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1.
Med Mycol ; 58(6): 779-788, 2020 Aug 01.
Article in English | MEDLINE | ID: mdl-31758173

ABSTRACT

Quantitative real-time PCR (qPCR) is increasingly used to detect Pneumocystis jirovecii for the diagnosis of Pneumocystis pneumonia (PCP), but there are differences in the nucleic acids targeted, DNA only versus whole nucleic acid (WNA), and also the target genes for amplification. Through the Fungal PCR Initiative, a working group of the International Society for Human and Animal Mycology, a multicenter and monocenter evaluation of PCP qPCR assays was performed. For the multicenter study, 16 reference laboratories from eight different countries, performing 20 assays analyzed a panel consisting of two negative and three PCP positive samples. Aliquots were prepared by pooling residual material from 20 negative or positive- P. jirovecii bronchoalveolar lavage fluids (BALFs). The positive pool was diluted to obtain three concentrations (pure 1:1; 1:100; and 1:1000 to mimic high, medium, and low fungal loads, respectively). The monocenter study compared five in-house and five commercial qPCR assays testing 19 individual BALFs on the same amplification platform. Across both evaluations and for all fungal loads, targeting WNA and the mitochondrial small sub-unit (mtSSU) provided the earliest Cq values, compared to only targeting DNA and the mitochondrial large subunit, the major surface glycoprotein or the beta-tubulin genes. Thus, reverse transcriptase-qPCR targeting the mtSSU gene could serve as a basis for standardizing the P. jirovecii load, which is essential if qPCR is to be incorporated into clinical care pathways as the reference method, accepting that additional parameters such as amplification platforms still need evaluation.


Subject(s)
Molecular Diagnostic Techniques/standards , Pneumocystis carinii/genetics , Pneumonia, Pneumocystis/diagnosis , Real-Time Polymerase Chain Reaction/standards , Bronchoalveolar Lavage Fluid/microbiology , DNA, Fungal/genetics , Humans , Molecular Diagnostic Techniques/methods , Pneumonia, Pneumocystis/microbiology , Sensitivity and Specificity
2.
Future Microbiol ; 13: 1557-1563, 2018 11.
Article in English | MEDLINE | ID: mdl-30417667

ABSTRACT

AIM: To establish the prevalence and antifungal susceptibilities of Aspergillus cryptic species from respiratory samples. Methods: Retrospective susceptibility data on Aspergillus species cultured between 2015 and 2017 by 'high volume culture' (HVC) versus 'conventional' culture techniques. RESULTS: Fifty-six (2.5%) isolates were identified as Aspergillus cryptic species by sequencing of ITS, BenA and CalM gene loci. Recovery was higher in HVCs compared to conventional cultures. Common cryptic species were Aspergillus montevidensis (n = 15), A. creber (n = 11), A. sydowii (n = 5) and A. calidoustus (n = 4). Eighteen (32.1%) isolates had minimum inhibitory concentration (MIC) values ≥4 mg/l to amphotericin B, and 19.1-60.1% had MIC values ≥8 mg/l to the triazoles. CONCLUSION: HVC increases the likelihood of recovery of cryptic species. MIC values to antifungals were high.


Subject(s)
Aspergillosis/microbiology , Aspergillus flavus/genetics , Aspergillus fumigatus/genetics , Aspergillus nidulans/genetics , Sequence Analysis, DNA , Amphotericin B/pharmacology , Antifungal Agents/pharmacology , Aspergillus flavus/isolation & purification , Aspergillus fumigatus/isolation & purification , Aspergillus nidulans/isolation & purification , Genes, Fungal/genetics , Genetic Loci/genetics , Humans , Microbial Sensitivity Tests , Retrospective Studies , Sputum/microbiology , Triazoles/pharmacology
3.
Med Mycol Case Rep ; 21: 23-25, 2018 Sep.
Article in English | MEDLINE | ID: mdl-30094128

ABSTRACT

We report a case of Cryptococcus gattii infection in the UK in a 76-year-old woman on biologic therapy for intra-abdominal non-Hodgkin lymphoma. An incidental nodular lung lesion was found on a chest imaging and histology, culture and molecular mycology studies of the lobectomy specimen revealed the presence of C. gattii.

4.
Ther Adv Med Oncol ; 10: 1758835918766189, 2018.
Article in English | MEDLINE | ID: mdl-29636827

ABSTRACT

BACKGROUND: Urogenital atrophy (UA) is a common treatment-limiting side effect of endocrine therapies. Topical estrogen is effective but systemic absorption may counter aromatase inhibitor efficacy. Numerous complementary approaches are marketed for use in UA without rigorous testing of their estrogenicity. We tested multiple essential oils in cancer cell growth and estrogen reporter assays in vitro and assessed clinical outcomes with the essential oil pessaries (EOPs) in breast cancer survivors with UA. METHODS: Effects on cell growth were tested in hormone-dependent (MCF-7) and -independent (MDA-MB-231) cell lines using the sulforhodamine-B assay. An estrogen response element (ERE) luciferase reporter assay was used to assess estrogenicity directly. Antifungal activity against two common pathogenic yeasts was assessed using standard microdilution methods. EOPs were offered to breast cancer survivors with symptomatic UA and the service evaluated using serial questionnaires. RESULTS: Two essential oils, Cymbopogon martinii and Pelargonium graveolens, demonstrated marked estrogenicity, stimulating ER+ cell growth and ERE-luciferase reporter activity to levels seen with premenopausal estradiol concentrations. Additional oils were screened for estrogenicity and Lavandula angustifolia and Chamaemelum nobile identified as non/minimally estrogenic. The antifungal activity of this combination of oils was confirmed. A second cohort of breast cancer survivors with UA received the second generation EOP with comparable improvement in symptom scores suggesting that estrogenicity may not be required for optimal therapy of UA. CONCLUSION: Certain essential oils demonstrate profound estrogenicity and caution should be exercised before their use in breast cancer survivors. Our minimally estrogenic pessary will be formally tested in clinical trials.

6.
PLoS One ; 9(7): e101859, 2014.
Article in English | MEDLINE | ID: mdl-24991987

ABSTRACT

The ability of C. albicans to form biofilms is a major virulence factor and a challenge for management. This is evident in biofilm-associated chronic oral-oesophageal candidosis, which has been shown to be potentially carcinogenic in vivo. We have previously shown that most Candida spp. can produce significant levels of mutagenic acetaldehyde (ACH). ACH is also an important mediator of candidal biofilm formation. We have also reported that D,L-2-hydroxyisocaproic acid (HICA) significantly inhibits planktonic growth of C. albicans. The aim of the present study was to investigate the effect of HICA on C. albicans biofilm formation and ACH production in vitro. Inhibition of biofilm formation by HICA, analogous control compounds or caspofungin was measured using XTT to measure biofilm metabolic activity and PicoGreen as a marker of biomass. Biofilms were visualised by scanning electron microscopy (SEM). ACH levels were measured by gas chromatography. Transcriptional changes in the genes involved in ACH metabolism were measured using RT-qPCR. The mean metabolic activity and biomass of all pre-grown (4, 24, 48 h) biofilms were significantly reduced after exposure to HICA (p<0.05) with the largest reductions seen at acidic pH. Caspofungin was mainly active against biofilms pre-grown for 4 h at neutral pH. Mutagenic levels (>40 µM) of ACH were detected in 24 and 48 h biofilms at both pHs. Interestingly, no ACH production was detected from D-glucose in the presence of HICA at acidic pH (p<0.05). Expression of genes responsible for ACH catabolism was up-regulated by HICA but down-regulated by caspofungin. SEM showed aberrant hyphae and collapsed hyphal structures during incubation with HICA at acidic pH. We conclude that HICA has potential as an antifungal agent with ability to inhibit C. albicans cell growth and biofilm formation. HICA also significantly reduces the mutagenic potential of C. albicans biofilms, which may be important when treating bacterial-fungal biofilm infections.


Subject(s)
Antifungal Agents/pharmacology , Biofilms/drug effects , Candida albicans/drug effects , Caproates/pharmacology , Acetaldehyde/pharmacokinetics , Acetaldehyde/toxicity , Candida albicans/genetics , Candida albicans/physiology , Caspofungin , Echinocandins/pharmacology , Fungal Proteins/genetics , Gene Expression Regulation, Fungal/drug effects , Lipopeptides , Microscopy, Electron, Scanning
7.
PLoS One ; 9(5): e97864, 2014.
Article in English | MEDLINE | ID: mdl-24867320

ABSTRACT

The ability of C. albicans to form biofilms is a major virulence factor and a challenge for management. This is evident in biofilm-associated chronic oral-oesophageal candidosis, which has been shown to be potentially carcinogenic in vivo. We have previously shown that most Candida spp. can produce significant levels of mutagenic acetaldehyde (ACH). ACH is also an important mediator of candidal biofilm formation. We have also reported that D,L-2-hydroxyisocaproic acid (HICA) significantly inhibits planktonic growth of C. albicans. The aim of the present study was to investigate the effect of HICA on C. albicans biofilm formation and ACH production in vitro. Inhibition of biofilm formation by HICA, analogous control compounds or caspofungin was measured using XTT to measure biofilm metabolic activity and PicoGreen as a marker of biomass. Biofilms were visualised by scanning electron microscopy (SEM). ACH levels were measured by gas chromatography. Transcriptional changes in the genes involved in ACH metabolism were measured using RT-qPCR. The mean metabolic activity and biomass of all pre-grown (4, 24, 48 h) biofilms were significantly reduced after exposure to HICA (p<0.05) with the largest reductions seen at acidic pH. Caspofungin was mainly active against biofilms pre-grown for 4 h at neutral pH. Mutagenic levels (>40 µM) of ACH were detected in 24 and 48 h biofilms at both pHs. Interestingly, no ACH production was detected from D-glucose in the presence of HICA at acidic pH (p<0.05). Expression of genes responsible for ACH catabolism was up-regulated by HICA but down-regulated by caspofungin. SEM showed aberrant hyphae and collapsed hyphal structures during incubation with HICA at acidic pH. We conclude that HICA has potential as an antifungal agent with ability to inhibit C. albicans cell growth and biofilm formation. HICA also significantly reduces the mutagenic potential of C. albicans biofilms, which may be important when treating bacterial-fungal biofilm infections.


Subject(s)
Acetaldehyde/metabolism , Antifungal Agents/pharmacology , Biofilms/drug effects , Candida albicans/drug effects , Candidiasis/drug therapy , Caproates/pharmacology , Mutagens/metabolism , Biofilms/growth & development , Candidiasis/microbiology , Candidiasis/pathology , Cell Proliferation/drug effects , In Vitro Techniques , Microbial Sensitivity Tests , Microscopy, Electron, Scanning , Real-Time Polymerase Chain Reaction
8.
Cancer Epidemiol Biomarkers Prev ; 22(1): 69-75, 2013 Jan.
Article in English | MEDLINE | ID: mdl-23155139

ABSTRACT

BACKGROUND: Esophageal cancer is unusually frequent in Western Kenya, despite the low prevalence of classical risk factors such as heavy drinking and tobacco smoking. Among Kenyans consumption of fermented milk is an old tradition. Our hypothesis is that alcohol and acetaldehyde are produced during the fermentation process and that their carcinogenic potential contributes to the high incidence of esophageal cancer. METHODS: Eight samples of mursik milk starter cultures were collected from different Kalenjin families in the Rift Valley province, Western Kenya. A protocol provided by the families was used for milk fermentation. Ethanol and acetaldehyde levels were measured by gas chromatography. The microbial flora in starter cultures was identified by 16S and 18S sequencing. RESULTS: 7/8 starter cultures produced mutagenic (>100 µmol/L) levels of acetaldehyde and 4/8 starter cultures produced more than 1,000 µmol/L of acetaldehyde. The highest alcohol levels (mean 79.4 mmol/L) were detected in the four fermented milks with highest acetaldehyde production. The mean number of microbial species in the starter cultures was 5 (range 2-8). Yeasts were identified in all starter cultures (mean 1.5 species/milk) but their proportion of the total microbial count varied markedly (mean 35%, range 7%-90%). A combination of yeast and lactobacilli, especially Candida krusei with Lactobacillus kefiri, with the exclusion of other species, seemed to correlate with higher acetaldehyde and ethanol levels. CONCLUSIONS: Significant levels of ethanol and acetaldehyde were produced during mursik fermentation. IMPACT: When ingested several times daily the repeated exposure to carcinogenic levels of acetaldehyde may contribute to esophageal carcinogenesis.


Subject(s)
Acetaldehyde/adverse effects , Esophageal Neoplasms/epidemiology , Esophageal Neoplasms/etiology , Ethanol/adverse effects , Fermentation , Milk/adverse effects , Animals , Candida/growth & development , Carcinoma, Squamous Cell/epidemiology , Carcinoma, Squamous Cell/etiology , Colony Count, Microbial , Developing Countries , Female , Humans , Incidence , Kenya/epidemiology , Lactobacillus/growth & development , Male , Milk/chemistry , Risk Assessment , Sampling Studies , Yeasts/growth & development
9.
Biochem Biophys Res Commun ; 360(4): 857-62, 2007 Sep 07.
Article in English | MEDLINE | ID: mdl-17631273

ABSTRACT

Snu13p is a Saccharomyces cerevisiae protein essential for pre-messenger RNA splicing and pre-ribosomal RNA processing. Snu13p binds U4 snRNA of the spliceosome and box C/D snoRNAs of the pre-ribosomal RNA processing machinery to induce assembly of each ribonucleoprotein complex. Here, we present structural and biochemical analysis of Snu13p. The crystal structure of Snu13p reveals a region of the protein which could be important for protein interaction during ribonucleoprotein assembly. Using the structure of Snu13p we have designed the first temperature-sensitive mutants in Snu13p, L67W and I102A. Wild-type and mutant Snu13p proteins were assayed for binding to U4 snRNA and U3 snoRNA. Both temperature-sensitive mutants displayed significantly reduced RNA binding compared to wild-type protein. As the temperature-sensitive mutations are not in the known RNA binding region of Snu13p this indicates that these mutants indirectly influence the RNA binding properties of Snu13p. This work provides insight into Snu13p function during ribonucleoprotein assembly.


Subject(s)
Mutation , RNA Precursors/genetics , RNA, Messenger/genetics , RNA, Ribosomal/genetics , Ribonucleoproteins, Small Nuclear/genetics , Saccharomyces cerevisiae Proteins/genetics , Cloning, Molecular , Models, Molecular , Ribonucleoproteins, Small Nuclear/chemistry , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae Proteins/chemistry
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