Comparison of Löwenstein-Jensen and BACTEC MGIT 960 culture for Mycobacterium tuberculosis in people living with HIV.

OBJECTIVES: The aim of the study was to clarify how HIV infection affects tuberculosis liquid and solid culture results in a resource-limited setting. METHODS: We used baseline data from the Study on Outcomes Related to Tuberculosis and HIV Drug Concentrations in Uganda (SOUTH), which included 268 HIV/tuberculosis (TB)-coinfected individuals. Culture results from Löwenstein-Jensen (LJ) solid culture and mycobacteria growth indicator tube (MGIT) liquid culture systems and culture-based correlates for bacillary density from the sputum of HIV/TB-coinfected individuals at baseline were analysed. RESULTS: Of 268 participants, 243 had a CD4 cell count available and were included in this analysis; 72.2% of cultures showed growth on solid culture and 82.2% in liquid culture systems (P < 0.015). A higher CD4 cell count was predictive of LJ positivity [adjusted odds ratio (OR) 1.14; 95% confidence interval (CI) 1.03-1.25 per 50 cells/µL increase; P = 0.008]. The same, but insignificant trend was observed for MGIT positivity (adjusted OR 1.09; 95% CI 0.99-1.211 per 50 cells/µL increase; P = 0.094). A higher CD4 cell count was associated with a higher LJ colony-forming unit grade (adjusted OR 1.14; 95% CI 1.05-1.25 per 50 cells/µL increase; P = 0.011) and a shorter time to MGIT positivity [adjusted hazard ratio (HR) 1.08; 95% CI 1.04-1.12 per 50 cells/µL increase; P < 0.001]. CONCLUSIONS: In a resource-limited setting, the MGIT liquid culture system outperformed LJ solid culture in terms of culture yield and dependence on CD4 cell counts in HIV/TB-coinfected individuals. We therefore suggest considering an adaptation of diagnostic algorithms: when resources allow only one culture method to be performed, we recommend that MGIT liquid culture should be used exclusively in HIV-positive individuals as a first-line culture method, to reduce costs and make TB culture results accessible to more patients in resource-limited settings.

Persistence of the efficacy of zoster vaccine in the shingles prevention study and the short-term persistence substudy.

BACKGROUND: The Shingles Prevention Study (SPS; Department of Veterans Affairs Cooperative Study 403) demonstrated that zoster vaccine was efficacious through 4 years after vaccination. The Short-Term Persistence Substudy (STPS) was initiated after the SPS to further assess the persistence of vaccine efficacy. METHODS: The STPS re-enrolled 7320 vaccine and 6950 placebo recipients from the 38 546-subject SPS population. Methods of surveillance, case determination, and follow-up were analogous to those in the SPS. Vaccine efficacy for herpes zoster (HZ) burden of illness, incidence of postherpetic neuralgia (PHN), and incidence of HZ were assessed for the STPS population, for the combined SPS and STPS populations, and for each year through year 7 after vaccination. RESULTS: In the STPS as compared to the SPS, vaccine efficacy for HZ burden of illness decreased from 61.1% to 50.1%, vaccine efficacy for the incidence of PHN decreased from 66.5% to 60.1%, and vaccine efficacy for the incidence of HZ decreased from 51.3% to 39.6%, although the differences were not statistically significant. Analysis of vaccine efficacy in each year after vaccination for all 3 outcomes showed a decrease in vaccine efficacy after year 1, with a further decline thereafter. Vaccine efficacy was statistically significant for the incidence of HZ and the HZ burden of illness through year 5. CONCLUSIONS: Vaccine efficacy for each study outcome was lower in the STPS than in the SPS. There is evidence of the persistence of vaccine efficacy through year 5 after vaccination but, vaccine efficacy is uncertain beyond that point.

The urinary antibiotic 5-nitro-8-hydroxyquinoline (Nitroxoline) reduces the formation and induces the dispersal of Pseudomonas aeruginosa biofilms by chelation of iron and zinc.

Since cations have been reported as essential regulators of biofilm, we investigated the potential of the broad-spectrum antimicrobial and cation-chelator nitroxoline as an antibiofilm agent. Biofilm mass synthesis was reduced by up to 80% at sub-MIC nitroxoline concentrations in Pseudomonas aeruginosa, and structures formed were reticulate rather than compact. In preformed biofilms, viable cell counts were reduced by 4 logs at therapeutic concentrations. Complexation of iron and zinc was demonstrated to underlie nitroxoline's potent antibiofilm activity.

International Society of Cardiovascular Infectious Diseases Guidelines for the Diagnosis, Treatment and Prevention of Disseminated Mycobacterium chimaera Infection Following Cardiac Surgery with Cardiopulmonary Bypass.

Mycobacterial infection-related morbidity and mortality in patients following cardiopulmonary bypass surgery is high and there is a growing need for a consensus-based expert opinion to provide international guidance for diagnosing, preventing and treating in these patients. In this document the International Society for Cardiovascular Infectious Diseases (ISCVID) covers aspects of prevention (field of hospital epidemiology), clinical management (infectious disease specialists, cardiac surgeons, ophthalmologists, others), laboratory diagnostics (microbiologists, molecular diagnostics), device management (perfusionists, cardiac surgeons) and public health aspects.

High-throughput screening of bacterial pathogens in clinical specimens using 16S rDNA qPCR and fragment analysis.

Molecular-based detection of bacterial pathogens directly from clinical specimens permits rapid initiation of effective antimicrobial treatment and adequate patient management. Broad-range polymerase chain reaction (PCR) amplification of the 16S rRNA gene (16S rDNA qPCR) is used in many diagnostic laboratories as a complement to cultural identification of bacterial pathogens. However, efforts for automation of 16S rDNA PCR workflows are needed in order to reduce turnaround times and to enhance reproducibility and standardization of the technique. In this retrospective method evaluation study, clinical specimens (N = 499) from patients with suspected bacterial infections were used to evaluate 2 diagnostic semiautomated workflows for rapid bacterial pathogen detection. The workflows included automated DNA extraction (QIASymphony), 16S rDNA qPCR, fragment or melting curve analysis, and amplicon sequencing. Our results support the use of the 16S rDNA qPCR and fragment analysis workflow as it enabled rapid and accurate identification of bacterial pathogens in clinical specimens.

Evaluation of Lightmix Mycoplasma macrolide assay for detection of macrolide-resistant Mycoplasma pneumoniae in pneumonia patients.

OBJECTIVES: Rapid detection of macrolide resistance-associated mutations in Mycoplasma pneumoniae is crucial for effective antimicrobial treatment. We evaluated the Lightmix Mycoplasma macrolide assay for the detection of point mutations at nucleotide positions 2063 and 2064 in the 23S ribosomal RNA (rRNA) gene of M. pneumoniae that confer macrolide resistance. METHODS: Samples from 3438 patients with a respiratory tract infection were analysed by M. pneumoniae real-time PCR, and 208 (6%) of them were tested positive. In this retrospective study, 163 M. pneumoniae real-time PCR-positive samples were analysed by the Lightmix assay, and results were compared to targeted 23S rRNA sequencing. RESULTS: Macrolide-resistant M. pneumoniae were found in 15 (9%) of 163 retrospectively analysed samples. The Lightmix assay showed a sensitivity of 100% (95% confidence interval, 78.2-100) and a specificity of 100% (95% confidence interval, 97.5-100) as the detected M. pneumoniae genotype (148 wild type and 15 non-wild type) was confirmed by 23S rRNA sequencing in all samples. CONCLUSIONS: The Lightmix assay is an easy-to-use and accurate molecular test that allows rapid determination of macrolide resistance in M. pneumoniae.

A vaccine to prevent herpes zoster and postherpetic neuralgia in older adults.

BACKGROUND: The incidence and severity of herpes zoster and postherpetic neuralgia increase with age in association with a progressive decline in cell-mediated immunity to varicella-zoster virus (VZV). We tested the hypothesis that vaccination against VZV would decrease the incidence, severity, or both of herpes zoster and postherpetic neuralgia among older adults. METHODS: We enrolled 38,546 adults 60 years of age or older in a randomized, double-blind, placebo-controlled trial of an investigational live attenuated Oka/Merck VZV vaccine ("zoster vaccine"). Herpes zoster was diagnosed according to clinical and laboratory criteria. The pain and discomfort associated with herpes zoster were measured repeatedly for six months. The primary end point was the burden of illness due to herpes zoster, a measure affected by the incidence, severity, and duration of the associated pain and discomfort. The secondary end point was the incidence of postherpetic neuralgia. RESULTS: More than 95 percent of the subjects continued in the study to its completion, with a median of 3.12 years of surveillance for herpes zoster. A total of 957 confirmed cases of herpes zoster (315 among vaccine recipients and 642 among placebo recipients) and 107 cases of postherpetic neuralgia (27 among vaccine recipients and 80 among placebo recipients) were included in the efficacy analysis. The use of the zoster vaccine reduced the burden of illness due to herpes zoster by 61.1 percent (P<0.001), reduced the incidence of postherpetic neuralgia by 66.5 percent (P<0.001), and reduced the incidence of herpes zoster by 51.3 percent (P<0.001). Reactions at the injection site were more frequent among vaccine recipients but were generally mild. CONCLUSIONS: The zoster vaccine markedly reduced morbidity from herpes zoster and postherpetic neuralgia among older adults.

Molecular detection of fungal pathogens in clinical specimens by 18S rDNA high-throughput screening in comparison to ITS PCR and culture.

The rising incidence of invasive fungal infections and the expanding spectrum of fungal pathogens makes early and accurate identification of the causative pathogen a daunting task. Diagnostics using molecular markers enable rapid identification of fungi, offer new insights into infectious disease dynamics, and open new possibilities for infectious disease control and prevention. We performed a retrospective study using clinical specimens (N = 233) from patients with suspected fungal infection previously subjected to culture and/or internal transcribed spacer (ITS) PCR. We used these specimens to evaluate a high-throughput screening method for fungal detection using automated DNA extraction (QIASymphony), fungal ribosomal small subunit (18S) rDNA RT-PCR and amplicon sequencing. Fungal sequences were compared with sequences from the curated, commercially available SmartGene IDNS database for pathogen identification. Concordance between 18S rDNA RT-PCR and culture results was 91%, and congruence between 18S rDNA RT-PCR and ITS PCR results was 94%. In addition, 18S rDNA RT-PCR and Sanger sequencing detected fungal pathogens in culture negative (N = 13) and ITS PCR negative specimens (N = 12) from patients with a clinically confirmed fungal infection. Our results support the use of the 18S rDNA RT-PCR diagnostic workflow for rapid and accurate identification of fungal pathogens in clinical specimens.

Detection limit of Mycobacterium chimaera in water samples for monitoring medical device safety: insights from a pilot experimental series.

BACKGROUND: A growing number of Mycobacterium chimaera infections after cardiosurgery have been reported by several countries. These potentially fatal infections were traced back to contaminated heater-cooler devices (HCDs), which use water as a heat transfer medium. Aerosolization of water contaminated with M. chimaera from HCDs enables airborne transmission to patients undergoing open chest surgery. Infection control teams test HCD water samples for mycobacterial growth to guide preventive measures. The detection limit of M. chimaera in water samples, however, has not previously been investigated. AIM: To determine the detection limit of M. chimaera in water samples using laboratory-based serial dilution tests. METHODS: An M. chimaera strain representative of the international cardiosurgery-associated M. chimaera outbreak was used to generate a logarithmic dilution series. Two different water volumes, 50 and 1000mL, were inoculated, and, after identical processing (centrifugation, decantation, and decontamination), seeded on mycobacteria growth indicator tube (MGIT) and Middlebrook 7H11 solid media. FINDINGS: MGIT consistently showed a lower detection limit than 7H11 solid media, corresponding to a detection limit of ≥1.44 × 104cfu/mL for 50mL and ≥2.4cfu/mL for 1000mL water samples. Solid media failed to detect M. chimaera in 50mL water samples. CONCLUSION: Depending on water volume and culture method, major differences exist in the detection limit of M. chimaera. In terms of sensitivity, 1000mL water samples in MGIT media performed best. Our results have important implications for infection prevention and control strategies in mitigation of the M. chimaera outbreak and healthcare water safety in general.

Cloning and expression of cDNA encoding antistasin, a leech-derived protein having anti-coagulant and anti-metastatic properties.

As a factor Xa inhibitor, antistasin is a potent anti-coagulant and anti-metastatic agent that is found in the salivary gland of the Mexican leech Haementaria officinalis. cDNA clones that encode antistasin have been isolated. Subsequent sequence analysis and comparison with the amino acid sequence of the mature protein indicates that antistasin is produced as a pre-protein containing a 17-amino acid signal peptide. Antistasin exists as at least two variants. By sequence analysis of multiple cDNA clones, we found two additional sites for amino acid substitutions, confirming variants that differ from each other by amino acid changes at a minimum of four residues. These sequence variations appear to be the result of allelic variation rather than gene duplication as deduced from DNA blot analyses. Sequence data suggest that antistasin may have evolved from a smaller ancestral gene by a duplication event giving rise to a two-fold structural homology between the N- and C-terminal halves of the molecule. Insect cells transfected with a recombinant baculovirus expressed antistasin which was biologically active and had an electrophoretic mobility identical to that of the native molecule.

Modifications on the heterocyclic base of acyclovir: syntheses and antiviral properties.

A group of compounds was prepared in which variations of the ring portion of the acyclovir (ACV) structure were made. These modifications included monocyclic (isocytosine, triazole, imidazole), bicyclic (8-azapurine, pyrrolo[2,3-d]pyrimidine, pyrazolo[3,4-d]pyrimidine) and tricyclic (linear benzoguanine) congeners. The derivatives were evaluated against herpes simplex virus type 1 (HSV-1) by the plaque-inhibition and plaque-reduction methods with only the 8-azapurine analogue 28 showing some activity. In a test measuring the ability of these compounds to inhibit the HSV-1 thymidine kinase, 28 and the tricyclic derivative 38 exhibited competition with ACV for binding to the enzyme. The inability of the group to exert significant antiherpetic action is attributed to their lack of phosphorylation to the requisite triphosphate stage.

Potent and selective nonpeptide inhibitors of caspases 3 and 7.

5-Dialkylaminosulfonylisatins have been identified as potent, nonpeptide inhibitors of caspases 3 and 7. The most active compound within this series (34) inhibited caspases 3 and 7 in the 2-6 nM range and exhibited approximately 1000-fold selectivity for caspases 3 and 7 versus a panel of five other caspases (1, 2, 4, 6, and 8) and was at least 20-fold more selective versus caspase 9. Sequence alignments of the active site residues of the caspases strongly suggest that the basis of this selectivity is due to binding in the S2 subsite comprised of residues Tyr204, Trp206, and Phe256 which are unique to caspases 3 and 7. These compounds inhibit apoptosis in three cell-based models: human Jurkat T cells, human chondrocytes, and mouse bone marrow neutrophils.

One-step fluorescent probe product-enhanced reverse transcriptase assay.

Recently developed PCR-based reverse transcriptase (RT) assays are useful in the detection of retroviruses since they are approximately a millionfold more sensitive than conventional RT assays. However, these assays are both labor- and time-intensive. The previously described product-enhanced reverse transcriptase (PERT) assay involves a two-step RT-PCR followed by detection and quantitation of PCR products by either Southern blot or enzyme-linked immunosorbent assay (ELISA). We have modified the PERT assay to be a one-step, fluorescent probe, PCR-based RT assay that can be completed from sample dilution to final quantitative assay results in approximately 5 h without loss of assay sensitivity or specificity. The assay has a dynamic range of 6 logs, and therefore, extensive sample dilution is not necessary for quantitation. This newly enhanced fluorescent PERT assay can play an important role in the high-throughput detection of retroviral infection and characterization of RT activity.

Anti-human immunodeficiency virus type 1 human monoclonal antibodies that bind discontinuous epitopes in the viral glycoproteins can identify mimotopes from recombinant phage peptide display libraries.

A phage display library screening approach was used to identify peptide sequences that could bind to anti-HIV-1 MAbs whose binding specificities are complex. Most of the antibodies used recognize discontinuous epitopes in gp120 and one recognizes gp41. Both a 15-mer and a 21-mer display library (each with a complexity of greater than 60 x 10[6]) and two constrained, V3 region-biased libraries, all expressed as recombinant pIII protein of filamentous phage, were used. The unmapped anti-gp120 human MAb A32 recognized a set of related linear sequences and repeatedly identified a single phage sequence that could form a cyclic disulfide structure. Selection methods were also developed so that phage could be obtained by competition selection in the presence of antibody bound to native, monomeric gp120 antigen (used with MAb IgG1b12 and the anti-gp120 V3 region MAb 447-52D) or gp120 variable region 3 synthetic peptides (used with anti-gp120 V3 region MAb 19b). The potent, virus-neutralizing MAb IgG1b12 recognized numerous sequences and, when used in competition with gp120, recognized only one sequence. These studies extend the range of antibody determinant studies that can be performed with display phage libraries, demonstrate a workable experimental strategy for use of competition ligands to discriminate among phage mimotopes, and provide a large number of mimotopes that bind potent virus-neutralizing MAbs for HIV-1 vaccine studies.

3-Deazaadenosine 5'-triphosphate: a novel metabolite of 3-deazaadenosine in mouse leukocytes.

Evidence has been obtained for the metabolic formation of small amounts (1-2% of the ATP pool) of 3-deazaadenosine 5'-triphosphate (c3ATP) from 3-deazaadenosine (c3Ado) in mouse cytolytic lymphocytes and mouse resident peritoneal macrophages. With intact leukocytes, pharmacological evidence was obtained that adenosine kinase was not the enzyme chiefly responsible for the phosphorylation of c3Ado. Moreover, in the presence of MgCl2, NaCl and IMP, purified rat liver 5'-nucleotidase catalyzed the phosphorylation of c3Ado to 3-deazaadenosine 5'-monophosphate (c3AMP). Two lines of evidence suggest that the metabolic formation of c3ATP is not involved in the inhibition of leukocyte function caused by c3Ado. First, the inhibitory action of c3Ado on antibody-dependent phagocytosis and lymphocyte-mediated cytolysis was reversed markedly upon removal of the drug from the medium. However, the intracellular content of c3ATP remained constant in lymphocytes and macrophages after removal of c3Ado. Second, in macrophages and in lymphocytes, similar intracellular amounts of c3ATP were formed from both c3Ado and 3-deazaadenine under conditions in which the former was biologically active and the latter was essentially inactive. Thus, it appears unlikely that the novel c3ATP metabolite is of relevance for the mechanism of action of c3Ado in mouse leukocytes.

Purification of individual varicella-zoster virus (VZV) glycoproteins gpI, gpII, and gpIII and their use in ELISA for detection of VZV glycoprotein-specific antibodies.

We have utilized monoclonal antibodies in immune affinity chromatography to purify each of the 3 major glycoproteins of varicella-zoster virus (VZV), gpI, gpII, and gpIII, in immunologically active form. Upon injection into guinea pigs, each preparation elicited the production of specific antibodies capable of immunoprecipitating the homologous glycoprotein and of neutralizing VZV infectivity in vitro. Also, total glycoproteins from VZV-infected cells have been purified by lectin affinity chromatography. Each of the individual purified glycoproteins, as well as total VZV glycoproteins and appropriate uninfected cell protein controls, have been employed as solid-phase reagents in enzyme-linked immunosorbent assay (ELISA) for the detection of antibodies directed against specific VZV glycoproteins. The specificity of the purified glycoproteins as ELISA reagents was verified by the ability of individual monoclonal antibodies to bind specifically to individual glycoprotein preparations. We have demonstrated the utility of the glycoprotein-specific ELISA by detecting antibodies in sera from post-zoster and post-varicella patients. The assay detects antibodies directed against each of the 3 major glycoproteins and is sensitive enough to detect antibodies in a 1:320 000 dilution of some sera. This assay, as well as the purified individual glycoproteins per se, should prove to be very useful reagents in understanding the role of each of gpI, gpII, and gpIII in immunity to VZV.

Production of purified hepatitis B surface antigen from Alexander hepatoma cells grown in artificial capillary units.

An artificial capillary system was devised for growth of hepatoma cells that yields very high titers of hepatitis B surface antigen (HBsAg). High yield of antigen was facilitated by slowing cellular metabolism through reduction of incubation temperature and addition of 0.1 mM caffeine. Deletion of serum from the medium did not reduce the yield of antigen. HBsAg prepared from the culture fluid by affinity chromatography and additional chemical and enzymatic steps was essentially pure and was indistinguishable from HBsAg prepared from infected human plasma. Preparation of HBsAg from the cell culture source presents advantages over that of human plasma and might be a source of HBsAg for vaccine preparation.

Role of caspases in human renal proximal tubular epithelial cell apoptosis.

In the present study, we have used an in vitro model of apoptosis using primary human renal proximal tubular epithelial (RPTE) cells to investigate the mechanisms involved in renal cell apoptosis. Treatment of RPTE cells with okadaic acid for 24-48 h induced apoptosis in a concentration-dependent manner. Apoptosis was accompanied by the activation of the p38 mitogen-activated protein kinase (MAPK) pathway followed by the activation of caspase-9, -3, and -7. The induction of caspase activity correlated with the proteolytic cleavage of beta-catenin, suggesting that beta-catenin is a caspase substrate. The caspase inhibitor, Z-Val-Ala-Asp-fluoromethylketone (Z-VAD-fmk), resulted in a dose-dependent inhibition of apoptosis and beta-catenin cleavage. These data suggest that okadaic acid-induced apoptosis is p38 MAPK and caspase-dependent and that proteolytic cleavage of beta-catenin by caspases is likely to be a downstream molecular event associated with the morphological and cytoskeletal changes induced during apoptosis.

Inhibition of caspase-3-like activity prevents apoptosis while retaining functionality of human chondrocytes in vitro.

Apoptosis was induced in a human chondrocyte cell line, T/C 28a4, by treatment with various stimuli, including camptothecin, tumor necrosis factor-alpha, staurosporine, okadaic acid, and reduced serum conditions. All stimuli induced a cytosolic DEVDase activity, coincident with apoptosis. Caspase activities in the lysates were characterized and quantitated with peptide cleavage profiles. To confirm that the results were not related to the immortalized nature of the cell line, primary human chondrocytes also were shown to undergo apoptosis under similar conditions, which resulted in increased cytosolic DEVDase activity. There was little or no caspase-1 (interleukin-1beta-converting enzyme) or caspase-8-like activity in the apoptotic cells. In all cases, the irreversible nonselective caspase inhibitor, Z-VAD-FMK, and the caspase-3-selective inhibitor, Ac-DMQD-CHO, inhibited DEVDase activity and apoptosis, whereas the caspase-1-selective inhibitor, Ac-YVAD-CHO, had no effect. Human chondrocytes were stably and transiently transfected with a type-II collagen gene (COL2A1) regulatory sequence driving a luciferase reporter as a specific marker of chondrocyte gene expression. Treatment of the cells with camptothecin or tumor necrosis factor-alpha plus cycloheximide significantly inhibited COL2A1 transcriptional activity. Significantly, cotreatment with Z-VAD-FMK or Ac-DMQD-CHO maintained COL2A1-reporter gene activity, indicating that the prevention of apoptosis by caspase-3 inhibition was sufficient to maintain cell functionality as assessed by the retention of type-II collagen promoter activity.