Molecular Typing Methods Research Articles

Molecular characterization of invasive Neisseria meningitidis isolates collected in Lithuania (2009-2019) and estimation of serogroup B meningococcal vaccine 4CMenB and MenB-Fhbp coverage.

Neisseria meningitidis causes invasive meningococcal disease (IMD), which is associated with significant mortality and long-term consequences, especially among young children. The incidence of IMD in Lithuania was among the highest in European Union/European Economic Area countries during the past two decades; however, the characterization of meningococcal isolates by molecular typing methods has not yet been performed. In this study, we characterized invasive meningococcal isolates (n=294) recovered in Lithuania from 2009 to 2019 by multilocus sequence typing (MLST) and typing of antigens FetA and PorA. The more recent (2017-2019) serogroup B isolates (n=60) were genotyped by analyzing vaccine-related antigens to evaluate their coverage by four-component (4CMenB) and two-component (MenB-Fhbp) vaccines using the genetic Meningococcal Antigen Typing System (gMATS) and Meningococcal Deduced Vaccine Antigen Reactivity (MenDeVAR) Index methods, respectively. The vast majority (90.5%) of isolates belonged to serogroup B. MLST revealed a predominance of clonal complex 32 (74.02%). Serogroup B strain P1.19,15: F4-28: ST-34 (cc32) accounted for 64.1% of IMD isolates. The overall level of strain coverage by the 4MenB vaccine was 94.8% (CI 85.9-98.2%). Most serogroup B isolates (87.9%) were covered by a single vaccine antigen, most commonly Fhbp peptide variant 1 (84.5% of isolates). The Fhbp peptides included in the MenB-Fhbp vaccine were not detected among the analyzed invasive isolates; however, the identified predominant variant 1 was considered cross-reactive. In total, 88.1% (CI 77.5-94.1) of isolates were predicted to be covered by the MenB-Fhbp vaccine. In conclusion, both serogroup B vaccines demonstrate potential to protect against IMD in Lithuania.

Molecular typing methods & resistance mechanisms of MDR Klebsiella pneumoniae.

The emergence and transmission of carbapenem-resistant Klebsiella pneumoniae (CRKP) have been recognized as a major public health concern. Here, we investigated the molecular epidemiology and its correlation with the mechanisms of resistance in CRKP isolates by compiling studies on the molecular epidemiology of CRKP strains worldwide. CRKP is increasing worldwide, with poorly characterized epidemiology in many parts of the world. Biofilm formation, high efflux pump gene expression, elevated rates of resistance, and the presence of different virulence factors in various clones of K. pneumoniae strains are important health concerns in clinical settings. A wide range of techniques has been implemented to study the global epidemiology of CRKP, such as conjugation assays, 16S-23S rDNA, string tests, capsular genotyping, multilocus sequence typing, whole-genome sequencing-based surveys, sequence-based PCR, and pulsed-field gel electrophoresis. There is an urgent need to conduct global epidemiological studies on multidrug-resistant infections of K. pneumoniae across all healthcare institutions worldwide to develop infection prevention and control strategies. In this review, we discuss different typing methods and resistance mechanisms to explore the epidemiology of K. pneumoniae pertaining to human infections.

Comparison of pulsed-field gel electrophoresis and a novel amplified intergenic locus polymorphism method for molecular typing of Campylobacter jejuni.

Campylobacter is regarded as the leading cause of zoonotic diseases and Campylobacter jejuni (C. jejuni) is one of the predominant pathogenic species. To track C. jejuni infections, various genotyping methods have been used. In this study, amplified intergenic locus polymorphism (AILP) was used to type C. jejuni for the first time. To confirm its feasibility, pulsed-field gel electrophoresis (PFGE) was performed as a control, and the results obtained by the AILP and PFGE methods were compared. Fifty-one isolates were resolved into 34 and 29 different genotypes with Simpson's indices of 0.976 and 0.967 using the AILP and PFGE methods, respectively. The adjusted Rand coefficient of the two approaches was as high as 0.845. In summary, the data showed that the two genotyping methods were similar for discriminating isolates and were both appropriate methods to distinguish whether two isolates were indistinguishable, but the AILP was faster and less costly than PFGE. Therefore, the AILP is a reliable, rapid, and highly discriminative method to genotype C. jejuni collected from poultry meat, which is helpful to effectively monitor C. jejuni.

Molecular Characterization of Enterococcus faecalis and Enterococcus faecium Isolated from a Meat Source in Shahrekord Local Markets, Iran.

Enterococcus faecalis (E. faecalis) and E. faecium (E. faecium) are commensals of the gastrointestinal biota of humans and animals and are considered opportunistic pathogens. This study aimed to improve the knowledge about E. faecalis and E. faecium isolated from meat. To this end, 104 meat samples were collected from sheep, goats, cattle, and calves from Shahrekord local markets in Iran. Presumptive colonies of E. faecalis and E. faecium were isolated from the samples and subjected to identification tests. Antimicrobial susceptibility was determined using the Kirby-Bauer disc diffusion method, and polymerase chain reaction (PCR) was performed to detect some virulence genes. Furthermore, randomly amplified polymorphic DNA typing and repetitive element sequence-based PCR fingerprinting were used to show the clonal relatedness of the isolates. The results revealed that enterococci were present in 90 out of 104 samples. Specifically, E. faecalis and E. faecium were the commonly isolated species, with the predominance of E. faecalis, which exhibited high resistance to streptomycin (95%) but was susceptible to vancomycin (85.6%). Virulence genes detection showed that ccf and cpd genes were the most prevalent genes in both species. In addition, the molecular typing method indicated that the isolates belonged to separate subgroups. This study shows the contamination of meat products by potential pathogens and resistant enterococci. There is a need to implement regular surveillance to monitor the emergence of antimicrobial-resistant E. faecalis and E. faeciumin food, particularly in meat production.

Distribution and Characteristics of Bacteria Isolated from Cystic Fibrosis Patients with Pulmonary Exacerbation.

Cystic fibrosis (CF) is an inherited recessive disorder characterized by recurrent and persistent pulmonary infections, resulting in lung function deterioration and early mortality. A cross-sectional study was conducted on the bacterial profile and antibiotic resistance pattern of 103 respiratory specimens from CF patients with signs of pulmonary exacerbation. Antibiotic susceptibility testing and biofilm formation of Staphylococcus aureus and Pseudomonas aeruginosa isolates were performed by the Kirby-Bauer disc diffusion method and microtiter plate assay, respectively. Molecular typing of S. aureus and P. aeruginosa isolates was carried out by spa typing and repetitive extragenic palindromic element PCR. In a total of 129 isolates, the most prevalent organisms were S. aureus (55.3%) and P. aeruginosa (41.7%). Other less prevalent bacterial isolates include coagulase-negative staphylococci, Escherichia coli, klebsiella spp., Enterobacter spp., and Achromobacter xylosoxidans. The highest rate of resistance for S. aureus was observed to azithromycin and erythromycin (80%), ciprofloxacin (52.3%), clindamycin (44.6%) and tetracycline (43%). Twenty percent of S. aureus isolates were methicillin-resistant S. aureus (MRSA) and 47.6% were MDR S. aureus. For P. aeruginosa isolates the highest resistance was to cefepime (38.3%) and levofloxacin (33.3%) and 20% showed MDR phenotype. Our study demonstrated a significant decline in the prevalence of P. aeruginosa infections in comparison to previous studies. We found S. aureus to be more prevalent in younger patients, whereas mucoid P. aeruginosa showed a shift in prevalence toward older ages. Molecular typing methods showed great diversity between isolates.

The Relative of Spa Gene Types, Prevalence and Antibiotic Resistance in Methicillin-Resistant Staphylococcus aureus.

Methicillin-resistant Staphylococcus aureus (MRSA) is the primary cause of nosocomial and animal, community-acquired infections. S. aureus is a common inhabitant of the upper respiratory tract. Molecular typing methods are vital for investigations of MRSA. The MRSA has significantly increased in Iraq. This study aimed to determine the prevalence of this bacteria and know the distribution of Spa type among antibiotics-resistant local isolates from different sources. A total of 150 samples were collected from three different sources: humans, animals and the environment. Among all these samples, 55 MRSA isolates were determined using the phenotypic method and the mecA gene. Antibiotic resistance profiles were screened using the disc diffusion method. Whereas Spa types were identified by using PCR technique and nucleotide sequencing analysis. The MRSA presence rates were 67.5%, 80 %, and 31.3% in bovine, human, and environmental sources, respectively. The highest sensitivity of MRSA was to vancomycin, and the lowest was to penicillin. Multi-drug resistance was found to be in all isolates. Molecular investigation showed that 100% of the tested MRSA isolates harboured a Spa gene; Spa gene typing assay reveals that the most repetitive spa type was t304, t8986, and t14870, which were reported in humans and animals, followed by type t 304 t14870 in environmental isolates. This study's findings could help identify the genetic variants responsible for the emergence and spread of these bacteria in the region.

Antibiotic resistance, virulence and genetic characteristics of Vibrio alginolyticus isolates from aquatic environment in costal mariculture areas in China.

Vibrio alginolyticus has been the second most common Vibrio species in the world and mainly grows in the ocean or estuary environment, which can induce epidemics outbreaks under marine organisms, and causing serious economic losses in aquaculture industry. In this study, the genetic populations and evolutionary relationship analysis of V. alginolyticus isolated from different geographical locations in China with typical interannual differences were exhibited originally genetic diversity. Then the virulence genes prevalence, antibiotic resistance phenotype, and antimicrobial resistance genes risk diversity of V. alginolyticus were analyzed by phenotypic and molecular typing methods. And they were complex correlations among antibiotic phenotypes, resistance and virulence genes under different genotype of V. alginolyticus. The results provide a theoretical foundation for further understanding the genetic and metabolic diversity among V. alginolyticus in China, and lay a theoretical foundation for the transmission risk assessment and regional diagnosis of Vibrio in aquatic animals.

Genomic Diversity of Campylobacter lari Group Isolates from Europe and Australia in a One Health Context.

Members of the Campylobacter lari group are causative agents of human gastroenteritis and are frequently found in shellfish, marine waters, shorebirds, and marine mammals. Within a One Health context, we used comparative genomics to characterize isolates from a diverse range of sources and geographical locations within Europe and Australia and assess possible transmission of food, animal, and environmental isolates to the human host. A total of 158 C. lari isolates from Australia, Denmark, France, and Germany, which included 82 isolates from human stool and blood, 12 from food, 14 from domestic animal, 19 from waterbirds, and 31 from the environment were analyzed. Genome-wide analysis of the genetic diversity, virulence, and antimicrobial resistance (AMR) traits was carried-out. Most of the isolates belonged to C. lari subsp. lari (Cll; 98, 62.0%), while C. lari subsp. concheus and C. lari urease-positive thermotolerant Campylobacter (UPTC) were represented by 12 (7.6%) and 15 (9.5%) isolates, respectively. Furthermore, 33 (20.9%) isolates were not assigned a subspecies and were thus attributed to distant Campylobacter spp. clades. Whole-genome sequence-derived multilocus sequence typing (MLST) and core-genome MLST (cgMLST) analyses revealed a high genetic diversity with 97 sequence types (STs), including 60 novel STs and 14 cgMLST clusters (≤10 allele differences), respectively. The most prevalent STs were ST-21, ST-70, ST-24, and ST-58 (accounting for 13.3%, 4.4%, 3.8%, and 3.2% of isolates, respectively). A high prevalence of the 125 examined virulence-related loci (from 76.8 to 98.4% per isolate) was observed, especially in Cll isolates, suggesting a probable human pathogenicity of these strains. IMPORTANCE Currently, relatedness between bacterial isolates impacting human health is easily monitored by molecular typing methods. These approaches rely on discrete loci or whole-genome sequence (WGS) analyses. Campylobacter lari is an emergent human pathogen isolated from diverse ecological niches, including fecal material from humans and animals, aquatic environments, and seafood. The presence of C. lari in such diverse sources underlines the importance of adopting an integrated One Health approach in studying C. lari population structure for conducting epidemiological risk assessment. This retrospective study presents a comparative genomics analysis of C. lari isolates retrieved from two different continents (Europe and Australia) and from different sources (human, domestic animals, waterbirds, food, and environment). It was designed to improve knowledge regarding C. lari ecology and pathogenicity, important for developing effective surveillance and disease prevention strategies.

Molecular typing and antibiotic resistance patterns among clinical isolates of Acinetobacter baumannii recovered from burn patients in Tehran, Iran.

Acinetobacter baumannii (A. baumannii) is now considered a highly resistant pathogen to various types of antibiotics. Therefore, tracking the source of its prevalence and continuous control is crucial. This study aimed to determine antibiotic resistance and perform various molecular typing methods on clinical isolates of A. baumannii isolated from hospitalized burn patients in Shahid Motahari Burn Hospital, Tehran, Iran. Hospital isolates were confirmed by phenotypic and molecular methods. Then the sensitivity to different antibiotics was determined using the minimum inhibitory concentration (MIC) method. In order to perform molecular typing, three-locus dual assay multiplex polymerase chain reaction (PCR), multiple-locus variable-number tandem repeat analysis (MLVA), and multilocus sequence typing (MLST) methods were used. Among the 60 isolates collected, the frequencies of multidrug-resistant (MDR) and extensively drug-resistant (XDR) isolates were 90 and 10%, respectively. The most effective antibiotics were colistin with 100% and tigecycline with 83.33% sensitivity. Isolates were 100% resistant to piperacillin/tazobactam and cephalosporins, and 68.3% were resistant to carbapenem. The results of multiplex PCR showed five groups that international clone I (IC I) and IC II were the most common. The MLVA method identified 34 MLVA types (MTs), 5 clusters, and 25 singletons. Multilocus sequence typing results for tigecycline-resistant isolates showed seven different sequence types (STs). Increasing antibiotic resistance in A. baumannii isolates requires careful management to control and prevent the occurrence of the pre-antibiotic era. The results of this study confirm that the population structure of A. baumannii isolates has a high diversity. More extensive studies are needed in Iran to better understand the epidemiology of A. baumannii.

Classification and Prognosis Analysis of Pancreatic Cancer Based on DNA Methylation Profile and Clinical Information.

Pancreatic adenocarcinoma (PAAD) has a poor prognosis with high individual variation in the treatment response among patients; however, there is no standard molecular typing method for PAAD prognosis in clinical practice. We analyzed DNA methylation data from The Cancer Genome Atlas database, which identified 1235 differentially methylated DNA genes between PAAD and adjacent tissue samples. Among these, 78 methylation markers independently affecting PAAD prognosis were identified after adjusting for significant clinical factors. Based on these genes, two subtypes of PAAD were identified through consistent clustering. Fourteen specifically methylated genes were further identified to be associated with survival. Further analyses of the transcriptome data identified 301 differentially expressed cancer driver genes between the two PAAD subtypes and the degree of immune cell infiltration differed significantly between the subtypes. The 14 specific genes characterizing the unique methylation patterns of the subtypes were used to construct a Bayesian network-based prognostic prediction model for typing that showed good predictive value (area under the curve value of 0.937). This study provides new insight into the heterogeneity of pancreatic tumors from an epigenetic perspective, offering new strategies and targets for personalized treatment plan evaluation and precision medicine for patients with PAAD.

A molecular study regarding the spread of vanA vancomycin-resistant Enterococcus faecium in a tertiary hospital in China

Vancomycin-resistant enterococci (VRE) are one of the most important bacterial causes of healthcare-associated infections (HAIs). In China, the detection rate of VRE Enterococcus faecium (VREfm) is low, although VREfm had a high prevalence in our hospitals between 2013-2015. In this study, we used molecular typing methods combined with epidemiological data to investigate the spread of VREfm in our hospital. The characteristics of E. faecium strains isolated from 89 patients with HAIs, including antibiotic susceptibility and virulence genes, were analyzed. This study analyzed 50 E. faecium strains isolated from 47 intensive care unit and Emergency ward patients using core genome Multilocus Sequence Typing and transposon typing. Epidemiological information about those patients was also analyzed. Twenty-seven E. faecium isolates containing the vanA gene were identified as VREfm in 89 non-duplicate E. faecium isolates. The major clonal VREfm strains that persisted from 2013-2015 were CT1/ST78/PFGE cluster A that contained transposon type Ⅰ. The other CT4/ST363/PFGE cluster of VREfm strains also contained transposon type Ⅰ. Three patients acquired different clonal E. faecium strains during the hospital period, and the VREfm strain infected one patient. In this study, we report the spread caused by vanA vancomycin-resistant E. faecium strains of different cluster types with the same type of transposon in a tertiary hospital. Our literature review revealed that this is the first report of the HAIs caused by ST363/CT4 VREfm strains.

Existence of viable Mycobacterium leprae in natural environment and its genetic profiling in a leprosy endemic region

IntroductionMolecular epidemiology of leprosy is very important to study leprosy transmission dynamics and to enhance our understanding of leprosy in endemic areas by utilizing the molecular typing method. Nowadays our understanding of leprosy transmission dynamics has been refined by SNP typing and VNTR marker analysis of M. leprae strains.ObjectiveThis study was carried out to find out the presence of viable M. leprae in the soil and water samples from residing areas of leprosy patients staying in different blocks of Purulia district of West Bengal, understanding their genotypes and compared with that of M. leprae present in patients.Material and methodsSlit-skin smear (SSS) samples (n=112) were collected from the active multibacillary leprosy patients from different blocks of leprosy endemic area. Soil samples (n=1060) and water samples (n=620) were collected from residing areas of leprosy patients. SNP subtyping was performed by PCR followed by sequencing. Multiplex PCR was performed using fifteen ML-VNTR loci and results were analysed.ResultsWe observed high PCR positivity in soil samples (344 out of 1060; 32%) and water samples (140 out of 620; 23%). These PCR positive samples when further screened for viability, it was observed that 150 soil samples (44%) and 56 water samples (40%) showed presence of 16S rRNA. SNP typing of M. leprae revealed presence of predominantly type 1. SNP subtype 1D (83%) was most prevalent in all the blocks of Purulia followed by subtype 1C (15%) and subtype 1A (2%). SNP subtype 2F was noted in only one sample. SNP and VNTR combination showed presence of similar strain type in certain pockets of Purulia region which was responsible for transmission.ConclusionPresence of viable M. leprae in the environment, and presence of SNP Type 1 M. leprae in patients and environment suggests both environment and patients play a role in disease transmission.

Comparative analysis of multiplexed PCR and short- and long-read whole genome sequencing to investigate a large Klebsiella pneumoniae outbreak in New York State

In 2017, the New York State Department of Health investigated a large Klebsiella pneumoniae outbreak in a health care facility. A retrospective analysis was conducted to compare the use of multiple molecular typing methods for characterizing the outbreak. Forty-four isolates were characterized using the rapid real-time PCR OpGen Acuitas® AMR Gene Panel. Additionally, short-read whole genome sequencing (WGS) analysis was used to identify antimicrobial resistance (AMR) genes and assess isolate relatedness. Long-read Oxford Nanopore MinION WGS was used to characterize the plasmid content of a subset of isolates. All methods showed overall concordance, identifying four clusters, with a few discrepancies in the clustering of individual isolates. Though short- and long-read WGS results provided a more nuanced understanding of the molecular epidemiology of this outbreak, this study highlights the utility of the Acuitas® PCR-based approach, which can more easily be performed by health care facilities, for rapid clustering of patient isolates.

A HOSPITAL INFECTION EXPLOSION: PAN DRUG-RESISTANT PSEUDOMONAS AERUGINOSA BIOFILM AND CONTAMINATED BRONCHOSCOPE

This study looks into a hospital infection epidemic that was linked to a contaminated bronchoscope and was caused by a pan-drug-resistant Pseudomonas aeruginosa (P. aeruginosa) biofilm. The issues presented by P. aeruginosa strains' resistance to antibiotics and the persistence of biofilm in hospital environments led to concerns being raised about the outbreak. By applying epidemiological analysis and molecular typing methods, we were able to pinpoint the contamination source and clarify the dynamics of transmission in the hospital setting. The study brought to light the vital significance that surveillance procedures and infection control strategies play in controlling and preventing nosocomial infections brought on by bacteria that are resistant to several drugs. The results also highlight the critical need for improved approaches to reduce the risks associated with infections linked to biofilms and antibiotic resistance in hospital settings.

The role of serological and molecular testing in the diagnostics and transfusion treatment of autoimmune haemolytic anaemia.

Autoimmune haemolytic anaemia (AIHA) is a rare autoimmune disease characterised by haemolysis associated with the presence of immunoglobulins and/or components of the complement system on red blood cells (RBCs). It is classified into warm or cold antibody-mediated AIHA according to the temperature at which autoantibodies bind optimally to RBCs. Clinicians should be familiar with the procedural tests used for a complete laboratory investigation of AIHA. Good collaboration between clinicians and laboratory specialists with correct sample handling and an exact diagnostic work-up is extremely important for the correct classification and proper therapeutic management of AIHA. Specialised serological test procedures are very complex. Problems with serological testing may be excluded with the molecular testing, which has now become a gold standard to predict a patient's phenotype in order to secure the right antigen-matched blood for AIHA patients. More recently, genotyping has been used instead of serological typing and complex adsorption tests. This paper offers a description of various tests for differentiating between types of AIHA. A diagnostic algorithm and the problems of laboratory investigation are also presented, and an application of molecular methods for the blood group typing in AIHA is elaborated.

Progress in research of genomic epidemiology of pathogenic fungi

Genomic epidemiology, based on whole-genome sequencing technology and bioinformatics analysis, can make up for the shortcomings of traditional molecular typing methods and provide a novel insight for the genetic evolution and transmission of pathogenic fungi. The combination of genetic information and epidemiological methods of pathogenic fungi can predict fungi transmission routes and risks, and provide a theoretical basis for the development of public health strategies for fungi infection prevention and control. This paper summarizes the development of molecular epidemiology and genomic epidemiology, as well as the application of genomic epidemiology methods in the analyses of genetic relationship, origin, evolution, drug resistance, virulence, and genome-wide association of pathogenic fungi, and discusses the development of pathogenic fungi genomic epidemiology.

Molecular Tools for Typing Mycoplasma pneumoniae and Mycoplasma genitalium

Mycoplasma pneumoniae and Mycoplasma genitalium are cell wall-less bacteria with strongly reduced genome content and close phylogenetic relatedness. In humans, the only known natural host, the microorganisms colonize the respiratory or genitourinary mucosa and may cause a broad range of clinical presentations. Besides fundamental differences in their tissue specificity, transmission route, and ability to cause prevalence peaks, both species share similarities such as the occurrence of asymptomatic carriers, preferred populations for infection, and problems with high rates of antimicrobial resistance. To further understand the epidemiology of these practically challenging bacteria, typing of strains is necessary. Since the cultivation of both pathogens is difficult and not performed outside of specialized laboratories, molecular typing methods with adequate discriminatory power, stability, and reproducibility have been developed. These include the characterization of genes containing repetitive sequences, of variable genome regions without the presence of repetitive sequences, determination of single and multi-locus variable-number tandem repeats, and detection of single nucleotide polymorphisms in different genes, respectively. The current repertoire of procedures allows reliable differentiation of strains circulating in different populations and in different time periods as well as comparison of strains occurring subsequently in individual patients. In this review, the methods for typing M. pneumoniae and M. genitalium, including the results of their application in different studies, are summarized and current knowledge regarding the association of typing data with the clinical characteristics of infections is presented.

Genome Sequence of Campylobacter Strain 19-13652, Isolated from Breeding Pheasants.

ABSTRACTWe report the whole-genome sequence of a Campylobacter strain that was isolated from breeding pheasants presenting “bulgy eyes” in Italy. Traditional molecular typing methods did not return any reliable result. Whole-genome sequencing and sequence comparison with known genomes did not meet the criteria for assignment to an existing species.

Prognostic significance of different molecular typing methods and immune status based on RNA sequencing in HR-positive and HER2-negative early-stage breast cancer

BackgroundThis study was conducted to evaluate the prognostic significance of different molecular typing methods and immune status based on RNA sequencing (RNA-seq) in hormone receptor (HR)-positive and human epidermal growth factor receptor 2 (HER2)-negative (HR + /HER2-) early-stage breast cancer and develop a modified immunohistochemistry (IHC)-based surrogate for intrinsic subtype analysis.MethodsThe gene expression profiles of samples from 87 HR + /HER2- early-stage breast cancer patients were evaluated using the RNA-seq of Oncotype Dx recurrence score (RS), PAM50 risk of recurrence (ROR), and immune score. Intrinsic tumor subtypes were determined using both PAM50- and IHC-based detection of estrogen receptor, progesterone receptor, Ki-67, epidermal growth factor receptor, and cytokeratins 14 and 5/6. Prognostic variables were analyzed through Cox regression analysis of disease-free survival (DFS) and distant metastasis-free survival (DMFS).ResultsSurvival analysis showed that ROR better predicted recurrence and distant metastasis compared to RS (for DFS: ROR, P = 0.000; RS, P = 0.027; for DMFS, ROR, P = 0.047; RS, P = 0.621). Patients with HR + /HER2- early-stage breast cancer was classified into the luminal A, luminal B, HER2-enriched, and basal-like subtypes by PAM50. Basal-like subgroups showed the shortest DFS and DMFS. A modified IHC-based surrogate for intrinsic subtype analysis improved the concordance with PAM50 from 66.7% to 73.6%, particularly for basal-like subtype identification. High level of TILs and high expression of immune genes predicted poor prognosis. Multi-factor Cox analysis showed that IHC-based basal-like markers were the only independent factors affecting DMFS.ConclusionsPrognosis is better evaluated by PAM50 ROR in early-stage HR + /HER2- breast cancer and significantly differs among intrinsic subtypes. The modified IHC-based subtype can improve the basal-like subtype identification of PAM50. High immunity status and IHC-based basal-like markers are negative prognostic factors.

Molecular epidemiology of hepatitis A outbreaks and sporadic cases, Latvia, 2017 to 2019.

BackgroundHepatitis A is an acute infection of the liver caused by hepatitis A virus (HAV). Molecular detection and typing of the HAV VP1/P2A genomic region is used for genotyping and outbreak investigations. After a large hepatitis A outbreak in Latvia in 2007–08, only sporadic cases were registered until 2017 when a rise in cases occurred. During 2017–19, 179 laboratory-confirmed hepatitis A cases were notified in Latvia.AimTo investigate the observed increase in hepatitis A cases during 2017 and to determine whether these cases were linked to one another, to risk groups, or to other outbreaks. The majority of HAV samples (69.8%) were typed.MethodsThe VP1/P2A genomic region of HAV was amplified and sequenced for 125 case serum samples. Information about hepatitis-related symptoms, hospitalisation, vaccination, a possible source of infection and suspected countries of origin of the virus were analysed for sequenced cases.ResultsMost HAV strains were subgenotype IA (n = 77), of which 41 were strains circulating among men who have sex with men (MSM) populations in Europe (VRD_521_2016 (n = 32), RIVM-HAV16–090 (n = 7) or V16–25801 (n = 2)). Forty-four cases were subgenotype IB and four cases subgenotype IIIA. However, other clusters and sporadic cases were detected with or without identifying the epidemiological link.ConclusionThis work represents molecular epidemiological data of hepatitis A cases in Latvia from 2017 to 2019. Molecular typing methods allow identification of clusters for public health needs and establishing links with other outbreaks, and to compare Latvian strains with reported strains from other countries.