Giuseppe Miragliotta

High Dose, Extended-Interval Colistin Administration in Critically Ill Patients: Is this the Right Dosing Strategy? A Preliminary Study

In critically ill patients with otherwise untreatable nosocomial infection due to gram-negative bacteria susceptible only to colistin, a high-dose, extended-interval colistin dosing regimen is, according to the pharmacokinetic/pharmacodynamic behavior of the drug, associated with low renal toxicity and high efficacy.

Prevalence and genotypes identification of human papillomavirus infection in a population of South Italy

BACKGROUND A limited number of human papillomavirus (HPV) types account for the majority of invasive cervical cancer cases. OBJECTIVES To assess, in a southern Italian region, where HPV infection had not yet been investigated, the prevalence of type-specific HPV infection. STUDY DESIGN Multiplex PCR was used to test cervical specimens from 871 asymptomatic women. RESULTS The HPV infection rate was 23.1%, with the highest prevalence being observed in women aged 20-30 years (32.6%). Type 16 was the most frequent HPV type detected either in mono-infected (39.8%) or in multi-infected (46.3%) women. CONCLUSIONS The HPV infection rate was higher than reported from other Italian areas. Our results further emphasise the importance of vaccinations to immunize females before they acquire HPV infection.

Detection of Mycobacterium tuberculosis DNA in blood of patients with acute pulmonary tuberculosis by polymerase chain reaction and non-isotopic hybridisation assay

The detection of Mycobacterium tuberculosis DNA in peripheral blood mononuclear cells (PBMC) by PCR and non-isotopic hybridisation assay was evaluated for the laboratory diagnosis of pulmonary M. tuberculosis infection. The PCR technique was based on the presence of IS6110, a DNA sequence specific for M. tuberculosis, and performed on PBMC from 30 patients belonging to the fifth group of the American Thoracic Society (ATS) classification of tuberculosis. The identification of amplification products was confirmed after electrophoresis by hybridisation with a non-isotopic probe in a DNA enzyme immunoassay (DEIA). Of the 30 blood samples studied by the PCR-DEIA technique, 26 gave positive results and four gave negative results. Blood samples from 30 subjects in a control group were negative by this technique. The data suggest that PCR-DEIA of blood may provide a sensitive, specific and useful means of diagnosing mycobacterial infection.

Polymerase chain reaction detection of Bartonella henselae bacteraemia in an immunocompetent child with cat-scratch disease

Abstract A case of Bartonella henselae bacteraemia is reported in an immunocompetent 8-year-old boy with cat-scratch disease. Serology to B. henselae, diagnosed by polymerase chain reaction, was positive. DNA was extracted from peripheral whole blood and amplified with specific primers targeting the htrA gene of B. henselae. A non-isotopic hybridization assay with a species-specific oligonucleotide probe was used to detect the amplified product. Conclusion The polymerase chain reaction can be used for the rapid laboratory diagnosis of bacteraemia in cat-scratch disease.

Detection of Bartonella henselae and Afipia felis DNA by polymerase chain reaction in specimens from patients with cat scratch disease.

Abstract Polymerase chain reaction (PCR) amplification and colorimetric identification of amplicons were performed to detect Bartonella henselae and Afipia felis DNA in specimens from patients who were clinically and histologically suspected of having cat scratch disease. PCR products were revealed using 2% ethidium bromide agarose-gel electrophoresis and identified with specific probes in a commercial colorimetric hybridization assay (DEIA) (GEN-ETI-K; DiaSorin, Italy). Six paraffin-embedded lymph node biopsies from 18 patients as well as 18 samples of peripheral whole blood and 18 sera were investigated. Bartonella henselae DNA was recovered from the whole blood of four patients, and Bartonella henselae and Afipia felis DNA were detected in one patients lymph node biopsy. This study suggests that PCR-DEIA is sufficiently sensitive to be considered feasible for the molecular diagnosis of cat scratch disease.

Prevalence of antibodies to Bartonella henselae in patients with suspected cat scratch disease (CSD) in Italy

Cat scratch disease (CSD) is a relatively new diagnosed illness with clinical signs of self-limiting regional lymphadenopathy accompanied by symptoms of fever and malaise, to encephalopathy and neuropathy, occurring after a cat scratch or flea bite. Bartonella henselae is now accepted as the etiologic agent of CSD. From January 1994 to September 1998, 412 patients were evaluated for suspect CSD in Italy. Sera were tested for antibodies to B. henselae by a commercially available indirect immunofluorescent assay (IFA), based on B. henselae-infected Vero-cells as the antigen substrate. Of the 412 patients, 26 (6.3%) were considered positive having titers of immunoglobulin G (IgG) to B. henselae of 64 or higher. In these patients CSD was indeed confirmed by either histopathologic examination of lymph nodes biopsy or fourfold raise in antibody titers. Nevertheless, sera were tested by IFA for Afipia felis and one showed a double reactivity to B. henselae and A. felis. Finally, three sera, negative to B. henselae serology, were positive to A. felis. Three hundred and eighty-six patients received alternative diagnoses. One hundred and twenty-five serum samples from control subjects were negative by IFA for either B. henselae or A. felis. Moreover, a cross-reactivity with sera from patients affected by other diseases was not observed. Our study shows that the ascertained cases of CSD are etiologically determined by B. henselae, IFA assay is confirmed as a useful tool in the laboratory diagnosis and, over a 5 years period of study, the incidence of CSD in Italy has been low.

Preliminary evidence of endotoxic activity of Bilophila wadsworthia

In this study we describe two properties of the Gram-negative bacterium Bilophila wadsworthia, namely the ability to clot Limulus lysate and the capacity to induce the production of tissue factor-like procoagulant activity by human mononuclear cells in vitro. Although exhibited at a lower degree when compared with those of typical Gram-negative bacteria or Gram-negative endotoxin those activities may account in part for Bilophilas pathogenicity. The capacity indeed to induce fibrin formation through the interaction with mononuclear cells suggests one mechanism by which the microorganism might cause abscess formation in the host. Moreover, since this activity is dependent on the number of Bilophila interacting with mononuclear cells, we hypothesize that this biological activity is closely influenced by growth environment.

Rapid and sensitive detection of blaKPC gene in clinical isolates of Klebsiella pneumoniae by a molecular real-time assay

BackgroundThe aim of this study was the rapid identification of blaKPC gene in 38 Klebsiella pneumoniae clinical isolates with reduced susceptibility to carbapenems. The modified Hodge Test (MHT) was carried out to phenotypically determine whether resistance to carbapenems was mediated by a carbapenemase. The detection of the blaKPC gene was performed by real-time acid nucleic sequence-based amplification (NASBA™™), specifically designed for the detection of KPC RNA target.ResultsThirty-two/38 isolates evaluated by MHT showed the production of carbapenemases, while all the strains exhibited the production of KPC by inhibition test with phenylboronic acid (the combined disk test with IPM/IPM plus phenylboronic acid). The detection of blaKPC gene by Nuclisens EasyQ KPC yielded positive results in 38/38 (100%) strains. The presence of blaKPC gene was confirmed in all K. pneumoniae isolates when tested by the gold standard PCR assay.ConclusionsIn consideration of the serious challenge represented by infections due to K. pneumoniae it appears necessary the rapid identification of carbapenemases in clinical settings as it is made possible by the use of NASBA™ assay.

Platelet aggregation and stimulation of leucocyte procoagulant activity by rickettsial lipopolysaccharides in rabbits and in man

The effects in vitro of 4 purified lipopolysaccharide (LPS) preparations from Rickettsiae on platelets and leucocytes were studied in rabbits and in man. All LPS induced aggregation in rabbit platelet-rich plasma but to differing degrees. This activity was abolished by inactivation of complement. None of the preparations induced aggregation of human platelets. Both rabbit and human leucocytes, when incubated with each of the rickettsial LPS preparations, generated a potent procoagulant activity (tissue factor). These findings add further support to the concept that rickettsial LPS behave as typical LPS from gram-negative bacteria and may be relevant to the understanding of the mechanism(s) responsible for triggering intravascular coagulation in rickettsial diseases.

Species Distribution and In Vitro Azole Susceptibility of Aspergillus Section Nigri Isolates from Clinical and Environmental Settings

ABSTRACT Aspergillus section Nigri includes species of interest for animal and human health, although studies on species distribution are limited to human cases. Data on the antifungal susceptibilities and the molecular mechanism of triazole resistance in strains belonging to this section are scant. Forty-two black Aspergillus strains from human patients (16 isolates), animals (14 isolates), and the environment (12 isolates) were molecularly characterized and their in vitro triazole susceptibilities investigated. Aspergillus tubingensis was isolated from humans, animals, and environmental settings, whereas Aspergillus awamori and Aspergillus niger were isolated exclusively from humans. Phylogenetic analyses of β-tubulin and calmodulin gene sequences were concordant in differentiating A. tubingensis from A. awamori and A. niger. Voriconazole and posaconazole (PSZ) were the most active triazoles. One A. tubingensis strain was resistant to itraconazole and PSZ and one A. niger strain to PSZ. Sequence analysis of the cyp51A gene revealed different sequence types within a species, and A. tubingensis strains were also phylogenetically distinct from A. awamori/A. niger strains according to the strain origin and susceptibility profile. Genetic analysis of the cyp51A sequences suggests that two nonsynonymous mutations resulting in amino acid substitutions in the CYP51A protein (changes of L to R at position 21 [L21R] and of Q to R at position 228 [Q228R]) might be involved in azole resistance. Though azole resistance in black Aspergillus isolates from animals and rural environments does not represent a threat to public health in Southern Italy, the use of triazoles in the clinical setting needs to better monitored. The cyp51A sequence is useful for the molecular identification of black Aspergillus, and point mutations in protein sequences could be responsible for azole resistance phenomena.