hepatitis c virus ( hcv ) is a major cause of post transfusion hepatitis and liver transplantation in many countries .
it is estimated that approximately 3% of the world 's population is infected with hcv and 60% to 85% of the patients develop chronic infection that leads to cirrhosis , and hepatocellular carcinoma , and need liver transplantation ( 1 , 2 ) .
three groups of hcv testing are available : 1 ) serological assays to detect anti - hcv .
hcv tests are used to diagnose hcv and to guide therapy and/or monitor the treatment of hcv infection ( 1 , 3 - 5 ) . the most common type of hcv antibody testing is the enzyme immunoassay by methods of enzyme linked immunosorbent assay ( elisa ) , and chemiluminescence immunoassay ( cia ) .
elisa testing is a semi - quantitative assay that its results are reported as positive or negative based on comparison of absorbance reading of each sample with a cut - off value defined for the lot used expressed as a signal to cutoff ratio ( s / co ) , and shows the quantity of antibody in blood ( 6 ) . although hcv antibody assays are highly sensitive and specific in patients with chronic hcv infection , there are frequent false positive in anti - hcv results .
quantitative viral load tests are used to measure the amount of hcv - rna in one milliliter of blood .
pcr - based hcv rna methods are used as the gold standard to confirm the hcv positivity in anti - hcv assays and monitor the treatment ( 1 , 4 , 7 - 9 ) .
the current study aimed to evaluate the relationship between quantitative anti - hcv ( s / co ratio ) and quantitative hcv - rna levels , also to determine a specific s / co ratio with a routine commercial hcv - ab kit used in the laboratories to identify viremic from non - viremic anti - hcv positive patients .
in the current cross sectional study , from may 2012 to october 2013 , 378 patients suspected with hcv who referred to iranian blood transfusion organization ( ibto ) research center were included .
patients had no history of hepatitis b virus ( hbv ) and human immunodeficiency virus ( hiv ) infections .
the written consent was signed by the patients to include in the study and the questioner was filled out for all subjects .
all subjects also were tested for anti - hcv assay ; hcv viral loads were performed on all anti - hcv positive patients .
all fresh serum samples were assayed for anti - hcv ( hepanostika hcv ultra , uk ) by elisa method , according to the manufacturer 's protocol .
the results were expressed as s / co ratio calculated by dividing the signal detected on each sample to the cut - off value .
briefly , viral rna was extracted from one milliliter plasma using the qiaamp ultrasense virus kit ( qiagen , germany ) according to the manufacturer 's protocol .
the extracted rna was eluted in elution buffer and used as the template for the quantitative rna pcr .
quantitative rna pcr was performed using artus hcv rg rt- pcr kit ( qiagen gmbh , qiagen strasse , germany ) with lower detection limit of 34 iu / ml .
20 l of the extracted rna was added to 30 l of master mix in each 0.1 ml microtube and test was performed in rotorgene - q apparatus .
a standard curve was automatically drawn with the rotorgene - q software using five quantification standard concentrations of hcv - rna to analyze the viral rna load .
the results expressed in iu/l were determined in iu / ml based on artus hcv rg rt- pcr kit handbook . in each run , plasma negative sample and internal control were used to prevent false positive and negative results , respectively .
quantitative group variables were described as mean , standard deviation , and range . to compare variables ,
in the current cross sectional study , from may 2012 to october 2013 , 378 patients suspected with hcv who referred to iranian blood transfusion organization ( ibto ) research center were included .
patients had no history of hepatitis b virus ( hbv ) and human immunodeficiency virus ( hiv ) infections .
the written consent was signed by the patients to include in the study and the questioner was filled out for all subjects .
all subjects also were tested for anti - hcv assay ; hcv viral loads were performed on all anti - hcv positive patients .
all fresh serum samples were assayed for anti - hcv ( hepanostika hcv ultra , uk ) by elisa method , according to the manufacturer 's protocol .
the results were expressed as s / co ratio calculated by dividing the signal detected on each sample to the cut - off value .
briefly , viral rna was extracted from one milliliter plasma using the qiaamp ultrasense virus kit ( qiagen , germany ) according to the manufacturer 's protocol .
the extracted rna was eluted in elution buffer and used as the template for the quantitative rna pcr .
quantitative rna pcr was performed using artus hcv rg rt- pcr kit ( qiagen gmbh , qiagen strasse , germany ) with lower detection limit of 34 iu / ml .
20 l of the extracted rna was added to 30 l of master mix in each 0.1 ml microtube and test was performed in rotorgene - q apparatus .
a standard curve was automatically drawn with the rotorgene - q software using five quantification standard concentrations of hcv - rna to analyze the viral rna load .
the results expressed in iu/l were determined in iu / ml based on artus hcv rg rt- pcr kit handbook . in each run , plasma negative sample and internal control were used to prevent false positive and negative results , respectively .
quantitative group variables were described as mean , standard deviation , and range . to compare variables ,
of the 265 hcv - ab positive patients , 204 ( 77% ) were male and the mean age was 43.53  13.17 years .
there were significant differences in s / co ratio between viremic and non - viremic patients .
the s / co ratio was higher in viremic group than the non - viremic group ( p < 0.05 ) ( table 1 ) .  
( % ) or mean  sd . the sensitivity , specificity , negative predictive and positive predictive values were 100% , 81.4% , 100% , and 77.2% , respectively in the s / co ratio of 2.7 ( table 2 ) .  
all of the data are presented as ( % ) except anti - hcv s / co ratio .
it is estimated that 20% of people with chronic hcv infection develop cirrhosis after 25 years ( 1 , 4 , 8 - 10 ) .
a positive result of anti - hcv by elisa method may represent active viremia , infection in the past or false positive .
although recombinant immunoblot assay ( riba ) is used to confirm results in elisa method , it can not detect viremia to follow treatment .
qualitative and quantitative assays for hcv - rna are introduced as gold standards to confirm viremia in patients with positive anti - hcv ( 1 , 4 , 8 , 9).quantification of hcv - rna is important to determine disease status and is used before and during anti - viral therapy ( 9 - 14 ) .
although using quantitative hcv - rna rt - pcr to detect and monitor the treatment of hcv infection is approved , it consumes time and money especially in patients with no viremia ; also , its high cost makes it unavailable in many laboratories .
recently , the necessity to use confirmatory testing in anti - hcv low s / co ratio was suggested by the centers for disease control ( cdc ) .
they also introduced s / co ratio  3.8 as a cut - off value and suggested that s / co ratio < 3.8 determines low positive .
an anti - hcv s / co ratio  3.8 determines a true anti - hcv positive result in 95% of cases .
in contrast , in patients with s / co ratio  3.8 the mentioned possibility is high ( 15 ) .
several studies conducted to detect a cut - off point to distinguish low positive from high positive subjects reported that the majority of subjects with low positive anti - hcv results , by elisa method , were negative in hcv - rna testing ( 6 , 12 , 13 ) .
several studies are conducted to introduce s / co value to distinguish viremic and non - viremic patients . in several published studies ,
different s / co values ranging from 3 to 34 were determined in the third generation of anti - hcv assays ( 6 , 10 , 13 , 16 - 20 ) .
the result of the current study showed that in low positive anti - hcv elisa results , the frequency of false positivity was high . according to the obtained result , using 2.7 as a cut - off for s / co ratio , the sensitivity was 100% .
the study found that all hcv - ab positive patients with s / co cutoff ratio < 2.7 were not detectable hcv - rna .
positive results in hcv - ab assay may represent a past infection or false positive result .
in the current study the majority of the patients with hcv - ab positive , s / co cut - off ratio  2.7 were viremic .
due to differences in sample size , the study population , and the kit used to detect hcv - rna , there are discrepancies in the s / co ratios introduced as cut - off point in different studies . further studies with common approaches are necessary to predict using anti - hcv s / co ratio as a cut - off value . in conclusion
the present study indicated that anti - hcv s / co ratio can be used as a useful tool to manage hcv infection .
a cut - off value of 2.7 can determine the need to hcv - rna testing .
therefore , for patients with s / co < 2.7 , hcv - rna viral load is not recommended .
it is suggested that laboratories should report s / co ratio along with anti - hcv results .
 background : hepatitis c virus ( hcv ) infection is diagnosed by antibody and rna based methods . 
 patients with anti - hcv sample rate / cutoff rate ( s / co ) ratios > 1 are reported as anti - hcv positive . 
 rna based methods are introduced to confirm positivity in seropositive samples.objectives:the current study aimed to assess relationship between s / co rates and hcv - rna levels in the laboratory to identify hcv viremia in patients with a positive anti-hcv.patients and methods : all serum samples were assayed for anti - hcv by elisa method . 
 a total of 265 anti - hcv positive patients were tested for hcv - rna testing by quantitative method using artus hcv rg real - time polymerase chain reaction ( rt- pcr ) kit . 
 statistical analysis was done by spss version 16.results:of the 265 patients with hcv infection , 204 ( 77% ) were male and the mean age was 43.53  13.17 years , ranging 1 - 81 years . 
 no correlation was found between s / co ratios and hcv - rna levels 
 . there was significant difference in s / co ratio between viremic and non - viremic subjects . 
 the sensitivity , specificity , negative predictive value , and positive predictive value were 100% , 81.4% , 100% , and 77.2% , respectively in the s / co ratio of 2.7.conclusions:the present study indicated that anti - hcv s / co ratio is useful to predict non - viremic patients . 
 a cut - off value of 2.7 can determine the usefulness of hcv - rna testing . 
 patients with s / co < 2.7 are not viremic ; therefore , hcv - rna testing is not recommended . 
 it is suggested that laboratories report s / co ratio along with anti - hcv results to manage hcv infection better , especially in countries that quantitative hcv testing is expensive or not available . 
1. Background
2. Objectives
3. Patients and Methods
3.1. Patients
3.2. ELISA Assay
3.3. RNA Extraction
3.4. Quantitative RNA PCR
3.5. Statistical Analysis
4. Results
5. Discussion