|Year : 2015 | Volume
| Issue : 5 | Page : 106-111
Utility of routine real time quantitative PCR monitoring of HCV infection in haemodialysis patients
S Datta, N Goel, C Wattal
Department of Clinical Microbiology, Sir Ganga Ram Hospital, Rajinder Nagar, New Delhi - 110 060, India
|Date of Submission||13-Nov-2013|
|Date of Acceptance||16-Jun-2014|
|Date of Web Publication||6-Feb-2015|
Department of Clinical Microbiology, Sir Ganga Ram Hospital, Rajinder Nagar, New Delhi - 110 060
Source of Support: None, Conflict of Interest: None
Background: Hepatitis C virus (HCV) infection causes significant morbidity and mortality in patients of end stage renal disease (ESRD) on haemodialysis (HD). Stringent screening methods can help in its early diagnosis. Objective: The study addresses the utility of real-time quantitative polymerase chain reaction (RQ-PCR) in the diagnosis and monitoring of HCV infection especially on seronegative and normal serum alanine aminotransferase (ALT) HD patients. Material and Methods: This retrospective study was carried out from January 2010 to December 2012. Patients of ESRD on maintenance HD and on whom all the three assays HCV antibody serology, PCR and ALT were done were included in the study (n = 123). Group 1 (n = 57), comprised of patients with negative serology and normal ALT, and Group 2 (n = 66), had either raised ALT and or a positive or equivocal serology. Results: Out of the 123 cases studied, HCV serology was positive in 36.5% (45), ALT raised in 18.6% (23) and PCR positive in 67.4% (83) cases. PCR positivity was significantly higher than serology and raised ALT. Group 2 had a significantly higher PCR positivity than Group 1 (P = 0.0004), but 50.9% patients of Group 1, were also PCR positive and 69% of them had a high viral count of >8 × 10 5 IU/ml at the time of detection. Conclusion: Regular routine screening of HCV by RQ-PCR in ESRD patients can help in early diagnosis of HCV infection in patients with low index of suspicion.
Keywords: Haemodialysis, hepatitis C virus, real time quantitative polymerase chain reaction
|How to cite this article:|
Datta S, Goel N, Wattal C. Utility of routine real time quantitative PCR monitoring of HCV infection in haemodialysis patients. Indian J Med Microbiol 2015;33, Suppl S1:106-11
|How to cite this URL:|
Datta S, Goel N, Wattal C. Utility of routine real time quantitative PCR monitoring of HCV infection in haemodialysis patients. Indian J Med Microbiol [serial online] 2015 [cited 2019 Nov 19];33, Suppl S1:106-11. Available from: http://www.ijmm.org/text.asp?2015/33/5/106/148832
| ~ Introduction|| |
Hepatitis C virus (HCV) infection is a major health problem and causes significant morbidity and mortality in patients with end stage renal disease (ESRD) on maintenance haemodialysis (HD).  The annual incidence of HCV infection in these patients is approximately 100-1000 times higher than the general population.  The Kidney Disease Improving Global Outcomes (KDIGO) guidelines published in 2008 proposes that 'In a low prevalence set up, serological screening for HCV should be done at the start of HD and 6 monthly, thereafter or earlier if they are transferred from another HD unit. A monthly screening of serum alanine aminotransferase (ALT) levels should also be done to rule out HCV infection. Nucleic-acid Amplification Test (NAT) should be performed in patients with unexplained abnormal ALT level.  In a HCV high prevalence set up, NAT should be considered as a screening method for HCV infection on admission to the HD unit.  A stringent screening protocol can help in an early diagnosis of HCV in HD units.
Screening for HCV antibodies by enzyme-linked immunosorbent assay (ELISA) is done regularly as recommended, but it under-estimates HCV prevalence.  ALT levels, though monitored monthly, are a poor predictor of hepatocellular damage in this population.  The NAT assay, Real-time quantitative polymerase chain reaction (RQ-PCR) is a sensitive diagnostic test that detects HCV RNA during window period and prior to the elevation of ALT. It also quantifies HCV RNA levels over a linear range of 10 IU/ml-100 million IU/ml. , We in our facility follow the KDIGO guidelines. NAT is usually done only when serology or ALT is deranged and once at the start of HD as recommended by KDIGO. This study addresses the utility of RQ-PCR for regular and routine HCV monitoring and also evaluates the baseline viral load in HD patients with negative serology and normal ALT level. It also correlates the putative roles of HCV serology, ALT monitoring in comparison with that of RQ-PCR in the diagnosis of HCV infection in this subset of high risk patients.
| ~ Material and Methods|| |
Patients and study design
This retrospective study was undertaken in a 650 bedded tertiary care hospital with an active organ transplant program at New-Delhi, North India, to evaluate the utility of HCV RQ-PCR for the diagnosis of HCV infection, especially in ESRD patients on HD.
A total of 343 patients with renal disease were screened for HCV infection by HCV RQ-PCR, serology and ALT levels between years 2010-2012 of which 123 cases were included in this study based on the following exclusion criteria. These 123 ESRD patients were on HD in our hospital during the study period. The blood samples of all these patients were screened as per the KDIGO recommendations.  PCR was done at the start of HD and subsequently following a seroconversion or a rise in ALT levels. Their serology and liver enzymes were routinely monitored as recommended.  PCR was also repeated on patients prior to renal transplant irrespective of the normal ALT level and seronegative status. The patient's demographics, liver function test and other clinical data were reviewed and recorded from the data repository of Hospital information system.
HD patients already diagnosed with Hepatitis B virus (HBV) and Human immunodeficiency virus (HIV) infection or any other cause of acute or chronic liver disease, patients on ATT or any hepatotoxic drugs, patients on anti-HCV therapy or undergoing dialysis for acute renal failure and those diagnosed with HCV before the study period were not included in the study.
The patient samples were grouped into Group 1 and 2 depending on their serology and ALT reports [Figure 1]. Group I comprised of 57 HCV seronegative samples with normal ALT levels and was candidate for renal transplant. This group helped us to evaluate the utility of RQ-PCR in normal ALT, seronegative cases. If PCR was positive in this group, a repeat assay was done with a fresh sample before they were reported as positive.
Group 2 comprised of 66 samples with either a raised ALT levels and or a positive or equivocal HCV serology. This group evaluated the correlation between the seropositivity and or raised ALT levels with PCR positivity. The viral counts of the PCR positive patients in both Groups 1 and 2 were compared.
A control group of 20 blood bank samples from healthy donors who were also screened by PCR and seroassays were included as healthy controls.
ELISA for HCV specific antibodies screening
HCV serology was done by CE-labelled commercial third generation enzyme-linked immunosorbent assay (ELISA) kit (Hepanostika ® HCV Ultra, Beijing united Biomedical Co. China). The blood samples of the patients on HD were screened for HCV specific antibodies third generation test as per the manufacturer's instruction. Specimens with absorbance values less than or more than the cut off value were considered non-reactive or reactive respectively. Low absorbance values close to or same as that of the cut off were reported as equivocal. An equivocal or reactive sample was retested in duplicate using Monolisa TM ANTI-HCV PLUS Version 2 (BIORAD, France) on a fresh sample from the same patient before reporting. The sensitivity and specificity of both the ELISA kits ranged from 99.8-100% respectively. Recombinant immunoblot assay (RIBA) was not used as a supplemental test.
HCV RNA RQ-PCR
COBAS ® TAQMAN ® HCV Test v2.0 (Roche Diagnostics, Indianapolis, IN, USA) was used not only to detect but also evaluate the level of viraemia by assessing the viral count present at the time of detection.
The patients' blood samples were collected in ethylenediaminetetraacetic acid (EDTA) vials; 500 μl of plasma was processed using COBAS Taqman HCV test with high pure extraction (Roche Diagnostics) as per the manufacturer's protocol. RQ-PCR assay was done using reverse transcription and PCR amplification based on dual fluorescent dye labelled hybridization probe targeting a defined sequence in the highly conserved 5′ un-translated region of HCV genome. Quantitation standards provided were included in the assay. Results were expressed as IU/ml according to the unit recommended by the WHO International Standard for HCV RNA NAT assays.  The lower limit for detection for this assay was 25 IU/ml and the linear detectable range for this assay was 25-3.91 × 10 8 IU/ml.
The ALT levels were analysed by Beckman System pack in UniCel ® Beckman DXI 800 autoanalyser. A level of >60 IU/L is considered as raised ALT.
Ethical approval was obtained from the SGRH institutional review board before the onset of the study (reference no. EC/09/12/415).
Descriptive and inferential statistics were computed with SPSS for Windows (Chicago IL, USA) version 17.0 software. Quantitative variables were expressed as median with range. The qualitative variables were expressed with numbers and percentage. A 95% confidence interval was calculated by using the modified Wald method. Pearson's Chi square was used to compare categorical variables as applicable. P values less than 0.05 were considered significant. The sensitivity, specificity and accuracy of the assays were also evaluated.
| ~ Results|| |
A total of 123 samples were investigated [Figure 1]. The mean age of the study group was 47.6 ± 13.0 (range: 18-75) years. Eighty two (66.6%) were males and 41 (33.3%) were females.
Irrespective of the group, a total of 45 samples were HCV seropositive (36.5%, CI 95= 0.285-0.453), 23 had raised ALT levels (18.6%, CI 95 = 0.127-0.265) and PCR was positive in 83 (67.4%, CI 95 = 0.587-0.751) samples. Five samples had an equivocal serology [Table 1]. The results of the second ELISA were in agreement with the primary kit. PCR positivity was significantly higher than positive serology and raised ALT (P< 0.0001). Seven of 123 samples (5.6%) were positive by all the three assays, and 28 (22.7%) samples remained negative by all 3 assays [Table 1]. Of these 123 samples, 57 belonged to Group 1 and the remaining 66 were in Group 2 [Table 1].
RQ-PCR results in Group 1 and 2
In Group I, 29/57 (50.9%) of the patient samples were PCR positive, though all the samples from this group belonged to seronegative ALT normal patients [Table 2].
In Group 2, a total of 54 (81.8%) samples were PCR positive. PCR positivity in Group 2 was significantly higher than that of Group 1 (P = 0.0005) [Table 2]. The 50.9% of the PCR positive samples belonging to Group I contributed to 23.5% of the total 123 samples [Table 1].
Considering PCR as the gold standard, the overall sensitivity of serology and ALT monitoring was 52.5% and 19.2% and the specificity was 92.1% and 82.5%, respectively. The accuracy of serology and ALT monitoring as compared to PCR was 65.2% and 39.8%, respectively.
Viral load in PCR positive cases in Group 1 and 2
The median HCV RNA viral load in the 83 PCR positive samples was 6.87 × 10 5 (range, 3.7 × 10 2 -1.14 × 10 8 ) IU/ml. The PCR positive samples of both Group 1 and Group 2 were grouped into low and high viral load with counts of ≤8 × 10 5 IU/ml and a count of >8 × 10 5 IU/ml, respectively.  There was no significant difference in the viral count of patients in Group 1 and Group 2. However, a total of 69.8% had a high viral load [Table 3], irrespective of the designated group.
All the 20 samples of healthy blood donors in this group were negative by all 3 assays.
| ~ Discussion|| |
HCV infection still remains an independent and significant risk factor of death in patients on maintenance dialysis.  HD continues to carry the risk of HCV transmission, with delayed seroconversion and normal ALT values, thus increasing the likelihood of undetected infection and resulting in uninterrupted spread of HCV. , Usually, after an exposure, HCV RNA can be detected within 1-2 weeks, whereas antibodies are detectable after 8 weeks in immunocompetent individuals. In ESRD patients on HD, the seroconversion may take several months and the median interval between NAT positivity and serology positivity may range from 154-246 days. 
However, the Kidney Disease: Improving Global outcomes (KDIGO) guidelines  as well as the Center of Disease Control and Prevention currently recommend serial testing of ALT and anti-HCV antibody as screening tests for HCV infection in patients on HD.  It is known that clinical and biochemical features of HCV are often silent in patients of ESRD on HD. Elevated levels of serum ALT are sensitive measure of hepatocellular injury, but so far, the relationship between anti-HCV and ALT among chronic HD patients was considered imperfect.  ALT levels are usually depressed, and ALT values in viraemic patients with histological disease often fall within normal reference level. Studies have reported a rise in only 12-31% of HCV RNA positive HD patients. ,, In our study also, only 18.6% of the 123 patients on HD had raised ALT. The sensitivity and specificity of ALT monitoring in our study as compared to PCR was 19.2% and 82.5%, respectively with a diagnostic accuracy of 39.8%. Several reasons are attributed to this imperfect relationship. A possible cause of decreased ALT levels may be due to subnormal serum levels of pyridoxal-5-phosphate (active form of B6) in the uremic serum, which serves as a co-enzyme for transaminases.  Coupled with this, there is often an intermittent rise and fall of ALT levels in HD patients which further decreases the sensitivity of ALT estimation in these patients. 
Serological tests are considered important tools to assess magnitude of HCV infection in HD patients. However, the time lag between HCV infection and seroconversion window is longer in ESRD patients on HD and anti-HCV antibodies may not be detected in infected cases in the acute phase of the disease. ,,
NAT assay, such as PCR, detects additional HCV cases that are otherwise negative by serology. , In our study, 67.47% cases were positive by PCR in comparison to 36.48% seropositive cases using ELISA technique. The sensitivity of serology as compared to PCR was 52.5% in our hands with a diagnostic accuracy of 65.2%. Similar observations were also reported by other studies.  Ours is a NABH and NABL accredited referral tertiary care centre, and we strictly adhere to infection control procedures. Despite taking stringent infection-control measures designed to prevent transmission of blood-borne pathogens including HCV by the treating facility along with the Hospital Infection Control committee, 29 of the total 123 samples (23.5%) that were seronegative and had a normal ALT level were positive by PCR [Table 1]. These PCR positive samples contributed to 50.9% of the Group 1 cases [Table 3]. Of these, 68.9% had a high viral count at the time of screening in spite of their normal ALT levels and seronegative status [Table 3]. It is known that cryptic HCV infections, (relating to patients infected chronically with HCV but are seronegative and have a positive HCV RNA) further reduces the sensitivity of serology in the diagnosis of HCV infection. This leads to undetected chronic carrier state in many individuals marked by the presence of HCV RNA and seronegative status.  Thus, the high HCV prevalence in HD patients in a developing nation emphasizes the need for aggressive monitoring of HD patients by PCR at regular intervals irrespective of the serological status.  There were 5 patients (4.06%) with equivocal serology of which 3 were positive by PCR [Table 1]. Though the number is not statistically significant but it underscores the fact that regular screening with PCR should be done irrespective of the serology. It is known that HCV core antigen detection by ELISA is a cheaper alternative and can also help in early HCV detection in HD patients but the detection of core antigen occurs a few days after the appearance of RNA and often becomes undetectable in large proportion of sero-coverted patients after the development of HCV antibody.  This can give a false impression of a non-viraemic state; moreover, the viral count cannot be assessed.
A quantitative PCR helps in early diagnosis and also ascertains the baseline viral load before initiation of therapy on these patients. There is an urgency to treat the infection while on HD prior to transplant as immunosuppressive drugs used after renal transplant can enhance HCV replication and accelerate hepatic disease.  Earlier clinical and therapeutic trials have shown that patients with baseline HCV RNA level of more than 8 × 10 5 IU/ml had 9% lower sustained virological response rate as compared to those with a viral load of less than 8 × 10 5 IU/ml.  It is also important to note that patients with low HCV RNA levels have 15-39% better response to therapy than those with high RNA levels.  Moreover, early source tracing can also prevent further transmission. The specificity of RT-PCR was 100% in our study. In our study, the healthy blood donors were all negative by PCR.
Our study had its limitations. Due to the retrospective nature of the study the outcome of PCR-positive patients could not be followed up nor could the source of HCV infection traced. The true incidence of HCV infection at our centre could not be assessed as many from the total patients screened got rejected by the exclusion criteria.
| ~ Conclusion|| |
However, this study highlights the need for aggressive monitoring of HCV infection on HD patients which can be done at regular definite intervals by RQ-PCR which is a sensitive and user friendly assay. Our study also highlights that more than 50% of the ESRD patients on HD with normal ALT and a negative serology had a high viral load further emphasizing the role of routine monitoring of these patients with RQ-PCR irrespective of their ALT levels and sero-status. There are no known studies of this nature to our knowledge that have evaluated the baseline HCV viral load of seronegative, ALT normal HD patients. Though the cost of the assay limits its utility as a screening test, its advantages far outweighs the profound damage which HCV infection can cause if left unnoticed. The study urges the need to review strategy and modify the existing guidelines which ensures regular monitoring of HCV infection by PCR at routine intervals in HD facilities in our country.
| ~ Acknowledgements|| |
We acknowledge Dr. Veronica Dinand and Ms. Parul Takkar for helping with the statistical analysis.
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[Table 1], [Table 2], [Table 3]