Tõnis Karki

Eradication of Salmonella Typhimurium infection in a murine model of typhoid fever with the combination of probiotic Lactobacillus fermentum ME-3 and ofloxacin.

BackgroundThe aim of the study was to detect whether in experimental Salmonella enterica Typhimurium infection the probiotic Lactobacillus fermentum ME-3 in combination with fluoroquinolone therapy would eradicate S. Typhimurium, prevent the development of liver and spleen granulomas and improve the indices of oxidative stress in the ileum mucosa.The selected bacteriological, histological and biochemical methods were applied.ResultsCombined treatment with L. fermentum ME-3 and ofloxacin eradicated Salmonella Typhimurium from blood, ileum and liver, decreased the number of animals with liver and spleen granulomas and reduced the value of lipid peroxides in the ileum mucosa. Higher total counts of intestinal lactobacilli in all experimental groups were associated with the absence of liver granulomas.ConclusionThe antimicrobial and antioxidative probiotic L. fermentum ME-3 combined with ofloxacin enhances the eradication of experimental S. Typhimurium infection. These observations on probiotic and antimicrobial co-action may serve as basis to develop new strategies for treatment of invasive bacterial infections of the gut.

CCL3L1 Copy Number Is a Strong Genetic Determinant of HIV Seropositivity in Caucasian Intravenous Drug Users

BACKGROUND A high copy number of CCL3L1, the most potent human immunodeficiency virus (HIV)-suppressive chemokine, associates with reduced HIV susceptibility. Whether CCL3L1 influences acquisition of multiple blood-borne infections (eg, hepatitis C virus [HCV], HIV, and hepatitis B virus [HBV] infections), which occur commonly among injection drug users (IDUs), is unknown. METHODS We determined CCL3L1 copy number by real-time polymerase chain reaction among 374 Caucasian IDUs from Estonia; 285 were HCV positive, 208 were HIV positive, 177 were HCV and HIV positive, and 57 were HCV and HIV negative. RESULTS In univariate and multivariate analyses, HCV and HBV seropositivity and duration of IDU each strongly predicted HIV seropositivity. A high CCL3L1 copy number (>2) was associated with an 80% reduced risk of acquiring HIV infection after adjusting for age, sex, HCV and HBV status, CCR5-Delta32 polymorphism, and IDU duration (odds ratio, 0.20; 95% confidence interval, 0.09-0.45). By contrast, CCL3L1 gene dose did not influence HCV seropositivity. Among HCV-positive IDUs, there was a 3.5-fold overrepresentation and 65% underrepresentation of a high CCL3L1 copy number among HCV-positive, HIV-negative subjects and HCV-positive, HIV-positive subjects, respectively. CONCLUSION Among IDUs with extensive exposure to HCV and HIV, CCL3L1 copy number is a major determinant of HIV seropositivity but not of HCV seropositivity. The contrasting distribution of a protective high CCL3L1 copy number among HCV-positive, HIV-negative IDUs versus HCV-positive, HIV-positive IDUs may reflect that HIV preferentially selects for subjects with a low CCL3L1 gene dose.

Antibacterial Susceptibility of Intestinal Lactobacilli of Healthy Children

We investigated the antibacterial susceptibility of intestinal lactobacilli of Estonian and Swedish children aged 1-2 y. Sixty isolates (10 species) of lactobacilli (29 Estonian and 31 Swedish strains) were tested against ampicillin, cefuroxime, cefoxitin, gentamicin, ciprofloxacin, tetracycline, vancomycin, metronidazole and erythromycin. We observed that intestinal lactobacilli do not display uniform susceptibility to antibiotics. None of the tested lactobacilli was resistant to ampicillin, gentamicin and erythromycin. Single strains were resistant to cefuroxime and tetracycline, about half of the strains to cefoxitin and ciprofloxacin and 73% of the strains to vancomycin. All studied strains were resistant to metronidazole. Most of the strains investigated were resistant to two or three antibiotics out of nine. Some differences in susceptibility were noted between strains belonging to different fermentation types. No differences in susceptibility were found between Estonian and Swedish isolates. Metronidazole, cefoxitin, vancomycin and ciprofloxacin seem to be safer for gastrointestinal lactoflora than other tested antibiotics in both countries.We investigated the antibacterial susceptibility of intestinal lactobacilli of Estonian and Swedish children aged 1?2 y. Sixty isolates (10 species) of lactobacilli (29 Estonian and 31 Swedish strains) were tested against ampicillin, cefuroxime, cefoxitin, gentamicin, ciprofloxacin, tetracycline, vancomycin, metronidazole and erythromycin. We observed that intestinal lactobacilli do not display uniform susceptibility to antibiotics. None of the tested lactobacilli was resistant to ampicillin, gentamicin and erythromycin. Single strains were resistant to cefuroxime and tetracycline, about half of the strains to cefoxitin and ciprofloxacin and 73% of the strains to vancomycin. All studied strains were resistant to metronidazole. Most of the strains investigated were resistant to two or three antibiotics out of nine. Some differences in susceptibility were noted between strains belonging to different fermentation types. No differences in susceptibility were found between Estonian and Swedish isolates. Metronidazole, cefoxitin, vancomycin and ciprofloxacin seem to be safer for gastrointestinal lactoflora than other tested antibiotics in both countries.

Absence of genotypic drug resistance and presence of several naturally occurring polymorphisms of human immunodeficiency virus-1 CRF06_cpx in treatment-naive patients in Estonia.

All non‐B HIV‐1 subtypes and circulating recombinant forms (CRFs) are characterized by several polymorphisms in protease (PR) region. In addition, in recent years the increasing use of antiretroviral treatment (ART) has rapidly raised the spread of transmitted drug resistance. We aimed to determine the presence of naturally occurring polymorphisms and transmitted drug resistance mutations (DRMs) in ART naïve HIV‐1‐positive subjects in Estonia. A total of 115 drug‐naive HIV‐1‐infected subjects (mean age 27 years; 70% male; 65% infected via intravenous drug use and 34% by heterosexual contact) were enrolled. Viral genomic RNA from plasma was directly sequenced in PR, revertase (RT), and envelope (env) regions. Phylogenetic analysis of RT and env regions revealed that 89% and 3% of sequenced viruses belonged to CRF06_cpx and subtype A1, respectively, and 6% were described as unique recombinants (signed A1‐06) between CRF06_cpx and subtype A1 viruses. No primary DRMs were found in PR or RT regions indicating the absence of transmitted drug resistance. The most common polymorphisms in the PR region were K14R, M36I, H69K, and L89M seen in 96%, 100%, 99%, and 100%, respectively. The clinical relevance of these polymorphisms in terms of success of ART has to be monitored in future clinical studies. J. Med. Virol. 81:953–958, 2009.

Association between TLR3 rs3775291 and resistance to HIV among highly exposed Caucasian intravenous drug users.

BACKGROUND TLR3 recognizes dsRNA and triggers immune responses against RNA and DNA viruses. A polymorphism in TLR3, rs3775291 (Leu412Phe), has been associated with the increased susceptibility to enteroviral myocarditis, protection against tick-borne encephalitis virus and HIV-1 infection. We investigated Caucasian intravenous drug users (IDUs) and blood donors in order to evaluate the associations between TLR3 genotypes and susceptibility to HIV infection. MATERIALS AND METHODS A total of 345 Caucasian IDUs were recruited, 50% of them were HIV positive, 89% HCV and 77% HBV positive. Based on their history of needle sharing, 20 of the HIV negative IDUs were classified as highly exposed HIV seronegatives (HESNs), 68 as non-HESNs and 85 as unexposed. The control group consisting of 497 blood donors tested negative for all three viruses. TLR3 rs3775291 were determined by using TaqMan Allelic Discrimination Assay. RESULTS The TLR3 rs3775291 T allele frequency was similar among the HIV negative and HIV positive IDUs and blood donors - 36%, 31% and 34%, respectively. The frequency of persons possessing at least one TLR3 rs3775291 T allele was significantly higher in HESNs compared with blood donors and HIV positive IDUs (80% vs. 55%; p=0.037 and 80% vs. 53%; p=0.031, respectively). In the univariate analysis, persons who possessed at least one T allele had reduced odds of being HIV seropositive (OR=0.29, 95% CI=0.09-0.90). This association remained significant (OR=0.25, 95% CI=0.07-0.87) after the adjustment for other co-variates (HCV, HBV serostatus and duration of intravenous drug use). CONCLUSIONS The TLR3 rs3775291 T allele has a protective effect against HIV infection among HESNs IDUs.

Emerging transmitted drug resistance in treatment-naïve human immunodeficiency virus-1 CRF06_cpx-infected patients in Estonia.

Abstract Human immunodeficiency virus (HIV)-1 transmitted drug resistance in the drug-naïve population is of growing relevance in Estonia, where the number of antiretroviral (ARV) treatment-experienced subjects has been exponentially increasing during the last 10 y. The aim of this study was to estimate the rate of transmitted drug resistance among newly diagnosed subjects in Estonia in 2008. Genotypic resistance testing for viral genomic RNA was conducted for 201 subjects tested HIV-positive between 1 April and 30 November 2008. Of 145 genotyped viral strains in newly diagnosed patients, 123 were CRF06_cpx, 2 were subtype A1 and 3 were subtype B; in 17 cases viral sequences revealed recombinant structures similar to CRF06_cpx, subtype A1 and CRF02_AG. Resistance mutations were found in 8 (5.5%) virus strains, and 3 strains were resistant to at least 2 ARV classes. A total of 2.8% of sequences harboured mutations indicating nucleoside/nucleotide reverse transcriptase inhibitor resistance (M41L, M184V, M184I, T215C and T215D), 2.1% non-nucleoside reverse transcriptase inhibitor resistance (K103N, P225H) and 2.8% protease inhibitor resistance (M46I, L90M). These data suggest the need to extend genotypic HIV-1 drug resistance testing to newly diagnosed HIV-positive subjects to prevent potential ARV treatment failure.

Influence of interleukin 10 polymorphisms -592 and -1082 to the HIV, HBV and HCV serostatus among intravenous drug users

BACKGROUND Interleukin 10 (IL-10) is a multifunctional cytokine produced by macrophages, monocytes, and T-helper cells. Two polymorphisms at positions -592 and -1082 have been associated with HIV susceptibility. However, their associations with susceptibility to HIV and its co-infections among intravenous drug users (IDUs) are largely unknown. METHODS A total of 345 IDUs were recruited. Of the 173 HIV negative IDUs, 20 were classified as highly exposed HIV seronegative subjects (HESNs). A control group consisted of 496 blood donors; all HIV, HCV, and HBV negative. The IL-10-592C/A and -1082A/G were determined using TaqMan allelic discrimination assay. RESULTS Of the IDUs, 50% were HIV positive, 89% HCV positive, 67% HBV positive and 41% had triple infection. IL-10-592C allele and -1082A allele were the most common and the -1082AG/-592CC was the most common genotype pair. All HESNs exhibited -1082A allele as compared to 81.4% of the HIV positive IDUs and 79% of donors (p=0.029 and p=0.019, respectively). None of HESNs had GG/CC genotype pair compared with 18.6% of HIV positive IDUs and 21.0% of donors (p=0.029 and p=0.019, respectively). The possession of -592AC and genotype pair AG/AC were associated with the decreased odds of HBV infection (OR=0.28; 95% CI 0.09-0.87; p=0.028 and OR=0.19; 95% CI 0.06-0.61; p=0.052, respectively). CONCLUSIONS The presence of low producing IL-10-1082A and -592A alleles and their containing genetic variants protect highly exposed IDUs against acquisition of HIV and HBV infections.

Characterization of Integrase Region Polymorphisms in HIV Type 1 CRF06_cpx Viruses in Treatment-Naive Patients in Estonia

Natural polymorphisms of HIV-1, often associated with drug resistance, are widely described in protease and reverse transcriptase regions but data on their presence in the integrase region, especially in non-B subtypes, are still very limited. We aimed to characterize naturally occurring polymorphisms in the integrase region in 104 treatment-naive and 10 treatment-experienced patients infected predominantly with HIV-1 CRF06_cpx and its recombinant with subtype A1 and/or CRF03_AB viruses. No primary drug resistance mutations against integrase inhibitors were found, but resistance-associated polymorphisms such as V72I, L74I, V201I, and T206S were seen in more than 90% of viruses. Substitutions E157Q and E157K, associated with raltegravir resistance, were found in only two CRF06_cpx strains. We conclude that similar to other HIV-1 non-B subtypes, the CRF06_cpx and its recombinants with subtype A1 and CRF03_AB are rich in integrase region natural polymorphisms, which may impact the development of resistance against integrase inhibitors.

Antibiotic Susceptibility Patterns of Community- and Hospital-acquired Staphylococcus aureus and Escherichia coli in Estonia

This study compares the susceptibility patterns of Staphylococcus aureus and Escherichia coli isolated from patients with hospital-acquired and outpatient infections. A total of 902 isolates of S. aureus and 1,114 of E. coli were collected in five different Estonian medical centers between January 1997 and November 1997. Strains were grouped into two different categories, depending on whether they had been obtained from inpatients or outpatients. Compared to S. aureus strains isolated from inpatients, the strains from outpatients were significantly more resistant to erythromycin (25.3% vs. 17.9%), tetracycline (33.5% vs. 22.4%) and trimethoprim-sulfamethoxazole (13.9% vs. 7.9%). The overall prevalence of oxacillin-resistant S. aureus was 10.4%, with no significant differences noted between isolates recovered from inpatients and outpatients. In the case of E. coli, significantly more isolates from inpatients (42.8%) than from outpatients (34.4%) were ampicillin-resistant. Inpatient isolates of E. coli were also more resistant to cefotaxime (9.3%) and nitrofurantoin (11.2%) than outpatient strains (0% and 3.1%, respectively). Analysis showed remarkable co-resistance among both inpatient and outpatient strains of S. aureus and E. coli. Multiple resistant S. aureus and E. coli strains represented 15.1% and 17.3%, respectively of the organisms examined in this study. With respect to E. coli, significantly more multiresistant isolates were found in inpatient than outpatient isolates (20.4% vs. 8.9%). Our results indicate that the distinction between community-acquired and hospital infections due to S. aureus and E. coli may not be valid in Estonia.This study compares the susceptibility patterns of Staphylococcus aureus and Escherichia coli isolated from patients with hospital-acquired and outpatient infections. A total of 902 isolates of S. aureus and 1,114 of E. coli were collected in five different Estonian medical centers between January 1997 and November 1997. Strains were grouped into two different categories, depending on whether they had been obtained from inpatients or outpatients. Compared to S. aureus strains isolated from inpatients, the strains from outpatients were significantly more resistant to erythromycin (25.3% vs. 17.9%), tetracycline (33.5% vs. 22.4%) and trimethoprim?sulfamethoxazole (13.9% vs. 7.9%). The overall prevalence of oxacillinresistant S. aureus was 10.4%, with no significant differences noted between isolates recovered from inpatients and outpatients. In the case of E. coli, significantly more isolates from inpatients (42.8%) than from outpatients (34.4%) were ampicillin-resistant. Inpatient isolates of E. coli were also more resistant to cefotaxime (9.3%) and nitrofurantoin (11.2%) than outpatient strains (0% and 3.1%, respectively). Analysis showed remarkable co-resistance among both inpatient and outpatient strains of S. aureus and E. coli. Multiple resistant S. aureus and E. coli strains represented 15.1% and 17.3%, respectively of the organisms examined in this study. With respect to E. coli, significantly more multiresistant isolates were found in inpatient than outpatient isolates (20.4% vs. 8.9%). Our results indicate that the distinction between community-acquired and hospital infections due to S. aureus and E. coli may not be valid in Estonia.

CCR5 haplotypes influence HCV serostatus in Caucasian intravenous drug users.

Background Up to 90% HIV-1 positive intravenous drug users (IDUs) are co-infected with HCV. Although best recognized for its function as a major co-receptor for cell entry of HIV, CC chemokine receptor 5 (CCR5) has also been implicated in the pathogenesis of HCV infection. Here, we investigated whether CCR5 haplotypes influence HIV-1 and HCV seropositivity among 373 Caucasian IDUs from Estonia. Methods Of these IDUs, 56% and 44% were HIV and HCV seropositive, respectively, and 47% were coinfected. 500 blood donors seronegative for HIV and HCV were also evaluated. CCR5 haplotypes (HHA to HHG*2) were derived after genotyping nine CCR2–CCR5 polymorphisms. The association between CCR5 haplotypes with HIV and/or HCV seropositivity was determined using logistic regression analysis. Co-variates included in the models were length of intravenous drug use, HBV serostatus and copy number of CCL3L1, the gene encoding the most potent HIV-suppressive chemokine and ligand for CCR5. Results Compared to IDUs seronegative for both HCV and HIV (HCV−/HIV-), IDUs who were HCV+/HIV- and HCV+/HIV+were 92% and 82%, respectively, less likely to possess the CCR5-HHG*1 haplotype, after controlling for co-variates (Padjusted = 1.89×10−4 and 0.003, respectively). This association was mostly due to subjects bearing the CCR5 HHE and HHG*1 haplotype pairs. Approximately 25% and<10% of HCV−/HIV- IDUs and HCV−/HIV- blood donors, respectively, possessed the HHE/HHG*1 genotype. Conclusions Our findings suggest that HHG*1-bearing CCR5 genotypes influence HCV seropositivity in a group of Caucasian IDUs.