Nino Manca

Development of a Real-Time PCR Assay for Detection of Plasmodium falciparum, Plasmodium vivax, and Plasmodium ovale for Routine Clinical Diagnosis

ABSTRACT A TaqMan-based real-time PCR qualitative assay for the detection of three species of malaria parasites—Plasmodium falciparum, P. ovale, and P. vivax—was devised and evaluated using 122 whole-blood samples from patients who had traveled to areas where malaria is endemic and who presented with malaria-like symptoms and fever. The assay was compared to conventional microscopy and to an established nested-PCR assay. The specificity of the new assay was confirmed by sequencing the PCR products from all the positive samples and by the lack of cross-reactivity with Toxoplasma gondii and Leishmania infantum DNA. Real-time PCR assay showed a detection limit (analytical sensitivity) of 0.7, 4, and 1.5 parasites/μl for P. falciparum, P. vivax, and P. ovale, respectively. Real-time PCR, like nested PCR, brought to light errors in the species identification by microscopic examination and revealed the presence of mixed infections (P. falciparum plus P. ovale). Real-time PCR can yield results within 2 h, does not require post-PCR processing, reduces sample handling, and minimizes the risks of contamination. The assay can therefore be easily implemented in routine diagnostic malaria tests. Future studies are warranted to investigate the clinical value of this technique.

Comparison of HIV-1 genotypic resistance test interpretation systems in predicting virological outcomes over time

Background Several decision support systems have been developed to interpret HIV-1 drug resistance genotyping results. This study compares the ability of the most commonly used systems (ANRS, Rega, and Stanfords HIVdb) to predict virological outcome at 12, 24, and 48 weeks. Methodology/Principal Findings Included were 3763 treatment-change episodes (TCEs) for which a HIV-1 genotype was available at the time of changing treatment with at least one follow-up viral load measurement. Genotypic susceptibility scores for the active regimens were calculated using scores defined by each interpretation system. Using logistic regression, we determined the association between the genotypic susceptibility score and proportion of TCEs having an undetectable viral load (<50 copies/ml) at 12 (8–16) weeks (2152 TCEs), 24 (16–32) weeks (2570 TCEs), and 48 (44–52) weeks (1083 TCEs). The Area under the ROC curve was calculated using a 10-fold cross-validation to compare the different interpretation systems regarding the sensitivity and specificity for predicting undetectable viral load. The mean genotypic susceptibility score of the systems was slightly smaller for HIVdb, with 1.92±1.17, compared to Rega and ANRS, with 2.22±1.09 and 2.23±1.05, respectively. However, similar odds ratios were found for the association between each-unit increase in genotypic susceptibility score and undetectable viral load at week 12; 1.6 [95% confidence interval 1.5–1.7] for HIVdb, 1.7 [1.5–1.8] for ANRS, and 1.7 [1.9–1.6] for Rega. Odds ratios increased over time, but remained comparable (odds ratios ranging between 1.9–2.1 at 24 weeks and 1.9–2.2 at 48 weeks). The Area under the curve of the ROC did not differ between the systems at all time points; p = 0.60 at week 12, p = 0.71 at week 24, and p = 0.97 at week 48. Conclusions/Significance Three commonly used HIV drug resistance interpretation systems ANRS, Rega and HIVdb predict virological response at 12, 24, and 48 weeks, after change of treatment to the same extent.

Genetic Polymorphisms Influence Plasmodium ovale PCR Detection Accuracy

ABSTRACT Detection of Plasmodium ovale by use of a nested PCR assay with a novel Plasmodium ovale primer set was superior to detection of Plasmodium ovale by real-time PCR assays. Nested PCR was also better at detecting P. malariae. The detection of P. ovale in many patients first admitted >2 months following their return to Italy indicated that P. ovale relapses are common.

Hepatitis C virus core antigen: Analytical performances, correlation with viremia and potential applications of a quantitative, automated immunoassay

BACKGROUND Testing for hepatitis C virus core antigen (HCV Ag) may represent a complementary tool to anti-HCV and HCV-RNA in the diagnosis and monitoring of HCV infection. OBJECTIVE To evaluate the performance characteristics of the automated Abbott ARCHITECT HCV Ag assay. STUDY DESIGN Five sites analyzed over 3000 routine serum samples from populations at different risk, comparing HCV Ag results with anti-HCV screening and supplemental assay results and with HCV-RNA. RESULTS The HCV Ag assay showed a specificity of 100%, a good precision (CV<10%) and excellent dilution linearity (r>0.999). The sensitivity (3 fmol/L) corresponds to 700-1100 IU/mL of HCV-RNA. A non-linear correlation with HCV-RNA was found: r=0.713 vs. Siemens bDNA (523 specimens), r=0.736 vs. Roche Cobas TaqMan (356 specimens) and r=0.870 vs. Abbott Real-Time PCR (273 specimens). HCV Ag quantitation was equally effective on different HCV genoypes (239 for genotype 1/1a/1b/1c, 108 for genotype 2/2a/2c, 86 for genotype 3/3a, 50 for genotype 4/4a/4c/4d). Testing of subjects at high risk for HCV and with potential or actual impairment of the immune system identified 2 cases negative for anti-HCV and positive for HCV Ag on 361 hemodialyzed (0.6%) and 7 cases on 97 (7.2%) among transplant recipients. HCV Ag positivity anticipated anti-HCV seroconversion in all three cases of acute hepatitis C. CONCLUSIONS HCV Ag may be used as reflex testing on anti-HCV positive individuals to confirm or exclude an active infection, and on subjects with acute hepatitis or belonging to high risk groups.

P. aeruginosa bloodstream infections among hematological patients: an old or new question?

Pseudomonas aeruginosa is a well-known cause of severe and potentially life-threatening infections among hematological patients. A prospective epidemiological surveillance program ongoing at our Hematology Unit revealed an increase over time of P. aeruginosa bloodstream infections (BSI). Their impact on outcome and antibiotic susceptibility was analyzed. BSI which consecutively occurred at our institution during a 70-month period were evaluated and correlated with type of pathogen, status of underlying disease, neutropenia, previous antibiotic therapy, resistance to antibiotics, and outcome. During the observation period, 441 BSI were recorded. Frequency of Gram-negative BSI was higher than that of other pathogens (57.3%). Overall, 66 P. aeruginosa BSI were recorded; 22 out of 66 were multiresistant (MR P. aeruginosa). Thirty-day mortality for all BSI was 11.3%; it was 27.3% for P. aeruginosa BSI and 36.4% for MR P. aeruginosa. At multivariate analysis, only active hematological disease and P. aeruginosa BSI were associated to an increased risk of death. For MR P. aeruginosa, BSI mortality was 83.3% vs. 18.8% when empiric therapy included or not an antibiotic with in vitro activity against P. aeruginosa (p = 0.011). Together with active disease, the emergence of P. aeruginosa BSI, particularly if multiresistant, was responsible for an increased risk of death among hematological patients at our institution. In this scenario, reconsidering the type of combination antibiotic therapy to be used as empiric treatment of neutropenic fever was worthwhile.

Evaluation of a real-time polymerase chain reaction assay for the laboratory diagnosis of giardiasis.

A real-time polymerase chain reaction (PCR) assay was evaluated in comparison with the combination of conventional methods (microscopic examination and antigen detection assay) during the period 2006 to 2008 on 771 fecal samples belonging to 386 patients to assess its usefulness for an accurate laboratory diagnosis of giardiasis. The real-time PCR assay detected Giardia intestinalis DNA in 195 samples (106 patients), including 26 samples (21 patients) negative by the conventional assays. Among the 21 patients, in 8 cases, giardiasis was previously diagnosed also by conventional methods in additional samples of the same patients, whereas in 13, it would have been undiagnosed if real-time PCR assay was not used. The real-time PCR assay demonstrated a detection limit of 2 cysts per reaction and 100% specificity and sensitivity compared to conventional methods. A genotype analysis targeting the beta-giardin gene allowed to identify 53 samples (23 patients) containing genotype A and 59 samples (45 patients) containing genotype B.

Recent increase in enterococci, viridans streptococci, Pseudomonas spp. and multiresistant strains among haematological patients, with a negative impact on outcome. Results of a 3-year surveillance study at a single institution

Abstract We prospectively analysed the microbiological isolates of all febrile/infectious episodes occurring at our haematology unit during 2 consecutive 18-month periods. Microbiologically documented infections (MDI) and antibiotic resistance were correlated with type and status of haematological disease, neutropenia, levofloxacin prophylaxis, central venous catheter and clinical outcome. Three hundred and ten MDI were observed and 369 pathogens were isolated. Gram-negative bacteria represented 49.3% and Gram-positive bacteria 40.9% of all pathogens. Fungal infections represented only 8.9% of MDI. A significant decrease in Staphylococcus aureus (p < 0.001) and an increase in enterococci, viridans streptococci and Pseudomonas spp. (p = 0.004) were observed during the second period. Four multiresistant (Multi-R) Pseudomonas were isolated, all during the last 12 months. The death rate in MDI was 8.7%, bacteria accounting for 70.4% of them. Enterococci, streptococci and Pseudomonas spp. infections were involved in 44.4% of MDI with an unfavourable outcome. Multi-R pathogens were involved in 4 cases (3 vancomycin-resistant enterococci and 1 Multi-R Pseudomonas), their death rate being 25%. Multivariate analysis showed that an infection due to a mycotic or a Multi-R pathogen was associated with an unfavourable outcome. The recent emergence of enterococci, viridans streptococci and Pseudomonas spp., particularly if Multi-R, is a major concern in haematological patients.

Avidity Index for Anti-HIV Antibodies: Comparison between Third- and Fourth-Generation Automated Immunoassays

ABSTRACT The development of assays for detecting recent HIV infections has become crucial for analyzing trends in infection in different populations, both for surveillance and prevention activities. The anti-HIV avidity index (AI), measured with third-generation immunoassays (which detect anti-HIV antibody), has been shown to be an accurate tool for discriminating recent HIV infections (<6 months) from established infections (≥6 months). We compared a third-generation immunoassay (AxSYM HIV 1/2 gO; Abbott Diagnostics) to a fourth-generation immunoassay (Architect HIV Ag/Ab Combo; Abbott Diagnostics; which detects anti-HIV antibody and p24 antigen) in terms of AI performance in distinguishing between recent and established HIV infections. A total of 142 samples from 75 HIV-infected individuals with an estimated date of seroconversion were assayed. The two assays showed the same accuracy in identifying a recent infection (91.5%), using an AI cutoff of 0.80, although Architect HIV Ag/Ab Combo was slightly more sensitive (89.4% versus 84.8%; P > 0.05) and yet less specific (93.4% versus 97.4%; P > 0.05). The correlation between assays was high (r = 0.87). When 20 specimens falling in the gray zone around the cutoff point (0.75 ≤ AI ≤ 0.84) were excluded, the accuracy of AI with Architect HIV Ag/Ab Combo was 94.7%, and the concordance between the two assays was 99.2%. The anti-HIV AI is a serological marker that accurately discriminates recent from established HIV infections. It can be successfully applied on fully automated fourth-generation HIV Ab/Ag immunoassays, which have several advantages, including increased throughput, high reproducibility, no need for specific technical skills, and easy comparability of results obtained in different settings.

Evaluation of a real-time polymerase chain reaction assay for the detection of Dientamoeba fragilis ☆

The diagnostic value of a real-time polymerase chain reaction (PCR) assay targeting the 5.8S rDNA of Dientamoeba fragilis was investigated as compared with conventional parasitologic methods including cultivation testing 959 fecal samples from 491 patients attending a tertiary-care hospital and suspected of having an intestinal parasitosis. The real-time PCR assay revealed 117 additional D. fragilis-positive samples as compared with conventional methods, showing 100% sensitivity and specificity in our experience. On the whole, D. fragilis infection was detected in 186 samples from 105 patients (21.4%, third in frequency among the diagnosed intestinal parasitoses). The evaluated real-time PCR assay represents an effective tool to obtain both an accurate diagnosis and a reliable epidemiologic picture of dientamoebiasis.

Rapid detection and identification of Brachyspira aalborgi from rectal biopsies and faeces of a patient

This study reports for the first time the detection of Brachyspira aalborgi in faeces and rectal biopsies of a female suffering for 3-4 months of abdominal pain with long-standing mucosal diarrhoea, rectal bleeding and suspected carcinoma of the rectum. After pre-treatment of samples (faeces and biopsies) with a liquid medium (trypticase soy broth-TSB) containing foetal calf serum (FCS, 10%) and spectinomycin and rifampicin (TSB-SR) the first detection of B. aalborgi isolate HBS1 was observed after 48 h in the primary plates of selective blood agar modified medium (BAM) containing spectinomycin and rifampicin (BAM-SR), where growth zones were signalled by a small weakly beta-haemolytic halo. Attempts to subculture spirochaetes in agar media failed. The new HBS1 isolate was only propagated in TSB broth and at electron microscopy it showed 4 endoflagella inserted at each tapered end. The phenotypic characterization of HBS1 demonstrated absence of hippurate hydrolysis, indole production, alpha-galactosidase, alpha- and beta-glucosidase activities in accordance with the B. aalborgi type strain. Rapid identification of B. aalborgi isolate HBS1 was performed directly from faeces and rectal biopsies and subsequently from pure cultures by a genetic method based on 16S DNA restriction fragment length polymorphism (RFLP)-polymerase chain reaction (PCR). The sequence of 16S DNA amplicon of the isolate HBS1 was found 99.2% corresponding to that of the B. aalborgi type strain. Our results encourage further investigations for the development of a suitable selective agar medium for the isolating and cultivating B. aalborgi from human specimens.