February 2011
Feature Story

Anne Paxton

Given that sexually transmitted infections are among the most common infections in the nation, it’s tragic but perhaps not surprising that young people are so often victims. Of the estimated 15.3 million new cases of sexually transmitted disease in the U.S. each year, 3 million occur in people between the ages of 13 and 19, and younger children can be victims as well, often through sexual abuse. Whether contracted through consensual sexual contact or sexual abuse, STIs in young people are a massive public health problem that becomes more serious by the year.

For the clinical diagnostic laboratory, these infections pose special problems because of children’s and adolescents’ physiology, the complications of using for children tests developed for adults, and the sensitive forensic and psychological issues the diseases often raise.

“STDs are a disease of adolescence and young adulthood, and the sequelae come later in life, so it’s very important to screen and treat them early,” says Amy L. Leber, PhD, associate director of clinical/molecular microbiology with Nationwide Children’s Hospital in Columbus, Ohio. “This is a problem that’s not addressed often in terms of lab testing and the complexities around that, yet it affects so many labs.” In screening guidelines, consent for testing, specimen collection, molecular assays versus culture, and the interface with child protective services and law enforcement, testing for STIs in children differs markedly from testing of adults.

Dr. Leber organized a panel of speakers at the American Society for Microbiology’s May 2010 meeting in San Diego, on the topic of sexually transmitted diseases in adolescents and children, and during those talks as well as in interviews with CAP TODAY, leading specialists in this area described the latest research findings, policy developments, and controversies. While the Centers for Disease Control and Prevention has just released new treatment guidelines that address some of the issues surrounding clinical and forensic testing of STIs in these populations, including potential sexual abuse in children (see “CDC sets new guidelines for STD diagnosis,”), many unresolved issues remain ahead for laboratories.

Because of the seriousness of STDs and the urgent need for accurate and sensitive diagnosis, they have been among the earliest targeted for molecular testing, Dr. Leber says, noting that tests for chlamydia, gonorrhea, and HPV are “some of the oldest FDA-approved molecular tests on the market.”

“The next wave will be other agents like Trichomonas vaginalis and herpes simplex virus testing,” she says.

The Food and Drug Administration has cleared one test for herpes simplex virus, she notes, but it is only for female vaginal swabs and excludes anyone under 18. “That’s a worry of mine —that we’re developing tests but many manufacturers have trouble including adolescents in clinical trials because of consent issues,” despite there being much more of these infections in younger people. As a result, “it’s up to each laboratory to verify performance of these tests in adolescents.” The FDA has changed its review process and is saying more and more often that there have to be limits around age, but she considers this policy an impediment to improving the health care of young patients.

Dr. Leber’s hospital runs the busiest pediatric emergency room in Ohio, and additional patients come through the adolescent medicine clinics and community physician practices. For the last five years, Nationwide Children’s has used the Gen-Probe Aptima system as the primary test to screen sexually active adolescents and young adults. “When we first brought it in-house, every positive was re-tested using the same test, which was a recommended algorithm at the time based on the CDC’s 2002 screening test guidance, but we looked at our data and found it added nothing to the precision of the result, so we stopped repeating routinely.”

However, suspected sexual abuse alters the stakes. “Culture had been considered the gold standard both legally and medically for those patients, but we know we’re not doing a good job at least medically by just using culture, so at our institution we use Aptima NAAT [nucleic acid amplification testing] and then the positives are sent for confirmation using a different molecular target.”

The legal standard for consenting to testing varies from state to state, but in Ohio, parental consent is not required for STD testing, and children and adolescents do not need to sign a separate consent; STD testing is covered by their consent for medical treatment. “For HIV testing you do have to be greater than 13 years, and in Ohio we have gone to the ‘opt-out’ model for HIV.” This is in line with CDC recommendations for universal screening, she notes.

But nationwide, states have been slow to adopt that model for HIV testing. “Many states have removed requirements about consenting and counseling, yet it’s very hard to get them to adopt universal screening. Even here, we’re not a model, and we’ve been implementing it in stages, in part because of the financial resources needed and clinician acceptance issues, and because it requires drawing serum, which patients don’t really want. So there are other sample types like oral fluids that we’re going to explore.”

The ED at Nationwide Children’s is currently pilot-testing an HIV screen whenever an STD test is done. “Even though it’s a point-of-care test, we’re doing it in the lab, so we assured the clinicians they’ll get the result in-house and can turn it around and use it the way it’s intended.” Anytime an adolescent is in a health care setting, Dr. Leber advises quick turnaround time. “If you’ve got them captured, you want to do as much as you can and follow through and get them into treatment, because if they leave you’ll lose them. They may never come back.”

The clinic and ER most often use urine as the sample specimen, “because adolescents are comfortable urinating, and there’s no need for a speculum exam,” Dr. Leber says. Chlamydia and gonorrhea testing are part of the standard screening, but other testing orders depend on the clinician. “My opinion right now is we do not screen enough for Trichomonas vaginalis, and that’s very often asymptomatic. So we’re missing a fair amount of it. We’re working to bring in-house a molecular test to screen for that on the urines we already test for chlamydia and gonorrhea, and then on other cervical swabs or vaginal swabs.”

Usually, if the clinician orders any screening beyond the urine test, the laboratory will test for hepatitis, HIV, and syphilis from a serum sample, she says. But fortunately, although there is a significant amount of syphilis in Franklin County where the hospital is located, “for some reason we don’t diagnose a lot of syphilis in our adolescent population.”

The same is not true of trichomoniasis, which is on a steep trajectory in the United States. “It’s probably more prevalent in many populations than N. gonorrhoeae, and many people, including me, would like to see it as a reportable disease,” Dr. Leber says. However, with a wet mount, labs are likely to miss Trichomonas because of low sensitivity. “Right now we’re in the midst of verifying new testing to include use of an amplified test in our ER and adolescent medicine clinic. We ‘d like a less subjective rapid test for use in those settings. We’d also like to have a molecular test that we could use on the same samples for chlamydia and gonorrhea.”

Cost, of course, is a factor when using molecular testing for screening. The other consideration is turnaround time. “Some clinicians feel the time to result is so important that they’re kind of wedded to a rapid test, such as wet mount. But usually if you show them the performance characteristics and they realize how low the sensitivity is, you can get them to change.”

Herpes simplex virus presents special difficulties, she says. There are no current guidelines for screening routinely for HSV antibodies. A special test that distinguishes between HSV-1, long considered HSV “above the waist,” and HSV-2, more associated with STDs, has been available for at least 10 years. “But for whatever reason, maybe because adolescents think oral sex is safer and they transfer HSV-1 to the genitals of their partners, HSV-1 is an STD and is actually on the rise.”

With HSV, PCR testing is particularly useful, and the laboratory has its own laboratory-developed assay. “Culture was once the recommended assay, but PCR is better, particularly when there are older, drier lesions, because there will be less active virus and PCR will be more sensitive.”

An FDA-cleared molecular assay is available for testing herpes, but only for vaginal lesions on females over 18. It is not cleared for spinal fluid, which is the sample type critical for testing neonates, but Dr. Leber believes other manufacturers are working on a test for this sample type.

The laboratory at Children’s has considered discontinuing culture for HSV, as other laboratories have already done. “PCR is more sensitive, there’s not a lot of need to have a viral isolate, we don’t do much viral susceptibility testing, and, honestly, culture is kind of diminishing in importance for labs in general.”

A chronic concern in the lab, however, has to do with the fact that genital lesions are often very “hot.” “They have a lot, a lot, a lot of virus. So we’re testing a lot of very hot samples intermixed with neonatal CSF samples. And we have to be scrupulously careful that we don’t cross-contaminate. Right now we do them separately, but if all the tests are done with PCR, it will pose some challenges in terms of workflow.”

What is the role of the clinical laboratory in the evaluation of cases of child sexual abuse? M. Ranee Leder, MD, associate professor of clinical pediatrics at Nationwide Children’s Hospital’s Center for Child and Family Advocacy, addressed this question in her ASM presentation on the medical evaluation of children suspected of being sexually abused.

“There were more than 3 million cases of sexual abuse reported in children and adolescents by DHHS in 2008, but this is likely the tip of the iceberg,” she says. Judging by interviews of adults who did not disclose abuse that occurred when they were children, “there are probably many more cases than these data reflect.”

“Of the cases that come to medical attention, probably less than five percent have a positive lab result for a sexually transmitted infection,” Dr. Leder says. But a positive lab result can be useful in cases such as with pre-pubertal children. “If a pre-pubertal child has a positive result for gonorrhea or chlamydia, that’s considered strong evidence of sexual abuse, because outside of neonatal cases, the only way to get those types of infection is through contact with infectious genital secretions, usually through sexual contact. That’s where the test can be valuable in diagnosing sexual abuse.”

Dr. Leder presented data from a study the center conducted between 2006 and 2008 using a specific NAAT, Aptima Combo 2, to identify chlamydia and gonorrhea infections. The objectives of the study were to confirm that NAAT is more sensitive than culture, and to discover if there is any difference between NAAT on urine and genital samples in sensitivity and specificity. “Urine is a much easier sample to obtain than a genital swab,” she notes. After evaluating about 400 girls for abuse, the researchers confirmed that urine NAAT and genital swab NAAT were both superior to culture in detecting chlamydia and gonorrhea infections. Sensitivity of urine and genital swab NAAT was nearly 100 percent compared with 24 percent sensitivity of chlamydia culture and 43 percent sensitivity of gonorrhea culture.

“One of the problems with culture, especially for chlamydia, is that the sample must have genital tract cells, because chlamydia is an intracellular organism, and that’s a very difficult specimen to obtain. Also, the required media may not be readily available in all clinical settings. While the media for gonorrhea may be more accessible, getting samples can be difficult, especially in emotionally traumatized patients, because it involves inserting a swab into the genital area.”

Following the study, at the end of 2008 the center discontinued use of genital culture in girls because the sensitivity was so low. “We now use urine NAAT as a screening test because it has been determined to have excellent sensitivity and specificity for both chlamydia and gonorrhea infection.” The center also found that using urine NAAT eliminated the need for swabbing the genital area in approximately 90 percent of patients who required STI testing.

A positive test result for chlamydia or gonorrhea in an adolescent sexual abuse patient may be of limited forensic significance. “The majority of positive test results at our center are in teenagers who are also consensually sexually active. In these cases, it is usually not possible to determine how the patient contracted the infection.” In Dr. Leder’s experience, for a case of suspected sexual abuse in an adolescent to proceed in the criminal court system, the patient usually has to give a clear statement of having been abused. “You cannot rely on the laboratory result alone.”

“Only about half of our pre-pubertal patients with a positive test for chlamydia or gonorrhea disclose sexual abuse in an initial interview,” Dr. Leder says. These cases are challenging for child protective services and law enforcement because the child has an infection that is diagnostic of sexual contact, but without a disclosure it is difficult to determine the identity of the alleged perpetrator. The child may be placed into counseling and eventually disclose sexual abuse. Law enforcement may use a positive result as an investigative “tool,” perhaps to ease a confession from an alleged perpetrator. But several data points are necessary, she adds. “We have not had a case involving a pre-pubertal child go to criminal court, to my knowledge, based on a positive lab test alone.”

“The legal gold standard for establishing a chlamydia or gonorrhea infection varies from region to region,” she says. Child abuse pediatricians in Arkansas, California, and Ohio, for example, have reported that the standard for infection is a positive NAAT confirmed with a second target. Other child abuse specialists in Alabama, Washington, DC, and southern Nevada report that the standard is genital culture. Similarly, chain-of-evidence requirements vary from state to state and are often different from forensic procedures used for adult samples.

Patients have the right to refuse exams, Dr. Leder notes, though if they are younger than three years, “we usually proceed because young children often protest any type of exam. When they’re old enough to reason with, we try to make them comfortable and demystify what we’re doing—explain it’s not painful, allow a caregiver in the room—and most of the time we can get them to comply.” Occasionally adolescents refuse. “The case can’t proceed very far if the teenager isn’t willing to cooperate,” but usually even an obstinate teenager is willing to give a urine specimen.

She considers the CDC’s new guidelines, which recommend the use of urine NAAT for chlamydia and gonorrhea detection in sexually abused girls, to be a positive step toward better evaluating these patients.

The quest for laboratory testing that is accurate yet noninvasive was one of the key motivations behind the CDC’s multicenter study of nucleic acid amplification tests in children under evaluation for sex abuse, said Carolyn M. Black, PhD, director of the Division of Scientific Resources of the CDC’s National Center for Emerging and Zoonotic Infectious Diseases. In her presentation, “Laboratory Testing for Chlamydial and Gonococcoal Infections in Children: Beyond the Culture Paradigm,” she outlined some of the important differences between testing for children and adults.

In 2008, 1.03 million cases of Chlamydia trachomatis were reported; for Neisseria gonorrhoeae, the number was 337,000. That 70 percent of chlamydia cases and 50 percent of gonorrhea cases are asymptomatic in females means substantial under-reporting is inevitable. In addition, Dr. Black said, “A paradox in testing children for NG has emerged: Even though culture is the gold standard, there has been a decline in laboratory capacity to perform culture and antimicrobial susceptibility testing, attributed to the rise in nucleic acid amplification testing.”

Culture has been the gold standard for testing children for chlamydia and gonorrhea, but molecular testing, particularly NAAT, is the gold standard for chlamydia in adolescents and adults. Tests for chlamydia and gonorrhea are commonly marketed and sold as combination tests, however, “so the drive for improved performance in CT testing has influenced the availability and performance of NG tests,” she explained. The result: Fewer laboratories are performing culture for NG.

In evaluating children for sexual abuse, the laboratory can play a critical role in deciding how likely it is that abuse occurred. A determination of possible abuse keeps the child and guardians in the same situation as when they started, Dr. Black said. But the laboratory can bump up a ‘possible’ to a ‘probable’ with certain results, and a ‘probable’ to a ‘definite’ with a positive lab test for gonorrhea, HIV, or syphilis.

Laboratories are faced with a dilemma in testing for chlamydia and gonorrhea, Dr. Black said. “Culture, the gold standard for diagnosing chlamydia, is very specific and therefore generates very few false-positive tests. However, the sensitivity of culture for CT in children is extremely low.” In part, this is due to a sampling issue. “Traditional sampling requires invasive sampling of the endocervix, but this cannot be used in children due to their age and the additional trauma collection of samples would cause, including occasional restraining.”

NAATs have replaced culture in screening of adolescents and adults for chlamydia, but sexual abuse allegations alter the picture, because clinical testing is for the purpose of treatment, while forensic evidence has a second objective: to provide legal evidence of a crime. “To conduct NAATs, we only need a few copies of the organism and no invasive specimen is required,” Dr. Black said, but the test has traditionally not been accepted in the U.S. for forensic testing.

To determine if NAAT is suitable as a new forensic standard of diagnosis for CT and NG testing in children, the CDC conducted a multicenter study of NAATs in children under evaluation for sexual abuse. In addition to evaluating the sensitivity and specificity of noninvasive NAAT relative to the forensic standard of culture, Dr. Black said, “we wanted to ask the important question: Does urine testing work as well in children as in adults, and is it accurate enough to become the new forensic standard in the U.S. for chlamydia testing?”

Researchers evaluated infants and children from 0 to 13 years in four cities to answer this question. The overall result, “NAAT detected more infections than culture, and this was particularly true for chlamydia. Urine testing resulted in 40 percent increased sensitivity for detection of CT and 13 percent increased sensitivity for NG, while urine NAAT and culture failed to detect infections in two children who were infected only at the rectal/anal site.”

Based on the study, CDC concluded urine NAAT tests reliably detect specific biomarkers of sexual activity and significantly increase the number of children with a positive STI diagnosis, Dr. Black said. The study “substantially strengthens the evidence that NAATs should become the new forensic standard of diagnosis of CT and NG in sexual abuse populations.” Among other findings, the study determined that if both CT and NG NAAT results are considered as evidence, “we could move eight children from categories of ‘probable’ to ‘definite’ abuse.” In addition, the study supported adoption of urine testing as a new forensic standard that would reduce discomfort and trauma and provide benefits in diagnosis, infection control, and protection of the child and other potential victims.

Related Links

• CDC sets new guidelines for STD diagnosis
• Finding multiple genotypes in adolescent females