Paolo Scarpellini

Diagnosis of central nervous system complications in HIV-infected patients: Cerebrospinal fluid analysis by the polymerase chain reaction

Neurological complications afflict the majority of people who are infected with HIV. These include opportunistic infections, tumours, vascular and metabolic complications, and also disorders that are unique to AIDS itself and associated with infection of the nervous system by HIV [1]. Although the use of different antimicrobial treatments has led to a significant increase in the survival of AIDS patients, many of the nervous system disorders still represent a diagnostic and therapeutic challenge.

Rapid Diagnosis of Mycobacterial Infections and Quantitation of Mycobacterium tuberculosis Load by Two Real-Time Calibrated PCR Assays

ABSTRACT Sensitive and specific techniques to detect and identify Mycobacterium tuberculosis directly in clinical specimens are important for the diagnosis and management of patients with tuberculosis (TB). We developed two real-time PCR assays, based on the IS6110 multicopy element and on the senX3-regX3 intergenic region, which provide a rapid method for the diagnosis of mycobacterial infections. The sensitivity and specificity of both assays were established by using purified DNA from 71 clinical isolates and 121 clinical samples collected from 83 patients, 20 of whom were affected by TB. Both assays are accurate, sensitive, and specific, showing a complementary pattern of Mycobacterium recognition: broader for the IS6110-based assay and restricted to the M. tuberculosis complex for the senX3-regX3-based assay. Moreover, the addition of a synthetic DNA calibrator prior to DNA extraction allowed us to measure the efficiency of DNA recovery and to control for the presence of PCR inhibitors. The mycobacterial burden of the clinical samples, as assessed by direct microscopy, correlates with the M. tuberculosis DNA load measured by the senX3-regX3-based assay. In addition, reduced levels of M. tuberculosis DNA load are present in those patients subjected to successful therapy, suggesting a potential use of this assay for monitoring treatment efficacy. Therefore, these assays represent a fully controlled high-throughput system for the evaluation of mycobacterial burden in clinical specimens.

Nested polymerase chain reaction for diagnosis and monitoring treatment response in AIDS patients with tuberculous meningitis.

ObjectiveTo investigate the usefulness of polymerase chain reaction (PCR) from cerebrospinal fluid (CSF) for rapid diagnosis, and assessing treatment response of tuberculous meningitis (IBM) in AIDS patients. PatientsForty-four CSF samples from 10 patients with TBM confirmed by autopsy or by a culture of CSF (41 samples), and from two patients with highly probable TBM (three samples) were analysed. CSF specimens were collected before, and during standard antituberculous treatment. CSF samples from 24 AIDS patients with autopsy evidence of other neurologic diseases were studied as controls. MethodsA nested PCR amplifying a 123 base-pair fragment of the IS6770 sequence was developed. Heating to 95±C for 15 min was used for pre-PCR treatment of samples. ResultsDetection limit was 102 colony-forming units per ml or 10fg purified Mycobacterium tuberculosis DNA. M. tuberculosis DNA was detected in CSF from all the 12 confirmed or highly probable TBM cases. CSF was positive by nested PCR in 17 of 17 (100%), and 18 of 27 (67%) samples collected before, and during therapy, respectively. Clinical, and microbiological follow-up ≥2 weeks was available for seven patients. PCR-positive CSF converted to M. tuberculosis DNA negative in four patients that showed improvement during treatment, but it remained positive in three patients who died of disseminated tuberculosis. All the CSF samples from the non-TBM controls were negative by nested PCR. ConclusionsNested PCR for detection of M. tuberculosis DNA is specific for diagnosis of TBM, and more sensitive than conventional bacteriology. Moreover, nested PCR could be a useful method for assessing treatment response in AIDS patients with TBM.

Selected Pool of Peptides from ESAT-6 and CFP-10 Proteins for Detection of Mycobacterium tuberculosis Infection

ABSTRACT We have validated a new test for detecting Mycobacterium tuberculosis infection. A pool of synthetic peptides derived from ESAT-6 and CFP-10 proteins was used to detect the number of specific gamma interferon-producing T cells by means of an enzyme-linked immunospot assay. Sixty-eight individuals positive for M. tuberculosis infection, either human immunodeficiency virus-seropositive or -seronegative, were studied. The test results were highly specific (87.5%) and sensitive (93.1%), more so than a classical lymphoproliferative assay (specificity and sensitivity of 77.27%), opening new possibilities for diagnosis and screening of tuberculosis. Moreover, the test allowed us to distinguish individuals infected with M. tuberculosis from those vaccinated with BCG.

Clostridium difficile PCR Ribotype 018, a Successful Epidemic Genotype

ABSTRACT Clostridium difficile infection (CDI) became a public health problem for the global spreading of the so-called hypervirulent PCR ribotypes (RTs) 027 and 078, associated with increases in the transmission and severity of the disease. However, especially in Europe, several RTs are prevalent, and the concept of hypervirulence is currently debated. We investigated the toxin and resistance profiles and the genetic relatedness of 312 C. difficile strains isolated in a large Italian teaching hospital during a 5-year period. We evaluated the role of CDI-related antibiotic consumption and infection control practices on the RT predominance in association with their molecular features and transmission capacity. Excluding secondary cases due to nosocomial transmission, RT018 was the predominant genotype (42.4%) followed by RT078 (13.6%), while RT027 accounted for 0.8% of the strains. RT078 was most frequently isolated from patients in intensive care units. Its prevalence significantly increased over time, but its transmission capacity was very low. In contrast, RT018 was highly transmissible and accounted for 95.7% of the secondary cases. Patients with the RT018 genotype were significantly older than those with RT078 and other RTs, indicating an association between epidemic RT and age. We provide here the first epidemiological evidence to consider RT018 as a successful epidemic genotype that deserves more attention in clinical practice.

An In-House RD1-Based Enzyme-Linked Immunospot-Gamma Interferon Assay Instead of the Tuberculin Skin Test for Diagnosis of Latent Mycobacterium tuberculosis Infection

ABSTRACT Identification of individuals infected with Mycobacterium tuberculosis is essential for the control of tuberculosis (TB). The specificity of the currently used tuberculin skin test (TST) is poor because of the broad antigenic cross-reactivity of purified protein derivative (PPD) with BCG vaccine strains and environmental mycobacteria. Both ESAT-6 and CFP-10, two secretory proteins that are highly specific for M. tuberculosis complex, elicit strong T-cell responses in subjects with TB. Using an enzyme-linked immunospot (ELISPOT)-IFN-γ assay and a restricted pool of peptides derived from ESAT-6 and CFP-10, we have previously demonstrated a high degree of specificity and sensitivity of the test for the diagnosis of TB. Here, 119 contacts of individuals with contagious TB who underwent TST and the ELISPOT-IFN-γ assay were consecutively recruited. We compared the efficacy of the two tests in detecting latent TB infection and defined a more appropriate TST cutoff point. There was little agreement between the tests (k = 0.33, P < 0.0001): 53% of the contacts with a positive TST were ELISPOT negative, and 7% with a negative TST were ELISPOT positive. Furthermore, respectively 76 and 59% of the ELISPOT-negative contacts responded in vitro to BCG and PPD, suggesting that most of them were BCG vaccinated or infected with nontuberculous mycobacteria. The number of spot-forming cells significantly correlated with TST induration (P < 0.0001). Our in-house ELISPOT assay based on a restricted pool of highly selected peptides is more accurate than TST for identifying individuals with latent TB infection and could improve chemoprophylaxis for the control of TB.

Detection of Pneumocystis carinii and Characterization of Mutations Associated with Sulfa Resistance in Bronchoalveolar Lavage Samples from Human Immunodeficiency Virus-Infected Subjects

ABSTRACT One hundred ninety-four bronchoalveolar specimens were evaluated by microscopic examination and by amplification of a sequence of a Pneumocystis carinii dihidropteroate synthase gene for identification of mutations linked to sulfa resistance. PCR sensitivity and specificity were 100 and 86.7%, respectively, compared to results of microscopic examination. However, 7 out of 19 microscopy-negative, PCR-positive samples were collected from subjects with a clinically high probability of P. carinii pneumonia, suggesting that PCR may be more sensitive than microscopic examination, although the absolute performance of PCR cannot be determined. Mutations were identified in 28 out of 70 (40%) PCR-positive specimens and were significantly more common in patients exposed to sulfa drugs (21 out of 29 [72.4%]) than in those not exposed to sulfa drugs (4 out of 35 [11.4%]).

Severe community-onset infections in healthy individuals caused by community-acquired MRSA in an Italian teaching hospital, 2006–2008

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Direct Detection of Helicobacter pylori Mutations Associated with Macrolide Resistance in Gastric Biopsy Material Taken from Human Immunodeficiency Virus-Infected Subjects

ABSTRACT One hundred forty gastric biopsies were tested by microbiological methods and by amplifying a sequence of 23S rRNA and identifying mutations associated to clarithromycin resistance. Seventy-six specimens were positive for Helicobacter pylori. Mutational analysis revealed alterations in 18 (39.1%) of 46 and 2 (8.7%) of 23 samples from human immunodeficiency virus-seropositive and -seronegative persons, respectively. The results of the mutational analysis fully correlated with those of the susceptibility tests.

Epidemic MRSA clone ST22-IV is more resistant to multiple host- and environment-related stresses compared with ST228-I

BACKGROUND ST22-IV is a successful hospital-associated MRSA clone. Due to its known ability to replace other MRSA clones in hospitals, it became a dominant clone in Europe and beyond. So far, there are no studies investigating the relationship between the epidemiological success of MRSA clones and their capacity to withstand commonly encountered stresses. METHODS We investigated the fitness of ST22-IV in comparison with the replaced clone ST228-I, evaluating its resistance to oxidative stress, autolytic activity, growth at high osmolarity and in acid and alkaline environments and survival under desiccation and heat shock. We also compared their phenotypic characteristics and examined the impact of antibiotic consumption on epidemiological success. RESULTS Here we demonstrate that the dominance of ST22-IV is linked neither to changes in antibiotic consumption nor to acquisition of additional resistances over time. Strong α-haemolysin activity, the production of β-haemolysin and the presence of an active agr could partly explain the virulence of ST22-IV previously observed in a murine model of pneumonia. Most importantly, we show that ST22-IV compared with ST228-I, besides retaining susceptibility to most antibiotics over time, has a superior capacity to survive under all stress conditions tested, which bacteria commonly face during their life cycle. CONCLUSIONS Our results support our hypothesis that ST22-IV has a fitness advantage over ST228-I. This fitness advantage could have allowed ST22-IV to displace ST228-I without acquiring additional resistances and could help explain its epidemic success in hospital settings and its spread in Europe and beyond.