Herpes virus culturing can make a timely addition to many hospitallaboratories’ inhouse test menus. For one thing, it is primarilyan outpatient service, exempt from prospective payment restrictions. Foranother, there’s growing demand from physicians’ offices forsuch 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 andoutpatient requests for the procedure, when we used to send out the workto reference laboratories, to volumes of 50 to 60 cultures a month at220-bed Northwest Hospital in Seattle and 80 or more a month at 230-bedGeneral Hospital in Everett, Wash.
That spells total net revenue of about $20,000 a year for the twocommunity hospitals and no more send-out costs. Just as important isthe service improvement. Turn-around time from specimen receipt tofinal report has been cut by as much as 50 per cent at our hospitalsthrough elimination of herpes culture send-outs. The microbiology staffalso benefits by learning a new method that meets current, pressingneeds. Ninety-five per cent of the testing is performed for outpatients.Most of the cultures come in from family physicians and ob/gyns, andpregnant patients account for about 60 per cent of the total workload. The incidence of genital herpes infections has risen considerablyover the past 10 years, and physicians face increasing public pressureto identify carriers of the virus as well as patients with activelesions.
Though a cure for herpes simplex remains to be discovered, newmedications can reduce morbidity. Equally important, sexually activepatients with lesions can be counseled to minimize the risk of spreadingthe disease to their partners. In addition, near-term expectant mothers, who may be asymptomatic vaginal or cervical carriers of herpes simplex, can be identified. Thentheir newborns can be protected from exposure to the virus byperformance of a cesarean section. Without this safeguard, one-third toone-half of babies born to women positive for primary herpes simplexinfection at time of delivery may die within a few days of birth;another 25 per cent may be moderately to seriously affected for life bythe residual sequelae. When we began considering inhouse testing three years ago, only tworeference laboratories in our area were capable of culturing for herpes.Our laboratories were able to provide evidence of the virus through Papor Tzanck smear examinations, but these smears can vary greatly insensitivity and specificity and thus run a poor second to tissue culturemethods, which are the gold standard for herpes virus identification. We surveyed physicians in our communities to determine the truelevel of interest in the proposed new service.
The response came backquickly–they would request much more herpes culturing if an accuratetest was available locally at a competitive fee. With that informationin hand, our pathologists encouraged us to go ahead. The process of getting suspected herpes culture material toreference laboratories was confusing and inconvenient for physicians.For example, one of the reference labs wanted the specimens shippedfrozen on dry ice, while the other lab insisted on wet ice packaging andrejected frozen specimens. Since specimens had to be transported forseveral miles, we spent considerable time repacking to help them survivethe trip. Physicians lost confidence when, despite our best efforts, virusviability was compromised and false-negative reports were generated. Ifwe were aware of a problem before the testing, we would call thephysician to request a new specimen.
That, however, meant inconveniencefor the patient as well as the expense of repeat processing by thephysician’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. Transportationcosts were high, particularly if special couriers had to be used forStat deliveries. On weekends and evenings, it seemed impossible toobtain prompt specimen handling. Getting results back represented the slowest phase of turn-aroundtime.
The practice at the distant laboratories was to observe tissuecultures for two weeks or more, to detect not only herpes but also otherviruses of interest in teaching and public health facilities. One labwas closed on weekends, which further delayed reports. After assessing all these factors, we were convinced that localtesting could reduce problems associated with the send-outs andstimulate many more culture requests. Although we lacked tissue culture experience, we were skilled insterile techniques and scientific methodologies.
We did have somereservations about working with the live virus and living cells used inculturing. We were comfortable enough with bacterial and fungaldeterminations and agar petri dishes, but this definitely was going tobe a change from the usual routine. Enthusiasm grew as we researchedthe need for branching into this new area and the possibilities itoffered.
Both labs already had most of the necessary equipment forprocessing tissue cultures: a -70 C freezer for specimen storage, amicroscope, a centrifuge, an incubator set at 35 to 37 C, and abiological safety cabinet for inoculation or transfer of specimens. Theonly outlay required was $420 for a roller drum in each lab, employed inincubation, and ongoing supply expenses. To learn about the procedure, we both attended workshops atAmerican Society for Microbiology meetings and a lecture and wetworkshop put on by a local supplier of the tissue culture cells andreagents. These sessions thoroughly covered the theory and practicalaspects of specimen collection, processing, virus identification, andlaboratory safety. We had anticipated that virus culturing would be a complex andtechnically difficult procedure, but just the opposite was true.
After afew hours of lecture plus hands-on experience–inoculating and examiningtissue culture tubes and confirming the presence of the virus by directstaining methods–we knew that with practice we could reliably detectand report these very common viral agents. Once were were comfortable with tissue culture basics, we trainedthe technologists who would help implement the new procedure. The nextstep was to educate our couriers about specimen handling and physiciansand their office staffs about the availability of the service andspecimen procurement techniques. Transportation under refrigerated conditions–not freezing–insuresthat a more viable virus reaches the laboratory. Couriers were told to collect specimens directly from clinicrefrigerators them in a cooler placed in each vehicle. We also madesure they were familiar with all pickup points and where to deliver thespecimens within the lab.
They were thoroughly checked out ondisinfecting accidental spills or broken specimen containers andreporting such incidents. We prepared informational handouts and mailed a letter about ournew test to the entire roster of local physicians. With help from thepathologists, we scheduled meeting in doctors’ offices, stagedslide/tape shows, gave informal lectures, and held impromptu sessions inthe doctors’ lounges at the hospitals. physicians had to appreciate the importance of collecting exudatesor cell scrapings from early lesions.
Old or crusted lesions do notyield live virus. Even using the wrong swab can affect the culture.Dacron swabs are better tolerated by the virus than cotton swabs. Ourplastic transport packs contained complete instructions, along with theproper swabs and a suitable transport medium in a leak-proof tube. During our one-month field trial, several physicians donatedspecimens from patients who had “typical” lesions and fromnormal or presumed negative patients. This gave us a good supply ofpositive and negative specimens upon which to sharpen our skills.
Wesplit them and sent some to a reference lab as a control. This exerciserapidly boosted our self-confidence. Any questionable results wererechecked with great care until we were satisfied that we couldrecognize and confirm herpes simplex. Since we could not afford the time and money needed to develop andmaintain our own herpes tissue cultures, we decided to purchasecommercial cells. The human fibroblastic cell line we chose issufficiently sensitive to detect low levels of herpes virus and also hasa very good shelf life. With a standing weekly order of 30 tissueculture tubes, fresh cells are always available. Technologists canrefeed any leftover cells, replacing the old maintenance medium with newmedium that prolongs their ability to absorb and grow the virus.
Cellscan be used for up to three weeks if maintained carefully. We vigorously vortex each specimen transport tube received in thelab to release the virus from intact patient epithelial cells.That’s followed by centrifuging to separate the virus from theresidual bacterial and cellular debris. A few drops of supernatant containing the virus are inoculated into tissue culture tubes, which arethen incubated for five to seven days at 35 C. This uncomplicatedprocedure can either be performed as specimens arrive, or they can beheld 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 andenlarge. This change is called the cytophatic effect (CPE) of virusinfection. Cells may coalesce, creating a multinucleated giant cellsimilar to those seen on Pap and Tzanck smears.
CPE is not specific, only presumptive evidence that the virus maybe in the herpes family. Other viruses, such as adenovirus, Coxsackie,herpes zoster, and varicella, can mimic herpes simplex. Herpes simplexmay also show atypical CPE, the cells becoming smaller than normal orjust lacking definition.
Encountering these unfamiliar formations canshake the confidence of newcomers to tissue culture. Fortunately, we don’t have to depend on the CPE alone toidentify the virus. While individual virus particles are too small tobe seen under an ordinary microscope, it is possible to detect masses ofthem present in infected cells by means of special stains thatincorporate a specific antibody and an indicator dye–for example,fluorescein for fluorescent microscopic observation or immunoperoxidaseconjugates that can be observed with a bright-field microscope.
Onlyherpes-infected cells will stain. To be of value to the physician, reports must be accurate, easilyinterpreted, and timely. We are specific when reporting the conditionof the specimen. After two to three days’ incubation of anunchanged cell sheet that has been challenged with patient sample, apreliminary report verifies the culture is negative for the presence ofvirus at that time. Since most of our cultures are positive or begin toshow cytophatic effect within 48 to 72 hours, a negative preliminaryreport is encouraging news and meets the need for rapid turnaround ofresults. When tissue culture tubes show evidence of virus, we reportimmediately by phone if a pregnant patient is near term or if we’reworking with neonatal or eye cultures.
All phoned reports are followedby a written report the same day. If tissue culture changes are typicalof herpes, the report reads: “Presumptive positive for herpessimplex.” If the cell changes are atypical or indeterminate, wemay report: “Cytophatic cell changes evident; tests in progress toconfirm or rule out herpes simplex” or “Cell changes nottypical of herpes simplex.” For physicians who want more than apresumptive report, final verification of herpes simplex by specificstaining procedures follows on the same or next day. If the confirmatory stain is positive, we report: “Herpessimplex (type 1 or 2) confirmed.
” With negative stains, the reportreads: “No herpes simplex isolated.” For the occasionalinconclusive culture, we either reprocess the specimen from the frozeninoculum or submit it to a reference laboratory with an appropriate noteto the physician–for example, “Specimen shows evidence of virus;tests in progress to rule out herpes simplex.” This covers theatypical small cells that are rounding but not enlarged, or a slowprogression of CPE uncharacteristic of herpes simplex, or delayedcytopathic effects of inoculum, which may signal that other viruses oragents are affecting cells. We take care to follow all cultures exhibiting CPE through passage(subculture) and with other tests, as necessary, to reach a firmconclusion. It certainly eases a patient’s concern to learn thatlesions are caused by herpes zoster or adenovirus, which does not havethe social ramifications of herpes simplex and does not carry the dangerof death in newborns. Our reports reach physicians an average of one week earlier thanthose formerly provided by area reference laboratories. For near-termpregnant patients, we post daily lists of positive and negative culturestatus in the laboratory, as a quick reference on the evening and nightshifts.
When delivery is imminent and the physician does not yet haveaccess to a report, these daily lists are truly lifesavers. They helpphysicians decide whether to perform a cesarean section or a vaginaldelivery. Quality control is essential to avoid false-negative andfalse-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 rollerdrum and confirm that the drum is still revolving (otherwise, some tubesmay be left cell-side up without the protection of maintenancemedium–causing cell death and viral inactivation). After we checked 400 lots of cells with a herpes stock culture, wedeveloped confidence in the cell supplier. As with microbiological agarculture media, tissue culture reagent manufacturers have the resourcesto do a much more thorough evaluation than we can of theirproducts’ 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, February1984.) We check each tube of cells in each new shipment to verify thatthe cells look normal prior to inoculation. We reserve at least onecell tube per lot to serve as a negative control for the entire time thelot remains in use.
That way we can monitor the effects of incubationon the cells as they age. Stains are checked with known positive andnegative controls upon receipt. During the first year of tissue culturing, we monitored sterilityof the refeeding medium used in our procedures. Finding no contaminated lots, we discontinued this practice and now rely on themanufacturer’s quality control.
Each new technologist is carefully instructed in herpes testing andmust correctly identify several blind unknowns. No laboratory test is ever without problems. The new herpes testoffered its share, but all were solvable. Here’s how we dealt withthe most common problems we encountered: * Life in the real world. Our first major problem was ourinexperience. We could recognize the distinctly positive and negativecell cultures in the workshops, but at the bench it was a lot harder todifferentiate between real CPE and normal cell changes. Dividingfibroblast cells become round just before division and resembleherpes-infected cells.
With continued observation, however, theseessentially normal cell sheets appear much different from truly infectedcell sheets. Herpes-infected cell sheets rapidly develop progressiveCPE, usually within 12 to 24 hours. * Contaminating super-bugs. Herpes lesions can be superinfectedwith Candida, and yeast lesions can mimic herpes. It is important toisolate the virus from the mat of pseudohyphae that sometimes overgrowsand obscures the tissue culture. We do this by filtering some of thetissue culture fluid through a sterile 0.
45 micron filter andreinoculating a few drops of the yeast-free fluid into a fresh tissueculture tube. * Unbathed cell cultures. Cell cultures that remain stationary fortoo long can develop a toxi imbalance of metabolic byproducts near thecell surface.
This causes the tissue culture to deteriorate or thevirus to progress more slowly in the cells. A roller drum is the idealway to prevent this; it makes increased cell quality possible withoutconstant technologist attention. If you are on a tight budget, you canachieve the same effect by rotating the tubes a few times each day. Butmakes sure the tissue culture is under the fluid when the tubes areplaced back in a slanted, stationary position.
* The overheated incubator. Excessive incubator temperaturesmarkedly affect tissue cultures. It is important to check the areanearest the tubes. Roller drum motors can raise tube temperature by adegree or more. * Old cells, cold cells.
Less sensitive cells often are agingcells, eight to 10 days old. Responsible suppliers control the age ofthe cell line by constantly refreshing the cells from low-passage(minimally subcultured) stocks. Cells that have been cold-shocked, asmay happen during winter transport, are less likely to be infected byvirus in the sample. For optimum sensitivity, we incubate cells afterovernight delivery to the lab before inoculating them with patientspecimens. * pH fiascos. A pH shift in the cell culture may cause thesurrounding 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 sodiumbicarbonate solution. Tubes that have become alkaline (dark red or darkpink) should be discarded. Was the program a success? Judge for yourself: During the initialtesting period, we processed about 20 patient specimens in the first 30days. When we offered the service to the community, volume doubled andthen tripled within three months. We are able to perform these tests for about 75 per cent less thanwhat we used to pay the reference laboratory, and we also save up to $18in long-distance courier fees for each specimen processed. A costcomparison is shown in Figure I.
Since we only screen for herpes virus, our methods are simpler thanthose used at a full-service reference laboratory. The need forextended incubation of the cultures–along with the concomitant taks ofpulling and reviewing tubes for several extra days–is minimal. Weestimate that it takes only 10 minutes, spread over five to seven days,to completely finalize a negative sample.
We need about 20 minutes toprocess and confirm a positive culture. Logging the specimen andpreparing reports for distribution requires another 7.8 minutes.
We have cut costs, but not corners. Overall, roughly 30 per centof the tests are positive, and 70 per cent are negative. We’reable to issue a preliminary report within 48 to 72 hours and send outfinal confirmation within five to seven days of specimen receipt. Our familiarity with tissue culturing opened up new possibilitiesfor expanded services. We now use the same fibroblast cells for anotherpractical and much-needed laboratory determination–Clostridiumdifficile toxin detection. The toxin of C. diffickle, a hardy survivorin the human gut following antibiotic therapy, causes pseudomembranouscolitis (PMC) and affects human fibroblast cells with characteristicCPE.
For our next project, we plan to institute direct diagnosis ofherpes simplex from lesion scraping using specific monoclonal antibody staining. This test is a more sensitive means of confirming lesionsthat might be too old to result in a positive tissue culture. On earlylesions, the direct test offers a turnaround time of less than 24 hoursfor confirmed diagnosis, versus two to seven days with the traditionalculture stain method. Asymptomatic carriers, such as near-term pregnantwomen with no visible lesions, are not candidates for this rapid method.Sensitivity will lag behind tissue culture in such cases. For manyother patients, though, the direct test represents a great improvementin specificity over the Pap and Tzanck smears.
Increasingly, hospital laboratories are performing tests in-housethat once were referred to other laboratories. Cultures for herpesvirus require a level of expertise traditionally associated with highlytrained technologists and thus are not practical for small-volumephysicians’ offices or laboratories. This gives clinic andhospital laboratories staffed with medical technologists an opportunityto provide new, quality services locally.
If you carefully plan the launching of a new test like ours, andmanage it through preliminary field trials to avoid excessive costs inimplementation, you to can succeed.