Herpes Simplex Virus (HSV) Diagnostics 2025: An Overview

TRANSCRIPT for Webinar with the title: Herpes simplex virus (HSV) diagnostics 2025: An overview

0:02
Hello everyone.

0:03
Welcome to our webinar on herpes simplex diagnostics.

0:06
My name is Bob Jones and I'm the Medical, the moderator for today's session and the

0:10
Senior Medical Director for Infectious Disease at Quest Diagnostics.

0:14
I've been with Quest for over 8 years after spending 25 years in clinical

0:19
infectious disease practice.

0:21
We have two outstanding speakers today who will review the current state of

0:26
herpes simplex virus diagnostics.

0:29
Our first speaker is Dr Beth Marlowe.

0:31
Beth is the Executive Scientific Director and leads research and development for

0:36
Quest Diagnostics, Infectious Diseases and Immunology.

0:39
She received her PhD from the University of Arizona followed by a postdoctoral

0:45
fellowship at UCLA.

0:47
Before joining the team at Quest, she worked for Genpro Kaiser Permanente

0:51
and held the position of Global Director of Medical Affairs at Roche Molecular

0:57
Systems.

0:57
She's a diplomat of the American Board of Medical Microbiology and has served on

1:03
several editorial boards.

1:06
Our second speaker is Dr Susan Realegeno.

1:10
She's currently a scientific director at Quest Diagnostics in San Juan Capistrano,

1:15
CA in the infectious diseases department.

1:18
She obtained her PhD from UCLA in microbiology,

1:21
immunology and molecular genetics and completed her clinical microbiology

1:26
fellowship at UCLA.

1:28
Susan is board certified in medical microbiology and currently overseas

1:33
serology testing and supports areas of molecular testing and research and

1:38
development at Quest Diagnostics.

1:42
Thank you, ladies, and looking forward to a great

1:44
conversation.

1:47
Thank you, Dr Jones, for that gracious introduction.

1:54
Thank you for attending our webinar today.

1:57
The objectives of our webinar today are to provide an overview of herpes simplex

2:03
virus or HSV, review the testing that's available for

2:07
HSV, highlight the current landscape for HSV

2:10
testing as well as diagnostic considerations for genital herpes as

2:15
outlined in the CDC guidelines, and discuss the diagnostic limitations

2:20
around that testing.

2:27
Herpes simplex virus is a member of the herpesviridae family.

2:32
There are 9 herpes virus types that are known to primarily infect humans,

2:36
and at least five of those are widespread among humans.

2:41
Herpes simplex virus 1 and herpes simplex virus 2 are the focus of today's talk.

2:46
HSV is the leading cause of genital ulcer disease worldwide,

2:51
predominantly due to HSV-2 and to a lesser extent, HSV-1.

2:57
It can cause chronic, lifelong latent infection with

3:00
asymptomatic or symptomatic reinfection.

3:03
As illustrated on the slide, these are large, enveloped,

3:07
double stranded DNA viruses.

3:10
The envelope makes up the outermost part of HSV and consists of a lipid bilayer

3:15
membrane which is studied with an array of 12 distinct types glycoproteins.

3:22
The glycoproteins are required for viral entry and elicit neutralizing antibodies.

3:27
It's the differences in glycoprotein G between HSV-1 and HSV-2 that have been

3:33
utilized in the development of HSV type specific serologic assays that Dr

3:38
Realegeno will be discussing later in the talk.

3:44
HSV-1 mostly spreads through oral contact and can cause infections around the mouth

3:49
such as cold sores, or it can cause genital herpes,

3:52
which we've seen an uptick of in the last few years due to changes in sexual

3:56
behavior.

3:58
HSV-2 spreads through sexual contact and causes genital herpes.

4:09
Let's take a look at the epidemiology of HSV according to the WHO.

4:14
Globally, an estimated 3. 8 billion people under the age of 50 have

4:19
HSV-1 infection, the main cause of oral herpes,

4:23
while an estimated 520 million people between the ages of 15 and 49 worldwide

4:29
have HSV-2 infection, the main cause of genital herpes.

4:34
Most HSV infections are asymptomatic or unrecognized.

4:39
Symptoms of herpes include painful blisters that can occur over time.

4:43
Genital herpes is among one of the most prevalent sexually transmitted diseases

4:47
in the United States.

4:49
Although both HSV-1 and HSV-2 can potentially cause genital infection,

4:54
most causes of genital herpes in the US are caused by HSV-2.

5:00
In 2018, there was an estimated 18. 6 million people between the ages of 18

5:05
and 49 years of age living with genital herpes caused by HSV-2,

5:10
an additional several millions others living with HSV.

5:16
In 2018 alone, it was estimated that almost 600,

5:19
000 persons 18 to 49 years of age had newly acquired HSV-2 infections in the

5:23
United States.

5:25
Since genital herpes is not a nationally notifiable condition,

5:29
the true prevalence of persons living with genital herpes and incidents of new

5:33
cases is difficult to determine.

5:36
The best seroprevalence data in the US is through the NHANES or National Health and

5:41
Nutrition Examination Survey.

5:44
Based on this data, which was collected between 2015 2016,

5:49
the prevalence rate was estimated at 12. 1%.

5:55
As you see from the graph, there's been a significant decline in

6:00
HSV-2 seroprevalence trends from 1999 - 2000,

6:04
where it was 18% to the more recent estimates of 12%.

6:09
Of note are the HSV-2 differences in gender,

6:13
where prevalences were nearly twofold higher among females compared to males.

6:20
As expected, seroprevalence notably increases with age

6:24
and disparities have been identified in HSV-2 prevalent rates with the highest

6:29
rates in non Hispanic black individuals and these disparities were persistence

6:33
among the time frames.

6:35
As seen in the graph, the clinical manifestations of genital

6:41
herpes vary significantly when comparing first clinical episodes and recurrent

6:50
outbreaks.

6:52
The severity, frequency of clinical manifestations,

6:56
and recurrence rates are influenced by the virus type as well as the immune

7:02
status of the host.

7:05
Persons who are serial positive for HSV-2 and not aware of their genital infection

7:10
account for the majority of genital HSV infections.

7:14
Initial HSV-2 genital infections in persons with a previous HSV-1 antibodies

7:20
are often asymptomatic.

7:23
Approximately 80% of people positive for HSV-2 have never received a diagnosis for

7:31
genital HSV evaluation.

7:33
Of people who have been undiagnosed with genitals,

7:37
HSV-2 have demonstrated that an estimated 20% have true asymptomatic infection or

7:43
they have a current of genital lesions and locations that maybe were not easily

7:49
identified, such as on the cervix.

7:53
The remaining 60% have mild, unrecognized symptoms.

7:58
Additionally, some symptoms caused by HSV may be

8:00
mistaken for other disorders such as vaginitis, hemorrhoids,

8:03
or allergic reactions.

8:06
When asymptomatic, HSV-2 seropositive individuals receive

8:10
education about the merit of symptoms caused by genital HSV infections.

8:15
It's been reported that approximately 2/3 will subsequently identify symptoms that

8:21
are consistent with genital HSV-2 infection.

8:30
The first clinical episode of HSV refers to the initial symptomatic occurrence of

8:35
herpes.

8:36
This can be an ulcer either on the oral, pharynx, skin, or genital mucosa.

8:42
Primary infection is defined as the first infection with either HSV-1 or HSV-2,

8:46
with the absence of antibodies to either.

8:50
Symptoms include bilateral genital ulcers, ulcers which are painful, itching,

8:56
dysphoria with vaginal or urethral discharge,

9:00
and tender equinal adenopathy without antiviral therapy.

9:05
Lesions can last two to three weeks with the evolution of lesions from a vascular

9:11
pustule to wet ulcers to dry crust.

9:14
Recurrent symptomatic herpes refers to the recurrence in a setting of known

9:20
diagnosis of genital HSVs.

9:23
This can happen with oral HSV.

9:26
Recurrent symptoms are characterized by mild localized symptoms that typically

9:31
resolve within two to five days after onset of prodromal symptoms like

9:36
localized tingling or burning due to HSV traveling along the nerve axons.

9:42
That's This is common and begins typically 12 to 24 hours before the 1st

9:46
lesions appear.

9:51
The frequency of symptomatic genital herpes reactivation to HSV-2 decreases

9:56
from a medium of four to five recurrences for the first year to three to four

10:01
recurrence in subsequent years.

10:04
For genital HSV-1.

10:05
The frequency of symptomatic genital herpes is a medium of one year,

10:09
one per year during the first year, and typically no outbreaks are seen in

10:14
subsequent years.

10:16
Reactive symptomatic infection is higher in women than in men and in persons who

10:21
experience prolonged symptoms associated with primary infection.

10:26
There are complications of HSV infection, and this includes karyentitis and social

10:31
nervous infections.

10:32
CNS infections, such as a aseptic meningitis,

10:35
account for approximately 10% of all cases of aseptic meningitis,

10:40
with most cases caused by HSV-2 infection.

10:45
HSV encephalitis is typically caused by HSV-1.

10:49
Uncommon complications of HSV include benign recurrent lymphatic meningitis,

10:55
also known as Mollaret's meningitis.

10:59
Neonatal HSV infection can also occur and is defined as HSV infection that develops

11:06
in a newborn during the first 28 days after birth.

11:11
It is a rare complication and occurs in about one in 3000 deliveries in the US.

11:16
The risk of transmission is highest in persons who acquire primary genital

11:20
herpes infection at or near the time of delivery.

11:24
Among neonatal transmission, 85% occur at delivery, 5% in utero,

11:30
and 10% postpartum.

11:35
Transmission of HSV usually occurs through close contact and direct contact

11:41
with people who are shedding virus at the mucosal or epithelial surface or in

11:46
genital or oral secretions.

11:50
The transmission of HSV too often involves asymptomatic shedding of the

11:56
virus in a person unaware that they have HSV.

12:00
As discussed earlier, HSV can be transmitted perinatally from

12:04
mother to child at the time of delivery, and fomite transmission of HSV is

12:10
unlikely, although auto inoculation can occur due

12:13
to viral particles from genital sites or other eucocutaneous sites from fingers,

12:19
usually during primary infection.

12:25
Clinical diagnosis of HSV can be challenging because many people are

12:30
asymptomatic and undiagnosed.

12:33
The clinical diagnosis of genital herpes should be confirmed by laboratory testing.

12:38
This slide is an overview of the generally available diagnostic test.

12:43
Diagnostics include molecular techniques such as PCR, cell culture, serology,

12:50
antigen detection and cytology.

12:54
The recommended test for direct detection of HSV and clinical symptoms samples are

13:00
molecular assays or cell culture.

13:03
Molecular methods are the most sensitive method of the two.

13:07
For serology, a 2-step type specific serology is

13:11
recommended for detecting antibodies while antigen and cytology methods are

13:16
not recommended due to low sensitivity.

13:19
Dr Realegeno

13:21
will go into further detail about the recommended diagnostics in the second

13:25
half of this webinar.

13:29
In terms of prevention and treatment, today there are no vaccines against HSV-1

13:35
or HSV-2.

13:36
Prevention includes minimizing close contact,

13:38
especially when lesions are present.

13:41
People should make sure that they practice good hygiene and wash their

13:44
hands and don't touch their eyes or mucosal area if they are infected.

13:48
If intercourse is involved, barrier protection is recommended and

13:52
caesarean delivery for mothers with active lesions.

13:56
There is antiviral treatment that's available for HSV.

14:00
HSV antiviral therapy includes 3 available nucleoside analog medications,

14:06
acyclovir, galicyclovir, and famciclovir.

14:11
Oral therapy is recommended when lesions occur,

14:14
and IV acyclovir treatment is available for more invasive infections,

14:18
such as those seen with central nervous infections or neonatal infections.

14:24
HSV may become resistant to first line and second line treatments,

14:27
although treatment susceptibility testing may be useful to help guide treatment

14:32
when patients aren't responding to medications.

14:35
This testing is available by phenotypic or genotypic susceptibility testing and

14:40
available through reference laboratories.

14:44
The history of HSV in acyclovir therapy is an interesting one if you've ever read

14:50
Ina Park's book Strange Bedfellows, which I highly recommend if you're

14:55
interested in a fascinating trip down history around STIs.

15:00
Is in 1982, the first FDA approved approval for

15:04
acyclovir became available and this was an acyclovir ointment for the treatment

15:10
of genital herpes.

15:12
It was a first of its kind antiviral drug able to suppress the virus but not

15:17
eradicate it and it later served as a prototype for antiviral drug development

15:21
against HIV.

15:24
Ironically, the marketing department predicted that

15:26
the sales of acyclovir would be modest and peak at about 10 million a year.

15:31
Well, this might seem like a good return on

15:32
investment.

15:33
It really dwarfed in comparison to other blockbuster drugs at the time like Zantac,

15:38
which had annual sales of $2 billion.

15:41
Ironically, it was a Time magazine article that

15:44
unexpectedly helped the cause around the uptake of acyclovir in August 1982 when

15:49
Time published a story entitled Today's Scarlet Letter with a giant H across the

15:54
cover of the magazine.

15:57
While Time never mentioned acyclovir or its manufacturer,

16:00
the public awareness of herpes grew as well as the interest in herpes treatment

16:04
and acyclovir was the only product on the market.

16:07
As a result, the media interest rose and from 1982 to

16:12
83, more than 1000 news articles were written

16:15
about the drug manufacturer and acyclovir.

16:19
Now this is really fascinating if you think about the fact that this was before

16:23
the age of digital communication.

16:25
Everything was still in print.

16:28
The ointment formulation of acyclovir was only approved for initials herpes

16:32
outbreaks and only reduced pain from outbreaks in men.

16:36
Acyclovir was later formulated into a capsule to be taken orally and speed up

16:40
the healing sores of initial infections and recurrent outbreaks.

16:45
By the end of January 1985, the FDA approved acyclovir capsules for

16:49
both men and women with herpes to be used for initial and recurrent outbreaks.

16:55
This was also around the time that direct consumer marketing was in its infancy.

17:00
The FDA did not explicitly forbid it, but it did call for a voluntary

17:04
moratorium on the practice between 83 and 85.

17:08
However, by 1986, the FDA lifted that moratorium and a

17:11
series of ads are run, which, by the way, had no mention of the product acyclovir

17:17
or the claims for the drug.

17:19
The ads increase attention to the disease and encourage consumers to get medical

17:23
attention.

17:24
Despite the subtlety of the ads of acyclovir capsules were just a big

17:28
success and shattered all previous predictions of sales and they reached

17:32
over 1 billion annually and eventually accounted for 1/3 of Burroughs welcomes

17:37
sales and half of its operating profits.

17:40
One of the pharmacologist Trudy Elion, was awarded the Nobel Prize in 1988 due

17:46
in part to the discovery of acyclovir.

17:49
The process.

17:50
The success of the drug later caught the attention of the pharmaceutical giant

17:55
Glaxo, which was acquired by the company in 85

17:57
for $14 billion.

18:00
Simultaneously, improvements in diagnostic testing were

18:03
occurring, which were allowing more diagnosis to be

18:06
made.

18:07
In the early 1980's, the diagnosis of herpes could only be

18:10
confirmed with viral culture as well as other direct methods such as DFA

18:15
techniques to detect HSV from active lesions.

18:19
This meant that only patients who had active symptoms could be certain of

18:23
diagnosis.

18:25
By the end of the 80's, the first herpes antibody test,

18:28
which could distinguish HSV-1 and HSV-2, began to hit the market and allowed

18:32
patients to begin to get blood tests regardless of the symptoms.

18:38
As we discussed earlier, undiagnosed, undiagnosed HSV makes up the large

18:43
portion of HSV infection due to asymptomatic and undiagnosed infection.

18:48
Today in 2026, we continue to see an evolution of

18:53
clinical diagnostics for HSV.

18:56
It's pretty amazing when you think about the fact that HSV has been overshadowed

19:01
for years by other STI's like syphilis and gonorrhea.

19:05
It wasn't even mentioned in the best selling sexual manuals in the 60s that

19:09
discussed venereal diseases.

19:11
In the 1977 VD handbook, which would by the way was a Canadian

19:16
manual, it devoted 14 pages to gonorrhea and two

19:19
to genital herpes.

19:21
Despite humans living with strains of HSV for hundreds of thousands of years and

19:26
descriptions of genital herpes appearing in scientific writing starting around

19:31
1736, the lack of attention was attributed to

19:34
the lack of treatment options for people with a diagnosis.

19:38
I think this further highlights the importance of having available

19:42
appropriate diagnostic testing.

19:44
And with that, I will hand it over to Dr Susan Realegeno

19:47
who will provide a more in-depth discussion of HSV diagnostics that are

19:51
available today.

19:53
Thank you, Dr Marlowe for that great introduction

19:56
and interesting history behind HSV.

19:58
It really brings attention to how important this infection is and why we're

20:03
talking about it.

20:04
So in this part of the talk, I'm going to focus on laboratory testing

20:08
like Dr Marlowe mentioned, and then I'll further go into utilization.

20:12
Limitations of certain types of testing, especially serological methods that are

20:17
used for genital herpes.

20:20
A diagnosis of HSV infection can be achieved through clinical evaluation and

20:24
further confirmed with laboratory testing using an appropriate specimen type.

20:29
So there are direct and indirect methods including biological tests and antibody

20:33
detection assays respectively.

20:36
These the type specific nucleic acid amplification tests such as PCR or TMA

20:41
are the most sensitive and preferred method for detection of HSV during acute

20:46
infection and also in patients with recurrent symptomatic infection.

20:51
There are other methods but they have lower sensitivity and that includes viral

20:56
culture and they may require additional testing for HSV specific typing.

21:01
Viral culture is also needed if phenotypic antiviral susceptibility

21:05
testing is performed.

21:07
Antigen and cytology examination overall also have lower sensitivity and are not

21:12
recommended for direct detection, but they may still be available at

21:16
certain institutions.

21:17
Finally, we have antibody detection tests which

21:21
include type specific HSV-1 and HSV-2 IgG test.

21:24
They may be used to help evaluate previous exposure.

21:28
However, serological testing when supporting

21:31
diagnosis of genital herpes is not recommended as a primary method,

21:35
but is recommended in certain scenarios due to the analytical limitations of

21:40
currently available methodologies.

21:47
When looking at the different clinical manifestations and what the preferred

21:52
laboratory method for confirming infection,

21:55
HSV NAAT is preferred across the board with several manifestations including

22:00
neonatal herpes, which requires a swab of multiple sites,

22:03
CNS infection, ocular involvement, and genital or herpes.

22:08
Collecting the appropriate specimen type is really important to ensure that you

22:12
have high sensitivity and specificity for these tests.

22:15
Viral culture is still listed in the guidelines as one of the acceptable

22:20
methods for recovering HSV from surface specimens and neonatal infection,

22:25
but it's not recommended for CSF due to the low viral yield and the risk of false

22:30
positives.

22:31
Genital herpes, in addition to having NAAT as the

22:35
preferred method for laboratory diagnosis, also include serological testing when

22:40
there are no lesions present.

22:46
Since NAATs are the preferred method for direct detection and diagnosis of herpes,

22:51
I wanted to give some examples of commercially available assays and their

22:55
intended use.

22:56
So there are several FDA cleared type specific HSV net acids that are available

23:01
and they come either as single Plex where they only contain HSV-1 and HSV-2 or they

23:06
can be found in Multiplex tests containing additional analytes.

23:11
For example, some of them may have VZV, while others may have an extensive list

23:15
of of additional analytes.

23:17
In the case of the meningitis encephalitis panels,

23:20
you'll see that the approved specimen types are limited to either cutaneous or

23:26
mucocutaneous lesions, sometimes restricted to the anogenital

23:30
region.

23:31
And then CSF samples for patients who have meningitis encephalitis or CNS

23:38
involvement.

23:40
And then these are specifically for patients who are symptomatic.

23:45
Several published studies have shown that the performance of these assays have

23:49
really high analytical sensitivity and specificity compared to other methods.

23:53
And it's important to note that random or blind swabs in the absence of lesions is

23:57
not recommended since this will decrease your clinical sensitivity and those

24:01
negative results may not rule out infection.

24:03
So that just is goes to show that you really need to use these tests

24:07
appropriately in order to have the highest clinical sensitivity and

24:12
specificity.

24:13
Some of these assays are not approved for patients under 18 years old or they

24:18
explicitly state that they're not for prenatal testing or I mean prenatal

24:23
screening.

24:23
So it's really important to know what test your platform you're using and what

24:27
the limitations are for those for those platforms and the instructions for use.

24:31
There are also additional qualitative and quantitative laboratory developed tests

24:36
that are available through commercial laboratories and these are used to

24:40
accommodate testing for additional specimen types that might not be FDA

24:45
cleared or for specific conditions that go beyond your typical HSV manifestations.

24:50
And some of those specimen types that are off label or are tested with LD TS might

24:56
include whole blood, serum, plasma tissue, maybe ocular fluid and they may not be

25:02
widely available.

25:08
Now I'm going to shift over to talking about type specific serological tests and

25:12
their performance characteristics specifically in the context of genital

25:16
herpes since I think this is a really important topic like Dr Marlowe had

25:20
highlighted previously.

25:22
So there's a variety of serological testing methods that are available and

25:26
used to support diagnosis or seroprevalence studies.

25:30
And these include type specific antibody assays that differentiate HSV-1 and HSV-2

25:35
antibodies using glycoproteins G1 and G2 as a target antigens.

25:41
Common commercially available methodologies include chemoluminescence

25:45
amino assays, which are really large platforms that are

25:48
FDA cleared that can accommodate high throughput testing.

25:52
There's also your traditional enzyme linked immunosorben assays, the ELISA.

25:57
There's electrochemoluminescence, amino assays, Multiplex flow immunoassays,

26:02
lateral flow, which come in point of care formats and

26:06
amino blot assays that could all be purchased from manufacturers and are FDA

26:11
cleared and vary in terms of of their complexity.

26:15
Western blot is considered the gold standard and many of these platforms have

26:20
been compared to Western blot.

26:22
And I'm going to go over a little more detail about some of these assays in

26:25
other slides.

26:27
One other important note that I want to make is that HSV IgM testing is no longer

26:33
available and that is because these assays lack the sensitivity to be used as

26:39
a reliable marker for active infection.

26:43
IgM antibodies, they can persist for months following

26:46
primary infection or they may or may not develop when you have recurrent infection,

26:51
whereas IgG will persist indefinitely.

26:53
So therefore IgM is is not is not something that you you want to use for

27:00
diagnosing acute or active infection.

27:07
When interpreting qualitative results from a HSV serological test,

27:11
there are certain limitations that need to be considered.

27:14
Since HSV-2 infections are usually sexually acquired,

27:18
the presence of HSV-2 IgG antibodies can indicate an anogenital herpes infection.

27:24
However, in contrast, the presence of IgG of IgG HSV-1 cannot

27:29
be used to support a genital herpes diagnosis.

27:32
Since HSV-1 infections are not limited to the anogenital region and can be acquired

27:38
during childhood so that one has limited utility when it comes to diagnosing

27:44
genital herpes.

27:46
Also, seroconversion can occur between two

27:49
weeks and three months with the majority of patients developing antibodies by 12

27:53
weeks.

27:53
So detection of if of antibodies can be also delayed in certain patients.

27:59
So knowing the that a negative result does not rule out infection.

28:04
If you recently suspect acquisition to be within that window,

28:08
then follow up testing may be indicated to to observe that sero conversion,

28:13
especially if it's recent recent infection.

28:18
And then detection of seroconversion can also vary depending on the type of method

28:22
or platform that you're using because they might they might have different

28:26
clinical sensitivities and I'll mention that in another slide.

28:34
So when is HSV serological testing indicated when being used for genital

28:40
herpes?

28:41
So the 2021 CDC STI Treatment Guidelines state that if a lesion is present,

28:46
then you want to swab that lesion and testing should be performed using a type

28:51
specific nucleic acid amplification test or viral cultures.

28:55
But what happens when you don't have a lesion present and you're still

28:59
suspecting genital herpes?

29:00
Well, the guidelines say that HSV-2 antibody

29:04
testing for IgG can be used, but only are under specific scenarios.

29:09
So those scenarios include if a patient has recurrent or atypical genital

29:15
symptoms or the lesion, you did swab A lesion and send it for

29:20
NAAT testing, but it comes back negative by either NAAT

29:25
or culture.

29:26
Or you have a person with a clinical diagnosis of genital herpes but without

29:30
any laboratory confirmation.

29:33
Or if you have a patient that's at higher risk for infection and for example,

29:38
one who has had a partner who has been diagnosed with genital herpes,

29:43
or if somebody is presenting for an STI evaluation,

29:46
those would be cases where HSV serological testing may be indicated.

29:56
When reviewing the US Preventative Services Task Force recommendations

30:01
specifically for screening that in conjunction with the CDC STI Treatment

30:07
Guidelines, both say that routine screening for a

30:10
genital HSV infection and asymptomatic individuals,

30:14
including pregnant women is not recommended.

30:18
This is due to the low specificity and high likelihood of false positives of

30:23
certain currently available assays when used for population based screenings,

30:28
which can lead to more harm than good in these in these patients.

30:33
Therefore, screening is not indicated in a person

30:36
with no known history, signs or symptoms of genital HSV

30:39
infection, including those with unrecognized

30:42
infection.

30:43
However, those at risk of acquiring infection,

30:46
like I mentioned before, regardless of pregnancy status,

30:49
including individuals with HIV or other immunosuppressive disorders,

30:53
can still be tested.

30:59
So many of the of the studies that have informed the HSV-2 IgG testing

31:04
recommendations for genital herpes have been using the HSV-2 IgG ELISA.

31:11
Performance characteristics such as sensitivities and specificities can vary

31:16
widely depending on the patient population tested,

31:19
the prevalence of that patient population, the timing of specimen collection,

31:24
and what the reference standard was used to characterize what a true positive is.

31:29
For example, some studies have shown a range of 80 to

31:34
98% for sensitivity and 57 to 98% for for specificity.

31:40
There have been also a number of studies that have shown poor specificity using

31:46
the ELISA and focusing on that lower range and showing that false positives

31:51
can occur more frequently at lower index values, specifically between 1.1 and 3.0.

31:58
More recently, there has been more published studies

32:03
using the FDA cleared more updated platforms.

32:07
And there's one study published by Dr Harry Prince,

32:10
actually 2 studies that compared the ELISA to the chemoluminescence platform,

32:16
which I refer to as CIA in these slides.

32:19
What these studies showed was that the discrepant result that there were

32:24
discrepant results between these two platforms and that false positives

32:28
actually can occur throughout the entire index range of the assay,

32:32
not just between 1.1 and 3.0.

32:34
For example, when looking at a cohort of over 2000 CIA

32:38
positive samples, 18% of them had ELISA negative results.

32:43
If you look at the breakdown by index value of those discordant results,

32:49
79% of them were between 1.1 and 3.0, and collectively 96% of the total

32:55
discordant samples were between 1.1 and 6. 0.

33:00
Conversely, when looking at the concordance samples

33:03
where both the CIA and the ELISA are positive,

33:06
those tended to be confirmed as true positives 97% of the time.

33:11
So the discrepancies between two methodologies may be due to either

33:15
differences in antigen used, clinical sensitivity of the platform,

33:19
or any analytical performance characteristics.

33:26
To further highlight differences in sensitivity between two methodologies

33:30
from a clinical perspective, there was one study looking at time to

33:34
seroconversion following HSV-2 primary infection by ELISA compared to Western

33:39
blot.

33:40
In this study, they tested paired samples collected from

33:43
a cohort of patients with newly acquired HSV-2 infection,

33:47
and they showed that the median number of days to seroconversion was shorter using

33:52
the ELISA compared to the Western blot.

33:55
So by six weeks, ELISA had detected 77% of the cohort,

33:59
while Western Blot had only detected 53%.

34:03
Because positivity for the Western Blot is based on reactivity to an array of HSV

34:08
antigens, they actually looked at HSV reactivity in

34:11
samples that did not meet the criteria for to be considered positive.

34:16
And they refer to this as a limited profile.

34:19
And there they showed a higher proportion of positivity at six weeks.

34:22
And this demonstrated that there was evidence of seroconversion.

34:27
So by 6 months, Western blot was able to detect 100% of

34:31
the seroconversion and ELISA 93% in this cohort.

34:35
And then what this study demonstrates is that timing of specimen collection and

34:39
methodology used for testing is important when interpreting results depending on

34:44
what stage of the infection you're in and when evaluating data from these studies

34:48
that use Western blot as a comparator for true positives.

34:55
More recently, there was a study published in the

34:58
Journal of Clinical Microbiology actually comparing 3 automated FDA cleared

35:03
platforms for the diagnosis of genital herpes using two characterized sample

35:07
cohorts.

35:08
The first cohort consisted of remnant samples submitted for Western blot

35:12
clinical testing and the second cohort included samples collected from persons

35:17
who had been diagnosed with genital herpes by either PCR or culture at least

35:22
six months prior.

35:23
So that would indicate that they should have their sero converted by then.

35:28
Concordance between the automated platforms in Western blot range from 78%

35:34
to 95% and HSV-2 positive and 92 to 100% for HSV-2 negative samples.

35:41
In addition, concordance was higher in samples that

35:46
were HSV-2 positive, HSV-1 negative compared to samples that

35:51
were HSV-2 and HSV-1 dual positive.

35:56
Additional analysis for sensitivity and specificity using NHANES

36:00
Population prevalence rates showed an overall range of about 95 to 98%

36:06
sensitivity and a 94 to 99% specificity when looking at all the samples on all

36:12
platforms.

36:14
So although there were select platforms that had higher rates of sensitivity and

36:19
specificity for HSV-2 compared to others depending on the cohort analyzed,

36:24
overall the data shows that the current FDA cleared platforms for testing HSV-2

36:29
have relatively high concordance compared to Western blot and to each other,

36:33
and high sensitivity and specificity in detecting antibodies and individuals with

36:38
confirmed genital herpes.

36:41
However, the consensus is that there still may be

36:44
gaps in room for improvement when it comes to HSV serologic testings.

36:48
And there's a lot more data in this paper.

36:50
So I encourage you to kind of go review it because it really depends how you

36:54
interpret the data and, and, and looking at the different platforms

36:58
and then getting an overall picture of how these different platforms performed

37:03
in what kinds of samples.

37:09
Because of the risk of false positives and the potential impact it may have on

37:13
patients, the 2021 STI Treatment Guidelines

37:16
actually recommend a 2-step testing algorithm when you're using an antibody

37:20
test to support diagnosis of genital herpes.

37:24
These guidelines were largely based on studies using ELISA,

37:27
which showed that the specificity was poor for these assays,

37:30
especially at the lower index values like I mentioned before.

37:34
Therefore, it's recommended that a positive IgG

37:37
sample should be confirmed using a second test that utilizes a method that's

37:42
distinct from the initial test and has a different antigen.

37:47
The Biokit and the Western blot assays were both provided as possible

37:51
confirmatory tests in the guidelines due to several studies showing that they can

37:55
improve overall accuracy of initial HSV results.

38:03
However, there are several limitations to

38:05
performing confirmatory testing.

38:08
One of the major limitations overall is that confirmatory testing is not readily

38:12
available.

38:14
For instance, the Western blot is available only at one

38:16
institution, the University of Washington.

38:19
And although it's considered the gold standard,

38:21
it's a very technically challenging and labor intensive assay.

38:26
And like I mentioned before, if it's early infection,

38:29
it might not be as sensitive as some of the other platforms available.

38:34
There's also a point of care device that can be purchased commercially.

38:38
It has been marketed under several different names.

38:41
The most commonly recognized is the Biokit.

38:43
The advantage of this of this device is that it's rapid and it can be performed

38:47
on site without any instrumentation.

38:49
However, reading can be subjective and there have

38:52
been supply challenges from the vendor throughout the years and it's not

38:56
amenable to high throughput testing.

38:59
There's also immunoblots that are commercially available that are easier to

39:03
perform compared to the Western blot the FDA cleared.

39:07
Reading is easy and standardized.

39:08
However, these were common antigens that could

39:11
potentially be the same as some of the screening assays and therefore not always

39:15
ideal for confirmatory testing.

39:17
Lastly, there is the HSV-2 IgG inhibition assay

39:21
that's available at Quest Diagnostics.

39:25
This is a reflex test that is intended to be used to confirm screen positive

39:30
samples with index values between 1. 1 and 6.0.

39:34
However, it's only offered at Quest and

39:37
occasionally if there is a sample with low IgG levels,

39:40
the inhibition results may not be determined,

39:43
and I'll go over this in more detail in subsequent slides.

39:47
I also want to highlight that the CDC guidelines point out that when

39:51
confirmatory testing is not available, patients should be counseled about the

39:56
limitations of serological testing, including the risk of false positive

40:00
results, before proceeding with with serologic

40:02
testing.

40:08
For comparison, I just wanted to give examples of how

40:12
confirmatory methods work.

40:14
The Western blot on the left utilizes proteins from lysates of HSV infected

40:20
cells and patient.

40:21
Antibodies bind to the viral proteins and depending on the reactivity to specific

40:26
bands, HSV-1 and HSV-2 antibody detection is

40:29
determined.

40:31
The immunoblot is similar but more straightforward.

40:33
It uses recombinant antigens that can be more clearly interpreted.

40:37
It does not have the wider way of banding pattern as the Western blot does though.

40:43
Contrast.

40:43
The HSV-2 rapid test is easy to perform and can be done as a point of care test.

40:49
But like I mentioned before, reading is very subjective and may be

40:52
variable depending on the reader.

40:55
Here I gave an example that any level of reactivity is considered positive based

41:00
on the instructions for use and interpretation may be impacted by any

41:04
deviations from the manufacturer's instructions.

41:07
So it could be over calling false positives potentially depending on the

41:12
reader.

41:14
Finally, the HSV-2 IgG inhibition test.

41:17
This is an ELISA based assay that utilizes HSV-1 and HSV-2 cultural lysate

41:22
for the differential absorption of type specific antibodies.

41:26
So this allows for us to identify the presence of HSV-2 specific antibodies.

41:35
The current 2-step testing algorithm at Quest begins with an HSV-2 IgG screen

41:41
using the DiaSorin CIA platform.

41:46
Since the CDC guidelines recommend confirming samples with low indices,

41:50
specifically below 3.0, we have set the reflex criteria beyond

41:55
this range to test positive samples with an index between 1.1 and 6.

41:59
0 with the inhibition assay.

42:02
This is also supported by the data that I was previously showing that over 96% of

42:07
the discord in CIA and ELISA results are within this range using this platform.

42:13
The inhibition assay uses the HerpeSelect ELISA kit.

42:18
The assay is set up as a three-wheel assay where the patient serial is

42:22
incubated with HSV-1 culture lysate, HSV-2 cultural lysate separately and then

42:27
tested for IgG reactivity with one well to determine the baseline IgG levels.

42:33
The results are determined based on a sequential evaluation of the results.

42:38
First we ask are there any detectable IgG antibodies above the cutoff in the sample

42:43
and this is because we need a positive IgG value to actually calculate the

42:47
percent inhibition.

42:49
Therefore, negative and equivocal results do not

42:52
have an inhibition result and this is indicated by a comment on the report.

42:57
If there is detectable IgG present, we check if there's substantial

43:01
inhibition with the HSV-1 lysate.

43:04
If so, then we cannot rule out inhibition due to

43:08
HSV-1 or the lysate itself and the results is reported as indeterminate.

43:14
However, if there is no substantial inhibition

43:18
present with the HSV-1 lysate, then HSV-2 specific percent inhibition is

43:23
calculated and cut off values are used to then report positive,

43:27
which indicate a true positive screen on the CIA negative,

43:32
which indicate an initial false positive on the screen,

43:36
which is what you'll see on the report.

43:44
The HSV-2 inhibition assay has been offered at Quest Diagnostics or Focus

43:49
Diagnostics since 2002.

43:51
It was initially developed in a collaboration between Focus Diagnostics

43:55
and the University of Washington to investigate discordant results between

44:00
ELISA and Western blot in a cohort of serum from an African population.

44:06
There are there is limited data comparing the inhibition test to the Western blot,

44:11
but the original publication reported a 95% agreement between Western blot and

44:16
the inhibition assay.

44:19
They also noted that the discordant results between these two assays were

44:24
mainly from samples collected from two specific geographical regions and that

44:29
those results had low index values.

44:33
The authors also suspected that the inhibition positive Western blot

44:37
negatives may have represented early stages of seroconversion that have may

44:42
have been detected by ELISA and not Western blot.

44:47
Also atypical results that were not reported as positive by Western blot

44:51
might have been picked up by the inhibition test.

44:55
There was also one follow up study that directly compared the inhibition test to

45:01
Western blot in a African population and the overall concordance was 74%.

45:06
But similar to the original study, there are certain nuances in this patient

45:10
population that may impact the performance of the assays.

45:15
More recently, the study that I previously mentioned by

45:19
Dr Harry Prince compared over 1800 samples tested with the CIA, ELISA,

45:25
and the inhibition assay.

45:27
And then when you look at true positives, over 99% of the inhibition positive

45:32
samples were also CIA and ELISA positive.

45:36
In addition, as part of the study, the inhibition assay also demonstrated

45:40
good agreement with the biokit in samples that were CIA positive and ELISA positive.

45:47
They also looked at the breakdown of index values from the CIA screening assay

45:51
and saw that unconfirmed results tend to be at the lower indexes very similar to a

45:57
lot of the published studies with the ELISAs.

46:00
And as the index value of the screen increases,

46:05
you have you're less likely to get a discrepant result or a false positive on

46:14
the ELISA and the inhibition assay.

46:18
To summarize the key points of this talk, we have several methodologies that are

46:24
available for HSV diagnosis and that is the preferred method for lesion swabs and

46:29
CSF.

46:30
However, when there is no lesion swabs, there are recommendations for using

46:35
serological testing.

46:36
However, serological screening is not recommended

46:39
in asymptomatic individuals and with no previous history of genital herpes and

46:44
that includes pregnant women due to the the risk of false positives in some of

46:49
these commercially available platforms.

46:52
There have been published studies that shown a range of performance depending on

46:58
the methodology and patient population for HSV-2, IgG assays,

47:02
and diagnosing genital herpes.

47:04
Some of these assay may yield false positive results especially at those low

47:08
indices, which is why confirmatory testing is

47:10
currently recommended.

47:12
However, testing options are limited for

47:15
confirmatory tests and the results can depend on timing,

47:20
methodology and pre-test probability.

47:24
Additional studies to compare the performance of newer automated platforms

47:29
are starting to emerge and starting to be compared to each other and older

47:34
methodologies.

47:36
But we do need additional studies for confirmatory tests and showing how they

47:40
can be used for how good they are in diagnosing genital herpes.

47:46
Most importantly I think is that appropriate diagnostic stewardship is

47:51
required for how these tests are utilized.

47:54
We want to ensure that the assays are being used in the right patient

47:58
population in order to reduce that risk of false positives and that not only the

48:03
provider is educated, but that the patient really understands

48:07
the limitations of the test and what that what the results actually mean.