Download Human Papillomavirus Genotypes in Women Living with Human Immunodeficiency Virus Infection ... PDF

TitleHuman Papillomavirus Genotypes in Women Living with Human Immunodeficiency Virus Infection ...
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Kathleen Korkor Glover (10191163)

Department of Microbiology, University of Ghana Medical School

College of Health Sciences, Korle-Bu


This thesis is submitted to the University of Ghana, Legon in partial fulfilment of

the requirement for the award of MPhil Microbiology degree.

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This is to certify that this thesis is the result of research undertaken by Kathleen Korkor

Glover towards the award of the Masters of Philosophy in Microbiology in the Department

of Microbiology, University of Ghana Medical School.

Signature---------------------------------------- Date----------------------------------

Kathleen Korkor Glover


Signature------------------------------------------ Date----------------------------------

Prof. J. A. A. Mingle.


Signature---------------------------------------- Date----------------------------------

Dr Charles Brown


University of Ghana

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each participant. Any further explanation was given to make sure that the patients really

understood what the project was all about.

A structured questionnaire (Appendix II) was administered to consenting female patients

in order to obtain relevant information that is useful for the project. These women were

ushered into an enclosed room where their pap smears were taken by a gynaecologist using

a pap kit which contained a glass slide, an Ayres spatula and a swab stick. The pap smears

were sprayed with a cytology spray fixative to preserve the cervical cells prior to pap

staining. The swab sticks were broken into the DNAgard (Biometrica Co, USA) which is

photosensitive and thus was placed in a bag to prevent direct sunlight in order to avoid

damaging the DNA of any HPV that maybe present in the cervical samples awaiting HPV

DNA extraction. Cervical swab samples stored in DNAgard were kept at room temperature

(25 -28oC).

All the cervical samples were assigned a pathological number for easy identification. The

recent CD4+ T cell count of the women were obtained from their folders.


The pap smears were sent to the cytology laboratory of the Department of Pathology of the

Medical School (KBTH) where they were stained with Papanicolau stain.

3.7.1 Papanicolau staining procedure

The slides were fixed into a paraffin based alcohol fixative for 15 min. Next they were

introduced into absolute alcohol, 70% alcohol and 50% alcohol, each for 2 min. They were

then immersed in distilled water for 3min. After 3min they were transferred into the nuclear

stain, Harris’ haematoxylin solution to stain for 3 min. The slides were then blued under a

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weak stream of tap water 3-5min. After blueing the slides, they were reintroduced into

increasing grades of alcohol namely 50 %, 70 %, 80 % and 96 %, for a minute each. The

cytoplasmic staining was next done using orange G solution for 3 min. The slides were

then washed with two changes of 96%alcohol. The slides were next stained in the

polychromatic stain (Eosin Azure solution) for 3 min. The slides were then dehydrated in

two changes of 96%alcohol.

Dehydration was continued in absolute alcohol for 5 min, then in equal parts of absolute

alcohol and xylene. Clearing was done with xylene for 2 min after which the slides were

mounted with DPX mountant. The stained Pap smears were then examined

microscopically by a cytologist to assess the severity of cervical neoplasia among HIV

infected women, using the Bethesda system of classification (Solomon et al., 2002).

3.7.2 HPV DNA extraction

DNA extraction was done using the QIAamp DNA mini kit (QIAGEN, Hilden, Germany)

according to the manufacturer’s instructions.

To a cervical swab sample stored in DNAgard was added 20µL Qiagen protease stock

solution (or proteinase K) and 300 µL Buffer AL. The tube was vortexed for 15 seconds

and then centrifuged at 5000rpm for 5 min after which the swab was removed from the

tube and discarded. The filtrate obtained was pipetted into a 2ml Eppendorf tube and

incubated at 56°C for 10 minutes, with occasional briefly centrifugation to remove drops

from inside the lid. After the incubation, 400 µL absolute ethanol was added to the tube,

followed again by vortexing for 15 seconds.

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Therefore V1= 10mM X 100µl


VI= 10µl

10µl of the dNTPs stock was pipetted from the whole primer stock after thawing it

from the -20°C. It was added to 90µl of the nuclease free water to make up the 10mM

primer working concentration. The primer working solution was then vortexed and

kept at -20°C to be used for the general PCR and HPV genotyping

3) 1X Tris acetate (TAE) Buffer.

Stock concentration of TAE buffer (C1) = 50X

Volume of the stock concentration used in making the working concentration (V1)

= x

Working stock concentration (C2) = 1X

Volume of the working solution to be used (V2) = 500ml

C1 X V1= C2 X V2

Therefore V1= 1 X 500ml


VI= 10ml

10ml of the stock 50x was transferred into a 500ml volumetric flask and topped up

to the 500ml mark of the flask with distilled water. The buffer was gently agitated

to mix and was kept at room temperature.

4) 2% agarose gel preparation.

2 grams of the agarose powder was weighed with a weighing balance and was

poured into a heat resistant bottle. 100ml of 1X TAE buffer was added to the

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agarose powder and mixed. The solution was then micro waved in order to dissolve

the agarose powder properly.

After microwaving the solution was allowed to cool under running water but no to

solidify after which 2µl of ethidium bromide was added and mixed. The solution

was then poured into a gel casting tray with the comb inserted in the tray in order

to create wells for loading PCR products.

University of Ghana

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