Immunodiffusion test for diagnosing and monitoring pythiosis in horses.

A practical, sensitive, and specific immunodiffusion test was developed for diagnosing and monitoring pythiosis in horses. Culture filtrates, a soluble cell mass, and trypsinized Pythium sp. antigens were evaluated against prepared rabbit anti-Pythium sp. serum and pythiosis horse case sera. The culture filtrate antigens demonstrated the greatest capacity for detecting precipitins and the greatest stability during storage. In contrast, the trypsinized antigens had the weakest capability for detecting multiple precipitins and the poorest stability. The 13 sera from horses with proven active pythiosis were positive in immunodiffusion tests with the culture filtrate antigens. Each serum contained from three to six precipitins. Treated horses lost precipitins, and some became antibody negative. No false-positive reactions were noted in tests with sera from normal horses and humans or with sera from a variety of heterologous horse and human infections.     

Immunodiffusion test for diagnosis and monitoring of human pythiosis insidiosi.

To facilitate the laboratory diagnosis of human cases of pythiosis insidiosi, an immunological test was evaluated. A soluble antigen was prepared from a human isolate of Pythium insidiosum, an aquatic, thermotolerant oomycete that causes infections in cattle, dogs, horses, and humans. Sera from seven proven cases of disseminated human pythiosis insidiosi were tested in an immunodiffusion test along with appropriate control sera from patients with a variety of actinomycotic, bacterial, and mycotic diseases as well as sera from uninfected individuals. Titers ranged from 1:1 to 1:32 in the seven serum samples from the disseminated cases of pythiosis insidiosi of varying severity. The heterologous sera gave negative reactions. The rapidity and specificity of the immunodiffusion test makes it a useful diagnostic tool for the serodiagnosis of P. insidiosum infections.     

Primary subcutaneous mucormycosis (zygomycosis): a case report

A case of mycormycosis presenting primarily as a subcutaneous mass of the left leg in an immunocompetent individual is described. The mass that was diagnosed initially as a non-specific foreign body granulomatous process recurred a year later. Histopathological examination of the primary and recurrent lesions revealed partly degenerated hyphae associated with acute necrotizing and chronic granulomatous inflammation. Histomorphological features of primary subcutaneous mucormycosis without predisposing factors have not been previously reported.     

Indirect enzyme-linked immunosorbent assay for zygomycosis.

A 2-h indirect enzyme-linked immunosorbent assay (ELISA) using homogenate antigens of Rhizopus arrhizus and Rhizomucor pusillus was developed and compared with the existing immunodiffusion (ID) test for zygomycosis, using homogenate antigens of R. arrhizus. Utilizing 1:400 as a minimally positive ELISA titer, 33 of 43 proven cases of zygomycosis were diagnosed. The sensitivity of the ELISA was 81%. The ID test, in contrast, detected only 21 cases and demonstrated a sensitivity of 66%. The specificity of the ELISA was 94%, whereas that of the ID test was 91%. Nonspecific ELISA reactivity was particularly evident with sera from patients with aspergillosis and candidiasis. With the antigens now available, the ELISA was unable to generically or specifically identify the etiologic agents.     

Immunodiffusion using tissue sections.

A simple radial immunodiffusion technique is described for determining relative antigen content of frozen tissue sections. Its use is illustrated with frozen sections of chick intestine containing the vitamin D-induced calcium-binding protein (CaBP). Diffusion of the intestinal CaBP from the sections placed on agar containing specific anti-CaBP anti-serum produced a precipitin ring, whose diameter was proportional to the CaBP content of the intestine. The technique allowed the determination of the relative antigen content in sections of tissue adjacent to others in which immunocytochemical localization or other histochemical studies were to be performed.     

Immunodiffusion method for identification of group A streptococci.

The development of a method suitable for identification of group A streptococci by microprecipitation in gels is described. The method is based on preparation of specific sera containing high antibody levels agains the antigenic determinant characteristic of group A streptococcal polysaccharide. In a comparative study with a counterimmunoelectrophoresis method, the proposed test proved to be specific, easily read, and less complicated. Results were obtained in 2 h.     

Comparison of ELISA antigen preparations alone or in combination for serodiagnosing Helicobacter pylori infections.

The immunoglobulin G antibody response to Helicobacter pylori was assessed in 78 patients with non-ulcer dyspepsia using five different antigen preparations. All patients were endoscoped and biopsied. The H pylori state was determined histologically on at least two endoscopic biopsy specimens using a modified Giemsa stain. The ultracentrifuged cell sonicate, acid glycine extract, and 120 kilodalton protein antigens were specific in diagnosing infection (95-98%), but had only moderate sensitivity (70-84%). By mixing either of the two complex antigens with the 120 kilodalton protein, the sensitivity of the test was increased to 97% without affecting the high specificity. The combination of ultracentrifuged sonicate or acid glycine extract with the 120 kilodalton protein therefore seems to be superior to the individual antigen preparations and is particularly suitable for the serodiagnosis of H pylori infection.     

Cutaneous zygomycosis

The prevalence of cutaneous and soft tissue zygomycosis appears to have increased in recent years. We reviewed 78 case reports of cutaneous zygomycosis published from 2004 through 2008. Most patients with cutaneous zygomycosis have underlying conditions such as haematological malignancies, diabetes mellitus or solid organ transplantation, but a large proportion of them are immunocompetent. Trauma is the most common predisposing factor leading to zygomycosis in immunocompetent patients. If the patient is immunocompromised, the infection may disseminate. Cutaneous zygomycosis may be localized, may extend to deep underlying tissues, or may be disseminated. The most common clinical presentation is induration of the skin with surrounding erythema, rapidly progressing to necrosis. Histological examination and culture of soft tissue are important for the diagnosis of cutaneous zygomycosis. Treatment consists of surgical debridement, administration of antifungal agents (amphotericin B formulations and/or posaconazole) and, occasionally, hyperbaric oxygen. Mortality rates are approximately 30%.     

Primary intranasal Fusarium infection. Potential for confusion with rhinocerebral zygomycosis

Fusarium species are saprophytic fungi that may colonize human skin and nails and may rarely cause invasive infections in traumatized tissue and in debilitated and immunocompromised patients. We report herein a case of invasive intranasal Fusarium oxysporum infection in a diabetic patient. This unusual presentation potentially can be confused with early rhinocerebral zygomycosis clinically and histologically. Distinguishing morphologic features and the possible role of diabetes in promoting this infection are discussed.     

Equine zygomycosis caused by Conidiobolus lamprauges.

A 15-year-old Arabian mare from southern Louisiana with a 2-month history of periodic epistaxis and severe weight loss had a large, fibrosing, granulomatous mass containing numerous nodules ("kunkers") projecting dorsally into the nasopharynx, and was euthanized at the owner's request. In addition to these kunkers, the mass contained a single trematode tentatively identified as Fasciola hepatica. Several kunkers were removed, washed thoroughly in sterile water and embedded in nutrient agars; the fungus that grew out of them was identified as Conidiobolus lamprauges Drechsler (Entomophthorales: Ancylistaceae). This is the first report of C. lamprauges from any vertebrate mycosis, and only the third Conidiobolus species reported from vertebrates. Unlike many other entomophthoraleans, the fungus isolated from this mycosis grew well at 37 degrees C. The possible means by which Conidiobolus species may infect vertebrates is discussed. These mycoses probably result most often from chronic exposure during sleep to conidia discharged from fungal growth on decaying plant material in the bedding.     

Primary renal zygomycosis due to Rhizopus oryzae.

Primary renal zygomycosis is a rare fungal infection, and only 45 cases have been reported in the literature. The major etiologic agents of the disease were Mucor spp., Rhizomucor spp., Rhizopus spp. and Absidia spp. Here we report a case of primary renal zygomycosis due to Rhizopus oryzae infection. The patient had systemic lupus erythematosus and was treated with corticosteroids. He had frequent micturition, urodynia and passed amorphous and membranous-looking masses through the urethra several times prior to admission. Histopathological examination of the mass showed numerous broad and nonseptate hyphae. Rhizopus oryzae was isolated from the mass. We also reviewed the clinical features of primary renal zygomycosis in the literature.     

Diagnostic challenge of zygomycosis in compromised hosts.

Rhinocerebral zygomycosis was classically associated with diabetes and diabetic ketoacidosis in the past. In recent years, hematological malignancies and immunocompromised states have become increasingly more frequent underlying conditions for patients with pulmonary and disseminated zygomycosis. In this study we identified 37 patients with a histopathologic diagnosis of zygomycosis and 21 patients with a positive culture for zygomycetes seen at the National Taiwan University Hospital, Taipei, during 1986-2003. Of these, 39 cases with probable or proven invasive zygomycosis were included in these studies. The major underlying diseases were immunocompromised states (74%), and diabetes mellitus (26%). The frequency of zygomycosis in immunocompromised hosts increased from 1.86 during 1986-1991 to 4.13 per 100,000 discharges during 1998-2003. Rhinocerebral involvement was the most common site (74%). An antemortem diagnosis by sinus biopsy was made in 93.1%. Immunocompromised patients were more likely to be younger than diabetics, to have an onset during hospitalization, a positive culture and a postmortem diagnosis. They were less likely than patients with diabetes to receive surgery and more likely to die in the hospital (p < 0.05). Of the 29 patients with invasive rhinocerebral zygomycosis, cerebral involvement (adjusted odds ratio [OR]: 31.7, 95% confidence interval [CI]: 2.4-426.8, p = 0.009) and positive cultures (adjusted OR: 23.8, 95% CI: 1.7-338.6, p = 0.019) were associated with in-hospital mortality by multivariate analysis. Hematological disease and steroid use have become the most important predisposing factors for zygomycosis. Aggressive diagnostic approaches, effective antifungal therapy and surgical debridement are essential for a successful outcome.     

Burn wound zygomycosis caused by Apophysomyces elegans.

A case of zygomycosis caused by Apophysomyces elegans in a patient having 25% full-thickness burns is described. Amputation of the leg was necessary to control rapid tissue invasion. The fungus was isolated from soil in the burn environment.     

Immunodiffusion studies of various structural preparations from mycobacterial cells.

Various structures and other preparations from mycobacterial cells were analyzed by immunodiffusion. The preparations were obtained from four strains referred to the species Mycobacterium bovis (BCG), Mycobacterium fortuitum, Mycobacterium phlei and Mycobacterium smegmatis. They represented cell walls (CW), culture filtrates (CF), artificially disintegrated cell material (XP), protoplasms (PP), crude ribosomes (CR), ribosomal 50S subunits (50S), ribosomal 30S subunits (30S), ribosomal 16S core particles (16S) and ribosomal-free fractions (UCS). The preparations were analyzed by reference precipitation systems based on CF, CR and 30S preparations. Serological comparisons were made with material from the same species and cross-testing between material from different species was not performed. It was shown that from the precipitinogenic point of view the CF and XP materials were very similar and that they both contained protoplasmic material to a large extent. Furthermore, it was found that ribosomal precipitinogens constitute an important part of the precipitinogens in PP as well as in the CF and the XP preparations. The ribosomal precipitinogens were found to represent the two subunits as well as the 16S core particle. One ribosomal precipitinogen, designated beta, was shown in all preparations except the CW and the UCS.     

Hyperbaric oxygen as an adjunctive treatment for zygomycosis

Zygomycosis is a rare but emerging mycosis. Because of the sub-optimal efficacy of the standard antifungal treatment for this disease, hyperbaric oxygen (HBO) has been used occasionally as an adjunctive therapeutic modality. A review of 28 published cases of zygomycosis indicates that adjunctive HBO may be beneficial in diabetic patients (94% survival), whereas its benefit in the small group of patients with haematological malignancies or bone marrow transplants is doubtful (33% survival; p 0.02). Prolonged courses of HBO were associated with a higher survival (100% survival; p 0.003). Additional studies are required to assess the optimal timing and dose for HBO treatment.     

Outbreaks of zygomycosis in hospitals

Zygomycosis refers to a group of uncommon and frequently fatal mycoses caused by fungi of the class Zygomycetes, the organisms of which are usually found in decaying organic matter. Disease can be transmitted by the inhalation of spores or by direct inoculation on disrupted skin or mucosa. For rare diseases such as zygomycosis, two or more cases occurring in a short time should be investigated as a probable epidemic. Twelve hospital outbreaks and two pseudoepidemics caused by Zygomycetes have been cited in the English literature. The first epidemic was recorded in 1977 and the last in 2008. Outbreaks have been reported in the USA, the UK and elsewhere in Europe. Cases have included cutaneous, disseminated, pulmonary and rhinocerebral disease. Species identified have included Rhizopus arrhizus , Rhizopus rhizopodiformis , Rhizopus microsporus , Rhizopus spp., Absidia corymbifera and Rhizomucor pusillius . Sources of infection have included Elastoplast adhesive bandage rolls, ventilation systems, wooden tongue depressors, karaya (plant-derived adhesive) ostomy bags, and water damage to a linen store and patient shower room. Patients have included cardiosurgery patients, renal transplant recipients, orthopaedic patients, adult leukaemia patients, intensive care unit neonates, immunocompromised haematology patients, and burn unit patients. Although zygomycosis outbreaks in the hospital environment are infrequent, a high index of suspicion should exist if necrotic lesions appear in proximity to a postoperative wound. Direct tissue examination and tissue culture and histopathology must be routinely performed.     

Myocardial infarction caused by cardiac disease in disseminated zygomycosis.

A case of disseminated zygomycosis is described, in which myocardial infarction rather than coincident coronary or heart disease was seen at necropsy. As zygomycosis is an opportunistic fungal infection, which tends to invade blood vessels, thereby causing thrombosis and infarction, it is surprising that cardiac disease is unusual and that premortem evidence of such disease has only rarely been reported.     

Hyperbaric oxygen therapy and other adjunctive treatments for zygomycosis

Zygomycetes are increasingly reported as a cause of life-threatening invasive fungal infections in profoundly immunocompromised patients and in those with diabetic ketoacidosis. Zygomycosis, typically presents as soft tissue, rhino-orbitocerebral, pulmonary or disseminated disease and is characterized by rapid clinical progression and high mortality rates. Treatment with amphotericin B lipid formulations in combination with surgery and, perhaps, the addition of caspofungin offers the best chance for survival; posaconazole, a new antifungal triazole, is increasingly used for consolidation or maintenance therapy. Because of the poor prognosis of zygomycosis, particularly in immunocompromised cancer patients, adjunctive treatments such as hyperbaric oxygen therapy, use of immunomodulatory cytokines, and in vivo iron starvation continue to be explored. However, although each of these modalities is based on a plausible scientific rationale and has been helpful in the management of individual patients, there is no clinical evidence for their general effectiveness as adjunctive treatments in patients with zygomycosis. Further experimental and clinical investigations are necessary to determine whether and how these treatments can impact on outcome and to determine which patients and which types of infection may benefit from them.