COLLEGE OF NATURAL AND APPLIED SCIENCES DEPARTMENT OF MOLECULAR BIOLOGY AND BIOTECHNOLOGY BL 390(RESEARCH PROJECT) RESEARCH PROPOSAL

TITLE: Assessing the hospital environment as a potential source of fungal nosocomial infections.

1.INTRODUCTION 

Hospital environment is a potential source of nosocomial pathogenic fungi due to overcrowding and poor environmental sanitation, increase of immunocompromised people attending the hospital for a visit or for other purposes. Patients are primarily admitted into hospital wards for proper management of their ailments, but while on admission some patients acquire other ailment than the one they were admitted for. These are called hospital associated infections (nosocomial infections) which can result from contact with a carrier directly or indirectly through inanimate objects or air. Fungal nosocomial infections is the one of nosocomial infections which is most observed in hospital areas these infections are aggravated by the reduced resistance of individual patients (1)

The quality of indoor air in terms of fungi microbial contamination in a given space at a given time period is said to be determined by the quality of air entering the space, the number of occupants in the space, number of patients in the hospital ward, their physical activities and resultant aerosol generation, human traffic and the degree of ventilation. Dust, which is a good vehicle of airborne contamination, may arise from human activities, such as sweeping, movement, waving of handkerchief and bed making. Sneezing has been described as the most vigorous mechanism of generating millions of droplet into the environment, and. While the larger droplets fall to the ground or on nearby objects, the smaller ones are rapidly evaporated to their non-volatile residual forms and remain suspended as droplet nuclei. and in this case they become infectious once they contain  fungi.

Measures frequently taken in preventing fungal nosocomial infections include effective use of antiseptics, disinfectants, adequate cleaning, and isolation of patients with highly infectious diseases. Sanitation protocols regarding uniforms, equipment sterilization, washing, and other preventive measures. Thorough hand washing and/or use of alcohol rubs (disinfectants)by all medical personnel before and after each patient contact is one of the most effective ways to combat nosocomial infections

Nevertheless, less attention is paid to indoor air as been a probable contributing factor to hospital acquired infections. Ishida et al reported that airborne bacteria in the hospital environment have been a major source of post-operative infection and a serious problem in the Intensive Care Unit. Many of these isolates (bacteria and fungi ) are shown to be resistant to common antiseptics used in hospital, hence the result into  possible infections

The organism that are more associated with fungal nosocomial infection include superficial infections, these fungal infections affect the skin or mucous membranes. Superficial fungal infections (e.g., yeast vaginitis, oral thrush, and athlete’s foot) affect millions of people worldwide. Although rarely life-threatening, they can have debilitating effects on a person's quality of life and may in some cases spread to other people or become invasive (systemic) and this is the case in Hospital environment. Most superficial fungal infections are easily diagnosed and can be treated effectively. Other organism are called systemic infections, these occur when fungi get into the bloodstream and generally cause more serious diseases. Systemic fungal infections may be caused either by an opportunistic organism that attacks a person with a weakened immune system, or by an invasive organism that is common in a specific geographic area, such as cocci and histoplasma. Unlike superficial infections, systemic fungal infections can be life-threatening. (3)

Infections are contagious and represent a significant public health problem in many parts of the world.  Dermatophytosis is not a reportable disease but is a matter of concern because of its contagiousness nature (4) correct identification of dermatophytes at the species level is useful for differentiating between Dermatophytosis and dermatomycosis (5), to control of environmental and animal sources of infection and help for developing the preventive strategies

Conventional identification techniques raises controversial issues like lack of evidence to confirm the isolated pathogenic fungi  types of species, Species-level identification of these fungi classically relies on macro and micro morphological features of the colonies on general and specific  culture media and on some biochemical and  physiological complementary tests (6). However,  in many circumstances phenotypic characteristics  overlap between species, and many isolates have  atypical nature in primary isolation thus attempt  for final identification is time consuming and  requires expertised personnel on macroscopical properties (7). By development of PCR  technology, a wide variety of molecular techniques  such as RAPD-PCR, Nested-PCR, PCR-RFLP,  PCR-EIA, Real-time PCR and microarray technology were employed as possible alternatives  for routine identification of fungi including dermatophytes (7).At the present study, the ITS1-5.8S-ITS2  fragment of ribosomal DNA gene (rDNA) in the  Dermatophyte species will be used as a reliable  marker for species identification. We will retrieve the reliable sequences of internal transcribed spacers (ITS) regions from GenBank, then omputationally (in-silico) and practically subjected them to a polymerase chain reaction-restriction enzyme (PCR-RE) assay for identifying nearly all pathogenic Dermatophyte species. Additionally, we will amplify and digest the DNA target in some reference Dermatophyte strains to confirm the method. We will   prepare a relatively perfect restriction fragment length polymorphism (RFLP) barcode by using only a single enzyme and believe that it could be useful for clinical and epidemiological aims .

Therefore this study will   be to aimed to  presents the findings about the possible source of nosocomial fungal infections in hospital environment  . it will also  investigate  the quality of indoor air of different wards and units of a chosen  Hospital, to ascertain their contribution to infection rate in the hospital. It will also provide a baseline information on the quality of indoor air, hospital wards which before was not available.

2. STATEMENT OF THE RESEARCH PROBLEM 

It has been proved that in hospitals pathogenic  fungi identifications can  facilitate  tracing the  sources of  infections in  hospitalized patients and then get raid on them ,get in control  of them .

Previous studies has shown that hospitalized patient are more likely to be affected by any pathogenic fungi even if in its mildest infection stage

There is still more to assess about hospital environment, like where is the highly infected environment that can raise contamination of the immunocompromised people 

And also how hospitalized people get infected by nosocomial fungal infections and who can possibly be responsible.

3. OBJECTIVES

3.1. GENERAL OBJECTIVE

Assessing the hospital environment as a potential source of fungal nosocomial infections.

3.2. SPECIFIC OBJECTIVES

• To determine molecular relatedness of isolates for epidemiologic investigation;
• To determine  the relationship  of infected areas and fungal infection rate in the Hospital;
• To determine  the diversity  and distribution  areas of  Pathogenic  fungi in Hospital

4. SIGNIFICANCE OF THE STUDY

Why assessing hospital environment as a source of nosocomial fungal infections?

Patients are primarily admitted for proper management instead they acquire nosocomial fungi infections

 Identification of the infected hospital areas by fungi would be advantageous and will help in getting raid of the nosocomial fungi infections

 Assessing hospitals environment will help the doctors to advice the patients and to educated the world how to behave when at the hospitals

5. LITERATURE REVIEW

6. STUDY AREA

7. HYPOTHESES/RESEARCH QUESTIONS

Ø  Can molecular relatedness of isolates for epidemiologic investigation be determined?

Ø  Can infected areas contribute to fungal infections rate in hospital?

Ø  Can the diversity and distribution areas of Pathogenic fungi in Hospitals be determined?

8. MATERIALS AND METHODS

8.1 MATERIALS

ü  Blunt scalpel

ü  sterile petriplate

ü  white envelopes

ü  Sabouraud’s dextrose Agar (Difco) (SDA)

ü  Chromo agar Candida

ü  Glass bead

ü  of  lysis buffer (100  mM Tris-Hcl pH 8,  10  mM  EDTA,  100  mM  NaCl,  1%  sodium  dodecyl  sulfate  (SDS),  2%  triton  X-100

ü  phenol  chloroform-isoamyl  alcohol  (25:24:1)

8.2. METHODS

Samples  will be  collected from the air ,hospital ward surfaces where patients have access to . The specimens will be  placed in clean white envelopes with each areas  code labeled and later  they will be  transferred to a sterile petriplate, we will also collect sample from indoor and outdoor environment (including  air,  medical  devices, clothes,  catheters  and  ) of the Hospital. We are expecting to get  a lot of   fungi  isolates  (including  yeasts and  moulds)  which will be collected  from  clinical  specimens consisting  of  urine,  surgical  wounds,  bedsores,  sputum  and  others,  and  some  locations  where  Fungi  are considered  as  normal  flora  such  as  skin,  oral  and nasal  cavities

Isolation medium:

The fungi will be allowed to grow on Sabouraud’s dextrose Agar (Difco) (SDA) with chloramphenicol (Chloramphenicol inhibits the growth of bacterial contaminants) and bromocresol purple milk glucose agar (Ellis et al.,2007)

Yeast, Fungi, Moulds   isolates  will  be  sub cultured  on  Chromo agar  Candida,  incubated  at  35^oC  for  2-4  days  and  harvested  yeast, fungi, moulds colonies will be  stored at -20°C

DNA extraction:

Further Genomic DNA will be extracted using glass bead disruption. Briefly,  300  mg  of  0.5  mm  diameter  glass  beads, 300µg of  lysis buffer (100  mM Tris-Hcl pH 8,  10  mM  EDTA,  100  mM  NaCl,  1%  sodium  dodecyl  sulfate  (SDS),  2%  triton  X-100)  and  300  µl  of  phenol  chloroform-isoamyl  alcohol  (25:24:1)  will be  added  to  about  1-5  mm of  a  fresh  yeast  colony  in  a  1.5  ml  tube. The samples  will be shaked  vigorously for 5 min,  centrifuged  for  5  min  at  5000  rpm,  and  the  supernatant will be  transferred to  a fresh tube. The supernatant  will be extracted  again  with  chloroform  and  DNA  will be   precipitated by  adding  the  equal  volume  of  isopropanol  and  0.1  volume  of  3  M  sodium  acetate  (pH  5.2).  The  solution  will be  vortexed  and  incubated  for  10  min  at  -20°C  and  centrifuged  for  15  min  at  12000  rpm

Restriction fragment length polymorphism( RFLP) analysis :

Fungal-specific universal primer pairs will be used to amplify internal  transcribed spacer 1 ( ITS1)—5.8SS- ITS2 regions of rDNA in the tested fungi, the amplification reaction will be  performed in a final volume of 25 µl 1 containing 1µl of extracted genomic DNA ( about 20mg),1,25 U of Taq  DNA polymerase,0.3Mm of each deoxynucleoside triphosphate mix( dATP,dTTP,dGTP,dCTP) 0.4µM of each  of  forward ITS1 ( 5`-TCC GTA GGT GAA CCT GCG G-3`) and reverse ITS4 (5`-TCC TCC GCT TAT TGA TAT GC-3`) primers, 1.5Mm of MgCl2 and 2,5µL OF 10X PCR buffer. PCR will be  carried out in a thermal cycler  with  the following  temperature profile : 1 cycle  of 5 min at 95 ^OC,followed by 35 cycles of 1 min at 94 ^OC, 1 min at 56 ^OC  and 1 min at 72 ^OC and a final extension step at 72 ^OC  for 10 min. PCR  products will  be  analyzed in 2% agarose gel with 0.5X  Tris - Borate-EDTA buffer, and it will b e stained with  ethidium bromide and be visualized in UV light.

Digestion with restriction enzymes (endonucleases):

Ten microliters of each PCR product will be separately digested with 10U of HinfI and HaeIII restriction    enzymes at 37 ^OC overnight(The amplified products were subjected to digestion with MvaI Fast digest (Fermentas Life Sci-ences, Lithuania) for 10 min at 37ºC. The reaction mixture contained 10 μl of PCR amplicons,  0.5 μl of the enzyme, 1.5 μl of 10X buffer and 3 µl of water to a final volume of 15 µl.). Restriction fragment  will be analyzed in 2.5% agarose gel electrophoresis in 0.5 X Tris Borate-EDTA buffer for about  2.5 h ATT 80V and visualized by staining with 0.5 µg Ml-1 of ethidium bromide.

Data analysis

The gels will be visualized using UV light system and  we will record photographically, then we will  compare  with the profiles  which will be  obtained in silico analysis.

Data of the isolates which will show growth  on SDA will be  analysed using SPSS^TM The results will be then presented in descriptive  statistics using frequency tables, cross tabulation and  bar charts.

REFERENCES

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3.      The Complete Idiot's Guide to Dangerous Diseases and Epidemics © 2002 by David Perlin, Ph.D., and Ann Cohen. 

4.      Anosike JC, Keke IR, Uwaezuoke JC, Anozie JC, Obiukwu CE, Nwoke BEB, Amajuoyi OU (2005).  Prevalence and distribution of ringworm infection in primary school children in parts of Eastern, Nigeria. J Appl Sci Environ Manage, 9(3): 21-25

5.      Panasiti V, Devirgiliis V, Borroni RG, Mancini M, Curzio M, Rossi M, Bottoni U, Calvieri S (2007). Epidemiology of dermatophytic infections in Rome, Italy: a retrospective study from 2002 to  2004. Med Mycol, 45(1): 57-60.

6.      Weitzman I, Summerbell RC (1995). The dermatophytes. Clin Microbiol Rev, 8(2): 240-59.

7.      De Baere T, Summerbell R, Theelen B, Boekhout T, Vaneechoutte M(2010). Evaluation of internal transcribed spacer 2-RFLP analysis for the identification of dermatophytes. J Med Microbiol, 59: 48-54.