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How to Diagnose this hepatitis A, B and C case?


Male patient who is working in the hospital and may be infected with contaminated blood had hepatitis with genome IA 5 years ago. His hepatitis A at that time was confirmed with the rise of anti.HAV IgM and later with anti--HAV IgG type antibodies. His wife is known to have antibodies against C hepatitits. The patient had icteric illness in about three weeks ago. Which are the best indicators in diagnosing his hepatitis?

Right answer is: Detection of HBsAg and anti-HCV antibodies.

I think this is because

  • to check if he has hepatitis B by HBsAg
  • to check if he has HepC by the antibody

Serum antigen of HB tells us about the levels of IgM and IgG. If vaccinated, then IgG should be positive, I think? What does HBsAg tell you?

Why do we need to detect anti-HCV? If they are found, what does it mean? I think that then it means if found that he does not have hepatitis C then.


The last sentence of the question doesn't make it clear what's being asked. But it seems to be asking:What version of Hepatitis did he get from the contaminated blood?

To that end…

Serum antigen of HB tells us about the levels of IgM and IgG. If vaccinated, then IgG should be positive, I think? What does HBsAg tell you?

HBsAg gives you the knowledge of whether or not there's an acute infection of Hep-B. Hep-B IgM antibodies indicate an acute infection, whilst IgG antibodies would show up due to his previous exposure/immunization. If the patient has Hep-B IgM, the icteric illness was probably a re-infection of Hep-B.

Why do we need to detect anti-HCV? If they are found, what does it mean? I think that then it means if found that he does not have hepatitis C then.

That would my assumption as well.

You run the HBsAg and can get two outcomes: IgM(+)/IgG(+) or IgM(-)/IgG(+). The former indicates the icteric illness was probably a reinfection of Hep-B.

You run the anti-HCV and get two outcomes: (+) or (-). The former indicates a present infection, the latter indicates no Hep-C.

What might throw you off is the wife's anti-HepC antibodies. If she was formerly infected and infected the patient (or vice versa) then you'd expect to find a (+) anti-HCV result.

I might come back to this after thinking on it a bit more, but that's the best I have for now.


Viral Hepatitis

Hepatitis refers to inflammation of the liver. Inflammation is a tissue’s reaction to irritation or injury which generally results in swelling and can cause pain.

There are many causes of hepatitis. Viral hepatitis is caused by a virus and can either be acute (lasting less than six months) or chronic (lasting more than six months). Viral hepatitis can be spread from person to person. Some types of viral hepatitis can be spread through sexual contact.

There are five known hepatitis viruses which are categorized by the letters A through E.

Several viruses are known to cause hepatitis. Common forms of viral hepatitis include:

  • Hepatitis A: According to the Centers for Disease Control and Prevention, there were about 2,007 instances of acute hepatitis A infections in the U.S. in 2016. This form of hepatitis does not lead to a chronic infection and usually has no complications. The liver usually heals from hepatitis A within several months. However, occasional deaths from hepatitis A have occurred due to liver failure, and some people have required a liver transplant for acute hepatitis A infection. Hepatitis A can be prevented by vaccination. : Around 22,000 new cases of hepatitis B occurred in 2017, and around 900,000 people are living with the disease in the US. Approximately 95% of adults recover from hepatitis B and do not become chronically infected. However, a few cases cause a life-long, chronic infection. The earlier in life hepatitis B is contracted, the more likely it is to become chronic. People can carry the virus without feeling sick but can still spread the virus. Hepatitis B can be prevented by getting a vaccine. : Hepatitis C is one of the most common causes of liver disease in the U.S., and used to be the number one reason for liver transplant. About 75% to 85% of patients with hepatitis C develop a chronic liver infection. Roughly 2.4 million people in the U.S. are estimated to have chronic hepatitis C infection. It often does not show any symptoms. No vaccine is yet available to prevent hepatitis C.
  • Hepatitis D: Hepatitis D only happens to people who are infected by the hepatitis B virus. If you are vaccinated against hepatitis B, you will be protected against hepatitis D virus.
  • Hepatitis E: This type of hepatitis is spread by ingesting contaminated food or water. Hepatitis E is common throughout the world. Even though vaccines exist, they are not available everywhere.

Healthcare providers might not be able to identify the virus causing hepatitis as one of these. Other viruses, such as CMV, EBV, and HSV can also cause hepatitis.

Most people recover from hepatitis, and the disease is often preventable. However, it is still considered a serious health risk because it can:

  • Destroy liver tissue.
  • Spread easily from person to person.
  • Weaken the body's immune system.
  • Cause the liver to fail.
  • Cause liver cancer.
  • Cause death (in rare cases).

What are the different types of hepatitis, and is there a vaccine?

There are six main types of viral hepatitis: A, B, C, D, E, and G. The three main types in the United States are hepatitis A, B, and C. Vaccines can prevent some viral hepatitis infections.

Hepatitis is an inflammation of the liver. It usually results from a viral infection, but drugs, toxins, and certain diseases, including autoimmune diseases, may also cause the condition.

Scientists have only recently discovered hepatitis G (HGV), and they know less about it than other forms of hepatitis.

Share on Pinterest To diagnose viral hepatitis, a doctor may order a blood test.

The following table shows the main types of hepatitis:

TypeTransmission Symptoms
A (HAV)consuming food or drink contaminated with the feces of someone with the infectionnausea, fatigue, dark urine, vomiting, fever, jaundice, and anorexia
B (HBV)making contact with the body fluids of a person with the infection — usually from the woman to the fetus at birth, but occasionally from using contaminated needles or having sex without a condom unease, fatigue, anorexia, and mild illness
C (HCV)making contact with blood containing the virus — mostly spread through contaminated needles of IV drug users 80% of people have no symptoms, but others have unease, fatigue, and anorexia
D (HDV)same as HBV unease, fatigue, anorexia, and mild illness
E (HEV)usually through consuming undercooked meat or food or drink contaminated with the feces of someone with the infection, but occasionally from a pregnant woman to a fetusmild illness
G (HGV)through blood containing the virusmild infection, but most people have no symptoms

The symptoms of hepatitis may be mild, but the condition can lead to severe complications. For example, each type of hepatitis can cause fulminant hepatic failure (FHF), which affects the liver.

Hepatitis may be acute or chronic . Acute hepatitis is short-term, lasting less than 6 months. Chronic hepatitis is long-term, lasting more than 6 months. Hepatitis C is the most common cause of chronic hepatitis.

Vaccines and vaccine schedules depend on the type of hepatitis.

Hepatitis A

The HAV vaccine can prevent hepatitis A. This shot is routine for infants between the ages of 12­ and 23 months. Unvaccinated children older than 23 months, adolescents, and adults should also have this shot.

A doctor will administer the vaccine over two doses, with 6 months between them.

Hepatitis B

The HBV vaccine is a routine shot that usually immunizes people for life. A person will typically have the first dose at birth and complete the schedule at 6 months old.

An unvaccinated child or adolescent below the age of 19 should also have the shot. Some adults who are not vaccinated should also receive the vaccine — for example, those traveling to areas where hepatitis B is common.

A doctor may administer the hepatitis B vaccine in two, three, or four doses.

Hepatitis C

There is currently no vaccine for hepatitis C, which causes 2 million new infections each year worldwide. However, scientists are carrying out trials to develop a vaccine.

Hepatitis D

The HBV vaccine can prevent hepatitis D, which is a coinfection of hepatitis B. However, the vaccine will not protect a person from hepatitis D if they already have chronic hepatitis B.


How to Diagnose Viral Hepatitis

This article was co-authored by Raj Vuppalanchi, MD. Dr. Raj Vuppalanchi is an Academic Hepatologist, a Professor of Medicine at Indiana University School of Medicine, and the Director of Clinical Hepatology at IU Health. With over ten years of experience, Dr. Vuppalanchi runs a clinical practice and provides care to patients with various liver disorders at the University Hospital in Indianapolis. He completed dual fellowships in Clinical Pharmacology and Gastroenterology-Hepatology at Indiana University School of Medicine. Dr. Raj Vuppalanchi is board certified in Internal Medicine and Gastroenterology by the American Board of Internal Medicine and is a member of the American Association for Study of Liver Diseases and the American College of Gastroenterology. His patient-oriented research is dedicated to finding new treatments for various liver disorders as well as the use of diagnostic tests for non-invasive estimation of liver fibrosis (transient elastography) and portal hypertension (spleen stiffness).

There are 21 references cited in this article, which can be found at the bottom of the page.

This article has been viewed 8,733 times.

Viral hepatitis is a type of liver disease that can be caused by several different viruses. The most common types of viral hepatitis are hepatitis A, B, and C, although there are other varieties as well, such as hepatitis D and E. These viruses may be acute (if they are quickly eradicated from the body) or chronic (if the virus continues to infect the individual for a long period of time). People with viral hepatitis may or may not present symptoms, so blood tests are the most reliable means of diagnosis. [1] X Expert Source


Hepatitis is inflammation of the liver. This inflammation may be due to viruses, medicines, alcohol, problems with the immune system, or not enough blood supply to the liver.

The liver is an essential organ in the body and carries out a wide range of functions, including removing poisons from the body, storing energy, and regulating blood clotting.

Although other viruses can involve the liver, viral hepatitis refers to inflammation of the liver due to one of several viruses that specifically attack the liver. These viruses are labeled with the letters A, B, C, D and E. The most important viruses in the United States are viral hepatitis A, B and C.

Viral hepatitis A and E enter the body by way of the gastrointestinal tract. One gets these viruses by eating food or drinking fluids contaminated with the virus, usually from the stool of an infected person. Hepatitis A may also be transmitted in areas where there is overcrowding, such as day care centers and correctional facilities, from eating raw shell fish that came from water contaminated with sewage, or by sexual (usually anal-oral) contact. Hepatitis E, although similar to hepatitis A, is not frequently seen in the United States. This virus is seen primarily in the Indian subcontinent and in certain areas of Africa. It is transmitted primarily by fecal-oral contamination (as mentioned above).

The hepatitis B and C viruses are generally transmitted by contact with infected blood or blood products, or by sexual contact. In the United States, intravenous drug use is the most frequent mode of transmission of hepatitis C. Heterosexual transmission is uncommon for hepatitis C though men having sex with men is being recognized increasingly as a risk factor for hepatitis C transmission. Hepatitis B is commonly transmitted sexually as well as through intravenous drug use, and can also be passed from a mother to her fetus, usually at the time of delivery.

Hepatitis D (sometimes referred to as Delta hepatitis) only afflicts individuals who have the hepatitis B infection. The mode of transmission of hepatitis D is similar to that of hepatitis B.

Acute viral hepatitis refers to inflammation of the liver associated with symptoms and abnormalities of liver enzymes, which last for a period less than six months. All of the viruses mentioned above can cause acute hepatitis. The symptoms of acute viral hepatitis can include low-grade fever, headaches, muscle ache, tiredness, loss of appetite, nausea, vomiting, diarrhea, dark-colored urine or light-colored stools. The most notable symptom of acute viral hepatitis is jaundice, which is a yellow discoloration of the skin and the whites of the eyes. Patients also usually complain of vague upper abdominal pain. Very few patients with acute viral hepatitis go on to develop liver failure that would require liver transplantation or lead to death. However, hepatitis E may have serious consequences if a person is infected during pregnancy.

Except for patients infected with hepatitis C, most cases of acute viral hepatitis will disappear on their own. With hepatitis C, more than 80% of patients will develop a persistent infection called chronic hepatitis. Although most of those with acute hepatitis B will resolve their infection, a significant number of infants and young children with hepatitis B will develop chronic hepatitis B.

Chronic viral hepatitis refers to ongoing inflammation and/or abnormalities of liver enzymes which last more than six months. Most patients who develop chronic hepatitis will have life-long disease unless they are treated. Chronic viral hepatitis in the United States is limited primarily to hepatitis B and C. Consequences of chronic viral hepatitis may include progressive liver disease, including cirrhosis and its complications, liver failure, liver cancer, and bleeding disorders, although many patients will never develop any of these complications. Furthermore, chronic hepatitis may be accelerated in people who use drugs that are toxic to the liver (including alcohol). Most of these complications can be prevented by early diagnosis and early treatment, and in the case of hepatitis B, vaccination will prevent acute infection in case of exposure to hepatitis B.

Viral hepatitis (both acute and chronic) can be easily diagnosed using simple blood tests. While a liver sample (called a biopsy) is not required in the vast majority of cases, it may sometimes be helpful, especially in difficult to diagnose cases or when deciding on treatment based on the severity of the liver scarring.

Acute viral hepatitis generally does not require treatment, as most patients will get better on their own. Acute hepatitis C is a notable exception, because only 15% of patients will clear the virus on their own and most people, if not treated, will develop chronic hepatitis C. In addition, there is a very high cure rate when patients are treated during the acute hepatitis infection stage.

Chronic hepatitis treatments are available for hepatitis B and C, the most predominant causes of chronic hepatitis. Chronic hepatitis B is usually treated by medications that interfere with the virus’ ability to multiply and replicate. These medications include oral medications as well as injectable medications. Approved medications for the treatment of chronic hepatitis B include the injectable pegylated interferons, as well as medications taken by mouth called directly acting antivirals (DAA’s). These DAA’s are virtually free of any side effects and are the mainstay of therapy. No medication is currently available which will rid the body completely free of the hepatitis B virus (cure). These medications keep the virus under excellent control, but will not completely eliminate the virus. The duration of treatment may vary from one year for the pegylated interferon to possibly indefinite for the oral DAAs. Research is ongoing on the medications that will completely eliminate the virus and achieve what scientists refer to as a functional cure.

Major advances have been made in the treatment of chronic hepatitis C infection. Currently, medications taken by mouth (called directly acting antivirals, or DAAs) have completely replaced the injectable medications (called pegylated interferons). These medications, which are taken by mouth, are specific for the type of virus the patient has, called genotype, of which there are six, (1-6). The oral medication ribavirin is still used in some patients with chronic hepatitis C. In the very near future, single tablet medications will be used to treat all of the six genotypes for hepatitis C! That cure rate with the currently available medications for hepatitis C is almost 100%. Although the cost of treatment for both viruses, especially hepatitis C have increased significantly, it is highly recommended that patients are treated as early as possible before they develop chronic liver disease and cirrhosis. The duration of treatment may be from eight weeks to 24 weeks.

Chronic viral hepatitis B and C should be treated by individuals who are knowledgeable of these conditions, the medications utilized to treat these conditions, and the side effects of these medications.

The main reason to treat chronic viral hepatitis is to prevent cirrhosis of the liver, during which the liver becomes scarred and gradually loses its ability to function. This can ultimately lead to liver failure or liver cancer. Another reason for treatment is to prevent the spread of infection. The longer a patient has chronic hepatitis, the more likely it is that cirrhosis will develop. Therefore, early diagnosis is crucial for initiating treatment as soon as possible. Unfortunately, since chronic viral hepatitis is usually not associated with symptoms, most patients remain undiagnosed.

As I have said previously, the injectable medications (interferon) are no longer recommended for patients with hepatitis C. The DAAs mentioned above may be used alone or in combination with ribavirin, both taken by mouth. The side effects are minimal for the DAA’s, and include a headache or difficulty with sleeping. Some of the medications may have interactions with other medications taken by the patient. Your doctor will advise you of these possible interactions. Ribavirin side effects may be more prominent, and include anemia, skin problems, or breathing problems. Difficulties with sleeping may also be seen with ribavirin. All of the side effects are minimal and are quite manageable.

Hepatitis B may be treated with injectable medications (as mentioned above), or oral medications (DAAs). The interferons have side effects, which include anemia, depression, and other problems and while they are a recommended therapy, are much less commonly used in the United States than the oral medications. The oral medications have virtually no side effects and are quite safe. It may be necessary to give them indefinitely. All of these medications, however, may require adjustments of doses and frequency in patients who have kidney disease. As mentioned previously, patients with chronic viral hepatitis should be treated in a center with a great deal of expertise in the management of these side effects.

Most cases of viral hepatitis can be prevented by using common sense precautions. Since hepatitis A and E viruses are transmitted through contaminated foods and liquids, patients should not eat or drink in areas with unsanitary conditions. Hence, international travelers should only consume bottled water, not use ice in beverages, and avoid food sold from street vendors. Also, individuals traveling to areas with high rates of hepatitis A should receive the hepatitis A vaccine in two (2) doses, six to 18 months apart.

Other people who should receive the hepatitis A vaccine include men who have sex with men, intravenous and other drug users, people with clotting factor disorders like hemophilia, people with chronic liver disease, and children who live in areas that have historically high rates of hepatitis A. For short term protection (for example, short term travel to endemic areas), immune globulin (IG) shots may be given. This is generally needed for people who are going to remain in those areas for a period of less than one month. This IG shot can temporarily prevent you from contracting the virus. An IG shot may also be given for protection within 2 weeks following exposure to a known hepatitis A carrier.

Most patients with acute hepatitis B will get better on their own, but this is a preventable disease. The best method of prevention is vaccination. The hepatitis B vaccine is highly effective in preventing the infection. Individuals who should receive this vaccine include those who use intravenous and other drugs, men who have sex with men, individuals with high-risk sexual behavior, and individuals with multiple sexual partners. As pregnant women who carry the virus are very likely to transmit it to their baby, newborns of hepatitis B infected mothers should receive the first dose of the hepatitis B vaccine within 12 hours of birth. It is also recommended that an IG (immune globulin) shot be given at the same time. This method is highly effective in preventing infection of the newborn. If the mother also has high amounts of the virus in her blood stream, it is recommended that she receive an oral antiviral agent after the second trimester of pregnancy to further reduce the risk of transmission to her baby.

Most states currently require hepatitis B vaccination of all newborns, as well as “catch-up” vaccination for children and adolescents. A combined hepatitis A and B vaccine (Twinrix®) is also available for people who need both vaccines. These vaccines offer lifelong immunity (as does recovery from infection) unless the patient has underlying immune disease or kidney failure.

Although some herbal medicines may briefly improve liver enzymes, there is currently no evidence that any medicines (other than those previously mentioned) can cure viral hepatitis. The most common herbal medicine, milk thistle showed no benefit in hepatitis C. One should never use any prescription or over-the-counter medications without consulting a physician first. Additionally, some herbal medications may seriously damage the liver.


Goals of Therapy

The goals for treatment of chronic HBV infection are to reduce inflammation of the liver prevent liver failure and cirrhosis and reduce the risk of hepatocellular carcinoma by suppressing HBV replication. Normalization of alanine transaminase (ALT), loss of HBeAg (seroconversion), decrease in serum HBV DNA level, and improvement in liver histology indicate treatment effectiveness.1 , 6 , 17 , 18 A recent systematic review found insufficient evidence to assess treatment effectiveness on patient-oriented outcomes, such as decreased mortality and improved quality of life.19 A disease-oriented outcome, suppression of HBV DNA levels, is often used as an end point of treatment.20

It is important to distinguish between patients who are HBeAg positive and those who are HBeAg negative because of a viral mutation. Seroconversion (i.e., conversion from HBeAg positive to HBeAg negative, followed by conversion from hepatitis B e antibody [HBeAb] negative to HBeAb positive) predicts long-term reduction in viral replication and is used as a response marker to therapy. Genotypes affect response and guide treatment choices. For example, genotype A is highly responsive to pegylated interferon alfa-2a (Pegasys) therapy.21 Certain populations (e.g., persons with renal insufficiency or decompensated liver disease, liver transplant recipients) require additional monitoring and expertise.


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References

Weinbaum C, Lyerla R, Margolis HS. Prevention and control of infections with hepatitis viruses in correctional settings. MMWR Recomm Rep. 200352(RR01):1-33. http://www.cdc.gov/mmwr/preview/mmwrhtml/rr5201a1.htm. Accessed January 4, 2008.

National Institutes of Health. NIH Consensus Statement on Management of Hepatitis C: 2002. NIH Consens State Sci Statements. 200219(3):1-46.

Strader DB, Wright T, Thomas DL, Seeff LB American Association for the Study of Liver Diseases. Diagnosis, management, and treatment of hepatitis C. Hepatology. 200439(4):1147-1171.

Citation

The viewpoints expressed in this article are those of the author(s) and do not necessarily reflect the views and policies of the AMA.

Author Information

Howard J. Worman, MD is a professor of medicine and pathology and cell biology at Columbia University College of Physicians and Surgeons in New York City, where he lectures about the liver and liver disease to first- and second-year medical students and mentors gastroenterology fellows in the Liver Clinic. His academic activities are divided between basic research, medical education, and the care of patients with liver diseases.


Occult or Latent HBV Infection

Other atypical HBV infections include seronegative occult or latent HBV infections. This heterogeneous group consists of patients who are HBsAg-negative who are either seronegative for all HBV markers or positive for anti-HBc and/or anti-HBs. 48 – 52 Many of these patients are positive for HBV DNA by polymerase chain reaction either in the liver or serum or both. Some of these patients have underlying liver disease, suggestive of ongoing hepatocellular injury from persistent HBV infection. Studies in animal models have demonstrated long-term persistence of viral genomes in the serum and/or liver of animals that have biochemical and serologic evidence of viral clearance and recovery from infection. 49 , 53 The important question is whether this observation represents ongoing viral replication and therefore clinically significant infection in terms of liver disease and transmission. Existing evidence supports the notion that it indeed indicates low-level viral replication, capable of transmission. 49 , 54 Studies in liver transplantation revealed transmission of HBV infection to recipients if the donors carried the anti-HBc marker. 55 In addition, reactivation of HBV infection in patients with serologic evidence of recovery undergoing immunosuppression or chemotherapy has been reported. 56 – 59 These observations, together with the immunologic studies described above, provide compelling evidence that one may not be able to completely eliminate HBV infection. Patients with serologic evidence of recovery probably have low-level viral replication that is effectively controlled by an active immune response. The possibility that these occult infections are caused by HBV mutants has been proposed. Although mutations have been reported in various regions of the viral genome, 60 – 63 definitive evidence in support of a pathogenic role of these mutants is lacking. Furthermore, whether liver disease can indeed result from these occult HBV infections is controversial. At present, there are no convincing studies in support of a causal relationship. Therefore, these occult HBV infections, other than the special situations described above, may not be clinically important.


How Many People Have Hepatitis A?

Since the release of the first vaccine in 1995, the rate of new HAV infections in the United States declined by more than 95% from 1996 to 2011. From 2012 through 2016, the number of hepatitis A cases fluctuated because large foodborne outbreaks occurred. From August 2016 through August 2020, 33 states reported hepatitis A outbreaks spread through person-to-person contact resulting in over 33,000 infections with high numbers of hospitalizations and deaths.

Globally, HAV infection is most common in countries with poor sanitary conditions and hygienic practices and transitional economies according to the World Health Organization .


PCR-Based Molecular Diagnosis of Hepatitis Virus (HBV and HDV) in HCV Infected Patients and Their Biochemical Study

Seroprevalence of HCV indicates that HCV is found in more than 10% of HBV- or HDV-infected patients worldwide leading to liver disease. Here we show HBV and HDV coinfection association with HCV infected Pakistani patients, study of disease severity, and possible interpretation of associated risk factors in coinfected patients. A total of 730 liver diseased patients were included, out of which 501 were found positive for HCV infection via PCR. 5.1% of patients were coinfected with HBV while 1% were coinfected with HBV and HDV both. LFTs were significantly altered in dually and triply infected patients as compared to single HCV infection. Mean bilirubin, AST, and ALT levels were highest (3.25 mg/dL, 174 IU/L, and 348 IU/L) in patients with triple infection while dual infection LFTs (1.6 mg/dL, 61 IU/L, and 74 IU/L) were not high as in single infection (1.9 mg/dL, 76 IU/L, and 91 IU/L). The most prominent risk factor in case of single (22%) and dual infection (27%) group was “reuse of syringes” while in triple infection it was “intravenous drug users” (60%). It is concluded that HBV and HDV coinfections are strongly associated with HCV infected Pakistani patients and in case of severe liver disease the possibility of double and triple coinfection should be kept in consideration.

1. Introduction

Chronic hepatitis is a common cause of liver related morbidity due to different hepatic viruses, where hepatitis B (HBV) and hepatitis C (HCV) have been identified as the main cause [1] and lead to many complications. Over a million persons die annually due to HBV related complications [2, 3]. HCV also leads to many complications including HCC in 32% of infected patients [4]. Infection with multiple viruses leads to management problems with higher incidence of morbidity and mortality [5]. Since hepatitis B, hepatitis C, and hepatitis D share almost the same modes of transmission, infection with more than one virus is possible [6]. Therefore, presence of dual and triple viral infections has been reported from various parts of the world [7].

HCV is a positive stranded RNA virus and has been classified into the genus Hepacivirus of the family Flaviviridae [8]. Lack of proper implementation of international standards in procedures like blood transfusion, reuse of injections, injecting drug users, tattooing, shaving from barbers, unsterilized dental reuse of needles for ear and nose piercing, and surgical instruments is the key factor of HCV transmission in Pakistan. Worldwide, there are about 170 million people infected with HCV, and three to four million individuals are diagnosed as new cases every year [9], while in Pakistan 10 million people are presumed to be HCV patients with 5% prevalence in general population [10].

Hepatitis B virus belongs to Hepadnaviridae family [11] with a circular genome of 3.2 kb composed of partially double-stranded DNA [12]. An average prevalence of hepatitis B antigen in Pakistan is 2.4% (range 1.4–11.0%) in healthy adults and 2.4% in pediatric population. The reuse of used syringes and unsafe blood transfusion are the major causes of spread of hepatitis B [10]. Hepatitis delta virus (HDV), first discovered by Rizzetto et al. [13] in a patient with chronic hepatitis B virus (HBV) infection, is a unique single-stranded negative circular RNA virus with genome of 1.7 kb that requires the helper function of HBV for infection [14]. It was originally thought to be a new nuclear antigen associated with HBV [5] but later proved to be a new virus that requires the surface antigens of HBV (HBsAgs) to support its life cycle and infectivity [15–17]. The prevalence of HDV accounts for 15–20 million people who are already infected with HBV [18]. Very limited data is available about the prevalence and epidemiology of HDV infection in Pakistan. HDV infection is present in 16.6% of hepatitis B infected patients in Pakistan [19]. In another study from Karachi 35.2% of the coinfection was reported. It was further explained that HBV/HDV coinfection resulted in the suppression of HBV DNA. A fair percentage of HBV/HDV coinfected patients with HBeAg negative had active hepatitis B infection and cirrhosis as compared to those with monoinfection [20]. Diagnosing multiple hepatitis viral infections is limited by low level of awareness among physicians and availability of simple diagnostic tests. Recently, good results were demonstrated by a single integrated protein microarray that could simultaneously determine in human sera two viral antigens (HBsAg and HBeAg) and seven antibodies (HBsAb, HBcAb, HBeAb, HCVAb, HDVAb, HEVAb, and HGVAb) of human hepatitis viruses within 20 minutes [21].

Treatment options are limited in triple infections as lamivudine alone or in combination with interferon has not shown much benefit in patients coinfected with HDV [22]. Since there is no report on triple infection caused by hepatitis viruses from Pakistani population [23], the aim of this study is to find the rate of coinfection of HBV and HDV in HCV infected Pakistani patients.

2. Material and Methods

2.1. Patients and Samples

Present study was conducted at Atta-ur-Rahman School of Applied Biosciences (ASAB), National University of Science and Technology (NUST), in collaboration with Capital Development Authority (CDA) Hospital, Islamabad, Railways Hospital (IIMCT), Rawalpindi, and Holy Family Hospital, Rawalpindi, Pakistan. The study was approved by the Institutional Review Board (IRB), ASAB, and NUST.

In the present study, a total of 730 patients, with liver disease, were included who were referred to ASAB diagnostics center during the period from July 2009 to March 2010. During the course of the study, venous blood samples were collected in the morning after 12-hour overnight fasting. 137 samples were collected from Capital Development Authority (CDA) Hospital, Islamabad, 257 samples from Government Holy Family Hospital, Rawalpindi, 155 samples from Railways Hospital (IIMCT), Rawalpindi, and 181 from Atta-ur-Rahman School of Applied Biosciences (ASAB) diagnostics.

2.1.1. Extraction of HCV RNA

Sera of the patients were subjected to viral RNA extraction by using QIAamp viral RNA extraction kit (Qiagen), according to the manufacturer’s protocol.

2.1.2. Qualitative Analysis (PCR-Based Detection)

All ELISA positive samples with chronic hepatitis C infection were further confirmed for HCV presence by PCR. Indirect ELISA method is used for the detection of antibodies to HCV in two-step incubation procedure (microLISA).

2.1.3. Detection of HCV RNA by Nested PCR

Viral RNA was taken to reverse-transcribe the 5′ NCR of HCV using Moloney murine leukemia virus reverse transcriptase (M-MLV RTase, Fermentas) in a total reaction volume of 20 μL. The reaction mixture contained 4 μL MMLV (5x) buffer, 1 μL M-MLV reverse transcriptase (RT) enzyme, 1 μL dNTPs (10 mM), 0.5 μL Rnase inhibiter (Fermentas), 1.5 μL RNase free water, 1 μL specific antisense primer P2 5′-ACTCGCAAGCACCTATCAGGCAGTAC-3′ (Macrogen, Korea), and 10 μL template (viral RNA). cDNA was synthesized using ABI Veriti 96-well thermocycler. Cycle conditions for cDNA were as follows: 42°C for 55 minutes followed by 70°C for 10 minutes. The cDNA produced was stored at 4°C for short-term storage or −20°C for prolonged storage. cDNA product was used for qualitative analysis of HCV infection. The first round PCR was performed using sense P1 5′-CCCTGTGAGGAACACTGTCTTCACGC-3′ and antisense P2 5′-ACTCGCAAGCACCTATCAGGCAGTAC-3′ primers (Macrogen, Korea) followed by second round PCR (nested PCR), using the first round product with inner sense P3 5′-GAAAGCGTCTAGCATGGCG-3′ and antisense P4 5′-CACAAGGCCTTTCCGACC-3′ primers (Macrogen, Korea). PCRs were carried out using Taq polymerase (Fermentas) for 35 cycles. The PCR product was visualized by 1.2% agarose gel and stored at −20°C until further use.

2.1.4. Detection of HBsAg in Serum Samples from Patients with HCV Infection

The sera of HCV samples were tested for infection of HBV. To analyze the HBV infection, strip device (ACON, USA) was used. For detection of HBsAg sandwich ELISA method was used (microLISA).

2.2. HBV DNA Detection through PCR

For the detection of hepatitis B virus, the specific portion of surface gene was amplified through PCR from HBV genomic DNA using specific forward and reverse primers.

2.3. Viral DNA Extraction

Viral DNA was isolated from the serum of HBV infected patient’s samples using Qiagen Kit (Germany) according to the manufacturer’s protocol.

2.4. Polymerase Chain Reaction

The extracted DNA was subjected to PCR for the detection of HBV. 20 μL PCR reaction mixture contain DNA, PCR Master Mix (50 units/mL of Taq DNA polymerase supplied in a proprietary reaction buffer pH 8.5, 400 μM dATP, 400 μM dGTP, 400 μM dCTP, 400 μM dTTP, and 3 mM MgCl2) (Promega Cat.# M7502), 10 mM of sense primer (Macrogen, Korea), 5′-CCGAATTCGCCACCATGCATCCTGCTGCTATGCCTCATCT-3′ and 10 mM of antisense primer (Macrogen, Korea), and 5′-CCCGAATTCGCCACCATGCGAACCACTGAACAAATGGCACT-3′. Thirty cycles of DNA amplification were performed. The condition of each cycle was denaturation at 94°C for 45 seconds, primer annealing at 64°C for 45 seconds, and elongation at 72°C for 45 seconds, followed by a final elongation at 72°C for 7 minutes. PCR product was further amplified with the inner nested primer set, 10 mM sense primer (Macrogen, Korea), 5′-CCCGAATTCGCCACCATGGGTATGTTGCCCGTTTGTCCTCT-3′, and 10 mM of antisense primer (Macrogen, Korea) 5′-CCCGAATTCGCCACCATGGGCACTAGTAAACTGAGCCA-3′ for an additional 30 cycles under the same reaction conditions. The PCR product was visualized by 1.2% agarose gel and stored at −20°C until further use.

2.5. Detection of Hepatitis Delta Virus (HDV) by ELISA

All the patient’s sera positive for HBV DNA by PCR were checked for anti-HDV antibodies using 3rd generation ELISA assay kit (Globe Diagnostics, Italy) using the methodology described in the manufacturer’s protocol. In brief, incubator was set to

. All the reagents were brought to room temperature before use (approximately 1 hour), without removing the plate from the bag. All components were shaken well. Then the plate was removed from the package. A 1/20 dilution of serum samples in tubes apart was prepared by adding 5 μL of sample to 95 μL of sample dilution solution (1/20 dilution). 80 μL of sample dilution solution was added into all wells except in those assigned to controls. 20 μL of the 1/20 dilutions of serum samples, 100 μL of positive control, 100 μL of cutoff (cutoff in duplicate), and 100 μL of negative control were added into the corresponding wells. Plate was covered with a sealing sheet and incubated at for 60 min. Then the antigen-conjugate complex was prepared. The seal was removed and aspirate liquid from all wells was washed five times with 0.3 mL of washing solution per well. Then Immediately 100 μL of reconstituted antigen-conjugate complex was added into each well, covered with a sealing sheet, and incubated in incubator for 60 min. The seal was removed and liquid from all wells was aspirated and washed five times with 0.3 mL of washing solution per well. Immediately after that 100 μL of substrate solution was added into each well, incubated at room temperature for 20 min, and protected from light and then immediately 50 μL of stopping solution was added into all wells, and finally read with ELISA Plate Reader at 450 nm within 1 hour of stopping.

2.6. Biochemical Factors

The following biochemical examinations were tested in present study. The level of fasting glucose, Total Cholesterol (TC), High-Density Lipoprotein (HDL), Alanine Amino Transferase (ALT), Aspartate Transaminase (AST), bilirubin, and fasting Triglyceride (TG) was measured by Microlab 300 apparatus (Merck). Sera of all 501 chronic HCV patients were analyzed for these parameters.

2.7. Statistical Analysis

In statistical analysis clinical variables presented as mean ± SD. Mean, SD,

value, and CI (95%) and standard error were calculated and compared by using independent

-test. For value, the level of significance was 5%. Data analysis was accomplished using the computer software, SPSS Version 16.0 for windows.

2.8. Data Collection for Risk Factors

For the evaluation of common risk factors associated with the HCV transmission, no provincial data collection system exists. Data was collected by conducting interviews with each of the patients and using questionnaire.

3. Results

3.1. Molecular Screening of Samples

A total of 730 samples, suspected for hepatitis C infection, were subjected to PCR-based detection for viral RNA, out of which 501 (with mean age of patients

) samples were proved as HCV positive, by giving a band size of approx. 227 bp in PCR, as shown in Figure 1.

3.2. Screening of HCV Positive Cases for HBV Infection (Coinfection)

A total of 501 HCV positive samples were screened for HBsAg through Immunochromatographic Technique (ICT HBsAg) out of 501 samples, 33 were (6.6%) found positive for HBsAg. These 33 samples were screened for HBV DNA through nested PCR technique and the results of nested PCR are shown in Figure 2. The positive samples gave a band size of approximate 242 bp. A total of 31 (6.2%) samples were found positive for HBV DNA and the data is given in Table 1.

3.3. Screening of HCV and HBV Coinfected Cases for HDV Infection

After the confirmation of samples for HBV DNA, 31 coinfected (HBV and HCV) samples were further screened for Hepatitis Delta (HDV) infection. Anti-HDV ELISA screening was performed in HBV and HCV coinfected samples and it was found that 5 (1% of the total HCV positive) samples were positive for anti-HDV antibodies showing that the 1% of the total 501 samples have triple infection of HCV, HBV, and HDV, with mean age of

3.4. Biochemical Parameters (LFTS) of Single, Double, and Triple Infection Patients

The 501 samples were divided into 3 groups: (1) the patients with single infection (HCV), (2) patients with the double infection (HCV and HBV), and (3) patients with the triple infection (HCV, HBV, and HDV) were subjected to the analysis of liver function tests, that is, ALT, AST, and bilirubin. In case of single infection, the mean levels of AST, ALT, and bilirubin were found to be 76 IU/L, 91 IU/L, and 1.9 mg/dL, respectively. These levels were found to be 61 IU/L, 84 IU/L, and 1.6 mg/dL in case of double infection for AST, ALT, and bilirubin, respectively. The mean values of LFTs for triple infection were 174 IU/L, 348 IU/L, and 3.25 mg/dL, respectively (Table 2).

3.5. Evaluation of Potential Risk Factors Associated with the Transmission of Single, Double, and Triple Infections

Various possible risk factors, for example, blood transfusion, injectable drug users, dental operations, razor sharing, observed in the current study responsible for infection transmission with each single, double, and triple infection are given in Figures 6, 7, and 8 and Table 3.

3.6. Statistical Analysis of the Data

All the 501 patients were included in the study 229 (46%) were females with mean age of and 229 (54%) were males with mean age of . A summary of gender and age association of HCV infected patients is given in Table 4. It is evident from Table 4 that there is no significant relation between gender/mean age of the patients and HCV infection.

In the first group with HCV single infection alone, there was no significant difference between the number of males (53%) and number of females (47%) as well as the mean age of males (

) and the mean age of females ( ) as shown in Table 5. Among the second group with dual hepatitis infection (HCV + HBV) the case was different from the first group. The numbers of males with dual infection were more than double (69%) as compared to females (31%). The mean age of females in this group was and that of males was

, as shown in Table 6. In the third group with triple hepatitis infection (HCV, HBV, and HDV) the observations were different. All of the patients in this group were males with mean age of .

3.7. Comparison of LFTs of Patients with Coinfection (HCV/HBV) and Single Infection (HCV)

LFTs of coinfected patients (HCV and HBV) were compared with HCV infected patients through independent -test. It was found that the LFTs of dually infected patients were significantly lower (closer to the normal values) as compared to the single infected patients with values 0.020, <0.05, and 0.052 for ALT, AST, and bilirubin, respectively, as shown in Figure 5.