Inconclusive Diagnosis of Cystic Fibrosis After Newborn Screening

Inconclusive Diagnosis of Cystic

Fibrosis After Newborn Screening

Chee Y. Ooi, MBBS, PhDa,b,c_{, Carlo Castellani, MD}d_{, Katherine Keenan, HBSc}e_{, Julie Avolio, RN}e_{, Sonia Volpi, MD}d_, Margaret Boland, MDf_{, Tom Kovesi, MD}g_{, Candice Bjornson, MD}h_{, Mark A. Chilvers, MD}i_{, Lenna Morgan, MD}j_,

Richard van Wylick, MDk_{, Steven Kent, MD}l_{, April Price, MD}m_{, Melinda Solomon, MD}n_{, Karen Tam, CGC}n_{, Louise Taylor, NP}n_, Kylie-Ann Malitt, MSca_{, Felix Ratjen, MD}e,o_{, Peter R. Durie, MD}c,e_{, Tanja Gonska, MD}c,e

abstract

OBJECTIVES:To prospectively study infants with an inconclusive diagnosis of cysticfibrosis (CF) identified by newborn screening (NBS;“CF screen positive, inconclusive diagnosis”[CFSPID]) for disease manifestations.

METHODS:Infants with CFSPID and CF based on NBS from 8 CF centers were prospectively evaluated and

monitored. Genotype, phenotype, repeat sweat test, serum trypsinogen, and microbiology data were compared between subjects with CF and CFSPID and between subjects with CFSPID who did (CFSPID→CF) and did not (CFSPID→CFSPID) fulfill the criteria for CF during thefirst 3 years of life. RESULTS:Eighty-two subjects with CFSPID and 80 subjects with CF were enrolled. The ratio of CFSPID to

CF ranged from 1:1.4 to 1:2.9 in different centers.CFTRmutation rates did not differ between groups; 96% of subjects with CFSPID and 93% of subjects with CF had 2 mutations. Subjects with CFSPID had significantly lower NBS immunoreactive trypsinogen (median [interquartile range]:77 [61–106] vs 144 [105–199]mg/L;P,.0001) than did subjects with CF.Pseudomonas aeruginosaandStenotrophomonas maltophiliawere isolated in 12% and 5%, respectively, of subjects with CFSPID. CF was diagnosed in 9 of 82 (11%) subjects with CFSPID (genotype and abnormal sweat chloride = 3; genotype alone = 4; abnormal sweat chloride only = 2). Sweat chloride was abnormal in CFSPID→CF patients at a mean (SD) age of 21.3 (13.8) months. CFSPID→CF patients had significantly higher serial sweat chloride (P,.0001) and serum trypsinogen (P= .009) levels than did CFSPID→CFSPID patients.

CONCLUSIONS:A proportion of infants with CFSPID will be diagnosed with CF within thefirst 3 years. Thesefindings underscore the need for clinical monitoring, repeat sweat testing at age 2 to 3 years, and extensive genotyping.

WHAT’S KNOWN ON THIS SUBJECT:Infants with an inconclusive diagnosis of cysticfibrosis after newborn screening may turn out to have cystic

fibrosis. However, little is known about the incidence, characteristics (phenotype and genotype), and outcomes of these infants to guide investigations and follow-up.

WHAT THIS STUDY ADDS:In this prospective longitudinal study, a proportion (11%) of infants with an initial inconclusive diagnosis were subsequently diagnosed with cysticfibrosis. This

finding underscores the need for follow-up of this population.

a_{Discipline of Pediatrics, School of Women’s and Children’s Health, Faculty of Medicine, University of New South}

Wales, Sydney, Australia;bSydney Children’s Hospital Randwick, Sydney, Australia;cDivision of Gastroenterology, Hepatology, and Nutrition,e_{Physiology and Experimental Medicine, Research Institute,}n_{Division of Clinical and}

Metabolic Genetics, ando_{Division of Respiratory Medicine, Department of Pediatrics, The Hospital for Sick}

Children, Toronto, Ontario, Canada;d_{Cystic Fibrosis Centre, Azienda Ospedaliera Universitaria Integrata di Verona,}

Verona, Italy;f_{Division of Gastroenterology, Hepatology, and Nutrition, and}g_{Division of Respirology, Department of}

Pediatrics, Children’s Hospital of Eastern Ontario, Ottawa, Canada;h_{Division of Respiratory Medicine, Department}

of Pediatrics, University of Calgary, Alberta Children’s Hospital, Calgary, Alberta, Canada;i_{Division of Pediatric}

Respiratory Medicine, Department of Pediatrics, BC Children’s Hospital, Vancouver, British Columbia, Canada;

j_{Pediatric Cystic Fibrosis Clinic, Windsor Regional Hospital, Windsor, Ontario, Canada;}k_{Department of Pediatrics,}

School of Medicine, Queen’s University, Kingston, Ontario, Canada;lVictoria General Hospital, Victoria, British Columbia, Canada; andm_{Children’s Hospital of Western Ontario, London, Ontario, Canada}

Cysticfibrosis (CF) is a life-shortening recessive genetic disease that is now commonly diagnosed via newborn screening (NBS). NBS is not only associated with false-positive results and identification of carriers of cystic

fibrosis transmembrane conductance regulator (CFTR) mutation but also identifies infants for whom confirmatory testing remain inconclusive with regard to a CF diagnosis.1–3

The US Cystic Fibrosis Foundation (CFF)1,2_{provided expert}

opinion–based recommendations in the interpretation and management of infants with an inconclusive diagnosis of CF. The term“CFTR-related metabolic syndrome”was proposed to label these infants.2_{However, this}

terminology has not been universally accepted because these infants are neither symptomatic nor have a“metabolic”disease, and it seems reasonable to speculate that an undefined number will never have any CFTR-related symptoms.3_More

recently, an international consensus undertaken by the European CF Neonatal Screening Working Group proposed the term“CF screen positive, inconclusive diagnosis”(CFSPID).4,5

Although NBS programs have been in existence since the 1980s, there have been no prospective longitudinal studies assessing infants with CFSPID. Consequently, little is known about its incidence and infants’characteristics (phenotype and genotype) and outcomes. Hence, we initiated a multicenter study to identify and prospectively evaluate infants with CFSPID. The phenotype, genotype, and clinical outcomes of infants with CFSPID over thefirst years of life were determined.

METHODS

Subjects

A clinical-research protocol, approved by the research ethics boards, was established to assess and monitor infants with CFSPID prospectively

from 7 CF clinics from 3 provinces in Canada (Ontario: Toronto, Kingston, London, Ottawa; British Columbia [BC]: Vancouver; and Alberta: Calgary, Edmonton) and 1 in Italy (Verona). Here, we report interim data from this ongoing study in subjects recruited between June 2007 and August 2013 during theirfirst 3 years of life.

The NBS strategies from different provinces/centers are summarized in Supplemental Table 4. Dried blood spot analysis for immunoreactive

trypsinogen (IRT) from all newborns was performed by using the

AutoDELFIA method (Perkin-Elmer Life Sciences, Boston, MA). An infant was considered to be NBS-positive if either (1) IRT exceeded the site-specific cutoff plus at least 1CFTRmutation (and/or increased meconium lactase in the Veneto and Trentino-Aldige region) or (2) IRT was.99.9th centile when no mutations were identified.

All NBS-positive infants underwent sweat testing and genotyping. NBS-positive infants were defined as CFSPID if there was (1) a CF-causing mutation on 1 allele and intermediate sweat chloride (30–59 mmol/L), (2)

CFTRmutations on both alleles (no more than 1 is known to be CF-causing) and normal (,30 mmol/L) or intermediate sweat chloride, or (3) no detected mutations but a very high IRT concentration (.99.9th centile) and an intermediate sweat test. Thus, with the exception of the third criterion, the CFSPID and CFTR-related metabolic syndrome definitions are equivalent.2

For comparison, NBS-positive infants with CF (sweat chloride$60 mmol/L and/or CF-causing mutations on both alleles) were enrolled during the same period. NBS-negative infants who presented with meconium ileus and fulfilled the diagnostic criteria for CF were also included. All subjects with CFSPID and CF were approached unselectively and consecutively for enrollment. Subjects with CFSPID were reviewed separately from patients with CF.

Sweat Testing

Sweat testing was performed by using the Gibson and Cooke method6_in

Vancouver and Verona and the Macroduct method7_{in the other}

clinics. Sweat chloride was determined in compliance with Clinical and Laboratory Standards Institute guidelines.8_{Serial sweat testing was}

performed at follow-up visits for subjects with CFSPID and at least annually for the subjects with CF.

Genotyping

Genotypes were obtained from NBS mutation analyses (Supplemental Table 4) and consequentCFTRgene sequencing. At the time of subject enrollment and throughout the study period, disease-causing mutations were defined on the basis of the 23 mutations described by the CFF consensus criteria.1_{At the time of}

data analysis, the number of identified disease-causing mutations had been increased by the CFTR2 project (www.cftr2.org).9

Clinical Characteristics

Anthropometric measurements and family history of CF and CFTR-related disorders were collected at

enrollment (baseline). Subjects were monitored every 6 months in thefirst 2 years of life, and annually thereafter for parent-reported respiratory and gastrointestinal symptoms (wheeze, cough, gastroesophageal reflux, constipation, and abdominal pain) by using a standardized questionnaire.

Microbiology

Samples for microbiologic testing were collected at follow-up visits for CFSPID and CF cohorts.

Oropharyngeal swabs were performed in Canada, whereas oropharyngeal suction was performed in Verona.

Exocrine Pancreatic Function

follows: fecal elastase-1,100mg/g10_;

fecal fat losses.15% and.7% of daily fat intake for infants,6 and$6 months old, respectively11–13_{; fecal}

chymotrypsin,5 U/g14_{; or serum}

trypsinogen,6 ng/mL.15,16_Serum

trypsinogen levels were determined annually in all subjects.

Statistical Analysis

Comparisons were made by using Student’sttest or Mann-WhitneyU

test for continuous variables and by Fisher’s exact test for categorical variables. A linear random-effects mixed model was conducted to evaluate longitudinal serum trypsinogen and sweat chloride concentrations to account for variations in the number and time interval of repeat measurements. SAS version 9.3 (SAS Institute, Cary, NC) and GraphPad Prism version 6.04 (GraphPad Software, La Jolla, CA) were used.Pvalues,.05 were considered significant.

RESULTS

Incidence of CFSPID in Infants

The numbers of NBS-positive infants and those who fulfilled the criteria for CF and CFSPID during the study

period are summarized in Fig 1. The ratios of CFSPID to CF in Ontario, BC, Alberta, and Verona were 1:1.5, 1:2.1, 1:2.9, and 1:1.4, respectively. Fifty-six subjects with CFSPID (42 in Ontario, 5 in BC, 9 in Alberta) and 71 subjects with CF (55 in Ontario, 12 in BC, 4 in Alberta) were enrolled in Canada, whereas 26 subjects with CFSPID and 9 subjects with CF were recruited from Verona (Fig 1).

Subject Characteristics

Eight-two subjects with CFSPID and 80 subjects with CF were enrolled. Among the 82 subjects with CFSPID, 79 had 2 mutations (no more than 1 is CF-causing) with intermediate (n= 40) or normal (n= 39) sweat chloride, and 3 had 1 mutation (CF-causing) with intermediate sweat chloride. Although both cohorts had elevated NBS IRT concentrations, subjects with CFSPID had significantly lower IRT levels than did the CF group (Table 1, section A). The weights for CF and CFSPID cohorts were not significantly different at birth, but by the time of initial assessment subjects with CF had significantly lower weight and height

zscores than did subjects with CFSPID (Table 1, section A). There was no difference in the head circumference

zscores between infants with CFSPID and CF. Subjects with CFSPID were seen at an older age than were subjects with CF atfirst assessment.

A family history of CF and

sinopulmonary disease was reported in 6% and 24% of CFSPID subjects, respectively, which were significantly less frequent than in subjects with CF (22% and 45%, respectively).

Symptoms and Microbiology

The median (interquartile range) age at the last clinical review for the CF and CFSPID cohorts were 24.8 (18.6–38.1) and 24 (17.4–35.1) months (P= .25). Although a subset of subjects with CFSPID had reported clinical symptoms, these were reported significantly less frequently than in patients with CF with respect to wheeze, cough, constipation, and abdominal pain (Table 1, section A). There was no significant difference in reported symptoms of

gastroesophageal reflux between patients with CF and CFSPID.

Microbiologic cultures positive for CF-associated bacteria were observed in both subjects with CF and CFSPID. There were significantly more positive cultures in subjects with CF

FIGURE 1

than in subjects with CFSPID for

Pseudomonas aeruginosa(31% vs 12%) andStaphylococcus aureus

(71% vs 40%) but not for

Haemophilus influenzaeand

Stenotrophomonas maltophilia

(Table 1, section A). Subjects with CFSPID were significantly more likely to have negative cultures than were patients with CF.

Serial Sweat Chloride Measurements

As expected, sweat chloride levels were significantly lower in subjects with CFSPID than in subjects with CF (P,.0001) (Fig 2A). Sweat chloride concentrations in both subjects with CFSPID and CF increased over time (P= .004). The mean (SD) sweat chloride levels for CFSPID and CF

were 27.3 (6.1) and 83.2 (10.8) mmol/L, respectively, at baseline and increased to 33.4 (7.6) and 97.5 (10.6) mmol/L, respectively, at 36 months of age.

Genotype

The genotypes and ethnic backgrounds of subjects with CFSPID are summarized in Table 2 and Supplemental Table 5. Seventy-nine of 82 (96.3%) subjects with CFSPID had 2CFTRmutations identified. Seventy-four of 80 (92.5%) subjects with CF carried 2CFTR

mutations (Supplemental Table 6). Two of 3 subjects with CFSPID and 4 of 6 subjects with CF with only 1 mutation identified were not extensively genotyped.

Exocrine Pancreatic Function

All of the subjects with CFSPID who had exocrine pancreatic function testing at baseline (n= 70) were pancreatic sufficient (PS). In contrast, 18 of 73 (24.7%) subjects with CF were PS (P,.0001). During the study period, there were no subjects with CFSPID who progressed to PI, whereas 6 of 18 PS subjects with CF developed PI at a median

(interquartile range) age of 10.8 (3.1–17.1) months. The genotypes of PS patients with CF, including those who subsequently progressed to PI, are described in the Supplemental Table 6.

Serial serum trypsinogen levels were significantly lower among subjects with CFSPID than among subjects with CF

TABLE 1 Comparison of Characteristics Between Subjects With CF and CFSPID and Between Subjects With CFSPID Who Met the Criteria for CF During the Study Period (CFSPID→CF) and Those Who Remained Inconclusive (CFSPID→CFSPID)

A B

CF (n= 80) CFSPID (n= 82) P CFSPID→CF (n= 9) CFSPID→CFSPID (n= 73) P

Newborn characteristics

Female gender,n(%) 41 (51) 43 (52) .99 5 (56) 34 (47) .99

Gestational age, mean (SD), wk 38.8 (2.6) 38.7 (2.1) .8 38.4 (2.0) 38.8 (2.7) .7

Birth weight, mean (SD), g 3212 (544) 3304 (670) .36 3345 (974) 3301 (651) .89

Newborn screening IRT, median (IQR),mg/L

144 (105–199) 77 (61–106) ,.0001 84 (67–128) 75 (60–103) .2

Family history,n/N(%)

CF 17/76 (22) 5/81 (6) .005 1 (11) 4/72 (6) .45

Sinopulmonary disease 21/47 (45) 16/67 (24) .026 2 (22) 14/58 (24) .99

Pancreatitis 2/46 (4) 2/68 (3) .99 1 (11) 1/59 (2) .25

Male infertility 1/49 (2) 1/70 (1) .99 0 1/62 (2) .99

Baseline anthropometrics, mean (SD)

Age at baseline assessment, mo 1.1 (0.9) 3.3 (5.1) .0002 4.9 (6.8) 3.2 (4.9) .40

Weight,zscore 20.94 (1.0) 0.03 (0.8) ,.0001 0.68 (0.9) 0.03 (0.9) .10

Height,zscore 20.83 (1.0) 0.04 (0.8) ,.0001 0.29 (0.6) 0.03 (0.9) .58

Head circumference,zscore 20.05 (1.0) 0.11 (2.0) .13 0.53 (1.4) 0.23 (2.0) .83

Symptoms (reported by parents), norn/N(%)

Wheeze 25 (31) 10/81 (12) .004 1 (11) 9/72 (13) .99

Cough 60 (75) 27/81 (33) ,.0001 3 (33) 24/72 (33) .99

Gastroesophageal reflux 14 (18) 7/81 (9) .11 1 (11) 6/72 (8) .53

Constipation 16 (20) 4/81 (5) .004 0 4/72 (6) .99

Abdominal pain 29 (36) 2/81 (3) ,.0001 0 2/72 (3) .99

Microbiology,n(%)

Pseudomonas aeruginosa 25 (31) 10 (12) .004 3 (33) 7 (10) .07

Staphylococcus aureus 57 (71) 33 (40) ,.0001 3 (33) 30 (41) .65

Haemophilus influenzae 34 (43) 33 (40) .87 2 (22) 31 (43) .24

Stenotrophomonas maltophilia 8 (20) 4 (5) .24 2 (22) 2 (3) .05

Aspergillusspecies 1 (1) 0 .49 0 0 .99

Streptococcus pneumoniae 7 (9) 4 (5) .37 0 4 (6) .99

Acinetobacter baumannii 7 (9) 1 (1) .03 0 1 (1) .99

Chryseobacterium indologenes 9 (11) 1 (1) .009 0 1 (1) .99

Coliforms 48 (60) 24 (29) .0001 1 (11) 23 (32) .20

No growth 7 (9) 22 (27) ,.0001 3 (33) 19 (26) .64

(P,.0001) (Fig 2B). Serum

trypsinogen decreased significantly over time in subjects with CF (P,.0001) and subjects with CFSPID (P,.0001).

Diagnosis of CF in Subjects With CFSPID

Nine of 82 (11%) subjects with CFSPID fulfilled the diagnostic criteria for CF (CFSPID→CF) during the follow-up period on the basis of genotype and/or abnormal sweat chloride ($60 mmol/L) (Table 3). All 9 subjects initially fulfilled the CFSPID criteria on the basis of the presence of 2CFTRmutations (no more than 1 is CF-causing) and intermediate sweat chloride. CFSPID→CF subjects

diagnosed on the basis of genotype had been originally identified as carrying 2CFTRmutations but were only subsequently recognized to carry 2 CF-causing mutations after the expansion of the number of CF-causing mutations by the CFTR2 project. Of the 9 CFSPID→CF subjects, 4 (4.9%) subjects were diagnosed on the basis of genotype alone, 3 (3.7%) on the basis of both genotype and abnormal sweat chloride, and 2 (2.4%) on the basis of abnormal sweat chloride only (see Supplemental Figure 4).

CFSPID→CF patients had significantly higher sweat chloride levels than did CFSPID→CFSPID subjects (P,.0001) (Fig 3A). There was also a significant

difference in the longitudinal sweat chloride trajectories between CFSPID→CF and CFSPID→CFSPID subjects (P= .017). Among those with abnormal sweat test levels, sweat chloride increased to abnormal levels (mean [SD] of 67 [5.1] mmol/L) at a mean (SD) age of 21.3 (13.8) months.

Characteristics of CFSPID→CF Subjects

The NBS IRT of CFSPID→CF was not significantly higher than

CFSPID→CFSPID (Table 1, section B). However, serial serum trypsinogen levels were significantly higher among CFSPID→CF than among CFSPID→CFSPID subjects (P= .009) (Fig 3B). Both longitudinal serum trypsinogen trajectories changed significantly over time (P,.0001). There were no significant differences between the clinical characteristics of both groups (Table 1, section B). CFSPID→CF subjects tended to be more likely to have positive cultures forP aeruginosa(33% vs 10%;

P= .07) andS maltophilia(22% vs 3%;P= .05) compared with CFSPID→CFSPID subjects, but these differences were not statistically significant.

DISCUSSION

Currently, there are limited data to guide investigations and follow-up of NBS-positive infants with an

equivocal diagnosis of CF, and to our knowledge, this study represents the

first prospective longitudinal evaluation of these infants. Infants with CFSPID are genotypically and phenotypically different from patients with CF at time of diagnosis, but additional diagnostic information may result in a diagnosis of CF later. In our cohort, abnormal repeat sweat test and updated functional mutation analysis by CFTR2, which identified 2 disease-causing mutations, led to the reassignment of the diagnosis of CF in 11% of infants with CFSPID. This

finding underlines current FIGURE 2

recommendations to follow children with CFSPID in clinics with CF expertise. There were other children with CFSPID who developed clinical features concerning for CF (eg,

P aeruginosaisolation), but these features did not lead to a change in their diagnosis. The majority of these children were well, and clinical

symptoms did not appear to be a significant discriminator for the subsequent diagnosis of CF during thefirst 3 years of life.

Current CFF guidelines recommend repeat sweat testing of infants with an equivocal diagnosis at 6 months of age.2_{Among CFSPID}→_{CF patients}

who were diagnosable by sweat

testing, the increase in sweat chloride

$60 mmol/L occurred at a mean (SD) age of 21.3 (13.8) months. This

finding suggests that repeat sweat testing should not only be performed at 6 months of age but also in the second to third years of life if the diagnosis remains inconclusive. It remains unclear whether individuals with CFSPID with a repeat

intermediate sweat test are at higher risk of future CF or CFTR-related disorder than those whose sweat test levels normalize or stay in the normal range. Current guidelines suggest yearly monitoring after the second year of life.2_{However, there are no}

recommendations for follow-up duration or who requires ongoing monitoring or can be discharged. Although current CF diagnostic criteria state that a positive newborn

screening test could substitute for clinical features, it is unclear to what age this applies to.1,2_{Until more data}

become available, parents of all infants with CFSPID should be educated on the risk of future disease.

In contrast to previous reports, the vast majority of subjects with CFSPID carried 2CFTR

mutations.17–20 _{The majority of}

subjects were compound

heterozygotes for 1 disease-causing mutation and 1CFTRvariant of variable or currently unknown consequence, with the F508del/ R117H-7T genotype being most common. In combination with a disease-causing mutation, R117H-7T has been associated with diagnostic uncertainties in CF,

TABLE 2 Genotypes of Subjects With CFSPID According to Initial Sweat Chloride Measurements

Sweat Chloride,30 mmol/L Sweat Chloride 30–59 mmol/L

Allele 1 Allele 2 n Allele 1 Allele 2 n

F508dela _{R117H (7T)}b _{9 F508del}a _R117Cd ₂c

F508dela _5Tb _{2 F508del}a _L206Wd ₂c

F508dela _D1152Hb _{2 F508del}a _P67Ld ₁c

F508dela _R117Hb _{1 F508del}a _5Tb ₈

F508dela D1270Nb 1 F508dela R117H (7T)b 3

F508dela L997F 3 F508dela R117Hb 3

F508dela 1716G.A 1 F508dela S1455X 1c

F508dela 621+3G.A 1 F508dela R170H 1

F508dela I1328T 1 F508dela I148T 1

F508dela L967S 1 F508dela L997F 1

F508dela M1137T 1 F508dela Q1476X 1

F508dela Y301C 1 F508dela S1235R 1

1717-1G.Aa D1152Hb 1 F508dela T1299I 1

2183AA.Ga 5Tb 1 2183AA.Ga R117Cd 1

2183AA.Ga S431G 1 2789+5G.Aa R117H (7T)b 1

3849+10kbC.Ta 3041-15T.G 1 3849+10kbC.Ta 3041-15T.G 1

621+1G.Ta R117H (7T)b 1 621+1G.Ta G1069Rb 1

711+1G.Ta _D1152Hb _{1 G542X}a _L206Wd ₁c

G542Xa _{R117H (7T)}b _{1 G542X}a _{C1410T 1}

G542Xa _D1152Hb _{1 G551D}a _5Tb ₁

G551Da _D1152Hb _{1 N1303K}a _5Tb ₁

N1303Ka _D1152Hb _{1 R1162X}a _{R117H (7T)}b ₁c

N1303Ka _{E527G 1 R553X}a _5Tb ₁

R117H (5T)a 5Tb 1 R553Xa L997F 1

R117H (7T)b R117H (7T)b 1 R560Ta G576A 1

R117H (7T)b 3041_71G.C 1 W1282Xa 5Tb 2

R117Hb Q1476X 1 F508dela — 2

R117H (5T)a — 1

—, no mutation identified on the second allele.

a_{CF-causing mutations.}

b_{Mutations of varying clinical consequences.} c_CFSPID→_{CF subjects.}

d_{Mutations that were initially classi}fi_{ed as uncertain signi}fi_{cance but subsequently rede}fi_{ned as CF-causing by CFTR2.}

TABLE 3 Characteristics of Subjects With CFSPID Who Later Met Diagnostic Criteria of CF

Subject Number Allele 1 Allele 2 Ethnicity NBS IRT,mg/L Initial Sweat Chloride, mmol/L Highest Sweat Chloride, mmol/L Country

1 F508del R117C White 105.8 36 61 Canada

2 F508del S1455X White 66.6 46 74 Canada

3 F508del P67L White 151.2 38 38 Canada

4 F508del L206W White 83.8 58 64 Canada

5 G542X L206W White 67 49 66 Canada

6 F508del L206W White 59.9 45 45 Canada

7 R1162X R117H-7T White 126 36 70 Italy

8 2183AA.G R117C White 129 32 32 Italy

including in newborn-screened infants with equivocal CF diagnosis and in older individuals with single-organ manifestations of CF.17,18,20–22_{As in the}

case of the 7 subjects who were initially classified as CFSPID but who were subsequently recognized to carry 2 disease-causing mutations on the basis of the CFTR2 project, the diagnostic consequences (benign versus disease-causing) of theCFTRmutations identified in all of the other subjects with CFSPID may not be apparent until later on, when new genetic information becomes available and classification of

CFTRmutations currently considered to be of “unknown”consequences is updated.

Apart from sweat chloride concentrations and genotype, only serum trypsinogen

concentrations were significantly different between CFSPID→CF and CFSPID→CFSPID subjects. This finding suggests an

association between IRT levels and CFTR dysfunction, and therefore the likelihood of having CF disease.16,23

In terms of symptoms, respiratory symptoms of wheeze and cough were common in subjects with CFSPID, but there was no difference between CFSPID→CF and CFSPID→CFSPID subjects, and these symptoms were less frequent than in subjects with CF.

Nonetheless, some subjects with CFSPID, especially CFSPID→CF, may have an increased risk of respiratory morbidity. Overall, patients with CFSPID showed a higher frequency of

P aeruginosa(12%) andS maltophilia

(5%) isolation compared with previously reported cross-sectional rates in healthy children without CF of 3.6% and 1% to 3.6%,

respectively.24,25_{Furthermore, there}

were more positiveP aeruginosaand

S maltophiliacultures among CFSPID→CF than CFSPID→CFSPID subjects, although thisfinding was not statistically significant. The

finding ofP aeruginosain 10% of CFSPID→CFSPID subjects is also higher than would be expected in healthy children. The rates ofS aureus

andH influenzaeisolation in subjects with CFSPID, including the CFSPID→CF subgroup, were comparable to those reported in healthy children (28%–48% and 11%–47%, respectively). The presence of CF-associated bacteria in infants with CFSPID warrants clinical and diagnostic concern. Ultimately, CF is a clinical diagnosis and treatment decisions need to be made irrespective of the diagnostic label.

The weight and height of subjects with CF were significantly lower compared with subjects with CFSPID by the time offirst assessment, even though birth weight was not different. This

finding suggests that a suboptimal nutritional state was already present in patients with CF at the time of diagnosis, despite detection by NBS. In contrast, there was no difference in anthropometric measurements between CFSPID→CF and CFSPID→CFSPID patients, which may be related to the fact that all patients with CFSPID were PS.

For every 3 infants diagnosed with CF, there were∼1 to 2 infants with CFSPID identified through NBS. This ratio is similar to the recent reported experience in New York.20_Verona

had a higher proportion of infants FIGURE 3

with CF and CFSPID than the Canadian provinces. Newborn screening (based on a higher IRT cutoff of the 99.5th percentile) in conjunction with CF carrier screening in Verona may have resulted in fewer false positives.26_{The differences in}

the screened populations, with different frequency and distribution ofCFTRmutations, may also account for this variation.

This study has several strengths and limitations. It is thefirst prospectively designed study in infants with CFSPID. Previous retrospective reports may have been limited by ascertainment bias due to selective reporting of patients returning or who were recalled for follow-up because of symptom development. Although our study commenced before the publication of the CFF

recommendations, our protocol was very similar to the guidelines and designed to monitor these subjects over many years. For ethical and logistical reasons, we were not able to include NBS-positive subjects who were discharged after a normal sweat test, which would have served as a comparator to the CFSPID cohort (especially serial sweat chloride concentrations and type and frequency ofCFTRmutations). In addition, the data presented in this study are interim and CF-like manifestations may not develop until adolescence and adulthood.21,22_{We anticipate that}

there would be additional

CFSPID→CFSPID subjects who would

fulfill the criteria for CF over time, due to moreCFTRgene variants being newly identified as disease-causing, abnormal sweat chloride, or by clinical criteria. Current comparisons between CFSPID→CFSPID and CFSPID→CF do not account for these potential changes over time. There are similarities in the genotypes of subjects with CFSPID and symptomatic adults who present later in life with single-organ manifestations of CF, including but not exclusive to mutations such as R117H-7T.21,22_A

long-term prospective study may shed more light into the proportion and characteristics of individuals with CFSPID who develop CF. Because this study was conducted in centers from different provinces and countries, there were not only variations in the NBS protocols and IRT cutoffs but also differences in methods for obtaining respiratory samples for microbiologic analysis and determining exocrine pancreatic function. Despite the multicenter approach, the CFSPID→CF sample size was small. Not all patients were recruited because of delays in ethics approval in some centers and decline in study participation; sampling bias may be inadvertently introduced. The psychosocial impact of CFSPID on families was not part of the current study.

CONCLUSIONS

NBS-positive infants with an inconclusive diagnosis of CF are not uncommon. These children are at risk

of positive cultures for CF-associated bacteria as well as fulfilling the diagnostic criteria of CF over time and thus require monitoring, ideally by CF clinicians. Repeat sweat

testing (including in the second or third year of life) and extensive genotyping may help clarify the diagnosis of CF over time. There may be a potential role for serum trypsinogen levels to predict a later CF diagnosis in CFPSID.

ACKNOWLEDGMENTS

We thank all of the doctors, research nurses, and coordinators: Ms Lori Fairservice and Ms Melissa Soles (Alberta Children’s Hospital, Calgary); Dr Peter Zuberbuhler, Ms Shannon Gregory, and Ms Angela MacDonald (Stoller Children’s Hospital,

Edmonton); Ms Adrianna Breen (Hotel Dieu Hospital, Kingston); Ms Monica Dawe, Ms Erin Fleischer, and Ms Jennifer Itterman (Children’s Hospital of Western Ontario, London); Ms Anne Smith (Children’s Hospital of Eastern Ontario, Ottawa); Ms Caroline Burgess and Ms Vanessa McMahon (British Columbia’s Children Hospital,

Vancouver); Dr Anna Tamanini, Dr Ciro D’Orazio, Ms Giovanna Amenta, Ms Elisa Calcaterra, Dr Nicoletta Vardaro, and Dr Ilaria Meneghelli (Azienda Ospedaliera Universitaria Integrata di Verona, Verona). We thank Ms Leslie Steele and Dr Peter Ray (Molecular Genetics, Hospital for Sick Children, Toronto) for their

involvement in genetic analysis.

www.pediatrics.org/cgi/doi/10.1542/peds.2014-2081

DOI:10.1542/peds.2014-2081 Accepted for publication Mar 9, 2015

Address correspondence to Tanja Gonska, MD, The Hospital for Sick Children, Division of Gastroenterology, Hepatology, and Nutrition, 555 University Ave, Toronto, ON M5G 1X8, Canada. E-mail: tanja.gonska@sickkids.ca

PEDIATRICS (ISSN Numbers: Print, 0031-4005; Online, 1098-4275).

Copyright © 2015 by the American Academy of Pediatrics

FINANCIAL DISCLOSURE:The authors have indicated they have nofinancial relationships relevant to this article to disclose.

FUNDING: This study was funded by Cystic Fibrosis Canada.

POTENTIAL CONFLICT OF INTEREST:The authors have indicated they have no potential conflicts of interest to disclose.

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DOI: 10.1542/peds.2014-2081 originally published online May 11, 2015;

2015;135;e1377

Pediatrics

Kylie-Ann Malitt, Felix Ratjen, Peter R. Durie and Tanja Gonska

van Wylick, Steven Kent, April Price, Melinda Solomon, Karen Tam, Louise Taylor,

Boland, Tom Kovesi, Candice Bjornson, Mark A. Chilvers, Lenna Morgan, Richard

Chee Y. Ooi, Carlo Castellani, Katherine Keenan, Julie Avolio, Sonia Volpi, Margaret

Inconclusive Diagnosis of Cystic Fibrosis After Newborn Screening

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DOI: 10.1542/peds.2014-2081 originally published online May 11, 2015;

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Kylie-Ann Malitt, Felix Ratjen, Peter R. Durie and Tanja Gonska

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Boland, Tom Kovesi, Candice Bjornson, Mark A. Chilvers, Lenna Morgan, Richard

Chee Y. Ooi, Carlo Castellani, Katherine Keenan, Julie Avolio, Sonia Volpi, Margaret

Inconclusive Diagnosis of Cystic Fibrosis After Newborn Screening

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