The Impact of Borderline Quantiferon-TB Gold Plus Results for Latent Tuberculosis Screening under Routine Conditions in a Low-Endemicity Setting.

Quantiferon-TB Gold Plus (QFT-Plus) is an interferon gamma release assay used to diagnose latent tuberculosis (LTB). A borderline range (0.20 to 0.99 IU/ml) around the cutoff (0.35 IU/ml) has been suggested for the earlier QFT version. Our aims were to evaluate the borderline range for QFT-Plus and the contribution of the new TB2 antigen tube. QFT-Plus results were collected from clinical laboratories in Sweden and linked to incident active TB within 3 to 24 months using the national TB registry. Among QFT-Plus results from 58,539 patients, 83% were negative (<0.20 IU/ml), 2.4% were borderline negative (0.20 to 0.34 IU/ml), 3.4% were borderline positive (0.35 to 0.99 IU/ml), 9.6% were positive (≥1.0 IU/ml), and 1.6% were indeterminate. Follow-up tests after initial borderline results were negative (<0.20 IU/ml) in 38.3%, without any cases of incident active TB within 2 years. Applying the 0.35-IU/ml cutoff, 1.5% of TB1 and TB2 results were discrepant, of which 52% were within the borderline range. A TB2 result of ≥0.35 IU/ml with a TB1 result of <0.20 IU/ml was found in 0.4% (231/58,539) of all included baseline QFT-Plus test results, including 1.8% (1/55) of incident TB cases. A borderline range for QFT-Plus is clinically useful as more than one-third of those with borderline results are convincingly negative upon retesting, without developing incident active TB. The TB2 tube contribution to LTB diagnosis appears limited.

Little difference between minimum inhibitory concentrations of Mycobacterium tuberculosis wild-type organisms determined with BACTEC MGIT 960 and Middlebrook 7H10.

The MIC wild-type (WT) distribution for Mycobacterium tuberculosis in BACTEC 960 MGIT is not defined, which may result in poor reproducibility for drug susceptibility testing (DST), as several DST methods with different breakpoints are in use. In a comparison between MGIT and Middlebrook 7H10 medium of seven first- and second-line drugs, including 133 MIC determinations of 15 WT isolates, we found an agreement of 91.7% within ± one MIC dilution step. The results confirm the agreement in MIC testing between 7H10 and MGIT and indicate that breakpoints could be harmonized in order to avoid misclassification.

Intra- and extracellular activities of trimethoprim-sulfamethoxazole against susceptible and multidrug-resistant Mycobacterium tuberculosis.

We investigated the activity of trimethoprim-sulfamethoxazole (SXT) against Mycobacterium tuberculosis, the pathogen that causes tuberculosis (TB). The MIC distribution of SXT was 0.125/2.4 to 2/38 mg/liter for the 100 isolates tested, including multi- and extensively drug-resistant isolates (MDR/XDR-TB), whereas the intracellular MIC90 of sulfamethoxazole (SMX) for the pansusceptible strain H37Rv was 76 mg/liter. In an exploratory analysis using a ratio of the unbound area under the concentration-time curve from 0 to 24 h over MIC (fAUC0-24/MIC) using ≥ 25 as a potential target, the cumulative fraction response was ≥ 90% at doses of ≥ 2,400 mg of SMX. SXT is a potential treatment option for MDR/XDR-TB.

A Clinical Scoring Algorithm for Determination of the Risk of Tuberculosis in HIV-Infected Adults: A Cohort Study Performed at Ethiopian Health Centers.

BACKGROUND: The World Health Organization (WHO) tuberculosis (TB) symptom screening instrument (WHO-TB) can identify human immunodeficiency virus (HIV)-infected individuals at low risk of tuberculosis (TB); however, many patients report WHO-TB symptoms and require further TB investigations. We hypothesized that further clinical scoring could classify subjects with a positive WHO-TB screening result (WHO-TB(+)) for the likelihood of TB. METHODS: HIV-infected adults eligible to initiate antiretroviral therapy (ART) were recruited and prospectively followed at 5 Ethiopian health centers. Irrespective of symptoms, all participants underwent sputum bacteriological testing for TB. Symptoms, physical findings, hemoglobin, and CD4 cell count results were compared between subjects with and those without bacteriologically confirmed TB. Variables associated with TB in WHO-TB(+) individuals were used to construct a scoring algorithm with multiple logistic regression analysis. RESULTS: Among 812 participants, 137 (16.9%) had TB. One hundred fifty-nine persons (20%) had a negative WHO-TB screen, 10 of whom had TB (negative predictive value [NPV], 94% [95% confidence interval {CI}, 90%-97.5%]). For WHO-TB(+) subjects, the following variables were independently associated with TB, and were assigned 1 point each in the clinical scoring algorithm: cough, Karnofsky score ≤80, mid-upper arm circumference <20 cm, lymphadenopathy, and hemoglobin <10 g/dL. Among subjects with 0-1 points, 20 of 255 had TB (NPV, 92% [95% CI, 89%-95%]), vs 19 of 34 participants with ≥4 points (positive predictive value, 56% [95% CI, 39%-73%]). The use of WHO-TB alone identified 159 of 784 (20%) with a low risk of TB, vs 414 of 784 (53%) using WHO-TB followed by clinical scoring (P< .001). The difference in proportions of confirmed TB in these subsets was nonsignificant (6.3% vs 7.2%; P= .69). CONCLUSIONS: Clinical scoring can further classify HIV-infected adults with positive WHO-TB screen to assess the risk of TB, and would reduce the number of patients in need of further TB investigations before starting ART. CLINICAL TRIALS REGISTRATION: NCT01433796.

Proficiency of drug susceptibility testing of Mycobacterium tuberculosis against pyrazinamide: the Swedish experience.

BACKGROUND: Pyrazinamide (PZA) is a key drug in the treatment of tuberculosis (TB), including multidrug-resistant TB. Drug susceptibility testing (DST) of Mycobacterium tuberculosis against PZA is not included in the World Health Organization's yearly proficiency testing. There is an increasing need to establish quality control of PZA DST. OBJECTIVE: To evaluate the performance of PZA DST and to introduce a quality assurance system for the test in Sweden. METHOD: Panels with PZA-susceptible and -resistant isolates were used in three rounds of proficiency testing in all five Swedish clinical TB laboratories and our reference laboratory. All laboratories used the MGIT 960 system. Minimum inhibitory concentrations (MICs) were determined and the pncA gene was sequenced to further characterise the 52 panel strains. RESULTS: Good agreement was seen between the phenotypic PZA DST and pncA sequence data, and MIC determination confirmed high levels of resistance. However, in contrast to other drugs, for which correct proficiency test results were observed, specificity problems occurred for PZA DST in some laboratories. CONCLUSIONS: In Sweden, using panel testing, differences were seen in the proficiency of TB laboratories in correctly identifying PZA susceptibility. Improved results were noted in the third round; PZA has therefore been included in yearly proficiency testing.

Reevaluation of the critical concentration for drug susceptibility testing of Mycobacterium tuberculosis against pyrazinamide using wild-type MIC distributions and pncA gene sequencing.

Pyrazinamide (PZA) is a potent first-line agent for the treatment of tuberculosis (TB) with activity also against a significant part of drug-resistant Mycobacterium tuberculosis strains. Since PZA is active only at acid pH, testing for susceptibility to PZA is difficult and insufficiently reproducible. The recommended critical concentration for PZA susceptibility (MIC, 100 mg/liter) used in the Bactec systems (460 and MGIT 960) has not been critically evaluated against wild-type MIC distributions in clinical isolates of Mycobacterium tuberculosis. Using the Bactec MGIT 960 system, we determined the PZA MICs for 46 clinical M. tuberculosis isolates and compared the results to pncA sequencing and previously obtained Bactec 460 data. For consecutive clinical isolates (n = 15), the epidemiological wild-type cutoff (ECOFF) for PZA was 64 mg/liter (MIC distribution range, ≤ 8 to 64 mg/liter), and no pncA gene mutations were detected. In strains resistant in both Bactec systems (n = 18), the PZA MICs ranged from 256 to ≥ 1,024 mg/liter. The discordances between pncA sequencing, susceptibility results in Bactec 460, and MIC determinations in Bactec MGIT 960 were mainly observed in strains with MICs close to or at the ECOFF. We conclude that in general, wild-type and resistant strains were clearly separated and correlated to pncA mutations, although some isolates with MICs close to the ECOFF cause reproducibility problems within and between methods. To solve this issue, we suggest that isolates with MICs of ≤ 64 mg/liter be classified susceptible, that an intermediary category be introduced at 128 mg/liter, and that strains with MICs of >128 mg/liter be classified resistant.

Wild-type distributions of seven oral second-line drugs against Mycobacterium tuberculosis.

OBJECTIVES: To determine wild-type minimum inhibitory concentration (MIC) distributions for Mycobacterium tuberculosis, as the background data for defining susceptibility breakpoints are limited. METHODS: We determined wild-type MIC distributions of M. tuberculosis using a 96-stick replicator in Middlebrook 7H10 (7H10) medium for ethionamide (ETH), prothionamide, thiacetazone, cycloserine, rifabutin (RFB), clofazimine and linezolid in consecutive susceptible clinical isolates (n = 78). RESULTS: Tentative epidemiological wild-type cut-offs (ECOFF) were determined for all investigated drugs where World Health Organization recommended critical concentrations for 7H10 are lacking, except for ETH. As the ECOFF was closely related to the non-wild-type strains for ETH and thiacetazone, the use of an intermediary (I) category in drug susceptibility testing could increase reproducibility. The cross-resistance between ETH and isoniazid was 21%. Applying 0.5 mg/l as a breakpoint for RFB classified two non-wild type and rpoB mutated isolates as susceptible for RFB and resistant against rifampicin. CONCLUSIONS: We propose that wild-type MIC distributions should be used as a tool to define clinical breakpoints against second-line drugs. This is increasingly important considering the rapid emergence of drug resistance.

Reducing blood culture contamination by a simple informational intervention.

Compared to truly negative cultures, false-positive blood cultures not only increase laboratory work but also prolong lengths of patient stay and use of broad-spectrum antibiotics, both of which are likely to increase antibiotic resistance and patient morbidity. The increased patient suffering and surplus costs caused by blood culture contamination motivate substantial measures to decrease the rate of contamination, including the use of dedicated phlebotomy teams. The present study evaluated the effect of a simple informational intervention aimed at reducing blood culture contamination at Skåne University Hospital (SUS), Malmö, Sweden, during 3.5 months, focusing on departments collecting many blood cultures. The main examined outcomes of the study were pre- and postintervention contamination rates, analyzed with a multivariate logistic regression model adjusting for relevant determinants of contamination. A total of 51,264 blood culture sets were drawn from 14,826 patients during the study period (January 2006 to December 2009). The blood culture contamination rate preintervention was 2.59% and decreased to 2.23% postintervention (odds ratio, 0.86; 95% confidence interval, 0.76 to 0.98). A similar decrease in relevant bacterial isolates was not found postintervention. Contamination rates at three auxiliary hospitals did not decrease during the same period. The effect of the intervention on phlebotomists' knowledge of blood culture routines was also evaluated, with a clear increase in level of knowledge among interviewed phlebotomists postintervention. The present study shows that a relatively simple informational intervention can have significant effects on the level of contaminated blood cultures, even in a setting with low rates of contamination where nurses and auxiliary nurses conduct phlebotomies.

Wild-type MIC distributions for aminoglycoside and cyclic polypeptide antibiotics used for treatment of Mycobacterium tuberculosis infections.

The aminoglycosides and cyclic polypeptides are essential drugs in the treatment of multidrug-resistant tuberculosis, underscoring the need for accurate and reproducible drug susceptibility testing (DST). The epidemiological cutoff value (ECOFF) separating wild-type susceptible strains from non-wild-type strains is an important but rarely used tool for indicating susceptibility breakpoints against Mycobacterium tuberculosis. In this study, we established wild-type MIC distributions on Middlebrook 7H10 medium for amikacin, kanamycin, streptomycin, capreomycin, and viomycin using 90 consecutive clinical isolates and 21 resistant strains. Overall, the MIC variation between and within runs did not exceed +/-1 MIC dilution step, and validation of MIC values in Bactec 960 MGIT demonstrated good agreement. Tentative ECOFFs defining the wild type were established for all investigated drugs, including amikacin and viomycin, which currently lack susceptibility breakpoints for 7H10. Five out of seven amikacin- and kanamycin-resistant isolates were classified as susceptible to capreomycin according to the current critical concentration (10 mg/liter) but were non-wild type according to the ECOFF (4 mg/liter), suggesting that the critical concentration may be too high. All amikacin- and kanamycin-resistant isolates were clearly below the ECOFF for viomycin, and two of them were below the ECOFF for streptomycin, indicating that these two drugs may be considered for treatment of amikacin-resistant strains. Pharmacodynamic indices (peak serum concentration [Cmax]/MIC) were more favorable for amikacin and viomycin compared to kanamycin and capreomycin. In conclusion, our data emphasize the importance of establishing wild-type MIC distributions for improving the quality of drug susceptibility testing against Mycobacterium tuberculosis.

Wild-type MIC distributions of four fluoroquinolones active against Mycobacterium tuberculosis in relation to current critical concentrations and available pharmacokinetic and pharmacodynamic data.

OBJECTIVES: To describe wild-type distributions of the MIC of fluoroquinolones for Mycobacterium tuberculosis in relation to current critical concentrations used for drug susceptibility testing and pharmacokinetic/pharmacodynamic (PK/PD) data. METHODS: A 96-stick replicator on Middlebrook 7H10 medium was used to define the MICs of ciprofloxacin, ofloxacin, moxifloxacin and levofloxacin for 90 consecutive clinical strains and 24 drug-resistant strains. The MICs were compared with routine BACTEC 460 susceptibility results and with MIC determinations in the BACTEC MGIT 960 system in a subset of strains using ofloxacin as a class representative. PK/PD data for each drug were reviewed in relation to the wild-type MIC distribution. RESULTS: The wild-type MICs of ciprofloxacin, ofloxacin, moxifloxacin and levofloxacin were distributed from 0.125 to 1, 0.25 to 1, 0.032 to 0.5 and 0.125 to 0.5 mg/L, respectively. The MIC data correlated well with the BACTEC 960 MGIT and BACTEC 460 results. PD indices were the most favourable for levofloxacin, followed by moxifloxacin, ofloxacin and ciprofloxacin. CONCLUSIONS: We propose S (susceptible)

Lectin histochemistry of the gastric mucosa in normal and Helicobacter pylori infected guinea-pigs.

Helicobacter pylori attaches via lectins, carbohydrate binding proteins, to the carbohydrate residues of gastric mucins. Guinea-pigs are a suitable model for a H. pylori infection and thus the carbohydrate composition of normal and H. pylori infected gastric mucosa was investigated by lectin histochemistry. The stomach of all infected animals showed signs of an active chronic gastritis in their mucosa, whereas no inflammation was present in the control animals. The corpus-fundus regions of the controls showed heterogeneous WGA, SNA-I, UEA-I and HPA binding in almost all parts of the gastric glands. While these lectins labelled the superficial mucous cells and chief cells heterogeneously, the staining of the parietal cells was limited to WGA and PHA-L. Mucous neck cells reacted heterogeneously with UEA-I, HPA, WGA and PHA-L. In the antrum, the superficial mucous cells and glands were stained by WGA, UEA-I, HPA, SNA-I or PHA-L. WGA, UEA-I, SNA-I and HPA labelled the surface lining cells strongly. The mucoid glands reacted heterogeneously with WGA, UEA-I, HPA, SNA-I and PHA-L. In both regions, the H. pylori infected animals showed similar lectin binding pattern as the controls. No significant differences in the lectin binding pattern and thus in the carbohydrate composition between normal and H. pylori infected mucosa could be detected, hence H. pylori does not induce any changes in the glycosylation of the mucosa of the guinea-pig. This unaltered glycosylation is of particular relevance for the sialic acid binding lectin SNA-I as H. pylori uses sialic acid binding adhesin for its attachment to the mucosa. As sialic acid binding sites are already expressed in the normal mucosa H. pylori can immediately attach via its sialic acid binding adhesin to the mucosa making the guinea-pig particularly useful as a model organism.

Infection with cagA- and vacA-positive and -negative strains of Helicobacter pylori in a mouse model.

To study the role of cytotoxin-associated protein (cagA) and vacuolating cytotoxin (vacA) in Helicobacter pylori infection in an experimental murine model, mice were infected with seven strains with different cagA and vacA status. Groups of 10 NMRI mice were challenged and were killed 5 weeks later. In a second study, 20 mice were challenged with a mixture of the same seven strains and killed 1, 3, 15 and 17 weeks post-inoculation. All seven strains were found to colonize the mice for the 5-week experimental period. Animals infected with vacA-positive strains, regardless of cagA status, showed an elevation of antibody titers. Two cagA-negative and vacA-positive strains and one cagA- and vacA-positive strain were found to 'take over' in the mixed infection as analyzed by the randomly amplified polymorphic DNA-polymerase chain reaction technique and in one mouse stomach we found coexistence of two of the strains. We found no evidence of the different strains colonizing different parts of the stomach.

Comparative study of Helicobacter pylori infection in guinea pigs and mice - elevation of acute-phase protein C3 in infected guinea pigs.

Eighteen Dunkin-Hartley guinea pigs and 50 NMRI mice were inoculated with Helicobacter pylori and the infection followed by culture, histopathology, antibody response, and plasma levels of the acute-phase proteins albumin, C3, and transferrin for up to 7 weeks. The immune response to H. pylori surface proteins was studied by an enzyme immunoassay (EIA) and Western immunoblot and the plasma levels of albumin, C3, and transferrin were analyzed by single radial immunodiffusion. Guinea pigs had a more severe gastritis and a higher EIA immune response than NMRI mice. Serum C3 levels were elevated in infected guinea pigs after 3 and 7 weeks indicating a systemic inflammatory response and a possible link between H. pylori infection and extragastric manifestations such as vasculitis associated with atherosclerosis. Serum cholesterol levels were analyzed in guinea pigs at 7 weeks and indicated a higher level in H. pylori-infected than in control animals, but this difference was not statistically significant.

High intake of selenium, beta-carotene, and vitamins A, C, and E reduces growth of Helicobacter pylori in the guinea pig.

PURPOSE: Helicobacter pylori is a human gastroduodenal pathogen associated with type-B gastritis and gastric cancer. Low gastric tissue antioxidant levels are believed to increase the risk of developing gastric cancer. We investigated whether dietary antioxidant levels protect against infection and type-B gastritis in H. pylori-infected guinea pigs. METHODS: Dunkin-Hartley guinea pigs infected for 6 weeks with H. pylori were fed diets with various antioxidant levels. Stomach specimens were cultured, and gastritis was graded from 0 to 3. RESULTS: Supplementation with vitamins A, C, and E and with selenium yielded H. pylori recovery from 17% of challenged animals, compared with 43% of those fed a control diet. Gastritis was scored at 0.33 and 0.93, respectively. Supplementation with only vitamin C or astaxanthin had less effect on gastritis and recovery rate. In a second experiment, gastritis score in a group given vitamins A, C, E, and selenium and beta-carotene was 2.25 and in a control group, it was 2.57. The H. pylori recovery rate was 75 and 100%, respectively, with fewer colonies from animals given antioxidant supplementation (P < 0.05). CONCLUSIONS: A combination of antioxidants can protect against H. pylori infection in guinea pigs. In animal studies, antioxidant intake should be low to optimize development of H. pylori-associated disease. Furthermore we established that H. pylori causes severe gastritis in guinea pigs.

The relationship between O-chain expression and colonisation ability of Helicobacter pylori in a mouse model.

The influence of lipopolysaccharide (LPS) O-polysaccharide chain production on the colonisation ability of Helicobacter pylori in four mouse models (NMRI, C57BL/6, CBA/Ca, and BALB/cA mice) was studied. H. pylori strains that produced smooth-form LPS (S-LPS) detectable in silver-stained electrophoretic gels colonised mice. In contrast, a laboratory-passaged strain G50 and the culture collection strain CCUG 17874 did not colonise mice; the former strain produced low amounts of O-chains only detectable in immunoblotting but not in silver-stained gels, whereas the latter produced rough-form LPS (R-LPS) without O-chains. Furthermore, a galE isogenic mutant, which produced R-LPS, did not colonise mice. However, after repeated broth culture, strains G50 and CCUG 17874 produced S-LPS detectable in silver-stained gels and were capable of colonising mice. Consistent with the production of O-chains, all colonising strains produced Lewis (Le) antigens, Le(x) and/or Le(y). Except for low expression of Le(y) by non-colonising G50, reflecting low production of O-chains, all other non-colonising strains and the galE mutant lacked expression of Le antigens consistent with their production of R-LPS. Lectin typing of strains supported these findings, and also showed that lectin types did not differ before and after colonisation. The low level of O-chain production and Le antigen expression by the non-colonising G50 may not be sufficient to aid colonisation. Examination of protein profiles of H. pylori strains before inoculation showed that protein expression was not significantly different between colonising and non-colonising strains. These results show that S-LPS production with O-chain expression is required by H. pylori for colonisation in a number of mouse models and that care should be taken with inoculating H. pylori strains that loss of O-chains does not occur during subculturing.

Severe gastritis in guinea-pigs infected with Helicobacter pylori.

An appropriate animal model is essential to study Helicobacter pylori infection. The aim of this study was to investigate if H. pylori can colonise the guinea-pig stomach and whether the infection causes gastritis and a serological response similar to that observed in man. Guinea-pigs were infected either with fresh H. pylori isolates from human gastric biopsies or with a guinea-pig passaged strain. When the animals were killed, 3 and 7 weeks after inoculation, samples were taken for culture, histopathology and serology. H. pylori was cultured from 22 of 29 challenged animals. All culture-positive animals exhibited a specific immune response against H. pylori antigens in Western blotting and gastritis in histopathological examination. Antibody titres in enzyme immunoassay were elevated among animals challenged with H. pylori. The inflammatory response was graded as severe in most animals and consisted of both polymorphonuclear leucocytes and lymphocytes. Erosion of the gastric epithelium was found in infected animals. These results suggest that the guinea-pig is suitable for studying H. pylori-associated diseases. Moreover, guinea-pigs are probably more similar to man than any other small laboratory animal as regards gastric anatomy and physiology.

Dietary factors influence the recovery rates of Helicobacter pylori in a BALB/cA mouse model.

The aim of this study was to assess the ability of different mouse diets to sustain an H. pylori infection in BALB/cA mice. Four commercially available mouse diets were compared. Experiment 1: Mice were fed the four diets for seven days before infection, infected three times at two-day intervals with 0.1 ml of 10(9) colony-forming units/ml H. pylori cells. H. pylori strains (n = 4) were cultured on GAB-Camp agar for 2 days, harvested and suspended in PBS. All animals were sacrificed at 2 and 4 weeks post inoculation. Experiment 2: Mice infected for 8 weeks were fed RM2, changed to the different diets for 10 days and sacrificed. Stomachs were collected, cultured on GAB-Camp agar to estimate H. pylori growth and stomach biopsies were analyzed by PCR. There were significant differences between diets in their ability to sustain growth of H. pylori. The range was from a few hundred colonies to no growth at all on the GAB-Camp agar. PCR signals showed good correlation with the culture results. All H. pylori-infected mice gave a significantly higher inflammation score compared to non-infected mice. The diet RM2, having the highest number of culturable H. pylori in the mouse stomach, also showed the highest inflammation. These results suggest that the dietary factors affect the amounts of H. pylori in an infection of BALB/cA mice.

Infection of BALB/c A mice by spiral and coccoid forms of Helicobacter pylori.

Helicobacter pylori exists in two different morphological forms, spiral and coccoid. This study demonstrated that both forms can infect BALB/c A mice. The animals were inoculated orally three times at 2-day intervals with 10(8) cfu of both spiral and coccoid forms of strain CCUG 17874 (NCTC 11637), strain 25 and strain 553/93. Infection was followed over a 30-week period by histological scoring of the grade of inflammation in gastric biopsies. At each time point sera were collected for analysis in ELISA and immunoblot analysis. Both spiral and coccoid forms of all H. pylori strains gave significantly higher inflammation scores than a control group of animals 1 week after inoculation. The histological evidence persisted throughout the entire 30 weeks. The inflammation was most severe in the pylorus and duodenum. Infection with strain 553/93 displayed the most severe gastritis. The spiral form of strain CCUG 17874 gave an immune response after only 4 weeks, whereas its coccoid form as well as strains 25 and 553/93 (spiral and coccoid forms) gave a significant increase in antibody response in ELISA and immunoblot after 16 weeks. It is concluded that both spiral and coccoid forms of H. pylori can cause acute gastritis in BALB/c A mice.