New chemotherapeutic strategies against malaria, leishmaniasis and trypanosomiases.

Due to the persistent lack of suitable vaccines, chemotherapy remains the only option for the treatment of patients infected by protozoan parasites. However, most available antiparasitic drugs have serious disadvantages, ranging from high cost and poor compliance to high toxicity and rapid induction of resistance. In recent decades basic research laboratories identified a considerable number of promising new molecules, but their development has not been pursued in depth by pharmaceutical firms because of poor prospects of economic return. The establishment of adequately funded public-private partnerships is currently reversing the trend. This review deals with new drugs against Plasmodium, Leishmania and Trypanosoma parasites, focusing on the molecules that are in the most advanced stage of development. The purpose of this article is to provide the reader with a panoramic view of the updated literature on the challenges and strategies of contemporary antiprotozoal drug research, paying the due attention to the already published reviews.

New antimicrobial frontiers.

New antimicrobials able to counteract bacterial resistance are needed to maintain the control of infectious diseases. The last 40 years have seen the systematic tailoring and refinement of previously identified antibiotics, to produce a multitude of semi-synthetic derivatives that share their mechanism of action with the original molecules. The major limit of this approach is the emergence of multi- and cross-resistant bacterial strains, favoured by the selective pressure inherent to the targeting of specific enzymes. The most promising new strategies aim to the development of molecules that, targeting essential bacterial structures instead of specific enzymatic activities, achieve infection control without enforcing a selective pressure on bacteria. This review, based on the consultation of the up-to-date literature, deals with antimicrobial peptides and some antivirulence factors.

Phenotypic and genotypic characterization of Pseudomonas aeruginosa from cystic fibrosis patients.

Pseudomonas aeruginosa accounts for about one half of all pulmonary infections of cystic fibrosis (CF) patients. In this study, we analyzed 135 P. aeruginosa strains isolated from the expectorations of 55 CF adult patients attending a CF referral center over a period of five years. We assessed the genotype of the strains by pulsed-field gel electrophoresis (PFGE) and analyzed some phenotypic characteristics, such as O serotype, enzyme and mucous production, antibiotics susceptibility, and motility. PFGE allowed the typification of 97.1% of strains, revealing the presence of nine different genomic patterns. The pattern indicated as B was the most frequent, whereas patterns H and I were the most uncommon. Serotyping failed to identify 37.8% of strains and 29 out of 55 patients harbored almost one non-typable (NT) strain. During the five years of the study, we observed a progressive reduction of O6 and O10 types, but an increase of the O1 type and of NT strains. Most strains produced protease, hemolysin, and gelatinase, and were mobile. Several patients harbored the same serotype or genotype in sequential isolates, though characterized by a different susceptibility to antimicrobials. We did not observe a relationship between bacterial genotype and phenotype. This could be due to the fact that PFGE is not sensitive enough to detect subtle genotypic differences. The epidemiological importance of the genotypic characterization of bacteria-colonizing CF subjects and the surveillance measures to be adopted in CF centers are briefly discussed.

Streptococcus agalactiae in pregnant women: phenotypic and genotypic characters.

OBJECTIVE: Streptococcus agalactiae (GBS) is considered a leading cause of neonatal sepsis. We evaluated the phenotypic and genotypic characters of 73 S. agalactiae strains isolated from different women at the 35-37 weeks of pregnancy. METHODS: Isolates were characterized by serotyping (direct agglutination) and by pulsed-field-gel-electrophoresis (PFGE). Resistance to antimicrobials (penicillin, macrolides, lincosamides, quinolones and tetracyclines) was assessed. RESULTS: All isolates were serologically typeable and ascribable to one of the six tested serotypes (Ia, Ib, II, III, IV, and V) and many strains of the same serotype were genetically heterogeneous. Strains belonging to serotypes III, V and Ia were the most prevalent and the most resistant to macrolides. CONCLUSIONS: This work reports GBS colonization rate (about 18%) and the prevalent capsular serotypes among pregnant women in Turin (Italy). Penicillin and erythromycin can be still considered the first and second choice drugs for prophylaxis and treatment of early-onset GBS infections in our district. The relevance of phenotypic and genotypic characterization of strains to monitor and control Streptococcus agalactiae infections is briefly discussed.

Pseudomonas aeruginosa and Burkholderia cenocepacia infections in patients affected by cystic fibrosis: serum resistance and antibody response.

Pseudomonas aeruginosa and Burkholderia cenocepacia are opportunistic pathogens causing important chronic pulmonary infections in patients affected by cystic fibrosis (CF). The interplay of bacterial and host factors involved in the establishment and evolution of these infections needs further clarification. We investigated the susceptibility of P. aeruginosa and B. cenocepacia derived from CF patients or from the environment to hyperimmune sera obtained from the same CF patients and evaluated the amount of specific antibodies present in these sera. Our data indicate that the bactericidal activity of human serum against these two bacteria is mostly complement-mediated, and that the mucous layer probably confers serum-resistance to B. cenocepacia. The mean amount of antibodies against P. aeruginosa was higher than that against B. cenocepacia. The contribution of these data to the assessment of the importance of the humoral immune response in CF pulmonary infections by Pseudomonas and Burkholderia is briefly discussed.

In vitro compared activity of telithromycin and azithromycin against northwest Italian isolates of Streptococcus pyogenes and Streptococcus pneumoniae with different erythromycin susceptibility.

AIMS: This study compared the in vitro activity of telithromycin with that of azithromycin against 438 Streptococcus pyogenes and 198 Streptococcus pneumoniae, isolated over the period 2005-2007 from specimens of different human origin obtained in three Piemonte Region's hospitals. METHODS AND RESULTS: The determination of antimicrobial activity was evaluated by the microdilution broth method and the erythromycin-resistant (Ery-R) phenotypes by the triple-disc test. Exactly 78.8% of S. pyogenes and 69.2% of S. pneumoniae were erythromycin-susceptible (Ery-S). Concerning S. pyogenes, telithromycin was active against M and inducible MLS(B), subtype-C, phenotypes but not against constitutive MLS(B) strains. Telithromycin acted well against all S. pneumoniae, irrespective of their mechanism of macrolide-resistance. On the contrary, the Ery-R isolates, both S. pyogenes and S. pneumoniae, were resistant to azithromycin. CONCLUSIONS: Our results indicate that macrolide resistance in streptococci still persist in northwest Italy (21.2% of S. pyogenes and 308% of S. pneumoniae) and that telithromycin is confirmed as being extremely active even against recent clinical Ery-R streptococcal isolates. SIGNIFICANCE AND IMPACT OF THE STUDY: The present study emphasizes that an active surveillance of the phenotype distribution and antibacterial resistance in streptococci is essential in guiding the effective use of empirical treatment option for streptococcal infections, also at regional level.

Evaluation of different compounds as quorum sensing inhibitors in Pseudomonas aeruginosa.

Quorum sensing (QS) controls systems affecting the pathogenicity of many microorganisms; its interruption has an anti-pathogenic effect and can be used in the treatment of bacterial infections. In this study we evaluated QS regulation by Pseudomonas aeruginosa strains and QS inhibition (QSI) by different compounds. The inhibitory activity of 3 macrolide and 3 lincosamide drugs, resveratrol, garlic extract and N-acetylcysteine was tested on 4 P. aeruginosa strains isolated from cystic fibrosis (CF) patients using Chromobacterium violaceum ATCC 12472 as biomonitor. One P. aeruginosa strain, lincomycin and N-acetylcysteine did not show QSI, contrary to other compounds and P. aeruginosa strains. These results indicate that QSI evaluation should be taken into account in the design of new therapeutic strategies to treat P. aeruginosa infections, especially in patients infected by antibiotic-resistant bacteria.

Burkholderia respiratory tract infections in Italian patients with cystic fibrosis: molecular characterization.

OBJECTIVES: To characterize by molecular techniques Burkholderia strains responsible for respiratory tract infections in cystic fibrosis (CF) patients (children and adults), to assign the Burkholderia cepacia complex (Bcc) isolates to a genomovar and to assess the presence of cblA and esmR genes in bacteria. Unique or sequential Burkholderia isolates (n=48) that had been collected from eight CF children and 17 adults over several (4-6) years were investigated; moreover 11 reference strains were analyzed. METHODS: The microorganisms were identified by using biochemical methods, genotyped by pulse field gel electrophoresis (PFGE) and random-amplified polymorphic DNA fingerprinting-PCR (RAPD-PCR), and assessed by PCR assays for the genomovar and cblA and esmR genes of Bcc. RESULTS: Among isolates 70.8% were identified as Bcc genomovar III-A; one child was infected by Burkholderia ambifaria and four adults were colonized with Burkholderia gladioli. The cblA gene was not detected in any of the isolates, while the esmR gene was detected in the 52.1% of the strains, all belonging to genomovar III-A. CONCLUSION: Molecular analysis of strains revealed in CF patients a colonization with a persistent Burkholderia flora with strains of one genotype. The prevalence of Bcc of genomovar III-A in the two categories of patients and of B. gladioli in four adults demonstrated that transmission may have occurred between subjects. Moreover the B. ambifaria infection demonstrated in a child may be environmentally derived.

Transmission of Burkholderia cepacia complex: evidence for new epidemic clones infecting cystic fibrosis patients in Italy.

To analyze national prevalence, genomovar distribution, and epidemiology of the Burkholderia cepacia complex in Italy, 225 putative B. cepacia complex isolates were obtained from 225 cystic fibrosis (CF) patients attending 18 CF centers. The genomovar status of these isolates was determined by a polyphasic approach, which included whole-cell protein electrophoresis and recA restriction fragment length polymorphism (RFLP) analysis. Two approaches were used to genotype B. cepacia complex isolates: BOX-PCR fingerprinting and pulsed-field gel electrophoresis (PFGE) of genomic macrorestriction fragments. A total of 208 (92%) of 225 isolates belonged to the B. cepacia complex, with Burkholderia cenocepacia as the most prevalent species (61.1%). Clones delineated by PFGE were predominantly linked to a single center; in contrast, BOX-PCR clones were composed of isolates collected either from the same center or from different CF centers and comprised multiple PFGE clusters. Three BOX-PCR clones appeared of special interest. One clone was composed of 17 B. cenocepacia isolates belonging to recA RFLP type H. These isolates were collected from six centers and represented three PFGE clusters. The presence of insertion sequence IS 1363 in all isolates and the comparison with PHDC reference isolates identified this clone as PHDC, an epidemic clone prominent in North American CF patients. The second clone included 22 isolates from eight centers and belonged to recA RFLP type AT. The genomovar status of strains with the latter RFLP type is not known. Most of these isolates belonged to four different PFGE clusters. Finally, a third clone comprised nine B. pyrrocinia isolates belonging to recA RFLP type Se 13. They represented three PFGE clusters and were collected in three CF centers.

Interaction of Bartonella henselae with the murine macrophage cell line J774: infection and proinflammatory response.

Bartonella henselae is the causative agent of cat scratch disease (CSD), a self-limiting condition characterized by a subacute regional lymphadenopathy that may develop into disseminated bartonellosis in immunocompromised subjects. Mice experimentally infected with B. henselae display typical liver and spleen granulomas rich in T cells and macrophages. So far there are no data on the interaction between bartonellae and macrophages. In order to clarify this topic, we investigated the interaction of B. henselae with J774, a mouse macrophage cell line. Analysis of bacterial uptake by functional assays and transmission electron microscopy indicates that bartonellae can enter and survive inside J774. Entry occurred within 30 min postinfection and reached a plateau at 160 min. Infection of J774 was followed by a dose-dependent release of the proinflammatory cytokines tumor necrosis factor alpha, interleukin 1beta (IL-1beta), and IL-6. Bartonellae persisted intracellularly without loss of viability for at least 8 h, and their number slightly decreased 24 h postinfection. Gamma interferon (IFN-gamma) treatment of J774 significantly decreased the number of recoverable bacteria at 8 and 24 h. This enhancement of macrophage bactericidal activity was associated with nitric oxide (NO) release and was prevented by the addition of the competitive inhibitor of NO synthesis N(G)-monomethyl L-arginine. These findings suggest that IFN-gamma-mediated activation of macrophages may be important for the clearing of B. henselae infection and that anti-B. henselae microbicidal activity of IFN-gamma-activated macrophages is mediated to a large extent by NO production.

Genetic diversity of Pneumocystis carinii isolated from human immunodeficiency virus-positive patients in Turin, Italy.

By DNA sequence analysis we identified two new strain types and five novel sporadic variations among 25 isolates of Pneumocystis carinii f. sp. hominis obtained from 19 human immunodeficiency virus-positive patients. Of these, 13 were infected with a single strain and 6 were coinfected. Fifteen different combination types were identified among the 18 strains for which complete molecular typing was accomplished.

In vitro activity of propolis against Streptococcus pyogenes.

Propolis, a multifunctional substance used by bees to maintain the safety of their hives, is popular for its therapeutic potential against some micro-organisms. Ethanolic extracts of two propolis specimens, collected from different areas within a region in the north-west of Italy, were examined to evaluate their antimicrobial activity against 46 Streptococcus pyogenes strains. By both agar dilution and agar diffusion methods, the minimal inhibitory concentration (MIC) and minimal bactericidal concentration (MBC) were

Macrolide resistance in group A streptococci.

Two hundred and twenty one Streptococcus pyogenes isolates collected from throat swabs of untreated children with uncomplicated pharyngotonsillitis living in two centres situated in the north of Italy were tested to evaluate their macrolide resistance phenotype. Isolates were also typed for T protein and assayed for opacity factor (OF) and protease production. Resistance to macrolides was found to be similar in the two centres. Fifty-one point two per cent of Torino strains and 43.5% of Pinerolo strains were not inhibited by erythromycin. Resistant strains belonged to one of three phenotypes: CR, constitutive resistance (37.9 and 42.5% in Torino and Pinerolo, respectively); IR, inducible resistance (40.9 and 17. 5%); NR, new resistance phenotype (21.2 and 40%). All the resistant and some of the susceptible strains were analysed by pulsed-field gel electrophoresis and genomic patterns were defined on the basis of band size and number. Five DNA profiles were found among erythromycin-resistant strains: three patterns characterized the NR resistance phenotype and one each the IR and CR phenotypes. The distribution of resistant strains according to their genomic patterns appears to be related to the resistance phenotype and only in some cases to the T serotype of bacteria. We conclude that the S. pyogenes strains analysed are genetically heterogeneous and therefore the high rate of erythromycin resistance observed is not caused by the spread of a single clone nor is it related to a particular serotype.

Streptococcus pyogenes collected in Torino (northwest Italy) between 1983 and 1998: survey of macrolide resistance and trend of genotype by RAPD.

We surveyed macrolide resistance in 1,086 isolates of Streptococcus pyogenes, collected between 1983 and 1998, from throat swabs of children with untreated pharyngotonsillitis living in Torino (northwest Italy). In 1983 and 1985, the frequency of erythromycin resistance was 10%, and from 1990 to 1992 it was 4%. However, it rose to 16.6% in 1994 and reached 51% in 1996 before decreasing to 38.5% in 1998. Characterization of the phenotype of resistant isolates revealed the prevalence of constitutive resistance (CR) in 1996, whereas the M phenotype, characterized by resistance to 14- and 15-membered macrolides with susceptibility to clindamycin and streptogramin B, prevailed in 1998. Moreover, in 1997 we observed an increase in the frequency of autoagglutinating bacteria and, in 1998, of OF-negative S. pyogenes. Meanwhile, penicillin tolerance, assessed in the isolates collected from 1990 to 1996, decreased and disappeared. Random amplification of polymorphic DNA (RAPD) was used to obtain the genomic profile of 32 S. pyogenes strains. Four main DNA profiles were demonstrated, generally related to the macrolide-resistance phenotype and for the major part to the T serotype. These results indicate that RAPD is reliable as a first screening method in the epidemiological characterization of resistant S. pyogenes.

Immunological investigation of malaria and new perspectives in paleopathological studies.

We applied a paleoimmunological investigation, using an immunoenzymatic assay revealing trophozoite derived Plasmodium falciparum histidine rich protein-2 antigen (PfHRP-2). The investigation was carried out on skin, muscle and bone samples. We examined predynastic egyptian mummies (3200 B.C.) from Gebelen, belonging to the Marro's Collection of the Anthropological and Ethnographic Museum of Turin, to assay the presence of malaria. The results obtained suggest an incidence of malaria of about 40% in the mummies of Gebelen group. Data are compatible with other observations effected on populations living in similar ecological conditions of malarial areas.

Interleukin-15 activates proinflammatory and antimicrobial functions in polymorphonuclear cells.

Interleukin-15 (IL-15) is a recently discovered cytokine produced by a wide range of different cell types including fibroblasts, keratinocytes, endothelial cells, and macrophages in response to lipopolysaccharide or microbial infection. This suggests that IL-15 may play a crucial role in the activation of phagocytic cells against pathogens. We studied polymorphonuclear leukocyte (PMN) activation by IL-15, evaluated as enhancement of PMN anti-Candida activity as well as IL-8 production, following stimulation with the cytokine. The PMN response to IL-15 depends on binding to the IL-15 receptor. Our experiments show that binding of a biotinylated human IL-15-immunoglobulin G2b IgG2b fusion protein was competed by the addition of human recombinant IL-15 (rIL-15) or of human rIL-2, suggesting that IL-15 binding to PMN might involve the IL-2Rbeta and IL-2Rgamma chains, which have been shown to be constitutively expressed by PMN. In addition, we show by reverse transcription-PCR and by flow cytometry with a specific anti-IL-15Ralpha chain monoclonal antibody that PMN express the IL-15Ralpha chain at the mRNA and protein levels. Incubation with IL-15 activated PMN to secrete the chemotactic factor IL-8, and the amount secreted was increased by costimulation with heat-inactivated Candida albicans. In addition, IL-15 primed the metabolic burst of PMN in response to formyl-methionyl-leucyl-phenylalanine but was not sufficient to trigger the respiratory burst or to increase the production of superoxide in PMN exposed to C. albicans. IL-15 also increased the ability of PMN to phagocytose heat-killed C. albicans organisms in a dose-dependent manner, without opsonization by antibodies or complement-derived products. In the same concentration range, IL-15 was as effective as gamma interferon (IFN-gamma) and IL-2 in increasing the C. albicans growth-inhibitory activity of PMN. Taken together, these results suggest that IL-15 is a potent stimulant of both proinflammatory and antifungal activities of PMN, activating several antimicrobial functions of PMN involved in the cellular response against C. albicans.

Detection of Pneumocystis carinii by DNA amplification in human immunodeficiency virus-positive patients.

The opportunistic pathogen Pneumocystis carinii (PC) is a frequent cause of a life-threatening pneumonia in human immunodeficiency virus (HIV)-infected individuals and in other immunocompromised hosts. Specimens obtained from 128 bronchoalveolar lavage (BAL) fluid samples from 123 HIV-positive patients with pulmonary disease and undergoing a diagnostic bronchoscopy were evaluated to detect this organism. We have developed a rapid DNA extraction procedure for nested polymerase chain reaction (PCR) using two sets of primers (pAZ102-E, pAZ102-H and P1 = 5'-CTAGGATATAGCTGGTTTTC-3' and P2 = 5'-TCGACTATCTAGCTTATCGC-3'). The results were compared using cytological techniques (direct wet mount, Giemsa, toluidine blue O) and related to the clinical follow-up of patients. The nested PCR had a 91% sensitivity and a 93% specificity. The effect of chemoprophylaxis and the evaluation of the follow-up of patients are discussed. Nested PCR may represent an important additional tool, along with current cytological methods, for the detection of P. carinii; however, at present it cannot replace routine microbiological methods more simple and less expensive.

[Recurrent urinary tract infections. Biological suppositions and clinical treatment with thymopentin]. / Infezioni delle vie urinarie recidivanti. Presupposti biologici e risultati clinici del trattamento con timopentina.

BACKGROUND: Urinary tract infections (UTI) are a common usual pathological event. They relapse often due to the periurethral colonization of microorganisms from the intestinal bacterial flora. They also constitute an important and considerable social and clinical problem. The absence of inducing organic conditions or an infective focus at the base of the pathogenetic mechanism suggests the existence of alterations of the immune response of the subject. MATERIALS AND METHODS: In this work we wanted to verify if, in those subjects with relapsing UTI (more than four events every year) cure with "biological" response modifiers, particularly "thymopentin", meaningfully reduced the number of events. RESULTS: The results obtained confirm that for those cases in which the chemo-antibiotic therapy did not have the expected results, it is rational to support it with an immune-modulating drug (thymopentin). In fact the post-therapy reduction of UTI observed during two years of follow-up is statistically significant when compared to the average of UTI before therapy. CONCLUSIONS: The "cost-benefits" analysis should prove to be a saving in favour of the use of thymopentin, taking into consideration the reduction of chemo-antibiotics consumption and the lower number of working hours lost every year.

Evaluation of the immune response in visceral leishmaniasis.

Detection of parasites in culture or by microscopy is still necessary to make diagnosis of visceral leishmaniasis (VL). Serological methods still need assessment, as they are quick but not very sensitive, especially in immunosuppressed subjects. This paper compares the results obtained with three serological methods (indirect immunofluorescence test (IFAT), direct agglutination test (DAT), and enzyme-linked immunosorbent assay (ELISA) and the specific cell-mediated immune response, evaluated as proliferation and IFN-gamma production by peripheral blood lymphocytes (PBL) following stimulation with heat-killed L. infantum promastigotes. PBL and sera were obtained from 10 healthy donors, 3 VL patients in acute phase, and 3 patients recovering after two glucantim treatment courses. No false positive results were observed with the serological methods. IFAT can be considered the most sensitive and best suited for follow-up, as it allowed a good discrimination between the acute and remission phase. DAT did not discriminate between healthy donors and remission-phase patients, whereas ELISA is unsuited for follow-up, as it did not show any significant difference between remission- and acute-phase patients. Assessment of the cellular response is not recommended for making a diagnosis, because false positive results are frequent. However, a strong cellular response in a patient stands for a successful treatment. IFN-gamma titration is preferable to the proliferation test, because it gives earlier results and does not require the use of radioactive isotopes.