Ultrasensitive Test Research Articles

The future of Clostridioides difficile diagnostics.

Although the epidemiology of Clostridioides difficile has changed, this organism continues to cause significant morbidity and mortality. This review addresses current and future approaches to the diagnosis of C. difficile disease. Over the last several years, large prospective studies have confirmed that there is no single optimal test for the diagnosis of C. difficile disease. The pendulum has swung from a focus on rapid molecular diagnosis during the years of the ribotype 027 epidemic, to a call for use of algorithmic approaches that include a test for toxin detection. In addition, diagnostic stewardship has been shown to improve test utilization, especially with molecular methods. Advances in testing include development of ultrasensitive toxin tests and an expansion of biomarkers that may be more C. difficile specific. Microbiome research may be leveraged to inform novel diagnostic approaches based on measurements of volatile and nonvolatile organic compounds in stool. As rates of C. difficile infection decline, emphasis is now on improving test utilization and a quest for improved diagnostic approaches. These approaches may involve implementation of technologies that improve toxin testing, predict patients likely to have disease and/or a severe outcome, and harnessing research on changes in the microbiome to advance metabolomics.

Turn-On fluorescence resonance energy transfer (FRET)-based electrospun fibrous membranes: Rapid and ultrasensitive test strips for on-site detection of Mercury (II) ion

Creation of a highly sensitive test strip that can provide simple, rapid, and high-throughput determination of Hg2+ levels in consumer goods and industrial waste is extremely challenging since the portability and convenience of the paper-based platform often come at the expense of inferior sensitivity. In this work, a fluorescent electrospun fibrous membrane that can report mercury contamination down to sub-ppb level in less than a minute is developed. The key success of this user-friendly device relies on the design of novel mercury-favored organic dye (NF06) that displays specific fluorescence enhancement – “Turn-On” positive results – through fluorescence resonance energy transfer (FRET) mechanism and exhibits extremely large Stokes shift of 248 nm. The surface chemistry of the cellulose acetate(CA)/polycaprolactone(PCL) electrospun fibers on which the NF06 sensor is immobilized has been found to efficiently facilitate the FRET operation of the sensor. As a result, the detection limit of this NF06 strip reaches 0.309 ppb (0.697 nM), approximately an order of magnitude lower than the maximum mercury level permitted in drinking water (2 ppb) recommended by the US Environment Protection Agency. Instrument-free visual readout of Hg2+ level-dependent fluorescence enhancement after dipping the strips in Hg2+-spiked drinking water and skin whitening serum demonstrates that these low-cost test strips could certainly be used by general consumers for safety evaluation as well as by regulatory agencies for large-scale screening of a variety of commercial products without the need for expensive and highly sensitive detectors.

Optofluidic multiplex detection of single SARS-CoV-2 and influenza A antigens using a novel bright fluorescent probe assay

The urgency for the development of a sensitive, specific, and rapid point-of-care diagnostic test has deepened during the ongoing COVID-19 pandemic. Here, we introduce an ultrasensitive chip-based antigen test with single protein biomarker sensitivity for the differentiated detection of both severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and influenza A antigens in nasopharyngeal swab samples at diagnostically relevant concentrations. The single-antigen assay is enabled by synthesizing a brightly fluorescent reporter probe, which is incorporated into a bead-based solid-phase extraction assay centered on an antibody sandwich protocol for the capture of target antigens. After optimization of the probe release for detection using ultraviolet light, the full assay is validated with both SARS-CoV-2 and influenza A antigens from clinical nasopharyngeal swab samples (PCR-negative spiked with target antigens). Spectrally multiplexed detection of both targets is implemented by multispot excitation on a multimode interference waveguide platform, and detection at 30 ng/mL with single-antigen sensitivity is reported.

Ultrasensitive Measurement of Both SARS-CoV-2 RNA and Antibodies from Saliva.

Tests for COVID-19 generally measure SARS-CoV-2 viral RNA from nasal swabs or antibodies against the virus from blood. It has been shown, however, that both viral particles and antibodies against those particles are present in saliva, which is more accessible than both swabs and blood. We present methods for highly sensitive measurements of both viral RNA and antibodies from the same saliva sample. We developed an efficient saliva RNA extraction method and combined it with an ultrasensitive antibody test based on single molecule array (Simoa) technology. We apply our test to the saliva of patients who presented to the hospital with COVID-19 symptoms, some of whom tested positive with a conventional RT-qPCR nasopharyngeal swab test. We demonstrate that combining viral RNA detection by RT-qPCR with antibody detection by Simoa identifies more patients as infected than either method alone. Our results demonstrate the utility of combining viral RNA and antibody testing from saliva, a single easily accessible biofluid.

Diagnostic performance of an ultrasensitive HRP2-based malaria rapid diagnostic test kit used in surveys of afebrile people living in Southern Ghana

BackgroundThe Alere™ Malaria Ag P.f Ultra-sensitive RDT (UsmRDT) kit is an HRP2-based malaria rapid diagnostic test (RDT) with enhanced sensitivity relative to the SD Bioline Malaria Ag P.f RDT (mRDT) kit. However, the diagnostic performance of the UsmRDT kit has not been evaluated in Ghana.MethodsA total of 740 afebrile participants aged between 3 and 88 years old were recruited from the Central and Greater Accra Regions of Ghana during the off-peak malaria season. Axillary body temperature was measured, and a volume of 1 ml venous blood was drawn from each participant. Prior to separating the blood into plasma and packed cell pellets via centrifugation, the blood was spotted onto one UsmRDT and one mRDT kit and also used to prepare thick and thin blood smears as well as filter paper blood spots. Plasmodium falciparum specific polymerase chain reaction (PCR) was performed on gDNA extracted from 100 µl of the whole blood.ResultsThe overall positivity rate for microscopy, PCR, UsmRDT and mRDT kit were 20.4%, 40.8%, 31.3% and 30.8%, respectively. Overall, the UsmRDT identified 9.3% (28/302) more PCR positive samples than the mRDT kits. All samples that were negative by the UsmRDT kit were also negative by the mRDT kit. Overall, the sensitivity and specificity of the UsmRDT was 73% (221/302) and 89% (388/436), respectively, while that for the mRDT kit was 58% and 90%, respectively.ConclusionAlthough the UsmRDT kit was not as sensitive as PCR at detecting asymptomatic P. falciparum carriage, it correctly identified P. falciparum in 9.3% of the study participants that were not captured by the mRDT kit. In malaria endemic settings, the UsmRDT would provide an added advantage by identifying more asymptomatic P. falciparum carriers than the mRDT kit for targeted treatment interventions.

Routine Molecular Screening of Patients with Advanced Non-SmallCell Lung Cancer in Circulating Cell-Free DNA at Diagnosis and During Progression Using OncoBEAMTM EGFR V2 and NGS Technologies

Background and ObjectivesThe use of ultra-sensitive diagnostic tests to detect clinically actionable somatic alterations within the gene encoding the epidermal growth factor receptor (EGFR) within circulating cell-free DNA is an important first step in determining the eligibility of patients with non-small cell lung cancer to receive tyrosine kinase inhibitors.MethodsWe present the clinical validation (accuracy, sensitivity, and specificity) of a highly sensitive OncoBEAMTM EGFR V2 test, which we compare to a custom next-generation sequencing assay, for the treatment of patients with non-small cell lung cancer with EGFR tyrosine kinase inhibitor therapies. The OncoBEAMTM digital-polymerase chain reaction method detects 36 different EGFR alterations in circulating cell-free DNA, whereas the next-generation sequencing assay covers major solid tumor oncodrivers. Of the 540 samples analyzed with the OncoBEAMTM EGFR V2 test, 42.4% of patients had undergone molecular testing at diagnosis (N = 229/540) and 57.7% of patients during disease progression (N = 311/540).ResultsThe sensitivity and specificity were measured for this BEAMing assay. The number of mutant beads and mutant allelic fraction were measured for each EGFR alteration and the level of detection was established at 0.1% for a median of 2861 genome equivalent (GE) in each reaction using HD780 horizon control DNA, as well as by an internal quality reference standard. Approximately 10%, 27%, and 63% of the 540 samples contained < 1500 GE, a range of 1500–3000 GE, and > 3000 GE, which corresponded to a maximal assay sensitivity of 2.0%, 0.5–0.1%, and 0.1–0.05% mutant allelic fraction, respectively. In a routine hospital setting, 11.4% of non-small cell lung cancer tumors were positive at diagnosis for EGFR alterations, while 43.7% samples harbored EGFR mutations at progression, among which 40.3% expressed EGFR resistance mutations after first-line tyrosine kinase inhibitor treatment with first- and second-generation drugs.ConclusionsThe OncoBEAMTM EGFR V2 is a sensitive, robust, and accurate assay that delivers reproducible results. Next-generation sequencing and BEAMing technologies act complementarily in the routine molecular screening. We show that using a next-generation sequencing assay, despite its lower sensitivity, enables the identification of rare EGFR alterations or resistance mechanisms (mutation, deletion, insertion, and copy number variation) to orient first- and second-line treatments.Supplementary InformationThe online version contains supplementary material available at 10.1007/s40291-021-00515-9.

A smartphone-read ultrasensitive and quantitative saliva test for COVID-19.

Point-of-care COVID-19 assays that are more sensitive than the current RT-PCR (reverse transcription polymerase chain reaction) gold standard assay are needed to improve disease control efforts. We describe the development of a portable, ultrasensitive saliva-based COVID-19 assay with a 15-min sample-to-answer time that does not require RNA isolation or laboratory equipment. This assay uses CRISPR-Cas12a activity to enhance viral amplicon signal, which is stimulated by the laser diode of a smartphone-based fluorescence microscope device. This device robustly quantified viral load over a broad linear range (1 to 105 copies/μl) and exhibited a limit of detection (0.38 copies/μl) below that of the RT-PCR reference assay. CRISPR-read SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2) RNA levels were similar in patient saliva and nasal swabs, and viral loads measured by RT-PCR and the smartphone-read CRISPR assay demonstrated good correlation, supporting the potential use of this portable assay for saliva-based point-of-care COVID-19 diagnosis.

Antigen tests for COVID-19.

PCR diagnosis has been considered as the gold standard for coronavirus disease 2019 (COVID-19) and other many diseases. However, there are many problems in using PCR, such as non-specific (i.e., false-positive) and false-negative amplifications, the limits of a target sample volume, deactivation of the enzymes used, complicated techniques, difficulty in designing probe sequences, and the expense. We, thus, need an alternative to PCR, for example an ultrasensitive antigen test. In the present review, we summarize the following three topics. (1) The problems of PCR are outlined. (2) The antigen tests are surveyed in the literature that was published in 2020, and their pros and cons are discussed for commercially available antigen tests. (3) Our own antigen test on the basis of an ultrasensitive enzyme-linked immunosorbent assay (ELISA) is introduced. Finally, we discuss the possibility that our antigen test by an ultrasensitive ELISA technique will become the gold standard for diagnosis of COVID-19 and other diseases.

A novel, rapid (within hours) culture-free diagnostic method for detecting live Mycobacterium tuberculosis with high sensitivity.

BackgroundNucleic acid amplification tests (NAATs) are widely used to diagnose tuberculosis (TB), but cannot discriminate live bacilli from dead bacilli. Live bacilli can be isolated by culture methods, but this is time-consuming. We developed a de novo TB diagnostic method that detects only live bacilli with high sensitivity within hours.MethodsA prospective study was performed in Taiwan from 2017 to 2018. Sputum was collected consecutively from 1102 patients with suspected TB infection. The sputum was pretreated and heated at 46°C for 1 h to induce the secretion of MPT64 protein from live Mycobacterium tuberculosis. MPT64 was detected with our ultrasensitive enzyme-linked immunosorbent assay (ELISA) coupled with thionicotinamide-adenine dinucleotide (thio-NAD) cycling. We compared our data with those obtained using a culture test (MGIT), a smear test (Kinyoun staining), and a NAAT (Xpert).FindingsThe limit of detection for MPT64 in our culture-free ultrasensitive ELISA was 2.0 × 10−19 moles/assay. When the criterion for a positive response was set as an absorbance value ≥17 mAbs, this value corresponded to ca. 330 CFU/mL in the culture method – almost the same high-detection sensitivity as the culture method. To confirm that MPT64 is secreted from only live bacilli, M. bovis BCG was killed using 8 μg/mL rifampicin and then heated. Following this procedure, our method detected no MPT64. Our rapid ultra-sensitive ELISA-based method required only 5 h to complete. Comparing the results of our method with those of culture tests for 944 specimens revealed a sensitivity of 86.9% (93/107, 95% CI: 79.0–92.7%) and a specificity of 92.0% (770/837, 95% CI: 89.9–93.7%). The performance data were not significantly different (McNemar's test, P = 0.887) from those of the Xpert tests. In addition, at a ≥1+ titer in the smear test, the positive predictive value of our culture-free ultrasensitive ELISA tests was in a good agreement with that of the culture tests. Furthermore, our culture-free ultrasensitive ELISA test had better validity for drug effectiveness examination than Xpert tests because our test detected only live bacilli.InterpretationOur culture-free ultrasensitive ELISA method detects only live TB bacilli with high sensitivity within hours, allowing for rapid diagnosis of TB and monitoring drug efficacy.FundingMatching Planner Program from JST (VP29117939087), the A-STEP Program from JST (AS3015096U), Waseda University grants for Specific Research Projects (2017A-015 and 2019C-123), the Precise Measurement Technology Promotion Foundation to E.I.

Basal glucose excretion in dogs: The impact of feeding, obesity, sex, and age.

BackgroundThe urine glucose (UG) measurements are an integral part of urinalyses, especially in dogs with polyuria and polydipsia. A positive dipstick result is considered pathologic for disease. This paradigm has been challenged by new ultrasensitive tests, where the manufacturers recommend tolerating slightly positive results. It implies that, as in other species, basal urine glucose losses can exceed the lower limits of detection using ultrasensitive glucose dipsticks in healthy dogs.ObjectivesWe aimed to determine whether glucose is routinely detectable using a sensitive quantitative wet chemistry method in the urine of nondiabetic, nonazotemic dogs, and investigate the impact of food intake, obesity, sex, castration status, and age.MethodsSerial UG measurements were performed in healthy clinic‐owned Beagle dogs that were randomly fasted or fed. Glucose was measured in morning urine samples from normal‐weight healthy and obese dogs, and the university's electronic database was searched for quantitative UG measurements (Gluco‐quant Enzyme Kit/Roche Diagnostics).ResultsSmall amounts of glucose were detected in 555 (99.1%) of 560 urine samples analyzed. All urine samples from the clinic‐owned Beagle dogs, as well as from privately owned obese and normal‐weight healthy dogs that tested positive for glucose. The median (range) UG concentration obtained from the university's electronic database was 0.39 (0‐1.55) mmol/L, and 2.2% of the samples tested negative. Feeding, obesity, gender, castration status, and age did not affect UG concentrations.ConclusionsStudies, including a larger number of healthy dogs, are warranted to define a cut‐off between physiologic and pathologic glucosuria.

Rapid label-free SERS detection of foodborne pathogenic bacteria based on hafnium ditelluride-Au nanocomposites

Two-dimensional (2D) nanomaterials have captured an increasing attention in biophotonics owing to their excellent optical features. Herein, 2D hafnium ditelluride (HfTe[Formula: see text], a new member of transition metal tellurides, is exploited to support gold nanoparticles fabricating HfTe2-Au nanocomposites. The nanohybrids can serve as novel 2D surface-enhanced Raman scattering (SERS) substrate for the label-free detection of analyte with high sensitivity and reproducibility. Chemical mechanism originated from HfTe2 nanosheets and the electromagnetic enhancement induced by the hot spots on the nanohybrids may largely contribute to the superior SERS effect of HfTe2-Au nanocomposites. Finally, HfTe2-Au nanocomposites are utilized for the label-free SERS analysis of foodborne pathogenic bacteria, which realize the rapid and ultrasensitive Raman test of Escherichia coli, Listeria monocytogenes, Staphylococcus aureus and Salmonella with the limit of detection of 10 CFU/mL and the maximum Raman enhancement factor up to [Formula: see text]. Combined with principal component analysis, HfTe2-Au-based SERS analysis also completes the bacterial classification without extra treatment.

Proposal of De Novo Antigen Test for COVID-19: Ultrasensitive Detection of Spike Proteins of SARS-CoV-2.

Polymerase chain reaction (PCR)-based antigen tests are technically difficult, time-consuming, and expensive, and may produce false negative results requiring follow-up confirmation with computed tomography. The global coronavirus disease 2019 (COVID-19) pandemic has increased the demand for accurate, easy-to-use, rapid, and cost-effective antigen tests for clinical application. We propose a de novo antigen test for diagnosing COVID-19 using the combination of sandwich enzyme-linked immunosorbent assay and thio-nicotinamide adenine dinucleotide (thio-NAD) cycling. Our test takes advantage of the spike proteins specific to the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus. The limit of detection of our test was 2.3 × 10−18 moles/assay. If the virus has ~25 spike proteins on its surface, our method should detect on the order of 10−20 moles of virus/assay, corresponding to ~104 copies of the virus RNA/assay. The detection sensitivity approaches that of PCR-based assays because the average virus RNA load used for PCR-based assays is ~105 copies per oro- or naso-pharyngeal swab specimen. To our knowledge, this is the first ultrasensitive antigen test for SARS-CoV-2 spike proteins that can be performed with an easy-to-use microplate reader. Sufficient sensitivity can be achieved within 10 min of thio-NAD cycling. Our antigen test allows for rapid, cost-effective, specific, ultrasensitive, and simultaneous multiple measurements of SARS-CoV-2, and has broad application for the diagnosis for COVID-19.

Sensitive and hook effect–free lateral flow assay integrated with cascade signal transduction system

The traditional lateral flow immunoassay (LFI) for detecting foodborne pathogens suffers from low sensitivity and hook effect that led false–negative result. Here, we established a novel LFI integrated with a cascade signal transduction system for the sensitive and hook effect–free detection of Escherichia coli O157:H7 (E. coli O157:H7). First, E. coli O157:H7 was captured by immunomagnetic nanoparticles (IMNPs) and monoclonal antibody–β–lactamase–gold nanoparticles (ILAuNPs) forming sandwich complexes. Second, penicillin solution was added and hydrolyzed by β–lactamase on the sandwich complexes. Last, the penicillin hydrolysate was transferred to an ultrasensitive penicillin immunochromatographic test strip (PIST). With the high β–lactamase activity of ILAuNPs (0.1 μL of ILAuNPs hydrolyzing 20 ng/mL of penicillin) and ultrasensitive PIST (cutoff value achieving 2 ng/mL), the detection sensitivity of this method was 1.37 × 102 CFU/mL for E. coli O157:H7, which prevailed 1000 times over that of the traditional LFI. This work also efficiently solved hook effect when the E. coli O157:H7 concentration was over 107 CFU/mL. Thus, the detection range of the novel method was 102–108 CFU/mL, thereby expanding by four orders of magnitude than that of the traditional LFI (105–107 CFU/mL).

Ultra-sensitive test detects a whisper of tumour DNA

Ultra-sensitive test detects a whisper of tumour DNA

Long-Term Delivery of an Anti-SIV Monoclonal Antibody With AAV.

Long-term delivery of anti-HIV monoclonal antibodies using adeno-associated virus (AAV) holds promise for the prevention and treatment of HIV infection. We previously reported that after receiving a single administration of AAV vector coding for anti-SIV antibody 5L7, monkey 84-05 achieved high levels of AAV-delivered 5L7 IgG1 in vivo which conferred sterile protection against six successive, escalating dose, intravenous challenges with highly infectious, highly pathogenic SIVmac239, including a final challenge with 10 animal infectious doses (1). Here we report that monkey 84-05 has successfully maintained 240–350 μg/ml of anti-SIV antibody 5L7 for over 6 years. Approximately 2% of the circulating IgG in this monkey is this one monoclonal antibody. This monkey generated little or no anti-drug antibodies (ADA) to the AAV-delivered antibody for the duration of the study. Due to the nature of the high-dose challenge used and in order to rule out a potential low-level infection not detected by regular viral loads, we have used ultrasensitive techniques to detect cell-associated viral DNA and RNA in PBMCs from this animal. In addition, we have tested serum from 84-05 by ELISA against overlapping peptides spanning the whole envelope sequence for SIVmac239 (PepScan) and against recombinant p27 and gp41 proteins. No reactivity has been detected in the ELISAs indicating the absence of naturally arising anti-SIV antibodies; moreover, the ultrasensitive cell-associated viral tests yielded no positive reaction. We conclude that macaque 84-05 was effectively protected and remained uninfected. Our data show that durable, continuous antibody expression can be achieved after one single administration of AAV and support the potential for lifelong protection against HIV from a single vector administration.

Survey and Diagnostic Challenges after Transmission-Stop: Confirming Elimination of Schistosomiasis haematobium in Morocco

Clinical cases of Moroccan residents have been recorded since 2004, indicating successful interruption of transmission of S. haematobium infection at national level. The first national survey initiated in 2009 for Schistosomiasis haematobium among children born after 2004, applied diagnostic test was the HAMA-EITB, based on the Western blot technology, and molecular malacological diagnostic tools clearly confirm transmission stop. In 2015, a recent, small survey utilizing an HAI, ELISA tests and an ultrasensitive antigen test, FTCUP CAA, in a group of individual with a past history of infection. However, obviously follow-up surveys to prevent reemergency and for certification of the schistosomiasis elimination require vigilant diagnosis strategies. Here we discuss diagnosis story line in the national laboratory and challenges based on the available tools in relation to their clinical parameters (sensitivity/specificity; Sn/Sp), practicability and associated costs. When transmission stop has been achieved, survey cost and speed are likely to benefit from cost effective pooling strategies and ultrasensitive assays indicating active infection in all potential risk groups. Similarly molecular pooling strategies to monitor infections in the snail vectors.

An ultrasensitive colorimetric test for the detection of somatic rare mutations in DNA.

Targeted therapies for cutaneous melanoma, such as those based on specific BRAF inhibitors, have improved the treatment and enhanced the survival rate of patients who harbor the V600E point mutation in the BRAF gene. However, tissue biopsies to characterize BRAF mutation status are prone to sampling bias, due to the intrinsic heterogeneity of a tumor mass. In contrast, blood biopsies, which analyze circulating tumor DNA (ctDNA), offer the most complete and sensitive characterization of the mutation status of a tumor, provide early and more accurate diagnosis, but they require instrumental and costly molecular tests. Therefore, the development of low-cost but highly sensitive tests for the non-invasive identification of BRAFV600E mutation in ctDNA would be of great clinical utility as a routine screening for the early identification of responsive patients and the follow-up of targeted therapy's response. The present work developed a naked-eye, inexpensive, yet very specific colorimetric assay, whose sensitivity is suitable for the detection of BRAFV600E rare mutation in ctDNA. Such test potentially may detect at an early stage the mutation in the tumor mass, when the first mutated cells appear in the blood, by using minimal instrumentation and thus enabling its widespread implementation in the clinics, even in local, minimally equipped laboratories. Indeed, the test detects 0.5% of BRAFV600E in an excess of BRAFWT DNA, which matches the sensitivity of some commercial instrumental assays. Such sensitivity is thus clinically relevant for diagnostic purposes, allowing the early identification of drug-sensitive patients.

N-Acety-L-Cysteine-Stabilized Pt Nanozyme for Colorimetric Assay of Heparin

N-Acety-L-cysteine (NAC), enriched with multiple electron-rich groups involving –SH, –C=O, and –COOH, exhibits strong affinities to transition metal ions. Herein, NAC was adopted as the stabilizing agent to synthesize a series of peroxidase-like Pt nanoclusters (NCs) with different physicochemical properties. Both the cluster size and the charge state of NAC–Pt NCs are highly dependent upon the molar ratio of [K2PtCl4]/[NAC], contributing to different peroxidase mimicking activities. The most active Pt nanozyme (~ 1.7 nm), composed of 60% metallic Pt0 and 40% positively charged Pt2+ respectively, exhibits the Km of 0.132 mM for 3,3′,5,5′-tetramethylbenzidine and 35 mM for H2O2. Based on the heparin-activated enzymatic activity of NAC–Pt at pH 6.0, an ultrasensitive test was established for quantitative determination of heparin, giving the linear response of 0.5–20 μg/mL as well as the limit of detection of 2 × 10−3 μg/mL. This proposed method is also applicable in biological fluid for practical applications. Such a colorimetric method possesses several advantages involving high sensitivity, short response, eco-friendly synthesis, low consumption of material and energy, free of expensive instrument, as well as simple operation.

J-108 Role of DNA methylation in prediction of human papillomavirus persistence and carcinogenesis

J-108 Role of DNA methylation in prediction of human papillomavirus persistence and carcinogenesis

Further evidence of Mycobacterium tuberculosis in the sputum of culture-negative pulmonary tuberculosis suspects using an ultrasensitive molecular assay

BackgroundRapid diagnosis of pulmonary tuberculosis (TB) is critical to TB control. However, many patients with paucibacillary TB disease remain undiagnosed. Current TB elimination goals require new tools to diagnose early disease. We evaluated performance of the Totally Optimized PCR (TOP) TB assay, a novel ultrasensitive molecular test. MethodsWe assessed analytical specificity against nontuberculous mycobacteria (NTM), and estimated the diagnostic accuracy of TOP in a pilot study in Brazil (n = 46) and a cross-sectional study in Boston (n = 60). We compared TOP results to culture and a composite reference standard (CRS). ResultsTOP exhibited no cross-reactivity against NTM. We tested 132 respiratory specimens from 106 patients with suspected pulmonary TB. The pilot demonstrated feasibility and 100% (95% CI 85–100) sensitivity in predominantly smear-positive specimens; TOP's specificity against solid media culture was low (58%, 37–77) but improved against a CRS (93%, 68–100). Similarly, when using the CRS in the Boston study, TOP (88%, 1–99) had greater sensitivity than solid or liquid media culture (25%, 3–65) and similar specificity (both 100%, 93–100). ConclusionsThe TOP assay enables detection of M. tuberculosis in culture-negative paucibacillary disease. While the use of TOP for the diagnosis of paucibacillary disease will require further clinical validation, its high sensitivity indicate a more immediate utility as a rule out TB test.