The notion of children as miniature adults may have faded long ago, but the magnitude of the differences between children and adults can still be surprising. And they are differences that can have a direct impact on the clinical laboratory. It’s not only children’s smaller size but also their developing immune systems, their microbial flora, and their exposure to pathogens that can significantly affect the diagnosis of infectious disease, say Alex J. McAdam, MD, PhD, director of the infectious diseases diagnostic laboratory at Children’s Hospital Boston and associate professor of pathology at Harvard Medical School, and Karen M. Frank, MD, PhD, associate professor of pathology at the University of Chicago Medical Center.
In a presentation at the American Society for Microbiology meeting earlier this year, and in recent interviews with CAP TODAY, Drs. Frank and McAdam mapped out some of the complexities of pediatric microbiology for six infectious diseases. Clinical laboratories, they say, need to be aware of and educate clinicians on the limitations and special significance of children’s diagnostic test results.
There are essentially four reasons why testing of children differs from testing of adults for infectious diseases, Dr. McAdam says.
“The biggest and most obvious factor is that children are much more vulnerable to pathogens. When children are born, they have immunoglobulin in their blood which they got from the mother across the placenta. But that antibody lasts only for about six months or a little longer; then it’s gone. So they have not encountered yet many of the pathogens they will encounter throughout their lives, and they have not built up antibodies or other specific responses.”
Second, bacterial flora change with age. For example, about half of two-week-old children are colonized with S. aureus, but the percentage declines steadily until it levels off at about age four, while the percentage with N. meningitidis climbs from age eight until peaking in a person’s early 20s, Dr. McAdam points out. As a result of their different flora, some tests are not sufficiently specific for children and thus yield false-positive values.
“The most common example is C. difficile, which causes colitis in people who have been treated with antibiotics. Young children are very frequently colonized with that organism. If you test their stool it’s positive for C. difficile in about four percent of healthy children, because you’re detecting normal flora. There aren’t good ways to distinguish between C. difficile that is a pathogenic or normal flora, so you have to work with the clinical staff and let them know the difficulties of interpreting the results in young children.”
Third, children’s smaller size means they have a smaller blood volume, which complicates sample collection. “We have to collect so many samples from inpatients that phlebotomy-induced anemia is surprisingly common in hospitalized children. People will try to collect small amounts of blood to avoid causing anemia, such as a mL or half mL, but the price is that the sensitivity of a blood culture goes down as you collect lower volumes.”
The last difference is the kind of pathogens to which children are exposed. As every parent of a toddler knows, “They just don’t act like us,” Dr. McAdam says. “They touch everything they encounter, and they put things into their mouths and eyes.” As an example, Baylisacaris is a worm that is normally a pathogen in raccoons. “It can be acquired by humans if they eat soil contaminated with raccoon feces, so nearly all cases in humans have occurred in toddlers.” Rabies is another example of an infectious disease to which children are more likely to be exposed because of their behavior. Of the roughly 55,000 cases of rabies in the world each year, nearly all leading to death, 30 percent to 50 percent are in children younger than 15. “That’s presumably because kids interact with dogs differently than adults interact with dogs.”
In pediatric care, an important laboratory issue is distinguishing rapidly between bacterial and viral meningitis, Dr. McAdam says. Patients with the latter disease—usually caused by enterovirus—will recover on their own, while mortality for bacterial meningitis ranges from one percent to five percent and significant sequelae, such as seizures and hearing loss, will afflict another 10 to 30 percent. “Patients with bacterial meningitis need to be admitted to the hospital for antibiotic treatment, but most patients with viral meningitis can be sent home,” he notes.
At Children’s Hospital Boston, “we do a number of PCR tests, and most are batched and performed two or three times a week.” But during two shifts a day, from 7 AM to 10 PM, enterovirus PCR is done as samples come in. “We thought it was very important that we do enterovirus much more quickly.”
Children’s Hospital plans to determine whether it will cut costs by performing PCR, because despite PCR’s added expense, it can help rule out several unnecessary hospital stays, Dr. McAdam says. “A modeling study predicted that if you had high enough prevalence, over 5.9 percent, then enterovirus PCR would save money, and we have a prevalence of 20.3 percent, so I think it really does save money. We’re now doing studies to determine if that’s the case.”
With PCR being developed so rapidly, many tests are becoming much easier to do, he points out. “Some systems have integrated sample processing and purification of nucleic acid with the PCR tests, so that they require much less hands-on technologist time and less technical expertise to perform.” For a small but growing number of PCR tests, turnaround time has become much faster, and enterovirus PCR is a leading example. “PCR is now the standard-of-care test for enterovirus, not only because it’s faster and much easier to do than viral culture, but also because it’s much more sensitive.”
Septic arthritis is another disease that appears much more commonly in children than adults, with approximately half of the pediatric cases occurring in children younger than two. “The interesting thing about it is that the pathogen called Kingella kingae, possibly the most common cause of septic arthritis, is not well detected by culturing joint fluid directly on solid agar plates. That’s a very insensitive way to detect this pathogen. It is much more sensitive to culture the joint fluid in a blood culture bottle.”
That procedure gives a big bump in sensitivity, Dr. McAdam says, “and you get a further bump in sensitivity by using PCR to detect the organism.” But the infectious disease physicians at Children’s feel the empiric therapy they use for joint infections (the treatment initiated before arriving at a firm diagnosis) would cover the organism, making PCR not worth the added work and expense.
The problem of children’s lower blood volume is particularly important in cases of bacterial sepsis, Dr. McAdam says. “Just over 20 percent of children who have bacteria in their blood have fewer than one colony-forming unit of bacteria per milliliter of blood—a very low concentration. Because of that, the more blood you culture, the more likely you are to detect the presence of bacteremia.”
In the past it was thought that children’s blood concentrations of bacteria were very high. “You still occasionally hear that,” he says. In fact, he points out, the 20th edition of Clinical Diagnosis and Management by Laboratory Methods states that “in infants and children, the concentration of microorganisms in blood is higher, and collection of 1 to 5 mL of blood per culture is adequate.”
But that statement is not correct. “It’s very clear that children can have very low levels of bacteria in their blood, and culturing low volumes makes it less likely that you’ll detect them.” That doesn’t mean high volumes of blood should be collected, though. “You just have to be aware you may be missing bacteria,” he says.
Despite its reputation, PCR does not solve this problem. “You use a small sample volume in PCR, so you’re still unlikely to detect very low levels of the organism. It’s surprising because PCR is the proposed ‘solution’ to nearly every problem in microbiology. Maybe it will be in the future, but right now, it’s not. The solution is to do your best to get an adequate volume of blood, and if you can’t, then educate clinicians that you may be getting a false-negative and they may want to consider prolonging the empiric therapy.”
Another difficulty with blood volume relates to anaerobic bottles. “With adults, when you collect blood cultures, you routinely collect aerobic and anaerobic bottles in paired sets. With kids, because of the volume issue, you want to be as efficient as possible with the limited blood available. And it turns out most of the time we don’t need to collect an anaerobic bottle, because anaerobes are a rare cause of sepsis in kids.” Some pediatric populations are exceptions, Dr. McAdam says. “You do want to do an anaerobic blood culture for patients who are immunocompromised with a lack of neutrophils, or in cases of other infections specifically associated with anaerobes, such as intra-abdominal and head-and-neck infections.”
In at least one area, he thinks, the differences between children and adults are perhaps not as important as people might think: pediatric blood culture bottles. “The manufacturers market these bottles and they have lower volumes of broth and some have slightly lower concentrations of the anticoagulant, SPS. So the reduced volume is supposed to give an optimum sample-to-broth ratio and allow the growth of pathogens, such as serum meningitis, that are inhibited by SPS.”
But in fact, these factors probably don’t matter very much, in Dr. McAdam’s view. “Unfortunately, we don’t have the head-to-head studies that would really answer this question, so the debate goes on and on. But in my opinion it’s probably not necessary to use pediatric blood culture bottles. For a large institution that serves both adults and children, it’s a reasonable practice to just use the adult bottles.”
Respiratory viruses also tend to affect children much more than adults, and respiratory syncytial virus, or RSV, is the most common cause of bronchiolitis and pneumonia in children under one year of age worldwide, Dr. Frank says. “Adults get RSV as well, but the implications of the disease for children are different. Children are more likely to get hospitalized for RSV, and morbidity can be significant in patients with any other conditions, such as asthma.”
The available methods to test for RSV are rapid antigen assay, immunofluorescence, cultures, and molecular tests. “The issue is what test is available in the lab, and many more labs can run the rapid test, which can be done in 15 to 20 minutes, than can run molecular tests,” she says. Despite the relatively low sensitivity of the rapid antigen assay, “physicians tend to prefer to have results as quickly as possible to determine their management of the patient.”
PCR tests for RSV are reported to have the highest sensitivity in some studies, and molecular assays with shorter turnaround times, such as the RVP Fast Assay, a streamlined version of the Luminex xTAG Respiratory Virus Panel, are still being evaluated. However, the majority of labs do not perform molecular tests, Dr. Frank notes; in a recent CAP Survey, 2,500 participating labs performed bacterial culture, 1,700 used rapid RSV antigen detection, and only 80 performed molecular testing.
Some of the laboratories that do use molecular assays, however, have made unexpected findings about children’s illnesses. “One thing relatively new in recent years is finding more than one virus in the patient,” Dr. Frank says. “In the past, you might not have even detected it, or if you had two positives you might have thought you had cross-reactivity in your antibody assays. But now, with the specificity of molecular assays, you can know that it’s real as opposed to a cross-reaction. There are a number of studies showing that mixed infections are more common in children than we realized.”
These mixed infections more often involve other viruses such as influenza A or B, metapneumovirus, or rhinovirus, she says. In such cases, “both may or may not be causing disease. But when you are detecting only one pathogen, it’s often RSV.” About 49 percent of the time, studies show, RSV is the sole pathogen. “So it really is causing the disease; the mixed infections were found more often with other combinations.”
For RSV, she summarizes, the recommended diagnostic test is validated molecular testing, if available, for the best sensitivity. Clinicians should be advised about the possibility of false-negatives with the antigen assays. And ideally, all negative RSV antigen assays should be reflexed to a secondary method. To avoid low-positive predictive values when RSV prevalence is low, she recommends that RSV be offered only during RSV season for the specific geographic region. For patients in critical care, a low threshold should be used to test for bacterial co-infections.
Collecting specimens to test children for RSV is an area with special complications. “What you actually want is nasal pharyngeal aspirates or nose/throat swabs for RSV, but for flu, nose/throat swabs are not recommended. Because we want to test a patient for a spectrum of pathogens, and there’s overlap between RSV and influenzas, nasal pharyngeal aspirates are the most commonly used. But there are particular problems with those collections with children: They don’t like it. You might argue it’s harder to get than a blood specimen. If the nurse or person collecting doesn’t do a good job, you won’t get a good specimen.”
Clinical laboratory testing plays a key role in controlling infection with adenovirus, which can cause severe and disseminated illness, especially in immunocompromised patients. Outbreaks in health care facilities are often associated with the ophthalmic equipment physicians use to check the eyes of newborns, Dr. Frank says. And because sometimes adenovirus is asymptomatic, rapid testing is needed to implement isolation. But no adenovirus detection method is clearly superior to other methods. Viral culture is considered the gold standard, but it is labor-intensive and prone to false-negatives, and it may take days or weeks to receive results.
For that reason, “If you have a suspicion, or as soon as you think you might have an outbreak, you might use multiple testing methods and possibly work with the state public health labs as well,” she recommends. “If you do have an outbreak, it’s a medical emergency and you have to get the test results to treat the patients. You basically implement precautions across the board, and then your testing may allow you to cohort groups or eventually to remove some patients from isolation if all their tests are negative after a period of time.”
Diagnosing tuberculosis in children presents other issues of potential misdiagnosis and problems of specimen collection. Though tuberculosis is less common in the United States than elsewhere (there were 12,904 cases here in 2008, 786 of them in children 14 and younger), it is definitely a disease that tertiary care centers have to deal with, Dr. Frank says.
“We have immunocompromised populations, we have people coming from other countries, we have untreated adults in urban centers, and children can get it from their parents.” Non-specific signs and symptoms sometimes make diagnosis difficult. But there are many clinical laboratory testing factors that can lead to misdiagnosis, including low bacillary load, the specimen recovery method, and the sensitivity of the diagnostic assay.
For example, “Depending on their age, children may not cooperate to cough up a specimen for you, so the recommended specimen is three samples of gastric aspirate.” However, the sensitivity of gastric aspirate is only about 20 percent to 50 percent. “In adults with pulmonary TB, the sputum smear could detect up to 75 percent of the cases, but in children who have TB, less than 10 percent were having a positive test result from either the sputum smear or gastric aspirate,” Dr. Frank notes. “Even if the stain for acid-fast bacilli is positive, the bacterial isolate may or may not be TB; you still have to go on and do additional testing to confirm the species present.”
As with adenovirus, the benefits to be gained from molecular testing for tuberculosis are not clear. “With viral testing such as for flu and RSV, when you compare culture, rapid antigen, DFA, and molecular, there was clear improvement with the molecular. But that’s not so clear with direct molecular TB methods. The sensitivity numbers are not all that great.”
However, she notes, there are molecular methods for isolate identification that are useful in diagnosing TB. “When we get a positive isolate for TB and grow the mycobacterium in the microbroth tube, we do the sequencing and we get identification very, very fast. We get it within a day after isolate growth, compared to earlier times with the old biochemistry methods in tubes, which took weeks. So molecular testing has been a huge benefit for identification of mycobacteria.”
Interferon gamma release assays are an effective means of testing adults for TB, but once again, studies have shown children are different, Dr. Frank says. “In a normal non-immunocompromised population, the number of indeterminate results is highest in very young children.” The Centers for Disease Control and Prevention recommends for immunocompetent adults that the interferon gamma release assays can be used anytime a skin TB test would have been used previously.
For children and immunocompromised populations, the benefits are murky. “So we do have a difference in practice in our hospital for pediatric versus adult patients with these assays,” Dr. Frank says. “With children, they do still want to do the skin test. Even though it has low specificity and is prone to false-positives, on the initial screen you would be more likely to pick up the children that have TB.”
With tuberculosis as well as the other infectious diseases, the means of sample collection, the laboratory tests selected, and the interpretation of results all may require different approaches when children are the patients being tested. While increasingly used, molecular testing doesn’t always offer a solution. Research continues on improved test methods, Drs. Frank and McAdam note, but it’s important that clinical laboratories remind clinicians treating children that the limitations of existing tests need to be taken into account—because children are a special case.
Anne Paxton is a writer in Seattle