Henrik Elvang Jensen

Successful treatment of ocular invasive mould infection (fusariosis) with the new antifungal agent voriconazole

Editor,—Voriconazole is a new, highly potent, triazole with broad spectrum activity against fungi, including moulds as well as fluconazole resistant Candida spp.1 Like other azole antifungal agents it interferes with ergosterole biosynthesis. Its antifungal activity has been shown in several experimental as well as clinical studies.2-5 ### CASE REPORT In November 1998, a 16 year old girl was transferred to the university eye hospital in Duesseldorf with a severe ulcerative hypopyon keratitis in the left eye, from which she had been suffering for 3 months after swimming in a lake in Italy. Smears, scrapings, and serology gave no hint of the aetiology. Despite intensive topical antibacterial, anti-acanthamoebal, antifungal, and antiherpetic therapy, as well as cryoapplication, her clinical situation had deteriorated continuously before admission to our hospital. As an optical rehabilitation was unlikely, owing to the severely …

Breakthrough Aspergillus fumigatus and Candida albicans Double Infection during Caspofungin Treatment: Laboratory Characteristics and Implication for Susceptibility Testing

ABSTRACT Caspofungin is used for the treatment of acute invasive candidiasis and as salvage treatment for invasive aspergillosis. We report characteristics of isolates of Candida albicans and Aspergillus fumigatus detected in a patient with breakthrough infection complicating severe gastrointestinal surgery and evaluate the capability of susceptibility methods to identify candin resistance. The susceptibility of C. albicans to caspofungin and anidulafungin was investigated by Etest, microdilution (European Committee on Antibiotic Susceptibility Testing [EUCAST] and CLSI), disk diffusion, agar dilution, and FKS1 sequencing and in a mouse model. Tissue was examined by immunohistochemistry, PCR, and sequencing for the presence of A. fumigatus and resistance mutations. The MICs for the C. albicans isolate were as follows: >32 μg/ml caspofungin and 0.5 μg/ml anidulafungin by Etest, 2 μg/ml caspofungin and 0.125 μg/ml anidulafungin by EUCAST methods, and 1 μg/ml caspofungin and 0.5 μg/ml anidulafungin by CLSI methods. Sequencing of the FKS1 gene revealed a mutation leading to an S645P substitution. Caspofungin and anidulafungin failed to reduce kidney CFU counts in animals inoculated with this isolate (P > 0.05 compared to untreated control animals), while both candins completely sterilized the kidneys in animals infected with a control isolate. Disk diffusion and agar dilution methods clearly separated the two isolates. Immunohistochemistry and sequencing confirmed the presence of A. fumigatus without FSK1 resistance mutations in liver and lung tissues. Breakthrough disseminated aspergillosis and candidiasis developed despite an absence of characteristic FKS1 resistance mutations in the Aspergillus isolates. EUCAST and CLSI methodology did not separate the candin-resistant clinical isolate from the sensitive control isolate as well as did the Etest and agar methods.

Diagnosis of systemic mycoses by specific immunohistochemical tests.

Immunohistochemistry has proved to be a powerful tool for the accurate diagnosis of a number of important mycoses in humans and animals, such as aspergillosis, candidosis, cryptococcosis, blastomycosis, coccidioidomycosis, histoplasmosis capsulati and duboisii, paracoccidioidomycosis, fusariosis, pseudallescheriosis (scedosporiosis), sporotrichosis, trichosporonosis, penicilliosis, and zygomycosis (mucormycosis). These techniques are also applicable to pneumocystosis and to non‐mycotic infections caused by algae such as protothecosis. Apart from the specificity of immunohistochemistry, the application of fluorochromes is highly effective for the localization of typical or atypical fungal elements in lesions with only few organisms present. Occasionally, a dual aetiology of fungal infections may be suspected on the basis of morphological study, and dual staining techniques have the capacity for resolving this question by simultaneous and differential staining of two fungal species present in a tissue specimen.

Mycotic and algal bovine mastitis in Denmark.

A one‐year examination of mammary secretions (n = 2,896) from Danish cattle with clinical or subclinical mastitis revealed 45 strains of fungi and algae. The strains originated from 44 mammary secretions of 42 cows in 40 herds. The following species of fungi were identified: Candida catenulata (n = 2), Candida kefyr (n = 6), Candida krusei (n=17), Candida rugosa (n = 6), Candida tropicalis (n = 3), Candida valida (n=1), Geotrichum capitatum (n = 5). The algal species Prototheca zopfii was demonstrated in five samples.

Development of murine monoclonal antibodies for the immunohistochemical diagnosis of systemic bovine aspergillosis

Murine monoclonal antibodies (MAbs) against water-soluble somatic antigens (WSSA) and the wall fraction (WF) from Aspergillus fumigatus were produced by fusion of splenocytes from immunized BALB/c mice with mouse myeloma X63-Ag 8.653 cells. The supernatants of in vitro cultured hybridomas were initially screened for reactivity with the WSSA and the WF from A. fumigatus and WSSA of other fungi in an enzyme-linked immunosorbent assay (ELISA). Supernatants reacting only with A. fumigatus antigens were subsequently screened for homologous and heterologous reactivity with immunohistochemical techniques using formalin-fixed, paraffin-embedded tissues from experimentally infected mice. Because of a high immunohistochemical reactivity with homologous fungi, 4 MAbs raised against A. fumigatus WSSA and WF were selected for a further evaluation of cross-reactivity (diagnostic specificity) in immunohistochemical and immunoblotting assays. In immunohistochemical assays, all MAbs raised against WSSA cross-reacted heavily with a number of other fungal species. All 4 MAbs (MAb-WF-AF-1-4) raised against the WF reacted strongly with hyphae of Aspergillus spp.; hyphae of Scedosporium apiospermum were also strongly labeled by MAb-WF-AF-3 and-4. The 2 specifically reacting MAbs (MAb-WF-AF-1 and-2) were of the IgM biotype and were precipitating, and in immunoblotting experiments both bound to a 106-kD antigen of the WF, whereas they did not bind to WSSA of A. fumigatus. One of the 2 aspergillosis-specific MAbs (MAb-WF-AF-1) was used to screen 145 mycotic lesions of cattle. The diagnoses on bovine lesions obtained by MAb-WF-AF-1 were compared with results based on reactivity with heterologously absorbed polyclonal antibodies and, for some lesions, to culture results. In the vast majority of lesions (n = 133), the MAb-WF-AF-1 and the polyclonal anti-Aspergillus antibodies reacted in a similar pattern, i.e., positively in 41 aspergillosis lesions and negatively in 92 zygomycotic lesions. Hyphae in 3 of 12 lesions that were not stained by the polyclonal antibodies reacted with the specific MAb-WF-AF-1; i.e., aspergillosis was diagnosed. The characteristics of the 2 MAbs (MAb-WF-AF-1 and-2) raised against the WF of A. fumigatus in ELISA and immunoblotting and immunohistochemical assays justify their application for the in situ diagnosis of systemic aspergillosis of cattle.

Enzyme immunohistochemistry with mono- and polyclonal antibodies in the pathological diagnosis of systemic bovine mycoses.

To improve the immunohistopathological diagnosis of systemic bovine mycoses we have evaluated the utility of antifungal polyclonal and monoclonal antibodies, and peroxidase and alkaline phosphatase staining techniques. A rabbit polyclonal antibody to mannan from Candida albicans was specific for candidosis. The diagnosis of aspergillosis was accomplished using a rat monoclonal antibody to the galactofuran side chains of Aspergillus galactomannan. A murine monoclonal antibody reacting with weakly Con‐A binding 41 and 46 kDa somatic antigens from Absidia corymbifera was used for immunostaining of zygomycetic hyphae. Peroxidase antiperoxidase (PAP) and alkaline phosphatase antialkaline phosphatase (APAAP) complexes were visualized using aminoethylcarbazole and fast red substrates. A green staining of PAP reactions with dioctyl sulfosuccinate sodium and 3,3′,5,5′‐tetramethylbenzidine (DONS/TMB) was effective for the demonstration of fungi in dual and triple infections. Tissue sections of experimentally infected mice were used to determine the sensitivity and specificity of the antibodies. Tisssues obtained from 161 bovine mycotic lesions previously studied by indirect immunofluorescence staining were further evaluated using the three antibodies. In all of 45 lesions solely affected by aspergillosis and in three solely affected by candidosis the diagnoses were confirmed by the new evaluation. In 85 of 96 cases of single infections with zygomycetes the diagnosis was confirmed, while none of the antibodies reacted with fungal elements in the remaining 11 lesions. Aspergillus hyphae were detected in all three lesions with dual aspergillosis and zygomycosis, whereas zygomycetic material was confirmed in only two of these cases. A mixed infection of candidosis and zygomycosis in a lymph node was confirmed too. In 13 cases in which a diagnosis had not hitherto been obtained, aspergillosis and zygomycosis were recorded each in three cases.

Some new aspects of the pathology, pathogenesis, and aetiology of disseminated lung lesions in slaughter pigs.

From 40 pigs rejected for human consumption at slaughter due to an apparent presence of pyemic lung lesions (defined as disseminated processes containing pus and/or necrotic material), the lungs, spleen, liver, and kidneys were subjected to an extended macroscopic examination. Several lung lesions were sampled from each animal for histological and bacteriological examination. Samples from the kidneys and spleens were also subjected to bacteriological examination. At gross level, four groups of lung lesions were identified: 1) disseminated foci with contents of pus and/or necrotic material (n=26); 2) disseminated or multifocally located ecchymoses with a central area of fibroplasia (n=9); 3) non‐pneumonic lesions, i.e., disseminated areas of atelectasis (n=1) or haemorrhagic areas developing due to the process of slaughter (n=1); and 4) suppurative lesions without a disseminated distribution pattern (n=3). Histologically, the disseminated suppurative/necrotic foci were identified as: A) abscesses (n=10); B) necrotic lesions (n=6); and C) ectatic or ectatic‐like bronchioles with contents of pus and necrotic material (n=10). The macroscopic observation of disseminated centres of fibroplasia with peripheral ecchymoses (n=9) was confirmed histopathologically. The livers of five pigs contained multiple areas of chronic interstitial fibrosis related to migration of Ascaris suum larvae (“milk spotted liver”). Such hepatic lesions were significantly (p<0.01) related to the simultaneous occurrence of disseminated pulmonary ecchymoses with a central area of fibroplasia. Generally, all lung lesions of each individual animal contained identical monocultures of bacteria following this pattern: Staphylococcus aureus (abscesses); Actinomyces hyovaginalis (necroses); S. aureus, A. hyovaginalis, and Arcanobacterium pyogenes (ectatic and ectatic‐like bronchioles). Areas with fibrosis were sterile or contained bacteria considered to be a result of contamination. Apart from one kidney, from which S. aureus was cultured, all other organs were sterile. It is concluded that difficulties exist in differentiating pulmonary pyemic lesions from non‐pyemic lesions at the gross level. Thus, it was not possible to distinguish between abscesses/necroses and ectatic bronchioles, the pathogenesis of the latter being uncertain. However, the chronic non‐pyemic lesions related to the migration of A. suum larvae should be identified by the absence of pus/necrosis. S. aureus was predominantly isolated from abscesses, whereas, and most surprisingly, A. hyovaginalis was the dominant bacterium isolated from the pulmonary necroses.

Serum amyloid A is expressed in histologically normal tissues from horses and cattle.

mRNA expression of the acute phase protein serum amyloid A (SAA) in histologically normal tissues derived from horses (n=13) and cattle (n=4) was investigated by quantitative reverse-transcriptase real-time polymerase-chain reaction. As expected, high constitutive SAA mRNA expression was demonstrated in hepatic tissue in both species. In horses, moderate (>1% of the hepatic expression) SAA mRNA expression was detected in the lung, mammary gland, pancreas, synovial membrane, thymus, thyroid gland and uterus. Other equine tissues and organs sampled included adipose tissue, adrenal gland, aorta, brain, different gastro-intestinal tissues, heart, kidney, lymph node, ovary, testis, prostate, skeletal and cardiac muscle, skin and spleen; all showed low (<1% of the hepatic expression) SAA mRNA expression. In cattle, SAA mRNA was expressed in moderate levels in adipose tissue, colon, jejunum, mammary gland, skeletal muscle, synovial membrane, thymus, thyroid gland, and uterus; expression was low in the remainder of the samples (same tissue panel as horses). The results confirm the liver as the main site of SAA production. Even though there was some inter-species variation in tissues expressing SAA mRNA, several organs communicating with the external environment (lung, mammary gland, uterus, and certain parts of the gastro-intestinal tract) showed SAA mRNA expression, which supports the hypothesis that SAA might possess a role in the innate defence against invading pathogens. The results of the study thus warrant further studies into functions of hepatically and extrahepatically produced SAA isoforms.

Bovine mammary protothecosis due to Prototheca zopfii

Mastitis due to Prototheca zopfii was diagnosed in three of 28 cows in a dairy herd. As two cows continued to shed algae after 45 days they were slaughtered and organs were examined by cultivation, histology, immunohistochemistry and electron microscopy. Algae were restricted to the mammary glands and regional lymph nodes in which a granulomatous inflammation was seen. Algae were predominantly seen in macrophages but neutrophils also contained organisms. In macrophages both sporangiospores and sporangia were found, suggesting that intracellular proliferation may be responsible for the failure to overcome the infection. Serum samples from all cows were assayed for antibodies against P. zopfii in an enzyme-linked immunosorbent assay (ELISA). Although the highest titre was found in an infected cow the difference between the mean values of the titre in infected and non-infected cows was not significant.

Abnormal growth plate function in pigs carrying a dominant mutation in type X collagen

Abstract. We have identified a naturally occurring, dominant mutation that causes dwarfism in domestic pigs (Sus scrofa). With a positional candidate gene approach, the dwarf phenotype was shown to be a result of a single amino acid change, G590R, in the α1(X) chain of type X collagen. Type X collagen is a homotrimer of α1(X) chains encoded by the COL10A1 gene, which is expressed in hypertrophic chondrocytes during the process of endochondral ossification. An amino acid substitution at the equivalent position in human type X collagen, G595E, has previously been shown to cause Schmid metaphyseal chondrodysplasia (SMCD), which is a relatively mild skeletal disorder associated with dwarfism and growth plate abnormality. Consistent with the clinical phenotype of SMCD patients, radiological and histological examination of the dwarf pigs revealed metaphyseal chondrodysplasia in the long bones. Yeast-based, two-hybrid protein interaction studies and in vitro assembly experiments demonstrated that the amino acid substitution interfered with the ability of the mutated collagen molecules to engage in trimerization. This work establishes that the chondrodysplastic dwarf pigs by genetic, biochemical, radiological and histological criteria provide a valid animal model of SMCD.