Clostridioides Difficile Carriage Rate Increases during Pediatric ALL Treatment

Background

Clostridioides difficile infection (CDI) is frequent in pediatric patients with acute lymphoblastic leukemia (ALL). Studies have shown upwards of 20% positivity rate in CDI testing among pediatric oncology patients, and up to several percent in pediatric ALL patients in the first 180 days of diagnosis. Antibiotic usage has been variably linked to CDI positivity in these populations. As CDI testing is usually done in symptomatic patients, the question of C. difficile carriage versus CDI has not been addressed. We and others have shown that microbiome is altered in pediatric ALL patients and survivors. We conducted a longitudinal stool microbiome study in pediatric ALL patients and tested the hypothesis that alteration of the microbiome during ALL treatment promotes C. difficile carriage.

Methods

Children with ALL were prospectively recruited on a rolling basis and stool samples were collected at diagnosis (Dx) and at the end of induction (EOI), consolidation (EOC), interim maintenance I (IMI), delayed intensification (DI), interim maintenance II (IMII), as well as approximately 3 months and 6 months into maintenance (M3, M6). Stool samples from healthy siblings were used as controls. TaqMan-based quantitative-PCR (qPCR) was performed on DNA extracted from stool samples to detect C. diff 16S rRNA, tcdA, (Toxin A) and tcdB (Toxin B) genes . Samples positive or either tcdA or tcdB, or both, were designated positive for toxigenic C. difficile. 16S rRNA hypervariable region V4 was sequenced and analyzed for microbiome diversity and relative abundance of microbiota.

Results

32 ALL patients age 3 months-19 years were included. The diagnoses were 12 standard risk and 14 high risk pre-B ALL, 5 T-ALL, and 1 relapsed pre-B ALL. Stool samples were collected from 18 healthy siblings. The numbers of samples tested at each treatment phase were: 29 Dx, 24 EOI, 23 EOC, 25 IMI, 21 DI, 6 IMII, 14 M3, 7 M6. No patient had symptoms suggestive of CDI, and no patient was clinically tested or treated for CDI.

Total number of stool samples tested was 149, of which 43 (29%) were positive for toxigenic C. difficile (Figure 1). At diagnosis, 2/29 (7%) patients were positive, compared to 2/18 (11%) in healthy siblings. At EOI, positivity rate increased to 17%, then up to 40% - 52% at EOC, IMI, and DI. C. difficile positivity were lower around 30% at M3 and M6, although few patients reached maintenance to contribute samples for analysis. Twenty-five patients (78%) were positive at some phase. Longitudinal analysis of individual patients showed that C. difficile positivity was intermittent through treatment phases; only 3 patients remained persistently positive. Seven patients (22%) were never positive.

Multivariate analysis showed that EOC, IMI, and DI treatment phases were significant risk factors for C. difficile carriage. Neither the number of antibiotics nor the number of antibiotic courses administered was significant. Leukemia risk stratification (high risk versus standard risk) also did not correlate with C. difficile positivity.

Microbiome analysis showed statistically significant differences in relative abundance of certain taxa between C. diff positive and negative samples at the class, order, and family levels (Figure 2). Examples include depletion of the class Verrucomicrobiae, which contains protective Akkermansia, and depletion of the common taxa Bifidobacteriaceae and Ruminococcaceae.

Conclusion

Longitudinal PCR testing of toxigenic C. difficile in pediatric ALL patients demonstrated increased C. difficile prevalence further into treatment phases. C. difficile carriage correlated significantly with depletion of several bacterial taxa, as microbiome diversity decreased overall with successive treatment phases. Our data lend support to the hypothesis that altered microbiome in ALL treatment allows permissibility for C. difficile carriage. In addition, no C. difficile positive patient had symptoms of CDI, therefore, caution must be taken in clinical testing, as there is a high asymptomatic carriage rate. Further longitudinal testing during maintenance and off-therapy is needed to see if C. difficile carriage rate returns to baseline and correlates with recovery of gut microbiome.

Figure 1

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Figure 1

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