Evaluation of an automated nucleic acid extractor for hepatitis C virus load quantification.

The increasing use of molecular methods strongly motivates clinical laboratories to introduce automated nucleic acid extractors. We compared the easyMAG (bioMérieux) with a manual extraction method for hepatitis C virus (HCV) load quantification (RealTime HCV; Abbott). Both methods were comparable, and, therefore, the easyMAG is suitable to be implemented in our laboratory for the management of HCV-infected patients.

Evaluation of a new assay in comparison with reverse hybridization and sequencing methods for hepatitis C virus genotyping targeting both 5' noncoding and nonstructural 5b genomic regions.

We report the evaluation of a new real-time PCR assay for hepatitis C virus (HCV) genotyping. The assay design is such that genotype 1 isolates are typed by amplification targeting the nonstructural 5b (NS5b) subgenomic region. Non-genotype 1 isolates are typed by type-specific amplicon detection in the 5' noncoding region (5'NC) (method 1; HCV genotyping analyte-specific reagent assay). This method was compared with 5'NC reverse hybridization (method 2; InnoLiPA HCV II) and 5'NC sequencing (method 3; Trugene HCV 5'NC). Two hundred ninety-five sera were tested by method 1; 223 of them were also typed by method 2 and 89 by method 3. Sequencing and phylogenetic analysis of an NS5b fragment were used to resolve discrepant results. Suspected multiple-genotype infections were confirmed by PCR cloning and pyrosequencing. Even though a 2% rate of indeterminates was obtained with method 1, concordance at the genotype level with results with methods 2 and 3 was high. Among eight discordant results, five mixed infections were confirmed. Genotype 1 subtyping efficiencies were 100%, 77%, and 74% for methods 1, 2, and 3, respectively; there were 11/101 discordants between methods 1 and 2 (method 1 was predominantly correct) and 2/34 between methods 2 and 3. Regarding genotype 2, subtyping efficiencies were 100%, 45%, and 92% by methods 1, 2, and 3, respectively; NS5b sequencing of discordants (16/17) revealed a putative new subtype within genotype 2 and that most subtype calls were not correct. Although only sequencing-based methods provide the possibility of identifying new variants, the real-time PCR method is rapid, straightforward, and simple to interpret, thus providing a good single-step alternative to more-time-consuming assays.

Effect of the administration of fermentable and non-fermentable dietary fibre on intestinal bacterial translocation in ascitic cirrhotic rats.

BACKGROUND: Bacterial infections are frequent in cirrhosis. Experimental studies suggest a pathogenic role of intestinal bacterial translocation in them. Both fermentable and non-fermentable fibre avoided intestinal bacterial translocation (IBT) in animal models of gut starvation and critical illness. AIM: To assess the effect of fermentable (pectin) or non-fermentable (lignin) fibre on IBT in ascitic cirrhotic rats. METHODS: Thirty-six rats induced to cirrhosis with oral CCl4 were randomized (6 weeks after the first CCl4 dose) to receive rat chow+5% lignin (LIG, n=13), rat chow+5% pectin (PEC, n=13), or rat chow only (CON, n=10). Once ascites developed, animals were laparotomized and samples of mesenteric lymph nodes (MLN), ascitic fluid, portal and peripheral blood and liver, were obtained for culture. RESULTS: IBT rate was: LIG=5/13, PEC=4/13, CON=5/10 (P=N.S.). The median amount of translocated bacteria in rats with IBT was lower in the PEC group (2 x 10(2) CFU/g MLN), than in LIG (10(5) CFU/g MLN) and CON (10(4) CFU/g MLN) groups (P<0.05). All other samples were sterile except for a portal blood sample (Enterococcus faecalis) of the LIG group. CONCLUSIONS: IBT incidence is not decreased by either pectin or lignin in ascitic cirrhotic rats, but pectin supplementation reduces the amount of translocated bacteria.

Passive serum therapy with polyclonal antibodies against Mycobacterium tuberculosis protects against post-chemotherapy relapse of tuberculosis infection in SCID mice.

We investigated the protective role of immune-sera against reactivation of Mycobacterium tuberculosis infection in SCID mice and found that passive immunization with sera obtained from mice treated with detoxified M. tuberculosis extracts (delivered in liposomes in a composition known as RUTI) exerted significant protection. Our SCID mouse model consisted of aerosol infection by M. tuberculosis, followed by 3 to 8weeks of chemotherapy with isoniazid+rifampicin (INH+RIF) (25 and 10mg/kg, respectively). After infection and antibiotic administration, two groups of mice were treated for up to 10weeks with intraperitoneal passive immunization using hyperimmune serum (HS) obtained from mice infected with M. tuberculosis, treated with chemotherapy (INH+RIF) for 8weeks and inoculated with RUTI (HS group) or with normal serum (CT group). Significant differences were found between HS and CT groups in the number of bacilli in the lungs (3.68+/-2.02 vs. 5.72+/-1.41log(10) c.f.u.), extent of pulmonary granulomatomous infiltration (10.33+/-0.67 vs. 31.2+/-1.77%), and percentage of animals without pulmonary abscesses (16.7% vs. 45.5%). These data strongly suggest a protective role of specific antibodies against lung dissemination of M. tuberculosis infection.

Intragranulomatous necrosis in lungs of mice infected by aerosol with Mycobacterium tuberculosis is related to bacterial load rather than to any one cytokine or T cell type.

Low dose aerosol infection of C57BL/6 mice with a clinical strain of Mycobacterium tuberculosis (UTE 0335 R) induced intragranulomatous necrosis in pulmonary granulomas (INPG) at week 9 postinfection. Infection of different knockout (KO) mouse strains with UTE 0335 R induced INPG in all strains and established two histopathological patterns. The first pattern was seen in SCID mice and in mice with deleted alpha/beta T receptor, TNF R1, IL-12, IFN-gamma, or iNOS genes, and showed a massive INPG with a high granulomatous infiltration of the lung, a large and homogeneous eosinophilic necrosis full of acid-fast bacilli, with marked karyorrhexis, coarse basophilic necrosis, and surrounded by patches delimited by partially conserved alveolar septum full of PMNs. The second pattern was seen in mice with deleted IL-1 R1, IL-6, IL-10, CD4, CD8 or gamma/delta T cell receptor genes, and showed more discrete lesions with predominant homogeneous eosinophilic necrosis with few bacilli and surrounded by a well-defined lymphocyte-based ring. Local expression of IFN-gamma, iNOS, TNF and RANTES showed no significant differences between these mouse strains generating a discrete INPG. Mouse strains showing a massive INPG showed higher, lower or equal expression values compared to the control strain. In conclusion, the severity of the INPG pattern correlated with pulmonary CFU counts, irrespective of the genetic absence or the infection-induced levels of cytokine mediators.

Catalase-peroxidase activity has no influence on virulence in a murine model of tuberculosis.

The capacity to generate a chronic and persistent infection in the experimental murine model of tuberculosis induced aerogenically by a low-dose inoculum was determined in eight isoniazid-resistant clinical strains of Mycobacterium tuberculosis showing different catalase-peroxidase (C-P) activities. Determination of bacillary concentration in lung and spleen and the percentage of pulmonary parenchyma occupied by granulomas were monitored. Data showed no relation between the lack of C-P activity and the ability to develop a persistent infection, highlighting the potential of C-P negative strains to spread through the community.

Elevated serum procalcitonin values correlate with renal scarring in children with urinary tract infection.

BACKGROUND: Urinary tract infection (UTI) in young children carries the risk of parenchymal damage and sequelae. The location of the infection within the urinary tract influences decisions regarding both therapeutics and follow-up. Because clinical features and laboratory markers of infection at an early age are not specific, it is difficult to make a distinction between lower UTI and acute pyelonephritis. Procalcitonin (PCT) has been studied as a marker of severe bacterial infection. The aim of this study was to test the usefulness of PCT concentration in serum to distinguish between uncomplicated UTI and severe acute pyelonephritis with renal scars. METHODS: PCT was measured by immunoluminometric assay in serum samples from children with microbiologically documented infection. Severe renal involvement was assessed by 99mTc-dimercaptosuccinic acid gammagraphy done 5 to 6 months after the episode to check for the presence of parenchymal scars. C-reactive protein (CRP) and leukocyte count were also measured. RESULTS: PCT at presentation showed a significant correlation (P < 0.001) with the presence of renal scars in children with UTI. Using a cutoff of 1 ng/ml for PCT and 20 mg/l for CRP, sensitivity and specificity in distinguishing between urinary tract infection with and without renal damage were 92.3 and 61.9%, respectively, for PCT and 92.3 and 34.4% for CRP. Positive and negative predictive values were 32 and 97.5%, respectively, for PCT and 23 and 95%, respectively, for CRP. CONCLUSIONS: A low PCT value at admission indicates a low risk of long term renal scarring. Increased PCT values at admission correlate with the presence of scars. PCT values have proved to be more specific than CRP and leukocyte count for identifying patients who might develop renal damage.

Procalcitonin, C-reactive protein and leukocyte count in children with lower respiratory tract infection.

BACKGROUND: Lower respiratory tract infection is the most common infection leading to unnecessary antibiotic treatment in children. Etiologic diagnosis is not immediately achieved, and the pathogen remains unidentified in a large number of cases. Neither clinical nor laboratory factors allow for a rapid distinction between bacterial and viral etiology. The aim of our study was to evaluate the reliability of procalcitonin (PCT), C-reactive protein (CRP) and leukocyte count in distinguishing pneumococcal, atypical and viral lower respiratory tract infection. METHODS: PCT, CRP and leukocyte count were measured in children with microbiologically documented diagnoses of lower respiratory tract infection. The results were compared of children with pneumococcal, atypical and viral etiologies. RESULTS: PCT and CRP showed significant correlation with a bacterial etiology of lower respiratory tract infection. No significance was found for leukocyte count. Using a cutoff point of 2 ng/ml for PCT and 65 mg/l for CRP, the sensitivities and specificities for distinguishing bacterial from viral lower respiratory tract infections were 68.6 and 79.4% for PCT and 79.1 and 67.1% for CRP. The sensitivities and specificities for distinguishing pneumococcal from other etiologies were 90.3 and 74.1% for PCT and 90.3 and 60% for CRP, respectively. CONCLUSIONS: High PCT and CRP values show a significant correlation with the bacterial etiology of lower respiratory tract infection. PCT and CRP show good sensitivity for distinguishing pneumococcal from other etiologies. PCT shows higher specificity than CRP. PCT and CRP can help make decisions about antibiotic therapy in children with lower respiratory tract infections.

Accuracy of a commercially available assay for HCV genotyping and subtyping in the clinical practice.

BACKGROUND: Hepatitis C virus (HCV) genotyping is mandatory for tailoring dose and duration of pegylated interferon-α plus ribavirin treatment and for deciding on triple therapy eligibility. Additionally, subtyping may play a role in helping to select future treatment regimens that include directly-acting antivirals. However, commercial assays for HCV genotyping fail to identify the genotype/subtype in some cases. OBJECTIVE: Our aims were (i) to determine the success rate of the commercial genotyping assay Abbott RealTime HCV Genotype II at identifying the genotype and the HCV-1 subtype; and (ii) to phylogenetically characterise the obtained indeterminate results. STUDY DESIGN: HCV genotyping results obtained between 2009 and 2012 in a Spanish reference hospital were reviewed. A total of 896 people were genotyped with the Abbott RealTime HCV Genotype II assay. Specimens with an indeterminate result were retrospectively genotyped using the reference method based on the phylogenetic analysis of HCV NS5B sequences. RESULTS: Using the commercially available assay, an indeterminate HCV genotype result was obtained in 20 of 896 patients (2.2%); these corresponded to genotypes 3a, 3k and 4d. Importantly, 8.6% of all cases where genotype 3 was detected were indeterminate. In addition, the HCV-1 subtype was not assigned in 29 of 533 cases (5.4%). CONCLUSIONS: The implementation in the clinical microbiology laboratory of the reference method for HCV genotyping allows indeterminate genotype/subtype results to be interpreted and may lead to the identification of previously uncharacterised subtypes.

Prevalence of human immunodeficiency virus, Chlamydia trachomatis, and Neisseria gonorrhoeae and risk factors for sexually transmitted infections among immigrant female sex workers in Catalonia, Spain.

OBJECTIVES: To determine the prevalence of human immunodeficiency virus (HIV), Chlamydia trachomatis (CT), and Neisseria gonorrhoeae (NG) among immigrant female sex workers (FSW) according to their geographic area of origin and identify possible risk factors independently associated with current infection with CT and/or NG. STUDY DESIGN: Cross-sectional study of 357 FSW in Catalonia in 2005. Information on sociodemographic and sex work characteristics, use of alcohol and drugs, sexual practices, and the use of social and health care services was collected. Oral fluid and urine samples were collected to determine the prevalence of HIV and CT/NG, respectively. Factors independently associated with CT/NG were assessed using multivariate logistic regression models. RESULTS: A total of 36.4% of women were from Eastern Europe, 34.5% from Latin America, and 29.1% from Africa. Overall CT and NG prevalence were 5.9% [95% confidence interval (CI): 3.7-8.9] and 0.6% (95% CI: 0.1-2.0), respectively. No differences were observed by geographic origin. Three African women were HIV positive (overall HIV prevalence was 0.8%, 95% CI: 0.2-2.4). In multivariate analysis, younger age and unprotected sex with clients were associated with the presence of CT/NG. CONCLUSIONS: The prevalence of sexually transmitted infections among FSW in Catalonia was lower than in other European countries. Even though the prevalence of HIV was only 0.8%, it could increase in the future given the high vulnerability of these women and their wide geographic mobility. It is necessary to continue with the work carried out by nongovernmental organizations (harm reduction programs, outreach programs, and safe sex workshops) as well as to facilitate the access to health centers, especially for the youngest women.

Evaluation of procalcitonin, neopterin, C-reactive protein, IL-6 and IL-8 as a diagnostic marker of infection in patients with febrile neutropenia.

Infectious complications in neutropenic patients are a major cause of morbidity and mortality. Clinical signs are unspecific and fever can be attributed to other causes. Inflammatory biomarkers have emerged as potentially useful in diagnosis of bacterial and fungal infection. Levels of several biomarkers were measured in patients with hematological malignancy at diagnosis and at the beginning of neutropenia due to cytostatic treatment or after hematopoietic stem cell transplantation, and daily until 6 days after presenting fever. Procalcitonin (PCT) and neopterin levels were not elevated at diagnosis or at the beginning of neutropenia. C-reactive protein (CRP) was moderately elevated. PCT levels were significantly higher in patients with Gram-negative bacteremia at 24-48 h after the onset of fever. Patients with probable fungal infection presented elevated PCT values when fever persisted for more than 4-5 days. CRP was more sensitive to predict bacteremia (both Gram-positive and Gram-negative) but the specificity was low. Neither neopterin, IL-6 nor IL-8 presented significant differences according to the origin or etiology of fever. Since it showed a high negative predictive value of Gram-negative bacteremia, clinical prediction rules that attempt to predict a high risk of severe infection might be improved by including measurement of PCT.

Intragranulomatous necrosis in pulmonary granulomas is not related to resistance against Mycobacterium tuberculosis infection in experimental murine models induced by aerosol.

Intragranulomatous necrosis is a primary feature in the natural history of human tuberculosis (TB). Unfortunately, this phenomenon is not usually seen in the experimental TB murine model. Artificial induction of this necrosis in pulmonary granulomas (INPG) may be achieved through aerosol inoculation of lipopolysaccharide (LPS) 3 weeks after Mycobacterium tuberculosis infection. At week 9 post-infection, the centre of primary granulomas became larger, showing eosinophilic necrosis. Interestingly, INPG induction was related to mice strains C57BL/6 and 129/Sv, but not to BALB/c and DBA/2. Furthermore, the same pattern was obtained with the induction of infection using a clinical M. tuberculosis strain (UTE 0335R) that naturally induces INPG. In all the mice strains tested, the study of pulmonary mRNA expression revealed a tendency to increase or to maintain the expression of RANTES, interferon-gamma, tumour necrosis factor and iNOS, in both LPS- and UTE 0335R-induced INPG, thus suggesting that this response must be necessary but not sufficient for inducing INPG. Our work supports that INPG induction is a local phenomenon unrelated to the resistant (C57BL/6 and BALB/c) or susceptible (129/Sv and DBA/2) background of mice strains against M. tuberculosis infection.

Comparison of the avidity index method and the serologic testing algorithm for recent human immunodeficiency virus (HIV) seroconversion, two methods using a single serum sample for identification of recent HIV infections.

A study was designed to compare an avidity index method to the serologic testing algorithm for recent human immunodeficiency virus (HIV) seroconversion (STARHS) for the detection of recent HIV infection. One hundred sixty HIV-positive sera were tested. Both techniques performed similarly in identifying recent infections, although STARHS tended to misclassify more individuals that had long-standing infection as being recently infected.

Immunotherapy with fragmented Mycobacterium tuberculosis cells increases the effectiveness of chemotherapy against a chronical infection in a murine model of tuberculosis.

Reduction of colony forming units by rifampicin-isoniazid therapy given 9-17 weeks post-infection was made more pronounced by immunotherapy with a vaccine made of fragmented Mycobacterium tuberculosis cells detoxified and liposomed (RUTI), given on weeks 17, 19 and 21 post-infection, in the murine model of tuberculosis in C57BL/6 and DBA/2 inbred strains. RUTI triggered a Th1/Th2 response, as demonstrated by the production of IgG1, IgG2a and IgG3 antibodies against a wide range of peptides. The histological analysis did not show neither eosinophilia nor necrosis, and granulomatous infiltration was only slightly increased in C57BL/6 mice when RUTI was administered intranasally.

Utility of a pneumonia severity index in the optimization of the diagnostic and therapeutic effort for community-acquired pneumonia.

The objective was to evaluate the utility of the Pneumonia Severity Index (PSI) developed by Fine et al. as a tool to streamline diagnostic and therapeutic effort. Site of care of patients was recommended in accordance with the PSI class: classes I and II underwent treatment at home and classes III, IV, and V were hospitalized. Class I comprised 37 patients; class II had 30, class III had 20, class IV had 31, and class V had 10 patients. 80 patients were admitted into the hospital, 3 of whom required admittance to the intensive care unit, and 48 were managed as outpatients from the emergency room. Overall mortality was 4 patients (3.1%). Of these, 3 belonged to class IV and 1 to class V. The aetiological diagnosis was obtained in 53.9% of the cases (69/128). If classes I to III are analysed together, the percentage of aetiological diagnoses was 47% (41/87), increasing to 68% (28/41) for patients in classes IV and V. In our experience Fine's PSI classification, with rationalization and adaptation to the particularities of each centre, is an effective tool for deciding on hospitalization for selecting the most suitable battery of diagnostic tests based on cost-benefit criteria. However, it is inadequate for young patients with hypoxia or pleural effusion. Therefore, although hospitalization of patients with pneumonia should be mainly based on clinical criteria, Fine's PSI classification could help physicians in making more rational decisions in this respect.

Widespread bronchogenic dissemination makes DBA/2 mice more susceptible than C57BL/6 mice to experimental aerosol infection with Mycobacterium tuberculosis.

We have used the murine model of aerosol-induced experimental tuberculosis to assess the effects of four clinical isolates and a reference strain of Mycobacterium tuberculosis on resistant C57BL/6 mice and susceptible DBA/2 mice. Histological studies and detection of 25 cytokines potentially involved in the infection were carried out. DBA/2 mice showed higher concentrations of bacilli in bronchoalveolar lavage fluid and lung tissue. Furthermore, these mice evidenced a larger granulomatous infiltration in the parenchyma due to an increased rate of emigration of infected foamy macrophages from the granulomas to the neighboring pulmonary alveolar spaces. The better control of bacillary concentrations and pulmonary infiltration observed in C57BL/6 mice from week 3 postinfection could result from their higher RANTES, ICAM-1, and gamma interferon (IFN-gamma) mRNA levels. On the other hand, the higher MIP-2 and MCP-3 mRNA levels seen in DBA/2 mice would result in stronger lung recruitment of macrophages and neutrophils. Additionally, DBA/2 mice showed increased inducible nitric oxide synthase expression, induced by the larger number of foamy macrophages, at weeks 18 and 22. This increment was a consequence of phagocytosed bacillary debris, was independent of IFN-gamma expression, and could exert only a bacteriostatic effect. The results of the study suggest that DBA/2 mice are more susceptible than C57BL/6 mice to M. tuberculosis infection due to a higher bronchial dissemination of bacilli inside poorly activated foamy macrophages.