RESEARCH

TITLE :Molecular Technique for the identification of diversity and distribution of pathogenic fungi  in Hospitals

1.  INTRODUCTION

1.1  General Introduction

Dermatophytes are the common sources of fungal infections, they cause   public health problem in informal settlements due to overcrowding and poor environmental sanitation, poor education which is the case in most areas in Dar es Salaam. Traditional identification techniques lack sensitivity & specificity, are slow, labor-intensive and require skilled personnel, these methods require specific culture media, and some biochemical and physiological complementary test methods the identifications of dermatophytes using the traditional are sometimes problematic because of atypical microscopic and macroscopic morphology. The shortcomings of   phenotypically based typing methods (generally these methods are viewed as being too time consuming and lacking in sufficient resolution amongst related strains) have led to the development of many DNA – based techniques.

By development of PCR  technology, a wide variety of molecular techniques  such as RAPD-PCR (Kim et al.,2001; Baeza et al.; 2006;Leibner-Ciszak et al.; 2010; Dobrowolska et al.; 2011; Spesso et al.;2013), Nested-PCR(Verrier et al.; 2013), PCR-RFLP(Yang et al., 2008; Rezaei-Matehkolaei et al.,2012, Samuel et al.; 2013),  PCR-EIA, Real-time PCR(Bergman’s et al., 2010)  and microarray technology were employed as possible alternatives  for routine identification of fungi including  dermatophytes   At the present study, the ITS1   and ITS 4 primer   fragment are used  to identify the dermatophyte species as a reliable marker for species identification. We  will retrieve  the reliable sequences of internal transcribed spacers (ITS) regions from GenBank, then  computationally (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 digested  the DNA target in some reference dermatophyte  strains to confirm the method.

RFLP,RAPD  have been adapted to detect dermatophytes from clinical specimens ,this study will presents the findings of the cause of the pathogenic fungi diseases, how to identify it by Molecular methods ,and how to control it.

The  random amplified  polymorphic  DNA  (RAPD) technique  relies  on  the  application  of  short  arbitrary primers  that  anneal  to  multiple  genomic  sites  using low  temperature  conditions.  This method detects strain polymorphisms and does not  depend  on  prior knowledge of species-specific sequence

The aim of this study will be to identify   the distribution and diversity of pathogenic fungi using these 2 molecular techniques (RAPD, RFLP)

The strain typing of the relevant isolates will facilitate tracing of probable source sand routes  of  transmission  of  infection  to  hospitalized  patients or non hospitalized patients  by using RAPD analysis

1.2  Statement of the Research Problem 

Previous studies have shown that the identification of dermatophytes by molecular techniques is more rapid.

It has been proved that in hospitals dermatophytes identifications can  facilitate  tracing the  sources of  infections in  hospitalized patients and can help in its control., On the  other hand,  there  is  a  need  for  recognizing  the  main  source of  the  infections;  whether  it  is  endogenous  or  acquire exogenously  from  other  patients  or  even  health  care workers. Therefore,  precise  identification  of  the fungi strains at the species and sub-species  levels are  highly demanded  to  perform  epidemiological  investigations as well as for controlling the outbreak

There is still more to investigate about that technique, like how many pathogenic fungi species can be detected at the same time.

·         Describes the research topic in more specific terms.

·         Clearly defines the research problem to be investigated

·         Briefly states what is already known about the topic

·         States what is yet to be investigated.

·         Concludes by stating the aim or general objective of the study, i.e., specifically what you are going to investigate about the problem

1.3  OBJECTIVES

GENERAL OBJECTIVE

To identify the diversity and distribution area  of pathogenic fungi  in Hospitals through the  use of Molecular Technique.

SPECIFIC OBJECTIVES

Does fungi isolates relate  epidemiologically?

• To determine molecular relatedness of isolates for epidemiologic investigation;

Does  Molecular technique effective in identifying pathogenic fungi?

• To determine how effective is Molecular technique to identify  pathogenic fungi;

How much can cost an identification of fungi?

• To estimate  the cost  for an identification of  fungi by molecular technique;

Does Pathogenic fungi differ depending on their distribution in hospital areas?

• To determine  the diversity  and distribution  areas of  Pathogenic  fungi in Hospital

1.4  SIGNIFICANCE OF THE STUDY

Why Molecular techniques to identify fungi?

Ø  Phenotypic Characters are unstable and can change with environmental change

After understanding the phenotypic characters of  each isolates of fungi from different environment  ,patients will be adviced how to behave once at the hospital, also the hospital staff will be advices  how they can control  depending on which area we will find more pathogenic fungi.

Ø   Identification methods based on Genotypic Characteristics would be advantageous and potentially more accurate, reproducible, simple and rapid

Using these genotypic characteristics identification will be helping a lot in finding more about fungi and further research can read  to producing the medicament  that are suitable for  the patient.

Ø   Molecular   techniques  will be cost effective and  will  help in preventing and reducing  nosocomial infections

We are expecting the molecular techniques  to be cost effective, because once it will be identified ,and once we will be able to recognize which area is it from,it will be easy to control it and also to eliminate possible nosocomial infections .

1.5  LITERATURE REVIEW

·         Gives details about what is already know about the overall research topic

·         Presents the findings of other researchers that are relevant to your study. 

·         Does not have to relate very directly to your specific problem, but can broadly review the literature about the general topic.

·         Can later be published as a review paper

·         You must write these findings in your own words,

·         Cite the author and year of publication in parentheses [e.g. (Clark, 1991) or (Clark and Roberts, 2003) or (Hanson et al., 1993)]. 

·         Should be divided in subsections

·         Can be considerable flexibility in the subsections

·         Subsections depend upon the topic

·         E.g., UV light, Increase in UV light through ozone depletion, the plastic being investigate (giving its properties and any previous studies done on the effect of UV light on this plastic

·         E.g. Properties of marble, economic importance of marble in Tanzania, Distribution of marble deposits in Tanzania (what is already known).

·         E.G. The BGA, Nitrogen fixation, Effects of phosphate on nitrogen fixation

·         Study Area (not Study Site)

§  Only included if yours is a field study

§  The last section of the literature review

§  Describes the general area around the sites where the study will be conducted

§  Location (may define the boundaries), climate, geography, environment, geo-physical conditions, biological conditions

§  Map

§  E.G., if you are doing a study in the Dar es Salaam harbour, the study area in the whole Dar es Salaam area.

1.6  HYPOTHESES

RESEARCH QUESTIONS

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

Ø  Are the molecular techniques effective to identify the pathogenic fungi?

Ø  What is the minimum  cost for the identification of fungi in Hospitals?

Ø  How to determine  the diversity  and distribution  areas of  Pathogenic  fungi in Hospitals?

2. MATERIALS AND METHODS

2.1 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)

ü   

2.2. METHODS

Samples  will be  collected  from  patients who  will be found at  Hospital, we will be  scrapping the  affected  skin of patients which will be done with the help of a  fairly blunt scalpel at the definite edge of  lesion. The specimens will be  placed in clean white envelopes with each participant’s code labeled and later  they will be  transferred to a sterile petriplate, we will also collected sample from indoor and outdoor environment (including  air,  medical  devices, clothes,  catheters  and  health  care  workers) 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  CHROMagar  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

PCR  amplification for rRNA  gene:

 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 diges-tion 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.

The species which will be identified as the same species, we will set primers to be applied in RAPD-PCR to find out the possible homogeny or variation within the isolated strains

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.

LITERATURE CITED (OR REFERENCES)

           

Ji Young Kim (Ann Dermatol Vol. 23, No. 3, 2011)Identification of Dermatophytes Using Multiplex  Polymerase Chain Reaction, M.D., Yong Beom Choe, M.D., Kyu Joong Ahn, M.D., Yang Won Lee, M.D.  Department of Dermatology, Konkuk University School of Medicine, Seoul, Korea

Sh Fahami P Kordbacheh (  September 2010) Species Identification and Strain Typing of Candida Isolates by PCR-RFLP and RAPD-PCR Analysis for Determining the Probable Sources of Nosocomial Infections-, M Moazeni, M Mahmoodi, H Mirhendi-  Iranian Red Crescent Medical Journal  Vol 12,

species from infected patients sumbul shamim*, s. waseemuddin ahmed,  shamim ara siddiqui and iqbal azhar akistan Journal of Pharmacology  Vol.22, No.1, January 2005, pp.41-46

Barbara J. Howard (1987). Clinical and Pathogenic Microbiology2nd ed. Mosby St.Louis, Boston, Chicago, London, pp.587-595.

A Rezaei-Matehkolaei¹  Use of Single-enzyme PCR-restriction Digestion Barcode Target-ing the Internal Transcribed Spacers (ITS rDNA) to Identify  Dermatophyte Species . Iranian J Publ Health, Vol. 41, No.3, 2012, pp.82-94