Electrochemical Immunoassay for Capturing Capsular Polysaccharide of Burkholderia pseudomallei: Early Onsite Detection of Melioidosis.

This study presented the detection and quantification of capsular polysaccharide (CPS) as a biomarker for the diagnosis of melioidosis. After successfully screening four monoclonal antibodies (mAbs) previously determined to bind CPS molecules, the team developed a portable electrochemical immunosensor based on antibody-antigen interactions. The biosensor was able to detect CPS with a wide detection range from 0.1pg/mL to 1 µg/mL. The developed biosensor achieved high sensitivity for the detection of CPS spiked into both urine and serum. The developed assay platform was successfully programmed into a Windows app, and the sensor performance was evaluated with different spiked concentrations. The rapid electro-analytical device (READ) sensor showed great unprecedented sensitivity for the detection of CPS molecules in both serum and urine, and results were cross-validated with ELISA methods.

Serum antibody levels to SARS-CoV-2 receptor-binding domain (RBD) in convalescent patients and vaccinated individuals of northern Nevada.

Antibodies reactive with the SARS-CoV-2 receptor-binding domain (RBD) of the spike protein are associated with viral neutralization, however low antibody titers, specifically against SARS-CoV-2 variants, may result in reduced viral immunity post naturally acquired infection. A cohort study comprised of 121 convalescent individuals from northern Nevada was conducted looking at anti-RBD antibody levels by enzyme-linked immunosorbent assay. Serum was collected from volunteers by staff at the University of Nevada, Reno School of Medicine Clinical Research Center and assessed for antibodies reactive to various SARS-CoV-2 RBD domains relevant to the time of the study (2020-2021). A nonpaired group of vaccinated individuals were assessed in parallel. The goal of the study was to identify antibody levels against the RBD subunit in convalescent and vaccinated individuals from northern Nevada.

Functional assays to screen and select monoclonal antibodies that target Yersinia pestis.

Yersinia pestis is a gram-negative bacterium that causes plague in animals and humans. Depending on the route of disease transmission, the bacterium can cause an acute, often fatal disease that has a narrow window for treatment with antibiotics. Additionally, antibiotic resistant strains have been identified, emphasizing the need for novel treatments. Antibody therapy is an appealing option that can direct the immune system to clear bacterial infections. Advances in biotechnology have made both engineering and producing antibodies easier and more affordable. In this study, two screening assays were optimized to evaluate the ability of antibodies to promote phagocytosis of Y. pestis by macrophages and to induce a cytokine signature in vitro that may be predictive of protection in vivo. We evaluated a panel of 21 mouse monoclonal antibodies targeting either the anti-phagocytic capsule F1 protein or the LcrV antigen, which is part of the type 3 secretion system that facilitates translocation of virulence factors into the host cell, using two functional assays. Anti-F1 and anti-LcrV monoclonal antibodies both increased bacterial uptake by macrophages, with greater uptake observed in the presence of antibodies that were protective in the mouse pneumonic plague model. In addition, the protective anti-F1 and anti-LcrV antibodies produced unique cytokine signatures that were also associated with in vivo protection. These antibody-dependent characteristics from in vitro functional assays will be useful in down-selecting efficacious novel antibodies that can be used for treatment of plague.

Digital image analysis for biothreat detection via rapid centrifugal microfluidic orthogonal flow immunocapture.

We report clear proof-of-principle for centrifugally-driven, multiplexed, paper-based orthogonal flow sandwich-style immunocapture (cOFI) and colorimetric detection of Zaire Ebola virus-like particles. Capture antibodies are immobilized onto nanoporous nitrocellulose membranes that are then laminated into polymeric microfluidic discs to yield ready-to-use analytical devices. Fluid flow is controlled solely by rotational speed, obviating the need for complex pneumatic pumping systems, and providing more precise flow control than with the capillary-driven flow used in traditional lateral flow immunoassays (LFIs). Samples containing the antigen of interest and gold nanoparticle-labeled detection antibodies are pumped centrifugally through the embedded, prefunctionalized membrane where they are subsequently captured to generate a positive, colorimetric signal. When compared to the equivalent LFI counterparts, this cOFI approach generated immunochromatographic colorimetric responses that are objectively darker (saturation), more intense (grayscale), and less variable regarding total area of the color response. We also describe an image analysis approach that enables access to rich color data and area statistics without the need for a commercial 'strip reader' or custom-written image analysis algorithms. Instead, our analytical method exploits inexpensive equipment (e.g., smart phone, flatbed scanner, etc.) and freely available software (Fiji distribution of ImageJ) to permit characterization of immunochromatographic responses that includes multiple color metrics, offering insights beyond typical grayscale analysis. The findings reported here stand as clear proof-of-principle for the feasibility of disc-based, centrifugally driven orthogonal flow through a membrane with immunocapture (cOFI) and colorimetric readout of a sandwich-type immunoassay in less than 15 minutes. Once fully developed, this cOFI platform could render a faster, more accurate diagnosis, while processing multiple samples simul-taneously.

Comparison of a Prototype SARS-CoV-2 Lateral Flow IMMUNOASSAY with the BinaxNOWTM COVID-19 Antigen CARD.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the virus responsible for the COVID-19 pandemic. From the onset of the pandemic, rapid antigen tests have quickly proved themselves to be an accurate and accessible diagnostic platform. The initial (and still most commonly used antigen tests) for COVID-19 diagnosis were constructed using monoclonal antibodies (mAbs) specific to severe acute respiratory syndrome coronavirus (SARS-CoV) nucleocapsid protein (NP). These mAbs are able to bind SARS-CoV-2 NP due to high homology between the two viruses. However, since first being identified in 2019, SARS-CoV-2 has continuously mutated, and a multitude of variants have appeared. These mutations have an elevated risk of leading to possible diagnostic escape when using tests produced with SARS-CoV-derived mAbs. Here, we established a library of 18 mAbs specific to SARS-CoV-2 NP and used two of these mAbs (1CV7 and 1CV14) to generate a prototype antigen-detection lateral flow immunoassay (LFI). A side-by-side analysis of the 1CV7/1CV14 LFI and the commercially available BinaxNOWTM COVID-19 Antigen CARD was performed. Results indicated the 1CV7/1CV14 LFI outperformed the BinaxNOWTM test in the detection of BA.2, BA.2.12.1, and BA.5 Omicron sub-variants when testing remnant RT-PCR positive patient nasopharyngeal swabs diluted in viral transport media.

Optimization of an Antibody Microarray Printing Process Using a Designed Experiment.

Antibody microarrays have proven useful in immunoassay-based point-of-care diagnostics for infectious diseases. Noncontact piezoelectric inkjet printing has advantages to print antibody microarrays on nitrocellulose substrates for this application due to its compatibility with sensitive solutions and substrates, simple droplet control, and potential for high-capacity printing. However, there remain real-world challenges in printing such microarrays, which motivated this study. The effects of three concentrations of capture antibody (cAb) reagents and nozzle hydrostatic pressures were chosen to investigate three responses: the number of printed membrane disks, dispensing performance, and microarray quality. Printing conditions were found to be most ideal with 5 mg/mL cAb and a nozzle hydrostatic pressure near zero, which produced 130 membrane disks in a single print versus the 10 membrane disks per print before optimization. These results serve to inform efficient printing of antibody microarrays on nitrocellulose membranes for rapid immunoassay-based detection of infectious diseases and beyond.

Detection and Quantification of the Capsular Polysaccharide of Burkholderia pseudomallei in Serum and Urine Samples from Melioidosis Patients.

Burkholderia pseudomallei is the causative agent of melioidosis, a life-threatening disease common in Southeast Asia and northern Australia. Melioidosis often presents with nonspecific symptoms and has a fatality rate of upwards of 70% when left untreated. The gold standard for diagnosis is culturing B. pseudomallei from patient samples. Bacterial culture, however, can take up to 7 days, and its sensitivity is poor, at roughly 60%. The successful administration of appropriate antibiotics is reliant on rapid and accurate diagnosis. Hence, there is a genuine need for new diagnostics for this deadly pathogen. The Active Melioidosis Detect (AMD) lateral flow immunoassay (LFI) detects the capsular polysaccharide (CPS) of B. pseudomallei. The assay is designed for use on various clinical samples, including serum and urine; however, there are limited data to support which clinical matrices are the best candidates for detecting CPS. In this study, concentrations of CPS in paired serum and urine samples from melioidosis patients were determined using a quantitative antigen capture enzyme-linked immunosorbent assay. In parallel, samples were tested with the AMD LFI, and the results of the two immunoassays were compared. Additionally, centrifugal concentration was performed on a subset of urine samples to determine if this method may improve detection when CPS levels are initially low or undetectable. The results indicate that while CPS levels varied within the two matrices, there tended to be higher concentrations in urine. The AMD LFI detected CPS in 40.5% of urine samples, compared to 6.5% of serum samples, suggesting that urine is a preferable matrix for point-of-care diagnostic assays. IMPORTANCE Melioidosis is very challenging to diagnose. There is a clear need for a point-of-care assay for the detection of B. pseudomallei antigen directly from patient samples. The Active Melioidosis Detect lateral flow immunoassay detects the capsular polysaccharide (CPS) of B. pseudomallei and is designed for use on various clinical samples, including serum and urine. However, there are limited data regarding which clinical matrix is preferable for the detection of CPS. This study addresses this question by examining quantitative CPS levels in paired serum and urine samples and relating them to clinical parameters. Additionally, centrifugal concentration was performed on a subset of urine samples to determine whether this might enable the detection of CPS in samples in which it was initially present at low or undetectable levels. These results provide valuable insights into the detection of CPS in patients with melioidosis and suggest potential ways forward in the diagnosis and treatment of this challenging disease.

Accuracy of 2 Rapid Antigen Tests During 3 Phases of SARS-CoV-2 Variants.

Importance: Variants of SARS-CoV-2 have sequence variations in the viral genome that may alter the accuracy of rapid diagnostic tests. Objective: To assess the analytical and clinical accuracy of 2 rapid diagnostic tests for detecting SARS-CoV-2 during 3 phases of variants. Design, Setting, and Participants: This diagnostic study included participants aged 18 years or older who reported onset of COVID-19-like symptoms within the prior 5 days and were tested at multiple COVID-19 testing locations in King County, Washington, from February 17, 2021, to January 11, 2022, during 3 distinct phases of SARS-CoV-2 infection (pre-Delta, Delta, and Omicron). Interventions: Two anterior nasal swab specimens were collected from each participant-1 for onsite testing by the SCoV-2 Ag Detect Rapid Self-Test and 1 for reverse transcriptase-polymerase chain reaction (RT-PCR) testing. Main Outcomes and Measures: The analytical limit of detection of the 2 rapid diagnostic tests (SCoV-2 Ag Detect Rapid Self-Test and BinaxNOW COVID-19 Ag Card) was assessed using Omicron (B.1.1.529/BA.1), Delta (B.1.617.2), and a wild-type (USA-WA1/2020) variant. Diagnostic sensitivity and specificity of clinical testing for the rapid antigen tests were compared with that of RT-PCR testing. Results: A total of 802 participants were enrolled (mean [SD] age, 37.3 [13.3] years; 467 [58.2%] female), 424 (52.9%) of whom had not received COVID-19 vaccination and presented a median of 2 days (IQR, 1-3 days) from symptom onset. Overall, no significant differences were found in the analytical limit of detection or clinical diagnostic accuracy of rapid antigen testing across SARS-CoV-2 variants. The estimated limit of detection for both rapid nucleocapsid antigen tests was at or below a 50% tissue culture infectious dose of 62.5, and the positive percent agreement of the SCoV-2 Ag Detect Rapid Self-Test ranged from 81.2% (95% CI, 69.5%-89.9%) to 90.7% (95% CI, 77.9%-97.4%) across the 3 phases of variants. The diagnostic sensitivity increased for nasal swabs with a lower cycle threshold by RT-PCR, which correlates with a higher viral load. Conclusions and Relevance: In this diagnostic study, analytical and clinical performance data demonstrated accuracy of 2 rapid antigen tests among adults with COVID-19 symptoms across 3 phases of SARS-CoV-2 variants. The findings suggest that home-based rapid antigen testing programs may be an important intervention to reduce global SARS-CoV-2 transmission.

Rapid Capsular Antigen Immunoassay for Diagnosis of Inhalational Anthrax: Preclinical Studies and Evaluation in a Nonhuman Primate Model.

Inhalational anthrax is a fatal infectious disease. Rapid and effective treatment is critically dependent on early and accurate diagnosis. Blood culture followed by identification and confirmation may take days to provide clinically relevant information. In contrast, immunoassay for a shed antigen, the capsular polypeptide gamma-d-polyglutamate (γDPGA), can provide rapid results at the point of care. In this study, a lateral flow immunoassay for γDPGA was evaluated in a robust nonhuman primate model of inhalational anthrax. The results showed that the time to a positive result with the rapid test using either serum or blood as a clinical specimen was similar to the time after infection when a blood culture became positive. In vitro testing showed that the test was equally sensitive with cultures of the three major clades of Bacillus anthracis. Cultures from other Bacillus spp. that are known to produce γDPGA also produced positive results. The test was negative with human sera from 200 normal subjects and 45 subjects with culture-confirmed nonanthrax bacterial or fungal sepsis. Taken together, the results showed that immunoassay for γDPGA is an effective surrogate for blood culture in a relevant cynomolgus monkey model of inhalational anthrax. The test would be a valuable aid in early diagnosis of anthrax, which is critical for rapid intervention and a positive outcome. Use of the test could facilitate triage of patients with signs and symptoms of anthrax in a mass-exposure incident and in low-resource settings where laboratory resources are not readily available. IMPORTANCE Patient outcome in anthrax is critically dependent on early diagnosis followed by effective treatment. We describe a rapid lateral flow immunoassay that detects capsular antigen of Bacillus anthracis that is shed into blood during infection. The test was evaluated in a robust cynomolgus monkey model of inhalational anthrax. Rapid detection of capsular antigen is an effective surrogate for the time-consuming and laboratory-intensive diagnosis by blood culture, direct fluorescent antibody staining, or other molecular testing. The test can be performed at the point of patient contact, is rapid and inexpensive, and can be used by individuals with minimal training.

Development of a dual antigen lateral flow immunoassay for detecting Yersinia pestis.

BACKGROUND: Yersinia pestis is the causative agent of plague, a zoonosis associated with small mammals. Plague is a severe disease, especially in the pneumonic and septicemic forms, where fatality rates approach 100% if left untreated. The bacterium is primarily transmitted via flea bite or through direct contact with an infected host. The 2017 plague outbreak in Madagascar resulted in more than 2,400 cases and was highlighted by an increased number of pneumonic infections. Standard diagnostics for plague include laboratory-based assays such as bacterial culture and serology, which are inadequate for administering immediate patient care for pneumonic and septicemic plague. PRINCIPAL FINDINGS: The goal of this study was to develop a sensitive rapid plague prototype that can detect all virulent strains of Y. pestis. Monoclonal antibodies (mAbs) were produced against two Y. pestis antigens, low-calcium response V (LcrV) and capsular fraction-1 (F1), and prototype lateral flow immunoassays (LFI) and enzyme-linked immunosorbent assays (ELISA) were constructed. The LFIs developed for the detection of LcrV and F1 had limits of detection (LOD) of roughly 1-2 ng/mL in surrogate clinical samples (antigens spiked into normal human sera). The optimized antigen-capture ELISAs produced LODs of 74 pg/mL for LcrV and 61 pg/mL for F1 when these antigens were spiked into buffer. A dual antigen LFI prototype comprised of two test lines was evaluated for the detection of both antigens in Y. pestis lysates. The dual format was also evaluated for specificity using a small panel of clinical near-neighbors and other Tier 1 bacterial Select Agents. CONCLUSIONS: LcrV is expressed by all virulent Y. pestis strains, but homologs produced by other Yersinia species can confound assay specificity. F1 is specific to Y. pestis but is not expressed by all virulent strains. Utilizing highly reactive mAbs, a dual-antigen detection (multiplexed) LFI was developed to capitalize on the diagnostic strengths of each target.

Characterization of a Centrifugal Microfluidic Orthogonal Flow Platform.

To bring to bear the power of centrifugal microfluidics on vertical flow immunoassays, control of flow orthogonally through nanoporous membranes is essential. The on-disc approach described here leverages the rapid print-cut-laminate (PCL) disc fabrication and prototyping method to create a permanent seal between disc materials and embedded nanoporous membranes. Rotational forces drive fluid flow, replacing capillary action, and complex pneumatic pumping systems. Adjacent microfluidic features form a flow path that directs fluid orthogonally (vertically) through these embedded membranes during assay execution. This method for membrane incorporation circumvents the need for solvents (e.g., acetone) to create the membrane-disc bond and sidesteps issues related to undesirable bypass flow. In other recently published work, we described an orthogonal flow (OF) platform that exploited embedded membranes for automation of enzyme-linked immunosorbent assays (ELISAs). Here, we more fully characterize flow patterns and cellulosic membrane behavior within the centrifugal orthogonal flow (cOF) format. Specifically, high-speed videography studies demonstrate that sample volume, membrane pore size, and ionic composition of the sample matrix significantly impact membrane behavior, and consequently fluid drainage profiles, especially when cellulosic membranes are used. Finally, prototype discs are used to demonstrate proof-of-principle for sandwich-type antigen capture and immunodetection within the cOF system.

Development of Immunoassays for Detection of Francisella tularensis Lipopolysaccharide in Tularemia Patient Samples.

Francisella tularensis is the causative agent of tularemia, a zoonotic bacterial infection that is often fatal if not diagnosed and treated promptly. Natural infection in humans is relatively rare, yet persistence in animal reservoirs, arthropod vectors, and water sources combined with a low level of clinical recognition make tularemia a serious potential threat to public health in endemic areas. F. tularensis has also garnered attention as a potential bioterror threat, as widespread dissemination could have devastating consequences on a population. A low infectious dose combined with a wide range of symptoms and a short incubation period makes timely diagnosis of tularemia difficult. Current diagnostic techniques include bacterial culture of patient samples, PCR and serological assays; however, these techniques are time consuming and require technical expertise that may not be available at the point of care. In the event of an outbreak or exposure a more efficient diagnostic platform is needed. The lipopolysaccharide (LPS) component of the bacterial outer leaflet has been identified previously by our group as a potential diagnostic target. For this study, a library of ten monoclonal antibodies specific to F. tularensis LPS were produced and confirmed to be reactive with LPS from type A and type B strains. Antibody pairs were tested in an antigen-capture enzyme-linked immunosorbent assay (ELISA) and lateral flow immunoassay format to select the most sensitive pairings. The antigen-capture ELISA was then used to detect and quantify LPS in serum samples from tularemia patients for the first time to determine the viability of this molecule as a diagnostic target. In parallel, prototype lateral flow immunoassays were developed, and reactivity was assessed, demonstrating the potential utility of this assay as a rapid point-of-care test for diagnosis of tularemia.

Development of an antigen detection assay for early point-of-care diagnosis of Zaire ebolavirus.

The 2013-2016 Ebola virus (EBOV) outbreak in West Africa and the ongoing cases in the Democratic Republic of the Congo have spurred development of a number of medical countermeasures, including rapid Ebola diagnostic tests. The likelihood of transmission increases as the disease progresses due to increasing viral load and potential for contact with others. Early diagnosis of EBOV is essential for halting spread of the disease. Polymerase chain reaction assays are the gold standard for diagnosing Ebola virus disease (EVD), however, they rely on infrastructure and trained personnel that are not available in most resource-limited settings. Rapid diagnostic tests that are capable of detecting virus with reliable sensitivity need to be made available for use in austere environments where laboratory testing is not feasible. The goal of this study was to produce candidate lateral flow immunoassay (LFI) prototypes specific to the EBOV glycoprotein and viral matrix protein, both targets known to be present during EVD. The LFI platform utilizes antibody-based technology to capture and detect targets and is well suited to the needs of EVD diagnosis as it can be performed at the point-of-care, requires no cold chain, provides results in less than twenty minutes and is low cost. Monoclonal antibodies were isolated, characterized and evaluated in the LFI platform. Top performing LFI prototypes were selected, further optimized and confirmed for sensitivity with cultured live EBOV and clinical samples from infected non-human primates. Comparison with a commercially available EBOV rapid diagnostic test that received emergency use approval demonstrates that the glycoprotein-specific LFI developed as a part of this study has improved sensitivity. The outcome of this work presents a diagnostic prototype with the potential to enable earlier diagnosis of EVD in clinical settings and provide healthcare workers with a vital tool for reducing the spread of disease during an outbreak.

Immunoglobulin G subclass switching impacts sensitivity of an immunoassay targeting Francisella tularensis lipopolysaccharide.

The CDC Tier 1 select agent Francisella tularensis is a small, Gram-negative bacterium and the causative agent of tularemia, a potentially life-threatening infection endemic in the United States, Europe and Asia. Currently, there is no licensed vaccine or rapid point-of-care diagnostic test for tularemia. The purpose of this research was to develop monoclonal antibodies (mAbs) specific to the F. tularensis surface-expressed lipopolysaccharide (LPS) for a potential use in a rapid diagnostic test. Our initial antigen capture ELISA was developed using murine IgG3 mAb 1A4. Due to the low sensitivity of the initial assay, IgG subclass switching, which is known to have an effect on the functional affinity of a mAb, was exploited for the purpose of enhancing assay sensitivity. The ELISA developed using the IgG1 or IgG2b mAbs from the subclass-switch family of 1A4 IgG3 yielded improved assay sensitivity. However, surface plasmon resonance (SPR) demonstrated that the functional affinity was decreased as a result of subclass switching. Further investigation using direct ELISA revealed the potential self-association of 1A4 IgG3, which could explain the higher functional affinity and higher assay background seen with this mAb. Additionally, the higher assay background was found to negatively affect assay sensitivity. Thus, enhancement of the assay sensitivity by subclass switching is likely due to the decrease in assay background, simply by avoiding the self-association of IgG3.

Immunoassay for Capsular Antigen of Bacillus anthracis Enables Rapid Diagnosis in a Rabbit Model of Inhalational Anthrax.

Inhalational anthrax is a serious biothreat. Effective antibiotic treatment of inhalational anthrax requires early diagnosis; the further the disease has progressed, the less the likelihood for cure. Current means for diagnosis such as blood culture require several days to a result and require advanced laboratory infrastructure. An alternative approach to diagnosis is detection of a Bacillus anthracis antigen that is shed into blood and can be detected by rapid immunoassay. The goal of the study was to evaluate detection of poly-γ-D-glutamic acid (PGA), the capsular antigen of B. anthracis, as a biomarker surrogate for blood culture in a rabbit model of inhalational anthrax. The mean time to a positive blood culture was 26 ± 5.7 h (mean ± standard deviation), whereas the mean time to a positive ELISA was 22 ± 4.2 h; P = 0.005 in comparison with blood culture. A lateral flow immunoassay was constructed for detection of PGA in plasma at concentrations of less than 1 ng PGA/ml. Use of the lateral flow immunoassay for detection of PGA in the rabbit model found that antigen was detected somewhat earlier than the earliest time point at which the blood culture became positive. The low cost, ease of use, and rapid time to result of the lateral flow immunoassay format make an immunoassay for PGA a viable surrogate for blood culture for detection of infection in individuals who have a likelihood of exposure to B. anthracis.

Genetic and pathological characteristics of Cryptococcus gattii and Cryptococcus neoformans var. neoformans from meningoencephalitis in autochthonous goats and mouflons, Sardinia, Italy.

In this study, we examined in Sardinia the brain of 555 autochthonous sheep, 50 goats, and 4 mouflons which were found affected by neurological signs. We found 6 goats and one mouflon with meningoencephalitis caused by Cryptococcus sp. There was no evidence of cryptococcal infections in any of the examined sheep. MLST genotyping on Cryptococcus sp. isolates identified Cryptococcus gatti genotype AFLP4/VGI and Cryptococcus neoformans var. neoformans genotype AFLP2/VNIV. Phylogenetically, all Cryptococcus gattii isolates fell within the autochthonous animal, human and environmental Mediterranean isolate cluster, forming a distinct branch along with environmental strains from Alicante, in the southern Mediterranean coast of Spain.

Serotype sensitivity of a lateral flow immunoassay for cryptococcal antigen.

To meet the needs of a global community, an immunoassay for cryptococcal antigen (CrAg) must have high sensitivity for CrAg of all major serotypes. A new immunoassay for CrAg in lateral flow format was evaluated and found to have a high sensitivity for detection of serotypes A, B, C, and D.

Large-scale evaluation of the immuno-mycologics lateral flow and enzyme-linked immunoassays for detection of cryptococcal antigen in serum and cerebrospinal fluid.

Cryptococcosis is a systemic infection caused by the pathogenic yeasts Cryptococcus neoformans and C. gattii. Detection of cryptococcal capsular antigen (CrAg) in serum and cerebrospinal fluid (CSF) plays an important diagnostic role. We prospectively compared the new Immuno-Mycologics Inc. (IMMY) lateral flow assay (LFA) and enzyme immunoassay (EIA) to our current CrAg test (Premier EIA; Meridian Bioscience Inc.). Discordant samples were retested with the latex-Cryptococcus antigen test (IMMY) and using serotype-specific monoclonal antibodies (MAbs). A total of 589 serum and 411 CSF specimens were tested in parallel. Qualitative agreement across assays was 97.7%. In all, 56 (41 serum and 15 CSF) samples were positive and 921 (527 serum and 394 CSF) samples were negative by all three assays. The 23 discrepant specimens were all Meridian EIA negative. Of 23 discordant specimens, 20 (87.0%) were positive by both the IMMY LFA and EIA, 2 were LFA positive only, and 1 was EIA positive only. Eleven discrepant specimens had adequate volume for latex agglutination (LA) testing; 8 were LA positive, and 3 were LA negative. LA-negative samples (2 CSF samples and 1 serum) had low IMMY LFA/EIA titers (≤1:10). Serotype-specific MAb analysis of the LA-positive samples suggested that these specimens contained CrAg epitopes similar to those of serotype C strains. In conclusion, the IMMY assays showed excellent overall concordance with the Meridian EIA. Assay performance differences were related to issues of analytic sensitivity and possible serotype bias. Incomplete access to patient-level data combined with low specimen volumes limited our ability to fully resolve discrepant results.

A defect in ATP-citrate lyase links acetyl-CoA production, virulence factor elaboration and virulence in Cryptococcus neoformans.

The interaction of Cryptococcus neoformans with phagocytic cells of the innate immune system is a key step in disseminated disease leading to meningoencephalitis in immunocompromised individuals. Transcriptional profiling of cryptococcal cells harvested from cell culture medium or from macrophages found differential expression of metabolic and other functions during fungal adaptation to the intracellular environment. We focused on the ACL1 gene for ATP-citrate lyase, which converts citrate to acetyl-CoA, because this gene showed elevated transcript levels in macrophages and because of the importance of acetyl-CoA as a central metabolite. Mutants lacking ACL1 showed delayed growth on medium containing glucose, reduced cellular levels of acetyl-CoA, defective production of virulence factors, increased susceptibility to the antifungal drug fluconazole and decreased survival within macrophages. Importantly, acl1 mutants were unable to cause disease in a murine inhalation model, a phenotype that was more extreme than other mutants with defects in acetyl-CoA production (e.g. an acetyl-CoA synthetase mutant). Loss of virulence is likely due to perturbation of critical physiological interconnections between virulence factor expression and metabolism in C. neoformans. Phylogenetic analysis and structural modelling of cryptococcal Acl1 identified three indels unique to fungal protein sequences; these differences may provide opportunities for the development of pathogen-specific inhibitors.

Phenotypic heterogeneity in expression of epitopes in the Cryptococcus neoformans capsule.

The opportunistic yeast Cryptococcus neoformans is surrounded by a polysaccharide capsule comprised primarily of glucuronoxylomannan (GXM). GXM is a key component of the antigenic character of the capsule. Expression of the epitope that allows for binding of mAbs that require O-acetylation of GXM for mAb recognition was greatly influenced by cell age, growth conditions and serotype. Yeast cells of serotype A grown in vitro under capsule induction conditions showed considerable cell-to-cell variability in binding of two O-acetyl-dependent mAbs, and such mAbs uniformly failed to bind to GXM that covers yeast buds. Expression of the O-acetyl-dependent epitope increased with cell age. In contrast, all serotype A cells harvested from brain tissue bound the same O-acetyl-dependent mAbs. The ability of the cryptococcal capsule to activate the complement cascade and bind C3 occurred uniformly over the surface of all yeast cells, including the bud. Finally, the cell-to-cell variability in binding of O-acetyl-dependent mAbs with strains of serotype A was not found with strains of serotype D; almost all cells of serotype D showed homogeneous binding of O-acetyl-dependent mAbs. These results indicate that variability in expression of antigenic epitopes by GXM should be considered in selection of mAbs used for immunodiagnosis or immunotherapy.