Chlamydia testing: have it your way.

The incidence of Chlamydia trachomatis infection is climbing rapidly, and so is the demand for rapid and accurate detection. Our 230-bed hospital laboratory began offering tests for the pathogen in 1981, following the success of another new in-house procedure, for herpes simplex virus ("Herpes Culturing: Expanding Outpatient Lab Services," MLO, October 1984).

Since then, requests for Chlamydia cultures and direct tests have climbed to 80 to 85 per month, generating monthly profits of up to $1,000. We expect the demand to keep increasing as the incidence continues to rise (nationally, estimates of Chlamydia infection among the adult population range from 6 to 12 per cent) and as more physicians become aware of the in-house tests.

Ours is the sole local laboratory offering Chlamydia testing. In a service radius of 15 miles, covering a population of 70,000 to 80,000, we do work for more than 250 physicians. Only about 50 of them send us specimens for Chlamydia detection, however.

Most physicians tend to treat suspected cases of Chlamydia infection presumptively. As more of them learn about the importance of identifying the organism prior to treatment, our roster of referring clinicians should grow significantly. The projected population increase for our area will expand the Chlamydia workload even further.

Chlamydia are bacterial organisms with the viral characteristic of requiring living cells in which to replicate. Long recognized as the most common cause of blindness in underdeveloped nations, they have been linked to other serious diseases in recent years. We have discovered that C. trachomatis causes urethritis, cervicitis, Proctitis, arthritic complications, and even childhood myocarditis. The organism also causes still-birth, infant pneumonia, and neonatal and adult inclusion conjunctivitis. In addition, more human infertility is now linked to infection with Chlamydia than with Neisseria gonorrhoeae.

Underscoring the importance of accurate detection is the fact that patients treated for an "uncomplicated" gonococcal infection sometimes relapse with a secondary chlamydial infection. In males, this may cause an obvious discharge. But the infection is often asymptomatic in both males and females, which contributes to spread of the infection by unsuspecting, sexually active individuals.

Chlamydia does not respond to the penicillins prescribe to treat gonorrhea. Instead, patients require broad-spectrum antibiotics for several days. Laboratory confirmation of the presence or absence of Chlamydia can prevent the underuse or overuse of these chemotherapeutic agents, save considerable health care dollars, and curb the spread of infection.

Although reliable serologic methods detect antibodies to Chlamydia, they are often of limited value in diagnosing active disease. Two sera are usually required--one collected within the first week of infection during the acute phase and another collected during the convalescent phase. Because chlamydial infections are often asymptomatic, the acute phase may be long past before the disease is suspected and serum is collected.

Without a good rapid test for the Chlamydia agent, weeks often elapse between initial suspicion and laboratory confirmation. During this interval, sexual partners may become infected, the organisms can invade even deeper into body tissues, and the patient--or a developing fetus--can face serious sequelae.

Culture or direct antigen detection methods are the best means of diagnosing active disease. Previously only a handful of specialty laboratories offered Chlamydia cultures. However, streamlined techniques and the availability of commercially packaged ready-to-use cell lines have brought the test within the reach of trained technologists in many laboratories.

To process Chlamydia cultures, a laboratory needs a microscope, a centrifuge, a biological safety cabinet, and a 35 to 37 C incubator. A -70 C freezer is helpful when specimens must be stored for later testing or for repeat testing in the event of, say, incubator failure.

We began preparing for this new test by attending workshops and seminars on Chlamydia culture procedures. Once we knew the basics, we solicited specimens from a local clinic and built up our confidence by performing controlled preliminary tests.

To recruit physicians, we supplied the appropriate containers, swabs, and transport fluids and instructed their staff members on the importance of proper collection and handling. We made a presentation with slides in physicians' offices to emphasize the need for a diffeent approach to culturing for this particular organism.

In isolating gonococcus, for example, purulent discharge is a productive specimen. Chlamydia isolation, on the other hand, requires epithelial cells or scrapings of the infected tissues. Pus cells do not yield viable Chlamydia and may carry contaimenants that will destroy the cell cultures. While endocervical specimens are necessary to isolate the organism from females, laboratory recovery is enhanced if the physician also obtains a urethral specimen.

Physicians store the specimens in their refrigerators pending pickup by our couriers and transport in ice chests. We batch process the Chlamydia specimens on the evening shift after the last courier run. On weekends, we start testing at 2 p.m.

For several weeks during the program's early days, our tissue cell supplier provided positive control cultures. These samples consisted of laboratory strains of Chlamydia in a transport medium. Both controls and patient specimens were handled the same way to develop the best method for our laboratory and establish a routine that was simple to follow but did not lead to false-negative or false-positive results. This also helped verify the freshness of the cell culture and its ability to support the organisms after transport from the manufacturer to the laboratory.

Here is our procedure:

Step 1: When the specimen arrives--usually a swab of epithelial cells in a Chlamydia transport tube--vortex the tube vigorously for a few seconds. The two or three glass beads in the transport tube help break up the epithelial cells and release the Chlamydia into the solution.

Step 2: Gently centrifuge down the heavy debris at 900 rpm for five minutes. The Chlamydia will remain suspended in the supernatant.

Step 3: Add just two or three drops of supernatant solution to the tissue culture vials. Contamination tends to overwhelm the organism if you add larger volumes of inoculum. Laboratories that process many samples at once can use microtiter-type multiwelled tissue culture plates and inoculate up to 96 samples and inoculate up to 96 samples and controls at one time. Single screw-capped vials are ideal for labs like ours, processing only a few specimens per day. These sterile containers come with a small coverslip in the bottom and are layered with fresh McCoy tissue cells--an ideal support cell for Chlamydia. To enhance infectivity, we pretreat the cells with a sterile solution of DEAE dextran for a few minutes before inoculation.

Step 4: Centrifuging is necessary to insure complete attachment of the organisms to the tissue culture cells. Although standard procedure is to spin the vials for one hour at 2,000 to 3,000 rpm and at 35 C or below, our centrifuge tends to heat up after an extended run--a potentially fatal occurrence for the organisms. We reduced the centrifuge time to 30 minutes and found that control samples showed no lack of infectivity when compared with those spun for the full 60 minutes. This modification also frees the centrifuge more quickly for other procedures.

Step 5: Carefully remove the vials from the centrifuge without agitating. Aspirate the excess inoculum, and add approximately 1 ml of fresh Chlamydia tissue culture maintenance medium to nourish the cells during the next two days of incubation.

Step 6: Incubate the vials at 35 C. Organisms are sometimes visible as early as 18 hours after inoculation, but we found that waiting 36 to 48 hours yields the best results and takes the least examination time. The inclusions are at the optimum size, and the cell culture is still in good condition.

Specimens incubated too long--80 to 96 hours or more--may be uninterpretable due to spontaneous death of the McCoy cells or an overgrowth of Chlamydia, which eventually kills the cells. Contaminating bacteria or yeast can also overgrow the cells during extended incubation and disrupt any inclusions.

Step 7: Carefully remove the coverslips from the vials and then stain with a single, high-quality monoclonal immunofluorescent antibody directed at the several serotypes of C. trachomatis. Less than 1 micron in diameter, Chlamydia is too small to be detected by ordinary Gram stains. But chlamydial "inclusion bodies," the clusters of the organisms within the cytoplasm of the cells, are often several microns in diameter and easily visualized.

Fluorescent methods allow slides to be read in a minute or two with great accuracy, as the inclusions stain brightly against a dark background. Laboratories without a fluorescent microscope can use newer immunoperoxidase stains to detect the inclusions in tissue culture. The accompanying photos illustrate the two approaches. We scan the coverslip under 250X magnification to locate suspicious bright areas and then switch to a magnification of 450X or greater to confirm the presence of inclusions in the cell cytoplasm.

An iodine stain has been used successfully to detect inclusions in cell culture. While this stain is less expensive, it is also less sensitive and less specific. Swab debris can mimic inclusions and other starchy deposits not related to the glycogen-rich inclusions and thus confuse technologists.

Giemsa stains have also been used to detect inclusions, particularly in direct patient scrapings. The inclusions often stain very poorly, however, so it may take several minutes to verify their presence. Using a darkfield condenser may make the Giemsa technique more sensitive.

Step 8: Report results promptly. Our standard procedure is to report the condition of the specimen as received in the laboratory as well as the results of the completed test. If a specimen is inappropriate--pus cells instead of epithelial cells, for example--or arrives in a leaking transport tube, we immediately tell the physician that re-collection is necessary for an accurate test report.

When tissue cultures are compromised by sample toxicity or contamination, which results in a suboptimal cell layer in the vials at the time of staining, we subculture the sample or repeat the inoculation from the original residual specimen, kept forzen at -70 C.

Positive specimens may contain only one inclusion or many having characteristics consistent with Chlamydia infections. Chlamydial inclusion bodies tend to be oval and sparkling. Most of the culture vials contain less than 10 inclusions, but we have observed some samples where every cell was infected, and the slide resembled a positive antinuclear antibody test.

Artifacts may be seen occasionally in negative specimens, even with fluorescent stains. An experienced microscopist will rule out them out as chalmydial inclusion bodies by noting their different color--yellow rather than apple green--or shape and size. On immunoperoxidase, RBCs may resemble Chlamydia. This artifact can be eliminated with pretreatment using equal amounts of 1 per cent hydrogen peroxide and absolute ethanol.

The rate of positives in our initial study was only about six out of 100 specimens tested. (In contrast, 28 to 30 per cent of our herpes cultures are positive.) If the laboratory processes only a few Chlamydia cultures each week, technologists may have to search several weeks before finding a positive specimen. Running positive specimen. Running positive specimen. Running positive control cultures at least weekly minimizes discouragement and maintains staff confidence.

Once physicians' offices became adept at collecting the proper type of specimen and minimizing contamination, our rate of positive specimens climbed to a current level of 7.5 to 9.3 per cent. The rate may be much higher or somewhat lower in other labs, depending on the patient population.

Figure 1 demonstrates how much we save by performing Chlamydia cultures in-house rather than sending them out. Reference lab fees plus transportation costs are four to six times our own $11.78 expense per test. We offer the test to physicians' offices for a maximum fee of $26, including courier costs. Positive tests are phoned directly to the clinician. Written reports for both positive and negative tests go out within 48 hours of specimen receipt, which trims at least a week off reference lab turnaround time.

Tissue culture procedures have their share of problems. the most common sources of error are false negatives due to improper collection and setup or transport delays. In addition, transport or storage at the wrong temperature may allow contaminating microflora to overgrow the delicate chlamydial organisms and ruin the tissue culture cells. There's still some debate about the optimum temperature for this organism, but we find that 4 to 8 C works best.

The very tissue upon which Chlamydia thrives can also be the organism's worst enemy. McCoy cells, for example, are a rapidly growing cell line. To prevent overgrowth and a resultant reduced sensitivity to Chlamydia attachment, the supplier adds cycloheximide to the monolayers, which slows cell growth. To further preserve the cells, we incubate for only one day upon receipt and then store at room temperature.

We also have the manufacturer ship only half our weekly order as "ready to use," since natural deterioration can affect sensitivity by the fourth or fifth day. The remaining vials come in with a "less-than-confluent" cell layer. These underinoculated sparse-celled vials keep growing and are ready to use, or at the peak of their ability to become infected, by the end of the week.

Tissue culturing isn't practical for all laboratories. Those located in remote areas sometimes have trouble obtaining supplies promptly or in good condition. Smaller labs may lack the equipment necessary to process tissue cultures or store the inoculum appropriately. If the demand for Chlamydia culturing is unpredictable, it is difficult to accurately anticipate tissue vial usage. This can mean wasting unused cells and reagents or facing shortages when supplies are most needed.

Fortunately, Chlamydia can now be detected directly in the patient sample. Physicians need only roll the specimen swab over a glass slide, which is subsequently stained in the lab using a monoclonal antibody tagged with a fluorescent dye. This non-culture method offers excellent sensitivity (93 to 95 per cent) on almost all patient specimens. surgical aspirates are still best done by culture, due to the dilution effect of body fluids.

The direct test has the added advantage of providing same-day results. It takes two days to complete a culture, while direct test results are available within one day of specimen receipt. technically, the tes takes only 30 minutes. While we don't see much demand for tha type of Stat response, an outpatient clinic might.

A good fluorescent microscope is the only laboratory equipment you need. Biological safety cabinets and centrifuges are unnecessary, and no freezer space is required.

We supply a convenient collection kit for the direct test. The kit contains instructions, a matted slide, proper swabs (broad-tipped and flexible-shaft, thin-tipped), and an acetone fixative vial. Once the specimen is collected and rolled on the slide, the vial of acetone fixative preserves the specimen at room temperature pending the laboratory test.

As with all direct tests, it is extremely important to collect an adequate amount of specimen. Many organisms must be present before even one can be seen in each oil immersion field under the microscope. We first scan the slide under a low-power objective (100X) to verify that the right type and number of patient cells are present. No cells or just a few cells mean the specimen must be re-collected--there is not enough to guarantee a true negative or positive result.

Assuming the specimen is adequate, we scan at a higher magnification (400X) and look for suspicious yellow-green fluorescing particles. We do not see intact inclusions, but instead find single elementary or reticulate bodies of the organisms. Usually, only a few are seen on each slide. On occasion, however, a heavily positive sample delights us with its "starry sky" appearance (see photo below).

The direct test is very reliable. About 10 per cent of our directs are positive for Chlamydia, a rate comparable to the more sensitive culture technique. Since we don't perform both a tissue culture and a direct test on every specimen, or send out split specimens anymore, I can't set a rate for false-positive and false-negative results. During the initial study, however, we isolated more organisms--true positives--than the reference laboratory found.

Our laboratory currently offers both the culture, which is particularly advantageous with surgical specimens when extremely scant organisms are present in the sample, and the direct test. The culture/direct mix now stands at about 70/30. We expect this ratio to flip in the next six months and the direct test eventually to replace the culture entirely.

The direct test is fast to perform, almost as accurate as culturing, and less expensive (Figure II). We offer it for $16.20.

By investing in a better oil immersion 40X objective for the fluorescent microscope, we have been able to meet the increasing test demand without unduly burdening our staff. The new objective halved the time needed to accurately scan each slide and greatly enhanced technologist confidence in the new procedure.

Recently, our laboratory has noted a drop in the number of cultures for group A beta hemolytic strep. With the current availability of rapid detection kits, this procedure has moved into physicians' offices. Adding Chlamydia detection to the microbiology workload allowed us to maintain our culture volume while providing a valuable service to the community.

In the future, we plan to aggressively pursue those tests that are truly needed and drop any procedure that either is no longer necessary or better done elsewhere. This strategy should help minimize the lab's dependence on distant facilities, particularly when the bill for send-outs exceeds the cost of accurately performing a test in-house.