Human recombinant antibody to HSP90: a natural partner in combination therapy.

Recent years have seen the development of the concept of combination therapy for treating severe fungal sepsis. The advantages of this approach are a potential improvement in patient survival and a reduction in the chance of resistance developing to each of the single agents. The disadvantage is that combining drugs may increase the chance of toxicity. Mycograb is a genetically recombinant antibody against fungal heat shock protein 90 (hsp90) which is poised to become the mainstay of combination therapy. This paper presents data on how hsp90 is important to fungi and what role it might play in human disease with possible interactions with interleukin 6 and nitric oxide. There is discussion of preclinical data demonstrating synergy in vitro between Mycograb and amphotericin B and caspofungin. The progress of Mycograb through a Phase II pharmacokinetic study when used in escalating doses with a liposomal amphotericin B preparation has also been reviewed. The concepts behind a Phase II pivotal study, where Mycograb or a placebo was given in combination with a liposomal amphotericin B drug for five days for the treatment of disseminated candidiasis are discussed.

Antibodies against Candida: potential therapeutics?

For many years, there has been controversy over the role of antibodies in immunity to Candida, but recently specific antibodies to mannoproteins and hsp90 have been shown to be protective against murine candidiasis. Combined with technical advances in antibody engineering, this raises the possibility of harnessing such antibodies into a new range of therapeutics.

Differential growth of epidemic methicillin-resistant Staphylococcus aureus in vancomycin.

This study examines the ability of isolates representing the 17 epidemic methicillin-resistant strains of Staphylococcus aureus to grow in increasing levels of vancomycin. Only EMRSAs 1, 2, 8, 11, 12 and 15 showed any growth and were designated EMRSAs 1A, 2A, 8A, 11A, 12A and 15A. On population analysis, these strains all produced clones that grew on 32 microg/mL vancomycin, while EMRSA 12A and 15A grew at 128 microg/mL. This was associated with increased resistance to lysostaphin and teicoplanin, a loss of phage sensitivity and an increase in cell wall diameter. Typing by pulsed-field gel electrophoresis following SmaI digestion showed no change in EMRSA 8A and 15A, while the other EMRSAs all lost or gained at least one band. EMRSAs 1A, 2A and 15A became more resistant to methicillin, and EMRSAs 8A, 11A and 12A became less resistant to methicillin. These results suggest that more than one mechanism is responsible for this phenomenon.

A reverse passive latex agglutination test for the diagnosis of systemic candidosis.

A new reverse passive latex agglutination test for the detection of serum antigen in systemic Candida albicans infection is reported. 1700 sera were examined from 91 patients who had either proven or suspected systemic candidosis, 183 patients who were colonized and 636 patients with no evidence of candidal infection. Thirty of the systemically infected patients had lymphoproliferative disorders and the rest a variety of surgical or medical diseases with no underlying neutropenia. The latex particles were sensitised with an antiserum raised in rabbits against a pressate of Candida albicans. The degree of antigenaemia was proportional to the likelihood of invasive disease such that a diagnostic cut-off point of 1 in 8 produced a test for systemic candidosis with a sensitivity of 90% and specificity of 80.4% in patients with lymphoproliferative disorders. In the remaining medical and surgical patients a diagnostic cut-off point of 1 in 10 produced a test with a sensitivity of 96.7% and specificity of 98.8%. The patients with lymphoproliferative disorders tended to produce lower serum antigen levels. The sera were also assayed for antibody using latex particles sensitised with pressate.

Antigen detection in invasive aspergillosis.

The application of a reverse passive latex agglutination test and dot-blot assay are reported in the diagnosis of 50 proven cases of invasive aspergillosis and 28 suspected cases. At a latex titre cut off of greater than or equal to 1 in 8 the test had a sensitivity of 29.4%, specificity of 96.3% and efficiency of 41.4%. This was in sera taken when the diagnosis was first suggested. The sensitivity rose to 55.1% when sera with the maximum level of antigen were examined. The dot blot was more sensitive with 33.3% of cases being positive in the initial sera. This increased to 61.5% when the serum with the maximum antigen level was taken.

Fingerprinting Candida albicans.

A new method of typing Candida albicans based on immunoblotting is described. Isolates were disrupted by a mixture of enzymic pretreatment with alpha-mannosidase followed by sonication. They were then stained using a modified ELISA system by a rabbit hyperimmune serum raised against a single isolate, C. albicans NCTC 3153. The 190 isolates examined from the London Hospital produced 16 different types. Type 1 accounted for 43% of the isolates and was the commonest type outside the intensive care unit. Type 2 caused an outbreak of systemic candidosis on the intensive care unit. The technique was much more sensitive than the serotyping and morphotyping methods and lacked the phenotypic variability of the biotyping procedure previously used to define the outbreak. The gel-to-gel variation precludes its use in large scale epidemiological work. Its value lies in identification of outbreaks so that they can be controlled by the introduction of measures to prevent cross-infection.

Immunoblot analysis: a new method for fingerprinting hospital pathogens.

Immunoblotting has recently become popular as a way of fingerprinting those hospital pathogens where other more conventional typing systems are deficient. Extracts of microorganisms are prepared by chemical or enzymic means, run on a standard SDS-PAGE gel and transferred onto nitrocellulose membrane. They are then probed either by a hyperimmune antiserum raised in a rabbit or by serum from a patient who has been previously infected by the organism. The pattern of antigenic bands which stain forms the basis of the method. This article discusses the limitations of the system, makes recommendations for further systems and outlines a typical fingerprinting protocol.

Diagnosing endocarditis with the cloned 112 kDa antigen of Enterococcus faecalis.

A new indirect ELISA is presented for the diagnosis of enterococcal endocarditis. It is based on the cloning of Enterococcus faecalis DNA into lambda gt11. The library was screened by antisera from three cases of E. faecalis endocarditis. Three positive clones were found, all of which cross-reacted with the 112 kDa antigen of E. faecalis. One of these was taken and lysogenised into E. coli Y1089 to produce a fusion protein of 125 kDa. This IPTG dependent protein was purified by affinity chromatography and used in an indirect ELISA. The test differentiated between five cases of enterococcal endocarditis (IgG ELISA optical density greater than 0.8) and patients with endocarditis due to staphylococci (IgG ELISA optical density less than 0.243) and other streptococci (IgG ELISA optical density less than 0.656). Patients with a simple E. faecalis septicaemia had a maximum IgG ELISA optical density of 0.636. The only major cross-reaction occurred in patients with Streptococcus bovis endocarditis.

The application of epitope mapping in the development of a new serological test for Helicobacter pylori infection.

Epitope mapping was applied to the derived amino acid sequences of the urease A and urease B genes of Helicobacter pylori. This identified 15 epitopes of which five were the most immunodominant. These were LTPKELD (Ure A), FISP, QIPTAF, EVGKVA and SIP (Ure B). Peptide 1 representing LTPKELD and peptide 2 representing EVGKVA were used to develop ELISA procedures for detecting antibody specific to H. pylori infection. The sensitivity, specificity and efficiency values for peptide 1 reactive IgM were 31.6, 92.8 and 52.5% and for peptide 1 IgG were 52.6, 35.7 and 45.4%. The corresponding values for peptide 2 IgM were 31.6, 100 and 60.6% and for peptide 2 IgG were 63.2, 71.4 and 66.6% respectively. When the tests were combined so that a positive for either peptide was counted as a positive overall the figures for IgM were 52.6, 92.8 and 69.6%. Thus epitope mapping delineated peptides against which specific IgM was produced in active H. pylori infection.

The application of epitope mapping in the development of a new serological test for systemic candidosis.

A new serological test for systemic candidosis was developed by raising a rabbit antiserum probe against a specific epitope on Candida albicans, hsp 90. A major fragment at the carboxy terminal end of this immunodominant candidal antigen was epitope mapped by Geysen's method. An epitope, recognised by all infected patients with antibody to the 47 kDa antigen, was synthesized and conjugated to keyhole limpet haemocyanin. A rabbit was successfully immunized against this synthesized peptide epitope and this antiserum was compared, in a dot-immunobinding assay, with unfractionated hyperimmune rabbit antiserum to C. albicans and an affinity-purified rabbit antiserum to the 47 kDa antigen. The epitope-specific antibody probe was more sensitive than the hyperimmune candidal antiserum but less sensitive than the affinity-purified antibody against the 47 kDa antigen, which recognised multiple epitopes. This probe is technically easy to prepare in large amounts and gives no false positives.

Immunoblot fingerprinting Aspergillus fumigatus.

A new technique for typing Aspergillus fumigatus is presented. This is based on immunoblot fingerprinting each isolate against a rabbit hyperimmune antiserum raised against A. fumigatus NCTC 2109. All isolates were typable and reproducibility for the 16 antigenic bands which formed the basis of the system was excellent. Discrimination was better than silver staining and revealed 11 types among the 21 isolates from eight patients with an aspergilloma. Each aspergilloma could be due to either a single or multiple types.

Antifungal antibodies: a new approach to the treatment of systemic candidiasis.

Antibody-based therapeutics have come of age, with advances in the genetic engineering of recombinant antibodies allowing application of a growing knowledge of the immunopathology of diseases to the development of novel drugs. For infections such as systemic candidiasis, which still have a mortality of 40 to 50%, antifungal antibodies could provide long-awaited novel therapies for use in combination with antifungal agents. They may also evolve into safe, broad-spectrum agents for prophylaxis in high-risk immunocompromised patients. Mycograb, a human genetically recombinant antibody against heat shock protein 90 (hsp90), has just started trials in patients with systemic candidiasis.

Immunoblotting and culture positive endocarditis.

Serum samples from patients with endocarditis due to Streptococcus mutans, Streptococcus pneumoniae, Streptococcus agalactiae, Streptococcus lactis and a "nutritionally" variant streptococcus were immunoblotted against antigenic extracts from all five species. In S mutans endocarditis there was an endocarditis specific pattern of IgM against bands of 220, 200, and 190 kilodaltons. In S pneumoniae IgM against antibody of a molecular weight greater than 150 kilodaltons was specific to endocarditis. In S agalactiae IgM against bands at 82, 71, and 66-67 kilodaltons was endocarditis specific. In S lactis endocarditis specific IgM was present against antigenic bands at 105, 66, 61 and 58 kilodaltons. With the "nutritionally" variant streptococcus it was impossible to distinguish between cases of endocarditis and controls.

Immunoblotting in the diagnosis of culture negative endocarditis caused by streptococci and enterococci.

AIM: To improve the diagnosis of culture negative endocarditis by diagnosing cases due to streptococci and enterococci. METHODS: Serum samples were immunoblotted against extracts of the commonest streptococci and enterococci. They were selected from patients with a cardiac murmur, persistent pyrexia and at least three negative blood cultures. The presence of patterns of endocarditis species specific antigenic bands was measured and correlated with clinical outcome. RESULTS: Negative serology was found in 28 patients where the diagnosis of endocarditis was rejected or, if proved, staphylococcal, yeast, Gram negative, systemic lupus erythematosus, due to Q fever or Chlamydia psittaci or nonbacterial thrombotic. Positive serology was found in 27 of the 34 patients where the response to antibiotics suggested streptococcal or enterococcal infection. In 22 of these there was objective evidence of endocarditis. Positive serology was also found in three of four further patients with vegetations at necropsy. CONCLUSION: The identification of patterns of antibody response on immunoblotting can be used to make a specific diagnosis of streptococcal or enterococcal endocarditis in the absence of positive blood cultures.

Immunoblot analysis of serological response to Pseudomonas aeruginosa septicaemia in man.

The occurrence of an outbreak of Pseudomonas aeruginosa septicaemias on an oncology ward permitted an analysis of antibody responses in patients who were all orally exposed to the same source of infection. Seven patients became septicaemic. Serial serum samples were immunoblotted against the homologous strain. Responses were compared with those of 16 other patients with septicaemias caused by other strains and 10 healthy controls. All 18 survivors produced increasing IgG or IgA antibody, or both, against a 35,000 dalton band, whereas these antibodies were usually absent or fell in titre in the five fatal cases. These antibodies were also lacking in sera taken just before a patient became septicaemic. This band had the electrophoretic characteristics of the outer membrane porin protein F.

The diagnosis of hepatosplenic candidiasis by DNA analysis of tissue biopsy and serum.

Hepatosplenic candidiasis is traditionally diagnosed by blood culture, magnetic resonance imaging (MRI), and histological analysis. The limitations of these methods include: low sensitivity (blood culture) and failure to isolate the organism (MRI/histology). This report describes a case of hepatosplenic candidiasis diagnosed by simultaneous polymerase chain reaction (PCR) analysis of liver biopsy and serum. PCR of biopsy and/or serum may offer a sensitive and specific diagnostic test for hepatosplenic candidiasis. Candida species can be identified, which helps guide antifungal chemotherapy, an important aspect in this difficult to treat infection.

Restriction endonuclease analysis of Aspergillus fumigatus DNA.

AIMS: To develop a genome based DNA fingerprinting system for Aspergillus fumigatus mould. METHODS: DNA was extracted from 21 isolates obtained from eight patients with an aspergilloma. This was with a freeze-dried mycelial extract fragmented in liquid nitrogen. DNA was subsequently purified by phenol-chloroform extraction followed by ultracentrifugation on a caesium chloride gradient. The DNA was restricted by EcoRI and Xba I. RESULTS: All isolates were identical when cut by EcoRI; Xba I delineated six DNA types. CONCLUSIONS: DNA fingerprinting can be used to type isolates of A fumigatus. Strains from within an aspergilloma which were morphologically distinct could either have the identical DNA fingerprint or produce a unique type.

Contamination of microscope slides with Aspergillus glaucus group A: a laboratory problem in the diagnosis of suspected mycotic infections.

The microscopic examination of lesions of patients with suspected mycotic infections using slides purchased from foreign countries often showed hyphae. The slides and their wrappings were cultured successfully on Sabouraud dextrose-agar medium. A heavy growth of suspected aspergillus colonies was obtained. These colonies were investigated further by culturing them on both Czapek's solution agar and Malt extract agar. After macroscopic and microscopic examination the fungus was identified as Aspergillus chevalieri from the Aspergillus glaucus group.

A polymerase chain reaction enzyme immunoassay for diagnosing infection caused by Aspergillus fumigatus.

AIM: To develop a polymerase chain reaction enzyme immunoassay (PCR-EIA) to measure levels of circulating aspergillus DNA in invasive aspergillosis caused by Aspergillus fumigatus. METHODS: The PCR reaction was based on primers from the 18s rRNA gene. Binding of the product to a streptavidin coated microtitration plate was mediated by a biotinylated capture probe. The product was digoxigenylated during PCR and this was the tag to which antibody was bound in the subsequent EIA. RESULTS: The optical density (OD) endpoint was < 0.1 in 10 sera from neutropenic patients with no evidence of invasive aspergillosis, and in 10 sera from nonneutropenic patients with bacterial pneumonia (group 1). The OD from five of 12 patients with allergic bronchopulmonary aspergillosis (ABPA) (group 2), three with an aspergilloma (group 3), and five with possible invasive aspergillosis (group 4) was > or = 0.1. In 63 sera from 33 cases of proven invasive aspergillosis (group 5) an OD > or = 0.1 was achieved in 48 sera from 30 patients. The maximum OD was 0.510. The level fell in survivors and gradually rose in fatal cases. CONCLUSIONS: This assay validated the concept of diagnosing invasive aspergillosis by measuring levels of circulating fungal DNA in serum.

Use of immunoblotting to identify antigenic differences between the yeast and mycelial phases of Candida albicans.

Western blotting was applied to the analysis of Candida albicans in the yeast and mycelial phases in an attempt to recognise mycelial specific antigens which might be of serodiagnostic value. The antisera were prepared in rabbits by immunising them with pressates of C albicans type A NCTC 3153 in the yeast phase or the mycelial phase. These were blotted against C albicans in the yeast and mycelial phases and the yeast phase of C parapsilosis, C krusei, C tropicalis, and Torulopsis glabrata. Cross reactivity was greatest against C parapsilosis. One yeast specific mannoprotein was identified with a molecular weight of 49 000. No mycelial specific antigens could be identified.