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Clinical Reviews


New screening tests: HSV, CMV, HBV, HCV, parvovirus, and HIV

Tips on choosing the right tests and getting valid results

October 2005 · Vol. 17, No. 10

Technology makes many things possible, but not without imposing new responsibilities. When it comes to viral infections, diagnosis through serology testing, antigen assays, or amplification techniques such as polymerase chain reaction (PCR) now is possible for a number of diseases, including:

  • herpes simplex virus (HSV),
  • cytomegalovirus (CMV),
  • hepatitis B and C viruses (HBV, HCV),
  • parvovirus B19 (B19), and
  • human immunodeficiency virus (HIV).

The responsibilities that come along with this ability: keeping up to date and selecting the most sensitive and specific test possible. This article reviews the latest tests and offers advice on their use in detecting 6 viruses.

How to pick the right test

When a patient’s presentation suggests viral infection, when something in her history raises a red flag, or when she reports possible exposure to a virus, the right test is critical to the diagnosis. The right test also helps prevent false positives and avoid confusion—but which test is best?

Which immunoglobulins matter?

Many of us have been taught that immunoglobulin M (IgM) correlates with acute infection, but that is not necessarily the case. Because of its high molecular weight, IgM is found most commonly in the intravascular compartment and is not transported to the fetus. IgM usually becomes apparent early during the course of infection. It has a half-life of 10 days and usually—but not always—regresses to undetectable levels over a few months.

The misconception that IgM is found only in acute infection and disappears within 3 months causes many clinicians to test for it and to misinterpret the results. In many cases, IgM fails to develop after acute infection. In others, it may persist for as long as 2 years after primary infection. It also can be detected with recurrent or reactivated infections.1

Immunoglobulin G (IgG) has a longer half-life (21 days) and is the most common immunoglobulin in humans. It is found in tissues and serum and readily crosses the placenta. It can be detected shortly after acute infection, exhibiting a steep rise and fall over several weeks after primary infection. IgG also is a sign of past infection.1

Telltale sign of acute infection

It now seems clear that an IgG antibody produced within the first months after primary infection binds to its antigen poorly. After this initial period, the IgG binds with greater intensity (ie, higher avidity) to that specific antigen (virus). Assays that measure this binding intensity are called avidity assays and are expressed as a percentage of IgG bound to the antigen after treatment with denaturing agents.

Avidity assays have been developed and studied for a variety of viruses.2 The detection of low-avidity IgG can be considered a more reliable sign of acute infection than IgM.

Herpes simplex virus

SERENA’S CASE

Monthly irritation and a vulvar lesion

Serena, 22, complains of irritation and pruritus that precede her period each month. She has tried treating herself with over-the-counter and prescription antifungal medication, without much relief. She presents to your office as an add-on patient and reports that the irritation started 1 day ago and usually lasts 7 days. Physical examination reveals a small area on the left vulva that is inflamed, with 2 small fissures. You obtain a vaginal pH, but it is normal, and there are negative findings on the wet prep.

In view of Serena’s history of recurrent symptoms and atypical lesions, recurrent herpes is a likely diagnosis, so you culture the lesion and order IgG type-specific serology for HSV 1 and 2. The culture is negative, but serology is positive for the HSV-2 antibody. Thus, serology confirms genital herpes.

Although Serena’s culture was negative, false-negative cultures are common with HSV, and serology testing usually is necessary to make the diagnosis of recurrent genital herpes.

HSV-2 is widespread: About 1 in 4 adults is infected. Of these, fewer than 1 in 10 is aware he or she has the virus. Thus, it makes sense to test for HSV-2 when physical findings suggest that it may be present.

Thanks to type-specific serology tests, HSV 1 and/or 2 infection can be diagnosed with confidence.3 Type-specific tests determine the IgG antibody response to an envelope glycoprotein (gG) of HSV. In the diagnosis of primary infection, the test detects seroconversion from a negative to a positive titer. The earliest time for antibody production is 3 weeks after infection—but 8 to 12 weeks should elapse prior to testing for seroconversion.4

The US Food and Drug Administration (FDA) has approved an office test for HSV-2 antibodies. The biokitHSV-2 rapid test (biokit USA, Lexington, Mass) requires blood or serum—though statistical analysis indicates that serum demonstrates higher sensitivity with this assay than does capillary whole blood—and can be performed in any outpatient setting, with excellent sensitivity and specificity.

Flaws of IgM testing and viral cultures

IgM testing for HSV is not advised, as the presence of IgM does not correlate with acute disease. As for cultures, laboratories have been slow to change from HSV cultures to PCR testing from suspected HSV lesions. Because viral cultures for HSV have extremely low sensitivity, only positive results are clinically useful.

Primary infection yields a higher positive culture rate because of increased viral shedding. In contrast, recurrent disease, with its low levels of viral shedding, yields significantly lower positive culture rates.5

Cytomegalovirus

CATHY’S CASE

Flu-like symptoms and plans to conceive

When Cathy, 28, comes in for her annual visit, she reports that she has been experiencing exhaustion, chills, and body aches for several weeks and wonders whether she might have mononucleosis. She also mentions that she and her husband are trying to have a child. She recently started a new job in a day care center, working directly with young children. You decide to test her for the Epstein-Barr virus. Since she is planning to conceive, you also test her for CMV, and the test is positive.

How do you counsel Cathy?

CMV infection usually is diagnosed after clinical findings in the individual or fetus (by ultrasound examination) suggest this infection, or when the clinician suspects it for other reasons.

In young adults, primary CMV infection can cause flu- or mononucleosis-like symptoms such as extreme fatigue, fever, chills, and/or body aches, though they generally resolve within several weeks without further morbidity. However, CMV can cause grave illness—even death—in immunocompromised patients, who may continue to experience the disease intermittently after the first outbreak. CMV also can seriously impair development in infants who are infected in utero. The vast majority of these infants appear normal at birth and only later exhibit problems.

Cathy should be counseled to postpone conception until the virus clears, to avoid jeopardizing her infant’s health.

Testing for CMV during pregnancy

Diagnosing primary infection in pregnancy can be difficult. In the United States, we do not screen for CMV prior to pregnancy, except for special circumstances such as Cathy’s, because the virus is widespread and causes few problems in healthy, nonpregnant individuals. This lack of screening renders the best diagnostic method (ie, seroconversion, or finding CMV IgG antibody in a previously seronegative woman) impossible in most cases. Rather, the clinician usually orders IgG and IgM antibody tests, hoping they will help determine whether a primary infection is present.

In these situations, detection of the IgM antibody during pregnancy is vital to diagnose primary infection.

IgG avidity test clarifies IgM findings

The enzyme-linked immunosorbent assay (ELISA) and the capture ELISA assay are used to detect CMV IgG and IgM antibodies. However, the ELISA for IgM can yield false-positive results due to the presence of:

  • IgG (especially at high titers),
  • rheumatoid factor of the IgM class (IgM-RF),
  • reaction between IgM antibodies and cellular antigens, and
  • primary Epstein-Barr viral infection, which can stimulate production of CMV IgM antibody in CMV-immune individuals.

Thus, a positive IgM finding in the serum of a pregnant woman can be caused by acute primary CMV infection, the convalescent phase of a primary CMV infection, or simply persistent IgM antibodies.6,7

That’s where the IgG avidity test comes in. It can help confirm and clarify the clinical significance of finding the IgM antibody and distinguish between primary and recurrent CMV infection. Using the IgM antibody test together with the IgG avidity test increases the likelihood of a correct diagnosis.

A positive IgM with a low avidity index is highly suggestive of a recent (less than 3 months) primary CMV infection.

Standardization of the IgG and IgM assays and the IgG avidity assay is urgently needed to reduce the likelihood of incorrect diagnosis and unwanted intervention in otherwise normal pregnancies.

Further testing can confirm fetal transmission

If primary CMV infection is detected in a pregnant woman, accurate prenatal diagnosis is possible to determine whether the infection has been transmitted to the fetus.

Sophisticated tests such as PCR make in utero diagnosis possible. In fact, PCR remains the most accurate means of diagnosing CMV, with sensitivities ranging from 80% to 100% in prospective studies of gravidas infected with cytomegalovirus.8

Hepatitis B

LIAN’S CASE

Pregnant, with no history of vaccination

Lian, 25, was born in Taiwan and has been in the United States 6 years. She presents for her first prenatal visit at 12 weeks’ gestation and reports no medical history, including no vaccination against hepatitis. This is her first pregnancy. Because testing for hepatitis B is routine in pregnancy, she undergoes a hepatitis B surface antigen test (HBsAg), which is positive.

What is your next step?

Lian’s positive test suggests she is a carrier for HBV. Additional testing can clarify her status and direct treatment and prevention.

Unlike some of the other viruses covered in this article, HBV has been well studied, and the identifying proteins have been described. This information has made it possible to produce sensitive and specific tests that can determine whether a patient is infected, a carrier, or immune to HBV.

Screening for carrier status is universal among gravidas in the United States. Standard ELISA assays have been developed for several HBV proteins as well as the antibody.

Signs of a carrier

The carrier state for HBV is determined by measuring HBsAg, the envelope protein of the virus, which circulates freely in the serum after acute infection (1–10 weeks) and in carrier patients. A woman is a carrier if HBsAg persists 6 months after primary infection.

The nucleocapsid core protein of the hepatitis B core antigen (HBcAg) and its secretory product e antigen (HBeAg) are produced after viral multiplication. There is no assay for HBcAg, but the hepatitis B surface antibody (HBsAb) develops after acute HBV infection. In the carrier state, no HBsAb is detected. However, HBcAb is detected in virtually all infected individuals, usually 4 to 8 weeks after infection.

HBeAg production is a sign of active production of the virus in the liver and is associated with more severe disease. It is usually cleared by 16 weeks post-infection. The antibody to the e protein (HBeAb) occurs more than 16 weeks after infection, and is a finding that suggests less severe disease.

The only finding in the serum of patients successfully vaccinated against HBV is HBsAb.9,10

How to qualify infection status

In Lian’s case, further testing should include liver function and other markers of HBV infection. The presence of hepatitis B e antibody (HBeAb) and e antigen (HBeAg) can define infection and the risk of transmitting the virus to the newborn. For example, when HBeAg is present, the patient has a higher risk of transmitting the virus to other people, including her fetus. When HBeAb is present, the patient has a lower risk of transmitting the virus to other people, including her fetus.

Continued...
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