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Tag Archives: PCR

– by Dr. Sameer M. Dixit and Prof. Dr. Meeta Singh

Cervical cancer is the most preventable cancer in women. Ironically, it is also the biggest killer amongst all cancers in Nepali women. All women that are sexually-active are at risk of contracting it. Although there are no clear reports, it is estimated that 20 percent of all female cancers is linked to cervical cancer, most of those being in advanced clinical stages. Annually, in Nepal, there are an estimated 1,100 deaths due to cervical cancer.

Research worldwide has shown that Human Papillomavirus (HPV) is the major cause of cervical cancer in women. Specifically, about 5 types of HPV (16, 18, 31, 32 and 45) among about 100 known strains have been linked to cervical cancer. According to literature, persistence infection with one of 15 high-risk HPV types is considered a necessary cause for cervical cancer. Estimates worldwide have suggested that types 16 and 18 account for 70 percent of all cervical cancers. In Asia, this has been liked to 67 percent of all cervical cancers, with Southern Asia (where Nepal is located) linked to 80 percent of all cervical cancers.

There are preventive vaccines already developed and being used in developed countries against at least four of the high-risk HPV groups. Gardasil (manufactured by Merck) protects against HPV 6, 11, 16 and 18. Cervarix (manufactured by Glaxo Smithkline) protects against 16 and 18. These are recommended for young girls in three shots (primary and booster dosage).

In the United States, the Center for Disease Control recommends that female patients start regular cervical cancer screening at the age of 21 or within three years after first having sexual intercourse. The first test for the screening test for cancer of the cervix is the pap test which detects precancerous or cancers cells. The second recommended test is the HPV test, which looks for the human papillomavirus that can cause these cell changes leading to cervical cancer. While the smear test can be carried out by most pathology laboratories, HPV test requires advanced laboratory setup. In US and other developed countries, early intervention is now made possible by molecular detection technologies such as polymerase chain reaction (PCR) or real-time PCR. These tests enable detection of high-risk HPV in the cervix rather than relying on histopathological results alone. Thus, by regular screening for HPV, it is now possible to reduce the risk of developing cancer in women worldwide.

For a developing country like Nepal, accurate screening of cases of cancer of the cervix in Nepal continues to be a major problem. Very little, if any, literature reports exist on the type of HPV present in the population, and in cervical cancer cases. Even World Health Organization (WHO) does not have data on Nepali context to come up with an effective solution to the problem. Even though vaccines exist, without the knowledge of the HPV type prevalent here, the usefulness of such vaccines in Nepal is not proven.

Women who are sexually-active should visit their gynecologist for cervical cancer screening. Cervical cancer is totally preventable, so there is no excuse for not getting tested for it.

Molecular detection of HPV and its strain identification has never been tried in Nepali laboratories before. The primary screening method used in Nepal is the Pap Test – Pap smears can detect cervical cancers if smears are taken properly and interpreted by an experienced expert cytologist. Pap smears may not provide accurate detection of all suspect cases if the smear sample is not taken properly. However, if we can even screen some of the early stage cervical cancer patients using novel molecular tools such as PCR, we could be saving many more lives. PCR could be the preferred and more efficient method of detection of HPV and thereby detect cancer cervix in our country where there are very few pathologists. It must be taken into account that just the presence of HPV in the cervix does not necessarily indicate cervical cancer risk. In most cases, the low-risk viruses are shed off by the body automatically. However, detection of the virus provides the gynecologist evidence to screen the patient at regular intervals, thereby enabling early detection of abnormal cellular morphology in the case of cancer. Identification of any of the high-risk HPV is thus the key to early intervention of cervical cancer.

It is, therefore, highly recommended for all women who are sexually-active to visit their preferred gynecologist for cervical cancer screening. HPV type molecular screening is also now available in the city. October of every year is dedicated to cancer, worldwide. Cervical cancer is totally preventable, so there is no excuse for not getting tested for it.

Dr. Dixit is associated with the Center for Molecular Dynamics Nepal (CMDN). Dr. Singh is Head of Department, Gynecology/Obstetrics, Tribhuvan University Teaching Hospital (TUTH). CMDN in collaboration with Dept of Gynecology/Obstetrics, TUTH, is in the process of initiating HPV type study into cervical cancer in Nepal.

s.dixit@cmdn.org

This article has been taken from The Republica National Daily http://myrepublica.com/portal/index.php?action=news_details&news_id=24761

DR SAMEER M DIXIT & PROF DR MEETA SINGH

Trainings that could fill the gap between academics and professional standards by producing skilled manpower in the field of research and diagnosis had been long awaited. Intrepid Nepal has taken an initiation to generate modern science and technology experts particularly in Molecular biology and associated fields. The training program entitled “Extensive training on Molecular and Immunological Techniques” which is tentatively scheduled for two weeks from 30th June to 17th July 2008 and covers latest molecular techniques plus immunological tests, considered the most rapid and confirmatory in the detection of pathogen of all sorts.

The training will be instructed by Ms. Elizabeth Cowan, research personnel from Harvard Medical School, United States. She will be conducting all molecular approaches and immunological techniques of modern days and will be sharing her life time experiences. The course, designed for the training is highly extensive and employ the latest knowledge and techniques including kit methods in detection and prevention of disease, comprise of following

1. PCR (Polymerase Chain Reaction)
2. ELISA (Enzyme- Linked Immuno sorbent Assay)
3. SDS PAGE (Sodium do-decilesulphate Polyacrylamide gel Electrophoresis)
4. Gel Electrophoresis
5. Quantitative Real Time PCR
6. Reagent and chemical preparation
7. Data analysis, Management and interpretation
8. Theories underlying behind each process and DNA sequencing too..
9. Many more

PCR (Polymerase chain reaction): technique that has enhanced sensitivity and accuracy will be covered in the training program. Trainees will be conducting hands on sample preparation such as Nucleic acid extraction from biological sources, pre-amplification procedures, amplification and finally post reaction analysis including verification of the product generated. Furthermore the courses include Protein extraction from different sources (Possibly from Microorganisms), analyzing them on Gel (SDS PAGE) and studying their characteristic bands against standard Protein markers (again another process for diagnosis of pathogens). They will also be taught about buffer and reagent preparation, plus their significances and limitations, whose role is immensely important in the process of experimentation.

Likewise, QPCR (Quantitative Real Time PCR): modified form of conventional PCR will also be included, at least to provide introduction on its role on quantitation of bacterial pathogens, viral load (such as HIV or HBV) in clinical samples and antiretroviral treatment efficacy, and detection of genetic alteration etc.

ELISA (Enzyme Linked Immunosorbent Assay): WHO recognized technique, will be provided to learn hands on for HIV-1 and HBV. The training will also include theory behind DNA sequence analysis and genetic profiling.

We believe that this opportunity provides everyone with a wide range of opportunities and trainees will get in-depth knowledge about modern techniques that would help individuals in seeking their career goals in research and diagnosis.