods for microRNA detection

Analytical Methods ◽

10.1039/c4ay02938k ◽

2015 ◽

Vol 7 (6) ◽

pp. 2258-2263 ◽

Cited By ~ 14

Author(s):

Hui-Ling Chen ◽

Meng-Meng Guo ◽

Hao Tang ◽

Zhan Wu ◽

Li-Juan Tang ◽

...

Keyword(s):

Nucleic Acid ◽

Nucleic Acid Amplification ◽

Detection Methods ◽

Basic Principles ◽

Microrna Detection

This review traces the basic principles of several nucleic acid amplification-based microRNA detection methods that have been developed in recent three years.

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Development of a Loop-mediated isothermal amplification (LAMP) technique for specific and early detection of Mycobacterium leprae in clinical samples

Scientific Reports ◽

10.1038/s41598-021-89304-2 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Nupur Garg ◽

Upasana Sahu ◽

Sudeshna Kar ◽

Farhan J. Ahmad

Keyword(s):

Early Detection ◽

Mycobacterium Leprae ◽

Negative Control ◽

Rapid Diagnosis ◽

Isothermal Amplification ◽

Detection Methods ◽

Diagnostic Tools ◽

Clinical Samples ◽

Rrna Gene ◽

Loop Mediated Isothermal Amplification

AbstractLeprosy, a progressive, mutilating and highly stigmatized disease caused by Mycobacterium leprae (ML), continues to prevail in the developing world. This is due to the absence of rapid, specific and sensitive diagnostic tools for its early detection since the disease gets notified only with the advent of physical scarring in patients. This study reports the development of a Loop-mediated isothermal amplification (LAMP) technique for fast, sensitive and specific amplification of 16S rRNA gene of ML DNA for early detection of leprosy in resource-limited areas. Various parameters were optimized to obtain robust and reliable amplification of ML DNA. Blind clinical validation studies were performed which showed that this technique had complete concurrence with conventional techniques. Total absence of amplification of negative control DNA confirmed the specificity of this test. Various visual detection methods viz. colorimetric, turbidity differentiation and bridge flocculation were standardized to establish easy-to-read and rapid diagnosis. This technique eliminates the lack of accuracy and sensitivity in skin smear tests in patients and the requirement for expensive lab equipments and trained technicians. The technique holds promise for further expansion and has the potential to cater to the unmet needs of society for a cheap, highly-sensitive and robust rapid diagnosis of ML.

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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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Association of One-Step Nucleic Acid Amplification Detected Micrometastases with Tumour Biology and Adjuvant Chemotherapy

International Journal of Breast Cancer ◽

10.1155/2017/4971096 ◽

2017 ◽

Vol 2017 ◽

pp. 1-5 ◽

Cited By ~ 1

Author(s):

Ghaleb Goussous ◽

Sadaf Jafferbhoy ◽

Niamh Smyth ◽

Lisette Hammond ◽

Sankaran Narayanan ◽

...

Keyword(s):

Nucleic Acid ◽

Adjuvant Chemotherapy ◽

Sentinel Node ◽

Survival Benefit ◽

Nucleic Acid Amplification ◽

Detection Rates ◽

Tumour Biology ◽

Number Of Patients ◽

One Step ◽

The Impact

One-step nucleic acid amplification (OSNA) is an intraoperative technique with a high sensitivity and specificity for sentinel node assessment. The aim of this study was to assess the impact of OSNA on micrometastases detection rates and use of adjuvant chemotherapy. A retrospective review of patients with sentinel node micrometastases over a five-year period was carried out and a comparison of micrometastases detection using OSNA and H&E techniques was made. Out of 1285 patients who underwent sentinel node (SLN) biopsy, 76 patients had micrometastases. Using H&E staining, 36 patients were detected with SLN micrometastases (9/year) in contrast to 40 patients in the OSNA year (40/year) (p<0.0001), demonstrating a fourfold increase with the use of OSNA. In the OSNA group, there was also a proportional increase in Grade III, triple-negative, ER-negative, and HER-2-positive tumours being diagnosed with micrometastases. Also on interactive PREDICT tool, the number of patients with a predicted 10-year survival benefit of more than 3% with adjuvant chemotherapy increased from 52 to 70 percent. OSNA has resulted in an increased detection rate of micrometastases especially in patients with aggressive tumour biology. This increased the number of patients who had a predicted survival benefit from adjuvant chemotherapy.

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