RELATIONSHIP BETWEEN HAEMODIALYSIS AND HEPATITIS C VIRUS TRANSMISSION IN SOUTH OF IRAQ

ABDULZAHRA K.M.¹*
¹Department of Laboratory Investigation, Faculty of Science, University of Kufa, Najaf, Iraq.
* Corresponding Author : abdulzahra.safarali@uokufa.edu.iq

Received : 09-09-2013     Accepted : 10-10-2013     Published : 21-12-2013
Volume : 2     Issue : 1       Pages : 12 - 14
J Pathol Res 2.1 (2013):12-14
DOI : http://dx.doi.org/10.9735/0976-8068.2.1.12-14

This project was designed to figure out the relation between dialysis and transmission of hepatitis C virus (HCV) to patients with kidney failure. 205 blood specimen were collected from patients with kidney failure living in south of Iraq (Najaf, Diwania, Nassiria, Babylon, Basrah, Messan, Muthanna) and submitted to anti-HCV Ab test. The results revealed that 10 patients (4.88%) with kidney failure were infected with HCV, tow of them (0.976%) were infected before they start dialysis. One patient (0.488) had infected after tow dialysis operations, and another patient (0.488%) infected after three dialysis operations. One patient (0.488) had infected after 32 dialysis operations. Tow patients (0.976%) had infected after 40 dialysis operations, one patients (0.488%) had infected after 64 dialysis operations, and two patients (0.976%) gain the infection after 104dialysis operation.

Haemodialysis, Hepatitis, Anti-HCV, Kidney failure, South of Iraq.

Patients with renal failure require a haemodialysis or peritone; haemodialysis requires direct access to the circulation [1] . The greatest variation in prevalence of HCV infection occurs among persons with different risk factors for infection, highest prevalence of infection is found among those with large or repeated direct percutaneous exposures to blood [2] . Moderate prevalence is found among those with frequent but smaller direct percutaneous exposures (e.g.: long term haemodialysis patients) [3] . Both incidence and prevalence studies have documented an association between anti-HCV positivity and increasing years of dialysis, independent of blood transfusion [4] . Investigation of dialysis - associated outbreaks of hepatitis C, indicate that transmission might occur among patients in a haemodialysis center as a result of incorrect implementation of infection-control particularly sharing of medication vial and supplies [5] . The hepatitis C virus is a member of flaviviridae family [6] . HCV particle consists of a core of genetic material (positive sense single stranded RNA), surrounded by an icosahedral protective shell of, protein capsid, and further enclosed in a two large (fatty) envelopes [7,8] . HCV replication is cytoplasmic, The genome acts as a mRNA [9] . Acute HCV infection refers to the first 6 months after infection with HCV, persons with acute HCV infection typically are either a symptomatic or have a mild clinical illness, 60-70% have no discernible symptoms, 20-30% might have jaundice, and 10-20% might have nonspecific symptoms (e.g. anorexia, malaise, or abdominal pain, fatigue, nausea, fever, muscle ache, joint pain, swollen legs/feet, diarrhea, vision loss, weakness) once HCV infection established, chronic infection can progress to scarring of the liver (fibrosis) and advanced (cirrhosis) which is generally apparent after many years [10] . After acute infection, 15-25% of persons appear to resolve their without sequels as defined by sustained absence of HCV RNA in serum and normalized of ALT levels [11] .
Hepatitis C virus is transmitted by transfusion of contaminated blood and blood products and organ transplantation. HCV can also be transmitted from infected mother to new born infant (vertical transmission), Sexual exposure was initially identified as a potent source of exposure to the hepatitis C virus [12] . Patients on haemodialysis are also at high risk for infection with HCV, tattooing, house hold persons, and sexual contact have also reported as a transmission route for HCV [1] . HCV is not spread through casual contact such as hugging, kissing, or sharing eating or cooking utensils [13] . World health organization (WHO) estimates that about 170 million people, or 3 percent of the world’s population, are infected with HCV. The geographical distribution, as measured by serological surveys, is lowest in Northern and Western Europe, the USA and Australia, and highest in Japan and Middle East. Egypt has the highest seroprevalence of HCV, up to 20% in some area, which is endemic inthat country [9,14] . Hepatitis C infection is treated by a combination of pegylated interferon -alpha-2a or pegylated interferon -alpha-2b and the antiviral drug ribavirin for a period of 24 or 48 weeks depending on hepatitis C virus genotype [15] . However, the majority of patients with chronic hepatitis C will not clear it without treatment [16] . No vaccine protects against contracting hepatitis C, or helps to treat it. Vaccines are under development and some have shown encouraging results. There is no hepatitis C vaccine for three basic reasons; The first is HCV has different genotype, the second HCV mutates very easily, and the third there is no effective small animal model or cell culture system [17] .

The study included 205 patients suffering from kiney failure, the specimen were collected from August 01, 2011 to April 01, 2012 from Al-Sadir teaching hospital and Al-Hakeem hospital.

ELISA kit, Anti-HCVAb. Biokit-Spain.

• Refrigerator (Concord, Lebanon)
• Centrifuge (Hittich, Germany)
• ELISA System
• Heating Block (BioTek, USA)
• Washer (BioTek, USA)
• Reader- Microwells pectrophotometer (BioTek, USA)
• Micropipette 2-20 µl, 20-200 µl, 1000 µl (BioHit, Finland).

Distilled water and washing buffer.

Tow ml of vein blood was drained using a disposable sterile syringe and dispensed in a sterile centrifuge tube, incubated at 37°C for 5 minutes until the blood clotted, then the specimen centrifuged to obtain patient’s serum so as to be tested for Anti-HCVAb.

The kit contain test strips that are consist of 8 wells, each well is coated with recombinant HCV antigens that represent the solid phase of the test.
1. 200 µl of sample diluent and 10 µl of each sample were dispensed to the designated wells, 8 wells for blank and controls were reserved, 200 µl of negative and positive and low positive control were dispensed to the rest of the wells.
2. 200 µl of negative control, 200 µl of positive control, 200 µl of low positive control were dispensed to 2 wells, 2 wells, and 3 wells respectively. One well were left empty for the substrate blank.
3. The microplate were covered with an adhesive seal, mixed gently and incubated for 1 hr. at 37°C.
4. The adhesive seal were removed and discarded. The contents of the wells were aspirated and the wells were filled completely (approximately 350 µl) with the diluted washing solution. The process of aspiration and washing were repeated 5 more times. Each column of wells were soaked for at least 15 seconds before the next aspiration cycle. After the last washing blot the microplate were absorbed on absorbent tissue to remove any excess liquid from the wells.
5. 100 µl of diluted conjugate were transported into each well, except the one reserved for the substrate blank.
6. The microplate were covered with an adhesive seal and incubated for 30 minutes at 37°C.
7. During the last 5-10 minutes of this incubation the substrate - chromogen solution were prepared by adding 280 µl of chromogen (TMB) to the bottle containing the substrate buffer (14 ml) and mixed well.
8. The adhesive seal were removed and discarded, and the wells were washed as in step 4.
9. 100 µl of substrate-TMB solution were added to each well, including the blank.
10. The plate were incubated for 30 minutes at room temperature (20-25°C).
11. The reaction was stopped by adding 100 µl of stopping solution in the same sequence and time intervals as for the substrate-TMB.
12. The reader was blanked at 450 nm with the blank well and the absorbance of each well were obtained within 30 minutes.
13. The cut-off value were calculated by multiplying the mean absorbance of the low positive controls (LPCx) by 0.9 (Cut-off = LPCx x 0.9).
14. The absorbance of the sample were divided by the cut-off value to obtain the ratio and determine the patient result as below.
• Positive: ratio absorbance/cut-off ≥ 1.0
• Negative: ratio absorbance/cut-off < 0.9
• Equivocal: ratio absorbance/cut-off ≥ 0.9 < 1.

205 sample of patients with dialysis were tested to Anti-HCV Ab, The results revealed that 10 patients (4.88%) with kidney failure were infected with HCV [Table-1] and [Table-2] , tow of them (0.976%) were infected before they start dialysis, only 3.9% of 205 patients were infected after starting dialysis.
[Table-1] Shows in details the percentages of HCV infection in total patients (250), the number of HCV negative patients where (195) and the HCV positive patients where only (10). This table also explains the percentages of HCV infection before starting dialysis (0.98%) and after starting dialysis (3.9%), then the total percentage of HCV infection became (4.88%).
[Table-2] Shows the percentages of appearance of infection for each patient associated with the number of dialysis operation after which HCV infection appears. One patient (0.488) had infected after two dialysis operations, another patient (0.488) infected after three dialysis operations. One patient (0.488) had infected after 32 dialysis operations. Two patients (0.976%) had infected after 40 dialysis operations. One patient had infected after 64 dialysis operations, and two patients (0.976%) gain the infection after 104 dialysis operations.
[Table-3] Demonstrates the average of the highest and the lowest number of the dialysis operation after which HCV infection appears. The lowest number of the dialysis operation 2 times, the highest number was 104 times, and the average of them was 40 times.

As in general population, prevalence of HCV infection among dialysis in patients varies world-wide. The results in [Table-1] were showed that the percentage of HCV infections after multiple operations was (3.9%) which is a low percentage comparing with registered percentage world-wide. HCV infection is highly prevalent among chronic dialysis patients (10-40%) [18] . In contrast, other study has indicated that the prevalence of HCV infections on long-term dialysis in northern Europe is below 5% around 10% in most of southern Europe and the USA, between 10% and 70% in many countries of developing world [19] So, results of this study reflects the accurate application of safety precautions in Iraqian hospitals. Hepatitis C virus (HCV) infection remains frequent in patients receiving long-term dialysis both in developed and les-developed countries, and the natural history of HCV infection in dialysis patient remains in completely understood [20] .
Results in [Table-2] and [Table-3] exhibited that HCV infection appears after tow dialysis operation in one patient, and after three dialysis operation in other patient which means that it is a nosocomial infection. To investigate the early events in HCV infection, two chimpanzees were infected with HCV and obtained blood and liver biopsy samples from them daily during the first 10 days of infection. In the early stage of infection when blood was tested by cDNA /polymerase chain reaction, HCV RNA became detectable in the serum as early as 3 days after inoculation, after HCV sequences were first detected in serum. Circulating anti-HCV antibodies appeared 13 weeks and 32 weeks after inoculation, respectively, in the chimpanzees. These data indicate a very early replicative phase for HCV and a potentially long period of infectivity before the appearance of anti-HCV [21] . Therefore, the screening for anti-HCV antibodies to diagnose HCV infection prior to dialysis operation is not sufficient to confirm that the patient is not infected because of the risk of being in the early stage of infection (false negative result).

Accordingly, this study strongly recommend the use of nucleic acid detection to check for HCV infection repeatedly before each dialysis operation. It is thought to be that using the molecular studies (considering the nucleic acids detections) may explore more accurate result in respect with hepatitis C virus infection patients.

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	Table 1- Percentage of HCV infection in patients with kidney failure before and after dialysis
	Table 2- Demonstrate the number of dialysis operations after which HCV infection appears for each patient
	Table 3- The highest, lowest, and average of dialysis operations number after which HCV infection appears