Correction: Performance of novel antibodies for lipoarabinomannan to develop diagnostic tests for Mycobacterium tuberculosis.

[This corrects the article DOI: 10.1371/journal.pone.0274415.].

A Reagent and Virus Benchmarking Panel for a Uniform Analytical Performance Assessment of N Antigen-Based Diagnostic Tests for COVID-19.

Rapid diagnostic tests (RDTs) that detect antigen indicative of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) infection can help in making quick health care decisions and regularly monitoring groups at risk of infection. With many RDT products entering the market, it is important to rapidly evaluate their relative performance. Comparison of clinical evaluation study results is challenged by protocol design variations and study populations. Laboratory assays were developed to quantify nucleocapsid (N) and spike (S) SARS-CoV-2 antigens. Quantification of the two antigens in nasal eluates confirmed higher abundance of N than S antigen. The median concentration of N antigen was 10 times greater than S per genome equivalent. The N antigen assay was used in combination with quantitative reverse transcription (RT)-PCR to qualify a panel composed of recombinant antigens, inactivated virus, and clinical specimen pools. This benchmarking panel was applied to evaluate the analytical performance of the SD Biosensor Standard Q COVID-19 antigen (Ag) test, Abbott Panbio COVID-19 Ag rapid test, Abbott BinaxNOW COVID-19 Ag test, and the LumiraDx SARS-CoV-2 Ag test. The four tests displayed different sensitivities toward the different panel members, but all performed best with the clinical specimen pool. The concentration for a 90% probability of detection across the four tests ranged from 21 to 102 pg/mL of N antigen in the extracted sample. Benchmarking panels provide a quick way to verify the baseline performance of a diagnostic and enable direct comparisons between diagnostic tests. IMPORTANCE This study reports the results for severe acute respiratory syndrome coronavirus-2 (SARS-COV-2) nucleocapsid (N) and spike (S) antigen quantification assays and their performance against clinical reverse transcription (RT)-PCR results, thus describing an open-access quantification method for two important SARS-CoV-2 protein analytes. Characterized N antigen panels were used to evaluate the limits of detection of four different rapid tests for SARS-CoV-2 against multiple sources of nucleocapsid antigen, demonstrating proof-of-concept materials and methodology to evaluate SARS-CoV-2 rapid antigen detection tests. Quantification of N antigen was used to characterize the relationship between viral count and antigen concentration among clinical samples and panel members of both clinical sample and viral culture origin. This contributes to a deeper understanding of protein antigen and molecular analytes and presents analytical methods complementary to clinical evaluation for characterizing the performance of both laboratory-based and point-of-care rapid diagnostics for SARS-CoV-2.

Performance of novel antibodies for lipoarabinomannan to develop diagnostic tests for Mycobacterium tuberculosis.

Lipoarabinomannan (LAM), a component of the Mycobacterium tuberculosis (MTB) cell wall, is detectable in the urine of MTB infected patients with active tuberculosis (TB). LAM-specific antibodies (Igs) have been developed by a variety of traditional and recombinant methods for potential use in a rapid diagnostic test (RDT). We evaluated the analytical performance of the TB LAM Igs to identify pairs that offer superior performance over existing urine LAM tests. We assessed 25 new and 4 existing Igs in a matrixed format using a multiplex electrochemiluminescence-based liquid immunoassay. A total of 841 paired Ig combinations were challenged with in vitro cultured LAM (cLAM) derived from MTB strains representing diverse phylogenetic lineages, alongside urinary LAM (uLAM) from the urine of adults with active pulmonary TB. Analytical sensitivity of down-selected Ig pairs was determined using MTB Aoyama-B cLAM, while diagnostic accuracy was determined using clinical samples. When testing cLAM, the reactivity of Ig pairs was similar across MTB lineages 1-4 but lineage 5:6 had significantly more reactivity among Ig pairs. Overall, 41 Ig pairs had a strong binding affinity to cLAM, as compared to the reference pair of S4-20/A194-01, and 28 Ig pairs therein exhibited a strong affinity for both cLAM and uLAM. Retrospective testing on clinical urine specimens demonstrated varying sensitivities (12-80%) and specificities (14-100%). The five top pairs had a similar analytical limit of detection to the reference pair but in four instances, the sensitivity and specificity with clinical uLAM samples was poor. Overall, epitopes presented by uLAM are different from cLAM, which may affect antibody performance when testing uLAM in patient samples. Several new Ig pairs had similar ranges of high sensitivity to cLAM but overall, there were no new candidate Ig pairs identified in this round of screening with increased performance with uLAM as compared to an existing optimal pair.

Performance of anterior nares and tongue swabs for nucleic acid, Nucleocapsid, and Spike antigen testing for detecting SARS-CoV-2 against nasopharyngeal PCR and viral culture.

OBJECTIVES: This study assesses and compares the performance of different swab types and specimen collection sites for SARS-CoV-2 testing, to reference standard real-time reverse transcriptase-polymerase chain reaction (RT-PCR) and viral culture. METHODS: Symptomatic adults with COVID-19 who visited routine COVID-19 testing sites used spun polyester and FLOQSwabs to self-collect specimens from the anterior nares and tongue. We evaluated the self-collected specimen from anterior nares and tongue swabs for the nucleocapsid (N) or spike (S) antigen of SARS-CoV-2 by RT-PCR and then compared these results with results from RT-PCR and viral cultures from nurse-collected nasopharyngeal swabs. RESULTS: Diagnostic sensitivity was highest for RT-PCR testing conducted using specimens from the anterior nares collected on FLOQSwabs (84%; 95% CI 68-94%) and spun polyester swabs (82%; 95% CI 66-92%), compared to RT-PCR tests conducted using specimens from nasopharyngeal swabs. Relative to viral culture from nasopharyngeal swabs, diagnostic sensitivities were higher for RT-PCR and antigen testing of anterior nares swabs (91-100%) than that of tongue swabs (18-81%). Antigen testing of anterior nares swabs had higher sensitivities against viral culture (91%) than against nasopharyngeal RT-PCR (38-70%). All investigational tests had high specificity compared with nasopharyngeal RT-PCR. Spun polyester swabs are equally effective as FLOQSwabs for anterior nasal RT-PCR testing. CONCLUSIONS: We found that anterior nares specimens were more sensitive than tongue swab specimens or antigen testing for detecting SARS-CoV-2 by RT-PCR. Thus, self-collected anterior nares specimens may represent an alternative method for diagnostic SARS-CoV-2 testing in some settings.

Screening Antibodies Raised against the Spike Glycoprotein of SARS-CoV-2 to Support the Development of Rapid Antigen Assays.

Severe acute respiratory coronavirus-2 (SARS-CoV-2) is a novel viral pathogen and therefore a challenge to accurately diagnose infection. Asymptomatic cases are common and so it is difficult to accurately identify infected cases to support surveillance and case detection. Diagnostic test developers are working to meet the global demand for accurate and rapid diagnostic tests to support disease management. However, the focus of many of these has been on molecular diagnostic tests, and more recently serologic tests, for use in primarily high-income countries. Low- and middle-income countries typically have very limited access to molecular diagnostic testing due to fewer resources. Serologic testing is an inappropriate surrogate as the early stages of infection are not detected and misdiagnosis will promote continued transmission. Detection of infection via direct antigen testing may allow for earlier diagnosis provided such a method is sensitive. Leading SARS-CoV-2 biomarkers include spike protein, nucleocapsid protein, envelope protein, and membrane protein. This research focuses on antibodies to SARS-CoV-2 spike protein due to the number of monoclonal antibodies that have been developed for therapeutic research but also have potential diagnostic value. In this study, we assessed the performance of antibodies to the spike glycoprotein, acquired from both commercial and private groups in multiplexed liquid immunoassays, with concurrent testing via a half-strip lateral flow assays (LFA) to indicate antibodies with potential in LFA development. These processes allow for the selection of pairs of high-affinity antispike antibodies that are suitable for liquid immunoassays and LFA, some of which with sensitivity into the low picogram range with the liquid immunoassay formats with no cross-reactivity to other coronavirus S antigens. Discrepancies in optimal ranking were observed with the top pairs used in the liquid and LFA formats. These findings can support the development of SARS-CoV-2 LFAs and diagnostic tools.

Correction to: Performance of an ultra-sensitive Plasmodium falciparum HRP2-based rapid diagnostic test with recombinant HRP2, culture parasites, and archived whole blood samples.

Following publication of the original article [1], the authors flagged an error concerning a reference to a product in the Methods section.

A new highly sensitive enzyme-linked immunosorbent assay for the detection of Plasmodium falciparum histidine-rich protein 2 in whole blood.

BACKGROUND: The detection of submicroscopic infections in low prevalence settings has become an increasingly important challenge for malaria elimination strategies. The current field rapid diagnostic tests (RDTs) for Plasmodium falciparum malaria are inadequate to detect low-density infections. Therefore, there is a need to develop more sensitive field diagnostic tools. In parallel, a highly sensitive laboratory reference assay will be essential to evaluate new diagnostic tools. Recently, the highly sensitive Alere™ Malaria Ag P.f ELISA (HS ELISA) was developed to detect P. falciparum histidine-rich protein 2 (HRP2) in clinical whole blood specimens. In this study, the analytical and clinical performance of the HS ELISA was determined using recombinant P. falciparum HRP2, P. falciparum native culture parasites, and archived highly pedigreed clinical whole blood specimens from Karen village, Myanmar and Nagongera, Uganda. RESULTS: The HS ELISA has an analytical sensitivity of less than 25 pg/mL and shows strong specificity for P. falciparum HRP2 when tested against P. falciparum native culture strains with pfhrp2 and pfhrp3 gene deletions. Additionally, the Z'-factor statistic of 0.862 indicates the HS ELISA as an excellent, reproducible assay, and the coefficients of variation for inter- and intra-plate testing, 11.76% and 2.51%, were acceptable. Against clinical whole blood specimens with concordant microscopic and PCR results, the HS ELISA showed 100% (95% CI 96.4-100) diagnostic sensitivity and 97.9% (95% CI 94.8-99.4) diagnostic specificity. For P. falciparum positive specimens with HRP2 concentrations below 400 pg/mL, the sensitivity and specificity were 100% (95% CI 88.4-100) and 88.9% (95% CI 70.8-97.6), respectively. The overall sensitivity and specificity for all 352 samples were 100% (CI 95% 96-100%) and 97.3% (CI 95% 94-99%). CONCLUSIONS: The HS ELISA is a robust and reproducible assay. The findings suggest that the HS ELISA may be a useful tool as an affordable reference assay for new ultra-sensitive HRP2-based RDTs.

Performance of an ultra-sensitive Plasmodium falciparum HRP2-based rapid diagnostic test with recombinant HRP2, culture parasites, and archived whole blood samples.

BACKGROUND: As malaria endemic countries shift from control to elimination, the proportion of low density Plasmodium falciparum infections increases. Current field diagnostic tools, such as microscopy and rapid diagnostic tests (RDT), with detection limits of approximately 100-200 parasites/µL (p/µL) and 800-1000 pg/mL histidine-rich protein 2 (HRP2), respectively, are unable to detect these infections. A novel ultra-sensitive HRP2-based Alere™ Malaria Ag P.f RDT (uRDT) was evaluated in laboratory conditions to define the test's performance against recombinant HRP2 and native cultured parasites. RESULTS: The uRDT detected dilutions of P. falciparum recombinant GST-W2 and FliS-W2, as well as cultured W2 and ITG, diluted in whole blood down to 10-40 pg/mL HRP2, depending on the protein tested. uRDT specificity was 100% against 123 archived frozen whole blood samples. Rapid test cross-reactivity with HRP3 was investigated using pfhrp2 gene deletion strains D10 and Dd2, pfhrp3 gene deletion strain HB3, and controls pfhrp2 and pfhrp3 double deletion strain 3BD5 and pfhrp2 and pfhrp3 competent strain ITG. The commercial Standard Diagnostics, Inc. BIOLINE Malaria Ag P.f RDT (SD-RDT) and uRDT detected pfhrp2 positive strains down to 49 and 3.13 p/µL, respectively. The pfhrp2 deletion strains were detected down to 98 p/µL by both tests. CONCLUSION: The performance of the uRDT was variable depending on the protein, but overall showed a greater than 10-fold improvement over the SD-RDT. The uRDT also exhibited excellent specificity and showed the same cross-reactivity with HRP3 as the SD-RDT. Together, the results support the uRDT as a more sensitive HRP2 test that could be a potentially effective tool in elimination campaigns. Further clinical evaluations for this purpose are merited.

Feasibility of utilizing the SD BIOLINE Onchocerciasis IgG4 rapid test in onchocerciasis surveillance in Senegal.

As effective onchocerciasis control efforts in Africa transition to elimination efforts, different diagnostic tools are required to support country programs. Senegal, with its long standing, successful control program, is transitioning to using the SD BIOLINE Onchocerciasis IgG4 (Ov16) rapid test over traditional skin snip microscopy. The aim of this study is to demonstrate the feasibility of integrating the Ov16 rapid test into onchocerciasis surveillance activities in Senegal, based on the following attributes of acceptability, usability, and cost. A cross-sectional study was conducted in 13 villages in southeastern Senegal in May 2016. Individuals 5 years and older were invited to participate in a demographic questionnaire, an Ov16 rapid test, a skin snip biopsy, and an acceptability interview. Rapid test technicians were interviewed and a costing analysis was conducted. Of 1,173 participants, 1,169 (99.7%) agreed to the rapid test while 383 (32.7%) agreed to skin snip microscopy. The sero-positivity rate of the rapid test among those tested was 2.6% with zero positives 10 years and younger. None of the 383 skin snips were positive for Ov microfilaria. Community members appreciated that the rapid test was performed quickly, was not painful, and provided reliable results. The total costs for this surveillance activity was $22,272.83, with a cost per test conducted at $3.14 for rapid test, $7.58 for skin snip microscopy, and $13.43 for shared costs. If no participants had refused skin snip microscopy, the total cost per method with shared costs would have been around $16 per person tested. In this area with low onchocerciasis sero-positivity, there was high acceptability and perceived value of the rapid test by community members and technicians. This study provides evidence of the feasibility of implementing the Ov16 rapid test in Senegal and may be informative to other country programs transitioning to Ov16 serologic tools.

What are the constraints and opportunities for HIVST scale-up in Africa? Evidence from Kenya, Malawi and South Africa.

INTRODUCTION: HIV self-testing (HIVST) has the potential to increase uptake of HIV testing among untested populations in sub-Saharan Africa and is on the brink of scale-up. However, it is unclear to what extent HIVST would be supported by stakeholders, what policy frameworks are in place and how variations between contexts might influence country-preparedness for scale-up. This qualitative study assessed the perceptions of HIVST among stakeholders in three sub-Saharan countries. METHODS: Fifty-four key informant interviews were conducted in Kenya (n=16), Malawi (n=26) and South Africa (n=12) with government policy makers, academics, activists, donors, procurement specialists, laboratory practitioners and health providers. A thematic analysis was conducted in each country and a common coding framework allowed for inter-country analysis to identify common and divergent themes across contexts. RESULTS: Respondents welcomed the idea of an accurate, easy-to-use, rapid HIV self-test which could increase testing across all populations. High-risk groups, such as men, Men who have sex with men (MSM), couples and young people in particular, could be targeted through a range of health facility and community-based distribution points. HIVST is already endorsed in Kenya, and political support for scale-up exists in South Africa and Malawi. However, several caveats remain. Further research, policy and ensuing guidelines should consider how to regulate, market and distribute HIVST, ensure quality assurance of tests and human rights, and critically, link testing to appropriate support and treatment services. Low literacy levels in some target groups would also need context-specific consideration before scale up. World Health Organization (WHO) policy and regulatory frameworks are needed to guide the process in those areas which are new or specific to self-testing. CONCLUSIONS: Stakeholders in three HIV endemic sub-Saharan countries felt that HIVST will be an important complement to existing community and facility-based testing approaches if accompanied by the same essential components of any HIV testing service, including access to accurate information and linkages to care. While there is an increasingly positive global policy environment regarding HIVST, several implementation and social challenges limit scale-up. There is a need for further research to provide contextual and operational evidence that addresses concerns and contributes to normative WHO guidance.

What should the ideal HIV self-test look like? A usability study of test prototypes in unsupervised HIV self-testing in Kenya, Malawi, and South Africa.

HIV self-testing (HIVST) is increasingly being sought and offered globally, yet there is limited information about the test features that will be required for an HIV self-test to be easy to use, acceptable to users, and feasible for manufacturers to produce. We conducted formative usability research with participants who were naïve to HIVST using five prototypes in Kenya, Malawi, and South Africa. The tests selected ranged from early-stage prototypes to commercially ready products and had a diverse set of features. A total of 150 lay users were video-recorded conducting unsupervised self-testing and interviewed to understand their opinions of the test. Participants did not receive a test result, but interpreted standardized result panels. This study demonstrated that users will refer to the instructions included with the test, but these can be confusing or difficult to follow. Errors were common, with less than 25% of participants conducting all steps correctly and 47.3% of participants performing multiple errors, particularly in sample collection and transfer. Participants also had difficulty interpreting results. To overcome these issues, the ideal HIV self-test requires pictorial instructions that are easy to understand, simple sample collection with integrated test components, fewer steps, and results that are easy to interpret.

Comparison of ThinPrep and SurePath liquid-based cytology and subsequent human papillomavirus DNA testing in China.

BACKGROUND: Liquid-based cytology (LBC) has been compared with conventional cytology in numerous studies. In the current study of 2 LBC systems, the accuracy, rates of unsatisfactory cytology, and sufficiency of residual LBC specimens for Hybrid Capture 2 (HC2) HPV DNA testing were compared. METHODS: Eligible women ages 30 to 49 years were recruited for this cross-sectional population-based study in rural China. Women were assessed by visual inspection with acetic acid (VIA), LBC, and high-risk HPV HC2 DNA assay. Cervical specimens were preserved according to SurePath or ThinPrep protocols. LBC results were manually read. HC2 testing was performed on specimens with sufficient residual volume. Colposcopies and biopsies were performed on women who were VIA positive at the time of initial screening. Women with abnormal LBC or HC2 test results were called back for colposcopies and 4-quadrant cervical biopsies. RESULTS: Of 2005 eligible women, 972 were tested by SurePath and 1033 by ThinPrep. Compared with SurePath samples, ThinPrep samples had higher rates of unsatisfactory cytology (0.2% for SurePath and 1.5% for ThinPrep) and insufficient residual volume for HC2 (0.0% for SurePath and 18.2% for ThinPrep). SurePath samples yielded higher sensitivities and similar specificities for LBC and HC2 testing of residual specimens, but these differences were not determined to be significant by area-under-the-curve analysis (LBC performance: 0.89 for SurePath and 0.85 for ThinPrep; HC2 performance: 0.91 for SurePath and 0.89 for ThinPrep). CONCLUSIONS: Both methods yielded similar validity in detecting significant cervical lesions. However, SurePath samples yielded higher rates of satisfactory LBC slides and sufficient residual volume for HC2.

Association of elevated E6 oncoprotein with grade of cervical neoplasia using PDZ interaction-mediated precipitation of E6.

OBJECTIVE: To determine the expression of human papillomavirus (HPV) type 16 E6 oncoprotein in cervical specimens of women with and without cervical intraepithelial neoplasia (CIN). MATERIALS AND METHODS: Cervical specimens from 2,530 unscreened women aged 30 to 54 years from Shanxi, China, were obtained. All women were assessed by liquid-based cytology, high-risk HPV DNA tests, and colposcopy with directed biopsy and endocervical curettage as necessary. Women with abnormal cytologic results or positive HPV DNA results were recalled for colposcopy, 4-quadrant cervical biopsies, and endocervical curettage. Women with biopsy-proven CIN and cancer and a convenience sample of HC2-positive, disease-negative women were tested for the presence of HPV-16 infection via HPV-16 E6 DNA-specific polymerase chain reaction. A PDZ interaction-mediated E6 oncoprotein precipitation method followed by E6-specific Western blot was performed on specimens from women with HPV-16 infections. Associations between elevated expression of E6 oncoprotein and CIN 2 and 3 were determined using logistic regression and a reference category of CIN 1 and disease-negative. RESULTS: A significant trend for the detection of HPV-16 E6 oncoprotein in specimen of women with proven HPV-16 infection was determined: 0% (0/12), 12.5% (1/8), 36.4% (4/11), and 42.9% (3/7) of those with negative findings, CIN 1, 2, and 3, respectively (p = .01). Compared with the category combining negative findings and CIN 1, detection of E6 oncoprotein was associated with CIN 2 (odds ratio = 10.9, p = .05) and CIN 3 (odds ratio = 14.3, p = .04). CONCLUSIONS: There is a significant association between elevated expression of E6 oncoprotein and grade of CIN. This finding seems consistent with the role played by E6 oncoprotein in carcinogenesis.

Feasibility study of a human papillomavirus E6 oncoprotein test for diagnosis of cervical precancer and cancer.

In a feasibility study using a prototype, lateral-flow test for human papillomavirus type 16, 18, and/or 45 (HPV16/18/45) E6 oncoproteins, 51 of 75 (68%; 95% confidence interval [95% CI] of 56 to 78%) of HPV16/18/45 DNA-positive specimens from women with a diagnosis of CIN3+ (cervical intraepithelial neoplasia grade 3+ or cervical cancer) tested positive for HPV16/18/45 E6 oncoprotein. None of 16 (95% CI of 0 to 37%) HPV16/18/45 DNA-positive cervical specimens from women with a negative or CIN1 diagnosis tested positive for HPV16/18/45 E6 oncoprotein.

A new HPV-DNA test for cervical-cancer screening in developing regions: a cross-sectional study of clinical accuracy in rural China.

BACKGROUND: A new test (careHPV; QIAGEN, Gaithersburg, MD, USA) has been developed to detect 14 high-risk types of carcinogenic human papillomavirus (HPV) in about 2.5 h, to screen women in developing regions for cervical intraepithelial neoplasia (CIN). We did a cross-sectional study to assess the clinical accuracy of careHPV as a rapid screening test in two county hospitals in rural China. METHODS: From May 10 to June 15, 2007, the careHPV test was done locally by use of self-obtained vaginal and provider-obtained cervical specimens from a screening population-based set of 2530 women aged 30 to 54 years in Shanxi province, China. All women were assessed by visual inspection with acetic acid (VIA), Digene High-Risk HPV HC2 DNA Test (HC2), liquid-based cytology, and colposcopy with directed biopsy and endocervical curettage as necessary. In 2388 women with complete data, 441 women with negative colposcopy, but unsatisfactory or abnormal cytology or who were positive on HC2 or the new careHPV test, were recalled for a second colposcopy, four-quadrant cervical biopsies, and endocervical curettage. An absence of independence between the tests was not adjusted for and the Bonferroni correction was used for multiple comparisons. FINDINGS: Complete data were available for 2388 (94.4%) women. 70 women had CIN2+ (moderate or severe CIN or cancer), of whom 23 had CIN3+. By use of CIN2+ as the reference standard and area-under-the-curve analysis with a two-sided alpha error level of 0.0083, the sensitivities and specificities of the careHPV test for a cut-off ratio cut-point of 0.5 relative light units, were 90.0% (95% CI 83.0-97.0) and 84.2% (82.7-85.7), respectively, on cervical specimens, and 81.4% (72.3-90.5) and 82.4% (80.8-83.9), respectively, on vaginal specimens (areas under the curve not significantly different, p=0.0596), compared with 41.4% (29.9-53.0) and 94.5% (93.6-95.4) for VIA (areas under the curve significantly different, p=0.0001 and p=0.0031, for cervical and vaginal-specimen comparisons for the careHPV test, respectively). The sensitivity and specificity of HC2 for cervical specimens were 97.1% (93.2-100) and 85.6% (84.2-87.1), respectively (areas under the curve not significantly different from the careHPV test on cervical specimens, p=0.0163). INTERPRETATION: The careHPV test is promising as a primary screening method for cervical-cancer prevention in low-resource regions.