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

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.

Purification and characterization of an antifungal thaumatin-like protein from Cassia didymobotrya cell culture.

A 23-kDa antifungal thaumatin-like protein was isolated and purified from Cassia didymobotrya (Fres.) cell cultures for the first time. The protein was secreted in the culture medium, but it could be also isolated after elution of whole cells with a 0.5 M CaCl(2) solution. Treatment of the cells with laminarin oligosaccharides or salicylic acid, but not with NaCl, resulted in enhancement of expression of the protein. A rapid purification protocol was used based on cationic exchange chromatography. The protein, with a highly basic character (pI 10), has an exact molecular mass of 23034 Da, as determined by MALDI-ToF mass spectrometry analysis. N-terminal sequencing of the intact polypeptide and the sequencing of two internal tryptic peptides indicated significant identity with other thaumatin-like proteins (TLP). The protein exerted antifungal activity towards some Candida species showing EC(50) values comparable to those of other antifungal TLPs. The collected data lead to classify this TLP as a new PR-5 protein.

Activity of quinupristin-dalfopristin in invasive isolates of Streptococcus pneumoniae from Italy.

Eighty-five recent isolates of Streptococcus pneumoniae from patients with invasive disease were examined for their susceptibility to erythromycin, clindamycin, penicillin and quinupristin-dalfopristin by E test. A novel duplex PCR assay was used to detect the presence of the erm(B) or mef(A) genes in all of the erythromycin-resistant isolates. All of the strains tested were susceptible to the combination quinupristin-dalfopristin, regardless of their susceptibility to penicillin or to erythromycin. By duplex PCR, two-thirds of the erythromycin-resistant strains harbored erm, and one-third harbored mef. The activity of quinupristin-dalfopristin was not influenced by the genetic determinant of erythromycin resistance. The in vitro susceptibility of S. pneumoniae to quinupristin-dalfopristin is promising for future use; however, it is important to monitor the possible emergence of resistance.

The efficacy and safety of imipenem/cilastatin in the treatment of severe bacterial infections.

We have assessed the efficacy and safety of imipenem/cilastatin in a non-comparative study of 27 immunocompromised patients suffering from severe bacterial infections. Moreover in two groups of 14 patients the efficacy of imipenem/cilastatin versus a standard broad spectrum antibiotic therapy has also been compared. Clinical and microbiological efficacy and side effects have been evaluated.

Pulmonary cryptosporidiosis in patients with acquired immunodeficiency syndrome.

Several reports have showed Cryptosporidium species as a cause of intractable diarrhea and malabsorption in patients with acquired immunodeficiency syndrome (HIV). A case of chronic diarrhea in a drug addict woman associated with a symptomatic interstitial pulmonary infection due to Cryptosporidium parvum is described. This unusual C. parvum spread into the bronchial tree is underlined and a survey of the literature is made.

[Mycobacterium tuberculosis drug resistance in patients with HIV and pulmonary tuberculosis infections in Rome: 1987-1996]. / La farmaco-resistenza di Mycobacterium tuberculosis nei pazienti con infezione da HIV e tubercolosi polmonare in Roma: 1987-1996.

A retrospective chart review was performed on 118 HIV infected patients with pulmonary tuberculosis hospitalized between 1987 and 1996 in a tertiary care center for Infectious Diseases in Rome. The aims of this study were: a) to evaluate global prevalence of and risk factors for drug-resistant Mycobacterium tuberculosis and multidrug resistant tuberculosis; b) to assess trends in prevalence of drug-resistant tuberculosis over the 10-year study period. Prevalence of drug resistance of first Mycobacterium tuberculosis isolates was tested on Lowenstein-Jensen medium with the proportional method. Of the 118 patients studied, 83 had never been treated for tuberculosis and 35 had already been treated for at least 1 month. The overall prevalence of resistance to one or more drugs was 25% (17% in never treated patients vs 46% in already treated patients; p = 0.002). Five percent of isolates were resistant to both isoniazid and rifampin (1% in never treated patients vs 14% in already treated patients; p = 0.008). Resistance rates to individual drugs were: isoniazid 14%, rifampin 8%, ethambutol 0%, streptomycin 13%. During the study period no significant variations in prevalence of drug-resistant tuberculosis were found. In our area, empiric therapy should include 4 drugs: as well as isoniazid, rifampin and pyrazinamide, we recommend ethambutol. Surveillance of drug-resistant tuberculosis is needed. Directly observed therapy should be considered for HIV patients in order to prevent increases in drug resistance, relapses, and treatment failures.

[Piperacillin in the therapy of severe bacterial infections: comparison of its efficacy and tolerance vs. ceftazidime]. / La piperacillina nella terapia delle infezioni batteriche gravi: confronto dell'efficacia e tollerabilità vs ceftazidime.

38 patients with severe acute bacterial systemic infections have been enrolled in this study: 19 patients were treated with piperacillin (100-200 mg/kg/day) and 19 with ceftazidime (45-90 mg/kg/day) by i.v. route. In both groups monotherapy has been found effective and well tolerated. Serious side-effects have not been observed. The high cure and eradication rates in both groups do not show statistically significant differences (chi 2 = 0.620 and chi 2 = 0.219, respectively, p greater than 0.05).

[Cefotetan vs ceftriaxone: clinical and bacteriological efficacy in complicated forms of urinary tract infection]. / Cefotetan vs ceftriaxone: efficacia clinica e batteriologica nelle forme complicate di infezione del tratto urinario.

Forty adult patients with UTI complicated by local and/or general diseases have been treated, 20 with Cefotetan and 20 with Ceftriaxone. Both treatments showed good clinical and bacteriological efficacy, with no statistically significant differences between the results. Cefotetan and Ceftriaxone were both well tolerated, without any local or systemic side effects.

Molecular analysis based on mtLSU-rRNA and DHPS sequences of Pneumocystis jirovecii from immunocompromised and immunocompetent patients in Italy.

Pneumocystis jirovecii is an opportunistic fungus predominantly reported in immunocompromised individuals, who develop severe interstitial pneumonia (PcP). However, it is known that asymptomatic or mild pulmonary infections, defined as colonization, are widely observed in the general adult population. So far, genetic and epidemiological data of P. jirovecii infections in Italy are rather scarce and limited to defined geographical regions, mainly regarding isolates from HIV-infected patients. The aim of this study was to evaluate the polymorphisms at the mtLSU-rRNA and the DHPS loci by the screening and genotyping of a cohort of patients from two major hospitals in Rome (Italy). The study included 263 patients divided into two groups, all enrolled consecutively from January 2006 to December 2010: (i) 38 immunocompromised subjects including 25 HIV-infected; (ii) 225 immunocompetent patients. Sixty-seven out of 263 patients (25.5%) were found positive after PCR amplification of the mtLSU-rRNA gene. Overall, genotyping at mtLSU-rRNA locus revealed that the genotype 2 was the most frequent. Sequences of the DHPS gene were obtained from 21 patients, 9 from immunocompromised patients (6 from HIV infected individuals), 12 from immunocompetent ones. Considering the most common DHPS mutations usually detected at amino acid positions 55 and 57 and potentially related to drug resistance, all samples analyzed showed the wild-type signatures. These are the first data in Italy on prevalence and genotypes of P. jirovecii regarding colonized immunocompetent adults. Further multicenter analyses on P. jirovecii infection will be necessary to better define the specific epidemiology of the disease in the Italian populations.

High rate of colistin resistance among patients with carbapenem-resistant Klebsiella pneumoniae infection accounts for an excess of mortality.

Carbapenem-resistant Klebsiella pneumoniae (CR-KP) is becoming a common cause of healthcare-associated infection in Italy, with high morbidity and mortality. Prevalent CR-KP clones and resistance mechanisms vary between regions and over time. Therapeutic approaches and their impact on mortality have to be investigated. We performed a prospective study of patients with CR-KP isolation, hospitalized in nine hospitals of Rome, Italy, from December 2010 to May 2011, to describe the molecular epidemiology, antibiotic treatment and risk factors for mortality. Overall, 97 patients (60% male, median age 69 years) were enrolled. Strains producing blaKPC-3 were identified in 89 patients, blaVIM in three patients and blaCTX-M-15 plus porin defects in the remaining five patients. Inter-hospital spread of two major clones, ST512 and ST258, was found. Overall, 36.1% and 20.4% of strains were also resistant to colistin and tigecycline, respectively. Infection was diagnosed in 91 patients who received appropriate antibiotic treatment, combination therapy and removal of the infectious source in 73.6%, 59.3% and 28.5% of cases, respectively. Overall, 23 different antibiotic regimens were prescribed. In-hospital mortality was 25.8%. Multivariate analysis adjusted for appropriate treatment, combination therapy and infectious-source removal, showed that Charlson comorbidity score, intensive-care unit onset of infection, bacteraemia and infection due to a colistin-resistant CR-KP strain were independent risk factors for mortality. The spread of clones producing K. pneumoniae carbapenemases, mainly ST258, is currently the major cause of CR-KP infection in central Italy. We observed a high rate of resistance to colistin that is independently associated with worse outcome.

Epidemic multidrug-resistant Acinetobacter baumannii related to European clonal types I and II in Rome (Italy).

The molecular epidemiology and the genetic basis of antibiotic resistance in 88 multidrug-resistant (MDR) Acinetobacter baumannii strains isolated during 18 months from infected patients in seven intensive care units (ICUs) in Rome were investigated. Random amplified polymorphic DNA and macrorestriction analysis identified two predominant clonal types, genetically related to the European epidemic clones I (type 2) and II (type 1), accounting for 98.9% of A. baumannii ICU isolates. Type 1 was isolated from all ICUs under survey. Class 1 integrons of 2.2 and 2.5 kb were detected in type 1 and type 2 isolates, respectively. The integron structures were similar to those previously determined for epidemic A. baumannii strains from various European countries, and suggestive of integron rearrangement/exchange among isolates related to the European epidemic clones I and II. Carbapenem resistance was associated with the presence of the bla(OXA-58) gene in type 1 isolates. The results indicate that the A. baumannii type 1 clone has a high potential of spreading among hospitals.

HIV-related bronchopulmonary infection by Pseudomonas aeruginosa in the HAART era: radiological findings.

PURPOSE: To evaluate whether highly active antiretroviral therapy (HAART) modifies radiographic appearances of Pseudomonas aeruginosa bronchopulmonary infection in HIV-infected patients. P. aeruginosa is increasingly reported as a respiratory pathogen in HIV+ patients with very low levels of CD4 lymphocytes. Few studies have analyzed the radiological presentation of bronchopulmonary disease that occurs in HAART-treated patients. MATERIAL AND METHODS: We retrospectively reviewed the chest radiographs of 46 HIV-infected patients with bronchopulmonary diseases in which P. aeruginosa was the sole respiratory pathogen that was isolated. All cases were community-acquired infection. Twenty-four of the patients were on HAART treatment, and 22 were not. Chest radiographs were assessed for the presence and distribution of parenchymal consolidation, reticular or reticulonodular infiltrates, bronchial wall thickening, ground-glass opacities, cavitation, pleural effusion, and adenopathies. Statistical analysis was done using Epi-Info version 6 (CDC, Atlanta, GA, USA). RESULTS: Normal chest radiographs were observed in 11 patients. Eight of these 11 (73%) were receiving HAART, and 3/11 (27%) were not. The most common radiographic abnormality was bronchopneumonia, present in 24 of 46 patients (52%): in 10 of 24 (42%) patients with HAART and 14 of 22 (64%) without. Cavitation was seen in 1 of 24 (4%) patients with HAART and in 5 of 22 (23%) without HAART. CONCLUSION: Cavitation was more frequent in patients that were not receiving HAART, and normal chest radiographs were more frequently seen in patients on HAART.

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.

Expanding drug resistance through integron acquisition by IncFI plasmids of Salmonella enterica Typhimurium.

We conducted a 30-year retrospective analysis of IncFI plasmids from Salmonella enterica serotype Typhimurium. These plasmids have been associated with the emergence of epidemic clones of multidrug-resistant Salmonella. Molecular and genetic evidence indicates that IncFI plasmids are evolving through sequential acquisition of integrons carrying different arrays of antibiotic- resistance genes.

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

BACKGROUND: 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 from 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.