Evaluation of the analytical performance of the 15-minute point-of-care DASH™ SARS-CoV-2 RT-qPCR test.

Accurate and timely diagnosis for COVID-19 diagnosis allows highly effective antiviral medications to be prescribed. The DASH™ Rapid PCR System is a sample-to-answer point-of-care platform combining state-of-the-art PCR kinetics with sequence specific hybridization. The platform's first assay, the DASH™ SARS-CoV-2/S test for anterior nares direct swab specimens, received FDA Emergency Use Authorization in March 2022 for point-of-care use. Here we report the analytical characteristics of the assay including limit of detection, dynamic range, and robustness of SARS-CoV-2 variant detection. The limit of detection was determined by testing swabs contrived with one hundred copies of wild type or Omicron BA.5 virus and detecting 20/20 and 19/20, respectively. The dynamic range was assessed with contrived swabs containing 102-106 copies; the log-linear relationship between Cq and copy input was plotted, and the qPCR efficiency calculated from the slope of the line was 101.4%. Detection of seven SARS-CoV-2 variants was demonstrated.

Hepatitis B Virus in West African Children: Systematic Review and Meta-Analysis of HIV and Other Factors Associated with Hepatitis B Infection.

While Hepatitis B virus (HBV) and the human immunodeficiency virus (HIV) are endemic in West Africa, the prevalence of HBV/HIV coinfection and their associated risk factors in children remains unclear. In this review, we sought to assess HBsAg seroprevalence among 0- to 16-year-olds with and without HIV in West African countries and the risk factors associated with HBV infection in this population. Research articles between 2000 and 2021 that reported the prevalence of HBV and associated risk factors in children in West Africa were retrieved from the literature using the Africa Journals Online (AJOL), PubMed, Google Scholar, and Web of Science databases as search tools. StatsDirect, a statistical software, was used to perform a meta-analysis of the retained studies. HBV prevalence and heterogeneity were then assessed with a 95% confidence interval (CI). Publication bias was evaluated using funnel plot asymmetry and Egger's test. Twenty-seven articles conducted across seven West African countries were included in this review. HBV prevalence among persons aged 0 to 16 years was 5%, based on the random analysis, given the great heterogeneity of the studies. By country, the highest prevalence was observed in Benin (10%), followed by Nigeria (7%), and Ivory Coast (5%), with Togo (1%) having the lowest. HBV prevalence in an HIV-infected population of children was (9%). Vaccinated children had lower HBV prevalence (2%) than unvaccinated children (6%). HBV prevalence with a defined risk factor such as HIV co-infection, maternal HBsAg positivity, undergoing surgery, scarification, or being unvaccinated ranged from 3-9%. The study highlights the need to reinforce vaccination of newborns, screening for HBV, and HBV prophylaxis among pregnant women in Africa, particularly in West Africa, to achieve the WHO goal of HBV elimination, particularly in children.

Development and clinical evaluation of a new multiplex PCR assay for a simultaneous diagnosis of tuberculous and nontuberculous mycobacteria.

BACKGROUND: The prevalence of non-tuberculous mycobacteria (NTM) has been increasing worldwide in both developed and developing countries. NTM infection is clinically indistinguishable from tuberculosis and therefore poses significant challenges in patient management, especially in patients chronically treated for pulmonary TB. In this study, we evaluated a new highly sensitive Multiplex MTB/NTM assay that can differentiate M. tuberculosis complex (MTBC) from all NTM, including the treatable and most common NTM, M. avium complex (MAC). METHODS: We developed and optimized a new open- Multiplex MTB/NTM assay with two gene-targets for MTBC (IS6110/senX3-regX3) and two targets for MAC (IS1311/DT1) with samples spiked with stored strains and testing 20 replicates. Patients with presumptive TB and NTM were enrolled at the Respiratory Disease Department of The University Teaching Hospital of Point G, in Mali. FINDINGS: In the development stage, the new assay showed a high analytic performance with 100% detections of MTBC and MAC at only 5 colony forming units (CFUs). Overall, without the treatment failure cases, the Multiplex assay and the Xpert showed a sensitivity, specificity, PPV and NPV of 83·3% [66·4-92·6], 96·6% [88·6-99·0], 92·5% [82·3-96·5] and 92·2% [82·7-96·5] and the Xpert had values of 96·7% [83·3-99·4], 80·0% [68·2-88·1], 70·7 [55·5-82·3] and 97·9% [89·3-99·6], respectively. The Multiplex assay successfully detected all (5/5) the MAC cases. INTERPRETATION: Our new Multiplex assay demonstrates better specificity than Xpert for all group studied, in addition to detecting potential NTM cases. The assay could therefore complement the widely used Xpert assay and enhance discrimination of TB and NTM infections. FUNDING: This work was supported by the National Institutes of Health (R03AI137674, U54EB027049, D43TW010350 and UM1AI069471) and Northwestern University's Institute for Global Health Catalyzer Fund.

C-THAN: A new research center for the development of point-of-care technology for HIV/AIDS.

The Center for Innovation in Point-of-Care Technologies for HIV/AIDS at Northwestern University (C-THAN) is a partner in the Point-of-Care Technologies Research Network (POCTRN) of the National Institutes of Biomedical Imaging and Bioengineering. POCTRN's mission is to drive the development of appropriate point-of-care (POC) diagnostic technologies through collaboration that merges scientific and technological capabilities with clinical need. C-THAN develops POC technologies for improved management of HIV/AIDS in low- and middle-income countries with a focus on sub-Saharan Africa. C-THAN incorporates clinical and user needs with technology expertise and resources to address commercialization and implementation barriers through: 1) assessment of unmet clinical needs in POC testing for HIV/AIDS and its comorbidities; 2) collaborations with physicians, researchers and engineers; 3) development of technical, clinical, industrial and regulatory partnerships; 4) clinical testing of prototype devices; and 5) creation of training opportunities for technology developers, evaluators, and other stakeholders. Technologies supported include tests for detection and monitoring of HIV/AIDS and its common comorbidities including tuberculosis, non-tuberculous mycobacteria, viral hepatitis and HIV-related malignancies. CTHAN relies on collaborations established by Northwestern University in Nigeria, South Africa, Mali and Tanzania, to have impact on the prevention and clinical management of HIV/AIDS.

Supporting Diagnosis and Management of HIV/AIDS Patients Through Point-of-Care Technology Development.

The HIV pandemic disproportionately impacts sub-Saharan Africa where in 2017, 71% of people living with HIV resided, 65% of new infections and 75% of deaths were reported. Prevention, screening and treatment strategies have led to progress in addressing this disease. HIV diagnostics have been crucial for prevention and treatment but more progress is required to reduce HIV infection. The Center for Innovation in Point-of-Care Technologies for HIV/AIDS at Northwestern University (C-THAN) is a vital partner in the National Institute of Biomedical Imaging and Bioengineering Point-of-Care Technologies Research Network. C-THAN's mission is to develop and commercialize a pipeline of point-of-care technologies critical for improved prevention and management of HIV in low- and middle-income countries with specific emphasis on sub-Saharan Africa.

Simultaneous diagnosis of tuberculous and non-tuberculous mycobacterial diseases: Time for a better patient management.

Tuberculosis (TB) is the deadliest infectious disease in the world which disproportionately affects low-and-middle-income countries (LMICs) where diagnostic resources and treatment options are limited. The incidence of pulmonary non-tuberculous mycobacteria (NTM) disease is also rapidly increasing in these regions traditionally dominated by TB infections. This poses significant diagnostic and treatment challenges, since these two diseases are often indistinguishable clinically or by sputum smear microscopy (SSM), the most commonly used TB diagnostic tool in LMICs. Consequently, NTM-infected patients usually receive unnecessary TB treatment for months. TB patients with NTM co-infections may also be treated incorrectly due to inaccurate SSM and Xpert™ MTB/RIF (M. tuberculosis./rifampin) results. These issues complicate the management of patients and contribute to the worsening of the current TB and NTM epidemiological features including development of drug resistant strains. It is therefore critical to develop improved diagnostic tools to accurately distinguish these two different pathogens that have many similar clinical and epidemiological features but have different treatment regimens. In this review, we will discuss limitations with current diagnostic tools and the need to develop novel techniques that can accurately and simultaneously diagnose TB and NTM disease._.

A simple and rapid DNA extraction method for Chlamydia trachomatis detection from urogenital swabs.

A highly sensitive and specific Chlamydia trachomatis (CT) diagnostic test was developed by combining filtration isolation of nucleic acid (FINA) extraction with quantitative polymerase chain reaction including an internal control to identify test inhibition. A pilot study of 40 clinical specimens yielded 100% sensitivity and specificity.

Immiscible phase filter extraction and equivalent amplification of genotypes 1-6 of hepatitis C RNA: The building blocks for point-of-care diagnosis.

The lack of hepatitis C virus (HCV) diagnostic tests designed for use in decentralized settings is a major obstacle for providing access to treatment and prevention services particularly in low and middle income countries. Here we describe the development and validation of two building blocks of the HCV Quant Assay, a test in development for point-of-care use: 1) an RT-qPCR assay with noncompetitive internal control that equivalently detects the 6 major HCV genotypes and 2) an automated sample prep method using immiscible phase filter technology. This novel assay has wide dynamic range of HCV quantification and a limit of detection of 30IU/ml with 200µl specimen volume. In a preliminary study of 61 clinical specimens, the HCV Quant Assay demonstrated 100% sensitivity and specificity and gave comparable viral load results across 4 logs of IU/ml when compared to the Abbott RealTime HCV Assay.

XtracTB Assay, a Mycobacterium tuberculosis molecular screening test with sensitivity approaching culture.

Nucleic acid amplification tests are increasingly used to diagnose tuberculosis (TB) due to their speed and sensitivity compared to sputum smear microscopy. However, these tests fail to equal culture's sensitivity with sputum smear microscopy negative specimens and therefore cannot be used to rule out TB disease. For molecular tests to match culture's sensitivity, they must detect ≤10 genomic copies of Mycobacterium tuberculosis (MTB) DNA, the limit of detection of culture, process ≥1 ml of sputum ensuring sufficient number of MTB are in the reaction, and efficiently remove sputum associated inhibitors from this large sample. Here we report the preliminary characterization of XtracTB Assay, a MTB testing protocol designed for inclusion in either an integrated point-of-care platform or a high throughput automated central laboratory system. The test combines DNA sequence specific sample prep to reduce the co-extraction of qPCR inhibitors with the amplification of two MTB specific loci (IS6110 and senX3-regX3) to increase test sensitivity and minimize the likelihood of false negatives. The analytical sensitivity of the XtracTB Assay was 5 genomic copies/ml of sputum rivaling that of culture. Furthermore, 142 valid test results yield clinical sensitivity of 94.9% (95% CI: 90.1-99.9) and specificity of 100% (95% CI: 90.0-100.0).

Highly sensitive sequence specific qPCR detection of Mycobacterium tuberculosis complex in respiratory specimens.

Nucleic acid amplification tests for Mycobacterium tuberculosis (MTB) detection from sputum are highly sensitive and specific with smear microscopy positive specimens, but their sensitivity with smear-negative/culture-positive specimens is much lower; therefore, these tests cannot rule out a tuberculosis diagnosis. Co-extraction of PCR inhibitors may be a cause of decreased test sensitivity. Here the design and early validation of a MTB screening assay with sample preparation and qPCR methods designed to specifically address this diagnostic gap is reported. First, human genomic DNA is identified as a significant qPCR inhibitor. To circumvent this problem, a novel, streamlined sample preparation method utilizing detergent and proteolysis to thin the sputum and DNA sequence specific MTB DNA isolation was developed. Additionally, a multiplexed qPCR assay targeting two MTB complex-specific loci: the potentially multi-copy IS6110 and the single-copy senX3-regX3, combined with the cotJC gene from Bacillus atrophaeus spores amplified as a process control was developed. The limit of detection of the test was estimated to be 20 cfu/ml which is significantly lower than the Xpert® MTB/RIF assay. In a preliminary field study of 60 de-identified blinded sputa, a test sensitivity of 96% and specificity of 100% was observed when compared to the Xpert® MTB/RIF assay.

Filtration Isolation of Nucleic Acids: A Simple and Rapid DNA Extraction Method.

FINA, filtration isolation of nucleic acids, is a novel extraction method which utilizes vertical filtration via a separation membrane and absorbent pad to extract cellular DNA from whole blood in less than 2 min. The blood specimen is treated with detergent, mixed briefly and applied by pipet to the separation membrane. The lysate wicks into the blotting pad due to capillary action, capturing the genomic DNA on the surface of the separation membrane. The extracted DNA is retained on the membrane during a simple wash step wherein PCR inhibitors are wicked into the absorbent blotting pad. The membrane containing the entrapped DNA is then added to the PCR reaction without further purification. This simple method does not require laboratory equipment and can be easily implemented with inexpensive laboratory supplies. Here we describe a protocol for highly sensitive detection and quantitation of HIV-1 proviral DNA from 100 µl whole blood as a model for early infant diagnosis of HIV that could readily be adapted to other genetic targets.

The nematode Caenorhabditis elegans displays a chemotaxis behavior to tuberculosis-specific odorants.

A simple, affordable diagnostic test for pulmonary tuberculosis (TB) is urgently needed to improve detection of active Mycobacterium tuberculosis. Recently, it has been suggested that animal behavior can be used as a biosensor to signal the presence of human disease. For example, the giant African pouched rats can detect tuberculosis by sniffing sputum specimens while trained honeybees respond to three of the volatile organic compounds (VOCs) detected in the breath of TB positive patients by proboscis extension. However, both rats and honeybees require animal housing facilities and professional trainers, which are outside the scope of most disease testing facilities. Here, we report that the innate olfactory behavioral response of the roundworm nematode Caenorhabditis elegans can be used to detect the TB-specific VOCs methyl p-anisate, methyl nicotinate, methyl phenylacetate and o-phenylanisole, in chemotaxis assays. Dauer larvae, a long-lived stress resistant alternative development state of C. elegans in which the animals can survive for extended periods of time in dry conditions with no food, were also demonstrated to detect the VOCs. We propose that exposing naive dauer larvae to TB-related VOCs and recording their response in this behavioral assay could lead to the development of a new method for TB diagnostics using breath as the sample type.

A simple and rapid DNA extraction method from whole blood for highly sensitive detection and quantitation of HIV-1 proviral DNA by real-time PCR.

Early diagnosis and access to treatment for infants with human immunodeficiency virus-1 (HIV-1) is critical to reduce infant mortality. The lack of simple point-of-care tests impedes the timely initiation of antiretroviral therapy. The development of FINA, filtration isolation of nucleic acids, a novel DNA extraction method that can be performed by clinic personnel in less than 2 min has been reported previously. In this report, significant improvements in the DNA extraction and amplification methods are detailed that allow sensitive quantitation of as little as 10 copies of HIV-1 proviral DNA and detection of 3 copies extracted from 100 µl of whole blood. An internal control to detect PCR inhibition was also incorporated. In a preliminary field evaluation of 61 South African infants, the FINA test demonstrated 100% sensitivity and specificity. The proviral copy number of the infant specimens was quantified, and it was established that 100 microliters of whole blood is required for sensitive diagnosis of infants.

Immiscible phase nucleic acid purification eliminates PCR inhibitors with a single pass of paramagnetic particles through a hydrophobic liquid.

Extraction and purification of nucleic acids from complex biological samples for PCR are critical steps because inhibitors must be removed that can affect reaction efficiency and the accuracy of results. This preanalytical processing generally involves capturing nucleic acids on microparticles that are then washed with a series of buffers to desorb and dilute out interfering substances. We have developed a novel purification method that replaces multiple wash steps with a single pass of paramagnetic particles (PMPs) though an immiscible hydrophobic liquid. Only two aqueous solutions are required: a lysis buffer, in which nucleic acids are captured on PMPs, and an elution buffer, in which they are released for amplification. The PMPs containing the nucleic acids are magnetically transported through a channel containing liquid wax that connects the lysis chamber to the elution chamber in a specially designed cartridge. Transporting PMPs through the immiscible phase yielded DNA and RNA as pure as that obtained after extensive wash steps required by comparable purification methods. Our immiscible-phase process has been applied to targets in whole blood, plasma, and urine and will enable the development of faster and simpler purification systems.

Rapid, point-of-care extraction of human immunodeficiency virus type 1 proviral DNA from whole blood for detection by real-time PCR.

PCR detection of human immunodeficiency virus type 1 (HIV-1) proviral DNA is the method recommended for use for the diagnosis of HIV-1 infection in infants in limited-resource settings. Currently, testing must be performed in central laboratories, which are usually located some distance from health care facilities. While the collection and transportation of samples, such as dried blood spots, has improved test accessibility, the results are often not returned for several weeks. To enable PCR to be performed at the point of care while the mothers wait, we have developed a vertical filtration method that uses a separation membrane and an absorbent pad to extract cellular DNA from whole blood in less than 2 min. Cells are trapped in the separation membrane as the specimen is collected, and then a lysis buffer is added. The membrane retains the DNA, while the buffer washes away PCR inhibitors, which get wicked into the absorbent blotter pad. The membrane containing the entrapped DNA is then added to the PCR mixture without further purification. The method demonstrates a high degree of reproducibility and analytical sensitivity and allows the quantification of as few as 20 copies of HIV-1 proviral DNA from 100 microl of blood. In a blinded study with 182 longitudinal samples from infants (ages, 0 to 72 weeks) obtained from the Women and Infants Transmission Study, our assay demonstrated a sensitivity of 99% and a specificity of 100%.

Structure-function analysis of the heat shock factor-binding protein reveals a protein composed solely of a highly conserved and dynamic coiled-coil trimerization domain.

Heat shock factor-binding protein (HSBP) 1 is a small, evolutionarily conserved protein originally identified in a yeast two-hybrid screen using the trimerization domain of heat shock factor (HSF) 1 as the bait. Similar in size to HSF1 trimerization domain, human HSBP1 contains two arrays of hydrophobic heptad repeats (designated HR-N and HR-C) characteristic of coiled-coil proteins. Proteins of the HSBP family are relatively small (<100 residues), comprising solely a putative coiled-coil oligomerization domain without any other readily recognizable structural or functional motif. Our biophysical and biochemical characterization of human HSBP1 reveals a cooperatively folded protein with high alpha-helical content and moderate stability. NMR analyses reveal a single continuous helix encompassing both HR-N and HR-C in the highly conserved central region, whereas the less conserved carboxyl terminus is unstructured and accessible to proteases. Unlike previously characterized coiled-coils, backbone 15N relaxation measurements implicate motional processes on the millisecond time scale in the coiled-coil region. Analytical ultracentrifugation and native PAGE studies indicate that HSBP1 is predominantly trimeric over a wide concentration range. NMR analyses suggest a rotationally symmetric trimer. Because the highly conserved hydrophobic heptad repeats extend over 60% of HSBP1, we propose that HSBP most likely regulates the function of other proteins through coiled-coil interactions.