Disclosed herein are methods of detecting microbial infection in mammalian subjects comprising treatment of a sample and detection of polysaccharide antigenic components. The methods disclosed provide for pretreatment of biological samples, such as urine samples, to maximize detection of galF - containing antigens and improvement of sensitivity of galF antigen detection assays. The methods include minimizing Intelectin-1 binding to galF - containing antigens and improvement of monoclonal antibody binding. The detection methods are useful for identifying the presence of microbial antigens related to Streptococcus pneumoniae, Klebsiella pneumonia, Escherichia coli, Mycobacteria species, Malassezia species, Aspergillus species, Fusarium species, Alternaria species, Coccidioides species, Cryptococcus species, Mucormycetes, Histoplasma species, Neosartorya species, Fusarium species, Paracoccidioides species, or combinations thereof.
Marr, Kieren A.
Datta, Kausik
Mehta, Seema
Ostrander, Darin B.
Rock, Michelle
Francis, Jesse
Feldmesser, Marta
Background. Establishing rapid diagnoses of invasive aspergillosis (IA) is a priority tests that detect galactomannan and beta-D-glucan are available, but are technically cumbersome and rely on invasive sampling (blood or bronchoalveolar lavage). Methods. We optimized a lateral flow dipstick assay using the galactofuranose-specific monoclonal antibody (mAb476), which recognizes urine antigens after Aspergillus fumigatus pulmonary infection in animals. Urine samples were obtained from a cohort of 78 subjects undergoing evaluation for suspected invasive fungal infections, and stored frozen until testing. Urine was processed by centrifugation through desalting columns and exposed to dipsticks. Reviewers blinded to clinical diagnoses graded results. Western blots were performed on urine samples from 2 subjects to characterize mAb476-reactive antigens. Results. Per-patient sensitivity and specificity for diagnosis of proven or probable IA in the overall cohort was 80% (95% confidence interval [CI], 61.4%-92.3%) and 92% (95% CI, 74%-99%), respectively. In the subgroup with cancer, sensitivity was 89.5% (95% CI, 66.7%-98.7%) and specificity was 90.9% (95% CI, 58.7%-99.8%); among all others, sensitivity and specificity were 63.6% (95% CI, 30.8%-89.1%) and 92.9% (95% CI, 66.1%-99.8%), respectively. Eliminating lung transplant recipients with airway disease increased sensitivity in the noncancer cohort (85.7% [95% CI, 42.1%-99.6%]). Semiquantitative urine assay results correlated with serum galactomannan indices. Western blots demonstrated mAb476-reactive antigens in urine from cases, ranging between 26 kDa and 35 kDa in size. Conclusions. Urine testing using mAb476 may be used as an aid to diagnose IA in high-risk patients.
Fungal organisms are ubiquitous in the environment. Pathogenic fungi, although relatively few in the whole gamut of microbial pathogens, are able to cause disease with varying degrees of severity in individuals with normal or impaired immunity. The disease state is an outcome of the fungal pathogen's interactions with the host immunity, and therefore, it stands to reason that deep/invasive fungal diseases be amenable to immunotherapy. Therefore, antifungal immunotherapy continues to be attractive as an adjunct to the currently available antifungal chemotherapy options for a number of reasons, including the fact that existing antifungal drugs, albeit largely effective, are not without limitations, and that morbidity and mortality associated with invasive mycoses are still unacceptably high. For several decades, intense basic research efforts have been directed at development of fungal immunotherapies. Nevertheless, this approach suffers from a severe bench-bedside disconnect owing to several reasons: the chemical and biological peculiarities of the fungal antigens, the complexities of host-pathogen interactions, an under-appreciation of the fungal disease landscape, the requirement of considerable financial investment to bring these therapies to clinical use, as well as practical problems associated with immunizations. In this general, non-exhaustive review, we summarize the features of ongoing research efforts directed towards devising safe and effective immunotherapeutic options for mycotic diseases, encompassing work on antifungal vaccines, adoptive cell transfers, cytokines, antimicrobial peptides (AMPs), monoclonal antibodies (mAbs), and other agents. =20
Limjunyawong, Nathachit
Fallica, Jonathan
Ramakrishnan, Amritha
Datta, Kausik
Gabrielson, Matthew
Horton, Maureen
Mitzner, Wayne
The mouse is now the primary animal used to model a variety of lung diseases. To study the mechanisms that underlie such pathologies, phenotypic methods are needed that can quantify the pathologic changes. Furthermore, to provide translational relevance to the mouse models, such measurements should be tests that can easily be done in both humans and mice. Unfortunately, in the present literature few phenotypic measurements of lung function have direct application to humans. One exception is the diffusing capacity for carbon monoxide, which is a measurement that is routinely done in humans. In the present report, we describe a means to quickly and simply measure this diffusing capacity in mice. The procedure involves brief lung inflation with tracer gases in an anesthetized mouse, followed by a 1 min gas analysis time. We have tested the ability of this method to detect several lung pathologies, including emphysema, fibrosis, acute lung injury, and influenza and fungal lung infections, as well as monitoring lung maturation in young pups. Results show significant decreases in all the lung pathologies, as well as an increase in the diffusing capacity with lung maturation. This measurement of lung diffusing capacity thus provides a pulmonary function test that has broad application with its ability to detect phenotypic structural changes with most of the existing pathologic lung models. =20
Datta, Kausik
Bartlett, Karen H.
Baer, Rebecca
Byrnes, Edmond
Galanis, Eleni
Heitman, Joseph
Hoang, Linda
Leslie, Mira J.
MacDougall, Laura
Magill, Shelley S.
Morshed, Muhammad G.
Marr, Kieren A.
Cryptococcus gattii has emerged as a human and animal pathogen in the Pacific Northwest. First recognized on Vancouver Island, British Columbia, Canada, it now involves mainland British Columbia, and Washington and Oregon in the United States. In Canada, the incidence of disease has been one of the highest worldwide. In the United States, lack of cryptococcal species identification and case surveillance limit our knowledge of C. gattii epidemiology. Infections in the Pacific Northwest are caused by multiple genotypes, but the major strain is genetically novel and may have emerged recently in association with unique mating or environmental changes. C. gattii disease affects immunocompromised and immunocompetent persons, causing substantial illness and death. Successful management requires an aggressive medical and surgical approach and consideration of potentially variable antifungal drug susceptibilities. We summarize the study results of a group of investigators and review current knowledge with the goal of increasing awareness and highlighting areas where further knowledge is required.
Marr, Kieren A
Sun, Yifei
Spec, Andrej
Lu, Na
Panackal, Anil
Bennett, John
Pappas, Peter
Ostrander, Darin
Datta, Kausik
Zhang, Sean X
Williamson, Peter R
The role of B cells and antibody-mediated immunity (AMI) is poorly understood regarding infections with the encapsulated yeast species, Cryptococcus. Human cryptococcal disease, or cryptococcosis, generally occurs in the setting of immune suppression, including deficits of T cells and other components of cell-mediated immunity (CMI), as observed in HIV/AIDS, cancer, solid-organ transplant, and similar conditions. The protective role of CMI is, therefore, well-described in the literature. However, CMI deficiencies alone cannot adequately explain the quantum of cryptococcal disease noted in human and animal populations, and a wealth of clinical and experimental data, mostly spanning the past several decades, has shed light upon a significant role of AMI in anticryptococcal immunity. Recent evidence suggests that rather than functioning discretely, these two host immune compartments work synergistically, with the AMI modulating CMI functions in order to provide a critical balance for host benefit. We describe what is currently known.
The prevalence of specific serotypes of Cryptococcus neoformans in a given area bears on regional epidemiological patterns, the expected spectrum of clinical disease, and predicted response to therapy. In this retrospective study we analyzed the serotypes of 45 C. neoformans isolates from 36 North Indian patients with varied clinical presentations. The majority of the isolates were serotype A (87%), and surprisingly, a significant number were scrotype B (five isolates, 11%), which caused infection in patients both positive and negative for HIV One unusual isolate was not typable with factor sera. Study of scrotype distribution in patients showed serotypes A and B to be present, respectively, in 92% and 8% of 36 patients. In one apparently immunocompetent patient two serotypes, A and B, were isolated simultaneously from two different sites, lung and scalp abscess. This is the first reported case in which an individual was infected with two serotypes at the same time. In one HIV-infected child serotype A was isolated from blood. Our results suggest that the distribution of serotypes in Indian clinical isolates is different than that found in other regions.
Schneeweiss, Sebastian
Carver, Peggy L.
Datta, Kausik
Galar, Alicia
Johnson, Melissa D.
Johnson, Matthew G.
Marty, Francisco M.
Nagel, Jerod
Najdzinowicz, Maryann
Saul, Melissa
Shoham, Shmuel
Silveira, Fernanda P.
Varughese, Christy A.
Wilck, Marissa
Weatherby, Lisa
Auton, Tim
Walker, Alexander M.
Although echinocandins are generally well tolerated, there is little information on the frequency with which renal and hepatic adverse effects occur during use of micafungin or other parenteral antifungal (PAF) agents in clinical practice. MYCOS is a multicentre cohort study of adult and paediatric patients who received micafungin or other PAFs between 2005 and 2012 at seven tertiary care hospitals from six centres in the USA. PAF cohort controls were selected through propensity score (PS) matching to micafungin recipients using clinical characteristics, other treatments, procedures and hospital service where PAF treatment was initiated. Analysis was restricted to patients without chronic liver and kidney conditions at the time of cohort entry. Treatment-emergent hepatic and renal injury was documented by changes in liver enzymes or estimated glomerular filtration rate through 30 days following completion of PAF treatment. Comparisons were quantified using the HR from a proportional hazards analysis. There were 2970 micafungin recipients PS matched to 6726 recipients of comparator PAFs. Balance was achieved in all baseline covariates between treatment groups. There were similar rates of hepatic injury (micafungin, 13 events per 100 patients and other PAF, 12 per 100; HR=3D0.99; 95% CI 0.86-1.14) and lower rates of renal injury (micafungin, 63 events per 100 patients and other PAF, 65 per 100; HR=3D0.93; 95% CI 0.87-0.99) for micafungin recipients versus PAF comparators. For a wide spectrum of underlying conditions, we observed no increase in liver injury by micafungin and possibly a reduced risk of renal dysfunction in comparison with other PAF medications.
Datta, Kausik
Bartlett, Karen H.
Baer, Rebecca
Byrnes, Edmond
Galanis, Eleni
Heitman, Joseph
Hoang, Linda
Leslie, Mira J.
MacDougall, Laura
Magill, Shelley S.
Morshed, Muhammad G.
Marr, Kieren A.
Subramaniam, Krishanthi S.
Datta, Kausik
Marks, Matthew S.
Pirofski, Liise-anne
Cryptococcus neoformans causes severe, and often fatal, disease (cryptococcosis) in immunocompromised patients, particularly in those with HIV/AIDS. Although resistance to cryptococcosis requires intact T-cell immunity, a possible role for antibody/B cells in protection against natural disease has not been definitively established. Previous studies of the antibody response to the C. neoformans capsular polysaccharide glucuronoxylomannan (GXM) have demonstrated that patients who are at increased risk for cryptococcosis have lower serum levels of GXM-reactive IgM than those who are not at risk, leading to the hypothesis that IgM might contribute to resistance to cryptococcosis. To determine the influence of IgM on susceptibility to systemic cryptococcosis in a murine model, we compared the survival of mice deficient in serum IgM (secretory IgM deficient [sIgM(-/-)]) and C57BL/6 x 129Sv (control) mice after intraperitoneal infection with C. neoformans strain 24067 and analyzed the splenic B- and T-cell subsets by flow cytometry and the serum and splenic cytokine/chemokine and serum antibody profiles of each mouse strain. The results showed that sIgM(-/-) mice survived significantly longer than control mice when challenged with 10(5) CFU of C. neoformans 24067. Naive sIgM(-/-) mice had higher levels of B-1 (CD5(+)) B cells, proinflammatory mediators (interleukin-6 [IL-6], IL-1 beta, MIP-1 beta, tumor necrosis factor alpha [TNF-alpha], and gamma interferon [IFN-gamma]), and anti-inflammatory mediators (IL-10 and IL-13) and significantly higher titers of GXM-specific IgG2a 3 weeks postinfection. In addition, CD5(+) splenocytes from both mouse strains had fungicidal activity against C. neoformans. Taken together, these results suggest that the inflammatory milieu in sIgM(-/-) mice might confer enhanced resistance to systemic cryptococcosis, stemming in part from the antifungal activity of B-1 B cells.
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