Awatar S. Sekhon

Ascocarp production by Nannizzia and Arthroderma on keratinous and non-keratinous media

Ascocarp production by the 22 known species of Nannizzia and Arthroderma was compared on 2 keratinous and 3 non-keratinous agar media. Other factors influencing ascocarp production, such as the medium used for maintenance of the tester strains, the age of the cultures, and the technique used in crossing the strains, were also studied.Ascocarps were regularly produced by all but 1 of the species on the keratinous media. Six of 8 Nannizzia species and 12 of the 14 Arthroderma species also formed gymnothecia on Oatmeal salts agar and on Pablum cereal agar. Diluted Sabouraud dextrose agar supported ascocarp production in only 3 species of each genus. Using conidial suspensions for inoculum rather than small cubes cut out of colonies was found superior for ascocarp production. Inocula originating from young (10-day) colonies resulted in a larger number of gymnothecia than inocula from old (20-day) colonies. Media containing high content of sugars and peptones were found unsuitable for maintenance of tester str...

In vitro sensitivity of Penicillium marneffei and Pythium insidiosum to various antifungal agents

Ten isolates of Penicillium marneffei and eight of Pythium insidiosum were tested for their in vitro sensitivity to amphotericin B, hamycin (a polyene heptaene), two water-soluble analogs of amphotericin B and hamycin, namely, JAI-Amb, and JAI-hamycin, 5-fluorocytosine, fluoconazole, itraconazole, ketoconazole and miconazole. Itraconazole manifested the strongest activity against all of the 10 isolates of P. marneffei and would be the drug of choice in the treatment of penicilliosis due to P. marneffei. The polyene antibiotics amphotericin B and hamycin and their water-soluble analogs showed no appreciable activity against P. insidiosum. Pytium insidiosum isolates were sensitive to fluconazole, ketoconazole, and miconazole.Miconazole exhibited the strongest in vitro activity against all of the 8 isolates of P. insidiosum, followed by ketoconazole.

In vitro Sensitivity of Medically Significant Fusarium Species to Various Antimycotics

Sixteen isolates belonging to Fusarium chlamydosporum (n = 4), Fusarium equiseti (n = 1), Fusarium moniliforme (n = 2), Fusarium oxysporum (n = 3), Fusarium proliferatum (n = 1), and Fusarium solani (n = 5) were tested against amphotericin B, 5-fluorocytosine, fluconazole, itraconazole, ketoconazole, JAI-amphotericin B (water-soluble compound), hamycin and amphotericin B combined with 5-fluorocytosine, using antibiotic medium M3, high-resolution broth (pH 7.1), Sabourauds dextrose, and yeast-nitrogen broth media (1 ml/tube). The minimal inhibitory and minimal fungicidal concentrations of 5-fluorocytosine and fluconazole for all species were > 100 micrograms/ml. All Fusarium isolates, except F. equiseti (3.125 micrograms), gave minimal inhibitory concentrations of 12.5-100 micrograms/ml for hamycin. The values for amphotericin B, itraconazole, ketoconazole, JAI-amphotericin B, and amphotericin B combined with 5-fluorocytosine were 1.56-100, 0.78-50, 3.125-100,50-100, and 1.56 to > 100 micrograms/ml, respectively. Although a wide range of minimal inhibitory concentrations was recorded for most of the isolates studied, it appears that some--F. solani, F. oxysporum, F. chlamydosporum, F. equiseti, and F. moliniforme--were more susceptible to amphotericin B, itraconazole, ketoconazole, hamycin, and amphotericin B in the presence of 5-fluorocytosine. All isolates showed resistance to 5-fluorocytosine and fluconazole. The minimal fungicidal concentrations were either the same or several times higher than the minimal inhibitory concentrations.

Penicillosis marneffei: serological and exoantigen studies.

An immunodiffusion (ID) test has been developed to diagnose infections caused byPenicillium marneffei. A 20 X concentrated culture-filtrate of six-week-old shake cultures (25 C) ofP. marneffei was employed as an antigen. This preparation was found to be better in quality than that from still cultures of the same age (30 C). Anti-P. marneffei rabbit sera were produced by injecting rabbits with increasing dosages of the inoculum for at least six weeks. These sera demonstrated two to three precipitin lines following their reaction with their antigens for 24–48 h at 25 C. TheP. marneffei antigenic preparations did not react with rabbit antisera to five species ofAspergillus, which commonly cause aspergillosis, or to antisera for four dimorphic systemic fungi. Similarly, the antigens of these other fungi did not react against the anti-P. marneffei rabbit serum. However, the anti-P. marneffei rabbit sera demonstrated antibody titres (1∶32 to 1∶64) to histoplasmin, blastomycin and coccidioidin in the complement fixation test. Cross-reactions were not observed with any of the human sera in the suspected or proven cases of opportunistic or systemic mycotic infections. Therefore, the ID test for penicillosis marneffei is considered to be highly specific. Exoantigen studies demonstrated that, of a total of 34 isolates of ten species ofPenicillium tested, only the extracts ofP. marneffei (6 isolates) and one isolate (PLM 771) of aP. species reacted positively with the anti-P. marneffei rabbit serum, giving at least two lines of identity with reference reagents. Based on this analysis, theP. species (PLM 771) was identified as P. marneffei. The exoantigen test is considered to be a specific and rapid method for the identification and confirmation ofP. marneffei isolates.ZusammenfassungEine Immun-Verbreitung (ID) Test war ausgearbeitet fur die Diagnose der Infektionen diePenicillium marneffei verursachen. Die 20 X conzentrierte Kultur-Fieltrier von der sechs Wochen alten schuttelten Kulturen (25 C) vonP. Marneffei war gebraucht fur Antigen. Diese Preparation war besser in Qualitat denn die stillen Kulturen von dem selben Alter (30 C). Die Anti-P. Marneffei Kaninchen Sera wurde produziert durch die Injektion der Kaninchen mit immer vergrosserden Dosen von dem Imfstoff-mindestebs wahrend sechs Wochen. Diese Sera zeigte zwei — drei Ubere sturzung Linien durch die Reaktion von ihrer Antigenen wahrend 24–48 Stunden im 25 C. Die Antigen-Preparationen vonP. Marneffei gaben keine Reaktionen mit Kaninchen Anti-sera fur funf Species vonAspergillus, die stellen an gewohnlich Aspergillosis, oder zu der Antisera fur fier dimorpische systemische Pilze. Gleichweis, die Antigenen von diese Plize gaben keine Reaktionen gegen die Kaninchen Antisera vonP. Marneffei. Jedoch die Kaninchen Antiserum vonP. marneffei zeigte Anti-Korper Titer (1∶32 bis 1∶64) zu Histoplasmin, Blastomycin und Coccidioidin in der Complement (Erganzung) Fixing Test. Kreuz-Reaktionen wurden nicht observiert mit keine menschlichen Sera in verdachtigen oder erwiesenen Krankengeschichten von passenden oder systematischen mycotischen Infectionen. Deswegen die ID Test fur Penicillosis marneffei ist sehr spezifisch. Die Untersuchungen haben demonstriert, dass 34 isolationen von zehn Species vonPenicillium — die untersucht waren — nur der Auszug vonP. Marneffei (6 Isolationen) und ein Isolation (PLM 771) vonP. Species eine positive Reaktion mit der Kaninchen Anti-Serum vonP. Marneffei, und es gab mindestens zwei identische Linen mit die Referenz Reagenten. An diese Analyse grunden wir dass, dieP. Species (PLM 771) war identifiziert wieP. Marneffei. Die Exo-Antigen Test ist anbetrachtet wie eine spezifische und schnelle Methode fur die Identifikation und Bestatigung der Isolierten vonP. Marneffei.

Pulmonary penicillosis marneffei: Report of the first imported case in Canada

A 57-year-old female, born in Laos and who had lived in Thailand prior to immigrating to Canada in 1989, was seen by her physician with a chief complaint of cough and dyspnea. Her chest X-ray showed bilateral pulmonary air fluid levels. A fungus, with a diffusible red pigment, tentatively identified asPenicillium marneffei, was isolated from the patients bronchial washings and sputum specimens. At 37 °C, the fungus converted to a yeast form when cultured on brain heart infusion agar. Microscopic examination of this culture revealed yeast cells that reproduced by fission. The identity of the patients isolate was confirmed asP. marneffei with an exoantigen test. The patients serum demonstrated specific antibodies toP. marneffei antigen. Treatment with amphotericin B and ketoconazole resulted in clinical improvement, clearing of chest X-rays and conversion to sero-negativity. Our case is the first recorded diagnosis of imported penicillosis marneffei in Canada. The minimal inhibitory concentrations recorded for the patients isolate to fluconazole, 5-fluorocytosine, itraconazole and miconazole were 12.5, 0.39, <0.195 and <0.195 µg/ml, respectively.

Blastomycosis: report of three cases from Alberta with a review of Canadian cases.

Approximately 120 cases of blastomycosis have been reported from Canada to-date. The great majority of these occurred in the Eastern provinces. Since 1970, three cases of blastomycosis have been seen in Alberta. The first case, with meningeal and pulmonary involvements, was diagnosed at post-mortem. The second case was that of a 75-year-old male with a history of pancytopenia, aortic arteriosclerosis, exposure to mercury, and fever. KOH and periodic-acid schiff (PAS) stained smears of the lung tissue, received after autopsy, showed numerous budding yeast cells of Blastomyces dermatitidis along with some hyphal filaments. Similarly, budding cells of B. dermatitidis and hyphal segments were observed in large numbers in the PAS and Gomoris methenamine-silver (GMS) stained sections made from adrenals, lung, kidney, and spleen tissues. Attempts to culture the fungus on a variety of selective and non-selective media were unsuccessful, due to heavy bacterial contamination. The indirect fluoroscent antibody results were 2+ with the B. dermatitidis conjugate. The third case was that of a 31-year-old male, who was admitted to the hospital with the chief complaint of chest pain. Biopsy tissue sections, stained with the GMS procedure revealed a few foci with B. dermatitidis yeast cells. The immunodiffusion and complement fixation (CF) tests gave positive results against B. dermatitidis antigen (titre, 1∶16). The CF titre declined following treatment with amphotericin B and the immunodiffusion test became negative after the institution of antifungal therapy. Except for the last patient, the other two patients had no history of travel in any known endemic areas. In addition to these cases, a survey of blastomycosis occurring in this country has been presented along with on the disease in dogs and a cat.

Evaluation of a commercial enzyme immunoassay for the detection of cryptococcal antigen

Summary. A total of 143 cerebrospinal and serum samples, from proven and suspected cases of cryptococcosis, were concurrently examined using a recently introduced enzyme immunoassay (EIA Premier, Meridian Diagnostics, Inc., Cincinnati, OH, USA) and three latex agglutination (LA) procedures (Immunomycologics, Inc., Norman, OK, USA; IBL, Inc., Cranbury, NJ, USA and a non‐commercial LA test). Of these 143 specimens, 115 were negative for cryptococcal antigen (CrAg) with the EIA and LA tests. The remaining 28 specimens were evaluated by the LA tests, and all were positive for CrAg (with titres ranging from 1:2 to 1:8192). Of these 28 LA‐positive specimens, 26 were also tested by the EIA. This procedure detected CrAg in 23 specimens (88.5%), with antigen levels ranging from 1:4 to 1:266,857. There were 3 LA‐positive specimens (titres 1:4 to 1:32) which were negative by the EIA procedure (10.7%). One LA‐negative specimen demonstrated CrAg (titre 1:30) by the EIA procedure. The sensitivity of the EIA and LA tests was 85.2 and 100%, respectively. The specificity of the LA test was 100%, whereas that of the EIA was 97%. The agreement among laboratories for testing the specimens with the three LA tests was 100%.

Hyalohyphomycosis caused by Paecilomyces variotii in an obstetrical patient

A case of hyalohyphomycosis, caused by Paecilomyces variotii, has been described in a 31-year-old female, who had undergone a cesarean section in her 39th week of pregnancy for a trial of labour. Five days following delivery, she complained of sharp, cramp-like pains, localized to the incisional site. She became febrile (38.2 °C). An ultrasound examination revealed a complex mass and fluid within the pelvis and upper abdomen. The fluid was drained by a needle aspiration and the patient was administered a regimen of antibacterial drugs. Microscopic examination did not reveal any bacteria in a gram stained preparation and cultures were negative as well. However, the fluid demonstrated a few segments of septate, hyaline hyphae, with cultures yielding a pure growth of P. variotii. An exoantigen procedure, currently under development, was helpful in confirming the identity of the patients fungus. The patients condition improved following needle aspiration and her recovery was uneventful. It is reiterated that certain infections, attributed to low-grade opportunistic pathogens, such as P. variotii, may be cured by proper surgical drainage.

Blastomycosis: report of the first case from Alberta Canada.

The first known case of laboratory confirmed blastomycosis in Alberta occurred in 1970. The patient, who is believed never to have left Alberta, presented with of headaches, sore neck and impaired intellect. Initially, tuberculous or cryptococcal meningitis was suspected, but laboratory findings did not support the diagnosis. A fungus resembling Blastomyces dermatitidis was isolated from the venticular cerebrospinal fluid and lung at autopsy. A few yeast cells suggestive of B. dermatitidis were seen in lung and brain tissue sections. Initial attempts at in vitro conversion of the mycelial form of the isolate into yeast form on several enriched media were unsuccessful. The fungus gave ± to ++ reactions with B. dermatitidis specific conjugate by the direct fluorescent antibody technique, it was not pathogenic for mice and guinea pigs, and no asexual spores were produced in slide cultures. Further investigation indicated that the mycelial form of the fungus converted into its yeast form when an actively growing inoculum was used, although the yeast cells varied considerably in size. The yeast form produced disseminated infection in mice within 10 days. Exoantigenic analysis demonstrated an ‘A’ antigen specific for B. dermatitidis, which revealed the identity of this organism as an atypical strain of B. dermatitidis.ZusammenfassungDer erste Fall in Blastomycosis von einem Laboratorium wurde 1970 in Alberta bestätigt. Der Patient — der wahrscheinlich Alberta nie verlassen hat — klagte über Kopfschmerzen, Genickschmerzen und zeigte beeinträchtigte Verstand. Anfänglich wurde TBC — oder cryptococcal meningitis vermutet, laborfunde unterstutzten jedoch nicht die Diagnose. Ein Fungus — ahnlich zum Blastomyces dermatitidis wurde von venticular cerebrospinal Flüssigkeit und von der Lunge nach der Autopsie isoliert. Wenige Hefezellen hinweisend auf B. dermatitidis wurden gewebschnitten von Lunge und Gehirn festgestellt. Anfängliche Versuche die isolierten mycel Form in die Hefe Form in vitro mit Hilfe verschiedener angereicherter Nahrboden was erfolglos. Der Fungus ergab in der ‘Direct Fluorescent Antibody Technik.’ ± zu ++ Reaktion mit dem B. dermatitidis specifische Konjugate; es war nicht pathogen fur Mause und Meerschweinchen, asexual Sporen wurden in der objecttrage Kultur produziert. Weitere Untersuchungen zeigten, dass sich die mycel Form des Fungus in die Hefeform umwandelte wenn ein gut wachsendes Inoculum gebraucht wurde. Die Hefezellen zeigten jedoch beträchtliche Grossenunterschiede. Die Hefeform verursachte in Mauseneine generallisierte Infektion in inerhalb von 10 Tagen. Exoantigenik Analyse zeigte ein ‘A’ Antigen, specifisch für B. dermatitidis, welches die Identität des Organismus als atypischen Stamm des B. dermatitidis aufwies.

In vitro susceptibility of mycelial and yeast forms of Penicillium marneffei to amphotericin B, fluconazole, 5-fluorocytosine and itraconazole.

The mycelial (25°C) and yeast-like (37°C) forms of Penicillium marneffei clinical and type strains were investigated for their in vitro susceptibility to amphotericin B (AmB), 5-fluorocytosine (5-FC), fluconazole (FLU) and itraconazole (ITZ), using Bacto antibiotic medium 3, yeast-nitrogen, Sabourauds dextrose (pH 5.7) and high resolution (pH 7.1) broth media (1ml/tube), respectively. Results indicated that the minimal inhibitory and minimal fungicidal concentrations (MICs and MFCs) for the mycelial cultures of P. marneffei to AmB were in the range 0.78–1.56 and 0.78–3.125 μg/ml, respectively, as against 3.125–25 μg (MICs) for the yeast form cultures. The MFCs to AmB for the yeast form were one dilution higher. The MICs to FLU were generally lower for the yeast form (6.25–25 μg) than the mycelial form (25–50 μg/ml), whereas MFCs for the mycelial cultures were > 100 μg as compared to 6.25–100 μg for their yeast form. The MICs for the mycelial form to 5-FC ranged from < 0.195–0.39 μg. Higher MICs (6.25 μg) were recorded for their yeast form. The MFCs to 5-FC for the yeast form were 25–100 μg/ml. The MICs for the mycelial form to ITZ ranged from < 0.195 to 3.125 μg/ml. Higher values (< 0.195–50 μg) were recorded for their yeast-like form. The MFCs to ITZ for mycelial and yeast forms ranged from < 0.195–0.39 and 25–100 μg/ml, respectively. Results indicate that P. marnefeis yeast form is more sensitive to FLU and ITZ (8 of 10 strains) while the mycelial form displayed greater susceptibility to AmB and 5-FC. The MICs for ITZ remained steady in SD medium, pH 5.7 to 7.1. However, some strains gave higher MIC values (0.39–1.56 μg/ml) when tested in the HR.