Evaluation of real-time PCR targeting the lipL32 gene for diagnosis of Leptospira infection.

BACKGROUND: Different diagnostic methods have been used for the laboratory confirmation of leptospirosis. Molecular diagnostic techniques are not only faster and more sensitive than culture analysis, but can also detect a Leptospira infection before the appearance of antibodies. The aim of the present study was to analyze and compare two different PCR approaches applied to blood and urine specimens obtained from patients with clinical manifestations that were suggestive of leptospirosis. Furthermore, the results of these different PCR approaches were compared with the results of culture and serology analyses. RESULTS: A total of 400 samples (234 blood or 58.5% and 166 urine of 41.5%) from 310 Slovenian patients with clinical manifestations suggestive of leptospirosis were tested using conventional PCR assays targeting the rrs gene and RT-PCR targeting the lipL32 gene. Additionally, culture, serology and sequence analysis were performed for the majority of these samples. The PCR and RT-PCR results were concordant in 376 out of 400 of these samples (94.0%). Conventional PCR was positive for 27 out of 400 samples (6.8%) and RT-PCR was positive for 47 out of 400 samples (11.8%). Culture and microscopic agglutination tests supported these diagnoses. CONCLUSIONS: A comparison of the two PCR methods indicated that the RT-PCR targeting of the lipL32 gene was faster, more sensitive and more specific for the determination of Leptospira DNA in these clinical samples.

Impact of added fluvastatin to standard-of-care treatment on sustained virological response in naïve chronic hepatitis C Patients infected with genotypes 1 and 3.

OBJECTIVES: The combination of pegylated interferon-α and ribavirin is a standard-of-care (SOC) treatment for chronic hepatitis C (CHC), and it achieves a sustained virological response (SVR) in 41-52% of genotype 1 and in 73-79% of genotype 3 patients. In a few clinical trials, the combination of fluvastatin and SOC increased the SVR in genotype 1 patients. METHODS: This prospective study enrolled 179 naïve CHC patients. In the fluvastatin group patients received the combination of SOC and fluvastatin 80 mg daily; historical controls matching the study group in genotype, age and gender were treated with the SOC treatment only. RESULTS: On-treatment viral responses as well as the SVR did not differ significantly between the two groups, except for the genotype 1 patients with a high viral load presenting a significantly higher SVR rate in the fluvastatin group (75%) compared to the control group (41%; p = 0.024). Multivariate logistic regression identified hepatitis C virus (HCV) genotype 3 infection (p < 0.001), age ≤40 years (p < 0.001), liver steatosis <5% (p < 0.01) and low viral load (p < 0.001) as independent predictors of an SVR. CONCLUSION: A combination of fluvastatin and SOC significantly improved the SVR in naïve CHC patients infected with HCV genotype 1 and high viral load, but it did not improve the SVR in patients infected with HCV genotype 3.

First Report on Leptospira Species Isolated from Patients in Slovenia.

Leptospirosis is an important worldwide zoonosis, and it has also been reported in Slovenia. The cultivation of Leptospira from human material is difficult. Despite that, we successfully isolated 12 human Leptospira strains isolated from patients between 2002 and 2020 and used various methods for the phenotypic and genotypic characterization of the strains, including matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF) using our own MALDI-TOF data library, melting temperature analysis of the amplified lfb1 gene, determination of Leptospira serogroups using rabbit immune sera, NotI-RFLP of the whole Leptospira genome, multilocus sequence typing (MLST) of seven housekeeping genes, and whole-genome sequencing (WGS)-based typing. We confirmed the presence of four pathogenic Leptospira species (L. kirschneri, L. interrogans, L. borgpetersenii, and L. santarosai) and three serogroups: Grippotyphosa, Icterohaemorrhagiae, and Sejroe. MALDI-TOF identified three of seven isolates at the species level and four isolates at the genus level. Serovars of 8 of the 10 strains were determined using NotI-RFLP. MLST showed that the clinical isolates belonged to sequence types ST17, ST110, and ST155. WGS confirmed the analysis of Leptospira strains using conventional methods. In addition, WGS provided better taxonomic resolution for isolate DDA 10944/10.

A Patient with Severe Leptospirosis Treated with Cytokine Removal and High-Dose Corticosteroids.

Leptospirosis is an ubiquitous zoonosis with significant morbidity and mortality. Approximately 10 percent of human infections evolve into a severe form, with a sepsis-like disease, multiorgan failure, and significant mortality rate. The cornerstone of treatment of severe disease is antibiotic therapy, with the aims of preventing complications, reducing the duration of disease, and ultimately reducing mortality. The initiation of antibiotic chemotherapy can precipitate a febrile inflammatory reaction, also known as a Jarisch-Herxheimer reaction. We present a case report of a patient with severe leptospirosis, complicated by multiorgan failure with severe circulatory failure of distributive and cardiogenic etiology, possibly as a consequence of the Jarisch-Herxheimer reaction. The patient was treated with antimicrobial therapy and other supportive measures along with high-dose corticosteroid therapy, long-term mechanical ventilation, high-dose vasopressor therapy, and continuous veno-venous hemodiafiltration with extracorporeal cytokine removal.With this case, we would like to report on a patient presenting with two neglected diseases in our part of Europe, who was treated with novel therapeutic strategies.

Hepatitis C virus genotypes in 1,504 patients in Slovenia, 1993-2007.

In order to identify the main routes of hepatitis C (HCV) transmission and to determine the HCV genotype distribution and its dynamics during a 15-year period in Slovenia, HCV genotypes were detected using the INNO-LiPA HCV II (Innogenetics) test for serum samples obtained from 1,504 patients representing 72.6% of all patients with chronic hepatitis C diagnosed from 1993 to 2007. HCV genotype 1 was predominant (56%), followed by genotypes 3, 2, and 4, with a prevalence of 37.8%, 5%, and 1.2%, respectively. HCV genotypes 5 and 6 were not detected in any patient. Patients infected with HCV genotype 3 were significantly younger (mean age 28.9 +/- 8.5 years) than those infected with genotype 1 (mean age 38.9 +/- 14.8 years; P < 0.0001) and those infected with HCV genotype 2 (mean age 50.3 +/- 18.2 years; P < 0.0001). Intravenous drug use was identified as the most frequent possible HCV transmission route (34.3%), followed by medical-related transmission such as transfusion of HCV-contaminated blood or blood products, and hemodialysis (12.5%). Being an intravenous drug user was found to be strongly associated with HCV genotype 3 (OR, 3.71 [95% CI, 2.97-4.65]; P < 0.0001) and reporting infection by transfusion of blood or blood products was found to be strongly associated with HCV genotype 1 (OR, 3.28 [95% CI, 2.18-4.95]; P < 0.0001). During the 15-year period, the proportion of genotype 3 increased substantially, reflecting the fact that the HCV epidemic in Slovenia is driven mostly by intravenous drug use.

False-positive result of a confirmatory human immunodeficiency virus line immuno assay in an apparently healthy individual--a case report.

A case of a false-positive result of human immunodeficiency virus (HIV) confirmatory immunoblot-based assay is described. Repeatedly borderline reactive anti-HIV screening enzyme immunoassay result obtained in a local hospital resulted in directing the sample to the Slovenian HIV/AIDS Reference Laboratory. In the Reference Laboratory, both anti-HIV screening assays and confirmatory Western blot were negative, while a confirmatory test INNO-LIA HIV I/II Score (Innogenetics, Ghent, Belgium) was anti-HIV-1 positive due to sgp120 and gp41 reactivity. The results of serological testing of the second sample obtained three weeks later were completely identical, while in the third sample obtained 5 months later, seroreversion was observed. Due to a negative dynamics in anti-HIV serological profile and repeatedly negative results of the molecular tests for HIV-1 and HIV-2, HIV infection was excluded and the results of test INNO-LIA HIV I/II Score were finally interpreted as false positive.

Evaluation of the immunochromatographic (Leptocheck) test for detection of specific antibodies against leptospires.

BACKGROUND: Leptospirosis is a febrile worldwide zoonosis. Routine diagnosis of leptospiral infection is based on demonstration of specific antibodies with serological tests. Performance of the reference serological test, the microscopic agglutination test (MAT), requires significant expertise. The aim of our study was to find out if leptospiral infection can be proven with simple, rapid, commercially available immunochromatographic Leptocheck test in order to introduce it for the first level diagnosis in emergency cases with less specialized laboratory staff. METHODS: In all, 590 serum samples of patients with clinical manifestations suggestive of leptospirosis were collected and tested with MAT and Leptocheck test. For confirmation of the results some other diagnostic methods such as polymerase chain reaction (PCR) and Leptospira isolation were performed. RESULTS: Results of both serological tests were consistent in 576/590 (97.63%) cases but Leptocheck gave more positive results in comparison to MAT (36 and 12, respectively) at first patient's testing. Following up the patient, MAT became positive in majority of Leptocheck positive patients at first visit. Leptospiral DNA was detected in nine blood and six urine samples belonging to thirteen different patients while only two samples were culture positive. CONCLUSION: In comparison with serological tests, PCR and culture have low sensitivity. According to our findings we conclude that Leptocheck test can prove leptospiral infection and could be used for rapid diagnosis of leptospirosis, later the sample should be confirmed with MAT.

Prevalence of HIV, hepatitis B, C and G virus infections among injecting drug users on methadone maintenance treatment in Maribor.

The prevalence of human immunodeficiency virus (HIV) infection and hepatitis B, C, and G virus infections was determined in a selected group of 40 intravenous drug addicts on methadone maintenance treatment. No drug addict was infected with HIV. Seven drug addicts (17.5%) had serologic markers of hepatitis B infection but only one among them (2.5%) exhibited signs of active infection. 16 drug addicts (40%) had serologic markers of hepatitis C infection and 13 (32.5%) of them had an active infection with positive viral tests. 22 addicts (55%) had signs of hepatitis G infection, 9 (22.5%) of them had signs of active infection and 13 (32.5%) had signs of past infection. Addicts infected with the hepatitis B and C viruses were older and had also been taking drugs longer than those who were not infected with these two viruses. The proportion of those susceptible to infection with hepatitis B (70%) is high, reflecting a low prevalence of this disease and at the same time revealing the failure of preventive measures and the unacceptably low vaccination rate.