Early virological response (EVR) to different interferon-based regimens plus ribavirin and its ability to predict the outcome of therapy in patients with chronic hepatitis C were investigated. The study design was as follows: 64 naive patients were considered, 32/64 received pegylated interferon alpha-2b (Peg-IFN-alpha2b) plus ribavirin and the remaining 32 received leucocyte interferon alpha (IFN-alpha) plus ribavirin. At week 4 of treatment, EVR was present in 68.7% and 37.5% of patients treated with Peg-IFN-alpha2b plus ribavirin, and with leucocyte interferon alpha (IFN-alpha) plus ribavirin, respectively (p = 0.024). At week 12, the cumulative EVR rates did not differ between the two groups (71.9% vs 56.2%, p >0.05) because a higher proportion of patients achieved EVR for the first time after more than 4 weeks of therapy in the standard IFN-alpha group. Sustained virological response (SVR) rates, however, resulted significantly higher in the Peg-IFN-alpha2b group (65.6% vs 37.5%; p = 0.045) since a higher proportion of patients who received standard IFN-alpha relapsed during the follow-up. In the standard IFN-alpha group, HCV genotype 1 (p = 0.035), high baseline viral load (p = 0.035) and the presence of bridging fibrosis/cirrhosis (p = 0.011) were closely associated with significantly lower SVR rates. In the Peg-IFN-alpha2b group, only bridging fibrosis/cirrhosis (p = 0.02) negatively influenced the outcome of treatment. Overall, 33/41 (80.5%) patients with EVR at week 12 were sustained responders, yielding a positive predictive value (PPV) of 0.80. However, when SVR was related to the time taken to reach EVR, 32/34 (94.1%) patients with EVR at week 4 of therapy (PPV = 0.94) versus 1/7 (14.3%) patients who had EVR after more than 4 weeks of therapy (PPV = 0.14) resulted sustained responders (p = 0.000057). In conclusion, EVR at week 4 of treatment is strongly associated with the likelihood of achieving SVR, regardless of the therapeutic regimen. However, when compared with standard IFN-alpha plus ribavirin, treatment with Peg-IFN-alpha2b plus ribavirin significantly increases the probability of viral clearance within the first 4 weeks of treatment. Finally, patients who do not clear the virus within the first 12 weeks of treatment have no chance of achieving SVR, justifying discontinuation of therapy in these patients.
Performance of chromogenic medium (ORSAB) for routine detection of methicillin resistant S. aureus (MRSA) was evaluated on 510 specimens collected from patients suspected of MRSA infection or colonization. Addition of ORSAB plates to the routine protocol allowed MRSA identification in 24 hours from samples plating. In 18 samples MRSA colonies were identified only on ORSAB plates, those cases would have been missed by routine protocol alone.
Starting in the 1960, electron microscopy (EM) became widely applied also in viral diagnosis. During the 1970th and 80th, many new agents were characterized from diagnostic cell cultures and clinical specimens. The wide introduction of ELISA- and PCR-techniques as well as cost-arguments recently reduced the role of EM in routine viral diagnosis. Compared to other diagnostic techniques, however, EM excells by speed and "open view", i.e. by the ability to detect also the "un-expected" without the need for specific reagents. As shown in 2003 by the elucidation of the SARS pandemia and the human monkeypox outbreak in US, EM is well suited as a safe, front-line diagnostic method in infectious diseases emergencies and/or in possible bioterrorist attacks.
A group of subgingival isolates of C. albicans recovered from Italian HIV-positive (HIV+) subjects were characterized both phenotypically and genotypically. Phenotyping of the isolates was carried out by a biotyping method based on the enzyme profiles, carbohydrate assimilation patterns and boric acid resistance of the yeasts. Genotyping was performed through randomly amplified polymorphic DNA (RAPD) analysis. Five biotypes were found among the 29 subgingival C. albicans strains examined. The predominant biotypes were A1R (55.17%), A1S (24.14%), and A2R (13.79%), while the biotypes A11R and A13R were represented by a single isolate each. RAPD profiles identified 15 genotypes among the 29 isolates. Almost every individual harboured his/her own specific isolate and in three out of the six subjects with multiple isolates (two to six each) more than one genotype (two to six) was found. The biotype distribution we found is consistent with previous reports on C. albicans isolates from other oral sources, whereas the resistance to boric acid was highly frequent in subgingival strains. RAPD analysis showed high genetic heterogeneity within subgingival isolates, also when isolates were phenotypically identical. These findings, obtained from HIV+ subjects living in Southern Italy, may be useful as baseline information on subgingival C. albicans colonization in the Mediterranean area.
The diagnosis of cutaneous Mycobacterium marinum infection is frequently presumptive, as detection by conventional methods is difficult. We describe a patient with granulomatous skin lesions on the right dorsal hand and forearm. Histological examinations were presumptive for mycobacterium lesions. We identified Mycobacterium marinum directly in the patient's lesional skin biopsy combining polymerase chain reaction (PCR) amplification using Mycobacterium genus-specific primers, and subsequent restriction enzyme analysis enabling identification to the species level. The symptoms were no longer present after specific therapy, thereby confirming the initial diagnosis.
Methicillin resistant Staphylococcus aureus (MRSA) is an emerging problem. We studied 71 MRSA strains for the presence of mecA gene by PCR, for the enterotoxins production and susceptibility to antimicrobials. In addition, the suitability of Random Amplified Polymorphic DNA analysis (RAPD) and Hypervariable Region (HVR)--PCR as molecular tools for typing MRSA was also tested. All the 71 strains previously found MRSA with conventional methods, presented the gene mec A. By molecular typing five distinct amplicons were found. MRSA with two DRUS were the most common type. RAPD analysis clustered MRSA in 8 groups, three of which were the most common. 26.8% of MRSA produce enterotoxins with a prevalence of type A. MRSA exhibited resistance to all quinolones tested and to gentamycin. Our data suggest that a typing method based on RAPD combined with HVR-PCR may be useful to compare MRSA isolated in a hospital environment, whereas PFGE may be used for further analysis.
This prospective open-label randomized trial of chronic hepatitis C genotype-1b patients compared compared the efficacy and safety of peg-interferon alfa-2b administered once-weekly versus interferon alfa-2b thrice-weekly or daily, both in combination with ribavirin. Seventy-eight previously untreated patients, with biopsy-documented genotype 1 chronic HCV and persistently elevated ALT levels and detectable HCV RNA, were randomized (26 subjects each) to receive: interferon alfa-2b at 6MIUs.c./three-times-weekly (group A) or interferon alfa-2b, 3MIUs.c./daily (group B) or peg-interferon alfa-2b 1.5mcg/Kg s.c./once-weekly (group C). All regimens included standard weight-based doses of ribavirin (800, 1,000 or 1,200 mg/day) administered for 52-weeks. Patients in the three groups were comparable for age, sex, viral load, ALT value and histological-activity-index (HAI). Therapy was completed by 22, 20 and 23 patients in groups A, B and C, respectively. At the end of treatment, a complete (biochemical and virological) response was observed in 50.0% patients of group A, 57.7% of group B and 65.4% of group C. After an additional 24-weeks of follow-up, a sustained response was observed in 26.9%, 46.1% and 50.0% of patients in groups A, B or C, respectively. Therapy was discontinued by 4, 6 and 2 patients because of adverse events in the above three groups. In naive patients with chronic genotype-lb hepatitis C, a 48 week therapy with peg-interferon or interferon at daily doses combined with ribavirin were both more effective than treatment with thrice-weekly interferon in inducing end of treatment and sustained response. Peg-interferon treatment was better tolerated and provoked significantly fewer therapy discontinuations.
This study investigated the onset and development of the immune response to Borrelia burgdorferi infection in 30 Italian patients with culture-confirmed Lyme Borreliosis in the stage of erythema migrans (EM). All patients received antimicrobial treatment when entering the study and were prospectively evaluated monthly for up to 30 days after enrolment. A total of 60 serially collected serum samples were tested by using two different commercial enzyme-linked immunosorbent assays (ELISAs): Anti-Borrelia plus VlsE ELISA, Euroimmun, and the synthetic peptide-based ELISA, Quick ELISA C6, Immunetics. Sixty-five potentially cross-reacting sera were also tested. Anti-Borrelia plus VlsE ELISA IgG was far more sensitive than Quick ELISA C6 (56.6% and 33.3%, respectively). Moreover, considering that 17 additional sera from the first bleeding group of Lyme disease patients were IgM positive when tested by Anti-Borrelia plus VlsE IgM, the sensitivity of Anti-Borrelia plus VlsE as a whole system rose to 85.0%. Nevertheless, due to the specificity values of Anti-Borrelia plus VlsE ELISA identified in this study (98.5% for IgG and 78.5% for IgM), the need of a confirmatory test for the diagnosis of Lyme disease remains. All the sera were also tested by two different commercial Western Blot (WB) assays: Euroline-WB against Borrelia, Euroimmun, and Qualicode B. burgdorferi WB, Immunetics, in comparison with a multispecies "home made" WB. Performances of the three WB methods for the detection of IgM were very similar. On the contrary, these WBs performed with different values of sensitivity and specificity when IgGs were evaluated. The most sensitive method was the "home-made" WB IgG (71.7%), followed by the Euroline-WB IgG against Borrelia (68.3%). Qualicode B. burgdorferi WB IgG demonstrated to be only 26.6% sensitive. Both "home-made" WB IgG and Qualicode B. burgdorferi WB IgG were 100% specific, whereas Euroline-WB IgG against Borrelia scored 12 cross-reacting samples as borderline, showing a specificity value of 80.0%.
BDProbeTec ET (Becton Dickinson, Sparks, Md, USA) is a fully automated walkaway system based on strand displacement amplification (SDA) technology that provides a method for the direct detection of Mycobacterium tuberculosis complex (MTBC) target sequence. The purpose of this study was to evaluate the ability of BDProbeTec ET system to detect MTBC directly from clinical specimens and compare the results with staining and culture. From February 2002 through December 2003 a total of 1521 [pulmonary (n=1329) and extrapulmonary (n=192)] specimens from 1518 patients were examined by BDProbeTec ET system for the detection of MTBC and the results were compared to those obtained by microscopy and liquid culture (BACTEC 9000 MB, Becton Dickinson). MTBC was cultivated from 65 specimens (60 pulmonary and 5 extrapulmonary) of which 43 (66.2%) (42 pulmonary and 1 extrapulmonary) were smear positive and 22 (33.8%) (18 pulmonary and 4 extrapulmonary) were smear negative. BDProbeTec ET detected MTBC in 58 (55 pulmonary and 3 extrapulmonary) of the 65 culture-positive specimens. Although the BDProbeTec ET system gave five false-negative results among the 18 smear-negative culture-positive pulmonary specimens, our results demonstrate that the BDProbeTec ET system is a reliable tool in smear-positive samples and given its technical characteristics it can be used for the rapid detection of MTBC in either pulmonary or extrapulmonary samples.
We report the epidemiology, clinical disease spectrum, treatment, and outcome of Plesiomonas shigelloides bacteraemia in our hospital over a nine-year period and compare the characteristics of patients with P. shigelloides bacteraemia in our hospital with those reported in the literature. During the nine-year period (1995--2003), a total of seven patients developed P. shigelloides bacteraemia. All cases occurred during the late spring, summer, and early autumn (May-October). All patients were over 75 and had underlying diseases (biliary tract diseases in four and malignancies in three). Six had acute cholangitis and five had polymicrobial bacteraemia. Overall two patients died. Compared with patients with P. shigelloides bacteraemia reported in the literature, patients with P. shigelloides bacteraemia in our hospital were associated with old age (P<0.001), underlying biliary tract diseases (P<0.001), acute cholangitis (P<0.001), and polymicrobial bacteraemia (P<0.001). There are major geographical differences of disease association in P. shigelloides bacteraemia. The sources of P. shigelloides in our patients were probably in the gastrointestinal tract.
Cloacal swabs collected from mallard ducks (Anas platyrhynchos) experimentally infected with a H7N1 avian influenza strain were examined by Reverse Transcription Polymerase Chain Reaction to detect the influenza A virus. Reverse Transcription Polymerase Chain Reaction was compared with standard methods: inoculation of embryonated chicken eggs and inoculation of three established cell lines: Newborn Swine Kidney cells, Newborn Pig Trachea cells and Madine Darby Canine Kidney cells. Reverse Transcription Polymerase Chain Reaction was performed using a set of primers based on conserved regions of the matrix and nucleoprotein genes.
In this study, we aimed to evaluate the frequency of non-tuberculous mycobacteria (NTM) isolated from clinical specimens using Polymerase Chain Reaction-Restriction Enzyme Analysis (PCR-REA) and to investigate the patients who had clinically significant NTM infections in our hospital through the five year period from May 1997 to June 2002. A total of 364 mycobacterial strains isolated from clinical specimens which gave positive growth index in the BACTEC 460 radiometric system in Hacettepe University Hospital Clinical Microbiology Laboratory were evaluated by PCR-REA and clinical data were obtained from the patient records. Three hundred and one of the strains (82.7%) were identified as Mycobacterium tuberculosis and 63 (17.3%) were identified as nontuberculous mycobacteria. Seven (11.1%) of 63 NTM patients were regarded as having clinical mycobacteriosis. Chronic obstructive pulmonary disease and other pre-existing lung diseases were seen in 39 (61.9%) of the patients, 11 (17.5%) of'the patients had chronic renal failure. Four (6.3%) and 9 (14.3%) of them had AIDS and carcinomas, respectively. PCR-REA was found to be a reliable method for typing of our mycobacterial isolates to the species level. These data may shed light on the epidemiology of the mycobacterial species and help to select a proper treatment regimen.
Crohn's disease is a non-specific chronic transmural inflammatory disease. The disease was associated with a frameshit mutation in the NOD2 gene. Nevertheless, other researchers associated the presence of M. paratuberculosis within the intestinal tissues of patients with the disease. An adapted "in situ hybridization" technique was used to detect IS900 M. paratuberculosis DNA in paraffin embedded tissue from Crohns tissue disease samples. We were able to identify M. paratuberculosis DNA in around 69% of the paraffine embedded intestinal samples of Crohn's disease patients analysed. The presence of M. paratuberculosis DNA in the intestinal samples analysed does not necessarily mean that M. paratuberculosis is responsible for Crohn's disease. Our results support the hypothesis that infection may be caused by cell wall defective M. paratuberculosis since no bacteria were detected by Ziehl Neelsen stain.
Gene fadD33 of Mycobacterium tuberculosis, one of the 36 homologues of gene fadD of Escherichia coli identified in the M. tuberculosis genome, predictively encodes an acyl-CoA synthase, an enzyme involved in fatty acids metabolism. The gene is underexpressed in the attenuated strain M. tuberculosis H37Ra relative to virulent H37Rv and plays a role in M. tuberculosis virulence in BALB/c mice by supporting mycobacterial replication in the liver. In the present paper, we investigated the role of fadD33 expression in bacterial growth within the hepatocyte cell line HepG2, as well as in human monocyte-derived THP-1 cells and peripheral blood mononuclear cells. M. tuberculosis H37Rv proved able to grow within HepG2 cells, while the intracellular replication of M. tuberculosis H37Ra was markedly impaired; complementation of strain H37Ra with gene fadD33 restored its replication to the levels of H37Rv. Moreover, disruption of gene fadD33 by allelic exchange mutagenesis reduced the intracellular growth of M. tuberculosis H37Rv, and complementation of the fadD33-disrupted mutant with gene fadD33 restored bacterial replication. Conversely, fadD33 expression proved unable to influence M. tuberculosis growth in human phagocytes, as fadD33-disrupted M. tuberculosis H37Rv mutant, as well as fadD33-complemented M. tuberculosis H37Ra, grew within THP-1 cells and peripheral monocytes basically at the same rates as parent H37Rv and H37Ra strains. The results of these experiments indicate that gene fadD33 expression confers growth advantage to M. tuberculosis in immortalized hepatocytes, but not in macrophages, thus emphasizing the importance of fadD33 in liver-specific replication of M. tuberculosis.
Leptospirosis is a zoonosis with a worldwide distribution very common in most countries. In Italy this acute febrile illness is more frequent in the Northern than in the Southern regions. In the period 1994-1996, the number of cases of Leptospirosis in Sicily was lower with respect to the northern-central regions (7.2% and 73.4% respectively). Between January 1990 and December 1999, a total of 9 leptospirosis cases were observed in the Regional Centre for Leptospirosis of Palermo. The patients were all males (age between 22 and 59 years) and their occupations varied. Laboratory diagnosis is performed by the classical microagglutination microscopical (MAT) but this test is very complex and time-consuming. This study compared the classical MAT with ELISA IgM by using 19 serum samples from 9 patients with confirmed leptospirosis. We also tested 23 serum samples from blood-donors and 29 serum samples from patients with other infectious diseases. By the MAT and the PanBio IgM ELISA all sera from patients were found to be positive. Our results indicate that MAT represents the test with the highest degree of specificity (100%), but ELISA is simpler to perform, considering the favourable degree of sensitivity (100%) and specificity (95.9%).
Rickettsial diseases have been reassessed in recent years since they represent an important field in today's medicine. New agents have been described: some are non-pathogenic agents and the others are associated with well-defined or peculiar clinical patterns. In addition, different species of rickettsiosis are found in relation to the geographic areas of the world. Some agents may be defined as variants of older diseases whereas most of the newly described forms of rickettsiosis represent distinct entities with unique epidemiologial and clinical features. Probably the main news regards the group of the spotted fevers. An additional new aspect is linked to the medicine of travellers and tourists. However, this aspect may not be significant for the rickettsial diseases in relation to other human illnesses, such as malaria. Therefore, an investigation into the geographical origin of patients has to enter our routine medical work.
Verocytotoxin-producing Escherichia coli O157 (VTEC) is an important food-borne pathogen of humans. The serious complications of VTEC infection and the established reservoir of VTEC in cattle used for mass food production are a public health concern. In this study 500 samples of hamburger and minced meat were examined for presence of E. coli O157. For E. coli detection, Tryptic Soy Broth supplemented (with novobiocin and bile salts) and Sorbitol Mc Conkey agar were used; an automated rapid enzyme linked fluorescent immunoassay (VIDAS E. coli O157) was also evaluated. E. coli O157 was found in 5 samples of hamburger, 2 strains were found to be positive for verocytotoxin production on Vero cells.
The aim of this study was to evaluate outcome after a genotype guided change of therapy in 18 patients failing HAART. Patients were divided into two groups according to the response to therapy: immune responders (12 patients with immune recovery defined as having more than 100 CD4 cells compared to baseline value), and 6 failing patients (without immune recovery). At month 12 after genotype change of therapy a significant difference in the decrease of HIV-RNA viral load between the two groups of patients was detected (mean -1.95 and +0.04 log HIV-RNA copies/ml, p=0.04). One year after the change of therapy, all but one patients experienced a decrease in the replication capacity of HIV strains. Particularly, the HIV replication capacity of HIV strains decreased from 52% (range 14-98%) to 15.2% (range 0.1-74.5%). The HIV strains of patients failing HAART showed a progressive impaired replication capacity. In patients failing HAART the impaired replication capacity of HIV strains could justify the persistence of an immune recovery.
