Herpes virus culturing can make a timely addition to many hospital
laboratories’ inhouse test menus. For one thing, it is primarily
an outpatient service, exempt from prospective payment restrictions. For
another, there’s growing demand from physicians’ offices for
such service, especially if it is available locally.
Our laboratories have successfully implemented and marketed herpes culturing. We have gone from a handful of monthly inpatient and
outpatient requests for the procedure, when we used to send out the work
to reference laboratories, to volumes of 50 to 60 cultures a month at
220-bed Northwest Hospital in Seattle and 80 or more a month at 230-bed
General Hospital in Everett, Wash.
That spells total net revenue of about $20,000 a year for the two
community hospitals and no more send-out costs. Just as important is
the service improvement. Turn-around time from specimen receipt to
final report has been cut by as much as 50 per cent at our hospitals
through elimination of herpes culture send-outs. The microbiology staff
also benefits by learning a new method that meets current, pressing
Ninety-five per cent of the testing is performed for outpatients.
Most of the cultures come in from family physicians and ob/gyns, and
pregnant patients account for about 60 per cent of the total workload.
The incidence of genital herpes infections has risen considerably
over the past 10 years, and physicians face increasing public pressure
to identify carriers of the virus as well as patients with active
lesions. Though a cure for herpes simplex remains to be discovered, new
medications can reduce morbidity. Equally important, sexually active
patients with lesions can be counseled to minimize the risk of spreading
the disease to their partners.
In addition, near-term expectant mothers, who may be asymptomatic vaginal or cervical carriers of herpes simplex, can be identified. Then
their newborns can be protected from exposure to the virus by
performance of a cesarean section. Without this safeguard, one-third to
one-half of babies born to women positive for primary herpes simplex
infection at time of delivery may die within a few days of birth;
another 25 per cent may be moderately to seriously affected for life by
the residual sequelae.
When we began considering inhouse testing three years ago, only two
reference laboratories in our area were capable of culturing for herpes.
Our laboratories were able to provide evidence of the virus through Pap
or Tzanck smear examinations, but these smears can vary greatly in
sensitivity and specificity and thus run a poor second to tissue culture
methods, which are the gold standard for herpes virus identification.
We surveyed physicians in our communities to determine the true
level of interest in the proposed new service. The response came back
quickly–they would request much more herpes culturing if an accurate
test was available locally at a competitive fee. With that information
in hand, our pathologists encouraged us to go ahead.
The process of getting suspected herpes culture material to
reference laboratories was confusing and inconvenient for physicians.
For example, one of the reference labs wanted the specimens shipped
frozen on dry ice, while the other lab insisted on wet ice packaging and
rejected frozen specimens. Since specimens had to be transported for
several miles, we spent considerable time repacking to help them survive
Physicians lost confidence when, despite our best efforts, virus
viability was compromised and false-negative reports were generated. If
we were aware of a problem before the testing, we would call the
physician to request a new specimen. That, however, meant inconvenience
for the patient as well as the expense of repeat processing by the
physician’s staff, our staff, and the reference lab’s staff.
We also found ourselves spending a good deal of time juggling taxi,
bus, and courier connections to out-of-town laboratories. Transportation
costs were high, particularly if special couriers had to be used for
Stat deliveries. On weekends and evenings, it seemed impossible to
obtain prompt specimen handling.
Getting results back represented the slowest phase of turn-around
time. The practice at the distant laboratories was to observe tissue
cultures for two weeks or more, to detect not only herpes but also other
viruses of interest in teaching and public health facilities. One lab
was closed on weekends, which further delayed reports.
After assessing all these factors, we were convinced that local
testing could reduce problems associated with the send-outs and
stimulate many more culture requests.
Although we lacked tissue culture experience, we were skilled in
sterile techniques and scientific methodologies. We did have some
reservations about working with the live virus and living cells used in
culturing. We were comfortable enough with bacterial and fungal
determinations and agar petri dishes, but this definitely was going to
be a change from the usual routine. Enthusiasm grew as we researched
the need for branching into this new area and the possibilities it
Both labs already had most of the necessary equipment for
processing tissue cultures: a -70 C freezer for specimen storage, a
microscope, a centrifuge, an incubator set at 35 to 37 C, and a
biological safety cabinet for inoculation or transfer of specimens. The
only outlay required was $420 for a roller drum in each lab, employed in
incubation, and ongoing supply expenses.
To learn about the procedure, we both attended workshops at
American Society for Microbiology meetings and a lecture and wet
workshop put on by a local supplier of the tissue culture cells and
reagents. These sessions thoroughly covered the theory and practical
aspects of specimen collection, processing, virus identification, and
We had anticipated that virus culturing would be a complex and
technically difficult procedure, but just the opposite was true. After a
few hours of lecture plus hands-on experience–inoculating and examining
tissue culture tubes and confirming the presence of the virus by direct
staining methods–we knew that with practice we could reliably detect
and report these very common viral agents.
Once were were comfortable with tissue culture basics, we trained
the technologists who would help implement the new procedure. The next
step was to educate our couriers about specimen handling and physicians
and their office staffs about the availability of the service and
specimen procurement techniques.
Transportation under refrigerated conditions–not freezing–insures
that a more viable virus reaches the laboratory.
Couriers were told to collect specimens directly from clinic
refrigerators them in a cooler placed in each vehicle. We also made
sure they were familiar with all pickup points and where to deliver the
specimens within the lab. They were thoroughly checked out on
disinfecting accidental spills or broken specimen containers and
reporting such incidents.
We prepared informational handouts and mailed a letter about our
new test to the entire roster of local physicians. With help from the
pathologists, we scheduled meeting in doctors’ offices, staged
slide/tape shows, gave informal lectures, and held impromptu sessions in
the doctors’ lounges at the hospitals.
physicians had to appreciate the importance of collecting exudates
or cell scrapings from early lesions. Old or crusted lesions do not
yield live virus. Even using the wrong swab can affect the culture.
Dacron swabs are better tolerated by the virus than cotton swabs. Our
plastic transport packs contained complete instructions, along with the
proper swabs and a suitable transport medium in a leak-proof tube.
During our one-month field trial, several physicians donated
specimens from patients who had “typical” lesions and from
normal or presumed negative patients. This gave us a good supply of
positive and negative specimens upon which to sharpen our skills. We
split them and sent some to a reference lab as a control. This exercise
rapidly boosted our self-confidence. Any questionable results were
rechecked with great care until we were satisfied that we could
recognize and confirm herpes simplex.
Since we could not afford the time and money needed to develop and
maintain our own herpes tissue cultures, we decided to purchase
commercial cells. The human fibroblastic cell line we chose is
sufficiently sensitive to detect low levels of herpes virus and also has
a very good shelf life. With a standing weekly order of 30 tissue
culture tubes, fresh cells are always available. Technologists can
refeed any leftover cells, replacing the old maintenance medium with new
medium that prolongs their ability to absorb and grow the virus. Cells
can be used for up to three weeks if maintained carefully.
We vigorously vortex each specimen transport tube received in the
lab to release the virus from intact patient epithelial cells.
That’s followed by centrifuging to separate the virus from the
residual bacterial and cellular debris. A few drops of supernatant containing the virus are inoculated into tissue culture tubes, which are
then incubated for five to seven days at 35 C. This uncomplicated
procedure can either be performed as specimens arrive, or they can be
held at 4 C and batch-processed at a later time.
Cell cultures are observed daily. When infected with herpes virus,
the normally spindle-shaped human fibroblast cells become round and
enlarge. This change is called the cytophatic effect (CPE) of virus
infection. Cells may coalesce, creating a multinucleated giant cell
similar to those seen on Pap and Tzanck smears.
CPE is not specific, only presumptive evidence that the virus may
be in the herpes family. Other viruses, such as adenovirus, Coxsackie,
herpes zoster, and varicella, can mimic herpes simplex. Herpes simplex
may also show atypical CPE, the cells becoming smaller than normal or
just lacking definition. Encountering these unfamiliar formations can
shake the confidence of newcomers to tissue culture.
Fortunately, we don’t have to depend on the CPE alone to
identify the virus. While individual virus particles are too small to
be seen under an ordinary microscope, it is possible to detect masses of
them present in infected cells by means of special stains that
incorporate a specific antibody and an indicator dye–for example,
fluorescein for fluorescent microscopic observation or immunoperoxidase
conjugates that can be observed with a bright-field microscope. Only
herpes-infected cells will stain.
To be of value to the physician, reports must be accurate, easily
interpreted, and timely. We are specific when reporting the condition
of the specimen. After two to three days’ incubation of an
unchanged cell sheet that has been challenged with patient sample, a
preliminary report verifies the culture is negative for the presence of
virus at that time. Since most of our cultures are positive or begin to
show cytophatic effect within 48 to 72 hours, a negative preliminary
report is encouraging news and meets the need for rapid turnaround of
When tissue culture tubes show evidence of virus, we report
immediately by phone if a pregnant patient is near term or if we’re
working with neonatal or eye cultures. All phoned reports are followed
by a written report the same day. If tissue culture changes are typical
of herpes, the report reads: “Presumptive positive for herpes
simplex.” If the cell changes are atypical or indeterminate, we
may report: “Cytophatic cell changes evident; tests in progress to
confirm or rule out herpes simplex” or “Cell changes not
typical of herpes simplex.” For physicians who want more than a
presumptive report, final verification of herpes simplex by specific
staining procedures follows on the same or next day.
If the confirmatory stain is positive, we report: “Herpes
simplex (type 1 or 2) confirmed.” With negative stains, the report
reads: “No herpes simplex isolated.” For the occasional
inconclusive culture, we either reprocess the specimen from the frozen
inoculum or submit it to a reference laboratory with an appropriate note
to the physician–for example, “Specimen shows evidence of virus;
tests in progress to rule out herpes simplex.” This covers the
atypical small cells that are rounding but not enlarged, or a slow
progression of CPE uncharacteristic of herpes simplex, or delayed
cytopathic effects of inoculum, which may signal that other viruses or
agents are affecting cells.
We take care to follow all cultures exhibiting CPE through passage
(subculture) and with other tests, as necessary, to reach a firm
conclusion. It certainly eases a patient’s concern to learn that
lesions are caused by herpes zoster or adenovirus, which does not have
the social ramifications of herpes simplex and does not carry the danger
of death in newborns.
Our reports reach physicians an average of one week earlier than
those formerly provided by area reference laboratories. For near-term
pregnant patients, we post daily lists of positive and negative culture
status in the laboratory, as a quick reference on the evening and night
shifts. When delivery is imminent and the physician does not yet have
access to a report, these daily lists are truly lifesavers. They help
physicians decide whether to perform a cesarean section or a vaginal
Quality control is essential to avoid false-negative and
false-positive results. Daily equipment checks are mandatory.
Temperatures above 37 C in the incubator can render the virus nonviable.
We monitor the temperature of the incubator environment near the roller
drum and confirm that the drum is still revolving (otherwise, some tubes
may be left cell-side up without the protection of maintenance
medium–causing cell death and viral inactivation).
After we checked 400 lots of cells with a herpes stock culture, we
developed confidence in the cell supplier. As with microbiological agar
culture media, tissue culture reagent manufacturers have the resources
to do a much more thorough evaluation than we can of their
products’ ability to perform as specified in the product inset.
(For more extensive discussion of this issue, see our article,
“Media Quality Control: An Unnecessary Evil,” MLO, February
1984.) We check each tube of cells in each new shipment to verify that
the cells look normal prior to inoculation. We reserve at least one
cell tube per lot to serve as a negative control for the entire time the
lot remains in use. That way we can monitor the effects of incubation
on the cells as they age. Stains are checked with known positive and
negative controls upon receipt.
During the first year of tissue culturing, we monitored sterility
of the refeeding medium used in our procedures. Finding no contaminated lots, we discontinued this practice and now rely on the
manufacturer’s quality control.
Each new technologist is carefully instructed in herpes testing and
must correctly identify several blind unknowns.
No laboratory test is ever without problems. The new herpes test
offered its share, but all were solvable. Here’s how we dealt with
the most common problems we encountered:
* Life in the real world. Our first major problem was our
inexperience. We could recognize the distinctly positive and negative
cell cultures in the workshops, but at the bench it was a lot harder to
differentiate between real CPE and normal cell changes. Dividing
fibroblast cells become round just before division and resemble
herpes-infected cells. With continued observation, however, these
essentially normal cell sheets appear much different from truly infected
cell sheets. Herpes-infected cell sheets rapidly develop progressive
CPE, usually within 12 to 24 hours.
* Contaminating super-bugs. Herpes lesions can be superinfected
with Candida, and yeast lesions can mimic herpes. It is important to
isolate the virus from the mat of pseudohyphae that sometimes overgrows
and obscures the tissue culture. We do this by filtering some of the
tissue culture fluid through a sterile 0.45 micron filter and
reinoculating a few drops of the yeast-free fluid into a fresh tissue
* Unbathed cell cultures. Cell cultures that remain stationary for
too long can develop a toxi imbalance of metabolic byproducts near the
cell surface. This causes the tissue culture to deteriorate or the
virus to progress more slowly in the cells. A roller drum is the ideal
way to prevent this; it makes increased cell quality possible without
constant technologist attention. If you are on a tight budget, you can
achieve the same effect by rotating the tubes a few times each day. But
makes sure the tissue culture is under the fluid when the tubes are
placed back in a slanted, stationary position.
* The overheated incubator. Excessive incubator temperatures
markedly affect tissue cultures. It is important to check the area
nearest the tubes. Roller drum motors can raise tube temperature by a
degree or more.
* Old cells, cold cells. Less sensitive cells often are aging
cells, eight to 10 days old. Responsible suppliers control the age of
the cell line by constantly refreshing the cells from low-passage
(minimally subcultured) stocks. Cells that have been cold-shocked, as
may happen during winter transport, are less likely to be infected by
virus in the sample. For optimum sensitivity, we incubate cells after
overnight delivery to the lab before inoculating them with patient
* pH fiascos. A pH shift in the cell culture may cause the
surrounding medium to adversely affect viral attachment and replication.
Acid shifts (indicated by medium changes from pink to yellow or orange)
can be corrected by adding one drop of sterile 8.8 per cent sodium
bicarbonate solution. Tubes that have become alkaline (dark red or dark
pink) should be discarded.
Was the program a success? Judge for yourself: During the initial
testing period, we processed about 20 patient specimens in the first 30
days. When we offered the service to the community, volume doubled and
then tripled within three months.
We are able to perform these tests for about 75 per cent less than
what we used to pay the reference laboratory, and we also save up to $18
in long-distance courier fees for each specimen processed. A cost
comparison is shown in Figure I.
Since we only screen for herpes virus, our methods are simpler than
those used at a full-service reference laboratory. The need for
extended incubation of the cultures–along with the concomitant taks of
pulling and reviewing tubes for several extra days–is minimal. We
estimate that it takes only 10 minutes, spread over five to seven days,
to completely finalize a negative sample. We need about 20 minutes to
process and confirm a positive culture. Logging the specimen and
preparing reports for distribution requires another 7.8 minutes.
We have cut costs, but not corners. Overall, roughly 30 per cent
of the tests are positive, and 70 per cent are negative. We’re
able to issue a preliminary report within 48 to 72 hours and send out
final confirmation within five to seven days of specimen receipt.
Our familiarity with tissue culturing opened up new possibilities
for expanded services. We now use the same fibroblast cells for another
practical and much-needed laboratory determination–Clostridium
difficile toxin detection. The toxin of C. diffickle, a hardy survivor
in the human gut following antibiotic therapy, causes pseudomembranous
colitis (PMC) and affects human fibroblast cells with characteristic
For our next project, we plan to institute direct diagnosis of
herpes simplex from lesion scraping using specific monoclonal antibody staining. This test is a more sensitive means of confirming lesions
that might be too old to result in a positive tissue culture. On early
lesions, the direct test offers a turnaround time of less than 24 hours
for confirmed diagnosis, versus two to seven days with the traditional
culture stain method. Asymptomatic carriers, such as near-term pregnant
women with no visible lesions, are not candidates for this rapid method.
Sensitivity will lag behind tissue culture in such cases. For many
other patients, though, the direct test represents a great improvement
in specificity over the Pap and Tzanck smears.
Increasingly, hospital laboratories are performing tests in-house
that once were referred to other laboratories. Cultures for herpes
virus require a level of expertise traditionally associated with highly
trained technologists and thus are not practical for small-volume
physicians’ offices or laboratories. This gives clinic and
hospital laboratories staffed with medical technologists an opportunity
to provide new, quality services locally.
If you carefully plan the launching of a new test like ours, and
manage it through preliminary field trials to avoid excessive costs in
implementation, you to can succeed.