Volume 6, Spring 2008 - Saddleback College

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Fall 2007 Biology 3A Abstracts Intestinal Candida albicans Overgrowth in Autistic Children with Food Allergies Crystine Gill and Samantha Lopez Department of Biological Science Saddleback College Mission Viejo, Ca 92692 Candida albicans overgrowth in the intestines has been proposed as a causative factor in a portion of autism cases. An overgrowth of C. albicans in the intestine can cause perforations in the intestinal walls that permit undigested substances to enter directly into the bloodstream, which can affect the central nervous system, and/or result in food sensitivities. Urine samples were collected from autistic and non-autistic children Homosapiens between the ages of 4-14 years old. Samples were plated on Bacto Candida BCG formula agar, for specific identification of Candida species. The plates were examined for fungal growth after 72 hours of incubation at 37° C. Variations in species of Candida were found, including Candida albicans, Candida stellatoidea, and/or Candida guilliermondii. Significant differences were not found in the frequency of Candida growth between specimens from autistic children and non-autistic children (p=0.234, one-tailed t- test). A correlation between children with food allergies and Candida growth was noted, but a significant difference was not supported (p=0.267, one tailed t-test). Introduction Autism is a condition, which can vary in degree of symptoms, characterized by impaired communication (delayed or lack of language), repetitive behaviors (preoccupations, motor mannerisms) and impaired social interaction (lack of eye gaze, peer relationships, sharing interests with others, reciprocity) (Vig and Jedrysek 1999). The diagnosis of Autism spectrum disorders has become an increasing syndrome among children within the United States. About one per 150 children is diagnosed with Autism Spectrum Disorders (CDC). Presently, the specific etiology of autism is unknown. Genetics and environmental combinative factors are lead potential causes of the disorder (Szatmari 2000). A higher frequency of gastrointestinal dysfunction is found in among people diagnosed with autism and other developmental delays (Erickson and others 2005). Studies have found evidence of immune response to intestinal fungus (Torrente and others 2002, Edelson 2000). Further studies have addressed the alleviation of the overproduction of immune responses that manifest as GI dysfunctions (Torrente and others 2004). The presence of epithelial IgG and other immunoglobulins in foveolar and glandular epithelium suggests the body is responding to an infection within the digestive tract. The focus of this study is the causative factor of GI dysfunction in autistic children, proposed as Candida albicans. C. albicans is known to cause diseases varying from simple mucocutaneous infections to fatal candidiasis. An overgrowth of C. albicans can invade intestinal tissue causing Spontaneous Intestinal Perforation (Robertson and others 2003). Undigested proteins or substances may enter directly into the bloodstream, possibly causing toxic effects possibly attributing to characteristics of autism, or causing food sensitivities (Andrutis 2000). A symptom of a Candida can be food allergy or food sensitivity. Those diagnosed with autism show cognitive dysfunctions, which may be a secondary result of by the toxic effects to a fungal infection. The presence of C. albicans in urine is expected to be found at a greater frequency in children diagnosed with autism and having food allergies than in nonautistic children having food allergies Materials and Methods Twenty-one children between the ages of four and fourteen years old participated in this study (mean age: 7); ten males and eleven females. Twelve, of the twenty-one participants, are diagnosed with autism, ranging from severe to high functioning. Food allergies are reported in six of the autistic children and two of the non-autistic children. All participants reside within Orange County, California. Urine samples were collected in sterile specimen cups between April 1-9, 2008. Urine was plated, via aseptic techniques, within 48 hours from collection 24 Saddleback Journal of Biology Spring 2008
Fall 2007 Biology 3A Abstracts onto Agar plates, prepared with bromcresol green (pH 6.1 ± 0.1; at 25 °C) according to a Bacto Candida BCG agar formula (Difco, NJ). Urine not plated within 1 hour was refrigerated at 10°C until plating. Inoculated plates were incubated at 37 °C. Examination for Candida albicans growth on the plates, was performed after 24 hours and after 72 hours of incubation. Difco Manual was used as a reference for morphology and pH indications to identify various Candida species. The morphology of the fungal colonies present was interpreted as Candida albicans, Candida stellatoidea and/or Candida guilliermondii (Difco reference manual). An unpaired, t-test and A-Nova test was performed in MS Excel (Microsoft Corporation, Sylmar, Ca) to assess the statistical differences between the quantity of specimens positive for Candida fungus in children diagnosed with Autism compared to children not diagnosed with Autism. Results After three days of incubation, inoculated agar plates were observed for growth and morphological identification. Twelve of the twenty-one plates had positive Candida growth (table 1). Fifty percent of the plates with specimen from children diagnosed with autism and sixty seven percent of the plates with specimen from non-autistic children were positive for Candida growth. Table 1. Results of urine samples plated on pH indicated agar, to indicate intestinal Candida growth in children with and without autism and food allergies. +Growth + Food allergies -Growth + Food allergies + Growth -Food allergies -Growth -Food allergies Autistic children 4 2 2 4 Non-autistic children 2 0 4 3 Plates that were positive for Candida growth varied in morphology most commonly found configuration was round with raised margin, smooth margins, and convexed elevation. Few of the colonies showed variations in morphology including one concentric configuration, undulate or irregular margins, and elevations ranging from raised to drop-like. All growths were characteristically deep green to blue with margins fading to a pale yellow (figure 1). A decrease in pH was also observed as the blue in the agar change d to green or yellow around colonial growths. Figure 1. Candida growth showing characteristic morphology round configuration with smooth (some undulated in this figure), raised margin, and convexed elevation. Deep green to blue coloring, with diminishing color to pale yellow toward margins. Interpretation of identification was ambiguous, due to less than 100% agreement between defined morphological characteristics and morphology characteristics observed on the plates. However, each colony was deduced to be one of two types of Candida. Eight of twelve growths were identified as either Candida albicans or Candida stelloidea. Three were identified as Candida albicans or Candida guilliermondi. One was deduced to be Candida albicans. There was not a significant difference in the number of positive plates of Candida growth between autistic and non-autistic children (p= 0.234, one-tailed t-test; n=21). Sixty-seven percent of the plates from children having food allergies were positive for Candida growth. There was not a significant difference in the number of plates positive for Candida growth between children with and without food allergies (p= 0.267, one-tailed t-test; n=6). Discussion This study did not support a significant difference in Candida growth between autistic children and any other group including autistic children without food allergies, non-autistic children with and without food allergies. The small number of participants yielded a limited result with narrow statistics in this study. False positives may be a variable, as independent collection of specimens may have been exposed to non-aseptic environments. Additionally, females are 25 Saddleback Journal of Biology Spring 2008
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