A sensitive and a rapid multiplex polymerase chain reaction for the identification of<i>Candida species</i>in concentrated oral rinse specimens in patients with diabetes

Sampath, Asanga; Weerasekera, Manjula; Gunasekara, T.D.C.P.; Dilhari, Ayomi; Bulugahapitiya, Uditha; Fernando, Neluka

doi:10.1080/00016357.2016.1265146

Cited by 9 publications

(13 citation statements)

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“…Each extraction was done in duplicate. The methods H1 and H2 involved heat treatment of tissue specimens (Asadzaheh et al 2010 ; Sampath et al 2016 ; Silva et al 2012 ), BP1 and BP2 were based on bead beater–phenol chloroform extraction (Sampath et al 2016 ; Walter et al 2001 ; Weerasekera et al 2013 ); method S was based on DNA precipitation at high salt concentration (Asadzaheh et al 2010 ) and method K used a commercial DNA extraction kit (Oates et al 2012 ).…”

Section: Methodsmentioning

confidence: 99%

“…Bead beater–phenol chloroform extraction method using STES buffer (BP1 method) was carried out according to the procedure described by Sampath et al in 2016 (Sampath et al 2016 ). A tissue debridement specimen was suspended in 100 µl STES buffer [200 mM Tris HCl (pH 7.6), 100 mM EDTA, 0.1% SDS] and 40 µl of TE buffer [10 mM Tris HCl (pH 8), 1 mM EDTA].…”

Section: Methodsmentioning

confidence: 99%

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Evaluation of the impact of six different DNA extraction methods for the representation of the microbial community associated with human chronic wound infections using a gel-based DNA profiling method

et al. 2017

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Infected chronic wounds are polymicrobial in nature which include a diverse group of aerobic and anaerobic microorganisms. Majority of these communal microorganisms are difficult to grow in vitro. DNA fingerprinting methods such as polymerase chain reaction-denaturation gradient gel electrophoresis (PCR-DGGE) facilitate the microbial profiling of complex ecosystems including infected chronic wounds. Six different DNA extraction methods were compared for profiling of the microbial community associated with chronic wound infections using PCR-DGGE. Tissue debris obtained from chronic wound ulcers of ten patients were used for DNA extraction. Total nucleic acid was extracted from each specimen using six DNA extraction methods. The yield, purity and quality of DNA was measured and used for PCR amplification targeting V2–V3 region of eubacterial 16S rRNA gene. QIAGEN DNeasy Blood and Tissue Kit (K method) produced good quality genomic DNA compared to the other five DNA extraction methods and gave a broad diversity of bacterial communities in chronic wounds. Among the five conventional methods, bead beater/phenol–chloroform based DNA extraction method with STES buffer (BP1 method) gave a yield of DNA with a high purity and resulted in a higher DGGE band diversity. Although DNA extraction using heat and NaOH had the lowest purity, DGGE revealed a higher bacterial diversity. The findings suggest that the quality and the yield of genomic DNA are influenced by the DNA extraction protocol, thus a method should be carefully selected in profiling a complex microbial community.

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Evaluation of the impact of six different DNA extraction methods for the representation of the microbial community associated with human chronic wound infections using a gel-based DNA profiling method

et al. 2017

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“…The main risk factors related to oral Candida infection in patients with diabetes mellitus are shown in Table 2. Eight studies [10,12,14,15,19,23,25,26] examined the possible influence of gender on the probability of Candida oral infection. Gender did not affect to fungal infection with no statistically significant association (OR = 1.40; 95% CI: 0.95 to 2.08; p = 0.09).…”

Section: Resultsmentioning

confidence: 99%

Candida species oral detection and infection in patients with diabetes mellitus: a meta-analysis

Rodríguez-Archilla¹,

Piedra-Rosales²

2021

Iberoam J Med

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Introduction: Diabetes mellitus is a chronic metabolic disorder that induces elevated plasma glucose levels. Diabetic patients are more susceptible to infections, especially fungal infections. There is a direct relationship between increased blood glucose levels and the number of Candida hyphae in the oral mucosa. This study aimed to evaluate oral candidiasis and the different Candida species found in patients with and without diabetes mellitus. Methods: A search for studies on oral candidiasis and diabetes mellitus was carried out in the following databases: PubMed (MEDLINE, Cochrane Library), Web of Science (WoS) and Google Scholar. For dichotomous outcomes, the estimates of effects of an intervention were expressed as odds ratios (OR) using the Mantel-Haenszel (M-H) method with 95% confidence intervals. Results: 25 studies were included in this meta-analysis. Diabetes Mellitus patients tripled the probability of being infected by Candida species (OR:3.16, p<0.001). Likewise, Candida species infections were more likely in patients with poor glycemic control (OR:2.94, p<0.001) and with dentures (OR:2.22, p<0.001). In contrast, neither gender nor diabetes mellitus type of diabetes conditioned fungal infections (p>0.05). The most prevalent Candida species in both diabetics and controls were C. albicans and C. tropicalis. Diabetics had significantly fewer C. non-albicans species oral infections than non-diabetics (p=0.04). Conclusions: Diabetics are more prone to oral candidiasis, especially C. albicans infections.

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“…Nas 34 amostras negativas para leveduras, ocorreu amplificação característica de alguma espécie do gênero em 14 delas, enquanto que em 20 amostras não houve detecção (Tabela 1). Recentemente, Sampath et al (2016) realizaram um estudo avaliando o uso de PCR multiplex na identificação de quatro espécies de Candida em amostras de enxágue oral concentrado. Para isso, foram utilizados quatro primers senso espécie-específicos e um primer antisenso universal, diferentemente do presente trabalho, no qual apenas um par de primers foi usado para identificação de ao menos cinco espécies de Candida.…”

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Desenvolvimento de primers para identificação e diferenciação de espécies de Candida em secreção vaginal por PCR em tempo real (qPCR)

Jackisch

Koehler

Toillier

et al. 2017

RJP

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RESUMOInfecções fúngicas são cada vez mais frequentes, principalmente relacionadas ao sistema geniturinário e causadas por Candida spp. A PCR mostra-se promissora para o diagnóstico e para sua aplicação faz-se necessário o desenvolvimento de primers. Para isso obteve-se sequências da região ITS do rDNA 18S de 5 espécies de Candida, alinhou-se e verificou-se os tamanhos dos amplicons e as Tms teóricas. Cepas padrão e cepas cultivadas foram utilizadas como referência. Além disso, 67 amostras de DNA de secreção vaginal foram submetidas a qPCR e os resultados foram confirmados em gel de agarose 2%. Os testes in silico demonstraram a capacidade do par de primers em diferenciar as cinco espécies, o que foi confirmado após a amplificação das cepas padrão e de amostras de secreção vaginal, identificadas em 20 amostras. O limite de detecção foi de 58 células/mL para as cepas padrão de C. albicans e C. tropicalis e 29 células/mL para C. parapsilosis. A sensibilidade da qPCR frente ao teste de crescimento microbiológico foi de 68%, com especificidade de 90%. A técnica possui potencial de diagnóstico, porém precisa ser otimizada para testes quantitativos.Palavras-chave: Infecção geniturinária. qPCR. Candida spp. Desing de primers. ABSTRACTFungal infections are increasingly frequent, mainly related to the genitourinary system and caused by Candida spp. The PCR is promising for the diagnosis and for its application it is necessary the development of primers. For this purpose 18S rDNA ITS region sequences were obtained from 5 Candida species, aligned and the sizes of the amplicons and the theoretical Tms were verified. Standard and cultivated strains were used as reference. In addition, 67 samples of vaginal secretion DNA were subjected to qPCR and the results were confirmed on 2% agarose gel. In silico tests demonstrated the ability of the pair of primers to differentiate the five species, which was confirmed after amplification of standard samples and vaginal secretion (it was possible to identify the species in 20 samples). The detection limit was 58 cells/mL for the standard C. albicans and C. tropicalis strains and 29 cells/mL for C. parapsilosis. The sensitivity of qPCR to the microbiological growth test was 68%, with specificity of 90%. The technique has diagnostic potential, but needs to be optimized for quantitative tests.

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A sensitive and a rapid multiplex polymerase chain reaction for the identification ofCandida speciesin concentrated oral rinse specimens in patients with diabetes

Abstract: Multiplex PCR is found to be rapid, sensitive and specific than phenotypic identification methods in discriminating multiple Candida species in oral rinse specimens.

Cited by 9 publications

References 31 publications

Evaluation of the impact of six different DNA extraction methods for the representation of the microbial community associated with human chronic wound infections using a gel-based DNA profiling method

Evaluation of the impact of six different DNA extraction methods for the representation of the microbial community associated with human chronic wound infections using a gel-based DNA profiling method

Candida species oral detection and infection in patients with diabetes mellitus: a meta-analysis

Desenvolvimento de primers para identificação e diferenciação de espécies de Candida em secreção vaginal por PCR em tempo real (qPCR)

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