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Home > Nucleic Acid Testing: Redefining Safety in Blood Screening

NUCLEIC ACID TESTING: REDEFINING SAFETY IN BLOOD SCREENING

Dr. Sangeeta Pathak, Dr. M. Chandrashekhar

INTRODUCTION:

Patient’s choice for an infection free recovery is of critical importance worldwide. Despite improvements in HIV and HCV and HBV serological tests in recent years, instances of viral transmission via transfusion still occur because of donations that take place while a donor is -

in the pre-seroconversion window phase,
infected with immunovariant viruses, or
a non-sero-converting chronic carrier.

Direct, sensitive detection of viral nucleic acid could substantially decrease the incidence of transfusion-induced infections.

In most countries, the safety of blood is ensured by donor selection, testing of donations for viral markers and in the case of blood products, the inclusion of viral inactivation steps during the manufacture of the blood products. However, transmission of HIV, HBV and HCV, continues to be a threat to safe blood transfusion, especially in developing countries. It is due to low numbers of voluntary donations, use of low sensitivity tests for viral screening and the high prevalence of these viruses. This has contributed to the high rate of transfusion transmitted infections compared with developed countries. In India, where the majority of donors are still replacement donors1, the risk of transfusion transmitted viral infections is much higher than in countries with a 100% voluntary donor base. The prevalence of post transfusion HBV and HCV in India is between 1 to 5%1. The prevalence of HIV varies from region to region, being particularly high in western and southern parts of India.

The implementation of nucleic acid testing (NAT) over the last decade into the blood screening process has added an extra layer of safety against transmission transfused viral infections. NAT was first introduced by the European plasma industry in 1995 and consequently for whole blood donation screening in some parts of Europe and then Asia. HCV NAT was first introduced between 1999 and 2001 in blood banks across France, Germany, Italy, Spain, Switzerland and the United Kingdom presumably as it was already mandatory for plasma screening in these countries.
OBJECTIVE OF NAT IMPLEMENTATION:
To reduce the serological Window Period and provide near zero risk Blood
Serological screening of blood donor have greatly reduced, but not eliminated, the risk of transmission of viral infections by transfusion of blood and blood products. Detection of blood borne viruses by conventional serology tests rely on the production of viral specific antibodies. The production of detectable levels of antibodies or antigen occurs several weeks after the initial infection. During this interval, also known as the serological window period, virus is present in the blood of the infected individual and may be transmitted although the serological test is negative. Studies have shown that Nucleic Acids Technology(NAT) based testing for viral nucleic acid can further reduce the transmission risk of these agents in blood donations made during the sero-conversion window period.

All countries that have introduced NAT have encountered a decrease in residual risk of these viral transmissions. Although, other safety measures such as more stringent selection of blood donors have contributed as well; a marked decrease is evident pre- and post NAT implementation. The residual risk for HCV transmission prior to NAT was 0.64 cases per million in France and 3.94 per million in Spain which decreased to 0.1 per million and 2.33 per million respectively after NAT was adopted. HIV NAT yield rates were estimated at 0.3 per million donations in France and Spain as opposed to 0.59 and 2.48 respectively preceding NAT2.

The primary benefit of NAT is the ability to reduce residual risk of infectious window period (WP) donations. The estimated reduction of the window period utilizing NAT for HCV is 70 to 12 days, HIV from 22 to 11 days and HBV from 59 to 25-30 days3. The NAT screening available ensures 99.99% blood safety. NAT is one of the best available technologies in which Polymerase Chain Reaction (PCR) is used.

NAT screening reduces window period as follows:
HIV> 11 Days post infections
HBV> 20 Days post infections
HCV> 15 Days post infections

The introduction of Nucleic Acid Technology testing has reduced the risk of transfusion transmitted HCV and HIV infections to approximately 1 in 1 million and 1 in 3 million respectively in Western Europe and the USA.

WHAT IS NAT & HOW DOES IT HELP?
NAT is a method of testing blood that is more sensitive than conventional tests that require the presence of antibodies to trigger a positive test result. While an infection occurs, NAT is used to detect the low levels of viral genetic material present in the body. However, it happens before the body begins producing antibodies in response to a virus, giving the ability to detect a disease at an earlier stage. NAT significantly reduces the 'window period' or the time between donor exposure to the virus and the appearance of antibodies. By decreasing the window period, it allows for earlier detection of the infection and thus further decreases the possibility of transmission via transfusion. NAT also detect mutants, occult cases and false negatives from serology.

The performance of the NAT assay is essentially dependent on both its analytical and clinical sensitivity and specificity.
The analytical sensitivity is generally determined by testing dilutions of standardized materials such as WHO International Standards and subsequent calculations of 95% Limit of Detection (LOD) by probit analysis. The analytical sensitivity for HBV NAT is particularly important due to its long doubling rate of 2.6 days during which the viral count is generally low. In comparison, the HCV and HIV-1 doubling rate of 14.9 hours and 20.5 hours respectively are shorter.
The majority of blood donations worldwide are currently subjected to screening by means of commercial testing which produces more standardized results. The use of automated systems to run such assays have shortened the assay run time, resulting in timely release of results which remains vital to blood banking.



PREVALENCE OF HIV, HBV AND HCV IN INDIA
Developing countries such as India which are endemic for both HIV and hepatitis pose a serious challenge for the blood banking community. India has the second highest pool of HIV persons estimated at 5.7 million and 3-5% HBV and 1-2% HCV cases reported in the population of almost 1.2 billion 4,5. Post-transfusion prevalence of hepatitis has been observed at 1-1.8% for hepatitis B and 0.28-0.5% for Hepatitis C and 0.23-0.47 for HIV-1 5,6.

Routine use of HBsAg as a serological marker has been established globally for the diagnosis of acute or chronic HBV infections and the screening of blood or organ donors. However, the emergence of Hepatitis B s mutant variants due to increased immunological pressure, by global immunization programs or viral escape in chronically infected patients have led to suboptimal performance of certain HBsAg assays. This is mainly due to natural variation and mutations in HBV S gene which can induce HBsAg conformational changes. Occult HBV is the major contributor for transfusion transmitted HBV infection in highly endemic countries such as India. Hence, the need for NAT in mini pools for HBV is necessary to circumvent the risk of missed positive cases during the window period, occult infections and HBsAg assays that do not detect all known s mutants. The use of Hepatitis B core antibody (Anti-HBc) testing in countries endemic for HBV is also not suitable due to the high prevalence of individuals who harbour these antibodies. In addition, the anti-HBc assay does distinguish between current and resolved HBV infections.

Occult HBV infection is defined as persistent HBV infection and presence of genomic particles in the liver tissue and/or serum of HBsAg negative individuals. Occult HBV infection may present itself as HBV DNA detected

In recovered individuals characterized by the presence of antibodies to HBV surface antigen
As chronic HBV infection caused by escape mutants and not detected by HBsAg assay used
As chronic HBV infection with anti-HBc as the only serological marker present
As chronic HBV infection with only HBV DNA present

A study in West Bengal of 1027 voluntary blood donors in 2004-2005 screened for HBV, reported occult HBV infection in 18.3% who were anti-HBc positive and of which 21% were positive for HBV DNA5. Another study conducted in a leading medical centre in New Delhi on a subset of 377 donors positive only for anti-HBc resulted in HBV DNA detection in 25% of the cases9.

High prevalence among the blood donors
Reliable nationwide figures for viral transmission are not available in India, but reports indicate that transfusion associated hepatitis B & C combined, is in the range of 7% for patients receiving between 1-8 units of blood. Per unit risk for HBV is estimated at 0.36% (Japan 0.000038%) and for HCV at 1.2%. Those getting repeated transfusions, such as patients of thalassemia or patients on renal dialysis, are distinctly worse off and show evidence of infection with HBV in 50% and HCV in 30% of the patients. Figures for HIV infections are more sketchy, but the National AIDS Control Organization's countrywide report for 1998-99 indicates that approximately 7% of individuals who are HIV positive got the infection from a Blood Transfusion. While the frequency of viral transmission, per unit of blood transfused in the USA, is estimated as 1 in 103,000 for HCV, 1 in 63,000 for HBV and 1 in 493,000 for HIV.

India has about 2000 Blood banks which are Fragmented & Poorly Regulated.
With Rapid and Elisa kits from multiple vendors, laboratory errors and focus on costs, there is great variation in the transfusion transmitted infection testing.
Different serological tests are used for screening from one blood bank to the next. Even after implementing the more sensitive, newest generation of serological tests, a considerable residual risk of transfusion transmission of these viruses remains due to long window period of available serological tests.

Authors :

Dr. Sangeeta Pathak, Senior Consultant & Head, Blood Bank, Max Super Speciality Hospital, 1-2 Press Enclave, Saket, New Delhi. Mobile: 9873081647;Email: sangeeta.pathak@maxhealthcare.com

Dr. M.Chandrashekhar, Director, Surgical Pathology & Blood Transfusion Services, Max Healthcare Institute Ltd.  New Delhi : Mobile: 9811896554: Email : m.chandrashekhar@maxhealthcare.com
 

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