Viesturs Baumanis

Mutations in the rpoB and katG Genes Leading to Drug Resistance in Mycobacterium tuberculosis in Latvia

ABSTRACT To characterize the genetic basis of drug resistance in Mycobacterium tuberculosis in Latvia, mutations involved in rifampin (rpoB gene) and isoniazid (katG gene) resistance in DNA from 19 drug-susceptible and 51 multidrug-resistant M. tuberculosis complex isolates were analyzed. The most frequent rpoB gene mutations found by the Line Probe assay were the S531L (14 of 34 isolates), D516V (7 of 34), H526D (4 of 34), and D516Y plus P535S (4 of 34) mutations. Direct sequencing of seven isolates with unclear results from Line Probe assay showed the presence of the L533P mutation and the Q510H plus H526Y (1 of 34) and D516V plus P535S (4 of 34) double mutations, neither of which has been described previously. Single-strand conformation polymorphism analysis showed strand mobility differences between the rifampin-susceptible and -resistant samples for the D516V, H526D, and D516Y plus P535S mutations but not for the S531L mutation. Nucleotide substitution at codon 315 (AGC→ACC) of the katG gene was found in 48 of 51 multidrug-resistant samples by sequencing. Furthermore, katG gene restriction fragment length polymorphism analysis with endonuclease AciI confirmed the nucleotide change in codon 315.

Vectors of tick-borne diseases and epidemiological situation in Latvia in 1993-2002.

During the period of 1993-2002 an increase and the remarkable changes in the incidence of tick-borne encephalitis (TBE) and Lyme borreliosis (LB) as well as annual activities of vector species were noticed. The highest increase of TBE morbidity in Latvia has been observed in 1994 and 1995, and less expressed also in 1998 which was followed by a significant decrease during subsequent years. Whereas the highest peak of LB morbidity has been noticed in 1998 with only a minor decrease during subsequent years. Two epidemiologically significant Ixodes tick species are common in Latvia. Ixodes ricinus L. spread in the western and central part of Latvia; but rarely and in small numbers also in the eastern part. Ixodes persulcatus P. Sch. seems to dominate only in the eastern part of the country. The changes of seasonal and annual activities of I. ricinus were observed in two types of monitoring sites--sylvatic and peridomestic, located in the central part of Latvia. Observations of I. persulcatus were made in the eastern districts. Comparing geographically the Tick-borne diseases (TBD) incidence data for three tick distribution regions (I. ricinus dominated, I. persulcatus dominated and mixed regions), it was observed that the trend of annual changes in LB incidence during the last decade is almost of the same kind for all regions. Whereas TBE incidence extremes in 1994 and 1995 were observed almost only in I. ricinus dominated region. The annual field-collected adult tick infection rate with TBE virus (TBEV) from 1993 to 2002 for I. ricinus adults varied between 1.7% and 26.6% and for I. persulcatus--between 0% and 37.3%. The infection level in ticks removed from humans was much higher and from 1998 to 2002 surpassed 30%. TBEV investigations of removed adults and nymphs in 2002 discovered a high TBEV prevalence also in I. ricinus nymphs (43%). Typing of TBE virus isolated from ticks and patient serum samples in collaboration with German and Swedish virologists revealed that all three TBEV subtypes were co-circulating in Latvia. The mean annual tick infection rate with Borrelia burgdorferi s.l. analysed by means of a nested polymerase chain reaction of OspA gene fragment during the period of 1999-2001 was 18%-38% for I. ricinus adults and 25%-51% for I. persulcatus adults. Typing of Borrelia-positive DNA samples indicated that B. afzelii was dominant in Borrelia-positive I. ricinus (64.9%) and I. persulcatus (81.2%) followed by B. garinii (I. ricinus: 24.3%; in I. persulcatus: 14.5%). Investigations of Ehrlichia phagocytophila genogroup (by nested PCR targeted the 16S rRNA gene) revealed the presence of Ehrlichia sp. in I. ricinus from the central part of Latvia. Obtained data on TBD vectors and the epidemiological situation during the last decade indicates the complex impact of different factors influencing TBE and LB morbidity in Latvia.

Mitochondrial DNA Portrait of Latvians: Towards the Understanding of the Genetic Structure of Baltic-Speaking Populations

Mitochondrial DNA (mtDNA) variation was investigated in a sample of 299 Latvians, a Baltic‐speaking population from Eastern Europe. Sequencing of the first hypervariable segment (HVS‐I) in combination with analysis of informative coding region markers revealed that the vast majority of observed mtDNAs belong to haplogroups (hgs) common to most European populations. Analysis of the spatial distribution of mtDNA haplotypes found in Latvians, as well as in Baltic‐speaking populations in general, revealed that they share haplotypes with all neighbouring populations irrespective of their linguistic affiliation. Hence, the results of our mtDNA analysis show that the previously described sharp difference between the Y‐chromosomal hg N3 distribution in the paternally inherited gene pool of Baltic‐speaking populations and of other European Indo‐European speakers does not have a corresponding maternal counterpart.

Prevalence of tick-borne pathogens in ticks collected from migratory birds in Latvia

Migratory birds act as hosts and long-distance vectors for several tick-borne infectious agents. Here, feeding Ixodes ticks were collected from migratory birds during the autumn migration period in Latvia and screened for the presence of epidemiologically important non-viral pathogens. A total of 93 DNA samples of ticks (37 larvae and 56 nymphs) removed from 41 birds (order Passeriformes, 9 species) was tested for Lyme borreliosis spirochaetes, Anaplasma phagocytophilum, Rickettsia spp., and Babesia spp. Borrelia burgdorferi DNA was detected in 18% of the tick samples, and a majority of infected ticks were from thrush (Turdus spp.) birds. Among the infected ticks, Borrelia valaisiana was detected in 41% of cases, Borrelia garinii in 35%, and mixed Bo. valaisiana and Bo. garinii infection in 24%. Anaplasma phagocytophilum DNA was detected in 2% of ticks, R. helvetica in 12%, and Babesia spp. pathogens in 4% of ticks. Among these samples, 3 Babesia species were identified: Ba. divergens, Ba. microti, and Ba. venatorum. Coinfection with different pathogens that included mixed infections with different Borrelia genospecies was found in 20% of nymphal and 3% of larval Ixodes ticks. These results suggest that migratory birds may support the circulation and spread of medically significant zoonoses in Europe.

Identification of Three Clinically Relevant Borrelia burgdorferi Sensu Lato Genospecies by PCR-Restriction Fragment Length Polymorphism Analysis of 16S-23S Ribosomal DNA Spacer Amplicons

ABSTRACT We report the results of a study of the prevalences of three clinically relevant Borrelia burgdorferi sensu lato genospecies (Borrelia burgdorferi sensu stricto, Borrelia afzelii, and Borrelia garinii) in 1,040 questing Ixodes ticks from all regions of Latvia, where Lyme borreliosis is endemic. The prevalences of Borrelia in Ixodes ricinus and Ixodes persulcatus were 22.6 and 27.9%, respectively. Molecular typing of B. burgdorferi from infected ticks was performed by restriction fragment length polymorphism (RFLP) analysis of PCR-amplified fragments of the 16S-23S (rrs-rrlA) rRNA intergenic spacer by using species-specific primers and subsequent sequencing. The dominant Borrelia species in both Ixodes species was B. afzelii. In addition, different restriction patterns of B. garinii and B. afzelii were also identified. This study demonstrates that the 16S-23S rRNA PCR-RFLP typing method is simple, sensitive, and fast and that it allows one to differentiate among B. burgdorferi species and subspecies with various degrees of pathogenic potential directly in ticks. These features are important in monitoring Lyme disease.

Spectrum of pncA Mutations in Multidrug-Resistant Mycobacterium tuberculosis Isolates Obtained in Latvia

Pyrazinamide (PZA) is an effective antituberculous agent ([1][1]) that becomes active when bacterial pyrazinamidase converts it to pyrazinoic acid, which is toxic to mycobacteria ([4][2]). In Mycobacerium tuberculosis , PZA resistance is associated with the loss of pyrazinamidase activity, mainly

Autochthonous canine babesiosis caused by Babesia canis canis in Latvia

This is the first report of confirmed canine babesiosis in Latvia supporting the observed geographical expansion of this disease. Between 2009 and 2011 three dogs which have not traveled outside of Latvia were diagnosed with babesiosis. Hematological analysis and serological tests for granulocytic anaplasmosis, ehrlichiosis and borreliosis were negative (Idexx SNAP 4Dx test). Peripheral blood erythrocytes of the three dogs contained large Babesia that were identified as Babesia canis canis by PCR. Sequences of partial 18S rRNA gene were 98-100% similar to the sequences of B. canis canis isolated from dogs in other European countries. We conclude that these are the first autochthonous canine babesiosis cases reported from Latvia.

Synthesis of recombinant atrial natriuretic peptide (rANP) using hybrid fusion protein-phage fr coat/ANP (CP/ANP).

Recombinant atrial natriuretic peptide (rANP) was expressed in and isolated from E. coli. rANP was purified using HPLC. Amino acid analysis, partial sequencing, and molecular mass were determined. Fused protein was used to rise polyclonal antibodies and to develop of immunoenzymatic assays of rANP and CP/ANP. Experiments were designed to study rANP effects on isolated rabbit aortic strips and to examine hypotensive, diuretic, and natriuretic activity, as well as renal creatinine clearance, in an in vivo rat model. Identity of recombinant and commercial ANP has been confirmed. Physiological activity of CP/ANP has allowed the investigators to predict the conformation of CP/ANP, pro-ANP processing, and the method by which fusion protein interacts with ANP receptors.

Genotypic and phenotypic characteristics of aminoglycoside-resistant Mycobacterium tuberculosis isolates in Latvia.

Mutations causing resistance to aminoglycosides, such as kanamycin (KAN), amikacin (AMK), and streptomycin, are not completely understood. In this study, polymorphisms of aminoglycoside resistance influencing genes such as rrs, eis, rpsL, and gidB in 41 drug-resistant and 17 pan-sensitive Mycobacterium tuberculosis clinical isolates in Latvia were analyzed. Mutation A1400G in rrs gene was detected in 92% isolates with high resistance level to KAN and diverse MIC level to AMK. Mutations in promoter region of eis were detected in 80% isolates with low-level MIC of KAN. The association of K43R mutation in rpsL gene, a mutation in the rrs gene at position 513, and various polymorphisms in gidB gene with distinct genetic lineages of M. tuberculosis was observed. The results of this study suggest that association of different controversial mutations of M. tuberculosis genes to the drug resistance phenotype should be done in respect to genetic lineages.

Structure of an outer surface lipoprotein BBA64 from the Lyme disease agent Borrelia burgdorferi which is critical to ensure infection after a tick bite.

Lyme disease is a tick-borne infection caused by the transmission of Borrelia burgdorferi from infected Ixodes ticks to a mammalian host during the blood meal. Previous studies have shown that the expression of B. burgdorferi surface-localized lipoproteins, which include BBA64, is up-regulated during the process of tick feeding. Although the exact function of BBA64 is not known, this lipoprotein is critical for the transmission of the spirochete from the tick salivary glands to the mammalian organism after a tick bite. Since the mechanism of development of the disease and the functions of the surface lipoproteins associated with borreliosis are still poorly understood, the crystal structure of the B. burgdorferi outer surface lipoprotein BBA64 was solved at 2.4 Å resolution in order to obtain a better insight into the pathogenesis of B. burgdorferi and to promote the discovery of novel potential preventive drugs against Lyme disease. In this study, the crystal structure of BBA64 was also compared with that of the paralogous protein CspA (also referred to as BbCRASP-1, CRASP-1 or BBA68). CspA is the complement regulator-acquiring surface protein-1 of B. burgdorferi; its structure is known, but its function apparently differs from that of BBA64. It is demonstrated that unlike the homologous CspA, BBA64 does not form a homodimer. Their differences in function could be explained by divergence in their amino-acid sequences, electrostatic surface potentials and overall tertiary structures. The C-terminal part of BBA64 has a different conformation to that of CspA; the conformation of this region is essential for the proper function of CspA.