Mechanisms of resistance to infection with Coccidioides immitis in mice.

Serum from vaccinated mice was ineffective in neutralizing the infectivity of arthrospores of Coccidioides immitis for recipient mice. However, a T-cell-enriched lymphocyte population was effective in preventing a lethal infection. Spleen cells from immune mice were passaged through nylon wool columns resulting in a T-cell enriched, B-cell-depleted population as shown by the susceptibility of the cell population to anti-theta serum and the inability of the cells to transfer adoptively an immune response to ovalbumin. Whereas transfer of 5 x 10(7) unfractionated immune spleen cells was required to protect 100% of the recipients against a lethal infection with C. immitis, 7 x 10(6) T-cell-enriched immune spleen cells were sufficient for the same level of protection. Thus, transfer of resistance to infection was achieved with fewer cells after the removal of B cells from the transferred spleen cells. The results confirm that T cells are crucial in transferring resistance against infection with C. immitis in mice.     

In vitro response of alveolar macrophages to infection with Coccidioides immitis.

Alveolar macrophages obtained from rhesus macaques (Macaca mulatta) by bronchial lavage were observed to phagocytize endospores and arthrospores of Coccidioides immitis. When the macrophages were subsequently maintained in vitro, the phagocytized spores developed into spherules. There was no significant reduction in the viability of C. immitis after phagocytosis by macrophages from normal macaques, nor was killing induced by the addition of immune serum, complement, or lung lining material obtained from the bronchial lavage fluid. The inability of the macrophages to kill C. immitis may in part be explained by the observation that C. immitis appeared to inhibit fusion of the phagosomes containing the fungal spores with the lysosomes within the macrophages.     

Extracellular ammonia at sites of pulmonary infection with Coccidioides posadasii contributes to severity of the respiratory disease

Coccidioides is the causative agent of a potentially life-threatening respiratory disease of humans. A feature of this mycosis is that pH measurements of the microenvironment of pulmonary abscesses are consistently alkaline due to ammonia production during the parasitic cycle. We previously showed that enzymatically active urease is partly responsible for elevated concentrations of extracellular ammonia at sites of lung infection and contributes to both localized host tissue damage and exacerbation of the respiratory disease in BALB/c mice. Disruption of the urease gene (URE) of Coccidioides posadasii only partially reduced the amount of ammonia detected during in vitro growth of the parasitic phase, suggesting that other ammonia-producing pathways exist that may also contribute to the virulence of this pathogen. Ureidoglycolate hydrolase (Ugh) expressed by bacteria, fungi and higher plants catalyzes the hydrolysis of ureidoglycolate to yield glyoxylate and the release CO₂ and ammonia. This enzymatic pathway is absent in mice and humans. Ureidoglycolate hydrolase gene deletions were conducted in a wild type (WT) isolate of C. posadasii as well as the previously generated Δure knock-out strain. Restorations of UGH in the mutant stains were performed to generate and evaluate the respective revertants. The double mutant revealed a marked decrease in the amount of extracellular ammonia without loss of reproductive competence in vitro compared to both the WT and Δure parental strains. BALB/c mice challenged intranasally with the Δugh/Δure mutant showed 90% survival after 30 days, decreased fungal burden, and well-organized pulmonary granulomas. We conclude that loss of both Ugh and Ure activity significantly reduced the virulence of this fungal pathogen.     

Persistent infection of L cells with an ovine abortion strain of Chlamydia psittaci.

L cells inoculated at multiplicities of infection greater than or equal to 1 inclusion-forming unit of the abortigenic chlamydial strain B577 were destroyed within 10 to 15 days. Upon continued incubation in fresh medium, a few surviving cells repopulated the flasks, and the reemerging cultures remained persistently infected. The persistent state was characterized by cycles of repopulation with a low ratio of infected cells and cycles of extensive cytopathic changes in which greater than 90% of the cells had chlamydial inclusions and which could be delayed or even terminated by penicillin treatment. Immunofluorescence and superinfection during the period of repopulation revealed that the persistently infected cells could adsorb chlamydiae but their multiplication was arrested. This nonpermissive state could be terminated by the specific action of cycloheximide. L cells spontaneously cured from a persistent infection exhibited no change in susceptibility to chlamydiae when compared with normal L cells. However, chlamydiae derived from L cells after 7.5 months of persistence destroyed L-cell monolayers more rapidly and at lower multiplicities of infection than the wild type. This state of chlamydia-host cell interaction could not be established with the arthropathogenic strain LW613 because chlamydial infectivity was lost after the first cytolytic burst of infection in the cell cultures. The persistence described for the strain B577-L-cell system appears to differ from previously described models involving other chlamydial strains.     

Persistent human parvovirus B19 infection following an acute infection with meningitis in an immunocompetent patient

A case in which parvovirus B19 infection persisted over a prolonged period of time in the blood of an immunocompetent patient following an acute infection with meningitis is reported. Using a nested polymerase chain reaction assay the viral genome was detected in cerebrospinal fluid as well as in blood at the time of overt disease and in consecutive blood samples collected for up to nine months.     

Systemic Coccidioides immitis infection in nude and beige mice.

The course of experimental systemic Coccidioides immitis infection was assessed quantitatively and histologically in beige mice, congenitally athymic nude mice, and their respective normal counterparts. After intravenous inoculation with 50 arthroconidia, the number of viable C. immitis cultured from the spleens, livers, and lungs progressively increased throughout the assay in the organs of all mice. During the first 2 weeks of infection, significantly greater numbers of CFU were recovered from the spleens and livers, but not the lungs, of nude mice than from the respective organs of their phenotypically normal littermates. Significantly greater numbers of CFU were cultured from the lungs and spleens of beige mice compared with the number recovered from their functionally normal littermates. After intranasal inoculation, extrapulmonary dissemination of C. immitis occurred at an equal rate and resulted in similar organ burdens in nude mice and their normal littermates. Histological examination of infected tissues revealed a characteristic mixed inflammatory cell infiltrate in euthymic mice; the response in nude mice was less severe, consisting predominantly, if not solely, of granulocytes. In addition, in tissue sections from nude mice, but not in those from their euthymic counterparts, mature spherules were frequently observed to be devoid of an associated inflammatory response. The inflammatory lesion in beige mice contained a predominance of mononuclear cells, whereas their littermates responded with a typical mixed granulomatous infiltrate. Collectively, these results provide evidence supporting the hypothesis that resistance to C. immitis infection involves two primary cell populations, one under the direct influence of T-cells and the other independent of T-lymphocytes.     

Inbred mouse strains differ in resistance to lethal Coccidioides immitis infection.

Inbred strains of mice were infected intraperitoneally with Coccidioides immitis, and the mean lethal dose was determined after 28 days. DBA/2N mice had a mean lethal dose of greater than 10(5) arthroconidia, whereas BALB/cAnN, C57BL/6N, and C57L/J mice had a mean lethal dose of less than or equal to 10(3). Since both BALB/c and DBA/2 mice are the H-2d haplotype, resistance is not primarily determined by the major histocompatibility locus. Resistance was the dominant phenotype. The pattern of C. immitis-resistant strains does not correspond to the strain distribution of the lsh gene or to the pattern of resistance to Blastomyces dermatitidis or Cryptococcus neoformans. Both resistant and susceptible mice, however, could be successfully immunized with a killed spherule vaccine, and susceptible BALB/cAnN mice were protected from an otherwise lethal infection by prior immunization with an attenuated mutant of C. immitis. Despite the evidence that BALB/cAnN mice could respond to immunization, nonimmune mice did not control the later phase of intraabdominal infection as well as DBA/2N mice. Dissemination of C. immitis to the lung occurred frequently in BALB/cAnN but not in DBA/2N mice. This suggests that BALB/cAnN mice cannot mount an effective immune response to C. immitis during the course of infection.     

Peripheral blood eosinophilia as a clue to the diagnosis of an occult Coccidioides infection

A marked peripheral blood eosinophilia is an uncommon finding in a complete blood count (CBC). According to Wardlaw and Kay (Eosinophils and Their Disorders. In: Beutler E, Lichtman MA, Coller BS, Kipps TJ, Seligsohn U, editors. Williams Hematology. 6(th) ed. New York: McGraw-Hill, 2001. p. 790-93), the most common causes are infection by helminthic parasites, atopic disease, and, less commonly, primary hypereosinophilic syndromes. Therefore, when eosinophilia is seen in a CBC, it can provide an important clue to the correct diagnosis. We present a case of a patient with a finding of pulmonary nodules in the setting of cancer and a CBC finding of profound peripheral blood eosinophilia. As a result of the high level of clinical suspicion for Coccidioides infection due in part to the eosinophilia, adequate steps were taken in the clinical laboratory not only to correctly diagnosis the patient, but also to protect the laboratory staff from work-related exposure to this easily aerosolizable infectious agent.     

Persistent infection with human cytomegalovirus in a lymphoblastoid cell line.

Cells from a line of human lymphocytes originating from a leukemic patient were persistently infected with human cytomegalovirus (HCMV). The infected culture has persistently yielded HCMV with titers ranging from 2 × 10⁴ to 3 × 10⁵ PFU/ml over a period of 1 year. Infectious center and fluorescent antigen assays and electron microscopic examination indicated that 1–10% of the cells were infected. It appears that persistent infection is due to a balance between release of virus and the growth of uninfected cells rather than to a defective or temperature-sensitive mutant of HCMV. The treatment of persistently infected cultures with anti-HCMV serum resulted in curing the virus infection. Cured cells in culture grew at the same rate as normal uninfected cells and became resistant to HCMV infection and relatively resistant to HSV infection.     

Persistent infection with bovine herpesvirus type 1: rabbit model.

Persistent infection with bovine herpesvirus type 1 (BHV-1) was established in all rabbits after conjunctival inoculation of virus. Spontaneous reactivations of BHV-1 with and without the appearance of recurrent ocular lesions were observed in persistently infected rabbits. BHV-1 was reactivated predictably and shed from all persistently infected rabbits after the administration of dexamethasone. During all reactivations, BHV-1 isolation was restricted to the inoculated eye.     

Human deep tissue infection with an entomopathogenic Beauveria species

Beauveria spp. are ubiquitous fungal entomopathogens that are commercially distributed as biological insecticides worldwide. In this paper we describe the clinical manifestation, diagnosis, and therapy of the first documented human deep tissue infection with an entomopathogenic Beauveria species in a patient receiving immunosuppressive therapy and describe the morphological and molecular characterization of the mold.     

Persistent infection of mouse fibroblasts with coxsackievirus

Summary Infection of fibroblast cell lines initiated from BALB/c or NFR mice with coxsackievirus B3 (CBV-3) or B4 (CBV-4) resulted in infections which persisted for a limited number of subpassages of the infected cells in most cases, but for over a year in one case. In all instances primary acute infections were characterized by cytopathology and release of infectious virus progeny. Viral antigen could be detected during the acute phase of infection, but not in subcultured infected cells. Infectious center assays showed that every cell was infected during the acute phase of infection, but that from the first subcultivation on, the numbers of cells which were able to initiate infection were greatly reduced. The long term persistent CBV-3 infection was characterized by wide fluctuations in titers of virus released into the supernatant fluids. Interferon did not appear to play a role in maintenance of the persistent infection. Information derived from studies on mechanisms of CBV persistence in thein vitro model may help to elucidate the role of CBV in chronic human diseases such as myocarditis.With 1 Figure     

Giant forms of Blastomyces dermatitidis in the pulmonary lesions of blastomycosis. Potential confusion with Coccidioides immitis

Typical yeast-phase cells of Blastomyces dermatitidis have a characteristic appearance in tissue sections. Fungal morphologic variation occurs infrequently in the lesions of blastomycosis, yet it can complicate the differential diagnosis, particularly if fresh tissue is not available for microbiologic culture. The authors report a case of pulmonary blastomycosis, confirmed by culture and direct immunofluorescence, in which some of the yeast-like cells were abnormally large. These giant yeast-like cells exceeded the size range accepted for the tissue forms of B. dermatitidis; therefore, coccidioidomycosis was considered initially in the differential diagnosis. Otherwise characteristic morphologic features of these cells, in particular multinucleation and the production of broad-based blastoconidia, helped resolve the differential diagnosis. The diagnosis can be confirmed by direct immunofluorescence or microbiologic culture.     

Coccidioides thyroiditis in an HIV-infected patient

We report a case of Coccidioides thyroiditis in an HIV-infected patient with a history of recent Coccidioides pneumonia but with negative Coccidioides serology determined by enzyme immunoassay at presentation. Diagnosis of Coccidioides thyroiditis was made based on histopathologic examination and culture of thyroid abscess material obtained by fine-needle aspiration biopsy.     

Isolation and identification of an exoantigen specific for Coccidioides immitis.

Results of previous studies have established that mycelial-phase cells of Coccidioides immitis produce a heat-stable (HS) exoantigen that is specific for this fungus. In the present study, the HS exoantigen was isolated from a heterogeneous culture filtrate of C. immitis mycelia by using a combination of physicochemical procedures. Affinity chromatography of the culture filtrate on concanavalin A yielded two fractions: an effluent fraction that did not bind to the lectin and an eluate fraction that eluted in alpha-2-methylmannoside. Antigenic analyses by two-dimensional immunoelectrophoresis established that of the seven antigens detected in the unfractionated culture filtrate preparation, only two were present in the column effluent, and of these, only one was stable to heating at 56 degrees C for 30 min. Reactivity in the immunodiffusion assay for the HS exoantigen was demonstrable with the column effluent fraction and not the column eluate. The detection of only one precipitinogen in two-dimensional immunoelectrophoresis of the heat-treated concanavalin A effluent fraction, coupled with the reactivity of this fraction in the immunodiffusion assay for the HS antigen, provides strong, if not definitive, evidence that this antigen is the HS exoantigen. Purification of the HS antigen and the production of monospecific antiserum will provide the necessary reagents for the development of a sensitive and specific immunoassay for detecting the HS antigen in C. immitis cultures.     

Isolation and characterization of an extracellular proteinase of Coccidioides immitis.

A proteinase isolated from the respiratory pathogen, Coccidioides immitis, was shown to have collagenolytic and elastinolytic activity, as well as the ability to cleave human serum immunoglobulin G and secretory immunoglobulin A. Proteolytic activity was demonstrated with a bovine casein digestion assay in conidial culture exudates, mycelial and spherule culture filtrates, conidial and spherule wall material, and Sephacryl S-300 fractions of the isolated soluble conidial wall material described previously. One of the latter fractions (fraction 2) demonstrated high proteolytic activity. The proteinase was purified from this chromatographic fraction by cold acetone extraction followed by Sephadex G-50 gel filtration and was identified as a polypeptide band of 36,000 Mr by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. By means of tandem two-dimensional immunoelectrophoresis, the proteinase was identified as antigen 11 on the basis of its reaction in the coccidioidin/anticoccidioidin reference system. The proteinase is characterized by a broad substrate specificity, optimal activity at 35 to 40 degrees C (pH 8.0) in the presence of human collagen, elastin, or hemoglobin, an isoelectric point of pH 4.5, and inhibition by organofluorides, N-tosyl-L-phenylalanine chloromethyl ketone, chymostatin, and alpha-1-antitrypsin. These features of the enzyme are comparable to those of chymotrypsinlike serine proteinases. Demonstration that the proteinase can cleave human immunoglobulins and digest ubiquitous tissue structural proteins (e.g., collagen and elastin) suggests that it may play a role in the virulence of the fungal pathogen.     

Detection of Coccidioides species in clinical specimens by real-time PCR

Coccidioides spp. are dimorphic fungal pathogens endemic to the semiarid regions of North, Central, and South America. Currently, direct smear and culture are the most common means of identifying Coccidioides spp. While these methods offer relatively sensitive and specific means of detecting Coccidioides spp., growth in culture may take up to 3 weeks, potentially delaying the diagnosis and initiation of appropriate antifungal therapy. In addition, growth of the organism represents a significant safety risk to laboratory personnel. The need for a rapid and safe means of diagnosing coccidioidomycosis prompted us to develop a real-time PCR assay to detect Coccidioides spp. directly from clinical specimens. Primers and fluorescent resonance energy transfer (FRET) probes were designed to target the internal transcribed spacer 2 region of Coccidioides. The assay's limit of detection is below 50 targets per reaction. An analysis of 40 Coccidioides sp. clinical isolates grown in culture demonstrated 100% sensitivity of the assay. A cross-reactivity panel containing fungi, bacteria, mycobacteria, and viruses was tested and demonstrated 100% specificity for Coccidioides spp. An analysis of 266 respiratory specimens by LightCycler PCR demonstrated 100% sensitivity and 98.4% specificity for Coccidioides spp. compared with culture. Analysis of 66 fresh tissue specimens yielded 92.9% sensitivity and 98.1% specificity versus those of the culture method. The sensitivity of the assay testing 148 paraffin-embedded tissue samples is 73.4%. A rapid method for the detection of Coccidioides spp. directly from clinical material will greatly assist in the timely diagnosis and treatment of patients, while at the same time decreasing the risk of accidental exposure to laboratory personnel.     

In-vivo selection of an azole-resistant petite mutant of Candida glabrata

Two isolates of Candida glabrata from the same stool sample from a bone marrow transplant recipient treated with fluconazole, and designated 1084-L for large colonies on yeast extract-peptone-dextrose-agar and 1084-S for small colonies, were analysed. In-vitro susceptibility tests with a commercially available disk diffusion procedure showed that isolate 1084-L had a susceptibility pattern typical of wild-type strains of C. glabrata with sensitivity to polyenes and the presence of resistant colonies randomly distributed within the inhibition zones for all azole compounds except tioconazole. In contrast, isolate 1084-S, which was found by pulsed-field gel electrophoresis and random amplification of polymorphic DNA to be genetically closely related to isolate 1084-L, exhibited cross-resistance to the azole compounds except tioconazole. Determination of MICs by the E-test method confirmed these results, showing that isolate 1084-S had greater sensitivity to amphotericin B and complete resistance to ketoconazole and fluconazole. Growth on agar plates containing glucose or glycerol as the sole carbon source suggested that the resistant isolate had a respiratory deficiency, which was further demonstrated by flow cytometric analysis of the fluorescence of rhodamine 123-stained blastoconidia. Restriction endonuclease analysis of mitochondrial DNA (mtDNA) established the mitochondrial origin of the respiratory deficiency. However, PCR amplification of the mtDNA with primers ML1 and ML6, as well as transmission electron microscopy, suggested a partial deletion of the mtDNA analogous to that described for rho- petite mutants of Saccharomyces cerevisiae. Together, these results provided evidence that the selection of azole-resistant petite mutants of C. glabrata may occur in vivo after fluconazole administration, which might explain, therefore, clinical failure of antifungal therapy.