Anna Majewska

A comparison of Api 20A vs MALDI-TOF MS for routine identification of clinically significant anaerobic bacterial strains to the species level.

Adequate identification of anaerobic bacteria still presents a challenge for laboratories conducting microbiological diagnostics. The aim of this study was to compare the use of Api 20A and MALDI-TOF MS techniques for identification of obligate anaerobes. The results indicate that MALDI-TOF MS ensures a rapid and accurate identification of the species isolated from patients.

New Alloferon Analogues: Synthesis and Antiviral Properties

We have extended our study on structure/activity relationship studies of insect peptide alloferon (H‐His‐Gly‐Val‐Ser‐Gly‐His‐Gly‐Gln‐His‐Gly‐Val‐His‐Gly‐OH) by evaluating the antiviral effects of new alloferon analogues. We synthesized 18 alloferon analogues: 12 peptides with sequences shortened from N‐ or C‐terminus and 6 N‐terminally modified analogues H‐X1‐Gly‐Val‐Ser‐Gly‐His‐Gly‐Gln‐His‐Gly‐Val‐His‐Gly‐OH, where X1 = Phe (13), Tyr (14), Trp (15), Phg (16), Phe(p‐Cl) (17), and Phe(p‐OMe) (18). We found that most of the evaluated peptides inhibit the replication of Human Herpesviruses or Coxsackievirus B2 in Vero, HEp‐2 and LLC‐MK2 cells. Our results indicate that the compound [3‐13]‐alloferon (1) exhibits the strongest antiviral activity (IC50 = 38 μm) among the analyzed compound. Moreover, no cytotoxic activity against the investigated cell lines was observed for all studied peptides at concentration 165 μm or higher.

Specific Character of Anaerobic Bacterial Infections in Patients Treated in Transplantation Wards at One of the Clinical Hospitals in Warsaw

Immunocompromised patients and patients undergoing invasive procedures are predisposed to bacterial infections, due to the possibility of micro-organism translocation from their physiological habitat. Infectious complications may occur both in the early and late post-transplantation periods. The purpose of this study was to evaluate the proportion as well as susceptibility profiles of obligatory anaerobes in the etiology of infections in patients hospitalized at transplantation wards of a large clinical hospital in Warsaw. A total of 104 strains of obligatory anaerobes derived from patients hospitalized in two transplantation clinics at a clinical hospital in Warsaw were evaluated. The strains were isolated from 87 clinical samples collected from 84 patients of two transplantation wards between 2007 and 2012. A total of 104 obligatory anaerobic bacterial strains were isolated and identified, with Gram-positive and Gram-negative bacteria constituting 60.6% and 39.4% of the isolates, respectively. Almost exclusively non-spore-forming anaerobes were detected in evaluated samples. The present study showed all isolated Gram-positive bacteria to be susceptible to ß-lactam antibiotics. Metronidazole-resistant bacteria were found among the genera Propionibacterium and Actinomyces. All Gram-negative rods were susceptible to imipenem and metronidazole. Among them, Bacteroides spp. and Parabacteroides distasonis showed resistance to penicillin G (100%). Because of their pathogenicity and altered antibiotic susceptibility profiles, the bacteria of the genera Bacteroides and Parabacteroides are of greatest clinical importance. Approximately 25% of isolates exhibit also resistance to clindamycin. Because of the growing rates of clindamycin resistance, the role of metronidazole in the treatment of Bacteroides spp. is of increasing importance.

Blood Infections in Patients Treated at Transplantation Wards of a Clinical Hospital in Warsaw

Establishment of the etiology in blood infection is always advisable. The purpose of this study was to evaluate the proportion of different bacterial species, including aerobic and anaerobic bacteria in blood cultures of patients hospitalized in transplantation wards of a large clinical hospital between 2010 and 2012. A total of 1994 blood samples from patients who were treated at one of two transplantation wards of a large hospital in Warsaw were analyzed using an automated blood culture system, BacT/ALERT (bioMerieux, France). The 306 bacterial strains were obtained from the examined samples. The highest proportion were bacteria from the family Enterobacteriaceae (112 strains; 36.6%) with Escherichia coli (61 strains), Klebsiella pneumoniae (30 strains), and Enterobacter cloacae (10 strains) most commonly isolated. The non-fermenting bacilli constituted 21.6% (66 strains), with most common Stenotrophomonas maltophilia (31 strains), Pseudomonas aeruginosa (14 strains), Achromobacter spp. (12 strains), and Acinetobacter baumannii (3 strains). Most frequent Gram-positive bacteria were staphylococci (25.2%). Of 77 staphylococcal strains, 56 were coagulase-negative staphylococci and 21 Staphylococcus aureus. Other Gram-positive bacteria included enterococci (14 strains) and Streptococcus pneumoniae (1 strain). Obligatory anaerobic bacteria were represented by 19 strains (6.2% of total isolates). Among all Enterobacteriaceae, 49 isolates (43.7%) produced extended-spectrum ß-lactamases (ESBLs). Resistance to methicillin was detected in 62% of S aureus isolates and in 46% of coagulase-negative staphylococci. Of 14 enterococci cultured from blood samples, 2 strains (14.3%) were resistant to vancomycin. Both were Enterococcus faecium. Resistant strains of Gram-negative and Gram-positive bacteria are significant problems for patients in the transplantation ward.

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.

Inhibition of adenovirus multiplication by inosine pranobex and interferon α in vitro

There are no specific antivirals designed for adenoviral infections. Due to many cases of adenovirus infections worldwide, epidemic nature of some types of adenoviruses, and growing number of patients with severe adenoviral infections resulting from dysfunction the immune system, the need for searching an effective and safe therapy is increasing. Inosine pranobex exerts antiviral effects which are both direct and secondary to immunomodulatory activity. In the present study we evaluated in vitro effect of inosine pranobex and interferon α (IFN-α) on replication of HAdV-2 and HAdV-5. The effectiveness of inosine pranobex under these conditions has not been previously reported. In conducted study we reported that inosine pranobex reduced the titer of infectious HAdV-2 and HAdV-5 in vitro. Higher concentrations of IP strongly inhibited multiplication of viruses. Combination of inosine pranobex and IFN-α display higher efficacy than either treatment alone and suggest that both agents may increase therapeutic effectiveness without augmenting toxic effects. Combination index calculations showed that inosine pranobex and INF-α synergistically inhibit HAdV-2 and HAdV-5 titers in A549 cells.

Antiviral Medication in Sexually Transmitted Diseases. Part II: HIV

This is a second part of a review under a main title Antiviral medication in sexually transmitted diseases. In the part we published in Mini Rev Med Chem. 2013,13(13):1837-45, we have described mechanisms of action and mechanism of resistance to antiviral agents used in genital herpes and genital HPV infection. The Part II review focuses on therapeutic options in HIV infection. In 1987, 6 years after the recognition of AIDS, the FDA approved the first drug against HIV--zidovudine. Since then a lot of antiretroviral drugs are available. The most effective treatment for HIV is highly active antiretroviral therapy--a combination of several antiretroviral medicines that cause a reduction of HIV blood concentration and often results in substantial recovery of impaired immunologic function. At present, there are over 20 drugs licensed and used for the treatment of HIV/AIDS, and these drugs are divided into one of six classes. Investigational agents include GS-7340, the prodrug of tenofovir and BMS-663068--the first in a novel class of drugs that blocks the binding of the HIV gp120 to the CD4 receptor.

Participation of Strictly Anerobic Bacteria in Infections among Hospitalized Transplant Patients in a Clinical Hospital in Warsaw

BACKGROUND Many strictly anerobic bacteria are a part of the human commensal microflora. Especially multitudinously they inhabit the skin, mucous membranes, gastrointestinal tract, respiratory, and genital tracts. Infections with these bacteria may occur after escape of the bacteria from their natural habitat. There are often mixed anerobic and aerobic infections. After rupture of the gastrointestinal tract or surgery, the organisms can cause significant pathology including abscesses and bacteremia. OBJECTIVE The aim of this study was to estimate the prevalence of gram-negative strictly anerobic bacteria isolated from different samples collected from patients on transplant wards. MATERIALS AND METHODS Samples from patients with suspected infections. Underwent isolation and identification of microorganisms under anerobic conditions using standard laboratory methods. RESULTS Gram-negative rods were observed in 46% of clinical samples, most frequently Bacteroides genus as well as Fusobacterium necrophorum/F.nucleatum and F.mortiferum (14%). Most species of the genus Bacteroides were accompanied by Escherichia coli, less frequently with other aerobic gram-negative rods or gram-positive cocci. CONCLUSION Gram-negative bacteria were frequently isolated in the samples. Because they are the part of the normal flora, this observation indicated the endogenous nature of infections resulting from bacterial translocation out of their natural habitat.

Infections due to alphaherpesviruses in early post-transplant period after allogeneic haematopoietic stem cell transplantation: Results of a 5-year survey

BACKGROUND Infections caused by human α-herpesviruses usually have a benign course with recurrencies. However, they may become dangerous in immunocompromised hosts. In this case, molecular methods constitute a reliable diagnostic tool enabling rapid assessment of the efficacy of antiviral treatment strategies. OBJECTIVES We estimated the frequency of alphaherpesviral DNAemia and the viral load during early post-transplantation period after alloHSCT; we also analyzed association of the DNAemia and chosen parameters of the patients. STUDY DESIGN A cohort of 190 alloHSCT recipients from two hospitals in Warsaw, Poland, was examined weekly during 100-day early post-transplantation period using quantitative real time PCR assays. A total of 2475 sera samples were evaluated for the presence of α-herpesviral DNA in patients, of whom 117 (62%) received unrelated grafts, while the remaining 73 (38%) received grafts from sibling donors. All patients received standard antiviral prophylaxis with acyclovir. In the examined group, anti-HSV-1, anti-HSV-2 and anti-VZV IgGs were examined prior to transplantation, RESULTS: Within the study period, DNA of α-herpesviruses was detected in 44 patients (23.2%). Most patients tested positive for HSV-1 DNA (43 patients, 22.6%), single patient for HSV-2, and no patient positive for VZV. Clinical symptoms such as pneumonia, skin changes, elevated levels of aminotransferases were observed in five patients, four of these patients presented symptoms of GvHD at the same time. (2,6%). Statistics shows that GvHD (P<0.001) and matched unrelated donor as a source of HSCT (P=0.048) are associated with the development of HSV-1 DNAemia. CONCLUSIONS Although our data demonstrate frequent reactivation of HSV-1 in the early post-transplant period, the rate of symptomatic infections was low. We did not find association between HSV-1 viremia and mortality, but significant association with GvHD and donor source was observed.

Difficulties in identifying the bacterial species from the genus Clostridium in a case of injury-related osteitis

Most Clostridium species are part of saprophytic microflora in humans and animals; however, some are well-known human pathogens. We presented the challenges in identifying the Clostridium species isolated from a patient with an infected open dislocation of the proximal interphalangeal joint of the fourth digit of the right hand. The clinical materials were intraoperative samples collected from a patient diagnosed with an injury-related infection, with soft tissue loss and tendon sheath involvement. The available biochemical, molecular, and genetic techniques were used in identifying the isolated bacteria. The isolated bacterium was shown to have low biochemical activity; hence, it was not definitively identified via biochemical tests Api 20A or Rapid 32A. Vitek 2 and mass spectrometry methods were equally inconclusive. Clostridium tetani infection was strongly suspected based on the bacterium’s morphology and the appearance of its colonies on solid media. It was only via the 16S rRNA sequencing method, which is non-routine and unavailable in most clinical laboratories, that this pathogen was excluded. Despite appropriate pre-laboratory procedures, which are critical for obtaining reliable test results, the routine methods of anaerobic bacterium identification are not always useful in diagnostics. Diagnostic difficulties occur in the case of environment-derived bacteria of low or not fully understood biological activity, which are absent from databases of automatic bacterial identification systems.