Genotype-phenotype of autosomal dominant polycystic kidney disease in Malta.

BACKGROUND: Autosomal dominant polycystic kidney disease (ADPKD) is characterized by the development of multiple renal cysts causing kidney enlargement and end-stage renal disease (ESRD) in half the patients by 60 years of age. The aim of the study was to determine the genetic aetiology in Maltese patients clinically diagnosed with ADPKD and correlate the clinical features. METHODS: A total of 60 patients over 18 years of age clinically diagnosed with ADPKD were studied using a customized panel of genes that had sufficient evidence of disease diagnosis using next generation sequencing (NGS). The genes studied were PKD1, PKD2, GANAB, DNAJB11, PKHD1 and DZIP1L. Selected variants were confirmed by bidirectional Sanger sequencing with specifically designed primers. Cases where no clinically significant variant was identified by the customized gene panel were then studied by Whole Exome Sequencing (WES). Microsatellite analysis was performed to determine the origin of an identified recurrent variant in the PKD2 gene. Clinical features were studied for statistical correlation with genetic results. RESULTS: Genetic diagnosis was reached in 49 (82%) of cases studied. Pathogenic/likely pathogenic variants PKD1 and PKD2 gene were found in 25 and in 23 cases respectively. The relative proportion of genetically diagnosed PKD1:PKD2 cases was 42:38. A pathogenic variant in the GANAB gene was identified in 1 (2%) case. A potentially significant heterozygous likely pathogenic variant was identified in PKHD1 in 1 (2%) case. Potentially significant variants of uncertain significance were seen in 4 (7%) cases of the study cohort. No variants in DNAJB11 and DZIP1L were observed. Whole exome sequencing (WES) added the diagnostic yield by 10% over the gene panel analysis. Overall no clinically significant variant was detected in 6 (10%) cases of the study population by a customized gene panel and WES. One recurrent variant the PKD2 c.709+1G > A was observed in 19 (32%) cases. Microsatellite analysis showed that all variant cases shared the same haplotype indicating that their families may have originated from a common ancestor and confirmed it to be a founder variant in the Maltese population. The rate of decline in eGFR was steeper and progression to ESRD was earlier in cases with PKD1 variants when compared to cases with PKD2 variants. Cases segregating truncating variants in PKD1 showed a significantly earlier onset of ESRD and this was significantly worse in cases with frameshift variants. Overall extrarenal manifestations were commoner in cases segregating truncating variants in PKD1. CONCLUSIONS: This study helps to show that a customized gene panel is the first-line method of choice for studying patients with ADPKD followed by WES which increased the detection of variants present in the PKD1 pseudogene region. A founder variant in the PKD2 gene was identified in our Maltese cohort with ADPKD. Phenotype of patients with ADPKD is significantly related to the genotype confirming the important role of molecular investigations in the diagnosis and prognosis of polycystic kidney disease. Moreover, the findings also highlight the variability in the clinical phenotype and indicate that other factors including epigenetic and environmental maybe be important determinants in Autosomal Dominant Polycystic Kidney Disease.

Quality control and external quality assessment for the independent clinic-based evaluation of point-of-care testing to detect Chlamydia trachomatis, Neisseria gonorrhoeae and Trichomonas vaginalis in eight countries.

BACKGROUND: Sexually transmitted infections caused by Chlamydia trachomatis (CT), Neisseria gonorrhoeae (NG) and Trichomonas vaginalis (TV) remain significant global health problems. The World Health Organization (WHO) has recently conducted a multi-faceted, multi-country validation study (ProSPeRo), which included an evaluation of the Xpert CT/NG and Xpert TV assays on the GeneXpert system (Cepheid, Sunnyvale, Ca., USA) in clinic-based settings across eight countries. To support the study, a training and quality management system was implemented and evaluated. METHODS: A comprehensive training program for the study was developed. Quality control (QC) and external quality assessment (EQA) samples were provided by an accredited quality assurance provider. QC testing was conducted at 14 point-of-care testing (POCT) clinics, while EQA samples were tested by the POCT sites and a reference laboratory supporting each clinic. RESULTS: For QC testing, concordance with the expected results for CT and NG was > 99% and rates of unsuccessful tests were < 4%. For TV testing, concordance was similar (97%), but rates of unsuccessful tests were high (18%), particularly in the 'TV negative' sample. For EQA testing initially conducted in 2018, concordance was 100% for CT and NG, and 90% for TV for the reference laboratory group (which used non-GeneXpert systems). Concordance for the POCT group was also high (> 94%) for all analytes, but this cohort (which used GeneXpert systems) exhibited a high rate of unsuccessful TV tests. All but one of these unsuccessful tests was subcategorised as 'invalid'. CONCLUSIONS: The high level of concordance for QC and EQA testing confirm that the trained operators at the POC clinical sites were competent to conduct POC testing and that the training and quality systems implemented for the ProSPeRo study were effective. The quality materials used were satisfactory for CT and NG but exhibited poor performance for TV testing on the GeneXpert system. The WHO should continue to work with industry and EQA providers to provide improved materials that are reliable, stable and cost effective for quality management, as it seeks to rollout molecular-based STI POCT in non-laboratory-based settings. TRIAL REGISTRATION: Ethics approval to conduct the ProSPeRo study was granted by the WHO Ethics Review Committee.

Seasonal and inter-seasonal RSV activity in the European Region during the COVID-19 pandemic from autumn 2020 to summer 2022.

Background: The emergence of the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) in early 2020 and subsequent implementation of public health and social measures (PHSM) disrupted the epidemiology of respiratory viruses. This work describes the epidemiology of respiratory syncytial virus (RSV) observed during two winter seasons (weeks 40-20) and inter-seasonal periods (weeks 21-39) during the pandemic between October 2020 and September 2022. Methods: Using data submitted to The European Surveillance System (TESSy) by countries or territories in the World Health Organization (WHO) European Region between weeks 40/2020 and 39/2022, we aggregated country-specific weekly RSV counts of sentinel, non-sentinel and Severe Acute Respiratory Infection (SARI) surveillance specimens and calculated percentage positivity. Results for both 2020/21 and 2021/22 seasons and inter-seasons were compared with pre-pandemic 2016/17 to 2019/20 seasons and inter-seasons. Results: Although more specimens were tested than in pre-COVID-19 pandemic seasons, very few RSV detections were reported during the 2020/21 season in all surveillance systems. During the 2021 inter-season, a gradual increase in detections was observed in all systems. In 2021/22, all systems saw early peaks of RSV infection, and during the 2022 inter-seasonal period, patterns of detections were closer to those seen before the COVID-19 pandemic. Conclusion: RSV surveillance continued throughout the COVID-19 pandemic, with an initial reduction in transmission, followed by very high and out-of-season RSV circulation (summer 2021) and then an early start of the 2021/22 season. As of the 2022/23 season, RSV circulation had not yet normalised.

Evolutionary and spatiotemporal analyses reveal multiple introductions and cryptic transmission of SARS-CoV-2 VOC/VOI in Malta.

IMPORTANCE: Our study provides insights into the evolution of the coronavirus disease 2019 (COVID-19) pandemic in Malta, a highly connected and understudied country. We combined epidemiological and phylodynamic analyses to analyze trends in the number of new cases, deaths, tests, positivity rates, and evolutionary and dispersal patterns from August 2020 to January 2022. Our reconstructions inferred 173 independent severe acute respiratory syndrome coronavirus 2 introductions into Malta from various global regions. Our study demonstrates that characterizing epidemiological trends coupled with phylodynamic modeling can inform the implementation of public health interventions to help control COVID-19 transmission in the community.

Microbial Dynamics in Newly Diagnosed and Treatment Naïve IBD Patients in the Mediterranean.

BACKGROUND: Microbial communities have long been suspected to influence inflammatory processes in the gastrointestinal tract of patients with inflammatory bowel disease. However, these effects are often influenced by treatments and can rarely be analyzed in treatment-naïve onset cases. Specifically, microbial differences between IBD pathologies in new onset cases have rarely been investigated and can provide novel insight into the dynamics of the microbiota in Crohn's disease (CD) and ulcerative colitis (UC). METHODS: Fifty-six treatment-naïve IBD onset patients (67.3% CD, 32.7% UC) and 97 healthy controls were recruited from the Maltese population. Stool samples were collected after diagnosis but before administration of anti-inflammatory treatments. Fecal microbial communities were assessed via 16S rRNA gene sequencing and subjected to ecological analyses to determine disease-specific differences between pathologies and disease subtypes or to predict future treatment options. RESULTS: We identified significant differences in community composition, variability, and diversity between healthy and diseased individuals-but only small to no differences between the newly diagnosed, treatment-naïve UC and CD cohorts. Network analyses revealed massive turnover of bacterial interactions between healthy and diseased communities, as well as between CD and UC communities, as signs of disease-specific changes of community dynamics. Furthermore, we identified taxa and community characteristics serving as predictors for prospective treatments. CONCLUSION: Untreated and newly diagnosed IBD shows clear differences from healthy microbial communities and an elevated level of disturbance, but only the network perspective revealed differences between pathologies. Furthermore, future IBD treatment is to some extent predictable by microbial community characteristics.

Treatment-naïve IBD onset patients from Malta show clear differences from healthy microbial communities and an elevated level of community disturbance, although differences between pathologies are only revealed by a network perspective. Furthermore, future IBD treatment is predictable by microbial community characteristics.

Hybrid immunity and protection against infection during the Omicron wave in Malta.

By December 2021, administration of the third dose of COVID-19 vaccinations coincided with the spread of the Omicron variant in Europe. Questions had been raised on protection against infection conferred by previous vaccination and/or infection. Our study population included 252,433 participants from the COVID-19 vaccination registry in Malta. Data were then matched with the national testing database. We collected vaccination status, vaccine brand, vaccination date, infection history, and age. Using logistic regression, we examined different combinations of vaccine dose, prior infection status and time, and the odds of infection during the period when the Omicron variant was the dominant variant in Malta. Participants infected with Sars-Cov-2 prior to the Omicron wave had a significantly lower odds of being infected with the Omicron variant. Additionally, the more recent the infection and the more recent the vaccination, the lower the odds of infection. Receiving a third dose within 20 weeks of the start of the Omicron wave in Malta offered similar odds of infection as receiving a second dose within the same period. Time since vaccination was a strong determinant against infection, as was previous infection status and the number of doses taken. This finding reinforces the importance of future booster dose provision especially to vulnerable populations.

The Maleth program: Malta's first space mission discoveries on the microbiome of diabetic foot ulcers.

The purpose of the Maleth Program, also known as Project Maleth, is Malta's first space program to evaluate human skin tissue microbiome changes in type 2 diabetes mellitus (T2DM) patients afflicted with diabetic foot ulcers (DFU). This was carried out in both ground-based models and spaceflight. The first mission (Maleth I) under this program was carried out to uncover the effects of spaceflight, microgravity and radiation on human skin tissue microbiome samples from six T2DM patients recruited into the study. Each patient human skin tissue sample was split in three, with one section processed immediately for genomic profiling by 16S typing and the rest were processed for longer term ground-control and spaceflight experiments. Ground-control and spaceflight human skin tissue samples were also processed for genomic profiling upon mission re-entry and completion. Maleth I's overall objective was achieved, as human skin tissue samples with their microbiomes travelled to space and back yielding positive results by both standard microbiology techniques and genetic typing using 16S rRNA amplicon sequencing. Preliminary findings of this mission are discussed in light of its innovative approach at DFU microbiome research, and the clinical implications that may emerge from this and other future similar studies.

Digitalizing and Upgrading Severe Acute Respiratory Infections Surveillance in Malta: System Development.

BACKGROUND: In late 2020, the European Centre for Disease Prevention and Control and Epiconcept started implementing a surveillance system for severe acute respiratory infections (SARI) across Europe. OBJECTIVE: We sought to describe the process of digitizing and upgrading SARI surveillance in Malta, an island country with a centralized health system, during the COVID-19 pandemic from February to November 2021. We described the characteristics of people included in the surveillance system and compared different SARI case definitions, including their advantages and disadvantages. This study also discusses the process, output, and future for SARI and other public health surveillance opportunities. METHODS: Malta has one main public hospital where, on admission, patient data are entered into electronic records as free text. Symptoms and comorbidities are manually extracted from these records, whereas other data are collected from registers. Collected data are formatted to produce weekly and monthly reports to inform public health actions. From October 2020 to February 2021, we established an analogue incidence-based system for SARI surveillance. From February 2021 onward, we mapped key stakeholders and digitized most surveillance processes. RESULTS: By November 30, 2021, 903 SARI cases were reported, with 380 (42.1%) positive for SARS-CoV-2. Of all SARI hospitalizations, 69 (7.6%) were admitted to the intensive care unit, 769 (85.2%) were discharged, 27 (3%) are still being treated, and 107 (11.8%) died. Among the 107 patients who died, 96 (89.7%) had more than one underlying condition, the most common of which were hypertension (n=57, 53.3%) and chronic heart disease (n=49, 45.8%). CONCLUSIONS: The implementation of enhanced SARI surveillance in Malta was completed by the end of May 2021, allowing the monitoring of SARI incidence and patient characteristics. A future shift to register-based surveillance should improve SARI detection through automated processes.

Unencapsulated penicillin-resistant Neisseria meningitidis ST-53 clonal complex strain causing symptomatic proctitis in an HIV-negative man who has sex with men: A case report.

We report a case of Neisseria meningitidis symptomatic proctitis in an HIV-negative man who has sex with men attending the genitourinary clinic in Malta. The proctitis was caused by a rare clinical unencapsulated penicillin-resistant N. meningitidis strain of the ST-53 clonal complex.

Dysbiosis in the Gut Microbiota in Patients with Inflammatory Bowel Disease during Remission.

Inflammatory bowel disease (IBD) is a chronic, relapsing, inflammatory disorder which comprises two main conditions: Crohn's disease (CD) and ulcerative colitis (UC). Although the etiology of IBD has not been fully elucidated, the gut microbiota is hypothesized to play a vital role in its development. The aim of this cross-sectional study was to characterize the fecal microbiota in CD or UC patients in a state of remission to reveal potential factors sustaining residual levels of inflammation and triggering disease relapses. Ninety-eight IBD patients in a state of clinical remission (66 UC, 32 CD) and 97 controls were recruited, and stool samples, as well as detailed patient data, were collected. After DNA extraction, the variable regions V1 and V2 of the 16S rRNA gene were amplified and sequenced. Patients with IBD had a decrease in alpha diversity compared to that of healthy controls, and the beta diversity indices showed dissimilarity between the cohorts. Healthy controls were associated with the beneficial organisms unclassified Akkermansia species (Akkermansia uncl.), Oscillibacter uncl., and Coprococcus uncl., while flavonoid-degrading bacteria were associated with IBD. Network analysis identified highly central and influential disease markers and a strongly correlated network module of Enterobacteriaceae which was associated with IBD and could act as drivers for residual inflammatory processes sustaining and triggering IBD, even in a state of low disease activity. The microbiota in IBD patients is significantly different from that of healthy controls, even in a state of remission, which implicates the microbiota as an important driver of chronicity in IBD. IMPORTANCE Dysbiosis in inflammatory bowel disease (IBD) has been implicated as a causal or contributory factor to the pathogenesis of the disease. This study, done on patients in remission while accounting for various confounding factors, shows significant community differences and altered community dynamics, even after acute inflammation has subsided. A cluster of Enterobacteriaceae was linked with Crohn's disease, suggesting that this cluster, which contains members known to disrupt colonization resistance and form biofilms, persists during quiescence and can lead to chronic inflammation. Flavonoid-degrading bacteria were also associated with IBD, raising the possibility that modification of dietary flavonoids might induce and maintain remission in IBD.

CYP2C19*2 genetic polymorphism and incidence of in-stent restenosis in patients on clopidogrel: a matched case-control study.

OBJECTIVES: The cytochrome P450 2C19*2 (CYP2C19*2) genetic polymorphism is associated with reduced clopidogrel bioactivation, increasing the risk of atherothrombotic complications after percutaneous coronary intervention (PCI). In-stent restenosis (ISR) is a complication that limits the long-term prognosis of PCI. The aim was to investigate the association between presence of the CYP2C19*2 allele and ISR within one-year after PCI in patients prescribed dual antiplatelet therapy with aspirin and clopidogrel. METHODS: Sixty patients with angiographically-confirmed drug eluting stent (DES)-ISR within 12 months post-PCI when on DAPT with aspirin and clopidogrel were retrospectively identified (Cases). Another 60 patients with no documented ISR post-PCI in the study period (Controls) were case-matched for age, gender, ethnicity, diabetes mellitus and estimated glomerular filtration rate value, and were invited for CYP2C19*2 genotyping. The association between presence of the CYP2C19*2 allele and ISR was analysed using the Fisher's exact test and binary logistic regression. RESULTS: Twenty-six (43.3%) cases and 5 (8.3%) controls were carriers of one or two CYP2C19*2 alleles. As to non-carrier status of the CYP2C19*2 allele, 34 (56.7%) cases and 55 (91.7%) controls were identified. The association between CYP2C19*2 carrier status and DES-ISR within one-year post-PCI was statistically significant (p<0.001) in both the univariate and multivariate analysis. CONCLUSIONS: The proportion of patients who were carriers of one or two CYP2C19*2 alleles who presented with DES-ISR within one-year post-PCI while on clopidogrel was significantly higher compared to patients with no documented ISR.

Identification of antibiotic resistance patterns in Helicobacter pylori strains isolated from gastric biopsies using real-time PCR and genotypic analysis.

BACKGROUND: Helicobacter pylori (H. pylori) is associated with dyspepsia, mucus-associated lymphoid tissue lymphoma, gastritis, and peptic ulcer disease. Treatment in Malta consists of triple therapy, which consists of a proton pump inhibitor and 2 of the antibiotics amoxicillin, clarithromycin, metronidazole and fluoroquinolones. We aimed to determine the resistance rates for clarithromycin and fluoroquinolones in patients with H. pylori, and its incidence, in patients undergoing an esophagogastroduodenoscopy (EGD) using real-time polymerase chain reaction (RT-PCR). METHODS: Patients undergoing an EGD were recruited. A rapid urease test (RUT) was performed, and 4 gastric biopsies were also taken (2 from antrum, 2 from corpus) and analyzed using RT-PCR. Positive samples were tested for antibiotic resistance using amplification and reverse hybridization techniques. RESULTS: Two hundred patients (mean age 53.6 [range 20-92] years; 53.1% female) were recruited; the majority were (78%) non-smokers. H. pylori was identified in 21.0% of the patients. Fluoroquinolone resistance was detected in 21.4% of the patients. Clarithromycin resistance was observed in 26.2%, with dual resistance identified in 4.8% of the patients. A high concordance was present with patients testing negative for H. pylori with both RUT and RT-PCR (94.3%). Only 57.6% of patients tested positive with both tests. However, 92.9% of RT-PCR positive patients had a positive genotype HelicoDR test. CONCLUSIONS: This data demonstrates a high rate of H. pylori resistance to both clarithromycin and fluoroquinolones. These should be avoided when treating H. pylori by utilizing different treatment regimes. Furthermore, we derived important data on the role of RT-PCR, which may be implemented in routine clinical practice.

Comparison of a rapid point-of-care and two laboratory-based CYP2C19*2 genotyping assays for personalisation of antiplatelet therapy.

BACKGROUND: A quick CYP2C19*2 genotyping assay can be useful in personalised antiplatelet-therapy. OBJECTIVE: To apply a rapid point-of-care (POC) CYP2C19*2 genotyping assay for personalisation of antiplatelet therapy in patients undergoing percutaneous coronary intervention (PCI) and to compare this POC assay to two laboratory-based CYP2C19*2 genotyping assays. SETTING: Cardiac Catheterisation Suite and Molecular Diagnostics Unit in a general hospital. METHODS: A buccal sample was collected for POC CYP2C19*2 genotyping with the Spartan™ RX system (Spartan Bioscience). A whole blood sample was collected from the same patients for laboratory-based CYP2C19*2 genotyping with a TaqMan® allelic discrimination assay (Life Technologies) using real-time quantitative PCR and with the GenID® reverse dot-blot hybridisation assay (Autoimmun Diagnostika GmbH). Each patient was genotyped as a non-carrier of CYP2C19*2 (*1/*1), a carrier of one CYP2C19*2 allele (*1/*2), or a carrier of two CYP2C19*2 alleles (*2/*2). Genotyping, interpretation and communication of genotype results (*1/*2, *2/*2) to the consultant cardiologist was undertaken by a clinical pharmacist researcher. Quantitative and qualitative comparison between the three assays was carried out. MAIN OUTCOME MEASURES: Application of a rapid POC CYP2C19*2 genotyping assay for antiplatelet therapy individualisation; comparison of the POC CYP2C19*2 genotyping assay to two laboratory-based assays. RESULTS: The total sample consisted of 34 Caucasian patients. With the POC assay, 21 patients were genotyped as non-carriers of CYP2C19*2, 12 patients as carriers of one CYP2C19*2 allele and one patient as a carrier of two CYP2C19*2 alleles. With both laboratory-based assays, the same 21 patients were genotyped as non-carriers of CYP2C19*2, however 13 patients were genotyped as carriers of one CYP2C19*2 allele and no patients were genotyped as carriers of two CYP2C19*2 alleles. Agreement in genotype results was 97 % (κ = 0.939) between the POC assay and both laboratory-based assays and 100 % (κ = 1.000) between the two laboratory-based assays. CONCLUSION: Compared to both laboratory-based genotyping assays, the POC assay is accurate and reliable, provides rapid results, can process single samples, is portable and more operator-friendly, however the tests are more expensive.