hcv infection is among life threatening public health problems worldwide , with over 170200 million infected people   including about 17 million from pakistan .
it has been estimated to cause approximately 27% of cirrhosis and 25% of hepatocellular carcinoma cases worldwide .
hcv is a small enveloped , positive sense single stranded rna virus and has been classified as a separate genus hepacivirus in the flaviviridae family .
the hcv genome is approximately 9.6  kb , encoding a polyprotein of about 3010 amino acids and is flanked by short untranslated regions ( utrs ) regions at the 5 and 3 terminus .
this polyprotein is posttranslationally processed by viral and cellular proteins to generate the structural proteins ( c , e1 , e2 , and p7 ) and nonstructural proteins ( ns2 , ns3 , ns4a , ns4b , ns5a , and ns5b ) , .
hcv shows high degree of genetic heterogeneity ; consequently six major genotypes and multiple subtypes of hcv have been identified so far in world .
the common subtypes found in north and south america , europe , russia , china , japan , australia , and new zealand are 1a , 1b , 2a , 2c , and 3a .
genotype 4 is predominant in egypt , north africa , central africa , and middle east .
genotype 5 in south africa   and genotype 6 in southeast asia   have been identified .
genotype 3 is the most prevalent genotype in india , bangladesh , pakistan , and nepal [ 1215 ] .
subtypes of genotypes 1 , 2 , 3 , and 6 have been found prevalent in thailand , vietnam , indonesia , and burma   respectively .
genotype 1b is the most prevalent genotype in china ; however genotype 2 has also been reported from some regions of china . in pakistan ,
the prevalence of hcv infection has been estimated to be 8% and is increasing gradually due to deficiency in basic health care recourses and lack of the general public awareness about safety measures .
some studies have been conducted on prevalence of hcv genotypes in khyber pukhtunkhwa region of pakistan [ 1720 ] .
however , little has been reported on prevalence of hcv genotypes in district bannu in khyber pukhtunkhwa region of pakistan .
therefore , this study was conducted to find out baseline information on the prevalence of hcv genotypes in district bannu .
accurate hcv genotyping can be used in better understanding of hcv infection , for creating awareness in the general public and subsequently for implementation of preventive and therapeutic strategies .
all the procedures used in this study were approved by the ethics committees of department of microbiology , kohat university of science and technology .
an inclusion criterion for patients was to be seropositive for anti - hcv by third generation enzyme linked immunosorbent assay ( elisa ) .
the information regarding age , gender , and possible routes of transmission was obtained from each participating patient .
total 117 blood samples were collected from patients attending district hospital bannu and khalifa hospital bannu .
serum was separated from each blood sample at 3000  g for 5  min and then labeled and stored deep - frozen at 20c .
for this 10  l of hcv extracted rna was incubated at 37c for 50  min along with primer specific for core region and 200  u of moloney murine leukemia virus reverse transcriptase ( m - mlv rtase ) ( fermentas usa ) , 5x first strand buffer ( mmulv buffer ) dntps and ddh2o . in the first round of nested pcr
the pcr program was as follows : initial denaturation was at 94c for 5  min , followed by 45 cycles , each of 45  sec denaturation at 92c , 45  sec annealing at 55c , and 1  min extension at 72c , with final extension at 72c for 10  min . in second round of nested pcr , genotype - specific pcr was performed by using allele specific primers for core region reported by ohno et al .   at same pcr program that was adopted for first round nested pcr .
the final pcr products obtained after each round of nested pcr were subjected to electrophoresis and separated on 2% agarose gel . after staining with ethidium bromide ,
out of 117 anti - hcv positive sera by elisa , 110 samples were found positive by pcr analysis , with greater representation of males 81 ( 73.6% ) as compared to females 29 ( 26.4% ) as shown in table 1 .
genotyping of 110 hcv - pcr positive samples determined four different genotypes including 1a , 2a , 3a , and 3b ( figure 1 ) .
however , hcv genotypes 1b , 2b , 4 , 5 , and 6 were not detected among patients studied .
genotype 3a was the most prevalent one detected in 42 ( 38% ) patients , followed by genotype 3b in 23 ( 21% ) patients and then genotype 2a in 13 ( 12% ) patients .
genotype 1a was the least prevalent and was detected in only in 1 ( 1% ) patient while in 8 ( 7% ) patients mixed genotypes of hcv were detected .
moreover , 23 ( 21% ) hcv - pcr positive samples could not be genotyped by using the method as described previously .
the studied patients were categorized in three different age groups and then prevalence of age associated hcv genotypes was determined ( table 1 ) .
genotype 3a was most prevalent genotype in all age groups patients and its prevalence was found high among patients with increasing age ( > 34 years ) .
similarly , high prevalence of genotype 2a was also observed in age group 5574 years .
moreover , genotype 1a was found in only one patient , who had age of 23 years and subsequently belonged to age group 1534 years . in our study , those patients who had history of visit to barber shop , intravenous drug addiction , and dental procedures were recorded major risk factors responsible for hcv transmission in district bannu as shown in table 2 .
patients with history of visit to barber shop and receiving multiple therapeutic injections accounted for 40 ( 36.4% ) and 39 ( 35.4% ) , respectively , followed by patients with history of dental procedures 22 ( 20% ) .
history of blood transfusion and tattooing was recorded in 7 ( 6.4% ) and 2 ( 1.8% ) patients , respectively . genotype 3a and genotype 3b
were found to be the most prevalent genotypes in patients with history of shaving by barbers , receiving multiple injections , and dental procedures .
however , genotype 2a and genotype 1a were more commonly found in patients with history of dental procedures ( table 2 ) .
the molecular epidemiological studies have reported that significant regional differences appear to be present in the frequency distribution of hcv genotypes . moreover ,
it has been reported that there are variations in disease outcome and response to antiviral therapy of hcv genotypes . however , in pakistan treatment of hcv infected patients is based on qualitative or quantitative viral detection and genotypes are not determined prior to treatment .
therefore variable response rates of hcv infected patients to antiviral therapy can not be detected .
the present study was conducted to determine baseline data on the prevalence of hcv genotypes in a district in khyber pakhtunkhwa region of pakistan .
the baseline information will help in better understanding of hcv infection , awareness in the general public and subsequent control strategies .
the genotype 3a was found to be the most prevalent genotype followed by 3b and 2a and genotype 1a was found to be less prevalent ( figure 1 ) .
results of the present study are in conformity with results of previous studies reported from different regions of the khyber pakhtunkhwa in pakistan [ 2025 ] .
previous studies conducted in india , bangladesh , and nepal also reported that the genotype 3 is the most prevalent genotype [ 1214 ] . in this
study the assay used could not determine hcv genotypes among a considerable number of hcv patients ( 23% ) .
however , there is a need to use more reliable and sensitive assay for genotyping of hcv in untypeable samples .
the distribution of hcv genotypes may be variable among the patients of different age groups .
studies have reported that genotypes 1b and 2 were more prevalent in older patients , whereas genotype 1a was observed more frequently in the younger population [ 26 , 27 ] .
another study reported that in france genotype 5 was frequently detected in patients aged more than 50 years .
in iran genotype 3a was the most frequently detected in patients less than 40 years . in this study , we observed the distribution of hcv genotypes among different age groups .
the prevalence of genotypes 2a and 3a was found increasing with increasing age of patients .
genotype 3b was found more prevalent in age groups more than 35 years . moreover , genotype 1a was least prevalent genotype detected in a patient of a younger age group less than 34 years .
analysis of possible routes in transmission of hcv genotypes in district bannu is shown in table 2 .
the hcv genotypes reported in present study were isolated from participating patients with known route of transmission . in our study
genotypes 3a and 3b were more frequently observed in patients with previous history of shaving by barbers followed by multiple injections received and dental procedures . whereas genotypes 1a and 2a were more commonly observed in patients who had history of dental procedures .
hcv genotype 2a was also common in patients with previous history of visit to barbers and receiving multiple injections .
the possible routes of transmission of hcv genotypes have also been reported in other studies .
the high prevalence of hcv genotype 3 is attributed to intravenous drug addicts in the united states and europe .
moreover present study and other studies from pakistan have also reported increased prevalence of genotype 3 in those patients who had received multiple therapeutic unsafe and unnecessary injections by untrained health practitioners particularly in rural areas .
these untrained health practitioners usually use nondisposable syringe or used syringe and needles for more than one patient at the public health - care centers [ 2432 ] .
however , high prevalence of genotypes 3a , 3b , and 2a among patients of district bannu with history of shaving by barbers and dental procedures has not been reported in the united states and europe . in present study
we observed in district bannu that uneducated barbers common practice is to reuse of unsterilized razors and scissors for multiple customers .
similarly untrained health practitioners at dental clinics are usually in practice of using used and unsterilized dental equipment for multiple individuals .
consequently , these barbers and health practitioners are promoting the risk of transmission of hcv infection from one person to another in this district . in a patient with genotype 1a
this is consistent with result of a previous study from pakistan where most of patients with genotype 1a had a history of dental procedures .
our study provides baseline information on the prevalence of hcv genotypes in district bannu in khyber pukhtunkhwa region of pakistan .
the most prevalent hcv genotype was 3a isolated from patients in district bannu , followed by genotypes 3b and 2a .
the frequency distribution of these genotypes was found variable according to the age groups of the patients studied .
the possible routes of transmission for these genotypes observed were shaving by barbers , receiving multiple injections , and dental procedures .
further studies are needed to investigate hcv genotypes in district bannu by using more sensitive assays and considering large population size .
 determination of an individual 's hepatitis c virus ( hcv ) genotypes prior to antiviral therapy has become increasingly important for the clinical management and prognosis of hcv infection . 
 therefore , this study was conducted to investigate the prevalence of hcv genotypes in hcv infected patients of district bannu in khyber pakhtunkhwa region of pakistan . 
 serum samples of 117 seropositive patients were screened for hcv - rna by using reverse transcriptase - nested polymerase chain reaction ( rt - nested pcr ) and then pcr positive samples were subjected to hcv genotyping . out of 117 seropositive samples , 
 110 samples were found positive by pcr analysis . 
 genotype 3a was the most prevalent one detected in 38% of patients , followed by genotype 3b in 21% of patients , and then genotype 2a in 12% of patients . 
 however 21% of hcv - pcr positive samples could not be genotyped by method used in this study . 
 genotype 3a was the most prevalent genotype in patients of all age groups and its prevalence was found high among patients with increasing age ( > 34 years ) . 
 moreover , genotypes 3a and 3b were found to be the most prevalent genotypes in patients with history of shaving by barbers , receiving multiple injections , and dental procedures . in conclusion 
 there is need of further investigation of genotypes of hcv by using more sensitive assays and considering large sample size in district bannu . 
1. Introduction
2. Materials and Methods
3. Results
4. Discussion
5. Conclusion