Nucleic Acid Amplification Technology Research Articles

Blood donor recruitment strategies and their impact on blood safety in Egypt

Because of the high incidence of HCV, blood safety presents a serious challenge in Egypt. Given the constrained economy which limits the implementation of nucleic acid amplification technology, proper recruitment of blood donors becomes of paramount importance. To evaluate the effectiveness of blood donor recruitment strategies, the seroprevalence of positive infectious markers among blood donors was studied. Donors' records covering the period from 2006-2012 were reviewed. Blood donations were screened for HCV antibodies, HBs antigen (HBsAg), HIV-1 and 2 and syphilis antibodies. Of 308,762 donors, 63.4% were voluntary donors (VD). VD of 2011-2012 were significantly younger than family replacement donors (RD) .The overall prevalences of HCV antibodies, HBsAg, HIV and syphilis antibodies were 4.3%, 1.22%, 0.07%, and 0.13%, respectively. All tested markers (except HIV) were significantly higher among RD, when compared to VD (P<0.0001). A consistent steady trend for decrease in HCV seropositivity was observed in RD and VD from 8.9% and 4.2% to 3.8% and 1.5%, respectively. A trend for decrease in HBsAg was demonstrated in VD from 1.2% to 0.53%. The decreasing trends in HCV antibody and HBs antigen is promising and may reflect the improved donor selection criteria.

Central Nervous System Tuberculosis: Challenges and Advances in Diagnosis and Treatment.

Mycobacterium tuberculosis is one of the most prevalent human infections. Although the largest share of the burden of disease is in Africa and Asia, tuberculosis has a global footprint due to travel and migration. Resource constraints in many low- and middle-income countries are hampering efforts to control new infections and to prevent drug resistance. Infection of the central nervous system by Mycobacterium tuberculosis includes meningitis, tuberculoma, and abscess and carries a high morbidity and mortality. High clinical suspicion, combined with cerebrospinal fluid analysis and brain imaging studies, can improve the diagnostic certainty. The recent scale-up of nucleic acid amplification technology may allow earlier diagnosis of tuberculous meningitis in many regions of the world. Treatment of tuberculous infection of the central nervous system is usually empirical and follows conventional regimens for pulmonary tuberculosis. The optimal treatment regimen is still being elucidated and has been the subject of recent clinical trials.

Routine screening of blood donations at Qingdao central blood bank, China, for hepatitis B virus (HBV) DNA with a real‐time, multiplex nucleic acid test for HBV, hepatitis C virus, and human immunodeficiency virus Types 1 and 2

The Roche cobas TaqScreen MPX test was used to evaluate the rate of hepatitis B surface antigen (HBsAg)-negative donations that were hepatitis B virus (HBV) DNA reactive from June 2010 to January 2011 in Qingdao, China. HBsAg-negative samples from 65,800 voluntary blood donors were tested with the cobas TaqScreen MPX test in pools of 6 on the Roche cobas s 201 blood screening platform. Samples positive for HBV DNA and negative for HBsAg were quantitated with the Roche COBAS AmpliPrep/COBAS TaqMan HBV test. In addition, serologic tests for HBsAg, hepatitis B surface antibody, anti-hepatitis B core antigen (anti-HBc), anti-hepatitis B e antigen (anti-HBe), and hepatitis B e antigen (HBe) were done using the Roche electrochemiluminescence immunoassay. A total of 80 nucleic acid amplification technology (NAT) test-reactive pools were identified and 59 pools (74%) resolved to a reactive sample. All samples were HBV DNA reactive and the viral load in each sample was quantitated. The viral loads of the samples ranged from less than 20 to 34,600 IU/mL; 13 samples (22%) had viral loads of more than 20 IU/mL, 27 samples (45.8%) had viral loads of less than 20 IU/mL, and 19 samples (32.2%) had undetectable viral loads. Of the 59 NAT-reactive samples, 40 (67.8%) were anti-HBc positive. Fifteen of the 59 samples could not be confirmed as NAT reactive either by an alternative NAT test or by serology. The HBV NAT yield in blood donors in Qingdao is 0.06% (38/65,800). This study confirmed the value of NAT for interdicting HBV-positive donations and preventing transfusion-transmitted HBV infections.

Degenerate polymerase chain reaction strategy with DNA microarray for detection of multiple and various subtypes of virus during blood screening

The risk of transferring blood-borne infections during transfusion is continually increasing because of newly emerging and reemerging viruses. Development of a rapid screening method for emerging viruses that might be transmitted by transfusion is required to eliminate such pathogens during blood donor screening. Owing to increased use of human materials in organ transplants and cell therapy, the risk of donor-transmitted viral infections is also increasing. Although nucleic acid amplification technology (NAT) is dedicated to blood screening, a small, convenient detection system is needed at the laboratory and hospital level. We developed a new pathogen detection system that can detect multiple viruses simultaneously, using originally designed degenerate polymerase chain reaction primers to amplify a wide range of viral genotypes. Amplified samples were identified using a DNA microarray of pathogen-specific probes. We detected very low copy numbers of multiple subtypes of viruses, such as human hepatitis C virus (HCV), human hepatitis B virus (HBV), human parvovirus B19 (PVB19), and West Nile virus (WNV), using a single plate. We also detected all genotypes of human immunodeficiency virus (HIV) but sensitivity was less than for the other viruses. We developed a microarray assay using novel primers for detection of a wide range of multiple pathogens and subtypes. Our NAT system was accurate and reliable for detection of HIV, HBV, HCV, PVB19, and WNV, with respect to specificity, sensitivity, and genotype inclusivity. Our system could be customized and extended for emerging pathogens and is suitable as a future NAT system.

Isothermal Strand-Displacement Polymerase Reaction for Visual Detection of the Southeast Asian–Type Deletion of α-Thalassemia

A rapid and reliable screening test for thalassemia carrier couples is the most effective strategy to decrease the risk of conceiving fetuses with severe thalassemia. We present an approach based on the isothermal strand-displacement polymerase reaction and the use of a lateral flow strip for the visual detection of an α-thalassemia Southeast Asian-type deletion. This assay was used to evaluate 86 clinical samples (72 cases of Southeast Asian-type deletions and 14 cases of other types of thalassemia), and the results obtained were 100% consistent with those obtained using conventional gap-PCR. The approach thus provides a simple, sensitive, rapid, and cost-effective method for the diagnosis of thalassemia genotypes.

Detection and identification of occult HBV in blood donors in Taiwan using a commercial, multiplex, multi‐dye nucleic acid amplification technology screening test

The ability of a new generation commercial, multiplex, multi-dye test from Roche, the cobas TaqScreen MPX test, version 2.0, to detect and identify occult HBV infections was evaluated using routine donor samples from Kaohsiung Blood Bank, Taiwan. A total of 5973 samples were tested by nucleic acid amplification technology (NAT); 5898 in pools of six, 66 in pools of less than six and nine samples individually. NAT-reactive samples were retested with alternative NAT tests, and follow-up samples from the donors were tested individually by NAT and for all the HBV serological markers. Eight NAT-only-reactive donors were identified, and follow-up samples were obtained from six of the donors. The results indicated that all eight donors had an occult HBV infection with viral loads <12IU/ml. The cobas(®) TaqScreen MPX test, version 2.0, has an advantage over the current Roche blood screening test, the cobas TaqScreen MPX test, for screening donations in countries with a high prevalence of occult HBV infections since the uncertainty associated with identifying samples with very low viremia is removed by the ability of the test to identify the viral target in samples that are reactive with the cobas TaqScreen MPX test, version 2.0.

Xpert MTB/RIF: a game changer for the diagnosis of pulmonary tuberculosis in children?

Xpert MTB/RIF: a game changer for the diagnosis of pulmonary tuberculosis in children?

Validation of the ANSR™ Salmonella Method for Detection of Salmonella spp. in Selected Foods and Environmental Samples

ANSR Salmonella is a new molecular diagnostic assay for detection of Salmonella spp. in foods and environmental samples. The test is based on the nicking enzyme amplification reaction (NEAR) isothermal nucleic acid amplification technology. The assay platform features simple instrumentation, minimal labor, and, following a single-step 10-24 h enrichment (depending on sample type), an extremely short assay time of 30 min, including sample preparation. Detection is real-time using fluorescent molecular beacon probes. Inclusivity testing was performed using a panel of 113 strains of S. enterica and S. bongori, representing 109 serovars and all genetic subgroups. With the single exception of the rare serovar S. Weslaco, all serovars and genetic subgroups were detected. Exclusivity testing of 38 non-salmonellae, mostly Enterobacteriaceae, yielded no evidence of cross-reactivity. In comparative testing of chicken carcass rinse, raw ground turkey, raw ground beef, hot dogs, and oat cereal, there were no statistically significant differences in the number of positive results obtained with the ANSR and the U.S. Department of Agriculture-Food Safety and Inspection Service or U.S. Food and Drug Administration/Bacteriological Analytical Manual reference culture methods. In testing of swab or sponge samples from five different environmental surfaces, four trials showed no statistically significant differences in the number of positive results by the ANSR and the U.S. Food and Drug Administration/ Bacteriological Analytical Manual reference methods; in the trial with stainless steel surface, there were significantly more positive results by the ANSR method. Ruggedness experiments showed a high degree of assay robustness when deviations in reagent volumes and incubation times were introduced.

Molecular virology in transfusion medicine laboratory.

and antibody to HBV core antigen (anti-HBc) testing began in the 80s to reduce transmission of non-A, non-B viral hepatitis. Then, the advent of the human immunodeficiency virus (HIV) epidemic and the rapidly following identification of hepatitis C virus (HCV) triggered unprecedented efforts to avoid the transmission of these new blood-borne viruses, and the implementation of antibody assays for both of these agents. To exclude individuals at risk for transmissible viral infection, donor screening process has been developed and implemented that includes the medical history of the candidate donor, a brief physical examination, and serologic testing. The next critical step in reducing viral TTI was achieved with the implementation of NAT technology 2 . Rapid progress and constant improvements in nucleic acid amplification technologies resulted in an unprecedented technological turning point in the molecular diagnostic and characterisation of viral infections. NAT combines the advantages of direct and highly sequence-specific detection of viral genomes (DNA or RNA) with an analytic sensitivity that is several orders of magnitude greater than that of antigen detection or virus isolation methods. Adequate specificity and high sensitivity combined with high throughput and the increasing availability of commercial assays, made NAT particularly suitable to screen blood donations for the presence of viral genomes. However, the considerable cost of NAT and challenges in automation led the users to develop strategies based on pooling of multiple donor samples

Rapid Clinical Bacteriology and Its Future Impact

Clinical microbiology has always been a slowly evolving and conservative science. The sub-field of bacteriology has been and still is dominated for over a century by culture-based technologies. The integration of serological and molecular methodologies during the seventies and eighties of the previous century took place relatively slowly and in a cumbersome fashion. When nucleic acid amplification technologies became available in the early nineties, the predicted "revolution" was again slow but in the end a real paradigm shift did take place. Several of the culture-based technologies were successfully replaced by tests aimed at nucleic acid detection. More recently a second revolution occurred. Mass spectrometry was introduced and broadly accepted as a new diagnostic gold standard for microbial species identification. Apparently, the diagnostic landscape is changing, albeit slowly, and the combination of newly identified infectious etiologies and the availability of innovative technologies has now opened new avenues for modernizing clinical microbiology. However, the improvement of microbial antibiotic susceptibility testing is still lagging behind. In this review we aim to sketch the most recent developments in laboratory-based clinical bacteriology and to provide an overview of emerging novel diagnostic approaches.

Yield of nat screening in Slovenia

Background: Safe blood supply is the main goal of blood transfusion services. Nucleic Acid Amplification Technologies (NAT) are the methods for direct detection of viral genoms in various biological samples. Introduction of NAT blood screening reduces the risk for transfusion transmitted diseases and improves the safety. Methods and Results: In Slovenia NAT screening with PCR (Polymerase Chain Reaction) method for testing pools of samples for hepatitis C virus (HCV) RNA was implemented in 2000. The screening is mandatory for all donations. Until 2007, 590,000 donated units had been screened and there were no HCV RNA-only positive result.In 2007, the testing was expanded to three viruses: HCV, hepatitis B virus (HBV) and human immunodeficiency virus (HIV). The testing method was TMA (Transcription Mediated Amplification) performed on individual donations. In five-year period, 458,000 blood units were screened and two window-period donations positive for HCV were detected (yield 1:524,000). HBV DNA-only was detected in 29 units. All the cases were donors with a resolved HBV infection, in one case the recent one, in others the occult hepatitis B infection (OBI) was confirmed. The yield of NAT testing for HBV is 1:15,600 units. There were no donations with HIV 1 RNA-only result. In all 5 HIV positive units in this period, anti-HIV antibodies were present as well. The yield of HIV NAT-only is thus 0:460,000. Conclusion: The implementation of NAT screening for three viruses has improved the blood safety. In the five-year period, 31 infectious units that were NAT–only positive were eliminated from the stock.

The detection of occult HBV infection in patients with HBsAg negative pattern by real-time PCR method

AimDiagnostic problems may be encountered in Hepatitis B virus (HBV) infections by serological tests and HBV DNA can be detectable in plasma and liver tissue while the HBsAg test is negative. This situation can be defined as occult or isolated Anti-HBc infections. Occult HBV infections may be divided into two categories by using hepatitis markers. One of them being that all hepatitis markers are negative and the other situation is having Anti-HBc +/- and Anti-HBs+patterns. These situations can be seen in isolated Anti-HBc cases. MethodIn this study, we aimed to detect the ratio of occult HBV infections by investigating HBV DNA in four different groups. These groups are: (1) 20 isolated Anti-HBc positive individuals, (2) 23 individuals naturally immune to HBV infection, (3) 20 individuals with seronegative hepatitis markers and high ALT levels, and (4) 23 vaccinated individuals against HBV. In order to detect HBV DNA the real-time PCR kit (QIAGEN, Artus HBV RG PCR Kit, Germany) with high analytical sensitivity (⩽3.8IU/ml) was used. ResultsThe reliability of the molecular methods was assessed by increasing the quantitation standards of internal, external and also positive controls. No HBV DNA was detected in any of the 86 individuals consisting of four study groups. ConclusionIn conclusion, we did not detect occult HBV infection in our four study groups by using a high sensitivity real-time (RT) PCR method, while occult HBV infections with various frequencies were detected in other large, serial international studies in which highly sensitive analytical molecular methods were used. Although we also used a high standard molecular kit to detect occult HBV infections, we suggest that the reason for the absence of detection of occult HBV infections may be due to the small number of cases included in this study. However, it was assumed that the use of a nucleic acid amplification technology (NAT) with high analytical sensitivity in blood banks to prevent HBV transmission by blood transfusion is controversial due to both costs and diagnostic efficacy and for this reason we suggest that it will be useful to perform large serial studies regarding occult HBV infections in the future.

Nucleic acid testing for tuberculosis at the point-of-care in high-burden countries

Early diagnosis of tuberculosis (TB) facilitates appropriate treatment initiation and can limit the spread of this highly contagious disease. However, commonly used TB diagnostic methods are slow, often insensitive, cumbersome and inaccessible to most patients in TB endemic countries that lack necessary resources. This review discusses nucleic acid amplification technologies, which are being developed for rapid near patient TB diagnosis, that are in the market or undergoing clinical evaluation. They are based on PCR or isothermal methods and are implemented as manual assays or partially/fully integrated instrument systems, with associated tradeoffs between clinical performance, cost, robustness, quality assurance and usability in remote settings by minimally trained personnel. Unmet needs prevail for the identification of drug-resistant TB and for TB diagnosis in HIV-positive and pediatric patients.

The distribution of hepatitis B virus genotypes in Thailand

Phylogenetic analysis was performed on hepatitis B virus (HBV) strains obtained from 86 hepatitis B surface antigen (HBsAg) positive donors from Thailand originating throughout the country. Based on the S gene, 87.5% of strains were of genotype C while 10.5% were of genotype B, with all genotype B strains obtained from patients originating from the central or the south Thailand. No genotype B strains were found in the north of Thailand. Surprisingly, one patient was infected with a genotype H strain while another patient was infected with a genotype G strain. Complete genome sequencing and recombination analysis identified the latter as being a genotype G and C2 recombinant with the breakpoint around nucleotide position 700. The origin of the genotype G fragment was not identifiable while the genotype C2 fragment most likely came from strains circulating in Laos or Malaysia. The performance of different HBsAg diagnostic kits and HBV nucleic acid amplification technology (NAT) was evaluated. The genotype H and G/C2 recombination did not interfere with HBV detection.

Evaluation of two, commercial, multi‐dye, nucleic acid amplification technology tests, for HBV/HCV/HIV‐1/HIV‐2 and B19V/HAV, for screening blood and plasma for further manufacture

The cobas TaqScreen MPX Test, version 2.0, a multiplex, multi-dye nucleic acid amplification technology (NAT) test from Roche was evaluated by two European Blood Banks, the German Red Cross Blood Donor Service, Frankfurt, Germany and Centro de Hemoterapia y Hemodonación de Castilla y León, Valladolid, Spain. In addition, the cobas TaqScreen DPX Test was evaluated for the simultaneous detection and quantitation of parvovirus B19 and the detection of hepatitis A virus (HAV). The performances of the two tests were evaluated regarding the analytical sensitivity, the reproducibility of the tests using samples containing low concentrations of each virus and cross-contamination using samples containing high titres of virus. The analytical sensitivity of the MPX Test, version 2.0, obtained by the German Red Cross Blood Donor Service was 1·1, 3·9 and 43·3 IU/ml for HBV, HCV and HIV-1, respectively. The comparable analytical sensitivity at Centro de Hemoterapia y Hemodonación de Castilla y León was 3·5, 17·6 and 50·6 IU/ml for HBV, HCV and HIV-1, respectively. The analytical sensitivity of the DPX test determined by the German Red Cross Blood Donor Service was 0·6 and 3·8 IU/ml for HAV and B19. These multiplex and multi-dye blood screening assays represent a flexible NAT screening system for mini-pools between 6 and 96 samples per pool and fulfil all requirements of the European Pharmacopoeia for HCV and B19V testing of plasma for fractionation. The inclusion of a new multi-dye technology means discriminatory assays are no longer required for either test thus improving workflow, turn-around time and minimize the risk of obtaining a reactive result for which the virus cannot be identified.

Chlamydia and Gonorrhea Screening Using Urine-Based Nucleic Acid Amplification Testing Among Males Entering New York City Jails

This article describes a pilot screening program to detect Chlamydia trachomatis (Ct) and Neisseria gonorrhoeae (Ng) sexually transmitted infections (STIs) in adolescent and adult males newly incarcerated in New York City jails using urine-based nucleic acid amplification technology (NAAT). Between December 8 and 22, 2003, 2,417 males were tested; 162 (6.7%) were found positive for Ct and/or Ng STIs, with 138 (86.8%) exhibiting no STI signs or symptoms and 102 (63%) treated prior to jail release. Younger age, positive urine leukocyte esterase test, and ≥11 recent sex partners were predictors of STI. Urine-based screening and treatment was feasible in this setting and identified STI that would otherwise have been undetected. Jails may thus be important venues for targeted male STI screening.

High false‐negative rate of anti‐HCV among Egyptian patients on regular hemodialysis

Routine serological testing for hepatitis C virus (HCV) infection among hemodialysis (HD) patients is currently recommended. A dilemma existed on the value of serology because some investigators reported a high rate of false-negative serologic testing. In this study, we aimed to detect the false-negative rate of anti-HCV among Egyptian HD patients. Seventy-eight HD patients, negative for anti-HCV, anti-HIV, and hepatitis B surface antigen, were tested for HCV RNA by reverse transcriptase polymerase chain reaction (RT-PCR). In the next step, the viral load was quantified by real-time PCR in RT-PCR-positive patients. Risk factors for HCV infection, as well as clinical and biochemical indicators of liver disease, were compared between false-negative and true-negative anti-HCV HD patients. The frequency of false-negative anti-HCV was 17.9%. Frequency of blood transfusion, duration of HD, dialysis at multiple centers, and diabetes mellitus were not identified as risk factors for HCV infection. The frequency of false-negative results had a linear relation to the prevalence of HCV infection in the HD units. Timely identification of HCV within dialysis units is needed in order to lower the risk of HCV spread within the HD units. The high false-negative rate of anti-HCV among HD patients in our study justifies testing of a large scale of patients for precious assessment of effectiveness of nucleic acid amplification technology testing in screening HD patient.

Transfusion transmission of HCV, a long but successful road map to safety

Improved blood banking practices and the development and implementation of increasingly sensitive serological and nucleic acid amplification technology assays for screening donors for HCV over the past few decades have helped minimize the residual risk from transfusion transmitted HCV in the developed world. Furthermore, studies of transfusion transmitted infections and of donors identified as infected by routine screening have provided significant insights into HCV transmission, epidemiology and pathogenesis. However, transfusion transmission of HCV is still a significant route of infection in the developing world. Key preventive mechanisms to ensure safe blood include elimination of paid donors and development of national donor pools comprising volunteer repeat blood donors, combined with implementation of standardized and maximally sensitive screening assays for HCV. There is also a need to develop up-to-date data on HCV disease burden on a global scale, in part, derived from systematic screening of donors for HCV infection. We suggest the creation of blood donor databases and specimen repositories, both at national and international levels, to facilitate epidemiological surveillance and pathogenesis and treatment studies in the future.

Isothermal nucleic acid amplification technologies for point-of-care diagnostics: a critical review

Nucleic Acid Testing (NAT) promises rapid, sensitive and specific diagnosis of infectious, inherited and genetic disease. The next generation of diagnostic devices will interrogate the genetic determinants of such conditions at the point-of-care, affording clinicians prompt reliable diagnosis from which to guide more effective treatment. The complex biochemical nature of clinical samples, the low abundance of nucleic acid targets in the majority of clinical samples and existing biosensor technology indicate that some form of nucleic acid amplification will be required to obtain clinically relevant sensitivities from the small samples used in point-of-care testing (POCT). This publication provides an overview and thorough review of existing technologies for nucleic acid amplification. The different methods are compared and their suitability for POCT adaptation are discussed. Current commercial products employing isothermal amplification strategies are also investigated. In conclusion we identify the factors impeding the integration of the methods discussed in fully automated, sample-to-answer POCT devices.

A specific statistical model and algorithm related to the detection of Mollicutes in contaminated biological samples by Real-Time Transcription Mediated Amplification

Among all nucleic acid amplification technologies, Real-Time Transcription Mediated Amplification (Real-Time TMA) is an isothermal method that can amplify RNA targets a billion-fold in less than one hour's time. By using this method, a new assay was developed for detecting the presence of Mollicutes in mammalian cell cultures and biologics. Production of amplicons is monitored in real time by measuring continuously a fluorescence signal during the reaction. The shape of this signal curve is a sigmoid, where an initial baseline phase precedes an exponential phase ending with a maximum followed by a linear decreasing phase.The aim of this study was to develop a curve-analysis tool to unambiguously assign a Mollicutes positive or negative status to a biological sample. In this context, a statistical analysis of the data combined with the selection of the best predictors allowed the development of an algorithm which removes erroneous data and applies the best predictors to generate the Mollicutes status prediction.Our results demonstrate that this algorithm assigns positive/negative/invalid status coming from Real-Time TMA fluorescence signal analysis with a confidence (true predictions) in the results close to 100%.