In vitro antifungal resistance of oral Candida albicans strains in non-AIDS Patients

Some cases of oral candidosis are refractory to antifungal treatment. This might be related to development of resistant Candida strains, but susceptibility testing is not standardized and not routinely available, and information related to this problem is scarce in non-AIDS patients. In this study, the in vitro antifungal resistance of oral Candida albicans strains was evaluated. The strains were obtained from a cohort of 72 HIV-negative patients with oral yeast carriage and clinical complaint. Laboratory identification revealed C. albicans in 93% of cases. None of these oral C. albicans isolates showed in vitro resistance to polyenes, but they showed varying resistance levels to fluorocytosine and azoles. This study confirms the usefulness of standardizing susceptibility testing so that it could be routinely available and of realizing a mycological diagnosis including an antifungigram when oral candidosis is suspected, whenever antifungal treatment with azoles is planned.     

In vitro antifungal susceptibility to six antifungal agents of 229 Candida isolates from patients with diabetes mellitus

The most common antifungal drugs in current clinical use for the treatment of oral candidosis are polyenes and azoles, mainly used topically. Poor glycaemic control in association with other local factors, such as the presence of oral dental prostheses, salivary pH, salivary flow rate and tobacco habits, may lead to the development of oral candidosis. Topical antifungal agents are frequently used to prevent the development of candidal infections in patients with poor metabolic control, particularly in the elderly wearing dentures. The aim of this study was to assess the antifungal susceptibility of Candida isolates to six antifungal agents using a commercially available kit, Fungitest. The isolated were collected from patients affected by diabetes mellitus from two different geographic localities (London, UK, and Parma, Italy) and from a group of healthy non-diabetic subjects. No differences in antifungal susceptibility to the six agents tested were observed between Candida isolates from diabetic and non-diabetic subjects. However, differences were observed between the two geographically different diabetes mellitus populations. Oral yeast isolates from diabetes mellitus patients in the UK more often displayed resistance or intermediate resistance to fluconazole (P=0.02), miconazole (P<0.0001), and ketoconazole (P=0.01) than did isolates from diabetes mellitus patients in Italy. In addition, more C. albicans isolates were found in diabetic and non-diabetic subjects that were susceptible to fluconazole (P=0.0008 and P=0.01, respectively) than non-albicans isolates. The difference in the antifungal resistance of isolates from the two populations of diabetes mellitus patients may be related to differences in the therapeutic management of candidal infections between the two centres.     

In vitro susceptibility of Candida albicans isolates from apical and marginal periodontitis to common antifungal agents

The susceptibility of a total of 70 Candida albicans strains to five common antifungal agents was determined. Thirty-five of the strains were isolated from persistent cases of apical periodontitis and 35 from cases of marginal periodontitis. The susceptibility of the strains to amphotericin B, 5-fluorocytosine and three azoles: fluconazole, miconazole and clotrimazole, was tested. The antifungal agents and yeast inoculums were prepared according to the NCCLS (National Committee for Clinical Laboratory Standards) recommendations. The yeasts were incubated with ten different concentrations of antifungal agents at 35 degrees C for 48 h. Yeast growth was measured spectrophotometrically. All strains from both isolation sources were susceptible to low concentrations of amphotericin B and 5-fluorocytosine, whereas the susceptibility to the three azoles varied, and three of the strains showed azole cross-resistance. These findings are in agreement with recent reports of increased azole resistance in Candida species in general and suggest the possibility that the oral cavity may act as a reservoir of resistant yeast isolates in systemic infections.     

五白汤组分体外抗白色念珠菌活性筛选

目的：筛选五白汤组分中具有抗白色念珠菌活性的药物。方法：采用白色念珠菌标准菌株（ATCC76615）为研究对象,应用煎煮法及水提醇沉淀法分别对五白汤各组成药物进行制备,参照NCCLS M27-A2推荐的方法测定五白汤各组成药物对白色念珠菌的最小抑菌浓度（MIC）。结果：白鲜皮水煎剂、苦参水煎剂和苦参醇提液对白色念珠菌的MIC分别为62.5 mg/mL、15.625 mg/mL、62.5 mg/mL。结论：白鲜皮水煎剂、苦参水煎剂以及苦参醇提液具有抗白色念珠菌活性。     

Prevalence of fluconazole-resistant strains of Candida albicans in otherwise healthy outpatients

BACKGROUND: In contrast to the immunosuppressed patient population, the prevalence of fluconazole-resistant strains of Candida albicans among healthy individuals has not been extensively studied. METHODS: Candida species were cultured form 50 healthy outpatients with clinical signs of oral candidiasis. Following one week of the recommended fluconazole regimen, post-treatment cultures were obtained. Both pre- and post-treatment yeasts were identified and in vitro susceptibility testing was performed using the NCCLS M-27A method. Strains were further differentiated using established cDNA probes. RESULTS: Forty-four patients (88%) had positive C.albicans cultures prior to treatment. Antifungal susceptibility testing of these strains demonstrated no in vitro resistance to fluconazole. At post-treatment evaluation, eight patients (18%) had persistent signs of infection and 10 patients (23%) had positive Candida sp. cultures despite no clinical signs of infection. DNA analysis confirmed that the same C. albicans strain was present both in the pre-treatment and the post-treatment cultures. CONCLUSIONS: Our results showed that the presence of fluconazole-resistant strains of C.albicans does not appear to be prevalent among healthy outpatients furthermore, in vitro antifungal susceptibility testing does not always predict successful therapy in these patients.     

Stable azole drug resistance associated with a substrain of Candida albicans from an HIV-infected patient

Oral candidiasis is one of the earliest and most frequent complications of a failing immune system in HIV-infected individuals. For several years, oral candidiasis has been treated effectively with azole drugs, the one most frequently used is fluconazole. Unfortunately, extensive use of the drug for treatment and prophylaxis has led to treatment failure in an increasing number of patients. In most of these cases, strains of C. albicans isolated from the infection are less susceptible to fluconazole. The development of azole resistance in strains of C. albicans has been studied biochemically and more recently with molecular techniques. One excellent example of the development of azole resistance in C. albicans has been documented in a series of 17 C. albicans isolates from a single patient over a 2-year period. During this time, the patient experienced 14 episodes of oral candidiasis and was treated with increasing doses of fluconazole. Molecular and biochemical analyses confirms that the isolates are the same strain of C. albicans and that the resistance in these isolates is stable over 600 generations, suggesting that the changes in this strain are genetic in nature. In addition, the development of resistance is correlated with the identification of a substrain or variant of the original strain, as identified by restriction fragment length polymorphism (RFLP) analysis with the moderately repetitive probe, Ca3. The analysis of this series of isolates demonstrates that azole drug resistance is associated with several small genetic changes, each of which contributes to the overall resistance of the strain. Clearly, continual use of azole drugs by a patient can select for genetic changes that render oral candidiasis refractory to treatment.     

Influence of antifungal polyenes on the adhesion of Candida albicans and Candida glabrata to human epithelial cells in vitro

Candidal adherence to mucosal surfaces is considered as the first step in the pathogenesis of oral candidiasis. We examined the effect of antifungal polyenes, amphotericin B, nystatin and natamycin, at sublethal and minimum inhibitory concentrations (MICs) on the adherence of Candida albicans and Candida glabrata to HeLa cervical carcinoma and HSC-3 oral squamous cell carcinoma cells. A total of six oral Candida isolates were used throughout the study. Two Candida strains, C. albicans (44990) and C. glabrata (MYA-275) were obtained from ATCC. Four Candida strains, C. albicans 19 and 24 and C. glabrata 15 and 21, were isolated from patients with documented Candida-associated denture stomatitis. Cells were either incubated with Candida in the presence of the drug, or pre-incubated with yeasts and exposed subsequently to the drug. In the drug-free controls, the mean number of C. albicans yeasts associated with HeLa cells obtained from all experiments (130.1±10.1 yeasts/mm²) was significantly greater than that for HSC-3 cells (114.7±10.1 yeasts/mm²; P<0.025). For C. glabrata, the mean adherence to HeLa and HSC-3 cells was 84.4±5.5 and 84.4±3.3 yeasts/mm², respectively, and these values were not statistically different (P>0.4). Candidal adherence was significantly reduced when the tested polyenes were present during the “adherence phase”. The obtained values were significantly different from the controls, except for the effect of nystatin at the MIC on the adherence of C. glabrata strain MYA-275 to HeLa cells (P<0.375). Amphotericin B had the highest effect against both Candida species, reducing adherence by 50 and 60%, at the MIC and sublethal concentrations, respectively. The susceptibility of cell-associated Candida to polyenes was decreased markedly and the treatment did not result in significant detachment of adherent yeasts. The reduction in adherence was between 2 and 10%, when compared to the drug-free controls. These findings suggest that sub-therapeutic levels of polyenes that are likely to persist in the oral cavity following topical treatment may modulate candidal colonization when present during the “adherence phase”.     

In vitro secreted aspartyl proteinase activity of Candida albicans isolated from oral diseases and healthy oral cavities

The in vitro secreted aspartyl proteinase (SAP) activity of Candida albicans isolated from a variety of oral conditions, including healthy oral cavities, was determined. SAP activity (units/10(6) cells/ml, +/-SD) was 0.28 +/- 0.33 for pseudomembranous candidosis isolates (n = 18), 0.35 +/- 0.46 for chronic erythematous candidosis isolates (n = 21) and 0.30 +/- 0.32 for chronic hyperplastic candidosis isolates (n = 50). SAP activity of 0.19 +/- 0.22 was recorded for isolates from squamous cell carcinoma (n = 18), 0.26 +/- 0.37 for burning mouth syndrome isolates (n = 29), 0.25 +/- 0.38 for isolates from xerostomia (n = 15) and 0.39 +/- 0.50 for isolates from lichen planus (n = 13). The SAP activity of isolates from oral disease states was significantly (P < 0.05) higher than that recorded for 28 isolates from healthy mouths (activity of 0.04 +/- 0.03). However, there was no significant difference in the SAP activity between the three forms of clinical oral candidosis (P > 0.05). SAP activity was inhibited in control samples containing the SAP inhibitor, pepstatin A. These results indicate that C. albicans strains associated with oral disease have inherently higher SAP activity.     

Antimicrobial susceptibility of oral isolates of Enterobacter cloacae and Klebsiella pneumoniae from a southern Chinese population

The antibiotic susceptibilities of 59 Enterobacter cloacae and 39 Klebsiella pneumoniae human oral isolates collected from a southern Chinese population in Hong Kong were investigated for their susceptibility to eight antibiotics: ampicillin, cephalothin, cefuroxime, ceftazidime, ciprofloxacin, gentamicin, tetracycline and trimethoprim/sulfamethoxazole using the E-Test method for direct quantification of minimum inhibitory concentrations. Most strains were sensitive to all antibiotics except ampicillin and cephalothin. Ampicillin resistance was exhibited by 82% of K. pneumoniae and 69% of E. cloacae isolates. Eighty-eight percent of E. cloacae isolates were resistant to cephalothin. Several strains fell within the intermediate category of sensitivity for ampicillin (E. cloacae and K. pneumoniae) , cefuroxime (E. cloacae) and tetracycline (K. pneumoniae). Comparison with other Hong Kong data suggests that resistance rates to cefuroxime, ceftazidime, ciprofloxacin, gentamicin, tetracycline and trimethoprim/sulfamethoxazole exhibited by the oral isolates are generally lower than in enterobacters and Klebsiella spp. isolated from urine, skin and soft tissues in Hong Kong populations.     

Prevalence of Candida species in AIDS patients and HIV-free subjects in Thailand

Teanpaisan R, Nittayananta W: Prevalence of Candida species in AIDS patients and HIV-free subjects in Thailand. J Oral Pathol Med 1998; 27: 4–7. © Munksgaard, 1998.
The purpose of this study was to examine the prevalence of Candida species among groups of HIV-infected and HIV-free subjects in Thailand and to ascertain whether particular Candida species were associated with HIV infection. Oral rinse specimens were collected from 45 AIDS patients (CDC stage IV), 74 HIV-free healthy subjects, and 42 HIV-free patients who had clinical candidiasis. Yeasts recovered in culture were identified and quantified. The mean ages of the cohorts were 30.75 ± 8.19 years (AIDS group), 28.50 ± 7.98 (HIV-free healthy group) and 41.83 ± 12.25 years (HIV-free candidiasis group). Yeasts were isolated from 30/45 (66.66%, range 6.6 ± 10²-5.7 × 10⁶ CFU/ml) of the AIDS group, 8/74 (10.81%, range 8.0 × 10¹-3.5 × 10⁴ CFU/ml) of the HIV-free healthy group, and 24/42 (57.14%, range l.0 × 10 10²-1.1 × 10⁵ CFU/ml) of the HIV-free candidiasis group. There were statistically significant differences in the Candida colony counts between the AIDS group without oral candidiasis and the healthy group ( P =0.0078) and between the AIDS group with candidiasis and the HIV-free, oral candidiasis group ( P = 0.0003). Candida albicans was the most common species recovered from AIDS patients (29 out of 30; 96.66%).     

Distribution and hydrolytic enzyme characteristics of Candida albicans strains isolated from diabetic patients and their non-diabetic consorts

Introduction:  The aim of this study was to investigate the oral colonization profile of Candida albicans strains isolated from diabetic patients and their non-diabetic consorts. In addition hydrolytic enzyme activity of these isolates was analysed.
Methods:  The genetic diversity of C. albicans oral isolates from 52 couples was established using isoenzyme marker and cluster analysis. Hydrolytic enzyme characteristics, namely secreted aspartyl proteinases (SAPs) and phospholipases (PLs) were also analysed.
Results:  Simultaneous colonization by C. albicans was observed in the consorts of 12 couples (23.1%). Patterns of monoclonal and polyclonal oral colonization by C. albicans strains were identified and the coexistence of identical or highly related strains was observed in both members of eight couples. The genetic diversity observed in the total yeast population revealed four large, genetically distinct groups (A to D) and the coexistence of strains in couples or consorts conjugally unrelated. SAP and PL activity was observed in the majority of C. albicans isolates without any association to particular strain, strain clusters (highly related isolates), or clinical characteristics of the consorts (diabetic, non-diabetic, and gender).
Conclusion:  Possible sources of transmission and oral propagation of groups (clusters) of strains of C. albicans can occur between diabetic and non-diabetic consorts. A conjugal genotypic identity exists in most C. albicans -positive couples, that is, both consorts share identical or highly related strains; however, this identity is not couple-specific as seen by the coexistence of clusters in couples and unrelated consorts.     

Biotypes of oral Candida albicans isolated from AIDS patients and HIV-free subjects in Thailand

This study was conducted to examine biotypes and antifungal susceptibility patterns of oral Candida albicans isolated from HIV-infected patients, HIV-free patients with candidiasis and healthy subjects. All isolates were biotyped using a typing system based on enzyme profiles, carbohydrate assimilation patterns and boric acid resistance. Thirty-eight biotypes were found amongst 218 oral C. albicans isolates. The major biotype found was A1S, which accounted for 32.6% of all isolates, and this biotype was the most common in all groups. There was a greater variety of biotypes of C. albicans in the HIV-infected group than in the other groups; however, there was no statistically significant difference between the groups. The minimum inhibitory concentrations (MICs) of a total of 118 isolates were determined for amphotericin B and for ketoconazole using the National Committee for Clinical Laboratory Standards (NCCLS) broth macrodilution method and the E-test. When the antifungal susceptibility patterns among the groups were compared, a statistically significant difference was found only with amphotericin B. The median MIC of amphotericin B in the HIV-infected group was higher than in the healthy group (P=0.013, NCCLS method; P=0.002, E-test). However, this difference in sensitivity was not restricted to any sub-type investigated. Our results showed that the biotype patterns of C. albicans isolates that colonize HIV-infected patients are similar to those of HIV-free subjects, and there is no relationship between antifungal susceptibility patterns and the biotypes.     

Distribution and hydrolytic enzyme characteristics of Candida albicans strains isolated from diabetic patients and their non‐diabetic consorts

Introduction: The aim of this study was to investigate the oral colonization profile of Candida albicans strains isolated from diabetic patients and their non‐diabetic consorts. In addition hydrolytic enzyme activity of these isolates was analysed. Methods: The genetic diversity of C. albicans oral isolates from 52 couples was established using isoenzyme marker and cluster analysis. Hydrolytic enzyme characteristics, namely secreted aspartyl proteinases (SAPs) and phospholipases (PLs) were also analysed. Results: Simultaneous colonization by C. albicans was observed in the consorts of 12 couples (23.1%). Patterns of monoclonal and polyclonal oral colonization by C. albicans strains were identified and the coexistence of identical or highly related strains was observed in both members of eight couples. The genetic diversity observed in the total yeast population revealed four large, genetically distinct groups (A to D) and the coexistence of strains in couples or consorts conjugally unrelated. SAP and PL activity was observed in the majority of C. albicans isolates without any association to particular strain, strain clusters (highly related isolates), or clinical characteristics of the consorts (diabetic, non‐diabetic, and gender). Conclusion: Possible sources of transmission and oral propagation of groups (clusters) of strains of C. albicans can occur between diabetic and non‐diabetic consorts. A conjugal genotypic identity exists in most C. albicans‐positive couples, that is, both consorts share identical or highly related strains; however, this identity is not couple‐specific as seen by the coexistence of clusters in couples and unrelated consorts.     

Inhibition of adherence of Candida albicans and Candida dubliniensis to a resin composite restorative dental material by salivary secretory IgA and monoclonal antibodies

OBJECTIVE: The attachment of Candida to oral surfaces is a crucial step in the colonization of the oral cavity and the eventual development of oral diseases caused by this microorganism. Inhibition of adhesion is one of the strategies currently studied to prevent Candida infections. The main objective of this study was to investigate the inhibitory effect of the human salivary components on the adherence of Candida albicans and C. dubliniensis to Herculite, a widely used resin composite restorative dental material. We have also investigated the influence on the adherence of three monoclonal antibodies (mAbs) directed against C. albicans cell wall antigens. DESIGN: The adhesion of three strains of C. albicans and one strain of C. dubliniensis was studied by a visual method after incubating the fungus and the resin in presence and in absence of human whole saliva, secretory immunoglobulin A (sIgA) and three mAbs directed against C. albicans cell wall surface antigens. RESULTS: Adherence of C. albicans was inhibited by whole saliva (41.7%), salivary sIgA (55.7%) and the salivary components that bind to the cell wall (36.7%). Whole saliva significantly reduced the adhesion of C. dubliniensis to Herculite to 45.3% of the control level. Saliva previously adsorbed with fungal cells or sIgA depleted saliva had no effect on adherence. An inhibition in the adhesion of C. albicans and C. dubliniensis to Herculite similar to that shown by whole saliva was also observed when mAbs C7 and 26G7 were used. However, mAb 21E6 increased adhesion of all the strains to Herculite. CONCLUSIONS: The results suggest that sIgA, as well as whole saliva, are important in blocking adherence of C. albicans and C. dubliniensis to Herculite and that this effect can be reproduced with mAbs directed against the cell wall surface of C. albicans.     

头颈部放化疗后口腔假丝酵母菌耐药性分析及抗真菌中药筛选

目的 研究头颈部放化疗患者口腔假丝酵母菌感染菌株的种类及耐药性,并依据美国国家临床实验室标准协会（NCCLS）的M27-A2标准方案筛选抗真菌中药。方法 采集头颈部放化疗患者口腔假丝酵母菌感染的临床分离菌株20株,经YBC Test Kit鉴定假丝酵母菌种类;依据NCCLS的M27-A2标准方案测定其对5-氟胞嘧啶、氟康唑、伊曲康唑、6种中药提取物（蒺藜、金银花、蒲公英、绿茶、松树皮、红三叶草提取物）及7种中药有效成分（苦参碱、苦豆碱、大黄素、甘草酸、白芍总苷、黄芩苷、盐酸小蘖碱）的药物敏感性。结果 20株放化疗患者口腔假丝酵母菌感染菌株中非白假丝酵母菌5株,对5-氟胞嘧啶全部敏感,对氟康唑和伊曲康唑的耐药比例分别为25%和40%;白芍总苷和盐酸小蘖碱具有抗假丝酵母菌活性。结论 头颈部放化疗患者口腔假丝酵母菌感染菌株中非白假丝酵母菌比例及对氟康唑和伊曲康唑的耐药比例较高;白芍总苷和盐酸小蘖碱是有临床应用前景的抗口腔假丝酵母菌药物;依据NCCLS的M27-A2标准方案筛选抗真菌中药是一种准确有效的方法。     

Genetic diversity and exoenzyme activities of Candida albicans and Candida dubliniensis isolated from the oral cavity of Brazilian periodontal patients

Objective

Mucosal surfaces are the primary oral reservoirs of Candida species, but these species can also be found in subgingival biofilm. The present study investigated the genetic diversity and production of exoenzymes of C. albicans and C. dubliniensis isolated from the oral cavity of systemically healthy patients with periodontitis.

Design

Fifty-three patients were analysed. Samples were collected from three oral cavity sites (periodontal pocket, gingival sulci and oral mucosa), plated and, after isolation, suspect strains of C. albicans and C. dubliniensis were identified by PCR. The genetic diversity of the isolates was evaluated by RAPD and the activities of the secreted aspartyl proteinases and phospholipases were evaluated by the agar plate method.

Results

Twenty-one patients showed positive results for Candida spp. There were no statistically significant differences between genders, or between sites. C. albicans was the most frequently found specie, while C. dubliniensis was isolated from the periodontal pocket of only one patient. Sixteen genotypes were detected among the C. albicans isolates, and one among the C. dubliniensis isolates. The similarity coefficient (S_SM) values among the C. albicans genotypes ranged from 0.684 to 1.0 with an average of 0.905 ± 0.074. All isolates produced high levels of Saps and most of them produced high levels of phospholipases. No relationship was found between the genotypes and the pattern of enzymatic production. There was no association between specific genotypes and their site of isolation.

Conclusions

The results of the present study suggest that genetically homogeneous strains of C. albicans are present in the oral cavity of patients with periodontitis and that these strains are capable of producing high levels of exoenzyme.     

Correlation between enzyme production,germ tube formation and susceptibility to fluconazole in Candida species isolated from patients with denture‐related stomatitis and control individuals

Introduction: Phospholipase and proteinase secretion in yeasts of the genus Candida has been described as a relevant virulence factor. Also, germ tube formation by Candida albicans is associated with its invasive capacity and is considered an important pathogenic mechanism. Methods: To link the production of hydrolytic enzymes with the capacity to produce infection, 232 clinical isolates of yeasts from the oral cavity of 140 individuals wearing removable maxillary protheses were studied. The sample was composed of 70 patients with denture‐related stomatitis (DRS) and 70 individuals with normal palatal mucosa. For strains identified as C. albicans, the correlation between germ tube formation and their capacity to cause infection was studied and the presence of Candida dubliniensis was investigated. Susceptibility to fluconazole was evaluated. Results: Candida albicans was the only species producing phospholipase and germ tube. We observed a higher level of production of phospholipase in cases of infection compared with commensals. Significant differences between the two groups of C. albicans isolates were observed as to germ tube production. Only, Candida glabrata showed lower susceptibility to fluconazole. Conclusion: The results reinforced the idea that C. albicans is the most frequent and can be the most pathogenic yeast in oral candidosis. However, the strains isolated from DRS patients and healthy individuals showed the same virulence factors. It seems that several virulence attributes are involved in the infective process but no single factor contributes to Candida virulence. Candida dubliniensis was absent in the oral cavity of individuals with and without DRS.     

Phenotypes and randomly amplified polymorphic DNA profiles of Candida albicans isolates from root canal infections in a Finnish population

A total of thirty-seven Candida albicans isolates from root canal infections in a Finnish population were subtyped using phenotypic and genotypic methods. A previously described biotyping method based on the presence of five different enzymes, assimilation of eleven different carbohydrates and boric acid sensitivity of the yeasts was used to determine the phenotype. Commercially available API ZYM and API 20 C test kits were used to determine the presence of enzymes and assimilation of carbohydrates. The sensitivity of the isolates to boric acid was tested by their ability to grow on yeast-nitrogen-agar with incorporated boric acid (1.8 mg. ml(-1)). Combination of the tests revealed a total of 14 different phenotypes. The majority of the isolates, 26 strains, were classifiable into three major phenotypes: 16 isolates (43.2%) belonged to phenotype A1R, six (16.2%) to A1S and four (10.8%) to B1S. The remaining 11 phenotypes represented only a single isolate each. The randomly amplified polymorphic DNA profiles were used to determine the genotypes. For this purpose two different primers, RSD6 and RSD12 were used to develop a combination randomly amplified polymorphic DNA profile for each isolate. Altogether 31 genotypes were noted among the 37 isolates, of which only three pairs of isolates presented with congruent phenotypic and genotypic profiles. The heterogeneity of both the phenotypic and randomly amplified polymorphic DNA profiles of C. albicans isolates from root canal infections is akin to previous reports from other oral and non-oral sources in different geographic locales.