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HEPATITIS C is an infectious disease affecting the liver caused by hepatitis virus Ryan et al 2004 hepatitis c virus a member of flaviviridy family was discovered as anew viral agent of non _A non _B, hepatitis virus by choo and coworkers in 1989 choo QC 1989
And is recognized as a major threat to global public health. (Lauer 2001; Booth 2001). Hepatitis C Virus has positive sense RNA genome that consists of single open reading frame of 9600 nucleoside bases (Hwang 2001; Muhammad 2005).
At is estimated that 200 million people word wide are infeted with the HCV, the overall prevalence of anti HCV antibodies was 2% in Spain, 3.9% in Jakarta, Indonesia and 1.25% Zakiuthos Greek.
The Seroprvalence of HCV in hospital based studies done in Nauritius, Ethiopia of South Indies showed a serprevalance of 5.9%, 6%, and 4.8% respectively,
WHO estimates indicate 10.24 million HCV in feted person in Ind and seroprevalance of 2.4% in Pakistan.
The seroprevalance of hepatitis "C" in different parts of country, reported in last 5 years is from2.2% to 13.5% the highest serporevalance of hepatitis "C" is reported from lahor 13.5% Jamshero and Mardan 9%,
Hpatitis C Virus HCV continues to be amajor disease burdenon the world. The who estimated prevalence of about 3% with the virus affecting 170 million people worldwide (world health organization, 2000). In developing countries where resource and facilities may be significantly limited the prevalence HCV is higher as compared to the developed world (wild and hall, 2000). The prevalence of chronic hepatitis C in the Asia pacific region is variable between 4% to 12% (Takahashi et al 1993). Approximately 80% of the individuals infected with HCV progress to chronic infection (Amarapurkar, 2000).
Hepatitis C virus is transmitted through contaminated blood transfusion, surgery, surgical instruments, dental surgery and excessive dental consultations, sexual contacts, durg abuses, sharing of the house hold items such as razors, tooth brushes and shaving from the barber (perrillo et al 1990; sato et al 1994; van staa 1997). Some health care procedures, i.e, surgical and dental treatments, have recentaly been indicated as risk factors for acute HCV (Mele and Marzolini, et al 2000). The prevalence of hepatitis C is more than hepatitis B the main risk factor is the dental surgery due to internalize instruments, reuse of syringes and refilling vials(Farid et al 2002).
In Pakistan, both HBV pose major risks as blood borne pathogens (shah et al, 2002). Widespread practices such as unsafe injections, improper disposal of hazardous waste, recycling of used syringes without proper sterilization, sharing of needles by injection drug users and unsafe sex are believed to facilitate the transmission of these infections, resulting in high prevalence rates in pakistan (Mujeeb, 2001and Khan et al, 2000). Nosocomial transmission of HCV is possible when disinfection and sterilization techniques are inadequate, and contaminated equipment is shared among patients. In particular, studies have indicated the possibility of an HCV infection occurring among patients on hemodialysis, due to poor infection control, and the sharing of contaminated medical vials and supplies (world health organization, 2000)
In Pakistan, blood transfusion is still a major source of HCV transmission. Possible reasons for this include lack of resources, weak infrastructure, ill-equipped resources, poorly trained staff, inadequate policy implementation, frequent power breakdown and infective screening of blood donors for anti-HCV antibody (Akhtar S, 2004). Regular blood transfusion in patients withy hereditary hemolytic anemia, particularly thalassemia, has improved their overall survival, but carries a definite risk of acquisition of blood - borne virus infection,s especially viral hepatitis ( Alavian et al 2005). Thalassemia patients are particularly at risk of acquiring HCV by blood transfusion (Akhtar, 2004)
Although advances have been made, a reliable culture system for HCV is not yet available (Bendinelli et al 2000). Currently, routine diagnosis of HCV is based on detecting antibodies (anti-HCV) in serum by enzyme immunoassay (EIA). However, these methods require venipuncture and expensive equipment, which may not be appropriate for mass detection proor developing countries (kuo et al, 1989). Other Laboratory assays that are available for the diagnosis and management of HCV infection include: (a) serological tests to detect HCV antibodies (enzyme linked immunosorbent assay (ELISA) and recombinant immunoblot assay (RIBA), (b) molecular tests to detect and quantitate HCV-RNA and (c) genotyping (Sandra,2002)
Objectives of the study are:
To identify point sources of HCV spread in Mardan.
To analyze nosocomial risk factors involved with the spread of HCV.
To analyze the efficiency of sterilization procedures and disinfectants used in the major hospitals of Mardan.
REVIEW OF LITERATURE
Piazza et al (1995) stated that environmental contamination of dental surgeries by HCV was investigated by following 35 anti-HCV and HCV-RNA-positive patients with chronic hepatitis through dental treatment; 328samples were collected from instruments and surfaces after their dental treatment. Twenty (6.1%) were positive for HCV RNA, including samples from work benches, air turbine handpieces, holders, suction units, forceps, dental mirrors and burs. The authors conclude that, these data indicate that there is extensive contamination by HCV of dental surgeries after treatment of Anti-HCV patients and that if sterilization and disinfection are inadequate there is the possible risk of transmission to susceptible individuals.
Gurevich et al. (1996) demonstrates that the potential for perso-to person transmission of infectious agents such as the human immunodeficiency virus HIV and hepatitis B and C viruses via inadequately sterilized dental instrument exists depending on the prevalence of HIV in the dental practices. Dental instruments and devices require sterilization or high-level disinfection. An evaluation of the implementation of such processes was undertaken. Eleven thousand questionnaires on methods used to sterilize and disinfect dental instruments were sent to dental practices and 1391(13%) were retuned for evaluation. Sixty eight percent of respondents believed they were sterilizing their instruments, however, some of the liquid chemical products used were not suitable for sterilizing instruments, and 12% of respondents used incorrect contact times. Forty nine percent of respondents did not challenge autoclaves with biological spores to check their function at a\n acceptable frequency.
Centers fro Disease Control and Prevention CDC(1998) published that Nosocomial transmission of HCV is possible in infection control techniques or disinfection procedures are inadequate and contaminated equipment is shared among patients. Reports from other countries do document nosoceomial HCV transmission, but such transmission rarely has been reported in the US, other than in chronic hemodialysis settings. Studies indicate that HCV transmission might occur among patients in a hemodialysis center because of incorrect implementation of infection control practices. It is recommended for all healthcare workers, those who are HCV positive should follow strict aseptic technique and standard precautions, including appropriate use of hand washing, protective barriers, and care in the use and disposal of needles and other sharp instruments.
Garcia et al (2000) stated that proper sterilization of invasive instruments and equipments is of great importance in the practice of dentistry, where various blood or saliva borne pathogens e.g. HCV and HBV could be easily transmitted to patients and dental staff via contaminated or inadequately sterilized instruments.
Mujeeb SA et al (2001) and Khan AJ et al (2000) concluded from their research that widespread practices such as unsafe injections, improper disposal of hazardous waste, recycling of used syringes without proper sterilization, sharing of needles by injecting drug users and unsafe sex are believed to facilitate the transmission of these HCV and other infections, resulting in high prevalence rates in the Pakistan.
BUTT et al. (2003) conducted the study to identify the role of dental surgical procedures in contributing to the transmission of hepatitis C.78 consecutive adult patients of both Sexes suffering form chronic hepatitis C Were evaluated. It was concluded that Dental procedures were the major source of exposure (39.7%) followed by injections (16.6%) surgical procedures (16.6%), diabetes (12.8%) family history of hepatitis (9%) blood transfusions (7%), tattooing (5.1%) and history of contact with a jaundiced patient (2.6%). There was a statistically significant difference in distribution of risk factor, with dental procedures being the commonest factor (p<0.001). The high prevalence of dental procedures in patients with chronic hepatitis C stresses the importance of ineffective infection control methods practiced by dental surgeons as a risk factor for acquiring hepatitis C and which were probably the source of infection.
Saeed et al. (2004) assessed HCV seroprevalence among polytransfused thalasssemic children registered at a charity clinic and blood bank of a nongovernmental organization (NGO) at Karachi for regular blood transfusion therapy. Between Noveber 1998 and January 1999, consecutive 256 registered children were included and a serum sample for evaluation of anti HCV from each participating child was obtained Anti HCV was detected by HCV microparticle enzyme immunoassay 3rd generation kit according to the manufacturer's instructions (Abbott, chicageo, USA) HCV ribonucleic acid RNA was tested in sera of a subset of anti-HCV positive patients using amplicor HCV RNA assay as suggested by the manufacturer (Roche diagnostic System, USA) Eighty nine of 256 (34.8%) thalassemic children were anti-HCV positive . HCV RNA was detected in 15 of 38 (39.5%) anti-HCV positive children who were tested for HCV RNA. The mean (±SD) age of HCV seropositive children was 11.9 ± 4.6 years.
Khan et al (2007) conducted a descriptive study carried out from July 2003 to July 2004 on 1630 patients admitted in the department of orthopedics Ayub Teaching Hospital Abbottabad . Patients of either sex, of all ages undergoing surgery were included in the study. All patients underwent screening for Hepatitis B and Hepatitis -C and confirmed by Elisa method in positive patients. Out of 1630 patients Hepatitis B and C was present in84(5.15%) patients.Out of 84 infected patients 51 (3.12%)
were suffering from hepatitis C and 33 (2.02%) were suffering from hepatitis B. In 2 (0.12%) patients both hepatitis B and C infections were present. Among the predisposing gactors previous history of surgery was positive in 18 (21.43%) patients; history of blood transfusion in 13 (15.47%) patients, dental procedure was in 7(8.33%) patients, and abroad visit in 4(4.76%) patients. It has been concluded that the prevalence of hepatitis B and C in orthopedic patients is quite high with the common risk factors.
E Girou et al. (2008) conduct ed a prospective observational study to assess the roles of environmental contamination and non-compliance with standard precautions in cross- transmission of HCV between patients in a hemodialysis unit. Patients undergoing long term kidney dialysis revealing 2 cases of HCV transmission. An investigation was then launched to determine whether the patients were infected in the hemodialysis unit. Environmental contamination by blood and HCV R NA was assessed, as was compliance with accepted infection control precautions such as hand washing and use of gloves. Blood contaminated surfaces may be a source of HCV cross transmission in a hemodialysis unit, " the study authors concluded.
MATERIALS AND METHODS
Blood samples will be collected from common population of district mardan, Samples from peoples of various age group will be collected in 1% PBS solution and transported to labs in Zoology Department Abdul Wali Khan University Mardan for further analysis.
Peoples who has a recent history of dental surgeries or scaling will also be screened and sampled for the presence of HCV,
Screening for HCV positive IDUs will be carried out with the help immuno- chromatographic (accurate) Blood samples will then be collected from HCV positive individuals for further analysis.
Liver Function Tests ( LFTs)
LFTs such as Alt, AST, Alkaline Phosphatase and bilirubin of ELISA positive samples will be done at Anwar Medical Center and Laboratory Mardan.
HCV RAN Extraction
HCV RNA will be extracted form 100 µl serum or samples collected from surgical instruments in PBS either manually or by using Anagen RNA extraction kit (Purescript, USA) according to the manufacturer's instruction.
Qualitative HCV RNA detection
A) Reverse Transcription PCR
Serum will be prepared in a laminar flow bench and will be frozen at 70C. Qualitative detection of serum HCV RNA will be performed by RTPCR. Briefly, after extraction of HCV RNA from 100 µl serum, cDNA will be synthesized using antisense primer at 37 C with M-MLV reverse transcriptase enzyme.
a) Regular PCR
The amplified cDNA will further be subjected to tow rounds of PCR amplification. The condition for the first round PCR will be as follows:
An initial denaturation step at 95C for 2 minutes followed by 30 cycles of 94C for 45 seconds. 54C for 45 secpmds. Amd 72C fpr 1 minute will be performed in Eppendorf thermal cycler (Eppendorf, Germany). The conditions for the 2nd round PCR will be the same except that a different set of inner primers will be used in the PCR mix in order to amplify the 1st round product.
The diagnosis of chronic HCV will be based on elevated serum ALT (SGPT) and AST (SGOT) levels, histological examination, and consistent detection of serum CVHhHhh HCV RNA.
Quantitative measure measurement of HCV RNA
Quantification of HCV RNA in pre treatment sera of patients will be performed by RT-PCR with an internal RNA standard derived from the 5 UT R.
Genotyping of HCV (according to the classification of Simmonds et al. (1993) will be performed by in house type-specific PCR at IBGE of Realtime PCR laboratory, peshawar. All contamination prevention measures suggested bb Kwok and Higuchi (1989) will be strictly followed.
Agarose gel electrophoresis and Gel documentation
All the PCR products(first and second rounds) will be analyzed on 1.8% agarose gel prepared in 0.5% TBE buffer, stained with ethedium bromide (10 ug/ml) as florescent dye. Gels will be photographed using alpha quant (Alpha innotech). 100-bp DNA ladder (Gibco BRL) will be used as DNA size marker.
The data will be analyzed and the summary statistic will be carried out by a statistical package, SPSS version 10.0 for windows.
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