Consider NAATs for detection of GBS in pregnant women

Group B streptococcus (GBS), a bacterium passed from mother to child during labor and childbirth, remains a considerable public health concern both in the United States and abroad.^1,2 In the U.S., the Centers for Disease Control and Prevention (CDC) has called for improvements of up to 90 percent in screening sensitivity; but culture-based methods have not yet approached this goal.^2-4

Our laboratory at The University of California, San Diego (UCSD) compared three polymerase chain reaction (PCR)-based nucleic acid amplification tests (NAATs) in their abilities to detect GBS samples from pregnant women, and found that all three have significantly greater sensitivities and reduced turnaround time (TAAT), relative to our standard of care culture-based testing. Therefore, considering NAATs as a new gold standard in the detection of GBS may be warranted.

GBS remains a global public health concern

Recent global estimates suggest GBS is responsible for 409,000 maternal/fetal/infant cases annually, including 205,000 cases of infants with early-onset disease (EOD), leading to septicemia, meningitis, or pneumonia within the first week after delivery.⁵ GBS is also estimated to be responsible for 147,000 stillbirths and infant deaths, and neurodevelopmental impairment in another 10,000 annually.^5,6

Vaginal-rectal GBS colonization has been reported to occur in about 18 percent of pregnant women globally and about 25 percent in the U.S., according to the CDC.^7,8 Transmission from mother to newborn occurs at an estimated rate of 40-73 percent with about one to two percent of colonized newborns developing EOD.^9-11 In the U.S., GBS infection is the leading cause of infant morbidity and mortality, and of bacterial meningitis and septicemia in a newborn’s first week of life.¹² Long-term disabilities may include retardation, hearing or vision loss, and potentially death.^10,11

Culture-based screening presents pros and cons

Since 2002, the CDC has recommended universal GBS screening at 35-37 weeks of pregnancy. The preferred choice since then has been culture-based methods using vaginal-rectal specimens, which has resulted in a dramatic decrease in the incidence of EOD, despite the carrier rate remaining steady at ~20 percent.²

The primary and highly effective strategy for preventing EOD is intravenous antibiotic administration during labor in women who test positive for GBS.² Still, EOD due to GBS remains a problem,^13,14 given a large percentage (81 percent) of neonates who develop EOD are born to GBS-negative mothers, which suggests an inadequate sensitivity of culture-based screening in the form of false-negatives.^3,14

A promising alternative to culture-based screening is PCR-based NAATs, several of which have been approved by the Food and Drug Administration (FDA). However, adoption of NAATs has not been widespread, and culture-based tests remain the gold standard.

Comparing three commercially available NAATs

We sought to determine the performance of three FDA-approved GBS NAATs: (1) the Hologic Panther Fusion GBS assay; (2) the Luminex Aries GBS assay; and (3) the Cepheid Xpert GBS LB assay; the goal to compare their sensitivity and specificity against one another and to culture. We collected 500 vaginal-rectal samples from women at 35-37 weeks of pregnancy and enriched them in Lim broth (Todd Hewitt broth, Copan) for 16-24 hours. After enrichment, an aliquot of the Lim broth was used for culture per UCSD’s standard of care testing. Residual enriched Lim broth specimens were aliquoted into multiple tubes for testing with each NAAT method.

NAATs exhibit superior sensitivity

All three NAAT tests were significantly more sensitive than the culture-based test. Culture was positive for 108 (21.6 percent) specimens, all of which were also positive by all three NAATs (apart from one specimen that was positive by culture and the Panther Fusion, but tested negative on the other NAATs). All three NAAT methods captured positives that were negative by culture: Initial positives for NAATs were 143 (28.6 percent) for the Panther Fusion, 147 (29.4 percent) for the Xpert, and 155 (31.0 percent) for Aries assays.

Next, we looked at how the tests compared to one another in terms of true positives, true negatives, false positives, and false negatives. To determine “true” results, we defined the consensus as two or more tests producing concordant results. Within this definition, 147 specimens were defined as true positives and 353 were defined as true negatives. Rate of GBS detection was 21.6 percent (108/500) with culture, 28.2 percent (141/500) for the Panther Fusion and Xpert, and 28.4 percent (142/500) for Aries assays.

Culture produced 39 false negative results, while the Panther Fusion, Aries, and Xpert produced 6, 5, and 6, respectively. No false positive results were observed with culture, while 2, 13, and 6 were recorded for the Panther Fusion, Aries, and Xpert, respectively. Based on consensus results, we calculated sensitivity for Panther Fusion and Xpert to be 95.9 percent; for Aries, 96.6 percent; and for culture, 73.5 percent. Specificities for each assay were 99.4 percent, 98.3 percent, and 96.3 percent for the Panther Fusion, Xpert, and Aries, respectively (for culture, 100 percent). No significant differences were identified in sensitivity or specificity between the three NAATs and the consensus result (McNemar’s chi-square test, P > 0.05), but all three NAATs would be significantly more sensitive than the culture method when using the consensus method (P < 0.0001).

Finally, to put into context the relative amounts of GBS nucleic acids present in specimens, we calculated threshold cycles (C_T) of all NAAT positive specimens. Threshold cycle is defined as the number of cycles required in a real time PCR assay for the fluorescent signal to accumulate and cross the threshold (i.e., exceed background level) to yield a positive result. Therefore, C_T levels are inversely proportional to the amount of target nucleic acid in the sample.

When we compared samples that were culture positive and positive by all three NAATs, the NAAT C_T values for these specimens ranged between 18.6 and 21.4, indicating high quantities of GBS DNA. When all three NAAT tests were positive but culture was negative, we found NAAT C_T was higher (ranging from 30.2 to 30.8), but nevertheless suggests a still high quantity of target DNA. Samples positive by two NAATs registered a low to moderate amount of target (36.2 to 39.0), and the average C_T values for samples positive by a single NAAT suggested the lowest target content is observed for false positives (37.5 to 39.4, respectively).

Workflow, throughput, and time comparisons

Some potentially meaningful differences exist in the instruments’ function and workflow with regard to random and continuous access, open channel functionality, throughput, and overall menu available. All instruments we evaluated have the potential to reduce TAT, compared to culture-based screening. Each laboratory should of course evaluate its own needs regarding patient volume and other relevant parameters when making equipment purchasing decisions. 

Implications

Our findings suggest that PCR NAATs are highly sensitive and should be considered the preferred method for GBS screening in the prenatal period. By reducing the number of false negatives, all three NAATs significantly increased the sensitivity of GBS screening relative to culture-based methods.

Compared to culture, NAATs also have the potential to reduce workload, including the time to obtain results. Though time savings may not be the central concern in GBS testing, accuracy of results is of critical importance. Our results suggest that NAATs may significantly reduce neonate morbidity and mortality associated with both EOD and late-onset disease, which occurs between one week and three months after birth.

Though earlier studies have illustrated the high sensitivity of NAAT testing, some hesitations may still exist among healthcare providers. One is cost: Molecular methods may be more expensive than culture, though they may save some time in labor costs due to efficiency, particularly in larger clinics. Additionally, hesitation among clinicians may be due to concerns that PCR is not sufficiently sensitive to detect GBS, though arguably, adequate evidence has by now accrued to counter that misapprehension. Finally, some clinicians may be concerned that NAAT tests will not allow for the determination of antibiotic susceptibility—but by requiring the broth enrichment step, a laboratory can identify positive samples via NAAT screening, and then go back to the broth and determine susceptibility if the organisms are viable.

Conclusion

Further studies will be necessary to address the full clinical impact of NAAT platforms compared to conventional cultures. However, our results make a strong case, illuminating the potential of NAATs to increase sensitivity for GBS detection and to reduce TAT, while holding the potential to reduce infant morbidity and mortality, particularly in large healthcare centers. Medical clinics and academic institutions running numerous cultures each day may find the prospect of switching to molecular methods daunting, but the added sensitivity of molecular testing is necessary to further reduce invasive GBS disease.

In summary, our findings strongly support the use of highly sensitive real-time PCR NAATs as the preferred method—and a new gold- standard—for GBS screening in the prenatal period.  

REFERENCES

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