Measles outbreaks in 2017-2019 - molecular surveillance started in the Czech Republic.

OBJECTIVE: Between 2017 and 2019, measles virus spread globally, causing a large measles epidemic that suddenly ended in 2020. Measles outbreaks also occurred in the Czech Republic (CR) as part of the global public health problem. In the recent alarming epidemiological situation, molecular surveillance is becoming increasingly important as it plays a vital role in the identification of imported cases and in the monitoring of virus transmission. Molecular surveillance makes it possible to obtain evidence of the discontinuation of the endemic spread and is indispensable for the verification of measles elimination. The study aim is to find out whether any of measles virus genotypes circulated in the CR for more than 12 months in order to either confirm or refute the endemic spread of measles virus in the country in relation to the recent loss of the measles elimination status. Another aim is to assess the current laboratory diagnosis from the perspective of recent measles outbreaks and the obligation to refer samples for confirmation and genotyping. MATERIAL AND METHODS: In total, 243 positive nasopharyngeal swabs collected from outbreak patients from all over the CR in 2018 and 2019 were analysed by molecular methods. The most variable part of the measles virus genome, the nucleoprotein gene (N-450), was sequenced according to the WHO protocol. The sequence analysis was performed by Sanger method using the Applied Biosystems 3 500 sequencer, and sequence data were analysed by the bioinformatics programe Geneious. RESULTS: In the CR, only two genotypes were found in measles outbreaks in 2018-2019, eight variants of the dominant D8 and six B3 variants, while genotype A was detected in eight samples. The dominant genotype of 2017 (D8, 4283) was identified for the first time in the CR in January 2018. Four months later, it was replaced by genotype D8, 4683, occurring in the CR from March 2018 to June 2019. This genotype was identified in 170 of 243 samples (70%). There was a 3-month window between the first and the second detection of this genotype, which does not imply that in the meantime the virus did not circulate in the population. The analysis of seven samples from 2017 conducted by the collaborating Regional Reference Laboratory at the Robert Koch Institute (RRL RKI) in Berlin assigned five samples from Ostrava to genotype B3 and detected two variants of genotype D8 (Praha, Liberec). Laboratory diagnosis was facilitated by a higher proportion of clinical specimens available for direct detection of the virus, which increased from 18% in 2017 to 43% in 2019. Samples were referred to the National Reference Laboratory (NRL) in Prague for sequencing in accordance with the set legal rules. Between 2018 - 2019, laboratories sent 424 samples. Two hundred and forty-three samples (60%) were successfully sequenced, while the sequencing of the remaining samples failed due to low viral load. CONCLUSIONS: Measles virus sequencing was introduced in the Czech Republic as a necessary part of molecular surveillance, and almost 60% of positive samples were analysed. The sequencing analysis confirmed the endemic spread of measles virus, with genotype D8, 4683 MVs/GirSomnath.IND/42.16 found to circulate in the CR for 16 months between 2018 and 2019. Laboratory diagnosis is recently focusing more on direct detection of the virus, which along with genotyping extended to include another part of the genome will improve molecular surveillance.

Primary resistance to integrase strand-transfer inhibitors in Europe.

OBJECTIVES: The objective of this study was to define the natural genotypic variation of the HIV-1 integrase gene across Europe for epidemiological surveillance of integrase strand-transfer inhibitor (InSTI) resistance. METHODS: This was a multicentre, cross-sectional study within the European SPREAD HIV resistance surveillance programme. A representative set of 300 samples was selected from 1950 naive HIV-positive subjects newly diagnosed in 2006-07. The prevalence of InSTI resistance was evaluated using quality-controlled baseline population sequencing of integrase. Signature raltegravir, elvitegravir and dolutegravir resistance mutations were defined according to the IAS-USA 2014 list. In addition, all integrase substitutions relative to HXB2 were identified, including those with a Stanford HIVdb score ≥ 10 to at least one InSTI. To rule out circulation of minority InSTI-resistant HIV, 65 samples were selected for 454 integrase sequencing. RESULTS: For the population sequencing analysis, 278 samples were retrieved and successfully analysed. No signature resistance mutations to any of the InSTIs were detected. Eleven (4%) subjects had mutations at resistance-associated positions with an HIVdb score ≥ 10. Of the 56 samples successfully analysed with 454 sequencing, no InSTI signature mutations were detected, whereas integrase substitutions with an HIVdb score ≥ 10 were found in 8 (14.3%) individuals. CONCLUSIONS: No signature InSTI-resistant variants were circulating in Europe before the introduction of InSTIs. However, polymorphisms contributing to InSTI resistance were not rare. As InSTI use becomes more widespread, continuous surveillance of primary InSTI resistance is warranted. These data will be key to modelling the kinetics of InSTI resistance transmission in Europe in the coming years.

HIV-1 drug-resistance patterns among patients on failing treatment in a large number of European countries.

BACKGROUND: Information about patterns of HIV-1 drug resistance among treatment-exposed patients is crucial for the development of novel effective drugs. Currently no system exists that monitors patterns of resistance in patients failing therapy. METHODS: The study included 1,988 HIV-1 sequences from patients experiencing therapy failure collected between 2000 and 2004 in 15 European countries. Genotypic resistance was interpreted using the ANRS algorithm. Phenotypic resistance was predicted using the Virco geno- to phenotype system. RESULTS: 80.7% of the sequences included at least one drug-resistance mutation. Mutations were found for NRTIs (73.5%), NNRTIs (48.5%), and protease inhibitors (35.8%). Ninety percent of sequences with genotypic resistance harbored M184V, M41L, K103N, D67N, and/or T215Y. Among NRTIs, resistance was most frequently predicted for lamivudine. About half of all sequences had reduced susceptibility for NNRTIs. Resistance to most boosted protease inhibitors was found in < 25%. No sequence had resistance to all currently available drugs. CONCLUSION: Levels of resistance among patients with therapy failure were high. The patterns of resistance reflect resistance to drugs available for a longer time. Fully suppressive regimens can be designed even for the most mutated HIV because boosted protease inhibitors have remained active against most circulating viruses and new drug classes have become available.

Long-term analysis of the resistance development in HIV-1 positive patients treated with protease and reverse transcriptase inhibitors: correlation of the genotype and disease progression.

In this study, 27 HIV-1-positive patients on long-term highly active antiretroviral therapy (HAART) in the Czech Republic were followed for a period of up to 7 years. Variability of the HIV-1 protease (PR) sequence common in the Czech Republic was observed. Under the pressure of inhibitors of protease (PRIs) and reverse transcriptase (RTIs) mutations in PR were detected. Development of resistance to PRIs was followed by a decrease in CD4 count and increase in viral load. The dynamics of viral load closely corresponded to the accumulation of specific primary mutations in PR and RT. Out of 27 patients 18 developed resistance to PRIs and the prolonged therapy led to the accumulation of a higher number of amino acid changes associated with the resistance and, consequently, cross-resistance to several PRIs was observed. These multi-resistant variants of HIV-1 with mutations in PR could not be inhibited sufficiently with PRIs that are currently available in clinical practice. Efficient yet temporary suppression of viral replication was achieved by a lopinavir (LPV) treatment.

Human immunodeficiency virus 1 strains resistant to nucleoside inhibitors of reverse transcriptase in isolates from the Czech Republic as monitored by line probe assay and nucleotide sequencing.

The genetic resistance to nucleoside inhibitors of the reverse transcriptase (RT) of human immunodeficiency virus I (HIV-1) isolates in the Czech Republic was examined by a line probe assay (LiPA) and nucleotide sequencing. The results of LiPA analysis of 294 blood specimens obtained from 156 patients revealed a high incidence of mutations in the RT gene related to resistance to various drugs (67.3%) in various combinations. Mutations in RT gene (M41L, K70R and T215Y/F) conferring the resistance to zidovudine (ZDV) were most frequent (62.6%), that (M184V) responsible for the resistance to lamivudine (3TC) was less frequent (33.7%), while those linked to the resistance to dideoxyinosine (ddl) and dideoxyinosine together with dideoxycytidine (ddl/ddC) were rather rare (6.5% and 5.1%, respectively). LiPA gave a high rate of uninterpretable results due to codon hybridization failure, especially in HIV-1 isolates of non-B subtype. Thirty-two specimens were analyzed also by direct sequencing of a part of RT gene. The results obtained by LiPA and the sequencing were highly concordant for codons successfully analyzed by both methods, but the sequencing provided information also about the codons that could not be analyzed by LiPA. A high prevalence of resistant strains in the Czech Republic and their heterogeneity justifies a regular HIV-1 resistance testing. LiPA turned out as a fast, powerful and most reliable tool for such a purpose. However, due to an increasing diversity of HIV-1 strains circulating in the Czech Republic, LiPA cannot replace the nucleotide sequence analysis.

Chemoraces and habitat specialization of Claviceps purpurea populations.

We studied genetic variability of 100 isolates of Claviceps purpurea by using randomly amplified polymorphic DNA (RAPD), an EcoRI restriction site polymorphism in the 5.8S ribosomal DNA (rDNA), the alkaloids produced, and conidial morphology. We identified three groups: (i) group G1 from fields and open meadows (57 isolates), (ii) group G2 from shady or wet habitats (41 isolates), and (iii) group G3 from Spartina anglica from salt marshes (2 isolates). The sclerotia of G1 isolates contained ergotamines and ergotoxines; G2 isolates produced ergosine and ergocristine along with small amounts of ergocryptine; and G3 isolates produced ergocristine and ergocryptine. The conidia of G1 isolates were 5 to 8 microm long, the conidia of G2 isolates were 7 to 10 microm long, and the conidia of G3 isolates were 10 to 12 microm long. Sclerotia of the G2 and G3 isolates floated on water. In the 5.8S rDNA analysis, an EcoRI site was found in G1 and G3 isolates but not in G2 isolates. The host preferences of the groups were not absolute, and there were host genera that were common to both G1 and G2; the presence of members of different groups in the same locality was rare. Without the use of RAPD or rDNA polymorphism, it was not possible to distinguish the three groups solely on the basis of phenotype, host, or habitat. In general, populations of C. purpurea are not host specialized, as previously assumed, but they are habitat specialized, and collecting strategies and toxin risk assessments should be changed to reflect this paradigm shift.

Sulfate assimilation in Aspergillus terreus: analysis of genes encoding ATP-sulfurylase and PAPS-reductase.

Two genes for the sulfate assimilation pathway in Aspergillus terreus were cloned. The genes sAT (coding for PAPS-reductase) and sCT (coding for ATP-sulfurylase) form a small gene cluster. Both genes are similar to their homologs in A. nidulans (sA and sC), Penicillium chrysogenum (aps) and Saccharomyces cerevisiae (MET3 and MET16). In the coding sequence of the sCT gene, a typical non-functional APS-kinase-like domain is present. The sCT gene is expressed in A. nidulans, but its expression there is less sensitive to methionine level than in the original species. Two regions 5' upstream of sAT were found to be similar to those of sA.

Polyketide synthase gene pksM from Aspergillus terreus expressed during growth phase.

The polyketide synthase gene pksM was detected in the genomic DNA library of Aspergillus terreus by hybridization with the 6-methylsalicylic acid synthase (6-MSAS) gene of Penicillium patulum as a probe. 9524 bp of the cloned DNA were sequenced and a 5.5 kb open reading frame was revealed. A single intron (62 bp) was identified in the conserved position. Two transcription start points were determined within the 5'-flanking region at 50 and 72 (major) bp upstream from the putative translation initiation codon ATG. The conserved active site motifs for ketosynthase, acyltransferase, dehydratase, ketoreductase and acyl carrier protein were found within the predicted polypeptide consisting of 1803 amino acids. Unlike the P. patulum 6-MSAS gene, the transcription of pksM from A. terreus was observed in the middle of the vegetative growth phase.

[Dexamethasone suppression test and the positive and negative symptoms of schizophrenia]. / Test hamowania deksametazonem, a objawy pozytywne i negatywne schizofrenii.

40 patients (26 males, 14 females), aged 18-50 years, with diagnosis of schizophrenia were studied before the treatment for recurrence of acute schizophrenic symptom was started. All patients had dexamethasone suppression test (DST) and BPRS and Hamilton Depression Scale tests. Initial cortisol plasma concentration correlated positively with the intensity of productive symptoms measured by BPRS. The cortisol plasma level measured 17 hour after dexamethasone administration correlated negatively with the global symptom intensity in the BPRS. Pathological DST results were observed in 14 patients (35%). These patients presented lower intensity of psychopathological symptoms in the BPRS as compared to the remaining patients. The intensity of depression measured by Hamilton Scale did not show correlation with DST results. We conclude that in the schizophrenic patients during acute phase of the disease, the intensity of psychopathologic symptoms (mainly productive symptoms) is related to increased activity of the hypothalamo-hypophyseal-suprarenal axis which manifests itself with increased basal levels of plasma cortisol. Reactivity of this axis, with good suppression in the DST results seems to be normal. The intensity of depressive symptoms did not show significant relation to activity of the axis judging from DST results.

[Monitoring of the treatment of endogenous depression with imipramine and amitriptyline (preliminary report)]. / Monitorowane leczenie depresji endogennej imipramina i amitryptylina (doniesienie wstepne).

Monitored treatment of a depressed phase of unipolar affective disorder was conducted in 11 female patients receiving imipramine and in 12 females taking amitriptyline. Patients were randomly assigned to one of the drug and in 6 patients the drugs were switched because of the lack of response to the first used compound. In the imipramine treated group a satisfactory response after 4 weeks of management (less than 6 points on Hamilton's depression scale) was observed in 6 patients and in amitriptyline treated group in 5 patients. Patients displaying a satisfactory response to amitryptyline had significantly higher--as compared to remaining patients in the group--plasma levels of the drug after two and four weeks of treatment. Such an association was not observed in patients treated wtih imipramine. Severity of depression and motor retardation before the treatment was similar both in patients with satisfactory and with poor response to imipramine as well as to amitriptyline. However the intensity of anxiety symptoms was higher in patients exhibiting poor response to treatment with amitriptyline and imipramine as well.

[Effect of neuroleptic treatment on positive and negative symptoms of schizophrenia and the results of the dexamethasone test]. / Wpøyw leczenia neroleptycznego na objawy pozytywne i negatywne schizofrenii oraz wyniki testu deksametazonowego.

20 schizophrenic patients (14 men and 6 women), aged 18-50, were studied using the BPRS, Hamilton scale and dexamethasone test before and after treatment with neuroleptic drugs. In the BPRS global rating and positive and negative symptoms rating were analyzed separately. The neuroleptic treatment improved mostly positive symptoms and slightly less depressive symptoms. The smallest effect was exerted on the negative symptoms. The neuroleptic treatment significantly reduced all cortisol levels measured during the dexamethasone test (most the cortisol level measured 17 hours after dexamethasone). The dexamethasone test result normalization depends probably on the neuroleptic treatment -- it did not correlate closely with the clinical improvement.

[Usefulness of standardized psychometric scales (BPRS, HDS)in the evaluation of schizophrenic symptoms (preliminary report)]. / Przydatnosc standardowych skal psychometrycznych (BPRS, HDS) w ocenie objawów schizofrenii (doniesienia wstepne).

The psychometric investigation using BPRS and HDS was made in 27 schizophrenics in both acute and remission phase. The control group consisted of 22 depressive patients. The schizophrenic patients showed significant higher score of 9 BPRS items respecting schizophrenia subscale of BPRS in Beech's modification. Among acute schizophrenic the significant correlation was found between the intensity of positive (productive) and negative (defective) symptoms. No correlation was found between the severity of depression in HDS, severity of positive/negative symptoms, and global BPRS score. Among schizophrenics during remission both total severity in BPRS and negative symptoms subscale score correlated significantly with the intensity of symptoms in Hamilton's scale. The results were confronted with the last data from the literature. The results show the usefulness of these scales for the diagnosis of schizophrenia and for the evaluation of importance of positive/negative symptoms in the course of schizophrenia.