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\begin{document}

\title{Increase in Sweat Chloride Concentration Is Associated with a Higher
Risk of CFSPID to CF Reclassification}



\author[1]{Danieli Salinas}%
\author[1]{Daniella  Ginsburg}%
\author[2]{Choo Phei Wee}%
\author[3]{Muhammad Saeed}%
\author[4]{John Brewington}%
\affil[1]{University of Southern California Keck School of Medicine}%
\affil[2]{University of Southern California Southern California Clinical and Translational Science Institute}%
\affil[3]{Kaiser Permanente Los Angeles Medical Center}%
\affil[4]{University of Cincinnati Department of Pediatrics}%


\vspace{-1em}



  
  \date{June 14, 2024}


\begingroup
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\selectlanguage{english}
\begin{abstract}
\textbf{Objectives:} Universal implementation of cystic fibrosis (CF)
newborn screening (NBS) has led to the diagnostic dilemma of infants
with CF screen positive, inconclusive diagnosis (CFSPID), for which
there is limited guidance regarding prognosis and standardized care.
Rates of reclassification from CFSPID to CF vary and risk factors for
reclassification are unknown. We investigated whether clinical
characteristics are associated with risk of reclassification from CFSPID
to a CF diagnosis. \textbf{Methods:} Children with a positive CF NBS
were recruited from two sites in California. Retrospective,
longitudinal, and cross-sectional data were collected. A subset of
subjects had nasal epithelial cells collected for CFTR functional
assessment. Multivariate logistic regression was used to assess the risk
of CFSPID-to-CF reclassification. \textbf{Results:} A total of 112
children completed the study (CF=53, CFSPID=59). Phenotypic
characteristics between groups showed differences in pancreatic
insufficiency prevalence, immunoreactive trypsinogen (IRT) levels, and
\emph{Pseudomonas aeruginosa} (PSA) colonization. Spirometry measures
were not different between groups. Nasal epithelial cells from 10
subjects showed 7-30\% of wild type (WT)-CFTR function in those who
reclassified and 27-67\% of WT-CFTR function in those who retained the
CFSPID designation. Modeling revealed that increasing sweat chloride
concentration (sw{[}Cl \textsuperscript{-}{]}) and PSA colonization were
independent risk factors for reclassification to CF.
\textbf{Conclusion:} Increasing sw{[}Cl \textsuperscript{-}{]} and
history of PSA colonization are associated with risk of reclassification
from CFSPID to CF in a population with high IRT and two \emph{CFTR}
variants. Close follow-up to monitor phenotypic changes remains critical
in this population. The role of CFTR functional assays in this
population requires further exploration.%
\end{abstract}%



\sloppy


\textbf{Introduction}

Newborn screening (NBS) for cystic fibrosis (CF) is a successful public
health strategy allowing for early recognition of affected infants. When
combined with early multidisciplinary care at CF centers, NBS improves
nutrition and cognitive outcomes, thereby reducing morbidity and
mortality.\textsuperscript{1,2} However, the universal implementation of
screening led to a new and complex diagnostic dilemma of infants who are
screen-positive with inconclusive sweat chloride tests and/or DNA
results.\textsuperscript{3} These infants have been designated as cystic
fibrosis transmembrane conductance regulator (CFTR) -- related metabolic
syndrome (CRMS) in the US and CF screen positive, inconclusive diagnosis
in other countries (CRMS/CFSPID).\textsuperscript{4} Although the first
cases of this new diagnosis were recognized in mid-2000s in US and
Europe and this population is now entering adolescence, there remains
limited guidance to families regarding prognosis, which is unsettling
for medical providers and parents.\textsuperscript{5,6}

Although most children with the CRMS/CFSPID designation will remain
healthy and asymptomatic, some will develop signs and symptoms of CF.
The rates of reclassification from CRMS/CFSPID to CF have been reported
from 6\% to 48\%, varying based on NBS algorithm, number of
\emph{CFTR}variants identified and their interpretations, population
heterogeneity, and length of follow up.\textsuperscript{3,7} The
criteria for reclassification include reassignment of \emph{CFTR}
variants as CF-causing, clinical features of CF that may surface with
age, and/or sweat chloride concentration (sw{[}Cl\textsuperscript{-}{]})
above the diagnostic threshold of 60 mmol/L in repeat assessments. Early
recognition of phenotypic patterns that are characteristic of
reclassification would promote early diagnosis and interventions;
however, risk factors that lead to reclassification to CF are unknown.

To determine the factors associated with reclassification from
CRMS/CFSPID to CF, we collected retrospective and cross-sectional data
in a cohort of children aged 2 months to 13 years old who had a positive
CF NBS in California, where extensive genetic testing is part of the NBS
algorithm. Findings from this work will help CF providers recognize risk
factors early, thereby minimizing long-term morbidity. These
observations will improve guidance to providers and decrease the
uncertainty for families of children with CRMS/CFSPID.

\textbf{Methods}

Participants

Participants were recruited from two medical centers in California. One
hundred and twelve children were recruited due to having had a positive
CF NBS and a designation of either CRMS/CFSPID or CF. As defined by the
IRT/DNA/DNA sequencing algorithm, participants had at least
two\emph{CFTR} variants identified.\textsuperscript{8} Protocol was
approved by the Institutional Review Boards of Children's Hospital Los
Angeles (CHLA) and Kaiser Permanente Los Angeles Medical Center (KP) and
informed consent was obtained from all participating families.

Procedures

As part of this research study, clinical data were collected
cross-sectionally and included height, weight, physical exam, and a
study-specific questionnaire related to relevant signs and symptoms in
the past 12 months (\textbf{Supplemental Figure 1} ). Sweat chloride
concentration (sw{[}Cl\textsuperscript{-}{]}) testing (ELItech
Macroduct\selectlanguage{ngerman}® system) was performed following Cystic Fibrosis Foundation
guidelines.\textsuperscript{9} Spirometry was performed using the
CareFusion Vmax Encore device (Care-Fusion, Yorba Linda, California,
USA). Forced expiratory volume in one second reported in percent
predicted (ppFEV\textsubscript{1}), forced vital capacity (FVC), and
FEV\textsubscript{1}/ FVC measurements were converted to global lung
function initiative (GLI).\textsuperscript{10} Lung Clearance Index
(LCI\textsubscript{2.5}) derived from nitrogen multiple breath washout
testing (MBW) was offered to all subjects \selectlanguage{english}[?]3 years old, and used the
Exhalyzer D(r) device (Eco Medics AG, Duernten, Switzerland) with
Spiroware software (version 3.1.6). Device calibration was performed
daily prior to testing. MBW test was performed per ATS/ERS consensus
statement and as described previously.\textsuperscript{11-13} Cystic
Fibrosis Questionnaires-Revised (CFQ-R) were applied for parents and
children \selectlanguage{english}[?]6 years old.\textsuperscript{14}

Electronic medical records (EMR) were reviewed from birth; data
collected included \emph{CFTR} genotype, immunoreactive trypsinogen
(IRT) value, sw{[}Cl\selectlanguage{english}\textsuperscript{-}{]} results, microbiology data
(focusing on \emph{Pseudomonas aeruginosa} {[}PSA{]}), fecal elastase
values, history of hospitalizations, history of lower respiratory tract
infections (Pneumonia) and pulmonary exacerbations, use of oral or
intravenous antibiotics in the past 12 months, and provider
documentation of crackles, chronic or recurrent respiratory symptoms,
constipation, signs of malabsorption, and relevant treatments.
Pancreatic insufficiency was defined as fecal elastase \textless{}200
\selectlanguage{greek}µ\selectlanguage{english}g/g. Chronic PSA was defined as two positive respiratory cultures in
the past 12 months.\textsuperscript{15} Both clinical research sites
used diagnostic criteria based on CF Foundation guidelines for CF and
CRMS/CFSPID.\textsuperscript{16} CRMS/CFSPID-to-CF reclassification
occurred when an individual's sw{[}Cl\textsuperscript{-}{]} rose to [?]60
mmol/L, one or more of an individual's \emph{CFTR} variants changed to a
CF-causing interpretation by CFTR2 (https://cftr2.org), and/or an
individual developed signs and symptoms of CF according to provider's
discretion.

Nasal epithelial samples were obtained from a subgroup of subjects to
explore the utility of Human Nasal Epithelial (HNE) assays in defining
baseline CFTR function, which could potentially identify risk for
reclassification. Subjects with one CF-causing variant and a copy of
R117H;7T, 5T;TG12, or 5T;TG13 were prioritized. Sampling and Ussing
chamber testing of planar respiratory cultures were performed as
previously described.\textsuperscript{17, 18}

Statistical analysis

Data are described in mean and standard deviation or median and
interquartile range for continuous variables, frequency and percentage
for categorical variables. Differences in key clinical variables between
sites, and demographic and clinical characteristics between diagnostic
groups (CF vs. CRMS/CFSPID) were examined using chi-squared or Fisher's
exact tests for categorical variables and Wilcoxon rank-sum or two
sample t-tests for continuous variables. A non-additive effect was
assessed using Firth logistic regression and generalized linear model to
evaluate whether the difference in CF and CRMS/CFSPID varied by site on
ever having a positive culture for PSA (PSA-ever), fecal elastase,
sw{[}Cl-{]}, and CFQ-R respiratory domain. Using longitudinal data
collected from EMR, sw{[}Cl-{]} progression was evaluated using a linear
median quantile mixed-effects model which time is modeled as age at
measurement and random effect at participant level. Individual
sw{[}Cl-{]} increases were estimated as a slope that was based on at
least three measurements (18 individuals were excluded for not having at
least 3 values). The association of clinical characteristics with the
risk of reclassification was assessed by Firth logistic regression model
to reduce bias due to the small number of reclassified patients.
Multivariate Firth logistic regression model further assessed the
association of clinical characteristics with the risk of
reclassification adjusting for site and demographic characteristics. All
statistical computations were performed in Stata 17 (StataCorp, College
Station, TX).

\textbf{Results}

Of 112 subjects who completed the study, 21 were from KP and 91 from
CHLA.

Sites were comparable on prevalence of reclassification from CRMS/CFSPID
to CF (\textasciitilde{}10\%). When sites were compared using specific
variables, the difference between CF and CRMS/CFSPID was not significant
(\textbf{Supplemental Table 1} ); therefore, stratification by site was
not necessary. Site was included, conservatively, as a covariate
throughout analysis to adjust for other possible site differences.
Demographic characteristics of the CF and CRMS/CFSPID groups showed no
significant differences in age, sex, race/ethnicity distribution, BMI,
and weight-for-length percentile (\textbf{Table 1} ). There were
significant differences in IRT levels, PSA-ever, pancreatic
insufficiency (PI), FEV\textsubscript{1}/FVC, and CFQ-R social
(\textbf{Table 1} \emph{).} \emph{CFTR} genotypes are summarized in

\textbf{Table 2.}

LCI\textsubscript{2.5} assessed by MBW was completed in 48 children ages
4 to 13 years. Median (IQR) values were comparable between the two
diagnostic groups (\textbf{Table 1} ). LCI\textsubscript{2.5} data were
classified based on two different upper limit of normal (ULN) cut-offs:
one defined by 26 healthy volunteers from CHLA ages 3 to 19 years and of
the same race/ethnicity distribution as study subjects (ULN =
8.02;\textbf{Figure 1} ); and the other defined from the literature (ULN
= 7.91, \textbf{Supplemental Figure 2} ).\textsuperscript{13} There is
no statistical difference in distribution between above and below cutoff
among CF, CRMS/CFSPID, and CF-classified based on healthy volunteers
from CHLA (p=0.140) and reference literature (p=0.345).

CFTR function was assessed by HNE assay in 10 selected subjects (8
CRMS/CFSPID, 2 re-classified to CF, \textbf{Figure 2} ). In the
CRMS/CFSPID group, wild type (WT)-CFTR (wtCFTR) function ranged from
27\% to 67\% (5.75 to 14 \selectlanguage{greek}µ\selectlanguage{english}A/cm\textsuperscript{2}; reference is 21
\selectlanguage{greek}µ\selectlanguage{english}A/cm\textsuperscript{2} for healthy controls), while their
sw{[}Cl\textsuperscript{-}{]} varied from 28 to 49 mmol/L. In the two
CF-reclassified subjects, CFTR function varied from 7\% to 30\% of
wtCFTR (1.52 to 6.25 \selectlanguage{greek}µ\selectlanguage{english}A/cm\textsuperscript{2}) while
sw{[}Cl\textsuperscript{-}{]} values were above the diagnostic threshold
of 60 mmol/L. There was no linear relationship between the HNE assay and
sw{[}Cl\textsuperscript{-}{]}.

Per California CF centers standard of practice, sites are encouraged to
perform sw{[}Cl\textsuperscript{-}{]} testing on the day of referral and
to repeat at 6 months, 1 year, and yearly thereafter if values are
\textless{}60 mmol/L. Repeated values (median {[}IQR{]} = 4 {[}2, 5{]}
repeated measures per participant) analyzed by quantile mixed-effects
showed distinct trajectories between children who retained the
CRMS/CFSPID designation compared to those who reclassified to CF
(p-value for difference between slopes = 0.013; \textbf{Figure 3} ).
Initial sw{[}Cl{]} values were also significantly different between
groups (Intercept difference, \textless{}0.0001). The CF-reclassified
population had an increase of 4.71 (95\% CI 2.45-6.97) mmol/L/year,
compared to 1.21 (95\% CI 0.4-2.02) mmol/L/year among those who did not
reclassify.

Firth logistic regression was used to detect factors associated with
CRMS/CFSPID-to-CF reclassification among 71 participants. On univariate
analysis, children with CRMS/CFSPID who reclassified had significantly
higher rates of respiratory cultures positive for PSA, crackles detected
on physical exam, respiratory symptoms, pneumonia, hospitalization for
respiratory illness, use of antibiotics in the previous 12 months,
growth concerns, and increasing (higher slope)
sw{[}Cl\textsuperscript{-}{]} (\textbf{Table 3} ). The multivariate
Firth logistic regression model included respiratory culture positive
for PSA, sw{[}Cl\textsuperscript{-}{]} trajectory (slope), history of
pneumonia, and study site. Children with CRMS/CFSPID who had a
respiratory culture positive for PSA were more likely to reclassify
(adjusted OR {[}95\% CI{]} = 6.43 {[}1.15 -- 36.07{]}, p=0.034).
Increase in sw{[}Cl\textsuperscript{-}{]} over time remained
significantly associated with the risk of reclassification (adjusted OR
{[}95\% CI{]} = 1.21 {[}1.02 -- 1.45{]}, p=0.033). A history of
pneumonia was not significantly associated with reclassification
(adjusted OR {[}95\% CI{]} = 1.87 {[}0.33 -- 10.63{]}, p=0.48). The
conclusion of modeling was that sw{[}Cl\textsuperscript{-}{]} increase
and history of PSA were independently associated with CF
reclassification.

\textbf{Discussion}

This study identified that increasing sw{[}Cl\textsuperscript{-}{]}
values and ever having a respiratory culture positive for PSA are
associated with risk of reclassification from CRMS/CFSPID to CF in a
population with high IRT and two \emph{CFTR} variants. These findings
emphasize that continuous follow up, allowing for observation of changes
in phenotype over time, will help define the prognosis for children with
CRMS/CFSPID. Multiple factors that could influence CF reclassification
were studied, and we found that children who reclassified to CF had more
frequent reporting of crackles, respiratory symptoms, and growth
concerns compared to those who retained the CRMS/CFSPID designation.
CF-reclassified children also had higher rates of pneumonia,
hospitalizations for respiratory symptoms, use of antibiotics, and a
steeper slope of rising sw{[}Cl\textsuperscript{-}{]} values. Measures
of ppFEV\textsubscript{1}, LCI\textsubscript{2.5}, IRT, and history of
asthma/reactive airways disease were \emph{not} significantly associated
with the risk of CRMS/CFSPID-to-CF reclassification; this is notable
considering the clinical relevance of these variables within the CF
population.

A rising sw{[}Cl\textsuperscript{-}{]} predicts CF reclassification,
confirming previous reports. The precise predictive threshold is unclear
and may vary by cohort. In prior studies, CRMS/CFSPID cohorts that have
reclassified to CF had sw{[}Cl\textsuperscript{-}{]} increases from
5.3-7.3 mmol/L/year and an increase in mean
sw{[}Cl\textsuperscript{-}{]} from 40 (\selectlanguage{ngerman}±11) to 82 (±26)
mmol/L.\textsuperscript{21} Our group and others have also reported that
healthy controls and those who retained the CRMS/CFSPID designation
through childhood did not have significant increase in
sw{[}Cl\textsuperscript{-}{]} over time.\textsuperscript{2021} Here, we
present that a rate of sw{[}Cl\textsuperscript{-}{]} increase of 4.71
mmol/L/year (95\%CI 2.45 -- 6.97) is an independent risk factor for CF
reclassification. These combined findings may guide providers to be more
attentive to children with CRMS/CFSPID showing increase
sw{[}Cl\textsuperscript{-}{]} \selectlanguage{english}[?]4 mmol/L/year, and more reasured by
others showing increase at \textasciitilde{}1 mmol/L/year. This also
suggests that there is more value in making clinical decision based on
multiple data points collected over time, rather than based on one data
point from an intial assessment. Notably, 50\% of those who reclassified
had an intial sw{[}Cl\textsuperscript{-}{]} \textless{}30 mmol/L, which
is considered ``CF unlikely'' by current guidelines.\textsuperscript{16}
Although an initial intermediate sw{[}Cl\textsuperscript{-}{]} (30-59
mmol/L) naturally raises concern for likelihood of CFTR
dysfunction,\textsuperscript{23} recent longitudinal data from the
CF-reclassified group suggests that providers should also focus on the
rate at which sw{[}Cl\textsuperscript{-}{]} rises as an indication for
change in diagnosis, even before it reaches the diagnostic threshold of
\selectlanguage{english}[?]60 mmol/L.\textsuperscript{21}

\emph{CFTR} genotypes from children who reclassified to CF included at
least one F508del (58\%) or a nonsense variant (G542X and
R1162X;\textbf{Table 2} )\textbf{.} Genotypes containing a CF-causing
variant and 5T;TG12 or 5T;TG13 were seen in 7 (58\%) of the 12 who
reclassified. Notably, as the CF NBS algorithm in California includes
\emph{CFTR}sequencing, it is unlikely that other variants influenced the
phenotypes in these individuals. As we previously reported, 38\% of
children with a CF-causing variant and 5T;TG13 reclassified to CF in
their first 8 years of life.\textsuperscript{20} This is aligned with
previous reports that determination of TG repeat number allows for more
accurate prediction of benign versus pathogenic 5T
alleles.\textsuperscript{24} Other variants observed in the second
allele of the CF-reclassified group were missense variants of varying
clinical consequence per CFTR2, and which are approved for CFTR
modulators: c.1853T\textgreater{}C (I618T), c.3454G\textgreater{}C
(D1152H) , and c.3154T\textgreater{}G (F1052V). (https://cftr2.org) The
non CF-causing variant c.3705T\textgreater{}G (S1235R) was observed as
part of a complex allele. Variants c.94C\textgreater{}A (L32M) and
c.1393-42G\textgreater{}A (1525-42G\textgreater{}A) are frequent in
California and are of unknown consequence.\textsuperscript{25}

LCI\textsubscript{2.5} was non-discriminatory between those with
CRMS/CFSPID who reclassified to CF and those who did not \textbf{(Figure
1, Supplemental Figure 2, Table 3)} and did not influence
reclassification when tested in the multivariate logistic regression
model. As children with CRMS/CFSPID may have abnormal
LCI\textsubscript{2.5} despite being
asymptomatic,\textsuperscript{11}longer follow-up will reveal if those
with abnormal measurements at a young age will progress to have symptoms
and abnormal ppFEV\textsubscript{1} later in life.\textsuperscript{26}
Munck and colleagues reported that asymptomatic children with
CRMS/CFSPID may have bronchiectasis;\textsuperscript{27} therefore, MBW
should continue to be explored as a sensitive and safe method to detect
early lung disease in this population.\textsuperscript{11,28}

Assessment of CFTR protein function was performed in a subgroup of
participants. This method was chosen because it directly estimates
protein function, is not expected to change over time, and is
an\emph{ex-vivo} test that can be tolerated by children. Our results
suggest greater overlap in HNE assay results between children who
reclassified to CF and those who retained the CRMS/CFSPID designation
compared to sw{[}Cl\textsuperscript{-}{]} values in these two groups
(\textbf{Figure 2} ). On the other hand, HNE assays show greater
distinction between the CRMS/CFSPID group and healthy controls than
sw{[}Cl\textsuperscript{-}{]}, as healthy controls and carriers may have
sw{[}Cl\textsuperscript{-}{]} from \textless{}30 to \textgreater{}60
mmol/L.\textsuperscript{29} Additionally, HNE assay can be performed at
any time, whereas sw{[}Cl\textsuperscript{-}{]} slope requires repeated
measurements over time. We recruited a selected genotype group to
evaluate using HNE assays based on higher risk of reclassification: a
CF-causing variant on one allele and R117H;7T, 5T;TG12, or 5T;TG13 on
the other.\textsuperscript{20,30} A larger sample is needed to draw
conclusions based on genotype groups. Additionally, a non-linear
association between HNE and sw{[}Cl\textsuperscript{-}{]} has been
reported in a cohort of individuals with CF \textsuperscript{18} that
was not found in this CRMS/CFSPID cohort. However, this association was
difficult to interpret when sw{[}Cl\textsuperscript{-}{]} was
\textless{}60 mmol/L. Finally, the HNE assay reported here was completed
in 10 subjects but a total of 17 underwent sampling (including some
individuals who were sampled twice; data not shown). The
\textasciitilde{}50\% test yield due to contamination and poor cell
growth may be a reflection of the discomfort caused by sampling, which
could be a barrier to utilizing this assay for young pediatric patients.
However, considering the potential benefit of having the accurate
estimation of CFTR function early in life, HNE assays should be explored
further in a larger sample of the CRMS/CFSPID population.

Microbiology characteristics included at least one positive respiratory
culture for PSA in 12\% of the CRMS/CFSPID group, similar to other US
reports and lower than in European cohorts (\textasciitilde{}10\% and
24\% respectively)\textsuperscript{3,27} (\textbf{Table 1} ). One child
with two positive cultures in the previous 12 months was placed in the
chronic category; in this case, the second positive culture was
immediately after 28 days of inhaled tobramycin (Tobi(r)), which triggered
a second course of treatment.

CFQ-R questionnaires were used as a patient reporting outcome for
patients \selectlanguage{english}[?]6 years of age.\textsuperscript{14} Among the different
domains, social showed significant higher results for children with
CRMS/CFSPID, likely because these children are asymptomatic and their
social lives are less impacted. Respiratory and emotional domains were
statistically similar, which may be a reflection of early interventions
related to early diagnosis of CF through NBS and to the young age group.

IRT results were statistically similar between the CF-reclassified
subjects and those who retained the CRMS/CFSPID designation
(\textbf{Table 3} ). This contrasts our previous findings, where an
IRT\textgreater{}80 ng/mL increased the likelihood of CF
reclassification.\textsuperscript{20} Others have reported no difference
in baseline IRT when reclassified and non-reclassified children with
CRMS/CFSPID were compared.\textsuperscript{21} Thus, it remains unclear
whether the baseline IRT measured at birth can be used as a predictor of
CF reclassification.\textsuperscript{31}

Other findings included no identification of CFTR-related disorder
(CFTR-RD) and no association of asthma or reactive airways disease with
reclassification to CF. CFTR-RD was defined in 2011 as a diagnosis
related to CFTR dysfunction that does not fulfill the diagnosis criteria
for CF and typically manifests in one affected organ system (congenital
bilateral absence of the vas deferens {[}CBAVD{]}, pancreatitis, or
bronchiectasis).\textsuperscript{32} As \emph{CFTR} variants associated
with CFTR-RD are also found in CRMS/CFSPID cohorts, it is possible that
some children with CRMS/CFSPID will eventually fulfill diagnostic
criteria for CFTR-RD but not CF. The CFTR-RD definition is under needed
revision, as it likely that a child with CRMS/CFSPID, two
\emph{CFTR}variants, and evidence of CFTR dysfunction (i.e.,
intermediate sw{[}Cl\textsuperscript{-}{]}) who develops chronic
respiratory symptoms and bronchiectasis will be diagnosed as
CF.\textsuperscript{4, 6}Chronic cough and wheezing can be the initial
manifestations of CF in childhood;\textsuperscript{33} however, they may
lead to the diagnosis of asthma/reactive airway disease instead of CF,
considering the increasing prevalence of asthma in
childhood.\textsuperscript{34}Interestingly, CFTR dysfunction is
associated with an increased likelihood of asthma, as seen among
\emph{CFTR}carriers.\textsuperscript{35} In this cohort, the asthma
prevalence was statistically similar between the CF-reclassified group
and those who kept the CRMS/CFSPID designation, and asthma did not
increase the risk of reclassification. It is unclear if the high
prevalence seen in both groups (42\% in CF-reclassified and 36\% in
CRMS/CFSPID; \textbf{Table 3} ) indicates misdiagnosis of CF, CFTR
dysfunction, or a public health trend. Nevertheless, primary care
providers should avoid misdiagnosis and refer a child with CRMS/CFSPID
and who has chronic respiratory signs and symptoms to a CF center for
re-evaluation.\textsuperscript{36}

This study is not without limitations. The recruitment was limited to
two sites, making the sample size relatively small and limited to a
specific geographic area. Both sites also had a high lost-to-follow-up
rate among CRMS/CFSPID children (51\% at CHLA and 34\% at KP),
potentially introducing bias to the population continuing to receive
care. We did attempt to address this by specifically recruiting from
this population -- 13 of the 59 (22\%) subjects in the CRMS/CFSPID group
were recruited from a lost-to-follow-up pool. Tobacco smoke exposure, a
known risk factor for CFTR dysfunction,\textsuperscript{37} could not be
studied because of the low prevalence of exposure to secondhand smoking.

A final thought is that although currently published data showed that
most children with CRMS/CFSPID designation have a favorable outcome,
data is limited to a small number entered in national registries and
short follow up time.\textsuperscript{27,31,38} The short follow up may
be attributed to young age of this population, early discharge per
physician's decision, and high rates of lost-to-follow
up.\textsuperscript{39} The second challenge for defining prognosis in
this population is high variability in duration of follow up, care
practices, and CF reclassification criteria despite improved
guidelines.\textsuperscript{40}

CF providers and investigators are still in the beginning of
understanding the intricacies of the CRMS/CFSPID population, and longer
longitudinal observational studies are needed to better define the risk
of reclassification to CF. Although recent literature suggests that most
persons with CRMS/CFSPID will not develop any symptoms, more current
findings indicate that some will develop CF, others CFTR-RD, and a
larger group may be at risk for a wide range of cystic fibrosis-related
conditions such as those seen in carriers. Key strengths of our work are
the size of the cohort, the diversity of fully-sequenced
\emph{CFTR}genotypes, and a wide age range with longitudinal data up to
13 years. We showed that the two independent risk factors for
manifesting signs and symptoms to fulfil CF diagnosis criteria are rapid
rising sw{[}Cl\textsuperscript{-}{]} values and the detection of PSA
from respiratory cultures. Yearly visits at the CF center and greater
awareness from the primary care providers will promote monitoring of
chronic respiratory symptoms and other signs and symptoms seen in early
CF disease, so treatment can start early fulfilling the preventive
purpose of NBS. The CF scientific community should continue to invest in
alternative CFTR function assays that can complement
sw{[}Cl\textsuperscript{-}{]} testing in defining risk of disease for
each unique combination of \emph{CFTR} variants. With all this put in
place we will better define each child's needs from infancy, which will
avoid unnecessary medical care for those at low risk and close follow up
for those at a higher risk of developing CF disease.

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