A.K. Garg

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.

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%.

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.

Evaluation of the “Yeast‐IDENT System” for the Identification of Medically Important Yeasts./Bewertung des “Yeast‐IDENT‐Systems” zur Identifizierung medizinisch wichtiger Hefen

Summary:  Ninety‐one coded cultures belonging to nine genera of medically important yeasts were used to evaluate the newly marketed “Yeast‐IDENT” (Y‐I) system for identification. The results were compared with conventional procedures which included microscopic morphology, carbohydrate assimilation and fermentation tests, other nutritional tests, and/or API 20C. The Y‐I system identified isolates of Candida albicans, C. guilliermondii, C. lusitaniae, C. pseudotropicalis, Cryptococcus albidus, Cr. neoformans, Rhodotorula rubra, Saccharomyces cerevisiae, Sporobolomyces salmonicolor, Torulopsis candida and T. glabrata with 100% accuracy. With the Y‐I system, other species identified with variable degree of accuracy (50–89%) included C. krusei, C. parapsilosis, C. rugosa, C. tropicalis, Cr. laurentii, Hansenula anomala and Trichosporon beigelii. One isolate each of Cr. luteolus, Cr. terreus and Prototheca wickerhamii (an achlorophyllous alga) were not identified with the Y‐I system. Overall accuracy of the Y‐I system was 87.2% when compared with the conventional and/or API 20C. The Y‐I system is rapid and requires only 4h of incubation at 35–37°C compared with 72h to 14 d necessary for the other two procedures.

Effects of Culture Media on the in vitro Susceptibility of Selected Opportunistic Fungi to Fluconazole and Itraconazole

The sensitivity of 23 isolates of opportunistic fungi, Aspergillus fumigatus (5), A. flavus (5), A. niger (5), Pseudallescheria boydii (5), Alternaria alternata (2) and Xylohypha bantiana (1), was investigated against fluconazole and itraconazole, using Sabourauds dextrose broth (SD) and a high-resolution (HR) medium (Pfizer, Inc.). The procedure followed was a standard tube dilution (1 ml/tube) method. Candida albicans Y01 09 was included as reference strain to monitor quality and reproducibility. Results indicated that the minimal inhibitory concentrations (MICs) of fluconazole for all Aspergillus spp. and C. albicans were greater than or equal to 100 micrograms/ml in SD medium, whereas, for P. boydi, A. alternata and X. bantiana, the MICs were 50 micrograms/l. The MICs of itraconazole in SD medium were less than 0.195-1.56 micrograms/ml for the fungi tested. In HR medium, the MICs of fluconazole for the Aspergillus spp, were greater than or equal to 100 micrograms/ml, and those for P. boydii, A. alternata and X. bantiana were 0.78, 50 and 50 micrograms/ml, respectively. The MICs of itraconazole for all fungi ranged from less than 0.198 to 0.78 micrograms/ml in the HR medium. The values for the reference strain were 1.56 and 100 micrograms/ml in the HR medium for fluconazole and itraconazole, respectively. The HR medium was more suitable for testing P. boydii against fluconazole. This culture medium did not appear to significantly affect the MICs of itraconazole as compared to those of fluconazole, for the fungi investigated.

Effect of culture media on in vitro susceptibility testing of fluconazole against some yeasts

Summary. Thirty clinical isolates, comprising six strains of Candida albicans, and four strains each of C. lusitaniae, C. parapsilosis, C. tropicalis, Cryptococcus neoformans, Torulopsis glabrata and Trichosporon beigelii were tested against fluconazole, using Sabourauds dextrose (SD) broth and a high resolution (HR) medium (Pfizer Central Research, Inc.). The procedure was a standard tube (1 ml/ tube) dilution, and C. albicansY01 09 was included as a reference strain to monitor quality and reprod‐ucibility. Results indicated that the minimum inhibitory concentrations (MICs) for all isolates of C. albicans, C. lusitaniae, C. tropicalis, and Tr. beigelii were 100 μg ml‐1 or greater in the SD medium. In the HR medium, however, the MICs for two isolates of C. albicans were 1.56 ug ml‐1, in other four gave higher values (> 100 μg ml‐1), and the results for C. lusitaniae and Tr. beigelii were in the range 1.56–3.12 μg ml‐1. The MICs for C. tropicalis were unaffected (100 μg ml‐1) by the medium used. All Cr. neoformans isolates yielded a uniform value (1.56 μg ml‐1) in HR medium as compared to somewhat more variable results (MICs 0.39–1.56 μg ml‐1) in SD broth. The MICs for T. glabrata in the SD and HR media were 3.12–12.5 and 6.25 μg ml‐1, respectively. The data indicated that the HR medium is preferable for the in vitro susceptibility testing of C. albicans, C. lusitaniae and Tr. beigelii to fluconazole. The MICs for other yeasts were not affected by the culture medium. The reference C. albicans isolate yielded an MIC of 1.56 μg ml‐1 throughout.

Reliability of exoantigens for differentiating Blastomyces dermatitidis and Histoplasma capsulatum from Chrysosporium and Geomyces species

A recent study suggested that Chrysosporium species have the same diagnostic antigens as Histoplasma capsulatum and Blastomyces dermatitidis and, thus, compromise the antigenic identification of these pathogens. In light of these findings, studies were undertaken to determine the reliability of the exoantigen tests for identifying B. dermatitidis and H. capsulatum organisms from cultures. Sixty-three slant or shake culture extracts, or both, were derived from C. asperatum, C. keratinophilum, C. parvum, C. pruinosum, C. parvum var. crescens, Geomyces (Chrysosporium) pannorus, B. dermatitidis, and H. capsulatum. These were analyzed by use of a commercial exoantigen kit and exoantigen test reagents obtained from a commercial source. The results of these analyses were compared with those obtained with Centers for Disease Control reagents. Many of the extracts derived from nonpathogenic fungi produced nonspecific precipitin bands when reacted with the kit and reference antisera, particularly the B. dermatitidis antisera. None, however, produced antigens identical to the specific B. dermatitidis A and H. capsulatum H and M antigens. Our findings indicate that the properly controlled immunoidentification procedure is 100% specific for B. dermatitidis and H. capsulatum, and that cross-reacting antigens derived from morphologically similar saparophytic fungi do not pose identification problems.