ECFS standards of care on CFTR-related disorders: Identification and care of the disorders.

This is the third paper in the series providing updated information and recommendations for people with cystic fibrosis transmembrane conductance regulator (CFTR)-related disorder (CFTR-RD). This paper covers the individual disorders, including the established conditions - congenital absence of the vas deferens (CAVD), diffuse bronchiectasis and chronic or acute recurrent pancreatitis - and also other conditions which might be considered a CFTR-RD, including allergic bronchopulmonary aspergillosis, chronic rhinosinusitis, primary sclerosing cholangitis and aquagenic wrinkling. The CFTR functional and genetic evidence in support of the condition being a CFTR-RD are discussed and guidance for reaching the diagnosis, including alternative conditions to consider and management recommendations, is provided. Gaps in our knowledge, particularly of the emerging conditions, and future areas of research, including the role of CFTR modulators, are highlighted.

The multi-faceted nature of 15 CFTR exonic variations: Impact on their functional classification and perspectives for therapy.

BACKGROUND: The majority of variants of unknown clinical significance (VUCS) in the CFTR gene are missense variants. While change on the CFTR protein structure or function is often suspected, impact on splicing may be neglected. Such undetected splicing default of variants may complicate the interpretation of genetic analyses and the use of an appropriate pharmacotherapy. METHODS: We selected 15 variants suspected to impact CFTR splicing after in silico predictions on 319 missense variants (214 VUCS), reported in the CFTR-France database. Six specialized laboratories assessed the impact of nucleotide substitutions on splicing (minigenes), mRNA expression levels (quantitative PCR), synthesis and maturation (western blot), cellular localization (immunofluorescence) and channel function (patch clamp) of the CFTR protein. We also studied maturation and function of the truncated protein, consecutive to in-frame aberrant splicing, on additional plasmid constructs. RESULTS: Six of the 15 variants had a major impact on CFTR splicing by in-frame (n = 3) or out-of-frame (n = 3) exon skipping. We reclassified variants into: splicing variants; variants causing a splicing defect and the impairment of CFTR folding and/or function related to the amino acid substitution; deleterious missense variants that impair CFTR folding and/or function; and variants with no consequence on the different processes tested. CONCLUSION: The 15 variants have been reclassified by our comprehensive approach of in vitro experiments that should be used to properly interpret very rare exonic variants of the CFTR gene. Targeted therapies may thus be adapted to the molecular defects regarding the results of laboratory experiments.

External quality assessment of laboratory performance in bacteriology in the Eastern Mediterranean Region, 2011-2019.

Background: Since 2007, national public health laboratories in the WHO Eastern Mediterranean Region (EMR) have participated in a regional external quality assessment scheme in bacteriology to improve testing proficiency. Aims: To assess laboratory performance in bacteriology in the EMR between 2011 and 2019 using the regional external quality assessment scheme. Methods: We analysed the accuracy of participant-reported data in bacterial identification, Gram stain microscopy, and antimicrobial susceptibility testing. For each category, we assessed the performance over time, the performance on multiple organisms, and whether a laboratory repeatedly failed to attain satisfactory results. Results: Between 2011 and 2019, 70% of laboratories achieved satisfactory performance for bacterial identification and antimicrobial susceptibility testing, and 85% performed satisfactory Gram stain microscopy. Testing did not improve on multiple organisms and results were consistently low for some pathogens and test categories. Twenty-nine percent of laboratories underperformed throughout the study period. Conclusion: The unchanged performance over time and underperformance of laboratories highlight the need for improvements in the regional external quality assessment scheme. Participating laboratories and WHO need to work more actively to strengthen the problem areas.

Performance of Eastern Mediterranean Region laboratories in the World Health Organization external quality assessment programme for arbovirus diagnostics.

BACKGROUND: Arboviruses such as dengue virus, yellow fever virus, Zika virus and chikungunya virus are major threats to human health globally, including countries in the Eastern Mediterranean Region (EMR). AIMS: This study aimed to assess laboratory proficiency in EMR countries for detection of dengue virus, yellow fever virus, Zika virus and chikungunya virus. METHODS: A global external quality assessment programme for arbovirus diagnostics was developed and run in 2016 and 2018. National-level public health laboratories were instructed to apply the polymerase chain reaction detection method on specimen panels containing dengue virus, yellow fever virus, Zika virus and chikungunya virus. RESULTS: Over both rounds of the programme, 100% of participating EMR laboratories correctly detected yellow fever virus and chikungunya virus, ≥ 84.6% detected dengue fever virus and ≥ 76.9% detected Zika virus. CONCLUSION: While participating EMR countries demonstrated good proficiency in detecting arboviruses, only half of them were enrolled in the global external quality assessment programme, providing an incomplete picture of regional capacity. Effort should be put into increasing participation in subsequent rounds.

Penetrance is a critical parameter for assessing the disease liability of CFTR variants.

BACKGROUND: Major issues of newborn screening (NBS) for CF are the assessment of disease liability of variants and of the penetrance of clinical CF, notably in inconclusive diagnosis. The penetrance of CF is defined as the risk of a particular genotype to lead to a CF phenotype. METHODS: We aimed to get insight into the penetrance of CF for fifteen CFTR variants: 5 frequent CF-causing and 10 classified as of varying clinical consequence (VCC) or associated with a CFTR-related disorder (CFTR-RD) in CFTR2 or CFTR-France databases. The penetrance was approached by: (1) comparison of variant allelic frequencies in CF patients (CFTR2) and in the general population; (2) estimation of the likelihood of a positive NBS test for the 14 compound heterozygous with F508del and the F508del homozygous genotypes, defined as the ratio of detected/expected number of neonates with a given genotype in the 2002-2017 period. RESULTS: A full penetrance was observed for severe CF-causing variants. Five variants were more frequently found in the general population than in CF patients: TG11T5, TG12T5, TG13T5, L997F and R117H;T7. The likelihood of a positive NBS test was 0.03% for TG11T5, 0.3% for TG12T5, 1.9% for TG13T5, 0.6% for L997F, 11.7% for D1152H, and 17.8% for R117H;T7. Penetrance varied greatly for variants with discrepant classification between CFTR2 and CFTR-France: 5.1% for R117C, 12.3% for T338I, 43.5% for D110H and 52.6% for L206W. CONCLUSION: These results illustrate the contribution of genetics population data to assess the disease liability of variants for diagnosis and genetic counselling purposes.

Variant classifications, databases and genotype-phenotype correlations.

Because CFTR gene studies now represent one of the most frequent genetic analyses routinely performed worldwide, the number of rare CFTR variants identified in various clinical situations, regularly increases. To provide appropriate diagnosis and prognosis to CF patients as well as appropriate genetic counseling to families, the clinical impact and the phenotypic spectrum of variants identified by diagnostic techniques need to be characterized. Three complementary locus specific databases, called CFTR1, CFTR2 and CFTR-France were developed to address these issues. Besides, the growing knowledge of the CF pathophysiology and the technical evolution in molecular biology allowed to identify candidate modifier genes, regulatory loci, epigenetic profiles and trans-regulators that could help to refine genotype-phenotype correlations at the individual level. These different factors may contribute to the large phenotypic variability between patients with CF, even when they carry identical CFTR variants, regarding lung function, meconium ileus susceptibility or the risk for developing CFTR-related diabetes and liver disease. Finally, the availability of new therapies that target the CFTR protein for numbers of CF patients led to the identification of 'good' and 'poor' responders, thus raising questions of pharmacogenetics factors that may influence treatment efficiency as a novel feature of the complexity of CF patients' management. © 2020 French Society of Pediatrics. Published by Elsevier Masson SAS. All rights reserved.

Functional characterization and phenotypic spectrum of three recurrent disease-causing deep intronic variants of the CFTR gene.

BACKGROUND: The CFTR genotype remains incomplete in 1% of Cystic Fibrosis (CF) cases, because only one or no disease-causing variants is detected after extended analysis. This fraction is probably higher in CFTR-Related Disorders (CFTR-RD). Deep-intronic CFTR variants are putative candidates to fill this gap. However, the recurrence, phenotypic spectrum and full molecular characterization of newly reported variants are unknown. METHODS: Minigenes and analysis of CFTR transcripts in nasal epithelial cells were used to determine the impact on CFTR splicing of intronic variants that we previously identified by next generation sequencing of the whole CFTR locus. Phenotypic data were collected in 19 patients with CF and CFTR-RD, in whom one of the deep intronic variants has been detected. RESULTS: Three deep-intronic variants promoted the inclusion of pseudo-exons (PE) in the CFTR transcript, hindering the synthesis of a functional protein. The c.2989-313A > T variant, detected in four patients with CF or CFTR-RD from three different families, led to the inclusion of a 118 bp PE. The c.3469-1304C > G variant promoted the inclusion of a 214 bp-PE and was identified in five patients with CF from four families. Haplotype analysis confirmed that this variant was associated with one CF chromosome of African origin. The most represented variant in our cohort was the c.3874-4522A > G, detected in 10 patients with various phenotypes, from male infertility to CF with pancreatic insufficiency. CONCLUSION: These three deep intronic CFTR variants are associated with a large phenotypic spectrum, including typical CF. They should be included in CF diagnostic testing and carrier screening strategies.

Guidelines for the clinical management and follow-up of infants with inconclusive cystic fibrosis diagnosis through newborn screening.

Neonatal screening for cystic fibrosis (CF) can detect infants with elevated immunoreactive trypsinogen (IRT) levels and inconclusive sweat tests and/or CFTR DNA results. These cases of uncertain diagnosis are defined by (1) either the presence of at most one CF-associated cystic fibrosis transmembrane conductance regulator (CFTR) mutation with sweat chloride values between 30 and 59mmol/L or (2) two CFTR mutations with at least one of unknown pathogenic potential and a sweat chloride concentration below 60mmol/L. This encompasses various clinical situations whose progression cannot be predicted. In these cases, a sweat chloride test has to be repeated at 12 months, and if possible at 6 and 24 months of life along with extended CFTR sequencing to detect rare mutations. When the diagnosis is not definite, CFTR functional explorations may provide a better understanding of CFTR dysfunction. The initial evaluation of these infants must be conducted in dedicated CF reference centers and should include bacteriological sputum analysis, chest radiology, and fecal elastase assay. The primary care physicians in charge of these patients should be familiar with the current management of CF and should work in collaboration with CF centers. A follow-up should be performed in a CF reference center at 3, 6, and 12 months of life and every year thereafter. Any symptom indicative of CF requires immediate reevaluation of the diagnosis. These guidelines were established by the "neonatal screening and difficult diagnoses" working group of the French CF society. Their objective is to standardize the management of infants with unclear diagnosis.

External quality assessment for arbovirus diagnostics in the World Health Organization Western Pacific Region, 2013-2016: improving laboratory quality over the years.

Arboviruses continue to pose serious public health threats in the World Health Organization (WHO) Western Pacific Region. As such, laboratories need to be equipped for their accurate detection. In 2011, to ensure test proficiency, the WHO Regional Office for the Western Pacific piloted an external quality assessment (EQA) programme for arbovirus diagnostics. By 2016, it had grown into a global programme with participation of 96 laboratories worldwide, including 25 laboratories from 19 countries, territories and areas in the Region. The test performance of the 25 laboratories in the Region in 2016 was high with 23 (92%) reporting correct results in all specimens for dengue and chikungunya viruses. For Zika virus, 18 (72%) of the 25 laboratories reported correct results in all specimens, while seven (28%) demonstrated at least one error. When comparing iterations of this EQA programme in the Region between 2013 and 2016, the number of participating laboratories increased from 18 to 25. The first round only included dengue virus, while the latest round additionally included chikungunya, Zika and yellow fever viruses. Proficiency for molecular detection of dengue virus remained high (83-94%) over the four-year period. The observed proficiency for arbovirus diagnostics between 2013 and 2016 is an indicator of laboratory quality improvement in the Region.

[Management of infants whose diagnosis is inconclusive at neonatal screening for cystic fibrosis]. / Recommandations pour la prise en charge et le suivi des nourrissons pour lesquels un diagnostic de mucoviscidose n'a pu être conclu après dépistage néonatal.

Neonatal screening for cystic fibrosis (CF) may detect infants with elevated immunoreactive trypsinogen (IRT) levels but with inconclusive sweat tests and/or DNA results. This includes cases associating (1) either the presence of at most one CF-causing mutation and sweat chloride values between 30 and 59mmol/L or (2) two CFTR mutations with at least one of unknown pathogenicity and a sweat chloride below 60mmol/L. This encompasses different clinical situations whose progression cannot be predicted. These cases require redoing the sweat test at 12 months and if possible at 6 and 24 months of life. This must be associated with extended genotyping. CFTR functional explorations can also help by investigating CFTR dysfunction. These infants must be initially evaluated in dedicated CF centers including bacteriological sputum analysis, chest radiology and fecal elastase dosage. A home practitioner must be informed of the specificity of follow-up. These infants will be reviewed in the CF center at 3, 6 and 12 months and every year. Any CF-related symptom requires reevaluation of the diagnosis. These guidelines were established by the "neonatal screening and difficult diagnoses" working group of the French CF Society. They aim to standardize management of infants with unclear diagnosis in French CF centers.

Non-invasive prenatal diagnosis (NIPD) of cystic fibrosis: an optimized protocol using MEMO fluorescent PCR to detect the p.Phe508del mutation.

BACKGROUND: Analysis of cell-free foetal DNA (cff-DNA) in maternal plasma is very promising for early diagnosis of monogenic diseases; in particular, cystic fibrosis (CF). However, NIPD of single-gene disorders has been limited by the availability of suitable technical platforms and the need to set up patient or disease-specific custom-made approaches. METHODS: To make research applications more readily accessible to the clinic, we offer a simple assay combining two independent methods to determine the presence or absence of paternally inherited foetal allele p.Phe508del (the most frequent mutation in CF patients worldwide). The first method detects the presence or absence of a p.Phe508del allele by Mutant Enrichment with 3'-Modified Oligonucleotide PCR coupled to Fragment Length Analysis (MEMO-PCR-FLA). The second method detects the p.Phe508del allele with classical Multiplex Fluorescent PCR including five intragenic and extragenic STR markers of the CFTR locus and a specific SRY sequence. RESULTS: We collected 24 plasma samples from 23 women carrying foetuses at risk for CF and tested each sample using both methods. Our new procedures were successfully applied to 10 couples where fathers carried the p.Phe508del mutation and mothers were carrying a different mutation in the CFTR gene. These simple tests provided clear positive or negative results from the maternal plasma of the pregnant women. We confirmed the presence of cff-DNA in the studied samples by the identification of a tri-allelic DNA profile using a miniSTR kit. All results were correlated with chorionic villus sampling or amniocentesis analyses. CONCLUSIONS: This NIPD approach, easily set up in any clinical laboratory where prenatal diagnosis is routinely performed, offers many advantages over current methods: it is simple, rapid, and cost-effective. It opens up the possibility for testing a large number of couples with offspring at risk for CF.

Preparedness for Zika virus testing in the World Health Organization Western Pacific Region.

On 1 February 2016, the World Health Organization (WHO) declared that clusters of microcephaly cases and other neurological disorders occurring in Zika virus (ZIKV)-affected areas constituted a public health emergency of international concern. Increased surveillance of the virus, including the requirement for laboratory confirmation of infection, was recommended. The WHO Regional Office for the Western Pacific therefore initiated a rapid survey among national-level public health laboratories in 19 countries and areas to determine regional capacity for ZIKV detection. The survey indicated that 16/19 (84%) countries had capacity for molecular detection of ZIKV while others facilitated testing through referral. These results suggest that robust laboratory capacity is in place to support ZIKV surveillance in the Western Pacific Region.

External quality assessment of dengue and chikungunya diagnostics in the Asia Pacific region, 2015.

OBJECTIVE: To conduct an external quality assessment (EQA) of dengue and chikungunya diagnostics among national-level public health laboratories in the Asia Pacific region following the first round of EQA for dengue diagnostics in 2013. METHODS: Twenty-four national-level public health laboratories performed routine diagnostic assays on a proficiency testing panel consisting of two modules. Module A contained serum samples spiked with cultured dengue virus (DENV) or chikungunya virus (CHIKV) for the detection of nucleic acid and DENV non-structural protein 1 (NS1) antigen. Module B contained human serum samples for the detection of anti-DENV antibodies. RESULTS: Among 20 laboratories testing Module A, 17 (85%) correctly detected DENV RNA by reverse transcription polymerase chain reaction (RT-PCR), 18 (90%) correctly determined serotype and 19 (95%) correctly identified CHIKV by RT-PCR. Ten of 15 (66.7%) laboratories performing NS1 antigen assays obtained the correct results. In Module B, 18/23 (78.3%) and 20/20 (100%) of laboratories correctly detected anti-DENV IgM and IgG, respectively. Detection of acute/recent DENV infection by both molecular (RT-PCR) and serological methods (IgM) was available in 19/24 (79.2%) participating laboratories. DISCUSSION: Accurate laboratory testing is a critical component of dengue and chikungunya surveillance and control. This second round of EQA reveals good proficiency in molecular and serological diagnostics of these diseases in the Asia Pacific region. Further comprehensive diagnostic testing, including testing for Zika virus, should comprise future iterations of the EQA.

First round of external quality assessment of dengue diagnostics in the WHO Western Pacific Region, 2013.

OBJECTIVE: Accurate laboratory testing is a critical component of dengue surveillance and control. The objective of this programme was to assess dengue diagnostic proficiency among national-level public health laboratories in the World Health Organization (WHO) Western Pacific Region. METHODS: Nineteen national-level public health laboratories performed routine dengue diagnostic assays on a proficiency testing panel consisting of two modules: one containing commercial serum samples spiked with cultured dengue viruses for the detection of nucleic acid and non-structural protein 1 (NS1) (Module A) and one containing human serum samples for the detection of anti-dengue virus antibodies (Module B). A review of logistics arrangements was also conducted. RESULTS: All 16 laboratories testing Module A performed reverse transcriptase polymerase chain reaction (RT-PCR) for both RNA and serotype detection. Of these, 15 had correct results for RNA detection and all 16 correctly serotyped the viruses. All nine laboratories performing NS1 antigen detection obtained the correct results. Sixteen of the 18 laboratories using IgM assays in Module B obtained the correct results as did the 13 laboratories that performed IgG assays. Detection of ongoing/recent dengue virus infection by both molecular (RT-PCR) and serological methods (IgM) was available in 15/19 participating laboratories. DISCUSSION: This first round of external quality assessment of dengue diagnostics was successfully conducted in national-level public health laboratories in the WHO Western Pacific Region, revealing good proficiency in both molecular and serological testing. Further comprehensive diagnostic testing for dengue virus and other priority pathogens in the Region will be assessed during future rounds.

Ebola preparedness in the Western Pacific Region, 2014.

West Africa is currently experiencing the largest outbreak of Ebola virus disease (EVD) in history with intense transmission in several affected countries. For non-affected countries, the best protective measures are adequate levels of preparedness including vigilant surveillance to detect cases early and well prepared health systems to ensure rapid containment of the virus and to avoid further spread. The World Health Organization Regional Office for the Western Pacific recently conducted two activities: a web-based EVD preparedness survey and an EVD simulation exercise to determine the overall level of EVD preparedness in the Region. The survey and exercise together demonstrate there is a good overall level of preparedness for a potential imported case of EVD in the Western Pacific Region. However, several areas still require further strengthening before the Region can efficiently and effectively respond to potential EVD events, including laboratory testing arrangements; clinical management and infection prevention and control; and public health intervention measures, particularly at points of entry. Importantly, the survey and exercise also highlight the unique situation in Pacific island countries and emphasize that special considerations are needed to better support these countries in EVD preparedness.

Proteasomes control caspase-1 activation in anthrax lethal toxin-mediated cell killing.

Activation of caspase-1 through the inflammasome protein Nalp1b controls anthrax lethal toxin (LT)-induced necrosis in murine macrophages. In this study we analyzed physiological changes controlled by caspase-1 in LT-treated murine macrophages. The caspase-1 inhibitor Boc-D-cmk blocked caspase-1 activity and membrane impairment in LT-treated cells. To determine the relationship between caspase-1 activation and membrane integrity, we added Boc-D-cmk to J774A.1 macrophages at different time points following LT exposure. Remarkably, Boc-D-cmk rescued LT-treated macrophages, even when added at the peak of caspase-1 activation. Late addition of the caspase-1 inhibitor reversed the losses of plasma membrane integrity and metabolic activity in these cells. Similar results were obtained with the proteasome inhibitor MG132, one of the most potent inhibitors of LT toxicity. LT-treated macrophages displaying evidence of membrane impairment recovered upon the addition of MG132, mirroring the Boc-D-cmk response. Strikingly, late addition of proteasome inhibitors also abrogated caspase-1 activity in LT-treated macrophages. Proteasomal control of caspase-1 activity and membrane impairment, however, was restricted to LT-induced cytolysis, because proteasome inhibitors did not block caspase-1 activation and cell death triggered by lipopolysaccharide and nigericin. Our findings indicate that proteasome inhibitors do not target caspase-1 directly but instead control an upstream event in LT-treated macrophages leading to caspase-1 activation. Taken together, caspase-1-mediated necrosis appears to be tightly controlled and differentially regulated by proteasomes depending on the source of caspase-1 induction.

Anthrax lethal toxin kills macrophages in a strain-specific manner by apoptosis or caspase-1-mediated necrosis.

Murine macrophages have been classified as either susceptible or nonsusceptible to killing by anthrax lethal toxin (LT) depending upon genetic background. While considered resistant to LT killing, we found that bone marrow-derived macrophages (BMMs) from DBA/2, AKR, and C57BL/6 mice were slowly killed by apoptosis following LT exposure. LT killing was not restricted to in vitro assays, as splenic macrophages were also depleted in LT-injected C57BL/6 mice. Human macrophages, also considered LT resistant, similarly underwent slow apoptosis in response to LT challenge. In contrast, LT triggered rapid necrosis and broad protein release in BMMs derived from BALB/c and C3H/HeJ, but not C57BL/6 mice. Released proteins included processed interleukin-18, confirming reports of inflammasome and caspase-1 activation in LT-mediated necrosis in macrophages. Complete inhibition of caspase-1 activity was required to block LT-mediated necrosis. Strikingly, minimal residual caspase-1 activity was sufficient to trigger significant necrosis in LT-treated macrophages, indicating the toxicity of caspase-1 in this process. IL-18 release does not trigger cytolysis, as IL-18 is released late and only from LT-treated macrophages undergoing membrane perturbation. We propose that caspase-1-mediated macrophage necrosis is the source of the cytokine storm and rapid disease progression reported in LT-treated BALB/c mice.