Nucleic Acid Amplification Technology Research Articles

Leapfrogging laboratories: the promise and pitfalls of high-tech solutions for antimicrobial resistance surveillance in low-income settings

The scope and trajectory of today’s escalating antimicrobial resistance (AMR) crisis is inadequately captured by existing surveillance systems, particularly those of lower income settings. AMR surveillance systems typically collate data...

Fabrication routes via projection stereolithography for 3D-printing of microfluidic geometries for nucleic acid amplification.

Digital Light Processing (DLP) stereolithography (SLA) as a high-resolution 3D printing process offers a low-cost alternative for prototyping of microfluidic geometries, compared to traditional clean-room and workshop-based methods. Here, we investigate DLP-SLA printing performance for the production of micro-chamber chip geometries suitable for Polymerase Chain Reaction (PCR), a key process in molecular diagnostics to amplify nucleic acid sequences. A DLP-SLA fabrication protocol for printed micro-chamber devices with monolithic micro-channels is developed and evaluated. Printed devices were post-processed with ultraviolet (UV) light and solvent baths to reduce PCR inhibiting residuals and further treated with silane coupling agents to passivate the surface, thereby limiting biomolecular adsorption occurences during the reaction. The printed devices were evaluated on a purpose-built infrared (IR) mediated PCR thermocycler. Amplification of 75 base pair long target sequences from genomic DNA templates on fluorosilane and glass modified chips produced amplicons consistent with the control reactions, unlike the non-silanized chips that produced faint or no amplicon. The results indicated good functionality of the IR thermocycler and good PCR compatibility of the printed and silanized SLA polymer. Based on the proposed methods, various microfluidic designs and ideas can be validated in-house at negligible costs without the requirement of tool manufacturing and workshop or clean-room access. Additionally, the versatile chemistry of 3D printing resins enables customised surface properties adding significant value to the printed prototypes. Considering the low setup and unit cost, design flexibility and flexible resin chemistries, DLP-SLA is anticipated to play a key role in future prototyping of microfluidics, particularly in the fields of research biology and molecular diagnostics. From a system point-of-view, the proposed method of thermocycling shows promise for portability and modular integration of funcitonalitites for diagnostic or research applications that utilize nucleic acid amplification technology.

Research progress on analysis of human papillomavirus by microchip capillary electrophoresis

Human papillomavirus (HPV), is a common spherical DNA virus that can lead to six types of cancers later in life, which has recently garnered human's attention. Microchip capillary electrophoresis (MCE) has provided simple, fast, portable, and sensitive HPV typing assay assisted by a variety of signal amplification technologies. This review presents the latest research progress of MCE in routine HPV typing assays, including both of the MCE techniques and MCE combined with the nucleic acid amplification techniques for HPV assay. The introduction on the former part concerns the MCE system, the MCE chips design and electrophoretic separation methods. The typical MCE system includes high voltage power supply, microfluidic chip of separation, sample injection, electrolyte cell, detection unit and so on. Four different chips are reviewed, containing straight separation channel, T-channel, serpentine channel and dual channel, because these microchips are the most used in the last decade. Furthermore, the high integration and high throughput on a single chip are often integrated the sample preparation unit on a chip. The advantages and disadvantages of different designed microchips are introduced at the same time. The separation methods of chip electrophoresis are briefly introduced. With the development and application of MCE for HPV detection, the separation time is greatly shortened from a few hours to several minutes. The review on the second part gives the comments on various kinds of nucleic acid amplification technologies coupled with MCE for HPV assay. Firstly, the comparative analysis is given on the polymerase chain reaction (PCR) combined with MCE, loop-mediated isothermal amplification (LAMP), PCR combined with restriction fragment length polymorphism (RFLP) for HPV DNA detection, and nucleic acid sequence based amplification (NASBA) for the detection of HPV mRNA, nested PCR and so on. Secondly, the reviews on the other methods beside MCE are also summarized, including the PCR coupled with Fourier transform-infrared spectroscopy (FT-IR spectroscopy), nanotechnology, DNA probes combined with electrochemical methods, reductive Cu(Ⅰ) particles catalyzed Zn-doped MoS2 quantum dots and T7 exonuclease with electrochemiluminescence, LAMP with CRISPR/Cas12a based lateral. In these non-MCE methods, the electrochemical sensing, e. g., impedimetric detection, pulse voltammetry method and flow biosensor, is an ideal method due to its low background signal and excellent time control ability. Finally, although MCE technologies have been developed and the developed instruments are applied in recent years, there are still some challenges in MCE techniques, methods and applications. The first challenge faced in the application of MCE technique in HPV typing assay is that the MCE device can not be well utilized for the detection of HPV with high resolution and high sensitivity, because MCE can not do signal amplification of HPV nucleic acid. The second challenge is that even though some researchers have successfully integrated PCR and MCE on one chip, the technique still faces difficulty for wide application and there is still no really integrated PCR-MCE chip for HPV detection. The third one is the MCE technique is lack for the manufacture of miniaturized and automatic instrument. At the end of review, the authors' insights are given on the development of automatic, fast, high stable and reliable detection in the HPV typing via the portable MCE device.

Reliability of CE-marked NATs for HIV-1 subtype C detection and quantitation.

We compared seven CE-marked HIV-1 RNA nucleic acid amplification technology (NAT) based assays for their detection efficiency and quantitation concordance in regard to HIV-1 subtype C. We used 398 plasma samples from South African repeat blood donors identified as HIV positive at occasion of routine screening NAT performed mainly during the years 2010-2013, with most plasma samples reflecting recent HIV-1 infections. All HIV-1 subtype C specimens were detected, independent of mono- or dual-target assay design. In the same time period new variants of HIV-1 subtype B had been identified which were missed by some mono-target assays, a finding which was not corroborated for subtype C in our study. A high level of concordance of HIV-1 subtype C quantitation was determined for the HIV-1 NATs, showing successful standardization in this diagnostic field.

Comparison of the World Pharmacopoeias’ Requirements for the Quality of Cell Lines

The cell line is one of the necessary components of a biomedical cell product (BMCP) which can include only viable human cells. In addition, human, animal, insect, or bacterial cell lines can be used as a substrate for the production of some biological drugs. The list of quality parameters and test methods for medicinal products quality control are specified in the State Pharmacopoeia of the Russian Federation, but it contains only a few general monographs on blood products and a few requirements for cell lines as substrates for the production of biological drugs (which cover all types of cells). Currently, there is no regulatory document comparable to the State Pharmacopoeia of the Russian Federation that would contain requirements and test methods for BMCP quality control in the Russian Federation. Thus, one of the issues that arises both during quality control and approval of BMCPs is the lack of a regulatory document defining requirements for BMCP quality parameters and test methods. However, some general monographs of the Russian Pharmacopoeia and other pharmacopoeias can be used for quality control of both cell lines and non-cellular components. The aim of the study was to analyse and compare different pharmacopoeial requirements for the quality of cell lines used as components in human cell- and tissue-based products (comparable to BMCPs), which could be used in BMCP quality control. The paper analyses general monographs of the United States Pharmacopoeia (USP), European Pharmacopoeia (Ph. Eur.), Japanese Pharmacopoeia, Pharmacopoeia of the Republic of Belarus, including general monographs on biological/biotechnological products, because their requirements apply to human cell lines included as components in products similar to BMCPs. The analysed approaches and methods of quality control of cell- and tissue-based products described in the USP and Ph. Eur. could form the basis for elaboration of general monographs for the Russian Pharmacopoeia, including identification, potency, viral safety, and mycoplasma tests that are based on the nucleic acid amplification technology and other tests for cell lines as components of BMCPs.

COVID-19 Clinical Diagnostics and Testing Technology.

Given the global nature of the coronavirus disease 2019 (COVID‐19) pandemic, the need for disease detection and expanding testing capacity remains critical priorities. This review discusses the technological advances in testing capability and methodology that are currently used or in development for detecting the novel coronavirus. We describe the current clinical diagnostics and technology, including molecular and serological testing approaches, for severe acute respiratory syndrome‐coronavirus 2 (SARS‐CoV‐2) testing as well as address their advantages and limitations. Nucleic acid amplification technology for molecular diagnostics remains the gold standard for virus detection. We highlight alternative molecular detection techniques used for developing novel COVID‐19 diagnostics on the horizon. Antibody response against SARS‐CoV‐2 remains poorly understood and proper validation of serology tests is necessary to demonstrate their accuracy and clinical utility. In order to bring the pandemic under control, we must speed up the development of rapid and widespread testing through improvements in clinical diagnostics and testing technology as well as access to these tools.

New Insights from Chemical Biology: Molecular Basis of Transmission, Diagnosis, and Therapy of SARS-CoV-2

Coronavirus disease 2019 (COVID-19) is caused by a novel strain of coronavirus, designated as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). It has caused a global pandemic rapidly s...

Ultra-sensitive detection of Pb2+ based on DNAzymes coupling with multi-cycle strand displacement amplification (M-SDA) and nano-graphene oxide

Lead is one of the toxic heavy metal ions which seriously threatens the environment and human health, even at ultra-trace level. Here, an innovative fluorescent sensor based on DNAzymes coupling with multi-cycle strand displacement amplification (M-SDA) and nano-graphene oxide (GO) was established for the first time to ultra-sensitively detect lead, which is the first attempt to combine biological nucleic acid amplification technology with GO to detect heavy metal ions. There are several advantages. Firstly, the high selectivity of DNAzymes endow the proposed sensor outstanding specificity. Secondly, the introduction of M-SDA let the proposed sensor own the high potential for ultra-sensitive detection of Pb2+. Compared with traditional SDA, the amplification efficiency of M-SDA is greatly enhanced due its intermediate products are able to induce another SDA. Thirdly, the using of GO in fluorescence ion sensor is simple and fast. Under the optimal experimental conditions, the proposed sensor exhibited a good linear from 0.01 nM to 850 nM (R2=0.99714) with the detection limit as low as 6.7 pM. Additionally, the proposed sensor could be applied to detect the real samples with good accuracy and reliability.

DNA Nanofirecrackers Assembled through Hybridization Chain Reaction for Ultrasensitive SERS Immunoassay of Prostate Specific Antigen.

Isothermal nucleic acid amplification technology has been widely adopted for analytical chemistry with the purpose of sensitivity improvement. Herein we present an ultrasensitive concatenated hybridization chain reaction (C-HCR) based surface-enhanced Raman scattering (SERS) immunoassay by forming antibody-antigen-aptamer heterosandwich structures with the model analyte of total prostate specific antigens (tPSA). In the C-HCR, two HCRs, one proceeds with two hairpins and the other with four biotin-modified hairpins, are coupled, making the formation of DNA nanofirecrackers with the lengths longer than 200 nm and more than four hundred million binding sites of streptavidin modified enzymes. These types of DNA nanofirecrackers through the aptamer encoded linker strand to form heterosandwich structures could provide a general signal application platform such as enzyme catalysis with high amplification efficiency. As a proof of concept, the Au@Ag core-shell nanostructure based SERS immunoassay with excellent signal amplification has been developed by employing the streptavidin modified alkaline phosphatase (SA-ALP) through its catalysis of 2-phospho-l-ascorbic acid trisodium salt (AAP) to form Au@Ag core-shell nanostructures via the formation of ascorbic acid (AA) to reduce AgNO3 and deposition of silver element on gold nanorods (AuNRs). The newly developed method has a detection limit as low as 0.94 fg/mL and has successfully achieved the detection of serum samples from clinical patients, which was consistent with the clinical test results, showing that this C-HCR strategy to form DNA nanofirecrackers has great potential in clinical applications.

LAMP assay is an auxiliary tool for rapid and sensitive pathogen diagnosis in veterinary areas

Molecular technique for nucleic acid amplification has become more advanced, especially isothermal nucleic acid amplification which can amplify DNA or RNA molecules at constant temperatures. This allows us to be independent of sophisticated equipment such as thermal cyclers or realtime-PCR. The feasibility of adopting molecular techniques for pathogen detection at point-of-care are approaching practical use. At present, there are various isothermal nucleic acid amplification technologies, including rolling cycle amplification (RCA), helicase-dependent amplification (HDA), recombinase polymerase amplification (RPA) and loop-mediated isothermal amplification (LAMP). LAMP is one of the most popular methods used for pathogen diagnosis in animals due to several readout platforms such as turbidity, fluorescence, lateral flow dipstick (LFD) and color change visualizable by naked eye. In addition, LAMP can be integrated with CRISPR-cas which in turn, augments pathogen detection at ultra sensitivity. This letter shows the implementation of LAMP for pathogen detection in veterinary areas with different readouts as well as a new perspective for LAMP adaptation.

Current and Future Perspectives on Isothermal Nucleic Acid Amplification Technologies for Diagnosing Infections.

Nucleic acid amplification technology (NAAT) has assumed a critical position in disease diagnosis in recent times and contributed significantly to healthcare. Application of these methods has resulted in a more sensitive, accurate and rapid diagnosis of infectious diseases than older traditional methods like culture-based identification. NAAT such as the polymerase chain reaction (PCR) is widely applied but seldom available to resource-limited settings. Isothermal amplification (IA) methods provide a rapid, sensitive, specific, simpler and less expensive procedure for detecting nucleic acid from samples. However, not all of these IA techniques find regular applications in infectious diseases diagnosis. Disease diagnosis and treatment could be improved, and the rapidly increasing problem of antimicrobial resistance reduced, with improvement, adaptation, and application of isothermal amplification methods in clinical settings, especially in developing countries. This review centres on some isothermal techniques that have found documented applications in infectious diseases diagnosis, highlighting their principles, development, strengths, setbacks and imminent potentials for use at points of care.

The Laboratory Diagnosis of Neisseria gonorrhoeae: Current Testing and Future Demands.

The ideal laboratory test to detect Neisseria gonorrhoeae (Ng) should be sensitive, specific, easy to use, rapid, and affordable and should provide information about susceptibility to antimicrobial drugs. Currently, such a test is not available and presumably will not be in the near future. Thus, diagnosis of gonococcal infections presently includes application of different techniques to address these requirements. Microscopy may produce rapid results but lacks sensitivity in many cases (except symptomatic urogenital infections in males). Highest sensitivity to detect Ng was shown for nucleic acid amplification technologies (NAATs), which, however, are less specific than culture. In addition, comprehensive analysis of antibiotic resistance is accomplished only by in vitro antimicrobial susceptibility testing of cultured isolates. As a light at the end of the tunnel, new developments of molecular techniques and microfluidic systems represent promising opportunities to design point-of-care tests for rapid detection of Ng with high sensitivity and specificity, and there is reason to hope that such tests may also provide antimicrobial resistance data in the future.

Manufacturing of plasma-derived C1-inhibitor concentrate for treatment of patients with hereditary angioedema.

Background: Replacement therapy with plasma-derived C1-inhibitor (C1-INH) has been used for decades to treat patients with hereditary angioedema (HAE) with C1-INH deficiency. Objective: This article reviewed the rationale for using C1-INH replacement therapy in patients with HAE and the process of manufacturing plasma-derived C1-INH. Methods: The manufacture of C1-INH is an involved and carefully monitored process that includes screening and selection of prospective donors, the collection of source plasma, and purification with dedicated pathogen reduction steps. Donor eligibility is determined by restrictive criteria established and monitored by regulatory agencies as well as voluntary standards implemented by plasma collection centers that exceed government regulations. Individual and pooled donations are tested for transfusion-transmissible infections, including hepatitis B virus, hepatitis C virus, human immunodeficiency virus, parvovirus B19, and hepatitis A virus, by using enzyme-linked immunosorbent assays or nucleic acid amplification technologies. Frozen plasma that is cleared for manufacturing undergoes controlled thawing and centrifugation, and the resulting supernatant (i.e., cryoprecipitate-depleted plasma) is used to manufacture several plasma-derived therapies, including C1-INH. In addition to chromatography steps, the manufacturing process consists of dedicated and effective pathogen reduction steps, including pasteurization, hydrophobic interaction chromatography or polyethylene glycol precipitation, and virus filtration. Manufacturers continuously monitor the safety profile of C1-INH products by robust pharmacovigilance processes that enable systematic collection and evaluation of all suspected adverse drug reaction reports as well as evaluation of safety information from all other sources. Results and Conclusion: These procedures used in donor screening, donation and manufacturing pool testing, manufacturing, and pharmacovigilance ensure that plasma-derived C1-INH products have the safety, quality, identity, potency, and purity that is necessary to provide the intended therapeutic effect.

2150. Detection and Characterization of Viral and Bacterial Pathogens in Tonsillar Tissues of Children Undergoing Tonsillectomy

BackgroundThe diagnosis of upper respiratory infections is commonly made using nucleic acid amplification technologies for viruses and bacteria. The impact of latency and colonization are not often appreciated. We aimed to detect viruses and bacteria present in tonsil and adenoid tissues from children during the absence of acute infection symptoms.MethodsRemnant tonsil and adenoid tissues were obtained from children undergoing tonsillectomy procedures. Nucleic acids of viruses and bacteria were detected using laboratory developed PCRs targeting Epstein–Barr virus (EBV), Adenoviruses (AdV), human herpes virus 6 (HHV6), human enteroviruses (HEV), Group A streptococcus (GAS), Kingella kingae (KKN), Staphylococcus aureus (SA), Streptococcus pneumoniae (SPN), Arcanobacterium haemolyticus (AHE), Fusobacterium necrophorum (FNE), Mycobacterium pneumoniae (MPN) and Neisseria meningitidis (NM). The genogroup of AdV and the type of HHV6 were determined as well. Demographics, clinical presentation and detection rates of these viruses and bacteria were analyzed.ResultsDuring the study period (April 2018 and March 2019), 239 samples were collected from patients <18 years with an average age of 5.1 years. More male subjects than female subjects were included (57.7% vs. 42.3%). Most of the patients underwent tonsillectomy due to adenotonsillar hypertrophy (93.3%). Thirty (12.5%) also had a history of tonsillitis, 224 (93.7%) sleep apnea, 36 (15.1%) otitis media, 35 (14.6%) Eustachian tube dysfunction and 46 (19.2%) had other conditions. The detection of the pathogens among each age group is presented in Table 1. The seasonal distributions of virus positivity are shown in Figure 1.ConclusionThe detection rates of each virus and bacterium in the tonsillar tissues from children absent of acute infection symptoms vary in each age group and fluctuate among seasons. In the molecular era when syndromic real-time multiplex PCR kits can provide sensitive and rapid results for a wide range of pathogens, it is important to understand the meaning of detection and differentiate between an infection and colonization or latency. Disclosures All authors: No reported disclosures.

Advances in Directly Amplifying Nucleic Acids from Complex Samples.

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.

Clinical evaluation of ID NOW influenza A & B 2, a rapid influenza virus detection kit using isothermal nucleic acid amplification technology - A comparison with currently available tests

In this study, we evaluated the performance of ID NOW Influenza A & B 2 (ID NOW 2), a rapid molecular point-of-care test for influenza within 13 min, in comparison with currently available tests. A total of 254 nasopharyngeal swabs (NPS) and 271 nasopharyngeal aspirates (NPA) collected from 373 children and 152 adults with influenza-like illness were tested using ID NOW 2, viral culture, rapid antigen detection test, and loop-mediated isothermal amplification test to evaluate the sensitivity and specificity compared with real-time reverse transcription polymerase chain reaction as the reference method. The sensitivities of ID NOW 2 for influenza A were 95.9% and 95.7% in NPS and NPA, respectively, and for influenza B were 100% and 98.7% in NPS and NPA, respectively. The specificity was 100% for both influenza A and influenza B in NPS and NPA. Sensitivity of each test method reflected the difference of analytical sensitivity among the tests, and ID NOW 2 was not affected by time after illness onset and patient age. In conclusion, ID NOW 2 demonstrated a high sensitivity and specificity that is useful for diagnosis of influenza in the clinical setting and infection control.

Construction of Lentivirus-Based Reference Material for RT-PCR Detection of Middle East Respiratory Syndrome Coronavirus and Its Application in External Quality Assessment.

Nucleic acid amplification technology (NAT) has been the most used one for rapid detection of Middle East Respiratory Syndrome coronavirus (MERS-CoV) since MERS-CoV was first detected in 2012. It is important to develop stable and safe reference materials for assessing the quality of NAT kits and performing an external quality assessment (EQA) in different laboratories. In this study, the MERS-CoV RNA fragments including upE, ORF1b, and N were packed within human immunodeficiency virus type 1 (HIV-1)-like particles. The lyophilized virus-like particles (VLPs) were found to be stable at 37 °C or below and safe to be used not only as the control material for PCR detection of MERS-CoV but also as the reference material for EQA. In an EQA organized by Ningbo International Travel Healthcare Center in China, 49 participating institutions achieved 100% agreement in detecting MERS-CoV using various commercial diagnosis kits and different extraction methods. However, different Ct values reported by different sites for the same sample implied that a need exists to standardize the RNA extraction method and/or the PCR detection conditions between the laboratories.

An efficient isothermal PCR method for on-site detection of nucleic acid.

Convective PCR(CPCR) is an isothermal nucleic acid amplification technology; however, natural convection exhibits a chaotic and multiplex flow state, resulting in low amplification efficiency and specificity. We placed a polycarbonate strip (p-strip) inside reaction tubes to induce circumfluence by blocking the inner ring that originally allowed fluid to flow at suboptimal temperatures. Moreover, we constructed a dual-temperature instrument to provide appropriate denaturing and annealing zones for CPCR. Tubes containing p-strips exhibited significantly improved efficiency, sensitivity and specificity. For real-time detection, the variation coefficients of three replicates having the same concentrations were less than 2% in more than half of the cases, indicating improved CPCR amplification and potential as a commercial on-site nucleic acid diagnosis tool.

Proceedings of the Food and Drug Administration public workshop on pathogen reduction technologies for blood safety 2018 (Commentary, p. 3026).

On November 29, 2018, experts in the field of infectious diseases, pathogen reduction technologies (PRTs) and other participants from blood centers, academia, and industry gathered at the Food and Drug Administration (FDA) White Oak Campus in Silver Spring, Maryland, for a 2‐day public workshop entitled “Pathogen Reduction Technologies for Blood Safety.” The workshop opened with welcome remarks from Dr. Nicole Verdun, Director, Office of Blood Research and Review (OBRR), Center for Biologics Evaluation and Research (CBER), FDA, followed by introductory remarks from Dr. Peter Marks, Director, CBER, FDA. The first day of the workshop focused on blood‐borne infectious agents and their impact on blood safety, experiences of the American Red Cross, and other blood establishments in implementing FDA‐approved pathogen inactivation (PI) technology for plasma and platelets (PLTs) in the United States and novel PRTs under consideration for whole blood (WB) and red blood cells (RBCs). The second day opened with welcome remarks from Dr. Chintamani Atreya, Associate Director for Research, OBRR, CBER, FDA. The focus was on emerging innovations relevant to PRTs and potential alternatives to PRTs. The workshop concluded with remarks on insights for future research and development in this area for blood and blood product safety from infectious agents. A brief introduction of each session by the session moderator followed by a summary of the speaker presentation as submitted by the moderator and speaker are reported here.

PREVALENCE AND GENOTYPING OF HEPATITIS C VIRUS IN HEMODIALYSIS PATIENTS AND EVALUATION OF HCV-CORE ANTIGEN TEST IN SCREENING PATIENTS FOR DIALYSIS IN SANA'A CITY, YEMEN

Hepatitis C virus infection is a constant worldwide public health concern. The prevalence of HCV infection is higher in patients on chronic haemodialysis (HD) than in the general population. Despite the control of blood products, hepatitis C virus transmission is still being observed among patients undergoing dialysis. Detection systems for serum HCV antibodies are insensitive in the acute phase because of the long serological window. Direct detection of HCV depends on PCR test but this test is not suitable for routine screening. The objective of this study was to determine prevalence of HCV, genotyping and if HCV core antigen test could be a better alternative to NAT techniques for the diagnosis of HCV infection during the window period and whether the sensitivity for antibody detection is preserved. We screened 159 patients on long-term dialysis by HVC antibodies test, PCR HCV-RNA and HCV core antigen test by commercial tests. The prevalence of HCV was 10.7% (17 patients) and genotype 4 was the most common one (64.7%). The sensitivity of HCV core antigen test was 94.1%, the specificity 100%, the positive predictive power 100%, and the negative predictive power 97.9%. In conclusions; patients on maintenance HD in Yemen have a high prevalence of HCV infection comparing with general population; and genotype 4 is predominant. The performance of serological detection of HCV core antigen was better than that of HCV antibodies test and may be an alternative to nucleic acid amplification technology (NAT) for routine monitoring of patients on chronic dialysis.