Viral pathogens
Abstract
Summary. Despite continuous improvement in safety and purity of blood products for individuals with haemophilia, transmissible agents continue to affect individuals with haemophilia. This chapter addresses three viral pathogens with significant clinical impact: HIV, hepatitis C and parvovirus B19. Hepatitis C is the leading cause of chronic hepatitis and the major co-morbid complication of haemophilia treatment. Clinically, asymptomatic intermittent alanine aminotransferase elevation is typical, with biopsy evidence of advanced fibrosis currently in 25%. Current treatment is effective in up to 70%, and many new agents are in development. For those progressing to end-stage liver disease, liver transplantation outcomes are similar to those in non-haemophilia subjects, although pretransplant mortality is higher. HIV infection, the second leading co-morbid condition in haemophilia, is managed as a chronic infection with highly active antiretroviral therapy (HAART). HAART also slows hepatitis C virus (HCV) progression in those with HIV/HCV co-infection. Viral inactivation and recombinant technologies have effectively prevented transfusion-transmitted viral pathogens in haemophilia. Human parvovirus B19 infection, typically associated with anaemia or, rarely severe aplastic crisis, is a non-lipid enveloped virus, for which standard inactivation techniques are ineffective. Thus, nucleic acid testing (NAT) to screen the blood supply for B19 DNA is currently under consideration by the Food and Drug Administration. To the extent, viral inactivation, recombinant, and NAT technologies are available worldwide, and the lifespan for those with haemophilia is approaching that of the normal population. The purpose of this chapter is to provide an update on three clinically significant transfusion-transmitted viral pathogens.
Introduction
Viral pathogens transmitted through the blood supply have been markedly reduced through the introduction of viral inaction and recombinant technologies. As a result, in countries in which these technologies are available, viral pathogens no longer infect young individuals with haemophilia, and their lifespan is approaching that of the general population. However, the consequences of past chronic hepatitis C virus (HCV) in the haemophilia population accounts for the major morbidity and mortality in this population. Highly active antiretroviral therapy (HAART) has converted HIV into a chronic treatable disease. Parvovirus B19, a non-lipid enveloped virus, is not readily inactivated by standard techniques. Thus, nucleic acid screening of the blood supply for B19 DNA is currently under consideration to rid the blood supply of this pathogen.
Outcomes of long-term HCV and HIV infection in haemophilia (Dr Ragni)
Hepatitis C virus is the major co-morbid condition in haemophilia, the most common cause of chronic liver disease and the leading cause of death in this population. In contrast to other at-risk populations, HCV infection in those with haemophilia was acquired early in life, with the first clotting factor exposure [1]. This is a unique feature of HCV in haemophilia, as they have longer duration HCV infection than other risk groups. Over 90% of those who infused clotting factor prior to the availability of recombinant and viral inactivation technologies became infected with HCV [2].
Chronic hepatitis C infection
Chronic HCV infection in individuals with haemophilia is typically asymptomatic, with intermittent transaminase elevation in up to 60% [3,4], yet the onset of thrombocytopenia, which may indicate the presence of cirrhosis with hypersplenism and may occur in up to 20% [3], may lead to mucosal bleeding, such as epistaxis or gastrointestinal bleeding, which may require factor replacement to manage. The occurrence of fatigue, disruption of the sleep-wake cycle, ascites, oedema, varices or encephalopathy may indicate progression to end-stage liver disease (ESLD), which may occur in up to 5–10%. Risk factors for progression to ESLD, after adjusting for age, include alcohol, hepatitis B surface antigenemia and HIV co-infection [3].
HIV co-infection
Approximately, 80% of HCV-infected men became co-infected with HIV through blood product exposure in the early 1980s [3]. In this group, it was shown that HIV accelerates HCV liver disease, leading to a higher HCV viral load [5] and a nearly fourfold greater rate of liver disease progression than in those with HCV alone [3]. HAART therapy significantly reduces that risk: the data from a cohort of HCV-infected haemophilic men demonstrated that ESLD-free survival was significantly better in co-infected men treated with HAART, and approached rates seen in HIV negative HCV mono-infected men [6].
Liver fibrosis
As HCV is usually asymptomatic until late in the disease, many haemophilic men do not seek treatment or undergo liver biopsy, although liver biopsy is the gold standard for determining the extent of liver damage. It is of note that liver biopsy is safe in individuals with haemophilia when performed by the transjugular route [7]. Rates of liver fibrosis were recently assessed in a large observational, multi-centre study of HCV(+) haemophilic men. Based on blinded review of liver biopsies from 220 haemophilic men from 34 U.S. HTCs, one-fourth of HCV(+) haemophilic men were found to have evidence of advanced fibrosis (Metavir F3), with a fibrosis score 1.4-fold greater in co-infected than in mono-infected haemophilic men [7]. Markers predictive of F3 fibrosis in multiple logistic regression and receiver operating curve analyses, included aspartate aminotransferase (AST), platelets, ferritin and alpha-fetoprotein [7]. These markers, similar to those in other risk groups, appear to be better predictors in HIV(−) than HIV(+) subjects, possibly related to the confounding effects of HIV on platelets and liver function [7].
Transplantation
Haemophilic men who develop ESLD now account for 10% of all liver transplants performed in HIV/HCV co-infected individuals in the U.S. [8,9]. Among those coming to liver transplantation, findings from the multi-centre HIV solid organ transplant study indicate that survival is comparable to that in non-haemophilic subjects [8,10]. However, pretransplant outcomes are worse: survival among co-infected haemophilic transplant candidates awaiting transplantation is significantly shorter than that in those without haemophilia [10]. The reason for this finding are not known, although it has been observed that longer duration of HCV infection in those with haemophilia is associated with faster progression to Model for Endstage Liver Disease (MELD) = 25 than in HCV(+) non-haemophilic candidates [10]. Hepatocellular cancer does not appear to affect these rates, nor does it differ between haemophilic and non-haemophilic transplant recipients.
MELD monitoring
The MELD score, which combines bilirubin, creatinine and international normalized ratio (INR) to predict posttransplant survival, was recently found also to predict pretransplant survival [11] and is now recommended for routine monitoring of pretransplant candidates. Sepsis and multi-organ failure may complicate both pretransplant [9,10] and posttransplant candidates and are the most common complications in pretransplant candidates and also seen in transplant candidates [9,10,12]. Among the latter, recurrent hepatitis C is a common and difficult complication in the setting of immunosuppressive antirejection agents [13], underscoring the need for more effective antiviral agents for hepatitis C.
HCV update: clinical and treatment issues (Dr Sherman)
The hepatitis C virus is an important pathogen associated with the development of chronic liver disease that progresses to cirrhosis in a high proportion of infected patients. Disease transmission is primarily through blood and blood product exposure. Thus, injection drug users and patients with inherited blood disorders who received contaminated blood products are at the highest risk of infection. The aetiological agent is a positive single strand RNA virus with approximately 10 000 bases. The virus replicates primarily in hepatocytes, although replication in extrahepatic sites is described [14]. Although some patients infected with HCV can spontaneously clear the virus, the majority will develop chronic infection, defined as continuous infection for more than 6 months after exposure. Chronic infection is characterized by fluctuating levels of serum alanine aminotransferase reflecting an ongoing tug-of-war between viral replication and cellular injury vs. both innate and specific immune responses. Interestingly, recent data suggest that viral proteins can modulate the immune response as well as the process of apoptosis (programmed cell death), thus facilitating the maintenance of chronic viral infection [14–18].
HCV replication
Hepatitis C virus viral replication is an area of intense scrutiny at this time. Briefly, extracellular virions appear to be highly associated with low density lipoproteins (LDL) in the serum. A number of surface receptors are involved with cell recognition and binding including the LDL receptor, CD81 and scavenger receptor B (SR-B). Following binding, there is an invagination and formation of a vesicle that is dependent upon claudin-1 and other proteins. Acidification of the vesicle leads to the release of the virion and its genetic material. The 5′ end of the viral RNA represents the internal ribosomal entry site, which binds to the ribosome and produces a polyprotein. This protein undergoes posttranslational cleavage into the structural (capsid) and functional (protease, polymerase, helicase, etc.) proteins. The RNA-dependent RNA polymerase catalyses the production of new positive RNA strands via a negative strand intermediate. The lack of proofreading function leads to the errors in the new RNA, which may affect replicative viability, but also permit rapid evolution from both immune and drug related pressure. It results in the formation of viral quasispecies (variants within an individual) and genotypes (variants in a population). Assembly of virus takes place at the endoplasmic reticulum, and the new virions are released to the extracellular space to start the process again [19,20].
HCV antiviral therapy
Treatment of HCV is currently based upon a two drug regimen of pegylated interferon alfa and the nucleoside analogue ribavirin. This combination has represented the standard of care for HCV for nearly a decade, although there have been considerable refinements in management related to treatment duration, drug dosing and individualization of treatment paradigms [21,22]. Today, patients with HCV genotype 1 are started on treatment with pegylated interferon alfa 2a or 2b using weekly s.c. injections plus weight-based ribavirin. Viral loads are obtained at baseline and at 4, 12, 24 and 48 weeks. An undetectable HCV RNA at 4 weeks is termed Rapid Viral Response (RVR) and suggests a strong chance of viral cure with 48 weeks of therapy [23]. Certain patients may achieve cure with only 24 weeks of treatment if they achieve RVR. At 12 weeks, patients are evaluated to see if they have had either viral clearance or a 2-log drop in viral load from baseline. This is termed complete Early Viral Response (cEVR) or Early Viral Response (EVR), respectively. Failure to achieve this is a negative prognostic indicator and often leads to early treatment discontinuation [24]. For subjects who achieve RVR or EVR, virus should be undetectable at weeks 24 and 48. A patient with negative virus at week 48 is said to have achieved End Treatment Response. At this point, drug therapy is discontinued and the patient is monitored for 24 weeks. If HCV RNA is not detected at that time, the patient has achieved Sustained Viral Response (SVR) or cure of their HCV infection. Patients with HCV Genotype 2 or 3 may be treated for shorter periods (24 weeks) and with lower doses of ribavirin. Overall, about 50% of patients will be cured of HCV, but rates vary with genotype. These with genotype 1 will achieve SVR in about 40–45% of cases, whereas those with more favourable genotypes may be cured >75% of the time.
Future therapeutic agents
The last decade has seen intensive research into new agents that affect a variety of potential targets in the HCV lifecycle. Agents in the development include entry inhibitors, protease and polymerase inhibitors, cyclophilin inhibits, NS5a inhibitors, interferon enhancers, immunomodulators and several other targeted strategies. Two agents have entered Phase III trials; namely telaprevir and bocepravir. Both are protease inhibitors that have demonstrated excellent efficacy against HCV, with multilog drops in viral load after oral dosing [25,26]. However, this class of agent is highly susceptible to mutant drug-resistant virus emergence, and viral breakthrough has been observed in <1 week when these drugs are used as single-agent therapies [27]. In the Phase II and III trials, both drugs have been combined with pegylated interferon and ribavirin, and SVR rates of 65–75% have been observed. Shorter duration of therapy may be possible for some patient/agent combinations. These agents are genotype targeted and have lower efficacy against non-genotypes 1. A number of agents in other classes are in human trials as well. Polymerase inhibitors have somewhat lower efficacy than protease inhibitors, but appear to have a higher intrinsic barrier to emergence of drug resistance [28]. Cyclophilin inhibitors target host proteins that the virus utilizes and appear quite potent as well with a good resistance profile, but need better characterization in terms of side-effect profiles [29]. NS5a inhibitors have also shown promising results in early trials. The opportunity to cure more patients with chronic HCV infection is now a reality, as multiple new agents become available. The next decade will see a rapid evolution of treatment modalities that will provide greater efficacy, less toxicity and shorter treatment duration. This will usher in the beginning of the end for chronic HCV infection.
Clinical impact of parvovirus B19 infection (Dr Jordan)
Human parvovirus B19 (B19) circulates worldwide. In temperate climates, epidemic manifestations occur more commonly in late winter, spring or early summer. B19 infection is commonly associated with rash-like illness in children [30]. B19 seroprevalence increases with age so that by the time one reaches adulthood, 50% of individuals have circulating B19-specific IgG.
B19 replication
B19 infects, replicates in and destroys the precursor cells that are responsible for producing mature red blood cells. In an infected individual, destroying the source of mature red blood cells will result in dramatically lower haematocrit levels and a temporary anaemic state [31,32]. For those individuals who have disorders that shorten their red cell half-life, B19 infection worsens the presentation producing a more severe transient aplastic crisis.
B19 clinical infection
Symptoms in a B19 infected individual will vary considerably from one person to the next. An individual can be infected and yet be completely asymptomatic, or have a mild flu-like illness, or a life-threatening illness. Despite the infected individual’s presentation, the viral load present within their bloodstream can be extremely high, at ∼1012 genome equivalents mL−1 (geq mL−1). This poses the risk that virus can be transmitted by transfusion of blood components obtained from asymptomatic viremic donors [33,34].
B19 and the blood supply
The incidence of B19 in the blood of healthy donors ranges from 1 in 20 000 to 1 in 50 000 donors [34,35]. The risk of transmission is greater when the blood components are made from pooled units compared with those made from single units. This fact places those individuals requiring repeated doses of any of these blood products at risk of becoming infected with B19 over time, including individuals living with haemophilia, who require life-long administration of clotting factor concentrate [34,36]. B19 DNA has also been detected in numerous batches of albumin, factor VIII, factor IX, clotting factor concentrates and immunoglobulin [37,38]. In general, there is a high likelihood that co-circulating neutralizing antibodies will be present in these pooled products, which may help to explain the lack of infectivity in pools with lower viral loads [33,36].
Inactivating non-enveloped viruses
When you combine the fact that asymptomatic individuals can have high levels of circulating virus with the fact that B19 is a non-enveloped DNA virus and as such is highly resistance to heat, solvent and detergent treatments, you begin to see the challenges facing the blood banking industry [39]. Solvent detergent treatment, which is highly effective for inactivating enveloped viruses like HIV, HBV and HCV, does not inactive non-enveloped viruses like B19 and HAV. As a result of this, the industry has had to turn to using more complicated and expensive dry-heat treatment and nano-filtration methods to reduce or eliminate the level of non-enveloped viruses.
Screening for non-enveloped viruses
In most countries, blood is not routinely screened for the presence of B19. Determining whether to screen blood and/or blood products for B19 and at what level, if any, B19 is considered a minimal or low risk for transmission is being actively addressed. As B19 cannot easily replicate in conventional cell or tissue culture methods, nucleic acid amplification testing (NAAT) has been developed and is the recommended method used to screen blood and blood products for the presence of B19 DNA.
NAAT testing
The Food and Drug Administration does not currently mandate screening the blood supply for B19, but is proposing that manufactured pools contain plasma B19 DNA levels consistently below 104 geq mL−1 [36]. Similarly, the Health Council for the Netherlands (2002/07; ISBN) considers 104 geq mL−1 the maximum permissible limit. The Health Council for the Netherlands has also recommended that a high-risk group approach be adopted for cellular blood products containing B19 DNA. In Europe, although there is no official guideline published for plasma pools, and screening of blood donations for B19 DNA is not routine, many manufacturers now voluntarily perform B19 polymerase chain reaction on plasma pools. The basis for the current recommended viral load cutoff came from observations of healthy volunteers. The findings of these studies suggest that acute B19 infection can occur from administration of blood components containing ≥107 geq mL−1 of B19 DNA. In contrast, patients receiving <104 geq mL−1 have not shown evidence of virus transmission [36,40]. A recent study linking donors and recipients was undertaken to assess the risk of transmission from B19 DNA-positive units containing <106 IU mL−1 into B19 susceptible recipients (B19-specific IgG negative). In this study, 105 B19 DNA-positive donations resulted in the transfusion of 112 B19-positive components into 107 recipients. None of the 24 susceptible cases resulted in a B19 infection [41]. Other investigators found that transmission did not occur in components containing <106 IU mL−1, transmission. In summary, it will be important to identify at-risk individuals and to establish an algorithm that can be used to minimize or eliminate their exposure to blood and blood products containing high levels of Human parvovirus B19.
Discussion and conclusions
Viral pathogens continue to be a potential risk for individuals with haemophilia treated with plasma derived blood products, and among these HIV, HCV, and parvovirus B19 have the greatest clinical impact.
Hepatitis C and HIV are arguably the major co-morbid condition in individuals with haemophilia. In contrast to other at-risk populations, HCV infection haemophilia was acquired early in life, with the first clotting factor exposure, and HIV about 10 years later, peaking in the early 1980s. HAART has not only changed the course of HIV, it has converted it into a treatable chronic infection, and further, has slowed HCV progression in co-infected individuals. Despite that, at least 25% of haemophilic men have evidence of fibrosis, and, unfortunately but may be reluctant to undergo antiviral treatment with combination peg-interferon and ribavirin, which is effective in upwards in 40–75%.
Better agents to treat HCV are sorely needed, especially for those with HIV co-infection, in whom HCV progression is greater and antiviral response is lower; and for those undergoing transplantation, in whom recurrent hepatitis C is more aggressive in the setting of immunosuppressive antirejection therapy. Potential alternatives will hopefully be identified among the more than 300 agents in development.
Highly active antiretroviral therapy is effective in suppressing HIV infection, and now that HIV has markedly changed the course of HIV infection from a lethal disease to a chronic infection, and importantly, has reduced the rate of HCV liver disease progression among those with HIV/HCV co-infection.
A third transfusion-transmitted viral pathogen, parvovirus B19, may cause anaemia, and rarely aplastic crisis, but is not likely to be eradicated from the blood supply by viral inactivation technologies as were HCV and HIV. Given its non-enveloped structure, NAAT of the blood supply is under consideration to eradicate this viral pathogen.
Disclosures
The authors stated that they had no interests which might be perceived as posing a conflict or bias.
