Nucleic Acid Amplification Assays Research Articles

GMO detection in food and feed through screening by visual loop-mediated isothermal amplification assays.

Isothermal DNA/RNA amplification techniques are the primary methodology for developing on-spot rapid nucleic acid amplification assays, and the loop-mediated isothermal amplification (LAMP) technique has been developed and applied in the detection of foodborne pathogens, plant/animal viruses, and genetically modified (GM) food/feed contents. In this study, one set of LAMP assays targeting on eight frequently used universal elements, marker genes, and exogenous target genes, such as CaMV35S promoter, FMV35S promoter, NOS, bar, cry1Ac, CP4 epsps, pat, and NptII, were developed for visual screening of GM contents in plant-derived food samples with high efficiency and accuracy. For these eight LAMP assays, their specificity was evaluated by testing commercial GM plant events and their limits of detection were also determined, which are 10 haploid genome equivalents (HGE) for FMV35S promoter, cry1Ac, and pat assays, as well as five HGE for CaMV35S promoter, bar, NOS terminator, CP4 epsps, and NptII assays. The screening applicability of these LAMP assays was further validated successfully using practical canola, soybean, and maize samples. The results suggested that the established visual LAMP assays are applicable and cost-effective for GM screening in plant-derived food samples.

Precision chemical heating for diagnostic devices.

Decoupling nucleic acid amplification assays from infrastructure requirements such as grid electricity is critical for providing effective diagnosis and treatment at the point of care in low-resource settings. Here, we outline a complete strategy for the design of electricity-free precision heaters compatible with medical diagnostic applications requiring isothermal conditions, including nucleic acid amplification and lysis. Low-cost, highly energy dense components with better end-of-life disposal options than conventional batteries are proposed as an alternative to conventional heating methods to satisfy the unique needs of point of care use.

Mycoplasma genitalium: update on diagnosis, treatment and resistance

Mycoplasma genitalium is a sexually transmitted bacterium and a major causative agent of non-chlamydial non-gonococcal urethritis (NGU) with approximately 20–35% of NGU cases attributed to this infection. In women it has been associated with cervicitis, endometritis, and pelvic inflammatory disease. There are a limited number of commercial nucleic acid amplification assays available for its detection, however none are yet FDA approved and most laboratories are either using these commercial or in-house developed assays to provide diagnostic results. Mycoplasma genitalium infection is primarily treated with macrolide antibiotic azithromycin. However, it has been shown that use of azithromycin in the treatment of NGU and other infections has resulted in increased macrolide resistance, attributed to mutations on the 23S rRNA gene. The currently recommended second line treatment, moxifloxacin, is a fluoroquinolone antibiotic. Moxifloxacin is relatively expensive, can result in side effects and is not suitable in pregnant women, which limits its widespread usage. Resistance to moxifloxacin has also been reported with its increasing use. Detection, appropriate treatment and test of cure of M. genitalium are important in control of this infection.

Methods of rapid diagnosis for the etiology of meningitis in adults.

Infectious meningitis may be due to bacterial, mycobacterial, fungal or viral agents. Diagnosis of meningitis must take into account numerous items of patient history and symptomatology along with regional epidemiology and basic cerebrospinal fluid testing (protein, etc.) to allow the clinician to stratify the likelihood of etiology possibilities and rationally select additional diagnostic tests. Culture is the mainstay for diagnosis in many cases, but technology is evolving to provide more rapid, reliable diagnosis. The cryptococcal antigen lateral flow assay (Immuno-Mycologics) has revolutionized diagnosis of cryptococcosis and automated nucleic acid amplification assays hold promise for improving diagnosis of bacterial and mycobacterial meningitis. This review will focus on a holistic approach to diagnosis of meningitis as well as recent technological advances.

Outbreaks of Kingella kingae infections in daycare facilities.

During the past decade, transmission of the bacterium Kingella kingae has caused clusters of serious infections, including osteomyelitis, septic arthritis, bacteremia, endocarditis, and meningitis, among children in daycare centers in the United States, France, and Israel. These events have been characterized by high attack rates of disease and prevalence of the invasive strain among asymptomatic classmates of the respective index patients, suggesting that the causative organisms benefitted from enhanced colonization fitness, high transmissibility, and high virulence. After prophylactic antibacterial drugs were administered to close contacts of infected children, no further cases of disease were detected in the facilities, although test results showed that some children still carried the bacterium. Increased awareness of this public health problem and use of improved culture methods and sensitive nucleic acid amplification assays for detecting infected children and respiratory carriers are needed to identify and adequately investigate outbreaks of K. kingae disease.

Potential of two nucleic acid amplification assays for quantifying mycobacterial load in respiratory and non-respiratory specimens: a prospective study

Quantification of bacillary load plays a critical prognostic role in tuberculosis patients. This study evaluated the potential of the Cepheid GeneXpert (GX) MTB/RIF and the BD ProbeTec system as quantitative assays. The time to positivity (TTP) measured by the Mycobacterial Growth Index Tube system was compared to the cycle threshold (Ct) of GX MTB/RIF and the Metric Other than Acceleration (MOTA) scores generated by the ProbeTec system. Out of 714 samples examined, 44 culture confirmed cases were identified. The Ct values in 21 respiratory samples showed a high linear fit with the TTP in liquid culture (Spearman's correlation coefficient r = 0.9, P value < 0.0001), which was not the case in 23 non-respiratory samples. In both types of specimens, the MOTA scores did not correlate with the TTP in liquid culture. This indicates the suitability of GX as a quantitative measurement of mycobacterial load in respiratory but not non-respiratory specimens.

Letter to the Editor: Another Look: Is There a Flaw to Current Hip Septic Arthritis Diagnostic Algorithms?

To the editor, I read with interest the article by Uzoigwe in which the author critically assessed the current diagnostic algorithms recommended for differentiating between pediatric septic arthritis and transient synovitis in children [3]. The six evaluated studies show wide variation in the risk posed by each of the clinical (a body temperature ≥ 38.5°C and inability to bear weight) and laboratory (white blood cell count > 12 × 109/L and erythrocyte sedimentation rate [ESR] ≥ 40 mm/hour) criteria included in the predictive scoring. Uzoigwe identified collinearity of the inflammatory predictors, inadequate sample size, and enrollment of an insufficient number of children with septic arthritis, as possible explanations for the incongruent between-studies results [3]. It should be noted, however, that the performance of any criterion for identifying diseased patients not only depends on the intrinsic properties of the investigated variable, but also on the reliability of the gold standard comparator. In the case of septic arthritis, the issue is complicated by the fact that, on average, in 33% (range, 16%–60%) of children with presumptive joint infections, the disease remains bacteriologically unconfirmed [2]. In full agreement with this concept, only 113 of 238 (47.5%) patients deemed to have septic arthritis in the studies reviewed by Uzoigwe grew an irrefutable pathogen in joint aspirates or blood cultures [3]. Therefore, children with symptoms of hip inflammation and synovial fluid (white blood cell count > 50 × 109/L), but no positive isolation of bacteria from a normally sterile site were included in the “septic arthritis” category. In recent years, Kingella kingae, a fastidious gram-negative member of the normal oropharyngeal flora has been recognized as the most common etiology of septic arthritis in children aged 6–36 months [4]. The organism requires inoculation of synovial fluid exudates into blood culture vials or the use of sensitive nucleic acid amplification assays for its detection [4], and it has been demonstrated that when these novel bacteriological methods are not used, many cases of joint infections caused by Kingella kingae will be missed and classified as “culture negative” septic arthritis [1, 4]. Additionally, children with Kingella kingae infections of the skeletal system frequently present with a mild clinical picture, requiring a high index of suspicion: the mean ± standard deviation body temperature is 38.4 ± 0.9°C, mean ESR is 44.1 ± 24.8 mm/hour, 21% of children have a blood white blood cell count < 12 × 109/L, and 23% have less than 50 × 109/L white blood cells in the synovial fluid aspirate, mimicking transient synovitis [1, 4]. Because none of the studies evaluated by Uzoigwe employed blood culture vials nor nucleic acid amplification tests, it is entirely possible that many children with K kingae septic arthritis presenting with mild clinical symptoms, unimpressive laboratory markers of inflammation, and negative cultures, could have been misclassified as cases of “transient synovitis.” Although Uzoigwe is correct in that well-controlled and adequately powered studies are in need to constructing a reliable diagnostic algorithm aimed to differentiate between pediatric joint infection and benign synovitis, use of improved bacteriological methods is also crucial for defining a more accurate gold standard for pediatric septic arthritis.

Comparison of Nucleic Acid Amplification Assays with BD Affirm VPIII for Diagnosis of Vaginitis in Symptomatic Women

A commercially available, nonamplified, nucleic acid probe-based test system (BD Affirm VPIII) was compared with nucleic acid amplification (NAA)-based assays for determining the etiology of vaginitis in a cohort of 323 symptomatic women. First, a semiquantitative, multiplexed PCR assay (BV-PCR) and the Affirm VPIII Gardnerellavaginalis test were compared with a unified bacterial-vaginosis (BV) reference standard incorporating both Nugent Gram stain scores and Amsel clinical criteria. In the evaluable population of 305 patients, BV-PCR was 96.9% (191/197) sensitive and 92.6% specific (100/108) for BV, while Affirm VPIII was 90.1% sensitive (179/197) and 67.6% specific (73/108). Second, a multiplexed PCR assay detecting Candida albicans and Candida glabrata (CAN-PCR) was compared with the Affirm VPIII Candida test using a reference standard for vulvovaginal candidiasis (VVC) of yeast culture plus exclusion of alternate vaginitis etiologies. In the population evaluated (n = 102), CAN-PCR was 97.7% sensitive (42/43) and 93.2% specific (55/59) and Affirm VP III was 58.1% sensitive (25/43) and 100% specific (59/59) for VVC. Finally, the results of a commercial NAA test (GenProbe Aptima Trichomonas vaginalis assay; ATV) for T. vaginalis were compared with the Affirm VPIII Trichomonas vaginalis test. In the absence of an independent reference standard for trichomonal vaginitis (TV), a positive result in either assay was deemed to represent true infection. In the evaluable cohort of 388 patients, the sensitivity of ATV was 98.1% (53/54) versus 46.3% (25/54) for Affirm VPIII. The diagnostic accuracy of the combined NAA-based test construct was approximately 20 to 25% higher than that of the Affirm VPIII when modeled in populations with various prevalences of infectious vaginitis.

A Handheld Point-of-Care Genomic Diagnostic System

The rapid detection and identification of infectious disease pathogens is a critical need for healthcare in both developed and developing countries. As we gain more insight into the genomic basis of pathogen infectivity and drug resistance, point-of-care nucleic acid testing will likely become an important tool for global health. In this paper, we present an inexpensive, handheld, battery-powered instrument designed to enable pathogen genotyping in the developing world. Our Microfluidic Biomolecular Amplification Reader (µBAR) represents the convergence of molecular biology, microfluidics, optics, and electronics technology. The µBAR is capable of carrying out isothermal nucleic acid amplification assays with real-time fluorescence readout at a fraction of the cost of conventional benchtop thermocyclers. Additionally, the µBAR features cell phone data connectivity and GPS sample geotagging which can enable epidemiological surveying and remote healthcare delivery. The µBAR controls assay temperature through an integrated resistive heater and monitors real-time fluorescence signals from 60 individual reaction chambers using LEDs and phototransistors. Assays are carried out on PDMS disposable microfluidic cartridges which require no external power for sample loading. We characterize the fluorescence detection limits, heater uniformity, and battery life of the instrument. As a proof-of-principle, we demonstrate the detection of the HIV-1 integrase gene with the µBAR using the Loop-Mediated Isothermal Amplification (LAMP) assay. Although we focus on the detection of purified DNA here, LAMP has previously been demonstrated with a range of clinical samples, and our eventual goal is to develop a microfluidic device which includes on-chip sample preparation from raw samples. The µBAR is based entirely around open source hardware and software, and in the accompanying online supplement we present a full set of schematics, bill of materials, PCB layouts, CAD drawings, and source code for the µBAR instrument with the goal of spurring further innovation toward low-cost genetic diagnostics.

P3.283 Trends and Associations of Neisseria Gonorrhoeae Infection in Men and Women with Genital Discharge Syndromes in Johannesburg, South Africa

BackgroundAnnual STI aetiological surveys undertaken at a primary healthcare facility in Johannesburg, South Africa, each year have determined the prevalence of gonorrhoea amongst men with male urethral discharge syndrome (MUDS)...

P5.062 An Easy to Use Real Time PCR Test For Chlamydia Trachomatis and Neisseria Gonorrhoeae Providing Results That Can Guide Treatment Choices Before the Patient Leaves the Clinic

BackgroundNew diagnostics for Chlamydia trachomatis (CT) and Neisseria gonorrhoeae (NG) that can provide accurate, rapid, and timely results are urgently needed so that patients can be treated at the time...

High-Risk Human Papillomavirus Messenger RNA Testing in Physician- and Self-Collected Specimens for Cervical Lesion Detection in High-Risk Women, Kenya

Little is known about the performance of physician-versus self-collected specimens for high-risk human papillomavirus (hrHPV) messenger RNA (mRNA) testing or risk factors for hrHPV mRNA positivity in physician- versus self-collected specimens. We compared the performance of hrHPV mRNA testing of physician- and self-collected specimens for detecting cytological high-grade squamous intraepithelial lesions or more severe (≥HSIL) and examined risk factors for hrHPV mRNA positivity in female sex workers in Nairobi. From 2009 to 2011, 344 female sex workers participated in this cross-sectional study. Women self-collected a cervicovaginal specimen. A physician conducted a pelvic examination to obtain a cervical specimen. Physician- and self-collected specimens were tested for hrHPV mRNA and sexually transmitted infections using APTIMA nucleic acid amplification assays (Hologic/Gen-Probe Incorporated, San Diego, CA). Cervical cytology was conducted using physician-collected specimens and classified according to the Bethesda criteria. Overall hrHPV mRNA prevalence was similar in physician- and self-collected specimens (30% vs. 29%). Prevalence of ≥HSIL was 4% (n = 15). Overall sensitivity of hrHPV testing for detecting ≥HSIL was similar in physician-collected (86%; 95% CI, 62%-98%; 13 cases detected) and self-collected specimens (79%; 95% CI, 55%-95%; 12 cases detected). Overall specificity of hrHPV mRNA for ≥HSIL was similar in both physician-collected (73%; 95% CI, 68%-79%) and self-collected (75%; 95% CI, 70%-79%) specimens. High-risk HPV mRNA positivity in both physician- and self-collected specimens seemed higher in women who were younger (<30 years), had Trichomonas vaginalis or Mycoplasma genitalium infections, or had more than 8 years of educational attainment. Self-collected specimens for hrHPV mRNA testing seemed to have similar sensitivity and specificity as physician-collected specimens for the detection of ≥HSIL among high-risk women.

Drug-resistant tuberculous meningitis

Drug-resistant tuberculosis, including drug-resistant tuberculous meningitis, is an emerging health problem in many countries. An association with Beijing strains and drug resistance-related mutations, such as mutations in katG and rpoB genes, has been found. The pathology, clinical features and neuroimaging characteristics of drug-resistant tuberculous meningitis are similar to drug-responsive tuberculous meningitis. Detection of mycobacteria in cerebrospinal fluid (CSF) by conventional methods (smear examination or culture) is often difficult. Nucleic acid amplification assays are better methods owing to their rapidity and high sensitivity. The Xpert® MTB/RIF assay (Cepheid, CA, USA) is a fully-automated test that has also been found to be effective for CSF samples. Treatment of multidrug-resistant tuberculous meningitis depends on the drug susceptibility pattern of the isolate and/or the previous treatment history of the patient. Second-line drugs with good penetration of the CSF should be preferred. Isoniazid monoresistant disease requires addition of another drug with better CSF penetration. Drug-resistant tuberculous meningitis is associated with a high mortality. HIV infected patients with drug-resistant tuberculous meningitis have severe clinical manifestations with exceptionally high mortality. Prevention of tuberculosis is the key to reduce drug-resistant tuberculous meningitis.

Rapid Antigen-Based Testing for Respiratory Syncytial Virus: Moving Diagnostics from Bench to Bedside?

Respiratory syncytial virus (RSV) is the most important cause of infantile bronchiolitis and pneumonia. It is ubiquitous, with most children acquiring their primary infection within the first year of life and with subsequent reinfection occurring in all age groups. Clinically, RSV is virtually indistinguishable from other viral respiratory infections. Traditionally, the microbiologic diagnosis of RSV has been based on moderate to complex techniques performed in a laboratory (cell culture, nucleic acid amplification and immunofluorescence assays); however, rapid antigen-detection tests offer potential advantages associated with point-of-care testing. This review seeks to familiarize the readers with RSV rapid antigen-detection tests, describe their performance characteristics and comment on their strengths and weaknesses. The authors will discuss the impact of rapid RSV testing on clinical practice, with a look to the future of what the field ultimately requires of a point-of-care diagnostic technique.

Dielectrophoretic liquid actuation on nano-textured super hydrophobic surfaces

Nano-textured super hydrophobic (SH) surfaces can achieve very high droplet contact angles (typically >150°) and are furthermore associated with reduced surface adsorption of enzymes and other macro-molecules. The advantages of such SH surfaces have already been explored for electrostatic based digital microfluidic (DMF) devices where the high CA and the reduced surface adsorption largely benefits the handling and manipulation of complex bio-chemical samples/reagents. Liquid dielectrophoresis (LDEP) based digital microfluidic (DMF) technology has demonstrated its capability for achieving combinatorial assays on standard hydrophobic surfaces. However, such devices suffer from similar issues as DMF devices when it comes to handling bio-reagents/samples where off-chip sample handling steps (washing/buffer exchange) are often required prior to the on-chip bio-assay. In this work, we have optimized a comprehensive fabrication protocol to develop a nano-textured SH surface topology for LDEP device applications. The static and transient behavior of LDEP actuation over the SH surface has been investigated using an improved lumped model and furthermore experimentally verified using specifically tailored liquid samples. The developed SH surface has also been analyzed for liquid actuation of TAQ DNA polymerase enzyme which is a key component of most nucleic acid amplification assays. The performance of LDEP actuation of tailored liquid samples is analyzed and compared for both hydrophobic and SH surfaces. The reported work demonstrates that specific textured SH surfaces can successfully achieve reliable LDEP actuation and furthermore dispense/manipulate TAQ DNA polymerase enzyme and various other aqueous samples.

Instrument-free exothermic heating with phase change temperature control for paper microfluidic devices.

Many infectious diseases, as well as some cancers, that affect global health are most accurately diagnosed through nucleic acid amplification and detection. There is a great need to simplify nucleic acid-based assay systems for use in global health in low-resource settings as well as in settings that do not have convenient access to laboratory staff and equipment such as doctors' offices and home care settings. In developing countries, unreliable electric power, inadequate supply chains, and lack of maintenance for complex diagnostic instruments are all common infrastructure shortfalls. Many elements of instrument-free, disposable, nucleic acid amplification assays have been demonstrated in recent years. However, the problem of instrument-free, low-cost, temperature-controlled chemical heating remains unsolved. In this paper we present the current status and results of work towards developing disposable, low-cost, temperature-controlled heaters designed to support isothermal nucleic acid amplification assays that are integrated with a two-dimensional paper network. Our approach utilizes the heat generated through exothermic chemical reactions and controls the heat through use of engineered phase change materials to enable sustained temperatures required for nucleic acid amplification. By selecting appropriate exothermic and phase change materials, temperatures can be controlled over a wide range, suitable for various isothermal amplification methods, and maintained for over an hour at an accuracy of +/- 1°C.

Performance of the Cepheid CT/NG Xpert Rapid PCR Test for Detection of Chlamydia trachomatis and Neisseria gonorrhoeae

Tests for Chlamydia trachomatis and Neisseria gonorrhoeae, which can provide results rapidly to guide therapeutic decision-making, offer patient care advantages over laboratory-based tests that require several days to provide results. We compared results from the Cepheid GeneXpert CT/NG (Xpert) assay to results from two currently approved nucleic acid amplification assays in 1,722 female and 1,387 male volunteers. Results for chlamydia in females demonstrated sensitivities for endocervical, vaginal, and urine samples of 97.4%, 98.7%, and 97.6%, respectively, and for urine samples from males, a sensitivity of 97.5%, with all specificity estimates being ≥ 99.4%. Results for gonorrhea in females demonstrated sensitivities for endocervical, vaginal, and urine samples of 100.0%, 100.0%, and 95.6%, respectively, and for urine samples from males, a sensitivity of 98.0%, with all estimates of specificity being ≥ 99.8%. These results indicate that this short-turnaround-time test can be used to accurately test patients and to possibly do so at the site of care, thus potentially improving chlamydia and gonorrhea control efforts.

Human Herpesvirus 6, 7 and 8 in Solid Organ Transplantation

Human Herpesvirus 6, 7 and 8 in Solid Organ Transplantation

P285: Comparison of two nucleic acid amplification assays, the probetec et assay and xpert mtb/rif assay, for detection of mycobacterium tuberculosis in respiratory specimens

Early diagnosis and management of TB is critical to reduce TB transmission in communities and health care facilities. In developing countries, TB diagnosis is largely based on smear microscopy of sputum samples. Nucleic acid amplification tests (NAATs) have greatly facilitated TB testing by lowering the turnaround time to a day or less from two or more weeks for a culture result.

Printing the Next Generation of Point-of-Care Diagnostic Tests

Two-dimensional paper networks (2DPNs) area new set of devices allow complex chemical processing in a very low-cost format. We have, for the last 5 years, been learning how to translate what we have learned about point-of-care diagnostic technologies in conventional microfluidics into the language of porous media. The wicking of fluids in porous materials (like paper, nitrocellulose membranes, etc.) allows us to discard pumps, which permits great savings in complexity and cost, and the potential to perform complex tests without any permanent instruments. However, there are many physical and chemical differences between open ducts and porous media. We have put a good deal of effort into understanding the performance and design rules of simple paper systems. Furthermore, application of reagents to the papers in liquid and dry form is central to device development, testing, and economical manufacture. Currently, the primary applications for this technology in our lab are highlysensitive multiplexed immunoassays and multiplexed isothermal nucleic acid amplification assays. All assays are designed with visible optical readout that can be captured and quantified using camera-equipped cellular phones.