Optimization of Turnaround Time for Group A Streptococcus PCR.
ABSTRACT: The use of some nucleic acid amplification tests (NAATs) for the diagnosis of group A Streptococcus (GAS) pharyngitis allows laboratories to adopt single-tiered testing without reflex culture. However, centralization may delay the delivery of actionable information to the bedside, particularly in the outpatient setting. We describe two novel workflows at our institution and their effect on in-lab turnaround time (TAT) at a tertiary care microbiology lab. Laboratory records were extracted, and relevant data were analyzed after the implementation of qualitative in vitro diagnostic testing for GAS with the Xpert Xpress Strep A assay, performed using the GeneXpert Infinity-48s. Workflow optimization steps studied included: (i) direct specimen submission to the microbiology laboratory via the pneumatic tube system and (ii) autoverification of GAS NAAT results in the laboratory information system. Between April 2018 and October 2018, 2,595 unique specimens were tested for GAS by PCR. Of these, 2,523 were included in the final analysis. Linear regression established that the total in-lab TAT was significantly reduced by direct specimen submission to the microbiology laboratory, autoverification, and processing during the night shift. We describe two workflow optimization methods that reduced the in-lab TAT for GAS NAAT. Although microbiology labs historically use manual processes, the advent of total laboratory automation and the adoption of on-demand NAATs will allow for more streamlined processing of microbiology specimens. It may be beneficial to consider instrument interfacing and specimen processing optimization during the early phases of implementation planning for NAATs in the microbiology laboratory.
Project description:BackgroundThe World Health Organization (WHO) recommends parasite-based diagnosis of malaria. In recent years, there has been surge in the use of various kinds of nucleic-acid amplification based tests (NAATs) for detection and identification of Plasmodium spp. to support clinical care in high-resource settings and clinical and epidemiological research worldwide. However, these tests are not without challenges, including lack (or limited use) of standards and lack of reproducibility, due in part to variation in protocols amongst laboratories. Therefore, there is a need for rigorous quality control, including a robust external quality assessment (EQA) scheme targeted towards malaria NAATs. To this effect, the WHO Global Malaria Programme worked with the UK National External Quality Assessment Scheme (UK NEQAS) Parasitology and with technical experts to launch a global NAAT EQA scheme in January 2017.MethodsPanels of NAAT EQA specimens containing five major species of human-infecting Plasmodium at various parasite concentrations and negative samples were created in lyophilized blood (LB) and dried blood spot (DBS) formats. Two distributions per year were sent, containing five LB and five DBS specimens. Samples were tested and validated by six expert referee laboratories prior to distribution. Between 37 and 45 laboratories participated in each distribution and submitted results using the online submission portal of UK NEQAS. Participants were scored based on their laboratory’s stated capacity to identify Plasmodium species, and individual laboratory reports were sent which included performance comparison with anonymized peers.ResultsAnalysis of the first three distributions revealed that the factors that most significantly affected performance were sample format (DBS vs LB), species and parasite density, while laboratory location and the reported methodology used (type of nucleic acid extraction, amplification, or DNA vs RNA target) did not significantly affect performance. Referee laboratories performed better than non-referee laboratories.ConclusionsGlobally, malaria NAAT assays now inform a range of clinical, epidemiological and research investigations. EQA schemes offer a way for laboratories to assess and improve their performance, which is critical to safeguarding the reliability of data and diagnoses especially in situations where various NAAT methodologies and protocols are in use.
Project description:<h4>Background</h4>Nucleic acid amplification tests (NAATs) are increasingly being used as diagnostic tools for soil-transmitted helminths (STHs; Ascaris lumbricoides, Trichuris trichiura, Necator americanus, Ancylostoma duodenale and A. ceylanicum), Strongyloides stercoralis and Schistosoma in human stool. Currently, there is a large diversity of NAATs being applied, but an external quality assessment scheme (EQAS) for these diagnostics is lacking. An EQAS involves a blinded process where test results reported by a laboratory are compared to those reported by reference or expert laboratories, allowing for an objective assessment of the diagnostic performance of a laboratory. In the current study, we piloted an international EQAS for these helminths (i) to investigate the feasibility of designing and delivering an EQAS; (ii) to assess the diagnostic performance of laboratories; and (iii) to gain insights into the different NAAT protocols used.<h4>Methods and principal findings</h4>A panel of twelve stool samples and eight DNA samples was validated by six expert laboratories for the presence of six helminths (Ascaris, Trichuris, N. americanus, Ancylostoma, Strongyloides and Schistosoma). Subsequently this panel was sent to 15 globally dispersed laboratories. We found a high degree of diversity among the different DNA extraction and NAAT protocols. Although most laboratories performed well, we could clearly identify the laboratories that were poorly performing.<h4>Conclusions/significance</h4>We showed the technical feasibility of an international EQAS for the NAAT of STHs, Strongyloides and Schistosoma. In addition, we documented that there are clear benefits for participating laboratories, as they can confirm and/or improve the diagnostic performance of their NAATs. Further research should aim to identify factors that explain poor performance of NAATs.
Project description:<h4>Background</h4>Concerns about false-negative (FN) severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) nucleic acid amplification tests (NAATs) have prompted recommendations for repeat testing if suspicion for coronavirus disease 2019 (COVID-19) infection is moderate to high. However, the frequency of FNs and patient characteristics associated with FNs are poorly understood.<h4>Methods</h4>We retrospectively reviewed test results from 15 011 adults who underwent ≥1 SARS-CoV-2 NAATs; 2699 had an initial negative NAAT and repeat testing. We defined FNs as ≥1 negative NAATs followed by a positive NAAT within 14 days during the same episode of illness. We stratified subjects with FNs by duration of symptoms before the initial FN test (≤5 days versus >5 days) and examined their clinical, radiologic, and laboratory characteristics.<h4>Results</h4>Sixty of 2699 subjects (2.2%) had a FN result during the study period. The weekly frequency of FNs among subjects with repeat testing peaked at 4.4%, coinciding with peak NAAT positivity (38%). Most subjects with FNs had symptoms (52 of 60; 87%) and chest radiography (19 of 32; 59%) consistent with COVID-19. Of the FN NAATs, 18 of 60 (30%) were performed early (ie, ≤1 day of symptom onset), and 18 of 60 (30%) were performed late (ie, >7 days after symptom onset) in disease. Among 17 subjects with 2 consecutive FNs on NP NAATs, 9 (53%) provided lower respiratory tract (LRT) specimens for testing, all of which were positive.<h4>Conclusions</h4>Our findings support repeated NAATs among symptomatic patients, particularly during periods of higher COVID-19 incidence. The LRT testing should be prioritized to increase yield among patients with high clinical suspicion for COVID-19.
Project description:OBJECTIVE:We evaluated the performance of nucleic acid amplification tests (NAATs) using vaginal specimens in comparison to specimens from the cervix or urine in their ability to detect chlamydia and gonorrhoea infection in women based on patient infection status (PIS). DESIGN:Systematic review. DATA SOURCES:EMBASE and Ovid MEDLINE databases were searched through 3 October 2017. ELIGIBILITY CRITERIA FOR SELECTING STUDIES:We included studies that tested samples from the vagina and ?1?other site (cervix and/or urine) with ?2 NAATs for chlamydia and ?2 NAATs or 1 NAAT and culture for gonorrhoea for each site. DATA EXTRACTION AND SYNTHESIS:Performance is defined as the sensitivity of a NAAT using a specimen site and PIS of the patient. We assessed risk of bias using modified QUADAS-2. RESULTS:Nine publications met the inclusion criteria (eight for chlamydia; six for gonorrhoea) and were narratively reviewed. Pooled summary estimates were not calculated due to the variable methodology and PIS definitions. Tests performed on vaginal specimens accomplished similar performance to cervical and urine specimens for chlamydia (range of performance estimates: vaginal 65%-100%, cervical 59%-97%, urine 57%-100%) and gonorrhoea (vaginal 64%-100%, cervical 85%-100%, urine 67%-94%). Vaginal specimens were estimated to have a performance >80% for chlamydia and gonorrhoea infections in all but one study. CONCLUSIONS:Performance of the NAATs for chlamydia and gonorrhoea detection using vaginal specimens was similar to that of cervical and urine specimens relative to PIS. As vaginal samples have a higher acceptability and lower cost, the study can support clinical testing guidelines by providing evidence that vaginal samples are a suitable alternative to traditionally used specimens.
Project description:<b>Background: </b>Nucleic Acid Amplification Tests (NAATs) are the recommended test type for diagnosing Chlamydia trachomatis (chlamydia). However, less sensitive diagnostic methods-including direct immunofluorescence (IF) and enzyme-linked immunoassay (ELISA)-remain in use in lower resourced settings. We estimate the risk of pelvic inflammatory disease (PID) following undiagnosed infection in women tested with non-NAATs and estimate the health gain from using accurate diagnostic tests.<br><br><b>Methods and findings: </b>We used Denmark's national Chlamydia Study dataset to extract all chlamydia tests performed in women aged 15-34 years (1998-2001). Tests were categorised as non-NAAT (IF/ELISA) or NAAT and limited to each woman's first test in the study period. We linked test data to hospital presentations for PID within 12 months from the Danish National Patient Register. The study included 272,105 women with a chlamydia test, just under half (44.78%, n = 121,857) were tested using NAATs. Overall, 6.38% (n = 17,353) tested positive for chlamydia and 0.64% (n = 1,732) were diagnosed with PID within 12 months. The risk of PID following a positive chlamydia test did not differ by test type (NAAT 0.81% [95% CI 0.61-1.00], non-NAAT 0.78% [0.59-0.96]). The risk of PID following a negative test was significantly lower in women tested with NAATs compared to non-NAATs (0.55% [0.51-0.59] compared to 0.69% [0.64-0.73]; adjusted odds ratio (AOR) 0.83 [0.75-0.93]). We estimate that 18% of chlamydia infections in women tested with a non-NAAT were undiagnosed and that the risk of progression from undiagnosed chlamydia infection to PID within 12 months was 9.52% (9.30-9.68). Using non-NAATs could lead to an excess 120 cases of PID per 100,000 women tested compared to using NAATs. The key limitations of this study are under ascertainment of PID cases, misclassification bias in chlamydia and PID exposure status, bias to the association between clinical presentation and test type and the presence of unmeasured confounders (including other sexually transmitted infection [STI] diagnoses and clinical indication for chlamydia test).<br><br><b>Conclusion: </b>This retrospective observational study estimates the positive impact on women's reproductive health from using accurate chlamydia diagnostic tests and provides further evidence for restricting the use of inferior tests. Women with a negative chlamydia test have a 17% higher adjusted risk of PID by 12 months if they are tested using a non-NAAT compared to a NAAT.
Project description:Isothermal nucleic acid amplification tests (NAAT) in a Lab-on-a-Chip (LoC) format promise to bring high-accuracy, non-instrumented rapid tests to the point of care. Reliable rapid tests for infectious diseases allow for early diagnosis and treatment, which in turn enables better containment of potential outbreaks and fewer complications. A critical component to LoC NAATs is the heating element, as all NAAT protocols require incubation at elevated temperatures. We propose a cheap, integrated, self-regulating resistive heating solution that uses 2xAAA alkaline batteries as the power source, can maintain temperatures in the 60-63°C range for at least 25 minutes, and reaches the target range from room temperature in 5 minutes. 4 heating element samples with different electrical characteristics were evaluated in a thermal mock-up for a LoC NAAT device. An optimal heating element candidate was chosen based on temperature profiling. The optimal candidate was further evaluated by thermal modelling via finite element analysis of heat transfer and demonstrated suitable for isothermal nucleic acid amplification.
Project description:Some U.S. nursing programs have considered or already have removed microbiology courses from their curriculum. In response to this, the American Society for Microbiology (ASM) published in 2018 the Microbiology in Nursing and Allied Health (MINAH) Undergraduate Curriculum Guidelines. Parts of these guidelines include competencies and skills, which are best taught in a laboratory setting. Recognizing that some programs face a burden of running nursing/allied health microbiology lab classes, we have designed a curriculum of seven laboratories that covers the concepts in the MINAH guidelines. These labs cover aseptic technique and proper specimen collection, microscopy, enumerating microorganisms, and diagnostic tests in identifying unknowns. In addition, we provide some tips and tools for keeping down the costs of offering a microbiology lab. It is our goal that these tips and the suggested guidelines will allow microbiology to remain in the nursing and allied health curriculum.
Project description:A prospective multicenter clinical study involving subjects from 21 sites across the United States was conducted to validate the performance of a new in vitro diagnostic nucleic acid amplification test (NAAT) for the detection of Mycoplasma genitalium Seven urogenital specimen types (n?=?11,556) obtained from 1,778 females, aged 15 to 74?years, and 1,583 males, aged 16 to 82?years, were tested with the Aptima Mycoplasma genitalium assay, an investigational transcription-mediated amplification (TMA) NAAT for the detection of M. genitalium 16S rRNA. Infected status for enrolled subjects was established using results obtained from testing either self-collected vaginal swab or clinician-collected male urethral swab specimens with a composite reference method consisting of three transcription-mediated amplification NAATs targeting unique regions of M. genitalium 16S or 23S rRNA. M. genitalium prevalence was 10.2% in females and 10.6% in males; prevalence was high in both symptomatic and asymptomatic subjects for both sexes. Compared to the subject infected status standard, the investigational test had sensitivity and specificity estimates, respectively, of 98.9% and 98.5% for subject-collected vaginal swabs, 92.0% and 98.0% for clinician-collected vaginal swabs, 81.5% and 98.3% for endocervical swabs, 77.8% and 99.0% for female urine, and 98.2% and 99.6% for male urethral swabs, 88.4% and 97.8% for self-collected penile meatal swabs, and 90.9% and 99.4% for male urine specimens. For all seven specimen types, within-specimen positive and negative agreements between the investigational test and the composite reference standard ranged from 94.2% to 98.3% and from 98.5 to 99.9%, respectively. These results provide clinical efficacy evidence for the first FDA-cleared NAAT for M. genitalium detection in the United States.
Project description:We present a prototype for conducting rapid, inexpensive and point-of-care-compatible nucleic acid amplification tests (NAATs) for tuberculosis (TB). The fluorescent isothermal paper-and-plastic NAAT (FLIPP-NAAT) uses paper-based loop mediated isothermal amplification (LAMP) for DNA detection. The cost of materials required to build a 12-test-zone device is $0.88 and the cost of reagents per reaction is $0.43. An inexpensive imaging platform enables filter-free fluorescence detection of amplified DNA using a cell-phone camera. FLIPP-NAAT can be operated by an untrained user and only requires a regular laboratory incubator as ancillary equipment. All reagents can be dry-stored in the device, facilitating storage and transportation without cold chains. The device design is modular and the assay demonstrated high specificity to Mycobacterium tuberculosis (Mtb), analytical sensitivity of the order of 10 copies of Mtb gDNA, and tolerance to complex samples. The clinical sensitivity and specificity of sputum-based FLIPP NAAT tests were 100% (zero false negatives) and 68.75% (five false positives), respectively (N?=?30), using Xpert MTB/RIF assay as the reference standard. FLIPP-NAAT has the potential to provide affordable and accessible molecular diagnostics for TB in low- and middle-income countries, when used in conjunction with an appropriate sample preparation technique. Although demonstrated for the detection of TB, FLIPP-NAAT is a platform technology for amplification of any nucleic acid sequence.
Project description:<h4>Objectives</h4>To estimate the costs and benefits of clinical pathways incorporating a point of care (POC) nucleic acid amplification test (NAAT) for chlamydia and gonorrhoea in genitourinary medicine (GUM) clinics compared with standard off-site laboratory testing.<h4>Method</h4>We simulated 1.2 million GUM clinic attendees in England. A simulation in Microsoft Excel was developed to compare existing standard pathways of management for chlamydia and gonorrhoea with a POC NAAT. We conducted scenario analyses to evaluate the robustness of the model findings. The primary outcome was the incremental cost-effectiveness ratio. Secondary outcomes included the number of inappropriate treatments, complications and transmissions averted.<h4>Results</h4>The baseline cost of using the point of POC NAAT was £103.9 million compared with £115.6 million for standard care. The POC NAAT was also associated with a small increase of 46 quality adjusted life years, making the new test both more effective and cheaper. Over 95 000 inappropriate treatments might be avoided by using a POC NAAT. Patients receive diagnosis and treatment on the same day as testing, which may also prevent 189 cases of pelvic inflammatory disease and 17 561 onward transmissions annually.<h4>Discussion</h4>Replacing standard laboratory tests for chlamydia and gonorrhoea with a POC test could be cost saving and patients would benefit from more accurate diagnosis and less unnecessary treatment. Overtreatment currently accounts for about a tenth of the reported treatments for chlamydia and gonorrhoea and POC NAATs would effectively eliminate the need for presumptive treatment.