The History of Cystic Fibrosis by Dr James Littlewood OBE

Edited and produced by Daniel Peckham

 

There are references, many from European sources, to bronchoscopy in children with CF most on the therapeutic use with bronchial lavage to reliieve bronchial obstruction. Also many more recent references where bronchoscopy is used to obtain material for culture and therapeutic purposes and even to detect changes in the bronchial epithelium with gene therapy.

 

1944 Menten ML, Middleton TO. Cystic fibrosis of the pancreas: report of 18 proved cases. Am J Dis Child 1944; 67:355-359. (included here as three patients had bronchoscopies)
Another paper based on a search through previous paediatric autopsies revealed 18 cases of CF from The Children’s Hospital, Pittsburgh; all had diffuse definite dilatation of the pancreatic acini and associated pneumonia. The authors state that radiological examination of the lungs showed a remarkable similarity in the unusual enlargement of the hilar shadows and a bilateral shadow decreasing to the periphery and suggest this as a diagnostic feature.

Three had bronchoscopy during life- in one showing a normal trachea and in two reddened thickened mucous membrane containing thick mucopurulent material.

This is the first mention of the bronchoscopic appearances seen in cystic fibrosis.

 

 

1948 Atkins JP. Bronchoscopic observations on the pulmonary aspects of fibrocystic disease of the pancreas. Ann Otol (St Louis) 1948; 57:791-801. [PubMed]
An early report of bronchoscopy in children with CF using the rigid bronchoscope – the only type available at that time. Atkins stated the expiratory intrusion of the posterior wall into the lumen produced crescenteric cross section of the bronchi not differing from that seen in bronchial asthma. Prior to this Menton & Middleton (1944 above), reporting 18 children with CF from Pittsburgh in 1944, incidentally described the bronchoscopic appearances in three patients who were so examined. Later Robert Wood in the USA pioneered the use of flexible bronchoscopy in children from 1975 onwards (See Wood et al, 1973 & 1975 below).

The tracheal abnormality is noted in later papers and also inrelations to the CF pigs.

 

1975 Wood RE, Wanner A, Hirsch J, Farrell PM. Tracheal mucociliary transport in patients with cystic fibrosis and its stimulation by terbutaline. Am Rev Respir Dis 1975; 111:733-8. [PubMed]
Robert Wood observed the movement of Teflon discs passing up the trachea through a bronchoscope in 14 adults with cystic fibrosis. The discs moved at 2.6 mm/min in CF and 20.1 mm/min in controls; terbutaline increased the speed of movement in CF to 5.5mm/min. The authors speculated, correctly as it has now become apparent, that this “may play a role in the pathogenesis of pulmonary disease”.


Robert Wood pioneered fibreoptic bronchoscopy in children with CF. The 3.5mm paediatric version of the bronchoscope he used in 1975 had no suction port which he circumvented by attaching a fine Teflon tube to the side allowing its use down to children aged 18 months; it was developed by the Olympus Corporation into the first flexible pediatric bronchoscope in 1978 at Dr Wood’s request.

 

 

1977 Mills RM, Young RC, Kulczycki LL. Validation of therapeutic bronchoscopic bronchial washing in cystic fibrosis. Chest 1977; 71:508-513.[PubMed]

Validation of rigid-tube bronchoscopy with small-volume (5-ml increments not to exceed 300 ml) bronchial washing as a therapeutic adjunct was performed on six patients with cystic fibrosis who had severe pulmonary involvement. The authors concluded that bronchoscopic bronchial washing may be effective in the management of patients with cystic fibrosis, by augmentation of their inadequate cleansing function of the conducting airways.

Later Lucas Kulczycki reported the results of 632 bronchial lavages on 173 patients over 16 years (1965-1980) below).

 

 

1977 Dahm LS, Ewing CW, Harrison GM, Rucker RW, Comparison of three techniques of lung lavage in patients with cystic fibrosis. Chest 1977; 72:593-596.[PubMed]

One hundred fifty-three lung lavage procedures were performed on 81 cystic fibrosis patients during the years 1963 to 1976. Local analgesia was used in 24 lavages done via a rigid bronchoscope and in a group of 73 lavages directed by a fiberoptic bronchoscope. No abnormalities in cardiac rate or rhythm were observed with the lavages directed by fiberoptic bronchoscope. Fifty-six lavages were performed under general anesthesia with 20 cardiac abnormalities being noted among this group. Subjective improvement was noted in 96% of the 56 patients who had lavage under general anesthesia. Objective improvement was apparent in 45% of the 24 patients who had lavage with the rigid bronchoscope and 64% of the 36 patients who had lavage by fiberoptic bronchoscopy.

The authors concluded that patients with cystic fibrosis of varying stages of disability can more safety (and with good improvement) have bronchial washouts with the fiber-optic bronchoscope than with either of the other two techniques.

 

 

1981 Kulczycki L. Experience with 632 bronchoscopic bronchial washings done on 173 cystic fibrosis patients during a 16-year period (1965-1980). In 1000 Years of Cystic Fibrosis. Collected Papers. Warwick WJ ed. University of Minnesota. 95-112.

A presentation at an invited meeting of CF experts. Dr Kulczycki notes that BBW may be harmful but needs special care in CF however the results indicate significant benefit in particular decreased morbidity and mortality, reduced hospitalisation, infant mortality prevented, tracheostomy not used, spininess pneumothoraces not seen, incidence of haemoptysis decreased and antibiotics usually limited to periods of hospitalisation or acute infection.

This is a very long and detailed report. There are some impressive X-rays in the article of improvement after bronchial lavage.

 

 

1980 Wood RE, Sherman JM. Pediatric flexible bronchoscopy. Ann Otol Rhinol Laryngol 1980; 89:414-416. [PubMed]
The first report of the use of the paediatric flexible fibreoptic bronchoscope in children by Robert Wood who pioneered the technique. He described a prototype flexible paediatric bronchoscope that he had used to perform both diagnostic and therapeutic procedures on pediatric patients ranging from infants of 840 g to children aged 14 years. Flexible bronchoscopy, with appropriate instrumentation and careful attention to physiological requirements of the patient, was found to be safe and effective in young patients. Wood correctly forecast that with this instrument, the indications for bronchoscopy in children would be considerably expanded.

 

(Also there is an excellent review of flexible bronchoscopy by Wood RE. J Pediatr 1988; 112:1-6 [PubMed] ; also Wood RE. “Pediatric flexible bronchoscopy: The inside story”. In: Doershuk CF, (Ed.). Cystic Fibrosis in the Twentieth Century. Cleveland: AM Publishing Ltd, 2001:112-119.With permission). Expansion in the use of bronchoscopy in CF continues and it has even been suggested that every infant identified by newborn screening should be bumptiousness to obtain cultures of the lower airways (Hilliard TN et al. Arch Dis Child 2007; 92:898-899.below [PubMed]).

 

 

2005 Davies JC, Davies M, McShane D, Smith S, Chadwick S, Jaffe A, Farley R, Collins L, Bush A, Scallon M, et al. Potential difference measurements in the lower airway of children with and without cystic fibrosis. Am J Respir Crit Care Med 2005; 171:1015-1019. [PubMed]
The potential difference measurements were made via a bronchoscope from the lower respiratory tracts of children as young as 1 year. Tracheal baseline values were significantly higher in children with CF than those without, although this was not the case more distally. In airways between the third and seventh generation, perfusion with a zero chloride solution containing isoprenaline led to a significant change in potential difference in children without CF, whereas no change was seen in those with CF. This measure provided a reliable distinguishing test between the two disease groups.

This is the first report of this technique in children

 

 

2007 Hilliard TN, Sukhani S, Francis J, Madden N, Rosenthal M Balfour-Lynn I, Bush A, Davies JC. Bronchoscopy following diagnosis with cystic fibrosis. Arch Dis Child 2007; 92:898-899. [PubMed]
The authors recently changed their practice and performed bronchoscopy following a diagnosis of cystic fibrosis. On a retrospective review of 25 children, Pseudomonas aeruginosa was detected in bronchoalveolar lavage for the first time in five children (20%) and Staphylococcus aureus in four (16%). Lavage culture was positive in eight of the 18 children without respiratory symptoms. The authors suggest that these findings highlight the potential of bronchoscopy following diagnosis, even in asymptomatic children.

 

Whether to recommend bronchoscopy in an asymptomatic screened infant with CF is dependent on many factors, not least, where the infant was born and the facilities and skill available for paediatric respiratory investigation. Also, the treatment policy of the unit responsible for the care of the infants. There is the potential danger of infecting an, as yet uninfected, infant with the instrument if sterilisation has been faulty; also hospitalisation does present a definite infection risk to infants with CF. If the infants identified by neonatal screening were started on prophylactic flucloxacillin from diagnosis (as is recommended by the UK CF Trust's expert Antibiotic Group 2009), it is very unlikely that S. aureus would have been cultured. Also units where screening has been routine for many years, such as Leeds, have managed to achieve very low levels of chronic Pseudomonas infection using only frequent throat cultures, cough swabs and serum antibody levels to recognise and treat early P. aeruginosa infection. If an infant with CF had repeatedly negative upper respiratory tract cultures and negative Pseudomonas antibody levels it would be very unlikely there would be positive bronchial cultures.

So bronchoscopy for all at diagnosis, although it may be decided is desirable, is definitely a policy that needs careful discussion before being applied generally.

The recent multicentre study completed in 2009 (summary below) comparing regular bronchoscopy with routine care led by Claire Wainwright of Brisbane does not show a significant advantage for those who have regular bronchoscopies.

However, a further report from the Brompton Hospital reaffirmed the value of early bronchoscopy in CF infants diagnosed by newborn screening (Stafler P et al. Bronchoscopy in cystic fibrosis infants diagnosed by newborn screening. Pediatr Pulmonol 2011; 46:696-700.[PubMed]).

 

 

2008 McLaughlin AM, McGrath E, Barry R, Egan JJ, Gallagher CG. Treatment of lobar atelectasis with bronchoscopically administered recombinant human deoxyribonuclease in cystic fibrosis? Clin Respir J 2008; 2:123-126. [PubMed]
The objective of this study was to describe experience in which rhDNase (Pulmozyme) was administered by bronchoscopic instillation into atelectatic lobes in five adults with CF. This method was successful in treating lobar atelectasis, which was resistant to conventional therapy with antibiotics and physiotherapy. In all but one of the cases described, administration of DNase in this manner resulted in a radiographic and clinical improvement of the atelectasis. The authors recommend that respiratory physicians consider this as a second line treatment in the management of atelectasis.

Although not the first to use this treatment for atelectasis, confirmation of the success of this method is useful for clinicians faced with resistant atelectasis.

 

 

 

2008 Wainwright CE, Grimwood K, Carlin JB, Vitamer S, Cooper PJ, Francis PW, Brines CA, Whitehead BF, Martin AJ, Robertson IF, Cooper DM, Da kin CJ, Masters IB, Massie RJ, Robinson PJ, Ranganathan S, Armstrong DS, Patterson LK, Robertson CF. Safety of bronchoalveolar lavage in young children with cystic fibrosis. Pediatr Pulmonol 2008; 43:965-972. [PubMed]
As part of Dr Claire Wainwright’s study of bronchoalveolar lavage (BAL) directed therapy, 333 BALs were carried out on 107 children median age 23.5 months (1.6 - 67.5 months); 170 (51%) were for exacerbations. 8.7% were followed by fever and 3% clinically significant episodes. 52% had minor adverse events.
The authors concluded that although adverse events were common they were usually transient and well tolerated. Parents should be warned that infants with respiratory infections had an increased risk of post-BAL fever.

 

This is an important ongoing study further progress of which was reported by Claire Wainwright at the 2009 NACFC in Minneapolis. The study concluded in 2009 and did not establish a case of managing the respiratory infections using regular bronchoscopies but did provide a vast amount of useful information - undoubtedly one of the studies of the decade! (SEE BELOW)

 

 

2009 Sly PD. Brennan S. Gangell C. de Klerk N. Murray C. Mott L. Stick SM. Robinson PJ. Robertson CF. Ranganathan SC. Australian Respiratory Early Surveillance Team for Cystic Fibrosis (AREST-CF). Lung disease at diagnosis in infants with cystic fibrosis detected by newborn screening. Am J Resp Crit Care 2009; 180:146-152. [PubMed]

Fifty-seven infants (median age, 3.6 mo) with CF underwent bronchoalveolar lavage and chest computed tomography (CT) using a three-slice inspiratory and expiratory protocol. MEASUREMENTS AND MAIN RESULTS: Despite the absence of respiratory symptoms in 48 (84.2%) of infants, a substantial proportion had lung disease with bacterial infection detected in 12 (21.1%), including Staphylococcus aureus (n = 4) and Pseudomonas aeruginosa (n = 3); neutrophilic inflammation (41. 4 x 10(3) cells/ml representing 18.7% of total cell count); proinflammatory cytokines, with 44 (77.2%) having detectable IL-8; and 17 (29.8%) having detectable free neutrophil elastase activity. Inflammation was increased in those with infection and respiratory symptoms; however, the majority of those infected were asymptomatic. Radiologic evidence of structural lung disease was common, with 46 (80.7%) having an abnormal CT; 11 (18.6%) had bronchial dilatation, 27 (45.0%) had bronchial wall thickening, and 40 (66.7%) had gas trapping. On multivariate analysis, free neutrophil elastase activity was associated with structural lung disease. Most children with structural lung disease had no clinically apparent lung disease. The authors suggested that these data support the need for full evaluation in infancy and argue for new treatment strategies, especially those targeting neutrophilic inflammation, if the promise of NBS for CF is to be realized.

 

The evidence is strong that lung infection and damage occurs very early in many CF infants. The use of prophylactic anti-staphylococcal treatment (as recommended in the UK) and agressive eradication treatment of Pseudomonas would certainly improve the situation revealed in these Australian studies. Certainly the findings support the advice that all screened CF infants should be treated at a CF Centre

 

 

2011 Stafler P, Davies JC, Balfour-Lynn IM, Rosenthal M, Bush A. Bronchoscopy in cystic fibrosis infants diagnosed by newborn screening. Pediatr Pulmonol 2011; 46:696-700. [PubMed]
There is evidence of early functional and structural changes in babies with cystic fibrosis (CF) diagnosed on newborn screening (NBS). The aim of the present study was to determine the yield of bronchoalveolar lavage (BAL) microbiology and cytology, and 24 hr pH monitoring in a group of CF infants diagnosed on NBS. Infants referred to the Brompton tertiary pediatric respiratory center between July 2007 and November 2009 underwent fiber-optic bronchoscopy (FOB), BAL, and insertion of a 24 hr dual pH probe under a single general anesthetic. The authors studied 33 infants, median age of 100 days (47-215 days) at the time of bronchoscopy. In 9 of 33 (27%) bacterial organisms were identified. Seven of the nine patients (78%) were asymptomatic and only one had had a positive cough swab prior to bronchoscopy. Neutrophilia was identified in 18/27 (67%) cases with a median of 11% (6-73%). 13/31 (42%) had an abnormal pH study with a pH index >12%. The authors concluded that the high yield of microbiology, cytology, and pH probe investigations in screened CF infants justifies invasive surveillance. Longitudinal studies to determine if early aggressive treatment results in improved outcome are awaited.

 

A study by the experienced paediatric team at the Royal Brompton in London. These results are impressive and confirm the early changes even in screened CF infants however, most paediatricians, even in CF Centres, would not opt for routine bronchoscopy in screened infants as the experience and facilities may be less satisfactory than at the Brompton. Many would rely on repeated throat and cough swabs.

Claire Wainwright's excellent Australian study of BAL- directed therapy failed to show a major advantage of periodic bronchoscopic surveillance when the infants are reviewed at 5 years (Wainwright CE et al. JAMA; 2011; 306:163-171.[PubMed] below). However, it is clear that lung infection occurs early and close follow up from diagnosis is mandatory for screened infants - it is noted that median age of the infants in the present report at the time of bronchoscopy was over 3 months.

 

 

2011 Wainwright CE, Vitamer S, Armstrong DS, Brines CA, Carlin JB, Sheena J, Cooper PJ, Grimwood K, Moo die M, Robertson CF, Tidiness HA, ACFBAL Study Investigators. Effect of bronchoalveolar lavage-directed therapy on Pseudomonas aeruginosa infection and structural lung injury in children with cystic fibrosis: a randomized trial. JAMA. 306(2):163-71, 2011 Jul 13. [PubMed]
Despite wide use of oropharyngeal cultures to identify pulmonary infection, concerns remain over their diagnostic accuracy. While bronchoalveolar lavage (BAL) is an alternative diagnostic tool, evidence for its clinical benefit is lacking. OBJECTIVE: To determine if BAL-directed therapy for pulmonary exacerbations during the first 5 years of life provides better outcomes than current standard practice relying on clinical features and oropharyngeal cultures. DESIGN, SETTING, AND PARTICIPANTS: The Australasian Cystic Fibrosis Bronchoalveolar Lavage (ACFBAL) randomized controlled trial, recruiting infants diagnosed with cystic fibrosis through newborn screening programs in 8 Australasian cystic fibrosis centers. Recruitment occurred between June 1, 1999, and April 30, 2005, with the study ending on December 31, 2009. INTERVENTIONS: BAL-directed (n = 84) or standard (n = 86) therapy until age 5 years. The BAL-directed therapy group underwent BAL before age 6 months when well, when hospitalized for pulmonary exacerbations, if Pseudomonas aeruginosa was detected in oropharyngeal specimens, and after P. aeruginosa eradication therapy. Treatment was prescribed according to BAL or oropharyngeal culture results. MAIN OUTCOME MEASURES: Primary outcomes at age 5 years were prevalence of P. aeruginosa on BAL cultures and total cystic fibrosis computed tomography (CF-CT) score (as a percentage of the maximum score) on high-resolution chest CT scan.

RESULTS: Of 267 infants diagnosed with cystic fibrosis following newborn screening, 170 were enrolled and randomized, and 157 completed the study. At age 5 years, 8 of 79 children (10%) in the BAL-directed therapy group and 9 of 76 (12%) in the standard therapy group had P. aeruginosa in final BAL cultures (risk difference, -1.7% [95% confidence interval, -11.6% to 8.1%]; P = .73). Mean total CF-CT scores for the BAL-directed therapy and standard therapy groups were 3.0% and 2.8%, respectively (mean difference, 0.19% [95% confidence interval, -0.94% to 1.33%]; P = .74). CONCLUSION: Among infants diagnosed with cystic fibrosis, BAL-directed therapy did not result in a lower prevalence of P. aeruginosa infection or lower total CF-CT score when compared with standard therapy at age 5 years.

 

This is a major study and the summary is reproduced in full. The results are clear - bronchoscopy guided antibiotic treatment during the first 5 years did not lead to a reduction in the number of infants who have Pseudomonas infection nor to a significant difference in CT scores. Perhaps the difference between this study and many previous ones comparing these two methods of bacteriological monitoring was that the children were followed over a prolonged period and it was not just a comparison of the findings of one isolated BAL and one oropharyngeal culture.

 

 

2012 Belessis Y, Dixon B, Hawkins G, Pereira J, Peat J, Macdonald R, Field P, Numa A, Morton J, Lui K, Jaffe A. Early cystic fibrosis lung disease detected by bronchoalveolar lavage and lung clearance index. Am J Resp Crit Care 2012; 185:862-873. [PubMed]

A study to determine whether the lung clearance index (LCI) is a sensitive and repeatable noninvasive measure of airway infection and inflammation in newborn-screened children with CF. Methods: Forty-seven well children with CF (mean age, 1.55 yr) and 25 healthy children (mean age, 1.26 yr) underwent multiple-breath washout testing. LCI within and between-test variability was assessed. Children with CF also had surveillance bronchoalveolar lavage performed.

Measurements and Main Results: The mean (SD) LCI in healthy children was 6.45 (0.49). The LCI was higher in children with CF (7.21 [0.81]; P < 0.001). The upper limit of normal for the LCI was 7.41. Fifteen (32%) children with CF had an elevated LCI. LCI measurements were repeatable and reproducible. Airway infection was present in 17 (36%) children with CF, including 7 (15%) with Pseudomonas aeruginosa. Polymicrobial growth was associated with worse inflammation. The LCI was higher in children with Pseudomonas (7.92 [1.16]) than in children without Pseudomonas (7.02 [0.56]) (P = 0.038). The LCI correlated with bronchoalveolar lavage IL-8 (R(2) = 0.20, P = 0.004) and neutrophil count (R(2) = 0.21, P = 0.001). An LCI below the upper limit of normality had a high negative predictive value (93%) in excluding Pseudomonas.

 

The authors of this study from Sydney concluded that the LCI is elevated (abnormal) early in children with CF, especially in the presence of Pseudomonas and airway inflammation. They suggest that LCI is a feasible, repeatable, and sensitive noninvasive marker of lung disease in young children with CF. The lung clearance index seems to be emerging as practical and accurate measure of airway function particularly useful in very young patients.

 

 

2012 Hoo AF, Thai LP, Nguyen TT, Bush A, Chudleigh J, Lum S, et al. London Cystic Fibrosis Collaboration. Lung function is abnormal in 3-month-old infants with cystic fibrosis diagnosed by newborn screening. Thorax 2012; 67:874-881. [PubMed](http://dx.doi.org/10.1136/thoraxjnl-2012-201747 for full text)
Long-term benefits of newborn screening (NBS) for cystic fibrosis (CF) have been established with respect to nutritional status, but effects on pulmonary health remain unclear. Lung clearance index (LCI) and functional residual capacity (FRC) using multiple breath washout (MBW), plethysmographic (pleth) FRC and forced expirations from raised lung volumes were measured in 71 infants with CF (participants in the London CF Collaboration) and 54 contemporaneous healthy controls age ∼3 months.
Compared with controls, and after adjustment for body size and age, LCI, FRC(MBW) and FRC(pleth) were significantly higher in infants with CF (mean difference (95% CI): 0.5 (0.1 to 0.9), p=0.02; 0.4 (0.1 to 0.7), p=0.02 and 0.9 (0.4 to 1.3), p<0.001, z-scores, respectively), while forced expiratory volume (FEV(0.5)) and flows (FEF(25-75)) were significantly lower (-0.9 (-1.3 to -0.6), p<0.001 and -0.7 (-1.1 to -0.2), p=0.004, z-scores, respectively). 21% (15/70) of infants with CF had an elevated LCI (>1.96 z-scores) and 25% (17/68) an abnormally low FEV(0.5) (below -1.96 z-scores). While only eight infants with CF had abnormalities of LCI and FEV(0.5), using both techniques identified abnormalities in 35% (24/68). Hyperinflation (FRC(pleth) >1.96 z-scores) was identified in 18% (10/56) of infants with CF and was significantly correlated with diminished FEF(25-75) (r=-0.43, p<0.001) but not with LCI or FEV(0.5).
The authors concluded that despite early diagnosis of CF by NBS and protocol-driven treatment in specialist centres, abnormal lung function, with increased ventilation inhomogeneity and hyperinflation and diminished airway function, is evident in many infants with CF diagnosed through NBS by 3 months of age.


This is an important study from the UK’s leading paediatric respiratory group confirming that many screened CF infants already have abnormal lung function - 61% of the screened infants had had some respiratory symptoms (52% mild, 9% severe), 23% a positive cough swab and 73% had received antibiotics in addition to their routine prophylactic medication. With the exception of a significantly lower FEV 0.5 in those who had received additional antibiotics for symptoms or positive cough swab, there was no significant association between LF outcomes and the infant’s genotype, clinical status, growth trajectory or treatment prior to the LFTs at 3 months of age.


It is perhaps encouraging that many infants who had been treated aggressively for respiratory exacerbations in the first few months had entirely normal LF by 3 months, whereas others with no prior symptoms or cause for concern had evidence of early lung disease. So damage from very early infection is not inevitable but does support the view that early specialised management is absolutely essential to achieve the maximum benefit from newborn CF screening; also lends some support for the case for prophylactic anti staphylococcal antibiotics from diagnosis.

 

 

2014 Brown PS. Pope CE. Marsh RL. Qin X. McNamara S. Gibson R. Burns JL. Deutsch G. Hoffman LR. Directly sampling the lung of a young child with cystic fibrosis reveals diverse microbiota. Ann Amer Thorac Soc 2014; 11:1049-55 [PubMed]
Tissue of a child who underwent lobectomy for severe, localised CF lung disease was examined using DNA methods and identified diverse, and anatomically heterogeneous, bacterial populations in the lung tissue that contained both culturable and non-culturable species, including abundant Haemophilus, Ralstonia, and Propionibacterium species whereas routine clinical cultures identified only Staphylococcus aureus.
The authors concluded that diverse and spatially heterogeneous microbiota, not necessarily dominated by "traditional CF pathogens," are present in the airways of young, symptomatic children with early CF lung disease.


Previous studies of this type have all been in lungs affected by advanced disease. The presence of these additional organisms now seems established in CF patients with even mild degrees of lung involvement but the significance is still not entirely clear.