Genomic and phenotypic inconsistencies in Pseudomonas aeruginosa resistome among intensive care patients.

Objective: Pseudomonas aeruginosa, a difficult-to-manage nosocomial pathogen, poses a serious threat to clinical outcomes in intensive care (ICU) patients due to its high antimicrobial resistance (AMR). To promote effective management, it is essential to investigate the genomic and phenotypic differences in AMR expression of the isolates. Methods: A prospective observational study was conducted from July 2022 to April 2023 at Liepaja Regional Hospital in Latvia. The study included all adult patients who were admitted to the ICU and had a documented infection with P. aeruginosa, as confirmed by standard laboratory microbiological testing and short-read sequencing. Since ResFinder is the only sequencing-based database offering antibacterial susceptibility testing (AST) data for each antibiotic, we conducted a comparison of the resistance profile with the results of phenotypic testing, evaluating if ResFinder met the US Food and Drug Administration (FDA) requirements for approval as a new AMR diagnostic test. Next, to improve precision, AST data from ResFinder was compared with two other databases - AMRFinderPlus and RGI. Additionally, data was gathered from environmental samples to inform the implementation of appropriate infection control measures in real time. Results: Our cohort consisted of 33 samples from 29 ICU patients and 34 environmental samples. The presence of P. aeruginosa infection was found to be associated with unfavourable clinical outcomes. A third of the patient samples were identified as multi-drug resistant isolates. Apart from resistance against colistin, significant discrepancies were observed when phenotypic data were compared to genotypic data. For example, the aminoglycoside resistance prediction of ResFinder yielded a major errors value of 3.03% for amikacin, which was marginally above the FDA threshold. Among the three positive environmental samples, one sample exhibited multiple AMR genes similar to the patient samples in its cluster. Conclusion: Our findings underscore the importance of utilizing a combination of diagnostic methods for the identification of resistance mechanisms, clusters, and environmental reservoirs in ICUs.

HLA class II DRB1, DQA1, DQB1 loci in patients with HIV infection and tuberculosis in a Latvian cohort group.

Introduction: Until the COVID-19 pandemic, tuberculosis (TB) was the leading cause of death from a single infectious agent, ranking above HIV/AIDS. It is also the key cause of death among people infected with HIV. Tuberculosis incidence in Latvia has decreased by 25% during the last 30 years, but the mortality level of TB remains significant. The HLA class II genes are responsible for antigen presentation and regulation of immune responses, which plays an important role in individual susceptibility to infection disease. Whether or not differential HLA polymorphism contributes to TB with HIV infection and TB without HIV infection in Latvian patients is unknown. Material and methods: For the detection of HLA class II DQA1, DQB1, and DRB1 alleles a total of 616 subjects were enrolled, including 80 primary active TB (PATB) patients, 168 HIV-1/TB patients, 168 HIV-1 patients and 200 HC individuals. Results: For immunodeficiency caused by TB, HIV-1 or HIV-1/TB coinfection, alleles DRB1*12:01, 14:01, 16:01, DQA1*01:02, 01:03, 02:01, 06:01, DQB1*03:03, 06:01 are identified as protective, but DRB1*07:01, 11:01, 15:01, DQA1*02:01, 03:01, DQB1*03:01, 05:01 are identified as risk alleles. Conclusions: The results of our experimental pilot studies demonstrated that HLA class II genes may contribute to the genetic risk of TB and HIV-1/TB co-infection, possibly by reducing the presentation of protective Mycobacterium tuberculosis antigens to T-helpers. It is necessary to conduct repetitive, multicentre, and large sample studies in order to draw more scientific conclusions and to confirm the relationship between TB, HIV and HIV-1/TB co-infection susceptibility and gene polymorphisms.

Multiplexed RT-qPCR Coupled with Whole-Genome Sequencing to Monitor a SARS-CoV-2 Omicron Variant of Concern in a Hospital Laboratory Setting in Latvia.

At the end of 2021, the SARS-CoV-2 Omicron variant of concern (VOC) displaced the previously dominant Delta VOC and enhanced diagnostic and therapeutic challenges worldwide. Respiratory specimens submitted to the Riga East University Hospital Laboratory Service by the central and regional hospitals of Latvia from January to March 2022 that were positive for SARS-CoV-2 RNA were tested by commercial multiplexed RT-qPCR targeting three of the Omicron VOC signature mutations: ΔH69/V70, E484A, and N501Y. Of the specimens tested and analyzed in parallel by whole-genome sequencing (WGS), 964 passed the internal quality criteria (genome coverage ≥90%, read depth ≥400×) and the Nextstrain's quality threshold for "good". We validated the detection accuracy of RT-qPCR for each target individually by using WGS as a control. The results were concordant with both approaches for 938 specimens, with the correct classification rate exceeding 96% for each target (CI 95%); however, the presumptive WHO label was misassigned for 21 specimens. The RT-qPCR genotyping provided an acceptable means to pre-monitor the prevalence of the two presumptive Omicron VOC sublineages, BA.1 and BA.2.

Hepatitis A virus subgenotypes in Latvia, 2008-2021.

BACKGROUND: In Latvia outbreaks of the HAV were observed between 2008 and early 2010 and again in 2017-2018. However, the risks of introducing and spreading infection still exist, as the virus spreads easily when personal hygiene is not followed. METHODS: To determine the spread of HAV subgenotypes in the territory of Latvia the VP1/P2A genomic region of HAV was amplified and sequenced for 259 case serum samples. The study carried out a molecular biological investigation and molecular epidemiological investigation. Demographic data (sex, age), disease data (hepatitis symptoms, hospitalization, vaccination) and epidemiology data (part of the outbreak, possible source of infection, recent travel) were collected. Based on the obtained sequences, the phylogenetic tree was built and analyzed for the homology and belonging to different isolated HAV clusters from other countries. RESULTS: From the obtained data, it was concluded that HAV subgenotype IA had 13 clusters and 12 sporadic cases, HAV subgenotype IB had eight clusters and 11 sporadic cases, HAV subgenotype IIIA had one cluster and nine sporadic cases. It was found that the sources of infection among the investigated cases were different, they were mostly associated with contact with a patient with HAV, travel, as well as between persons who inject drugs and men who have sex with men, and the prevalence of HAV similar sequences was observed in different years. It was concluded that patients with HAV subgenotype IA had the longest hospitalization duration and averaged 9.3 days, while patients with subgenotype IB - 7.3 days, subgenotype IIIA - 7.7 days. Analyzing the data on vaccination, it was found that mostly all were not vaccinated or had an unknown vaccination status. CONCLUSIONS: All of this has led to the conclusion that the application of molecular biological methods of the HAV and a careful analysis of epidemiological data can help to better understand the ways of spreading the infection, investigate local outbreaks, detect cases of imported infection and track the recirculation of the virus.

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.

First Report on the Latvian SARS-CoV-2 Isolate Genetic Diversity.

Remaining a major healthcare concern with nearly 29 million confirmed cases worldwide at the time of writing, novel severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) has caused more than 920 thousand deaths since its outbreak in China, December 2019. First case of a person testing positive for SARS-CoV-2 infection within the territory of the Republic of Latvia was registered on 2nd of March 2020, 9 days prior to the pandemic declaration by WHO. Since then, more than 277,000 tests were carried out confirming a total of 1,464 cases of coronavirus disease 2019 (COVID-19) in the country as of 12th of September 2020. Rapidly reacting to the spread of the infection, an ongoing sequencing campaign was started mid-March in collaboration with the local testing laboratories, with an ultimate goal in sequencing as much local viral isolates as possible, resulting in first full-length SARS-CoV-2 isolate genome sequences from the Baltics region being made publicly available in early April. With 133 viral isolates representing ~9.1% of the total COVID-19 cases during the "first coronavirus wave" in the country (early March, 2020-mid-September, 2020) being completely sequenced as of today, here, we provide a first report on the genetic diversity of Latvian SARS-CoV-2 isolates.