Question:
Inaccuracy of Certain Commercial Enzyme Immunoassays
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This article is taken from Medline, and requires a membership to access, so I am posting a 'reprint' of it here, for anyone who is interested. Sort of technical, though. :roll:

Am J Clin Pathol 120(6):839-844, 2003. ? 2003 American Society of Clinical Pathologists, Inc

Discussion
The recent CDC treatment guidelines for sexually transmitted diseases recommend that type-specific testing based on gG be used for diagnosing genital herpes.[3] Several manufacturers in the United States sell HSV tests that are not based on gG-1 for HSV-1 antibody detection and gG-2 for HSV-2 antibody detection. To our knowledge, the accuracy of such tests has not been determined directly in characterized patient groups. To study the ability of currently available HSV serologic test kits to accurately diagnose genital herpes due to HSV-1 or HSV-2, we selected serum samples from patients with culture-documented first episodes of genital HSV-1 or genital HSV-2 and from patients after episodes of culture-documented recurrent lesions. These serum samples were tested with kits based on crude antigen mixtures from Zeus, DiaSorin, and Wampole. For comparison, the same serum samples were tested using gG-based ELISAs (for HSV-1 and HSV-2) from Focus. The Focus tests (brand name, HerpeSelect) have received US Food and Drug Administration approval for type-specific serologic testing of adults and pregnant women.

We used single serum samples obtained 28 to 90 days after symptomatic, lesional outbreaks of genital herpes. The non-gG-based tests gave inaccurate results in a high proportion of patients seroconverting to HSV-1 or HSV-2. The Zeus and Wampole tests were insensitive for detecting early HSV infection (57% and 77%, respectively). The DiaSorin HSV-2 test seemed to have acceptable sensitivity for early episodes of HSV-2 (93%-95%) at the expense of specificity: 3 (19%) of 16 patients with primary HSV-1 infection had false-positive results for HSV-2. When the predominant antibody was calculated for DiaSorin, none of the patients with nonprimary HSV-2 were diagnosed correctly, in accord with previous results of other non-gG-based tests.[9] One of the main reasons to use a type-specific serologic test is to confirm infection in newly symptomatic patients without a virologic diagnosis. In our opinion, the use of any of these tests to supplement or replace virologic testing of patients with genital herpes would produce confusion rather than clarity.

The non-gG-based tests generally were more sensitive for detecting antibody after a recurrent HSV-2 episode than for diagnosing first episodes. An exception was the DiaSorin kit when the predominant antibody method was used: only 33% (16/49) of HSV-2 positive serum samples were detected, albeit with high specificity (98%). The HSV-2 specificities for the Zeus, Wampole, and DiaSorin tests compared with Western blot were low (54%-83%). Thus, positive HSV-2 tests with these kits have a high probability of being falsely positive.

In contrast with diagnosing HSV-2, the ability to diagnose antibody after recurrent HSV-1 episodes was low for all methods except DiaSorin-1. The Zeus test had a high false-negative rate for recurrent HSV-1 (10/26 [38%]) and a high false-positive rate for HSV-2 results (5/26 [19%]) in this group. The Wampole test had a false-negative rate of only 7% for HSV-1 (2/28), but 4 (14%) of 28 patients with recurrent HSV-1 had false-positive results for HSV-2. All 15 of the incorrect HSV-1 diagnoses with the DiaSorin kit were false-positive results for HSV-2 antibody. The high false-negative rate for HSV-1 by the non-gG-based tests would result in continued diagnostic uncertainty, at best, for a large proportion of patients with HSV-1 genital herpes. On the other hand, the high number of patients with HSV-1 with false-positive HSV-2 results could lead to incorrect counseling and treatment for patients and their partners.

The Focus HSV-2 ELISA was positive in 39 (87%) of 45 first episodes of HSV-2. The false-negative Focus test results occurred in serum samples obtained 28 to 76 days after infection (median, 48 days); false-negative findings of the other tests occurred in serum samples obtained 30 to 90 days after infection (median, 43 days for the DiaSorin test and 45 days for the Wampole and Zeus tests). Previous studies using various gG-based tests including the Western blot have shown that individual patients seroconvert over a range of times.[4,16] In a recent study, we found that 93% of patients with primary HSV-2 episodes had a positive result in the HSV-2 Focus ELISA by 3 months (90 days), as did 73% of patients with nonprimary HSV-2 episodes. The median time to seroconversion shown by the Focus HSV-2 test was 21 days for primary infection and 23 days for nonprimary infection.[16] Similar results were noted in the present study: 93% (26/28) of patients with primary HSV-2 infection were positive by 90 days (median, 45 days; range, 30-90 days), as were 76% (13/17) of patients with nonprimary HSV-2 infection (median, 39 days; range, 28-76 days). The longer median times to apparent seroconversion in the present study likely were due to our testing only serum samples obtained at least 4 weeks after infection, thereby missing earlier seroconversion.

Of 23 patients with subclinical HSV-2 infections, only the Wampole and Focus tests accurately identified all 23 as having HSV-2 antibodies. However, the specificity of the Wampole test for HSV-2 in patients without HSV-2 infection was only 83%, whereas the specificity for the Focus HSV-2 ELISA was 96% against the Western blot and 98% against infection status. Thus, false-positive test results in patients without a history of genital herpes are almost certainly reduced by using a gG-based test.

Our study was limited by its emphasis on symptomatic patients; however, this emphasis permitted the clinical and virologic status, rather than solely a serologic assay, to function as the gold standard for test performance. In isolated patient groups, individual non-gG-based tests functioned adequately; however, overall, these tests had inadequate specificity for an accurate diagnosis of either HSV-1 or HSV-2 in almost all clinical scenarios. In the light of the consistently more accurate performance of the Focus gG-based ELISAs and the widespread availability of this test, we consider the use of non-gG-based tests unnecessary and inadvisable for diagnosis of genital HSV infection.

Acknowledgements

Sera and patient data were collected by Lawrence Corey, MD, and Anna Wald, MD, MPH.

Funding Information

The study was supported, in part, by NIH Herpes Program AI-30731, National Institutes of Health, Bethesda, MD.

Reprint Address

Dr Morrow: Virology, Room G815, 8G-3, Children's Hospital and Regional Medical Center, 4800 Sand Point Way, NE, Seattle, WA 98105.

Answer:
Results
Diagnosis of New HSV-1 Infections
Samples from 66 patients with newly acquired HSV-1 (n = 17) or HSV-2 (n = 49) were tested. Equivocal test results occurred with all 4 of the tests in a few patients. Although such a result might indicate early seroconversion, the incidence of equivocal results was not higher in patients with new infections than in those with established infections. Thus, equivocal results were censored from analysis of accuracy for detecting antibodies in newly infected persons.

Table 1 gives the numbers of serum samples with correct results for each kit by category of infection. HSV-1 antibodies were present in most patients with primary HSV-1 according to the DiaSorin test (15/17 [88%]) and in more than half according to the Wampole test (10/16; 63%). The Zeus test was positive for HSV-1 antibody in only 3 (19%) of 16 patients with HSV-1. The gG-based Focus test was positive for HSV-1 in 13 (81%) of 16. Incorrect results of the Zeus, Wampole, and Focus tests were all false-negative, while the DiaSorin test showed antibodies to HSV-2 and HSV-1 in 1 of 17 and gave false-negative results in 1 of 17 by DiaSorin-1. When DiaSorin-2 results were used, 1 of 16 samples had false-negative results, and 2 of 16 were positive for HSV-1, but also for HSV-2.

Diagnosis of New HSV-2 Infections
HSV-2 infection was reflected by positive HSV-2 test results in most patients with primary HSV-2 by the Focus test (26/28 [93%]), while in 1 patient (4%), only HSV-1 antibody was detected. Predominant HSV-2 antibodies were detected by the DiaSorin-1 method in 22 (76%) of 29 patients; however, 6 patients (21%) were mistakenly given a diagnosis of HSV-1 with this method. DiaSorin-2 results were positive for HSV-2 in 27 (96%) of 28; however, 23 (85%) of the 27 serum samples were, in addition, falsely positive for HSV-1. The Wampole and Zeus tests detected far fewer HSV-2 seroconversions: 19 (70%) of 27 and 12 (50%) of 24, respectively (Table 1).

Nonprimary infections are first episodes of HSV-2 genital herpes that occur in patients with preexisting HSV-1 antibodies. Of 20 such patients, the Zeus and DiaSorin-2 tests showed high proportions of positive results for HSV-2: 19 (95%) of 20. Wampole test results were positive in all 18 serum samples that had definitive HSV-2 results. In contrast, the DiaSorin-1 predominant antibody method detected none of the 20 HSV-2 seroconverters (Table 1). The Focus ELISA showed HSV-2 seroconversion in 13 (76%) of 17.

Overall, correct serologic results (detecting antibody of the infecting type) were obtained in 57% (34/60) to 92% (59/64) of serum samples from first episodes of HSV-1 or HSV-2 when crude antigen-based tests were used. Correct serologic results were obtained in 85% (52/61) of first episodes when the gG-based Focus ELISAs were used (Table 1).

Diagnosis of Recurrent HSV-1 Episodes
All serum samples from culture-documented HSV-1 recurrences had HSV-1 predominant antibody to HSV-1 shown by DiaSorin-1. However, 15 patients had detectable antibody to HSV-2 so that when the predominant antibody calculation was not applied (DiaSorin-2), almost half of these HSV-1 subjects seemed to have dual antibody (Table 2). The Zeus test was positive for only HSV-1 in 11 (42%) of 26 of those with recurrent HSV-1, while 5 (19%) were positive for both HSV-1 and HSV-2 and 10 (38%) were negative for both HSV-1 and HSV-2. The Wampole test detected only HSV-1 antibody in 22 (79%) of 28 patients with only HSV-1 infection but was positive for both types of antibody in 4 (14%) and negative for both HSV-1 and HSV-2 antibodies in 2 (7%). The Focus test was positive for HSV-1 only in 27 (90%) of 30 subjects with recurrent HSV-1 but was negative for both HSV-1 and HSV-2 antibody in 2 (7%) and positive for both in 1 subject (3%).

Diagnosis of Recurrent HSV-2 Episodes
All HSV-2 kits detected antibodies to HSV-2 (Table 2) after recurrent episodes. However, if DiaSorin-1was used, only 33% (16/49) had predominant HSV-2 (Table 2).

Asymptomatic Patients With HSV-2 Antibody
The CDC has suggested that type-specific serologic testing be used to identify patients who lack a definitive clinical diagnosis of genital herpes.[3] Partners of persons with genital herpes also may benefit from testing to ascertain their infection status if they have not been diagnosed.[15] We tested 23 patients without a history of HSV-2 but with HSV-2 antibody shown by Western blot analysis who were shown to have true-positive results for HSV-2 infection by later polymerase chain reaction or culture of genital secretions.[14] All 23 had positive results for the HSV-2 antibody with the Focus and Wampole tests, while 22 (96%) of 23 had positive results with DiaSorin-2, and 22 (96%) of 23 had positive results with the Zeus test for the HSV-2 antibody. However, when DiaSorin-1 was applied, HSV-2 antibodies were identified correctly in only 5 (22%) of 23 serum samples.

Sensitivity and Specificity of ELISAs
To compare test performance of these kits against a single serologic standard, we calculated the sensitivity and specificity for each test against the results of Western blot testing without regard for the clinical status of the patient (Table 3). Sensitivity for HSV-1 antibody was high for the Wampole (91%) and both DiaSorin test methods (94%-98%). However, the specificity was low for each test (8%-70%), indicating a high probability of falsely positive HSV-1 test results with these kits. The Zeus test had low sensitivity (77%) and low specificity (53%) for HSV-1.

The gG-based Focus test had 83% sensitivity and 90% specificity for HSV-1 (Table 3). The false-negative HSV-1 results occurred in 3 patients with primary HSV-1 infection, in 1 patient with nonprimary infection, in 2 patients with recurrent type 1, and in 12 of 31 culture-documented HSV-2 recurrent episodes in patients with dual antibody shown by Western blot. The false-positive HSV-1 Focus results occurred in 3 patients with primary HSV-2 infection and in 2 patients with recurrent HSV-2, with HSV-2 shown only by Western blot. Serum samples from these subjects were barely above the cutoff for positive in the Focus HSV-1 test (median, 2.5; cutoff, 1.10).

The HSV-2 test sensitivities were 88%, 92%, and 96% for the Zeus, Wampole, and DiaSorin kits, respectively (Table 3), but the associated specificities were low (81%, 83%, and 54%, respectively). The predominant antibody method for DiaSorin detected only 43 (38%) of 112 serum samples with HSV-2 antibody shown by Western blot, but specificity was high (98%). The gG-based Focus test was 98% sensitive and 96% specific against the Western blot test. One of the 2 falsely positive Focus HSV-2 test results was in the nonprimary HSV-2 group; Western blot had not shown seroconversion in this serum sample; thus, by clinical criteria, these results were not falsely positive for HSV-2 but, rather, falsely negative by Western blot. For this group of patients, the Focus HSV-2 ELISA had 98% specificity when clinical data were considered.

Overall concordance with Western blot results is shown for each test in Table 3. This is a highly rigorous test because both the HSV-1 and HSV-2 test results had to be in accord with the respective Western blot result to be counted as concordant. About half (52%-63%) of the non-gG-based ELISA results were concordant with Western blot. The Focus test was concordant with Western blot in 83% of the serum samples.

http://www.medscape.com/viewarticle/465627_4

http://www.medscape.com/content/2003...65627_tab.html

Answer:
From http://www.fda.gov/fdac/features/2002/202_herp.html

"In the past several years, the FDA has cleared three blood tests that accurately determine if a person is infected with HSV-1 or HSV-2. The HerpeSelect ELISA Kits and the HerpeSelect Immunoblot Kit made by Focus Technologies of Herndon, Va., detect both types. The POCkit Rapid Test made by Diagnology Inc. of Cary, N.C., detects HSV-2 only. (For more information, see "Herpes Blood Tests".)

Another blood test is the Western Blot. Although not 100 percent accurate, the Western Blot is considered the "gold standard" of blood tests and is used to determine the accuracy of other herpes blood tests that are developed. The University of Washington is the premier institution for performing and interpreting the test. (See "For More Information" to find out how you can have your blood tested with a Western Blot.)

Many older FDA-cleared blood tests for herpes are still on the market, and many labs use these tests because they are widely available and inexpensive. Although they may be labeled type-specific (can determine whether the infection is HSV-1 or HSV-2), they are not reliable, says Simms.

But it's difficult for people to make sure they are getting one of the newer, accurate tests, says Wald. Doctors and even lab workers may not know what test they're using. "The patient needs to ask, but it's a very tall order. It will take a significant amount of work on their part and phone calls to the lab themselves."

This is where a herpes support group can help, says Adams, who facilitates a group called HELP of Washington. "We keep a list of doctors who are up to speed, knowledgeable, and know what the right tests are."
I called the Univ of Washington to inquire about the Western Blot. THey told me they could not recommend any particular doctor in my area of Southern Calif. They are the only lab in the country to process the Western Blot exam. They will send any MD the kit that it requires (but he may already have it). It's basically a red test tube and a cold pack to use, plus protective styrofoam packaging required by overnight courier companies for blody fluid safety. Your doctor in your state has to order the test, submit the paperwork and his or her nurse or tech can draw the blood. The Western Blot costs around $110.

http://depts.washington.edu/herpes/

If you are not in the Seattle area, you can have your blood tested with a Western Blot at the University of Washington Virology Lab. To do this, have your health care provider call #206-598-6066 to request the HSV Type-Specific Serology information packet.
Hope this helps someone -