Serum Cell-Free DNA-based Detection of Mycobacterium avium Complex Infection.

Rationale: Mycobacterium avium complex (MAC) is the most common cause of nontuberculous mycobacterial (NTM) pulmonary disease (PD), which exhibits increasing global incidence. Current microbiologic methods routinely used in clinical practice lack sensitivity and have long latencies, leading to delays in diagnosis and treatment initiation and evaluation. A clustered regularly interspaced short palindromic repeats (CRISPR)-based assay that measures MAC cell-free DNA (cfDNA) concentrations in serum could provide a rapid means to detect MAC infection and monitor response to antimicrobial treatment. Objectives: To develop and optimize a CRISPR MAC assay for MAC infection detection and to evaluate its diagnostic and prognostic performance in two MAC disease cohorts. Methods: MAC cfDNA serum concentrations were measured in individuals with diagnoses of MAC disease or who had bronchiectasis or chronic obstructive pulmonary disease diagnoses without histories of NTM PD or NTM-positive sputum cultures. Diagnostic performance was analyzed using pretreatment serum from two cohorts. Serum MAC cfDNA changes during MAC PD treatment were evaluated in a subset of patients with MAC PD who received macrolide-based multidrug regimens. Measurements and Main Results: The CRISPR MAC assay detected MAC cfDNA in MAC PD with 97.6% (91.6-99.7%) sensitivity and 97.6% (91.5-99.7%) specificity overall. Serum MAC cfDNA concentrations markedly decreased after MAC-directed treatment initiation in patients with MAC PD who demonstrated MAC culture conversion. Conclusions: This study provides preliminary evidence for the utility of a serum-based CRISPR MAC assay to rapidly detect MAC infection and monitor the response to treatment.

Disulfiram Is Effective against Drug-Resistant Mycobacterium abscessus in a Zebrafish Embryo Infection Model.

Mycobacterium abscessus is an emerging nontuberculous mycobacterium (NTM) pathogen infecting susceptible people with cystic fibrosis (CF) and non-CF bronchiectasis. Here, we demonstrated the activity of an FDA-approved drug, disulfiram, against drug-susceptible and drug-resistant M. abscessus strains utilizing in vitro and intracellular macrophage assays and a zebrafish embryo infection model. These data demonstrate effective antimicrobial activity of disulfiram against M. abscessus infection in vivo and strongly support further study of disulfiram in human NTM infections.

Detection of Mixed Populations of Clarithromycin-Susceptible and -Resistant Mycobacterium abscessus Strains.

Clarithromycin resistance in Mycobacterium abscessus subsp. abscessus, massiliense, and bolletii occurs through induction of erm(41) or mutations in rrl (23S rRNA) genes. Phenotypic detection of clarithromycin resistance is hindered by the need for extended incubation as well as co-occurrence of mixed populations of M. abscessus with different susceptibility profiles. We developed a quantitative EvaGreen-based droplet digital PCR (ddPCR) scheme for rapid detection of full-length or truncated erm(41) and a probe based ddPCR screening assay for assessment of 23S rRNA rrl mutational resistance. We tested 100 M. abscessus strains, synthetic mixes with different susceptibility profiles, and 13 positive MGIT samples. Truncated and full-length erm(41) genes were detected in 27/100 and 73/100 strains and 4/13 and 9/13 MGIT samples, respectively yielding a sensitivity and specificity of 100%. Clarithromycin resistance mutations in rrl were detected in 26/100 isolates, i.e., A2058G (18/100), A2058C (7/100), and A2059G (1/100), and in 3/13 MGIT samples, i.e., A2058G (2/13) and A2059G (1/13). A screening assay of rrl ddPCR (A2058A/A2058G probes) showed 100% sensitivity in detecting the wild type or A2058G mutation as well as identifying samples requiring further testing. Upon inclusion of additional ddPCR assays, we were able to detect A2058C and A2059G clarithromycin resistance-conferring mutations in the rrl gene. Our ddPCR scheme can differentiate between full-length and truncated erm(41) and identify clarithromycin resistance-conferring mutations in the rrl gene from clinical isolates and positive MGIT samples as well as deconvolute and quantitate mixed populations of M. abscessus with different clarithromycin resistance traits.

Disseminated Mycobacterium marinum in Human Immunodeficiency Virus Unmasked by Immune Reconstitution Inflammatory Syndrome.

Distinguishing disseminated Mycobacterium marinum from multifocal cutaneous disease in persons with human immunodeficiency virus/AIDS can present a diagnostic challenge, especially in the context of immune reconstitution inflammatory syndrome (IRIS). In this work, we demonstrate the utility of flow cytometry and whole genome sequencing (WGS) to diagnose disseminated M. marinum unmasked by IRIS following initiation of antiretroviral therapy. Flow cytometry demonstrated robust cytokine production by CD4 T cells in response to stimulation with M. marinum lysate. WGS of isolates from distinct lesions was consistent with clonal dissemination, supporting that preexisting disseminated M. marinum disease was uncovered by inflammatory manifestations, consistent with unmasking mycobacterial IRIS.

Pooled Saliva Specimens for SARS-CoV-2 Testing.

We evaluated saliva (SAL) specimens for SARS-CoV-2 reverse transcriptase PCR (RT-PCR) testing by comparison of 459 prospectively paired nasopharyngeal (NP) or midturbinate (MT) swabs from 449 individuals with the aim of using saliva for asymptomatic screening. Samples were collected in a drive-through car line for symptomatic individuals (n = 380) and in the emergency department (ED) (n = 69). The percentages of positive and negative agreement of saliva compared to nasopharyngeal swab were 81.1% (95% confidence interval [CI], 65.8% to 90.5%) and 99.8% (95% CI, 98.7% to 100%), respectively. The percent positive agreement increased to 90.0% (95% CI, 74.4% to 96.5%) when considering only samples with moderate to high viral load (cycle threshold [CT ] for the NP, ≤34). Pools of five saliva specimens were also evaluated on three platforms, bioMérieux NucliSENS easyMAG with ABI 7500Fast (CDC assay), Hologic Panther Fusion, and Roche Cobas 6800. The average loss of signal upon pooling was 2 to 3 CT values across the platforms. The sensitivities of detecting a positive specimen in a pool compared with testing individually were 94%, 90%, and 94% for the CDC 2019-nCoV real-time RT-PCR, Panther Fusion SARS-CoV-2 assay, and Cobas SARS-CoV-2 test, respectively, with decreased sample detection trending with lower viral load. We conclude that although pooled saliva testing, as collected in this study, is not quite as sensitive as NP/MT testing, saliva testing is adequate to detect individuals with higher viral loads in an asymptomatic screening program, does not require swabs or viral transport medium for collection, and may help to improve voluntary screening compliance for those individuals averse to various forms of nasal collections.

Quantification of circulating Mycobacterium tuberculosis antigen peptides allows rapid diagnosis of active disease and treatment monitoring.

Tuberculosis (TB) is a major global health threat, resulting in an urgent unmet need for a rapid, non-sputum-based quantitative test to detect active Mycobacterium tuberculosis (Mtb) infections in clinically diverse populations and quickly assess Mtb treatment responses for emerging drug-resistant strains. We have identified Mtb-specific peptide fragments and developed a method to rapidly quantify their serum concentrations, using antibody-labeled and energy-focusing porous discoidal silicon nanoparticles (nanodisks) and high-throughput mass spectrometry (MS) to enhance sensitivity and specificity. NanoDisk-MS diagnosed active Mtb cases with high sensitivity and specificity in a case-control study with cohorts reflecting the complexity of clinical practice. Similar robust sensitivities were obtained for cases of culture-positive pulmonary TB (PTB; 91.3%) and extrapulmonary TB (EPTB; 92.3%), and the sensitivities obtained for culture-negative PTB (82.4%) and EPTB (75.0%) in HIV-positive patients significantly outperformed those reported for other available assays. NanoDisk-MS also exhibited high specificity (87.1-100%) in both healthy and high-risk groups. Absolute quantification of serum Mtb antigen concentration was informative in assessing responses to antimycobacterial treatment. Thus, a NanoDisk-MS assay approach could significantly improve the diagnosis and management of active TB cases, and perhaps other infectious diseases as well.

Fatal Meningitis in Patient with X-Linked Chronic Granulomatous Disease Caused by Virulent Granulibacter bethesdensis.

Granulibacter bethesdensis is a pathogen reported to cause recurrent lymphadenitis exclusively in persons with chronic granulomatous disease. We report a case of fatal meningitis caused by a highly virulent G. bethesdensis strain in an adolescent in Europe who had chronic granulomatous disease.

Performance of RGM Medium for Isolation of Nontuberculous Mycobacteria from Respiratory Specimens from Non-Cystic Fibrosis Patients.

A new selective medium for rapidly growing mycobacteria (RGM medium) was evaluated on respiratory specimens from non-cystic fibrosis patients and compared to the mycobacterial growth indicator tube (MGIT) system and Middlebrook 7H11 agar for the isolation of all nontuberculous mycobacteria (NTM). A total of 203 mucolyzed respiratory specimens collected from 163 patients were inoculated on RGM medium and incubated at both 30°C (RGM30) and 35°C (RGM35) over a 28-day period. N-Acetyl-l-cysteine-sodium hydroxide (NALC-NaOH)-decontaminated specimens were inoculated into MGIT and Middlebrook 7H11 agar and incubated at 35°C for 42 days. NTM were identified by matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) or gene sequencing. A total of 133 NTM isolates were recovered overall from 101 (49.8%) specimens collected from 85 (52.1%) patients by a combination of all culture methods. The sensitivity of RGM30 for the recovery of NTM was significantly higher than that of either the MGIT system (76.7% versus 59.4%; P = 0.01) or Middlebrook 7H11 agar (76.7% versus 47.4%; P = 0.0001) alone, but it was not significantly different from that of an acid-fast bacillus culture (AFC) which includes both MGIT and Middlebrook 7H11 agar (76.7% versus 63.9%; P = 0.0647). RGM35 had significantly lower sensitivity than the MGIT system (49.6% versus 59.4%; P = 0.0367) and AFC (49.6% versus 63.9%; P = 0.0023). RGM medium was highly effective at inhibiting the growth of nonmycobacterial organisms in the respiratory specimens, with breakthrough contamination rates of 5.4% and 4.4% for RGM30 and RGM35, respectively.

Transcriptional Response of Respiratory Epithelium to Nontuberculous Mycobacteria.

The incidence of pulmonary nontuberculous mycobacteria (NTM) disease is increasing, but host responses in respiratory epithelium infected with NTM are not fully understood. In this work, we aimed to identify infection-relevant gene expression signatures of NTM infection of the respiratory epithelium. We infected air-liquid interface (ALI) primary respiratory epithelial cell cultures with Mycobacterium avium subsp. avium (MAC) or Mycobacterium abscessus subsp. abscessus (MAB). We used cells from four different donors to obtain generalizable data. Differentiated respiratory epithelial cells at the ALI were infected with MAC or MAB at a multiplicity of infection of 100:1 or 1,000:1, and RNA sequencing was performed at Days 1 and 3 after infection. In response to infection, we found down-regulation of ciliary genes but upregulation of genes associated with cytokines/chemokines, such as IL-32, and cholesterol biosynthesis. Inflammatory response genes tended to be more upregulated by MAB than by MAC infection. Primary respiratory epithelial cell infection with NTM at the ALI identified ciliary function, cholesterol biosynthesis, and cytokine/chemokine production as major host responses to infection. Some of these pathways may be amenable to therapeutic manipulation.

Severe BCG-osis Misdiagnosed as Multidrug-Resistant Tuberculosis in an IL-12Rß1-Deficient Peruvian Girl.

PURPOSE: Mendelian suceptibility to mycobacterial disease (MSMD) is a rare primary immunodeficiency predisposing to severe disease caused by mycobacteria and other intracellular pathogens. Delay in diagnosis can have an impact on the patient's prognosis. METHODS: We evaluated the IFN-γ circuit by studying IFN-γ production after mycobacterial challenge as well as IL-12Rß1 expression and STAT4 phosphorylation in response to IL-12p70 stimulation in whole blood of a 6-year-old Peruvian girl with disseminated recurrent mycobacterial infection diagnosed as multidrug-resistant tuberculosis. Genetic studies with Sanger sequencing were used to identify the causative mutation. Microbiological studies based on PCR reactions were used to diagnose the specific mycobacterial species. RESULTS: We identified a homozygous mutation in the IL12RB1 gene (p. Arg211*) causing abolished expression of IL-12Rß1 and IL-12 response. MSMD diagnosis led to a microbiological reevaluation of the patient, revealing a BCG vaccine-related infection instead of tuberculosis. Treatment was then adjusted, with good response. CONCLUSIONS: We report the first Peruvian patient with IL-12Rß1 deficiency. Specific mycobacterial species diagnosis within Mycobacterium tuberculosis complex is still challenging in countries with limited access to PCR-based microbiological diagnostic techniques. Awareness of MSMD warning signs and accurate microbiological diagnosis of mycobacterial infections are of the utmost importance for optimal diagnosis and management of affected patients.

The Complexities of Nocardia Taxonomy and Identification.

Nocardia species are a complex group of organisms considered to belong to the aerobic actinomycetes. Of the validly described species, many have been implicated as the cause of serious human infections, especially in immunocompromised patients. The genus has a complicated taxonomic history; this is especially true for Nocardia asteroides, the type species of the genus and previously the most frequently reported nocardial taxon from human specimens. We provide background on the current taxonomy of Nocardia, with a focus on clinically relevant species, and discuss the currently available methods used to accurately identify isolates to the species, complex, or group level.

VT-1161 protects mice against oropharyngeal candidiasis caused by fluconazole-susceptible and -resistant Candida albicans.

BACKGROUND: Candida albicans, the most common human fungal pathogen, causes chronic mucosal infections in patients with inborn errors of IL-17 immunity that rely heavily on chronic, often lifelong, azole antifungal agents for treatment. However, a rise in azole resistance has predicated a need for developing new antifungal drugs. OBJECTIVES: To test the in vitro and in vivo efficacy of VT-1161 and VT-1129 in the treatment of oropharyngeal candidiasis with azole-susceptible or -resistant C. albicans strains. METHODS: MICs of VT-1161, VT-1129 and nine licensed antifungal drugs were determined for 31 Candida clinical isolates. The drug concentrations in mouse serum and tongues were measured following oral administration. IL-17-signalling-deficient Act1-/- mice were infected with fluconazole-susceptible or fluconazole-resistant C. albicans strains, and the amount of mucosal fungal burden was determined after fluconazole or VT-1161 treatment. RESULTS: Fourteen isolates (45%) were not fluconazole susceptible (MIC ≥4 mg/L). VT-1161 and VT-1129 showed significant in vitro activity against the majority of the 31 mucosal clinical isolates (MIC50 0.03 and 0.06 mg/L, respectively), including Candida glabrata (MIC50, 0.125 and 0.25 mg/L, respectively). After oral doses, VT-1161 and VT-1129 concentrations in mouse serum and tongues were well above their MIC50 values. VT-1161 was highly effective as treatment of both fluconazole-susceptible and -resistant oropharyngeal candidiasis in Act1-/- mice. CONCLUSIONS: VT-1129 and VT-1161 exhibit significant in vitro activity against Candida strains, including fluconazole-resistant C. albicans and C. glabrata. VT-1161 administration in mice results in significant mucosal drug accumulation and eradicates infection caused by fluconazole-susceptible and -resistant Candida strains.

Antigen 85B peptidomic analysis allows species-specific mycobacterial identification.

BACKGROUND: Nontuberculous mycobacteria (NTM)-mediated infections are a growing cause of worldwide morbidity, but lack of rapid diagnostics for specific NTM species can delay the initiation of appropriate treatment regimens. We thus examined whether mass spectrometry analysis of an abundantly secreted mycobacterial antigen could identify specific NTM species. METHODS: We analyzed predicted tryptic peptides of the major mycobacterial antigen Ag85B for their capacity to distinguish Mycobacterium tuberculosis and three NTM species responsible for the majority of pulmonary infections caused by slow-growing mycobacterial species. Next, we analyzed trypsin-digested culture supernatants of these four mycobacterial species by liquid chromatography-tandem mass spectrometry (LC-MS/MS) to detect candidate species-specific Ag85B peptides, the identity of which were validated by LC-MS/MS performed in parallel reaction monitoring mode. RESULTS: Theoretical tryptic digests of the Ag85B proteins of four common mycobacterial species produced peptides with distinct sequences, including two peptides that could each identify the species origin of each Ag85B protein. LC-MS/MS analysis of trypsinized culture supernatants of these four species detected one of these species-specific signature peptides in each sample. Subsequent LC-MS/MS analyses confirmed these results by targeting these species-specific Ag85B peptides. CONCLUSIONS: LC-MS/MS analysis of Ag85B peptides from trypsin-digested mycobacterial culture supernatants can rapidly detect and identify common mycobacteria responsible for most pulmonary infections caused by slow-growing mycobacteria, and has the potential to rapidly diagnose pulmonary infections caused by these mycobacteria through direct analysis of clinical specimens.

Phellinus species: An emerging cause of refractory fungal infections in patients with X-linked chronic granulomatous disease.

Aspergillus spp. are a leading cause of mortality in chronic granulomatous disease (CGD), but other fungi have emerged in the era of mould prophylaxis. Of these, Phellinus spp. are an under-recognised cause of invasive fungal infections (IFIs) in CGD, and data on their presentation and management are scarce. We present a patient with CGD who developed disseminated IFI involving the lungs and brain. Surgical specimens grew a basidiomycete which was disregarded as a contaminant. After three months of progressive disease despite antifungals, he was diagnosed with Phellinus tropicalis by internal transcribed spacer (ITS) sequencing. He improved with amphotericin B and isavuconazole but required haematopoietic stem cell transplantation (HSCT). We review the literature on Phellinus infections in CGD and conclude that: (i) these infections emerge on mould-active prophylaxis and are indolent; (ii) they typically cause locally destructive disease but can disseminate; (iii) diagnosis is delayed and requires molecular methods; (iv) amphotericin B is most active in vitro; and (v) treatment is protracted and requires surgery and possibly HSCT. In conclusion, Phellinus spp. are emerging pathogens in CGD. Every effort should be made to establish the diagnosis of non-Aspergillus IFIs in patients with CGD by sending tissue specimens for molecular diagnostics.

Methylotroph Infections and Chronic Granulomatous Disease.

Chronic granulomatous disease (CGD) is a primary immunodeficiency caused by a defect in production of phagocyte-derived reactive oxygen species, which leads to recurrent infections with a characteristic group of pathogens not previously known to include methylotrophs. Methylotrophs are versatile environmental bacteria that can use single-carbon organic compounds as their sole source of energy; they rarely cause disease in immunocompetent persons. We have identified 12 infections with methylotrophs (5 reported here, 7 previously reported) in patients with CGD. Methylotrophs identified were Granulibacter bethesdensis (9 cases), Acidomonas methanolica (2 cases), and Methylobacterium lusitanum (1 case). Two patients in Europe died; the other 10, from North and Central America, recovered after prolonged courses of antimicrobial drug therapy and, for some, surgery. Methylotrophs are emerging as disease-causing organisms in patients with CGD. For all patients, sequencing of the 16S rRNA gene was required for correct diagnosis. Geographic origin of the methylotroph strain may affect clinical management and prognosis.

Genomic insights into the fate of colistin resistance and Acinetobacter baumannii during patient treatment.

Bacterial whole-genome sequencing (WGS) of human pathogens has provided unprecedented insights into the evolution of antibiotic resistance. Most studies have focused on identification of resistance mutations, leaving one to speculate on the fate of these mutants once the antibiotic selective pressure is removed. We performed WGS on longitudinal isolates of Acinetobacter baumannii from patients undergoing colistin treatment, and upon subsequent drug withdrawal. In each of the four patients, colistin resistance evolved via mutations at the pmr locus. Upon colistin withdrawal, an ancestral susceptible strain outcompeted resistant isolates in three of the four cases. In the final case, resistance was also lost, but by a compensatory inactivating mutation in the transcriptional regulator of the pmr locus. Notably, this inactivating mutation reduced the probability of reacquiring colistin resistance when subsequently challenged in vitro. On face value, these results supported an in vivo fitness cost preventing the evolution of stable colistin resistance. However, more careful analysis of WGS data identified genomic evidence for stable colistin resistance undetected by clinical microbiological assays. Transcriptional studies validated this genomic hypothesis, showing increased pmr expression of the initial isolate. Moreover, altering the environmental growth conditions of the clinical assay recapitulated the classification as colistin resistant. Additional targeted sequencing revealed that this isolate evolved undetected in a patient undergoing colistin treatment, and was then transmitted to other hospitalized patients, further demonstrating its stability in the absence of colistin. This study provides a unique window into mutational pathways taken in response to antibiotic pressure in vivo, and demonstrates the potential for genome sequence data to predict resistance phenotypes.

Performance of the Cryptococcal Antigen Lateral Flow Assay in Non-HIV-Related Cryptococcosis.

The cryptococcal antigen lateral flow assay (CrAg LFA) was evaluated for the diagnosis of cryptococcosis in HIV-negative patients. The sensitivity was excellent, suggesting that this assay can replace conventional testing based on latex agglutination (LA). CrAg LFA and LA titers were correlated but were not directly comparable, with implications for conversion between assays.

Multicenter Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry Study for Identification of Clinically Relevant Nocardia spp.

This multicenter study analyzed Nocardia spp., including extraction, spectral acquisition, Bruker matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) identification, and score interpretation, using three Nocardia libraries, the Bruker, National Institutes of Health (NIH), and The Ohio State University (OSU) libraries, and compared the results obtained by each center. A standardized study protocol, 150 Nocardia isolates, and NIH and OSU Nocardia MALDI-TOF MS libraries were distributed to three centers. Following standardized culture, extraction, and MALDI-TOF MS analysis, isolates were identified using score cutoffs of ≥2.0 for species/species complex-level identification and ≥1.8 for genus-level identification. Isolates yielding a score of <2.0 underwent a single repeat extraction and analysis. The overall score range for all centers was 1.3 to 2.7 (average, 2.2 ± 0.3), with common species generally producing higher average scores than less common ones. Score categorization and isolate identification demonstrated 86% agreement between centers; 118 of 150 isolates were correctly identified to the species/species complex level by all centers. Nine strains (6.0%) were not identified by any center, and six (4.0%) of these were uncommon species with limited library representation. A categorical score discrepancy among centers occurred for 21 isolates (14.0%). There was an overall benefit of 21.2% from repeat extraction of low-scoring isolates and a center-dependent benefit for duplicate spotting (range, 2 to 8.7%). Finally, supplementation of the Bruker Nocardia MALDI-TOF MS library with both the OSU and NIH libraries increased the genus-level and species-level identification by 18.2% and 36.9%, respectively. Overall, this study demonstrates the ability of diverse clinical microbiology laboratories to utilize MALDI-TOF MS for the rapid identification of clinically relevant Nocardia spp. and to implement MALDI-TOF MS libraries developed by single laboratories across institutions.

GATA2 deficiency: a protean disorder of hematopoiesis, lymphatics, and immunity.

Haploinsufficiency of the hematopoietic transcription factor GATA2 underlies monocytopenia and mycobacterial infections; dendritic cell, monocyte, B, and natural killer (NK) lymphoid deficiency; familial myelodysplastic syndromes (MDS)/acute myeloid leukemia (AML); and Emberger syndrome (primary lymphedema with MDS). A comprehensive examination of the clinical features of GATA2 deficiency is currently lacking. We reviewed the medical records of 57 patients with GATA2 deficiency evaluated at the National Institutes of Health from January 1, 1992, to March 1, 2013, and categorized mutations as missense, null, or regulatory to identify genotype-phenotype associations. We identified a broad spectrum of disease: hematologic (MDS 84%, AML 14%, chronic myelomonocytic leukemia 8%), infectious (severe viral 70%, disseminated mycobacterial 53%, and invasive fungal infections 16%), pulmonary (diffusion 79% and ventilatory defects 63%, pulmonary alveolar proteinosis 18%, pulmonary arterial hypertension 9%), dermatologic (warts 53%, panniculitis 30%), neoplastic (human papillomavirus+ tumors 35%, Epstein-Barr virus+ tumors 4%), vascular/lymphatic (venous thrombosis 25%, lymphedema 11%), sensorineural hearing loss 76%, miscarriage 33%, and hypothyroidism 14%. Viral infections and lymphedema were more common in individuals with null mutations (P = .038 and P = .006, respectively). Monocytopenia, B, NK, and CD4 lymphocytopenia correlated with the presence of disease (P < .001). GATA2 deficiency unites susceptibility to MDS/AML, immunodeficiency, pulmonary disease, and vascular/lymphatic dysfunction. Early genetic diagnosis is critical to direct clinical management, preventive care, and family screening.