Eugenio Bordi

Pulmonary tuberculosis in HIV-infected patients presenting with normal chest radiograph and negative sputum smear

AbstractBackground: HIV-infected patients with pulmonary tuberculosis exhibit atypical radiological presentation and negative sputum smear more frequently than their HIV-negative counterparts. Patients and Methods: We performed a retrospective study based on a chart review of 146 HIV-infected patients with pulmonary symptoms and culture-proven pulmonary tuberculosis. We compared clinical characteristics and the outcome in 71 patients (49%) with positive sputum smear (SS+), 62 patients (42%) with negative sputum smear/abnormal chest X-ray (SS−/CXR+) and 13 patients (9%) with negative sputum smear/normal chest X-ray (SS−/CXR−). Patients were enrolled form January 1987 to December 1998, and were followed up until December 1999. Results: On hospital admission the three groups of patients examined did not differ significantly in demographic characteristics, degree of immunosuppression or Mycobacterium tuberculosis drug-susceptibility pattern. SS−/CXR− patients were significantly less likely to present with prolonged fever and dyspnea. Median survival was shorter for SS−/CXR− patients (6.4 months vs. 20.2 and 18.8 months in the other two groups). In multivariate analysis, SS−/CXR− patients had a significantly increased risk of death (hazard ratio 3.0, 95% confidence interval, 1.4 to 6.4, p = 0.004) compared to SS+ patients. This increase in risk was no longer statistically significant when initiation of antituberculous therapy within 8 weeks from the collection date of the first specimen yielding M. tuberculosis was included in the multivariate model. Conclusion: Decreased survival was observed in HIV-infected patients with pulmonary tuberculosis and with both negative sputum smear and normal chest X-ray presentation. This may primarily be a result of delayed tuberculosis diagnosis and initiation of antituberculous therapy. The latter delay may also lead to a faster progression of HIV infection in SS−/CXR patients, in whom diagnostic oversight may be common.

Prevalence, determinants, and molecular epidemiology of Streptococcus pneumoniae isolates colonizing the nasopharynx of healthy children in Rome

The aim of this study was to determine the factors favouring Streptococcus pneumoniae nasopharyngeal colonization of healthy children attending daycare centres and to describe the circulation of penicillin-nonsusceptible strains using molecular techniques. A single nasopharyngeal swab was obtained from 610 children attending daycare centres in the southeast area of Rome. Streptococcus pneumoniae isolates were serotyped, and antibiotic susceptibility was assayed by the E test. The genetic determinants of erythromycin resistance were detected by a duplex polymerase chain reaction, and the penicillin-nonsusceptible isolates were typed by pulsed-field gel electrophoresis. The overall carriage rate of Streptococcus pneumoniae was 14.9%. Living with more than three persons in the same household was the only risk factor statistically associated with carriage. Sixteen of 85 (18.8%) strains were nonsusceptible to penicillin, and 44 (52%) were resistant to erythromycin. Of the erythromycin-resistant strains, the vast majority showed a high level of resistance and carried the erm(B) gene. The penicillin-nonsusceptible strains belonged to six different serotypes; molecular typing showed that in only one case (2 strains) was there a circulation of the same clone in the same daycare centre. In view of the high rate of resistant Streptococcus pneumoniae strains, risk factors for carriage of resistant strains were evaluated. Children who received macrolides in the previous month had a higher risk of being colonized by macrolide-resistant strains as well as by strains resistant to both penicillin and erythromycin. Limiting the use of antibiotics in children seems the most appropriate measure to control the spread of antibiotic-resistant strains.

Identification of clinically relevant yeast species by DNA sequence analysis of the D2 variable region of the 25–28S rRNA gene

Clinically relevant yeasts are conventionally identified by a combination of phenotypic tests, which occasionally provide ambiguous results for atypical isolates or uncommon species. In this study, we evaluate a direct polymerase chain reaction‐sequencing method, which exploits sequence divergence in the hypervariable D2 region of the large subunit of the 25–28S ribosomal RNA (rRNA) gene for identification of facultative pathogenic asco‐ and basidiomycota. A panel of 53 yeasts, including 40 clinical isolates and 13 reference strains representative of some clinically relevant taxa, was investigated by combining standard phenotypic tests with commercial identification systems (RapID, API 20C AUX), and results were compared with the taxonomic allocations inferred by D2 sequence analysis. Species‐level resolution was achieved for almost all (52/53) strains by combining internet‐based D2 sequence homology (BLAST and FASTA) searches in free‐access synchronised databases with phylogenetic analysis. The phylogenetic information carried by the short D2 sequence substantiates a pattern of molecular evolution, which is similar to that inferred from analysis of the larger D1/D2 region, and consistent with previously published 25–28S rRNA phylogenetic architectures of facultative pathogenic yeast, including recently identified species. Inconsistency between conventional and molecular identification results was observed for 11/53 strains, likely on account of the ambiguous interpretation of phenotypic tests.

Flavohemoglobin and nitric oxide detoxification in the human protozoan parasite Giardia intestinalis

Flavohemoglobins (flavoHbs), commonly found in bacteria and fungi, afford protection from nitrosative stress by degrading nitric oxide (NO) to nitrate. Giardia intestinalis, a microaerophilic parasite causing one of the most common intestinal human infectious diseases worldwide, is the only pathogenic protozoon as yet identified coding for a flavoHb. By NO amperometry we show that, in the presence of NADH, the recombinant Giardia flavoHb metabolizes NO with high efficacy under aerobic conditions (TN=116+/-10s(-1) at 1microM NO, T=37 degrees C). The activity is [O(2)]-dependent and characterized by an apparent K(M,O2)=22+/-7microM. Immunoblotting analysis shows that the protein is expressed at low levels in the vegetative trophozoites of Giardia; accordingly, these cells aerobically metabolize NO with low efficacy. Interestingly, in response to nitrosative stress (24-h incubation with 5mM nitrite) flavoHb expression is enhanced and the trophozoites thereby become able to metabolize NO efficiently, the activity being sensitive to both cyanide and carbon monoxide. The NO-donors S-nitrosoglutathione (GSNO) and DETA-NONOate mimicked the effect of nitrite on flavoHb expression. We propose that physiologically flavoHb contributes to NO detoxification in G. intestinalis.

Giardia intestinalis Escapes Oxidative Stress by Colonizing the Small Intestine: A Molecular Hypothesis

Giardia intestinalis is the microaerophilic protozoon causing giardiasis, a common infectious intestinal disease. Giardia possesses an O2‐scavenging activity likely essential for survival in the host. We report that Giardia trophozoites express the O2‐detoxifying flavodiiron protein (FDP), detected by immunoblotting, and are able to reduce O2 to H2O rapidly (∼3 μM O2 × min × 106 cells at 37 °C) and with high affinity (C50 = 3.4 ± 0.7 μM O2). Following a short‐term (minutes) exposure to H2O2 ≥ 100 μM, the O2 consumption by the parasites is irreversibly impaired, and the FDP undergoes a degradation, prevented by the proteasome‐inhibitor MG132. Instead, H2O2 does not cause degradation or inactivation of the isolated FDP. On the basis of the elevated susceptibility of Giardia to oxidative stress, we hypothesize that the parasite preferentially colonizes the small intestine since, compared with colon, it is characterized by a greater capacity for redox buffering and a lower propensity to oxidative stress.

In vitro activity of doripenem in combination with various antimicrobials against multidrug-resistant Acinetobacter baumannii: possible options for the treatment of complicated infection.

AIMS The aim of this study was to evaluate the in vitro activity of doripenem (DOR) alone and in combination with a variety of commonly used anti-Acinetobacter chemotherapeutic agents against 22 primary multidrug-resistant (MDR) Acinetobacter baumannii isolates (including 17 isolates that were resistant to DOR) from Intensive Care Unit patients. Antibiotic interactions were evaluated using the chequerboard method and the time-kill assay. RESULTS Considering all antimicrobials in combination with DOR, chequerboard analysis showed synergy in 13 A. baumannii strains (54.2%). Seven strains (29.2%) showed ≥2 synergistic interactions. DOR showed synergy in combination with tigecycline (TIG) (eight strains), colistin (COL) (eight strains), amikacin (AMK) (four strains), ampicillin/sulbactam (two strains), and rifampicin (one strain). Remarkably, synergistic effects were detected only in DOR nonsusceptible strains. Time-kill assays confirmed synergy in eight isolates (giving 10 synergistic interactions) for DOR in combination with TIG (n=4), COL (n=5), and AMK (n=1). No antagonistic interactions were observed with both methods. CONCLUSIONS This study demonstrates the in vitro synergistic activity of DOR in combination with TIG, COL, and AMK against DOR-resistant A. baumannii strains, opening the way to in vivo assessment of novel combination therapies for treatment of infections caused by MDR A. baumannii.

Cases of cryptosporidiosis co-infections in AIDS patients: a correlation between clinical presentation and GP60 subgenotype lineages from aged formalin-fixed stool samples

Abstract Nine cases of cryptosporidiosis co-infections in AIDS patients were clinically categorised into severe (patients 1, 3, 8 and 9), moderate (patients 4 and 5) and mild (patients 2, 6 and 7). Formalin-fixed faecal specimens from these patients were treated to obtain high quality DNA competent for amplification and sequencing of the 60-kDa glycoprotein (GP60) gene. Sequence analysis revealed that one patient was infected with Cryptosporidium hominis whereas the remaining eight patients were infected with C. parvum. Interestingly, the patients showing severe cryptosporidiosis harboured two subtypes within the C. parvum allelic family IIc (IIcA5G3 and IIcA5G3R2), whereas patients with moderate or mild infections showed various subtypes of the C. parvum allelic family IIa (IIaA14G2R1, IIaA15G2R1, IIaA17G3R1 and IIaA18G3R1). DNA extraction and genotyping of Cryptosporidium spp. is a challenging task on formalin-fixed stool samples, whose diagnostic outcome is age-dependent. The method herein reported represents a step forward routine diagnosis and improves epidemiology of HIV-related clinical cases. Due to the need to elucidate genetic richness of Cryptosporidium human isolates, this approach represents a useful tool to correlate individual differences in symptoms to subgenotyping lineages.

The superoxide reductase from the early diverging eukaryote Giardia intestinalis.

Unlike superoxide dismutases (SODs), superoxide reductases (SORs) eliminate superoxide anion (O(2)(•-)) not through its dismutation, but via reduction to hydrogen peroxide (H(2)O(2)) in the presence of an electron donor. The microaerobic protist Giardia intestinalis, responsible for a common intestinal disease in humans, though lacking SOD and other canonical reactive oxygen species-detoxifying systems, is among the very few eukaryotes encoding a SOR yet identified. In this study, the recombinant SOR from Giardia (SOR(Gi)) was purified and characterized by pulse radiolysis and stopped-flow spectrophotometry. The protein, isolated in the reduced state, after oxidation by superoxide or hexachloroiridate(IV), yields a resting species (T(final)) with Fe(3+) ligated to glutamate or hydroxide depending on pH (apparent pK(a)=8.7). Although showing negligible SOD activity, reduced SOR(Gi) reacts with O(2)(•-) with a pH-independent second-order rate constant k(1)=1.0×10(9) M(-1) s(-1) and yields the ferric-(hydro)peroxo intermediate T(1); this in turn rapidly decays to the T(final) state with pH-dependent rates, without populating other detectable intermediates. Immunoblotting assays show that SOR(Gi) is expressed in the disease-causing trophozoite of Giardia. We propose that the superoxide-scavenging activity of SOR in Giardia may promote the survival of this air-sensitive parasite in the fairly aerobic proximal human small intestine during infection.

Salmonella enterica ssp. arizonae infection in a 43-year-old Italian man with hypoglobulinemia: a case report and review of the literature

IntroductionSalmonella enterica ssp. arizonae is an uncommon human pathogen with serious infections reported in immunocompromised hosts. In Europe, only a few cases have been described. Patients with this infection usually have a history of contact with reptiles or travel abroad. We present a case report of infection in a patient with hypoglobulinemia and a literature review.Case presentationWe describe the case of a 43-year-old Caucasian Italian man with hypoglobulinemia who presented to our hospital with sepsis and diarrhea. A stool culture yielded S. enterica ssp. arizonae. Our patient was treated with oral ciprofloxacin and made a full recovery. We also present a review of the cases of S. enterica ssp. arizonae infections previously reported in Europe.ConclusionsThe majority of infections from S. enterica ssp. arizonae occur in patients who are immunocompromised. Data from the literature suggests that it may be difficult to eradicate the bacteria and thus, prolonged antibiotic courses are often used. It would be advisable for clinicians to investigate for pre-existing immune dysfunction if S. enterica ssp. arizonae is isolated. In Italy, although there have only been a few cases, the likely route of transmission remains unclear and requires further surveillance.

Drug Resistance Patterns among Tuberculosis Patients in Rome, 1990-1992

Prevalence of, and risk factors for, drug-resistance of Mycobacterium tuberculosis were assessed among 407 hospitalized patients with tuberculosis in Rome, Italy, during the period 1990-1992. Resistance to 1 or more drugs was detected in 106 isolates (26%). Resistance to streptomycin was the most common (18.4%), followed by isoniazid (10.3%) and rifampin (7.9%). 23 isolates (5.7%) were resistant to both isoniazid and rifampin. Resistance to at least 1 drug and resistance to both isoniazid and rifampin were significantly more common among recurrent cases (40.7% vs. 22.1%, p < 0.001; and 22.1% vs. 1.2%, p < 0.001). Sex, country of origin and HIV infection were not significantly associated with prevalence of drug resistance. Among recurrent cases, prevalence of resistance to at least 1 drug and of resistance to both isoniazid and rifampin, was higher in subjects who had had a previous episode of tuberculosis later than 1969. In the population studied the prevalence of drug-resistant tuberculosis was high, although the risk of initially becoming infected with a multidrug-resistant strain of M. tuberculosis in this area appears to be low. This study suggests the need for enhanced surveillance of drug-resistance of tuberculosis in our country and for implementation of intervention aimed to ensure adequate and complete therapy for patients with tuberculosis.