Impact of gas micro-nano-bubbles on the efficacy of commonly used antimicrobials in the food industry.

AIM: To study the impact of incorporating micro-nano-bubbles (MNBs) in commonly used food antimicrobials (AMs) against Escherichia coli O157:H7 (EC) and Listeria monocytogenes (LM). METHODS AND RESULTS: Air, carbon dioxide (CO2 ) and nitrogen (N2 ) were used to incorporate MNBs in city water. AM solution (with or without MNBs) of 9 ml was individually taken into sterile test tubes and mixed with 1 ml of inoculum grown in brain heart infusion (BHI) broth to get the net AM concentrations of 28·4 ppm peracetic acid (PAA), 200 ppm chlorine (Cl2 ), 5·4% citric acid (CA) and 4·5% lactic acid (LA). After treatment time of 1·5 and 3·0 min, 1 ml of sample was neutralized using Dey-Engley neutralizing broth and plated on BHI agar. For EC, Cl2 -CO2 solutions resulted in significantly greater log reductions (5·2 logs) compared to that of Cl2 solutions without MNBs (3·8 logs). For LM, PAA-CO2 solutions resulted in significantly greater log reductions (4·4 logs) compared to that of PAA solutions without MNBs (1·7 logs). CONCLUSIONS: This study demonstrated that the efficacy of Cl2 and PAA AM solutions could be increased by incorporating CO2 -MNBs against EC and LM in microbiological growth medium. SIGNIFICANCE AND IMPACT OF THE STUDY: Incorporation of CO2 -MNBs in AM solutions could increase the efficacy of AMs against pathogens on/in food matrices, which should be tested in future research.

Usefulness of a microimmunodiffusion test for the detection of Penicillium marneffei antigenemia, antibodies, and exoantigens.

Eight sera from culturally-proven cases of penicilliosis marneffei and their corresponding isolates were examined for circulating antibody(ies) and antigen, and exoantigens, respectively, using a microimmunodiffusion (MID) test. Two of the 8 sera produced strong precipitins (1-2) when reacted against control Penicillium marneffei antigen (5-week-old shaken cultures at 25 C) in the presence of control rabbit anti-P. marneffei serum. Five of the 8 sera produced a strong precipitin line when reacted against control hyperimmune serum to P. marneffei. These five sera, and one additional serum, which tested negative for antibody to P. marneffei, demonstrated the presence of antigen by reacting only against the anti-P. marneffei serum. Serological evaluations of the sera revealed that the MID test is capable of detecting antibody and antigen in AIDS patients having penicilliosis marneffei infections. Exoantigen analysis of the 8 P. marneffei isolates, which were previously identified using the conventional and time-consuming macro- and micro-morphological characteristics, showed the presence of 1 to 4 specific exoantigens in MID. With the exoantigen analysis, the identity of all of the isolates was confirmed as P. marneffei. Our studies indicated that the serological tests are useful for detecting circulating antibody and/or antigen in patients' sera, and that the exoantigen test is reliable for confirming the identity of P. marneffei cultures.

Antigenic relationships among pathogenic Beauveria bassiana with Engyodontium album (= B. alba) and non-pathogenic species of the genus Beauveria.

Exoantigenic extracts of 15 isolates belonging to hyalohyphomycosis-causing Beauveria bassiana (1), and Engyodontium album (1), as well as other species of the genus Beauveria (one isolate each of B. brogniartii, B. densa, B. stephanoderis, B. velata, B. vermiconia and six isolates of unknown Beauveria species) were studied. Aqueous-merthiolated extracts derived from 10-day-old Sabouraud's dextrose agar slant cultures (25 degrees C) were concentrated (25X), and reacted against rabbit anti-B. bassiana serum in the presence of partially purified homologous antigen (20X) prepared from 5-week-old shaken cultures (30 degrees C), using a microimmunodiffusion procedure. Beauveria bassiana reference antigen and antiserum reacted to produce four bands of identity. With the exception of E. album, which was negative, extracts of the isolates of B. brogniartii, B. densa, B. stephanoderis, B. velata, B. vermiconia and the unknown Beauveria species all produced 2-4 lines of identity against the homologous anti-B. bassiana serum. These results suggested that all the species of the genus Beauveria tested were antigenically related to B. bassiana. Engyodontium album demonstrated antigenic distinctness, however, from B. bassiana and thus supported the validity of this taxon.

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 degrees 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 patient's fungus. The patient's 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.

An exoantigen test for the rapid identification of medically significant Fusarium species.

The accurate identification of Fusarium species can take 2-3 weeks. Preliminary exoantigen studies indicate that a mature culture suspected of being a Fusarium species may be immunologically identified 48 h after receipt. Exoantigen extracts of 10-day-old slant cultures of Fusarium chlamydosporum, Fusarium moniliforme (= Fusarium verticilloides), Fusarium oxysporum, Fusarium proliferatum and Fusarium solani and partially purified reference homologous and heterologous shake culture extracts (6-week-old) were reacted against rabbit anti-F. chlamydosporum, F. moniliforme, F. oxysporum, F. proliferatum and F. solani sera, in a micro-immunodiffusion procedure. The results indicated that all the strains belonging to a given species produced 1-3 bands of identity only when tested against its homologous antiserum and reference antigen. No cross-reactions were observed with the heterologous antisera. Furthermore, extracts from isolates of Fusarium dimerum, Fusarium equiseti, Fusarium roseum complex, Acremonium species, Cylindrocarpon, Fonsecaea pedrosoi and Trichoderma species did not react with any of the prepared Fusarium species' antisera. Our data suggest that the exoantigen procedure is a rapid and reliable tool for the accurate immuno-identification of the medically important Fusarium species studied.

In vitro susceptibility of Mucor ramosissimus Samutsevitsch, a rare fungal pathogen, to antimycotics.

The in vitro susceptibility of a rare fungal pathogen, Mucor ramosissimus Samutsevitsch, to antimycotics in clinical use was determined. Minimal inhibitory concentrations (MICs) and minimal fungicidal concentrations (MFCs) were (MIC/MFC, microgram/ml): amphotericin B, 3.125/25; miconazole, 6.25/6.25; clotrimazole, 25/25; fluconazole, 5-fluorocytosine, itraconazole, and ketoconazole, > or = 100/100. Our results show that amphotericin B and miconazole are the most effective antimycotics.

The value of the Premier enzyme immunoassay for diagnosing Blastomyces dermatitidis infections.

One hundred and three sera drawn from 20 proven and 65 suspected cases of blastomycosis were examined concurrently with the enzymes immunoassay and microimmunodiffusion tests for the 'A' antibody specific for Blastomyces dermatitidis. Results indicated that all 20 proven sera were positive by both these tests. Thirteen of the 65 sera from suspected blastomycosis cases were positive by the enzyme immunoassay only, whereas none reacted positively in the micro-immunodiffusion test. Eighteen sera from apparently normal subjects, and patients with heterologous fungal and HIV infections were also tested by both tests. The sensitivity and specificity of the enzyme immunoassay test was 100% and 85.6%, respectively. The micro- immunodiffusion test was 100% sensitive and specific. In light of the fact that the enzyme immunoassay test is not entirely specific, a positive result should be confirmed by either a positive culture, histopathology or micro-immunodiffusion test.

Comparative evaluation of commercial Premier EIA and microimmunodiffusion and complement fixation tests for Coccidioides immitis antibodies.

A total of 409 serum and cerebrospinal fluid specimens from human subjects with proven coccidioidomycosis, with other infections, or with no apparent illness were tested for antibodies to Coccidioides immitis by the Premier EIA (Meridian Diagnostics, Inc., Cincinnati, Ohio), which tests for immunoglobulin G (IgG) and IgM responses to coccidioidal antigens, and by the conventional complement fixation (CF) or immunodiffusion (ID) assays for antibodies corresponding to those detected by the tube precipitin (TP) or CF tests. Of the 409 specimens, 47 were from persons with confirmed coccidioidomycosis and all were positive for C. immitis antibodies in IDCF tests and enzyme immunoassays (EIAs) for both IgG and IgM. The EIA for detecting both IgG and IgM antibodies proved to be sensitive for detecting coccidioidomycosis case sera positive by the IDCF, IDTP, and CF tests. Maximal sensitivity for diagnosing coccidioidomycosis is dependent upon detection of both IgG and IgM antibodies in the EIA. The EIA, however, was not absolutely specific, since some sera from patients with confirmed blastomycosis and some from patients with noncoccidioidal disease produced false-positive reactions.

Ovadendron sulphureo-ochraceum endophthalmitis after cataract surgery.

PURPOSE: We examined an 82-year-old woman with delayed-onset endophthalmitis caused by an opportunistic pathogen, Ovadendron sulphureo-ochraceum. METHODS: Tissue obtained during vitrectomy was cultured and examined by light and electron microscopy. An enucleation specimen was examined by light microscopy. RESULTS: The patient had fungal endophthalmitis, with O. sulphureo-ochraceum present in the lens capsule. The eye developed a necrotizing scleritis secondary to O. sulphureo-ochraceum. The patient failed to respond to intravitreous, subconjunctival, and systemic amphotericin B, and the eye was enucleated. CONCLUSION: In this case of O. sulphureo-ochraceum as a human pathogen, the organism caused endophthalmitis after cataract extraction.

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 as Penicillium marneffei, was isolated from the patient's bronchial washings and sputum specimens. At 37 degrees 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 patient's isolate was confirmed as P. marneffei with an exoantigen test. The patient's serum demonstrated specific antibodies to P. 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 patient's isolate to fluconazole, 5-fluorocytosine, itraconazole and miconazole were 12.5, 0.39, < 0.195 and < 0.195 micrograms/ml, respectively.

Comparative evaluation of the Premier enzyme immunoassay, micro-immunodiffusion and complement fixation tests for the detection of Histoplasma capsulatum var. capsulatum antibodies.

A total of 178 sera, including 68 from proven cases of histoplasmosis (65 positive for the presence of Histoplasma capsulatum var. capsulatum antibodies and three positive for antigen), 93 from patients with suspected histoplasmosis but with no laboratory evidence of H. capsulatum var. capsulatum infection, 14 from humans with heterologous fungal and non-fungal infections and three from normal individuals, were tested for IgG H. capsulatum antibodies and M or M and H precipitins by enzyme immunoassay (EIA) (Meridian Diagnostics, Cincinnati, OH, USA) and microimmunodiffusion (MID) respectively. Sixty-three of the 68 histoplasmosis case sera demonstrated IgG antibody, and 65 of 68 demonstrated the presence of specific precipitins in the MID test. Nine positive case sera, when tested with the Laboratory Branch complement fixation (LBCF) test, reacted positively to whole yeast and histoplasmin antigens (titres 1:8 to 1:512). Three histoplasmosis case sera repeatedly tested negative for IgG, specific precipitins and complement-fixing antibodies, whereas they were positive for Histoplasma antigen. Eighteen of 95 sera from patients without evidence of histoplasmosis demonstrated IgG antibody in the EIA only. Among these positive sera, three out of three cases of aspergillosis and three out of five cases of blastomycosis were confirmed. Sera from HIV-infected and healthy individuals did not show IgG or M and/or H antibodies to H. capsulatum. Ninety-three sera were negative by both EIA and MID. The EIA for IgG was less sensitive (97%) than MID (100%). The specificity of EIA and MID was 84% and 100% respectively.

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), Sabouraud's 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.

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

The mycelial (25 degrees C) and yeast-like (37 degrees 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, Sabouraud's 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 micrograms/ml, respectively, as against 3.125-25 micrograms (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 micrograms) than the mycelial form (25-50 micrograms/ml), whereas MFCs for the mycelial cultures were > 100 micrograms as compared to 6.25-100 micrograms for their yeast form. The MICs for the mycelial form to 5-FC ranged from < 0.195-0.39 microgram. Higher MICs (6.25 micrograms) were recorded for their yeast form. The MFCs to 5-FC for the yeast form were 25-100 micrograms/ml. The MICs for the mycelial form to ITZ ranged from < 0.195 to 3.125 micrograms/ml. Higher values (< 0.195-50 micrograms) 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 micrograms/ml, respectively. Results indicate that P. marneffei's 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 micrograms/ml) when tested in the HR.

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

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 (tires 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%.

The prevalence of congenital orthopaedic anomalies in a rural community.

A rural Indian population of 50,055 was studied for the detection of congenital orthopaedic anomalies by a door-to-door survey. An incidence of 2.25 cases per 1000 population was found. Club foot was the commonest anomaly at 0.9 per 1000, followed by polydactyly and syndactyly at 0.45 and 0.38 cases per 1000. The most common anomaly found in females was a congenital short 4th metatarsal (0.24 per 1000).

Evaluation of the Pro-Lab ID ring system for the identification of medically important yeasts.

We evaluated 151 coded isolates of medically important yeast species belonging to the genera Candida, Cryptococcus, Geotrichum, Rhodoturula, Saccharomyces and Torulopsis using the newly developed rapid Pro-Lab Identification Ring, PL 960 system (PLID-Ring). All isolates were concurrently identified by the API 20C and conventional procedures comprising macro- and micromorphology, assimilation and fermentation of various carbon and nitrogen compounds. The PLID-Ring system identified isolates of Candida albicans, C. kefyr, C. krusei, C. lusitaniae, C. parapsilosis, Rhodotorula rubra, and Torulopsis glabrata with 100% accuracy in 24 h. This system identified C guilliermondii and S. cerevisiae isolates with an accuracy of 90% and 86%, respectively, while those belonging to Cr. neoformans, T. candida (= C. famata), C. rugosa and C. tropicalis were identified with 38.4%, 50%, 12.5% and 50% accuracy, respectively. Three isolates of Cr. laurentii were not identified by the PLID-Ring system. The overall accuracy of the PLID-Ring system was 81.45% (123 of 151 isolates). However, the system does not include species such as Cr. laurentii in its data base. When these three Cr. laurentii isolates were excluded from the evaluation, the accuracy of the PLID-Ring system increased from 81.45% to 83.1%.

Cerebral phaeohyphomycosis caused by Xylohypha bantiana, with a review of the literature.

A 76-year-old male with chief complaints of back and right leg sciatica was hospitalized. His abdominal CT scan revealed lumber spondylitic stenosis. A laminectomy was performed. Postoperatively, he became febrile, aphasic and had grand mal seizure. A left craniotomy of the front abscess, seen in the CT scan, was performed. H and E stained smears of drainage revealed dematiaceous, septate hyphae. Cultures of the abscess drainage grew an olivaceous-grey fungus. Based on macro- and micro-morphological characters, growth at 42 degrees C, and exoantigenic analysis, the patient's fungus was identified as Xylohypha bantiana. Treatment with amphotericin B and 5-fluorocytosine was initiated. Despite surgical procedures and antifungal therapy, the patient's condition deteriorated and he died a few weeks later due to cerebral edema. The case reported here is the first microscopically, culturally, histopathologically and exoantigenically proven case of phaeohyphomycosis caused by X. bantiana in the province of Alberta and from Canada. A review of the literature on cases of X. bantiana infections has also been presented.

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, fluconazole, 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.