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  1. Gunasegar S, Himratul-Aznita WH
    FEMS Yeast Res., 2019 Mar 01;19(2).
    PMID: 30476044 DOI: 10.1093/femsyr/foy123
    Candida albicans ATCC 14053 and Candida parapsilosis ATCC 22019 hyphal-wall protein 1 (HWP1) are involved in hyphae formation and pathogenesis. The transcriptional agglutinin-like sequence 3 (ALS3) genes in both species are responsible for the development of biofilm and colonization on tooth surfaces. Therefore, we investigated the expression of HWP1 and ALS3 quantitatively in C. albicans and C. parapsilosis and examined the biofilm structure upon exposure to various nicotine concentrations. In vitro, biofilms of Candida species were developed directly on slides using the Lab-Tek Chamber Slide System and visualized by confocal laser scanning microscopy. Quantitative real-time polymerase chain reaction was used to measure HWP1 and ALS3 expression in C. albicans ATCC 14053 and C. parapsilosis ATCC 22019. The results indicated that nicotine multiplied the number of yeast cells and increased the extracellular polysaccharides of Candida species. We also found that 1-2 mg/mL nicotine could enhance the formation of biofilm. The findings also revealed that the expression of HWP1 and ALS3 in Candida species were increased as the nicotine concentration increased. Therefore, nicotine influences the biofilm development of oral-associated C. albicans ATCC 14053 and C. parapsilosis ATCC 22019.
  2. Zahir RA, Himratul-Aznita WH
    Yeast, 2013 Jan;30(1):13-23.
    PMID: 23208647 DOI: 10.1002/yea.2937
    This study aimed to determine the distribution of Candida species in the oral cavity and differentiate the species based on PCR amplification, including HinfI and MspI digestion, in order to assess the effectiveness of using the rDNA region for species identification. Samples from saliva as well as palate, tongue and cheek mucosa surfaces were collected from 45 individuals, consisting of three groups: periodontal disease patients; denture-wearers; and the control group. The samples were serially diluted, spread on BHI and YPD agar plates and scored for colony-forming units (CFUs). Fifteen random candidal colonies were isolated and subjected to genomic DNA extraction, based on glass beads disruption. Four primers were used to amplify regions in the rDNA, and the ITSI-5.8S-ITSII PCR product was digested by HinfI and MspI restriction enzymes. The microbial loads on all sites of the denture-wearers were found to be significantly higher than control, while in the periodontal disease group only the microbial loads on the tongue were significantly higher than control. Meanwhile, there was no significant difference at other sites. The restriction fragment lengths of the clinical samples were compared to those of seven control species, allowing the differentiation of all seven species and the identification of 14 species from the clinical samples. The MspI restriction digest was not able to distinguish between C. albicans and C. dubliniensis, whereas the HinfI digest could not distinguish between C. tropicalis and C. parapsilosis. It was concluded that PCR-RFLP of the candidal rDNA region has potential for species identification. This study demonstrates the potential use of candidal rDNA as a means for identifying Candida species, based on genotype. The results also indicate the possibility of constructing genetic probes that target specific restriction fragments in the ITSI-5.8S-ITSII region, enabling swift and precise identification of Candida species.
  3. Fathilah AR, Himratul-Aznita WH, Fatheen AR, Suriani KR
    J Dent, 2012 Jul;40(7):609-15.
    PMID: 22521700 DOI: 10.1016/j.jdent.2012.04.003
    C. tropicalis and C. krusei have emerged as virulent species causing oral infections. Both have developed resistance to commonly prescribed azole antifungal agents.
  4. Abdulbaqi HR, Himratul-Aznita WH, Baharuddin NA
    Arch Oral Biol, 2016 Oct;70:117-124.
    PMID: 27343694 DOI: 10.1016/j.archoralbio.2016.06.011
    OBJECTIVE: Green tea (Gt), leafs of Camellia sinensis var. assamica, is widely consumed as healthy beverage since thousands of years in Asian countries. Chewing sticks (miswak) of Salvadora persica L. (Sp) are traditionally used as natural brush to ensure oral health in developing countries. Both Gt and Sp extracts were reported to have anti-bacterial activity against many dental plaque bacteria. However, their combination has never been tested to have anti-bacterial and anti-adherence effect against primary dental plaque colonizers, playing an initial role in the dental plaque development, which was investigated in this study.

    METHODS: Two-fold serial micro-dilution method was used to measure minimal inhibitory concentration (MIC) of aqueous extracts of Gt, Sp and their combinations. Adsorption to hexadecane was used to determine the cell surface hydrophobicity (CSH) of bacterial cells. Glass beads were used to mimic the hard tissue surfaces, and were coated with saliva to develop experimental pellicles for the adhesion of the primary colonizing bacteria.

    RESULTS: Gt aqueous extracts exhibited better anti-plaque effect than Sp aqueous extracts. Their combination, equivalent to 1/4 and 1/2 of MIC values of Gt and Sp extracts respectively, showed synergistic anti-plaque properties with fractional inhibitory concentration (FIC) equal to 0.75. This combination was found to significantly reduce CSH (p<0.05) and lower the adherence ability (p<0.003) towards experimental pellicles.

    CONCLUSION: Combination between Gt and Sp aqueous extracts exhibited synergistic anti-plaque activity, and could be used as a useful active agent to produce oral health care products.

  5. Himratul-Aznita WH, Nor-Zulaila CO, Nurul-Fatihah K
    Springerplus, 2016;5(1):1696.
    PMID: 27757368
    Candida spp. is the most prevalent species causing systemic fungal infections. The effect of antifungal agents were screened in vitro and their synergism effect were determined between hydroxychavicol (HC) in association with commercialized antifungal drugs-amphotericin B (AMB), and 5-fluorocytosine (5-FC) alone and in combination against five different oral Candida spp. in their planktonic states at different ratio (1:1 v/v; 1:2 v/v and 2:1 v/v). In vitro susceptibilities of Candida spp. to HC and commercialized antifungal agents were investigated by broth microdilution method as described by Clinical and Laboratory Standards Institute M38-A2. The intensity of the interactions was evaluated by visual reading and spectrophotometric method in checkerboard microdiluton assay, and the nature of the interactions was assessed by fractional inhibitory concentration index. The minimum inhibitory concentration (MIC50) of HC, AMB and 5-FC alone against five different planktonic oral Candida spp. ranged from 240 to 120, 8 to 15, and 2 to 8 µg/mL respectively. Positive synergistic effect existed between HC and AMB at 1:1 ratio in all Candida spp. However, there was no synergy effect observed in the majority of Candida spp. for the combination of HC with 5-FC. The data of combination between HC with AMB may be useful in the treatment of systemic infections caused by oral Candida spp. instead of the combination of HC with 5-FC.
  6. Abdulbaqi HR, Himratul-Aznita WH, Baharuddin NA
    BMC Complement Altern Med, 2016 Dec 01;16(1):493.
    PMID: 27903262
    BACKGROUND: In the author's earlier in vitro investigation, a combination of 0.25 mg/ml green tea and 7.82 mg/ml Salvadora persica L. aqueous extracts was found to exhibit significant synergistic anti-bacterial and anti-adherence effects against primary plaque colonizers biofilm. A clinical trial was needed to support these preliminary in vitro results and to investigate its efficacy as a mouthwash in the control of dental plaque.

    METHODS: A 24 h plaque re-growth, double-blinded, randomized crossover trial was carried out. Participants (n = 14) randomly rinsed with test formulation, 0.12% chlorhexidine (control) and placebo mouthwashes for 24 h. A week before the trial, all participants received scaling, polishing and oral hygiene education. On the trial day, the participants received polishing at baseline and rinsed with 15 ml of randomly allocated mouthwash twice daily without oral hygiene measures. After 24 h, plaque index was scored and then the participants entered a 6-days washout period with regular oral hygiene measures. The same protocol was repeated for the next 2 mouthwashes.

    RESULTS: The results were expressed as mean (±SD) plaque index. The test mouthwash (0.931 ± 0.372) significantly reduced plaque accumulation when compared with placebo (1.440 ± 0.498, p  0.0167).

    CONCLUSIONS: The test mouthwash has an anti-plaque effect for a 24 h period. Longer-term clinical studies are highly encouraged to investigate its anti-plaque effect for longer periods.

    TRIAL REGISTRATION: This study was registered in ClinicalTrials.gov as NCT02624336 in December 3, 2015.

  7. Fahim A, Himratul-Aznita WH, Abdul-Rahman PS
    Pak J Med Sci, 2020 2 18;36(2):271-275.
    PMID: 32063973 DOI: 10.12669/pjms.36.2.1457
    Objective: Chlorhexidine mouthrinses are considered a gold standard as an adjunct treatment of oral infections. However, owing to its toxicity, discoloration of tooth surface and the emerging prevalence of drug-resistant species, attention is being given to exploring natural alternatives to the drug.

    Methods: The experiment was carried out in Azra Naheed Center for Research and Development (ANCRD), Superior University, Lahore, Pakistan from September 2018 till May 2019. Biofilms and planktonic cells of C. albicans alone and in combination with streptococci were subjected to chlorhexidine, allium sativum and bakuchiol individually and to allium-bakuchiol combination. Kirby-Bauer test, antifungal susceptibility testing, CFU count and drug synergy assessment was done on planktonic cells. Dynamic biofilms were formed to mimic conditions similar to oral cavity and CFU was determined.

    Results: MIC of all three agents was higher against mixed species when compared to single species planktonic cells and biofilm. Allium sativum and bakuchiol demonstrated synergistic effects. The decrease in CFU count and minimum biofilm reduction to salivary pellicle caused by allium sativum-bakuchiol was comparable to that of chlorhexidine.

    Conclusion: Thus, allium sativum-bakuchiol combination demonstrated antimicrobial effects similar to chlorhexidine against planktonic cells and dynamic biofilm. It could serve as a possible natural, economical alternative to chlorhexidine mouthrinses usually recommended in dental clinics. However, in vivo studies are required to determine the correct dosage of these agents.

  8. Fahim A, Himratul-Aznita WH, Abdul-Rahman PS, Alam MK
    PeerJ, 2022;9:e12251.
    PMID: 35036111 DOI: 10.7717/peerj.12251
    Background: Polymicrobial biofilms are notorious for causing intraoral tissue destruction. Streptococcus sanguinis and Streptococcus mitis, commensals of oral cavities, have been found co-existing with C. albicans in resistant oral infections. There is an urgent need to find alternative treatment options. This study aims to assess the efficacy of garlic (G) and bakuchiol (Bk) combination against candida virulent genes and their subsequently secreted proteins.

    Methods: In vitro single species biofilms of C. albicans, and mixed species biofilms formed in combination with streptococci were exposed to bakuchiol and garlic extract (Bk+G). Gene expression of agglutinin-like sequence (ALS1), (ALS3), adhesin-like wall proteins (HWP1) and aspartyl proteinases (SAP5) were determined using qPCR and their subsequent proteins were assessed through Western blotting.

    Results: Virulent genes were significantly downregulated in single species biofilms when they were treated with Bk+G combination. However, Bk+G did not have significant effect on ALS1 and HWP1 gene in polymicrobial biofilms. ALS3 and SAP5 were significantly downregulated in Bk+G treated polymicrobial biofilm. Similar results were portrayed in Western blotting.

    Conclusion: Bk+G combination exhibited antimicrobial effects against single and mixed species biofilms. The findings might provide insights for treating resistant candida infections. This combination could potentially serve as an herbal alternative to traditional antifungals following further research.

  9. Nordin MA, Abdul Razak F, Himratul-Aznita WH
    PMID: 26633986 DOI: 10.1155/2015/918624
    Bakuchiol is an active component of Psoralea glandulosa and Psoralea corylifolia, used in traditional Chinese medicine. The study aimed at investigating the antifungal activity of bakuchiol on planktonic and biofilm forms of orally associated Candida species. The antifungal susceptibility testing was determined by the broth micro dilution technique. Growth kinetics and cell surface hydrophobicity (CSH) of Candida were measured to assess the inhibitory effect of bakuchiol on Candida planktonic cells. Biofilm biomass and cellular metabolic activity were quantitatively estimated by the crystal violet (CV) and the 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-5-[(phenylamino)carbonyl]-2H-tetrazolium hydroxide (XTT) assays. All Candida strains have been shown to be susceptible to bakuchiol with the MIC ranges from 12.5 to 100 μg/mL. Significant decrease in specific growth rates and viable counts demonstrates the inhibitory effect of bakuchiol on Candida planktonic cells. A brief exposure to bakuchiol also reduced CSH of Candida (P < 0.05), indicating altered surface properties of yeast cells towards hydrophobic interfaces. Biofilm biomass and cell metabolic activity were mostly decreased, except for C. glabrata (P = 0.29). The antifungal properties of bakuchiol on Candida species in this in vitro study may give insights into the application in therapeutic strategy against Candida infections.
  10. B Abraham S, Abdulla N, Himratul-Aznita WH, Awad M, Samaranayake LP, Ahmed HMA
    PLoS One, 2020;15(12):e0244585.
    PMID: 33378378 DOI: 10.1371/journal.pone.0244585
    OBJECTIVE: The indiscriminate prescription of antibiotics has led to the emergence of resistance microbes worldwide. This study aimed to investigate the antibiotic prescribing practices amongst general dental practitioners and specialists in managing endodontic infections in the United Arab Emirates (UAE).

    DESIGN: General dental practitioners and specialists in the UAE were invited to participate in an online questionnaire survey which included questions on socio-demographics, practitioner's antibiotic prescribing preferences for various pulpal and periapical diseases, and their choice, in terms of the type, dose and duration of the antibiotic. The link to the survey questionnaire was sent to 250 invited dentists. Data were analyzed by descriptive statistics and chi-square tests for independence and level of significance was set at 0.05.

    RESULTS: A total of 174 respondents participated in the survey (response rate = 70%). The respondents who prescribed antibiotics at least once a month were 38.5% while 17.2% did so, more than three times a week; amoxicillin 500 mg was the antibiotic of choice for patients not allergic to penicillin (43.7%), and in cases of penicillin allergies, erythromycin 500 mg (21.3%). There was a significant difference in the antibiotic prescribing practices of GDPs compared to endodontists and other specialties especially in clinical cases such as acute apical abscesses with swelling and moderate to severe pre-operative symptoms and retreatment of endodontic cases (p<0.05). Approximately, three quarters of the respondents (78.7%) did not prescribe a loading dose when prescribing antibiotics. About 15% respondents prescribed antibiotics to their patients if they were not accessible to patients due to a holiday/weekend.

    CONCLUSIONS: In general, the antibiotic prescribing practices of UAE dentists are congruent with the international norms. However, there were occasions of inappropriate prescriptions such as in patients with irreversible pulpitis, necrotic pulps with no systemic involvement and/or with sinus tracts.

  11. Abraham SB, Al Marzooq F, Himratul-Aznita WH, Ahmed HMA, Samaranayake LP
    BMC Oral Health, 2020 12 01;20(1):347.
    PMID: 33256696 DOI: 10.1186/s12903-020-01347-5
    BACKGROUND: There is limited data on the prevalence of Candida species in infected root canal systems of human teeth. We attempted to investigate the prevalence, genotype, virulence and the antifungal susceptibility of Candida albicans isolated from infected root canals of patients with primary and post-treatment infections in a UAE population.

    METHODS: Microbiological samples from 71 subjects with infected root canals were aseptically collected, and cultured on Sabouraud dextrose agar, and C. albicans was identified using multiplex polymerase chain reaction, and the isolates were further subtyped using ABC genotyping system. Their relative virulence was compared using further four archival samples of endodontic origin from another geographical region, and four more salivary isolates, as controls. The virulence attributes compared were biofilm formation, and production of phospholipase and haemolysin, and the susceptibility to nystatin, amphotericin B, ketoconazole, and fluoconazole was also tested.

    RESULTS: 4 out of 71 samples (5.6%) yielded Candida species. On analysis of variance among the groups, the intracanal isolates, mainly Genotype A, possessed a high degree of phospholipase and haemolysin activity (p 

  12. B Abraham S, Al-Marzooq F, Samaranayake L, Hamoudi RA, Himratul-Aznita WH, Aly Ahmed HM
    PLoS One, 2024;19(7):e0305537.
    PMID: 39008450 DOI: 10.1371/journal.pone.0305537
    OBJECTIVE: Endodontic microbiota appears to undergo evolutionary changes during disease progression from inflammation to necrosis and post-treatment. The aim of this study was to compare microbiome composition and diversity in primary and post-treatment endodontic infections from a cohort of patients from the UAE.

    DESIGN: Intracanal samples were collected from primarily infected (n = 10) and post-treatment infected (n = 10) root canals of human teeth using sterile paper points. Bacterial DNA was amplified from seven hypervariable regions (V2-V4 and V6-V9) of the 16S rRNA gene, then sequenced using next-generation sequencing technology. The data was analyzed using appropriate bioinformatic tools.

    RESULTS: Analyses of all the samples revealed eight major bacterial phyla, 112 genera and 260 species. Firmicutes was the most representative phylum in both groups and was significantly more abundant in the post-treatment (54.4%) than in primary (32.2%) infections (p>0.05). A total of 260 operational taxonomic units (OTUs) were identified, of which 126 (48.5%) were shared between the groups, while 83 (31.9%) and 51 (19.6%) disparate species were isolated from primary and post-treatment infections, respectively. A significant difference in beta, but not alpha diversity was noted using several different indices (p< 0.05). Differential abundance analysis indicated that, Prevotella maculosa, Streptococcus constellatus, Novosphigobium sediminicola and Anaerococcus octavius were more abundant in primary infections while Enterrococcus faecalis, Bifidobacterium dentium, Olsenella profusa and Actinomyces dentalis were more abundant in post-treatment infections (p <0.05).

    CONCLUSION: Significant differences in the microbiome composition and diversity in primary and post-treatment endodontic infections were noted in our UAE cohort. Such compositional differences of microbiota at various stages of infection could be due to both intrinsic and extrinsic factors impacting the root canal ecosystem during disease progression, as well as during their therapeutic management. Identification of the key microbiota in primarily and secondarily infected root canals can guide in the management of these infections.

  13. Zanul Abidin Z, Mohd Salleh N, Himratul-Aznita WH, Ahmad SF, Lim GS, Raja Mohd N, et al.
    PeerJ, 2023;11:e15750.
    PMID: 37601266 DOI: 10.7717/peerj.15750
    BACKGROUND: The study's objective is to assess the adherence of C. albicans in different types of denture polymers and the effectiveness of eugenol and commercialized denture cleansers in the removal of C. albicans. Three types of denture base polymers (Lucitone® 199 (High-Impact PMMA), Impact® (conventional PMMA) and Eclipse® (UDMA)) and two hard denture reline materials (Kooliner® and Tokuyama® Rebase II Fast) were used in this study.

    METHODS: Three hundred samples were prepared (6 × 2 mm disc shape) and divided into five groups of denture polymers (n = 60) and further subjected into five treatment groups (Polident®, Steradent, distilled water, eugenol 5-minutes, and eugenol 10-min). Three samples were extracted from each treatment group for baseline data (n = 12). Baseline data were used to calculate the initial number of C. albicans adherence. A 0.5 ml immersion solution from each specimen was cultured on YPD agar and incubated for 48 h at 37 °C. Visible colonies were counted using a colony counter machine (ROCKER Galaxy 230).

    RESULTS: The result showed that the denture base polymer significantly affected the initial adherence (p = 0.007). The removal of C. albicans was also considerably affected by the denture base polymers and denture cleansers (p 

  14. Wong GR, Ha KO, Himratul-Aznita WH, Yang YH, Wan Mustafa WM, Yuen KM, et al.
    Oral Dis, 2014 Nov;20(8):762-7.
    PMID: 24320099 DOI: 10.1111/odi.12218
    The objective of the study was to determine the prevalence of HPV seropositivity among patients with oral squamous cell carcinoma (OSCC) and healthy individuals and to correlate the association between HPV 16 seropositivity and risk of OSCC.
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