Clinical Laboratory Strategies: August 12, 2010

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Detecting HSV Infection in Women in Labor with Rapid RT-PCR
How will this new assay be used in practice?
By Genna Rollins 

Neonatal Herpes Simplex Virus (HSV) infection is a serious complication of childbirth. Unfortunately, diagnostic procedures, like visual inspection of maternal herpetic lesions, have had limited success in identifying the disease when treatments can be most effective. Real-time quantitative polymerase chain reaction (RT-PCR) assays have proven to be sensitive and reproducible in the diagnosis and management of other viral diseases. This issue of Strategies examines a study reporting development and validation of a rapid HSV RT-PCR assay for use in women in labor.

Neonatal Herpes Simplex Virus (HSV) infection occurs in up to 1 in 3,200 live births and can cause devastating neurological complications and even death, despite antiviral therapy. Current prevention guidelines for the condition call for suppressive antiviral therapy in women with active recurrent genital herpes starting at 36 weeks of gestation. Otherwise, there should be visual inspection for maternal herpetic lesions at the time of labor, with cesarean delivery if lesions are found. However, this strategy has not significantly reduced the incidence of neonatal HSV. In addition, ongoing research has shown that most women with HSV do not have signs and symptoms of the infection and that the main means of transmission from mother to infant is exposure to infected maternal secretions during the baby’s passage through the birth canal. But there is no clinically useful method of detecting HSV in the genital tract during labor. For example, viral cultures collected at this time are not helpful because results are not possible within a timeframe that allows for effective treatment.

If the state of science around detecting maternal HSV infection in a timely manner appears to be less than ideal, real-time quantitative polymerase chain reaction (RT-PCR) assays have proven sensitive and reproducible in the diagnosis and management of other viral diseases, albeit usually in state-of-the-art clinical laboratories rather than at the point-of-care. The authors of a recent study sought to develop and validate a rapid HSV RT-PCR assay that could detect HSV-1 or HSV-2 from genital swab samples taken in the delivery room and be reported back within 2 hours (Obstet Gynecol 2010;115:1209-16).

“We were interested in developing the test because our research group has been working on neonatal herpes for more than 30 years. To date, there’s not been a fast, reliable means of detecting herpes in the genital tract of women in labor to guide management decisions about the mode of delivery and post-partum care,” said lead author Carolyn Gardella, MD, MPH, associate professor of obstetrics and gynecology at the University of Washington in Seattle. “We thought this assay would be an important tool we might be able to use in neonatal herpes prevention programs in the future.” The researchers hypothesized that results from their rapid HSV PCR assay would not vary significantly from those of a standard 4-hour HSV PCR assay.

In setting up the assay, the researchers used samples from a buffer-only solution as a negative control group, swab samples from nonpregnant patients with herpetic genital lesions, and mucotaneous genital swab samples from pregnant women presenting in labor. Experienced PCR technologists in the molecular diagnostic lab prepared and aliquoted reagents for the samples and controls. The investigators did this to make the rapid PCR procedure as easy as possible to perform by less-experienced technologists in a less-stringent lab setting.

The researchers extracted DNA using a kit with an automatic extraction machine and eluted it into 100 mL of buffer AVE from the manufacturer’s kit. Ten microliters of eluted DNA was then used for PCR, with one negative and one positive control included in each extraction run to control for contamination and DNA recovery. Following the rapid RT-PCR test, the researchers reanalzyed the samples using a previously validated HSV PCR assay as the comparator gold standard. The glycoprotein B gene primers and probe used in the study pick up both HSV-1 and HSV-2 and have been validated in other studies, according to the authors.

The researchers found that defining a positive test as positivity in both wells minimized the potential for false-positive results due to low-level contamination. In addition, they applied three different interpretation strategies using cutoffs from 50-500 copies/mL and found that requiring both wells to detect ≥150 copies/mL maximized specificity at 96.7% without appreciable loss of sensitivity, at 99.6%. The positive (PPV) and negative predictive values (NPV) at this cutoff were 96.7 and 99.6, respectively. This PPV and NPV also assumed a prevalence of 10.8%, which the authors had observed as the prevalence of HSV shedding in other studies. The 150 copies/mL cutoff also corresponds with the standard cutoff of the gold standard HSV PCR assay.

The rapid assay took a median time of 2 hours to perform, from sample collection to results. The researchers concluded that this timeframe would be sufficient to enable physicians to start appropriate interventions to reduce the risk of HSV transmission to the neonate. However, laboratorians not involved in the study questioned whether this turnaround time would be doable in actual practice. “I think it’s an optimistic timeline. A molecular person who is very experienced might be able to handle that if they work very diligently and fast, but on a routine basis it might be a challenge,” said Helen Fernandes, PhD, director of the molecular diagnostic lab at the University of Medicine and Dentistry of New Jersey in Newark. Dr. Gardella said the key to the median 2-hour time to complete the test was due to the hospital’s molecular lab preparing and aliquoting reagents ahead of time.

Although Gardella indicated that the study was an important step in establishing a rapid, accurate diagnostic tool for HSV, she emphasized the need for further research to understand how the test actually might be used. “We’re still working out how it would be put into clinical practice. We need more research about the cost-benefit and the number needed to treat to see how it would be applied,” she explained. “It’s likely that this test would be applied but then a serologic test also might be used to determine the risk for an individual woman with HSV.”

She also encouraged other researchers to study the assay. “We’d be happy to have people replicate it so we could confirm if the timeframe is generalizable,” she said. Further analysis of the assay bears watching, according to Barbara Romanowski, MD, FRCPC, clinical professor in the division of infectious diseases at the University of Alberta (Canada) in Edmonton. “Stay tuned. This test is not ready for use in the field and still requires much further testing in real clinical situations to determine if it would change or improve the management or outcome of pregnant women who present at term with possible genital herpes. The ultimate test would be, does it prevent neonatal herpes?”

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