Infections caused by Slackia exigua: A single-center experience and literature review. / Infekcje wywolane przez Slackia exigua. Przeglad literatury i obserwacje wlasne.

Slackia exigua, originally classified as Eubacterium exiguum, is a Gram-positive, asaccharolytic, rod-shaped anaerobic bacterium. The virulence factors of S. exigua have not been accurately identified. The objective of the study is to evaluate the pathogenic potential of S. exigua by presenting the cases of infections diagnosed at our hospital laboratory. Additionally, we reviewed the literature to summarize the experience with S. exigua infections to clarify, in the light of current knowledge, the clinical picture, diagnostic, and therapeutic issues related to this anaerobic bacterium. We reported eleven severe human infections caused by S. exigua. All patients required hospitalization. Nine of the cases involved chronic infections in the stomatognathic system, in two patients, skin infections were diagnosed. As it is known, S. exigua is a component of the human microbiota; however, it can cause opportunistic infections, particularly in the case of translocation outside its natural habitat. A critical literature analysis revealed that S. exigua can be responsible for bacteremia, meningitis, tissue necrosis, periprosthetic joint infection, and osteomyelitis. Several studies have been published regarding the determination of drug susceptibility of S. exigua. The isolated strains were susceptible to most antibiotics used for the treatment of anaerobic infections. The interpretation of antimicrobial susceptibility testing for some slow-growing in vitro, infrequently causing infections anaerobic bacteria, such as S. exigua, is based on The European Committee on Antimicrobial Susceptibility Testing (EUCAST) additional guidance taking into account the determination of drug susceptibility for groups of microorganisms for which cut-off values have not been developed.

New insights into posttranslational modifications of proteins during bull sperm capacitation.

BACKGROUND: Due to the unique nature of spermatozoa, which are transcriptionally and translationally silent, the regulation of capacitation is based on the formation of posttranslational modifications of proteins (PTMs). However, the interactions between different types of PTMs during the capacitation remain unclear. Therefore, we aimed to unravel the PTM-based regulation of sperm capacitation by considering the relationship between tyrosine phosphorylation and reversible oxidative PTMs (oxPTMs), i.e., S-nitrosylation and S-glutathionylation. Since reversible oxPTMs may be closely related to peroxyredoxin (PRDX) activity, the second aim was to verify the role of PRDXs in the PTM-based regulation of capacitation. METHODS: Cryopreserved bull sperm were capacitated in vitro with or without PRDX inhibitor. Qualitative parameters of sperm and symptoms characteristic of capacitation were analyzed. Posttranslational protein modifications (S-nitrosylation, S-glutathionylation, tyrosine phosphorylation) were investigated at the cellular level (flow cytometry, fluorescence microscopy) and at the proteomic level (fluorescent gel-based proteomic approach). RESULTS: Zona-pellucida binding proteins (ACRBP, SPAM1, ZAN, ZPBP1 and IZUMO4) were particularly rich in reversible oxPTMs. Moreover, numerous flagellar proteins were associated with all analyzed types of PTMs, which indicates that the direction of posttranslational modifications was integrated. Inhibition of PRDX activity during capacitation caused an increase in S-nitrosylation and S-glutathionylation and a decrease in tyrosine phosphorylation. Inhibition of PRDXs caused GAPDHS to undergo S-glutathionylation and the GSTO2 and SOD2 enzymes to undergo denitrosylation. Moreover, PRDX inhibition caused the AKAP proteins to be dephosphorylated. CONCLUSIONS: Our research provides evidence that crosstalk occurs between tyrosine phosphorylation and reversible oxPTMs during bull sperm capacitation. This study demonstrates that capacitation triggers S-nitrosylation and S-glutathionylation (and reverse reactions) of zona-pellucida binding proteins, which may be a new important mechanism that determines the interaction between sperms and oocytes. Moreover, TCA-related and flagellar proteins, which are particularly rich in PTMs, may play a key role in sperm capacitation. We propose that the deglutathionylation of ODFs and IZUMO4 proteins is a new hallmark of bull sperm capacitation. The obtained results indicate a relationship between PRDX activity and protein phosphorylation, S-glutathionylation and S-nitrosylation. The activity of PRDXs may be crucial for maintaining redox balance and for providing proper PKA-mediated protein phosphorylation during capacitation. Video Abstract.

Phenotypic and genotypic identification of carbapenem resistance in Bacteroides fragilis clinical strains.

Bacteroides fragilis is an important etiological agent of serious infections in humans. Rapid methods, readily adaptable to use in medical laboratories, are needed to detect antibiotic resistance and decrease the likelihood of therapy failure. The aim of this study was to determine the prevalence of B. fragilis cfiA-positive isolates. The second purpose was to investigate the carbapenemase activity in B. fragilis strains by Carba NP test. In the study, 5.2% of B. fragilis isolates are phenotypically resistant to meropenem. The cfiA gene was identified in 6.1% of B. fragilis isolates. The MICs of meropenem were significantly higher in cfiA-positive strains. The presence of the cfiA gene along with the IS1186 was detected in one B. fragilis strain which was resistant to meropenem (MIC 1.5 mg/L). The Carba NP test results were positive for all the cfiA-positive strains, including those susceptible to carbapenems based on their MIC values. A review of the literature revealed that the rate of B. fragilis with the cfiA gene varies from 7.6 to 38.9% worldwide. Presented results are in line with the other European studies. Phenotypic testing with the Carba NP test, it seems to be a viable alternative for the cfiA gene detection in B. fragilis isolates. The positive result obtained is of greater clinical importance than the detection of the gene cfiA.

Ulcerated skin evaluation by electrical impedance measurements.

In the presented study, the transdermal results from the areas surrounding the ulcerated skin areas were compared with those obtained from healthy skin tissue. The analysis of electrical parameters, such as the slope of the Nyquist plot, min. IM, min. RE, min. f, Imagine part index, Phase index, Real part index, and Magnitude index were conducted. Electrical parameters have been measured in the group without lower leg ulceration and in the group with lower leg ulcers. On the basis of the statistical analysis, it was determined that these parameters may be effective in the evaluation of the skin. In fact, the skin surrounding the ulceration was characterised by different values of electrical parameters as compared with healthy skin tissue. A statistically significant difference was found in the electrical parameters obtained for the healthy leg skin and the skin surrounding the ulceration. This study was to investigate the applicability of electrical parameters in the evaluation of the skin in lower leg ulcers. The electrical parameters can be used as an effective tool in assessing the condition of the skin, both healthy and surrounding the ulcerations. The most useful parameters in assessing skin condition using electrical parameters include min. IM, min. RE, min. f, Imagine part index, Phase index, and Magnitude index.

40 Years after the Registration of Acyclovir: Do We Need New Anti-Herpetic Drugs?

Herpes simplex virus types 1 and 2 HSV1 and 2, namely varicella-zoster VZV and cytomegalovirus CMV, are among the most common pathogens worldwide. They remain in the host body for life. The course of infection with these viruses is often asymptomatic or mild and self-limiting, but in immunocompromised patients, such as solid organ or bone marrow transplant recipients, the course can be very severe or even life-threatening. Unfortunately, in the latter group, the highest percentage of infections with strains resistant to routinely used drugs is observed. On the other hand, frequent recurrences of genital herpes can be a problem even in people with normal immunity. Genital herpes also increases the risk of acquiring sexually transmitted diseases, including HIV infection and, if present in pregnant women, poses a risk to the fetus and newborn. Even more frequently than herpes simplex, congenital infections can be caused by cytomegalovirus. We present the most important anti-herpesviral agents, the mechanisms of resistance to these drugs, and the associated mutations in the viral genome. Special emphasis was placed on newly introduced drugs such as maribavir and brincidofovir. We also briefly discuss the most promising substances in preclinical testing as well as immunotherapy options and vaccines currently in use and under investigation.

Evolution of Influenza Viruses-Drug Resistance, Treatment Options, and Prospects.

Viral evolution refers to the genetic changes that a virus accumulates during its lifetime which can arise from adaptations in response to environmental changes or the immune response of the host. Influenza A virus is one of the most rapidly evolving microorganisms. Its genetic instability may lead to large changes in its biological properties, including changes in virulence, adaptation to new hosts, and even the emergence of infectious diseases with a previously unknown clinical course. Genetic variability makes it difficult to implement effective prophylactic programs, such as vaccinations, and may be responsible for resistance to antiviral drugs. The aim of the review was to describe the consequences of the variability of influenza viruses, mutations, and recombination, which allow viruses to overcome species barriers, causing epidemics and pandemics. Another consequence of influenza virus evolution is the risk of the resistance to antiviral drugs. Thus far, one class of drugs, M2 protein inhibitors, has been excluded from use because of mutations in strains isolated in many regions of the world from humans and animals. Therefore, the effectiveness of anti-influenza drugs should be continuously monitored in reference centers representing particular regions of the world as a part of epidemiological surveillance.

Pandemic resilient cities: Possibilities of repairing Polish towns and cities during COVID-19 pandemic.

Cities, due to population density and the complexity of human interactions, experience specific problems arising from their functioning in the era of the Covid-19 pandemic. Many scientists who publish their findings in contemporary literature agree on the need to shape pandemic resilient cities by making changes to the functional-spatial structure and preparing cities for rapid revitalization [regeneration] in the post-pandemic period. The main objective of the research is to identify the most important problems regarding service infrastructure deficiencies in the nine medium-sized Polish cities selected for research located in the Warsaw agglomeration, which are well connected with Warsaw. The research was based on: analysis of selected GIS spatial data, demographic data from the Polish Central Statistical Office, surveys among residents, on their needs during pandemic constraints. The focus was on three important aspects of development in the era of the pandemic: access to first-need services, access to greenery and active recreation areas, accessibility and transport efficiency problems. The research revealed changes in the way people travel and do their essential shopping, inadequate cycling infrastructure and problems with the availability of essential services close to where they live. The research programme varies, although the diagnosis and the possibility of transforming the functional-spatial structure of the cities studied as typical medium-sized cities in the impact zone of a large centre dominate. The conclusions provide examples of proposals for tactical urbanism planning solutions that could rapidly improve the quality of residence in a low-budget way. Finally, the proposals and guidelines for creating sustainable pandemic resilient cities are described. The article investigates the aspect of urban resilience related to transport, services, relaxation, while using the method of tactical urbanism as a remedy for cities' pandemic problems.

Venous leg ulcers treated with fish collagen gel in a 12-week randomized single-centre study.

Introduction: Venous ulcers are difficult to heal chronic wounds. Most challenging are the non-healing and recurrent ulcers, because of which we are seeking for therapies that stimulate and support their healing. Aim: To assess the efficacy of fish collagen and its impact on the process of healing of venous leg ulcers. Material and methods: This 12-week randomized single-centre study included a total of 97 adults with venous leg ulcer, randomized into two groups: the study group (n = 48) and the control group (n = 49). All patients received specialist medical care including skin and limb care, wound development according to the TIME scheme (tissue debridement, infection and inflammation control, moisture balance, edge) and short-stretch bandage compression (compression class II). In addition, the study group received tropocollagen gel applied to the skin around the wound. Every 2 weeks we evaluated the wound surface and temperature distribution on the shin (average temperature, median, minimum and maximum) - symmetrically on the sick and healthy limb. Results: We observed a greater number of complete ulcer healing in the study group (week 12: 29.2% vs. 22.4%; week 24: 52.1% vs. 36.7%). Faster healing time in the study group was observed in both big and small ulcers. The thermographic analysis showed statistically significant reduction in periwound inflammation among patients from the study group (p < 0.05). Statistically significant reduction in mean as well as maximum values of limb temperature occurred only in the study group (Tx: R = 0.37, p < 0.001; Tmax: R = 0.40, p < 0.001). Conclusions: Thermographic analysis showed better wound healing effects in the group using tropocollagen. Significant impact of collagen on the healing process was observed only after 8 weeks of application.

Gel-based fluorescent proteomic tools for investigating cell redox signaling. A mini-review.

The specific chemical reactivity of thiol groups makes protein cysteines susceptible to reactions with reactive oxygen species (ROS) and reactive nitrogen species (RNS) resulting in the formation of various reversible and irreversible oxidative post-translational modifications (oxPTMs). This review highlights a number of gel-based redox proteomic approaches to detect protein oxPTMs, with particular emphasis on S-nitrosylation, which we believe are currently one of the most accurate way to analyze changes in the redox status of proteins. The information collected in this review relates to the recent progress regarding methods for the enrichment and identification of redox-modified proteins, with an emphasis on fluorescent gel proteomics. Gel-based fluorescent proteomic strategies are low-cost and easy-to-use tools for investigating the thiol proteome and can provide substantial information on redox signaling.

What we actually know about the pathogenicity of Bacteroides pyogenes.

The aim of the study was to evaluate the pathogenic potential of Bacteroides pyogenes, rarely identified in clinical laboratories anaerobic bacteria. To increase the knowledge about this poorly understood anaerobic microorganism, the study also includes cases of infections described so far in the literature. Only the use of 16S rRNA sequencing and mass spectrometry technique allowed the identification of B. pyogenes from clinical specimens. We reported 13 severe human infections caused by B. pyogenes. Bacteria were cultured from the wound after biting by animals, chronic infections within the oral cavity, from patients with histologically or radiological proven osteomyelitis, surgical site infection, and from urine sample collected after a urological procedure. Most (9/13) of the patients required hospitalization. Almost 70% of them needed urgent admission via the emergency room. Two inpatients due to a life-threatening condition were admitted to the intensive care unit. Almost 50% of isolates were resistant to penicillin. All resistant to penicillin strains were isolated from skin and mucous membrane infections.

Bull Sperm Capacitation Is Accompanied by Redox Modifications of Proteins.

The ability to fertilise an egg is acquired by the mammalian sperm during the complex biochemical process called capacitation. Capacitation is accompanied by the production of reactive oxygen species (ROS), but the mechanism of redox regulation during capacitation has not been elucidated. This study aimed to verify whether capacitation coincides with reversible oxidative post-translational modifications of proteins (oxPTMs). Flow cytometry, fluorescence microscopy and Western blot analyses were used to verify the sperm capacitation process. A fluorescent gel-based redox proteomic approach allowed us to observe changes in the level of reversible oxPTMs manifested by the reduction or oxidation of susceptible cysteines in sperm proteins. Sperm capacitation was accompanied with redox modifications of 48 protein spots corresponding to 22 proteins involved in the production of ROS (SOD, DLD), playing a role in downstream redox signal transfer (GAPDHS and GST) related to the cAMP/PKA pathway (ROPN1L, SPA17), acrosome exocytosis (ACRB, sperm acrosome associated protein 9, IZUMO4), actin polymerisation (CAPZB) and hyperactivation (TUBB4B, TUB1A). The results demonstrated that sperm capacitation is accompanied by altered levels of oxPTMs of a group of redox responsive proteins, filling gaps in our knowledge concerning sperm capacitation.

Effect of 2-Cys Peroxiredoxins Inhibition on Redox Modifications of Bull Sperm Proteins.

Sperm peroxiredoxins (PRDXs) are moonlighting proteins which, in addition to their antioxidant activity, also act as redox signal transducers through PRDX-induced oxidative post-translational modifications of proteins (oxPTMs). Despite extensive knowledge on the antioxidant activity of PRDXs, the mechanisms related to PRDX-mediated oxPTMs are poorly understood. The present study aimed to investigate the effect of bull sperm 2-Cys PRDX inhibition by Conoidin A on changes in oxPTM levels under control and oxidative stress conditions. The results showed that a group of sperm mitochondrial (LDHAL6B, CS, ACO2, SDHA, ACAPM) and actin cytoskeleton proteins (CAPZB, ALDOA, CCIN) is oxidized due to the action of 2-Cys PRDXs under control conditions. In turn, under oxidative stress conditions, 2-Cys PRDX activity seems to be focused on antioxidant function protecting glycolytic, TCA pathway, and respiratory chain enzymes; chaperones; and sperm axonemal tubulins from oxidative damage. Interestingly, the inhibition of PRDX resulted in oxidation of a group of rate-limiting glycolytic proteins, which is known to trigger the switching of glucose metabolism from glycolysis to pentose phosphate pathway (PPP). The obtained results are expected to broaden the knowledge of the potential role of bull sperm 2-Cys in both redox signal transmission and antioxidant activity.

Correction to: Multiresistant Neisseria gonorrhoeae: a new threat in second decade of the XXI century.

The article "Multiresistant Neisseria gonorrhoeae: a new threat in second decade of the XXI century", written by Beata Mlynarczyk­Bonikowska, Anna Majewska, Magdalena Malejczyk, Grazyna Mlynarczyk, Slawomir Majewski was originally published electronically on the publisher's internet portal (currently SpringerLink) on December 04, 2019 without open access.

Multiresistant Neisseria gonorrhoeae: a new threat in second decade of the XXI century.

Neisseria gonorrhoeae is an etiologic agent of gonorrhoea, one of the most common sexually transmitted diseases caused by bacteria. For many years, infections caused by N. gonorrhoeae were considered to be relatively easy to treat; however, resistance has emerged successively to all therapeutic agents used in treatment of the disease, e.g., penicillin, ciprofloxacin or azithromycin. Currently, the global problem is the emergence and a threat of spread of N. gonorrhoeae strains resistant to extended-spectrum cephalosporins (ESC), such as injectable ceftriaxone and oral-used cefixime. Especially, dangerous are multi-resistant strains resistant simultaneously to ESC and azithromycin. Three strains with high-level resistance to azithromycin and resistant to ESC were first time isolated in 2018. Moreover, in 2018, the first ESBL was described in N. gonorrhoeae and that makes the threat of appearing the ESBL mechanism of resistance in N. gonorrhoeae more real, even though the strain was sensitive to ceftriaxone. Molecular typing revealed that variants resistant to ESC occurred also among strains belonging to epidemic clonal complex CC1 (genogroup G1407) distinguished in NG-MAST typing system. The G1407 genogroup, in particular the ST1407 sequence type, is currently dominant in most European countries. The presence of different mechanisms of drug resistance significantly affects clinical practice and force changes in treatment regimens and introduction of new drugs.

Head and Neck Paragangliomas-A Genetic Overview.

Pheochromocytomas (PCC) and paragangliomas (PGL) are rare neuroendocrine tumors. Head and neck paragangliomas (HNPGL) can be categorized into carotid body tumors, which are the most common, as well as jugular, tympanic, and vagal paraganglioma. A review of the current literature was conducted to consolidate knowledge concerning PGL mutations, familial occurrence, and the practical application of this information. Available scientific databases were searched using the keywords head and neck paraganglioma and genetics, and 274 articles in PubMed and 1183 in ScienceDirect were found. From these articles, those concerning genetic changes in HNPGLs were selected. The aim of this review is to describe the known genetic changes and their practical applications. We found that the etiology of the tumors in question is based on genetic changes in the form of either germinal or somatic mutations. 40% of PCC and PGL have a predisposing germline mutation (including VHL, SDHB, SDHD, RET, NF1, THEM127, MAX, SDHC, SDHA, SDHAF2, HIF2A, HRAS, KIF1B, PHD2, and FH). Approximately 25-30% of cases are due to somatic mutations, such as RET, VHL, NF1, MAX, and HIF2A. The tumors were divided into three main clusters by the Cancer Genome Atlas (TCGA); namely, the pseudohypoxia group, the Wnt signaling group, and the kinase signaling group. The review also discusses genetic syndromes, epigenetic changes, and new testing technologies such as next-generation sequencing (NGS).

The presence of antibiotic resistance genes and bft genes as well as antibiotic susceptibility testing of Bacteroides fragilis strains isolated from inpatients of the Infant Jesus Teaching Hospital, Warsaw during 2007-2012.

The purpose of this study was to assess drug susceptibility of clinical B. fragilis strains and to determine any correlation between drug resistance and the presence of specific genes. Antimicrobial susceptibility was assessed using E-tests. All isolates were analyzed with the PCR technique for the presence of antibiotic resistance genes (cepA, cfxA, cfiA, ermF, ermB, ermG, nim), insertion sequences elements (IS1186, IS1187, IS1188, IS942), and enterotoxin-encoding genes (bft). Susceptibility tests yielded the following rates of resistance to the evaluated antibiotics: penicillin G (100%), clindamycin (22.5%), cefoxitin (6.3%), amoxicillin/clavulanic acid (1.8%). All strain were susceptible to imipenem, and metronidazole. The following antibiotic resistance genes were detected in the evaluated isolates: cepA (in 96.4% of isolates), cfxA (in 12.6%), cfiA (in 1.8%), and ermF (in 25.2%). Genes ermB, ermG, and nim were not found. The presence of the cepA gene showed no correlation with the penicillin G MIC. However, we observed a high correlation between cefoxitin MIC values and the presence of gene cfxA as well as a nearly complete correlation between clindamycin MIC values and the presence of gene ermF. The presence of a bft gene was detected in 14.4% of the analyzed B. fragilis isolates; with the bft-1 allele found in 75%, bft-2 in 25%, and bft-3 in none of the isolates. Antibiotic susceptibility profiles of enterotoxin gene-positive isolates in our study did not differ from those of enterotoxin gene-negative isolates.

Orthopedic infections caused by obligatory anaerobic Gram-negative rods: report of two cases.

Anaerobic bone and joint infections are uncommon, although the number of anaerobic infections is presumably underestimated because of difficulties with isolation and identification of obligate anaerobes. This study describes two cases of complicated Bacteroides fragilis peri-implant infection of the lumbar spine, infection of the hip and osteomyelitis. Bacteria were identified with the use of a mass spectrometer, VITEK MS system. Drug susceptibility was performed with the use of E-test. The EUCAST breakpoints were used for interpretation with B. fragilis ATCC 25285 as a control. In the two described cases clinical samples were collected for microbiological examination intraoperatively and simultaneously empirical treatment was applied. B. fragilis was isolated in monoculture or in a combination with other bacteria. The treatment was continued according to the susceptibility tests. In a case one clindamycin failure was observed and clindamycin resistance of the isolate was likely due to inadequate time of therapy. Difficulties in collecting an adequate samples and culturing anaerobic bacteria cause that not all infections are properly recognized. In a successful therapy, identification and determination of the susceptibility of the pathogen are essential as well as an appropriate surgical debridement.

Novel analogs of alloferon: Synthesis, conformational studies, pro-apoptotic and antiviral activity.

In this study, we report the structure-activity relationships of novel derivatives of the insect peptide alloferon (H-His-Gly-Val-Ser-Gly-His-Gly-Gln-His-Gly-Val-His-Gly-OH). The peptide structure was modified by exchanging His at position 9 or 12 for natural or non-natural amino acids. Biological properties of these peptides were determined in antiviral in vitro test against Human Herpes Virus 1 McIntrie strain (HHV-1MC) using a Vero cell line. The peptides were also evaluated for the pro-apoptotic action in vivo on hemocytes of the Tenebrio molitor beetle. Additionally, the structural properties of alloferon analogs were examined by the circular dichroism in water and methanol. It was found that most of the evaluated peptides can reduce the HHV-1 titer in Vero cells. [Ala(9)]-alloferon exhibits the strongest antiviral activity among the analyzed compounds. However, no cytotoxic activity against Vero cell line was observed for all the studied peptides. In vivo assays with hemocytes of T. molitor showed that [Lys(9)]-, [Phg(9)]-, [Lys(12)]-, and [Phe(12)]-alloferon exhibit a twofold increase in caspases activity in comparison with the native peptide. The CD conformational studies indicate that the investigated peptides seem to prefer the unordered conformation.

IN VITRO INfIBITION OF HHV-1 REPLICATION BY INOSINE PRANOBEX AND INTERFERON-α.

Key issues in the development of novel antivirals are the emergence of resistant strains. The development of new drugs effective against herpes diseases has proven to be both difficult and time-consuming. Some alternative may be to optimize the efficacy and selectivity of existing antiviral drugs or combining them with other well known agents. Inosine pranobex exerts a direct antiviral effect as well as secondary effect to its immunomodulatory activity. We found that increasing concentrations of inosine pranobex (50-400 µg/mL) produced progressively growing inhibitory effect on HHV-1 replication, following infection of different cell lines. The combination of 1000 IU/mL IFN-α and inosine pranobex also resulted in enhanced anti-HHV activity. Immunotherapy may be beneficial for patients from whom strains resistant to currently known antiviral drugs have been isolated.

[Comparison of three multiplex real-time PCR assays for the detection of herpes simplex viruses type 1 and type 2 DNA].

INTRODUCTION: Herpes simplex viruses type 1 and type 2 (HSV-1 and HSV-2) cause widespread infection worldwide with different course and intensity. Although the disease caused by this viruses mainly concern healthy children and adults, the HSV infections are much more dangerous for people with immunodeficiencies. The aim of this work was to compare the diagnostic value of two qPCR methods for detection HSV-1/2 DNA, based on TaqMan* and HybProbes chemistry with commercial HSV-1/2 Qual Kit. METHODS: DNA from 51 clinical samples was tested for presence of HSV-1/2 sequences on LightCycler 2.0 thermocycler, using two ,,in-house" developed multiplex real-time PCR assays and commercial test using SCORPIONSTM primers. RESULTS: The results showed high specificity and sensitivity of all molecular biology tests used. Statistically, there was no significant difference in the sensitivity of real-time PCR assays when using TaqMan* and HybProbes' chemistry and when using the commercial SCORPIONSTM based method (P>0.05). CONCLUSIONS: Obtained results show that all tested methods are highly specific and can possibly be used to simultaneously detect and differentiate HSV-1/2 DNA in clinical samples. The high detection rate and short duration of qPCR assayas has great importance for immunocompromised patients where quick application of effective and safe treatment is necessary. It is also important in the event of amorphous form of the infection and the occurrence of nonspecific and generalized symptoms.