Features of indeterminate results of QuantiFERON-TB Gold In-Tube test in patients with haematological malignancies.

BACKGROUND AND AIMS: The application of QuantiFERON-TB Gold in-Tube (QFT-GIT) in patients with haematological malignancies (HMs) has not been well studied. Therefore, we aimed to investigate the features of patients with HMs whose QFT-GIT results were indeterminate. METHODS: This study enrolled patients with HMs for the analysis of QFT-GIT tests and additional 2-year follow-up. The characteristics and predictors of QFT-GIT indeterminate results were identified. Mycobacterium tuberculosis (TB) incidence rate (IR) and incidence rate ratio (IRR) were also investigated. RESULTS: Of 89 participants, 27 (30.3%) had QFT-GIT indeterminate results. The QFT-GIT indeterminate patients were characterized with the diagnosis of leukaemia (63.0% versus 32.3%, p = 0.044), abnormal white blood count (WBC) (88.9% versus 14.5%, p = 0.001), abnormal lymphocyte percentage (81.5% versus 14.5%, p = 0.001) and lower lymphocyte count (×109/l) (0.5 versus 2.2, p = 0.000) when compared with those with determinate results. Meanwhile, abnormal WBC [odds ratios (OR): 15.18, p = 0.003] and lymphocyte percentage (OR: 6.90, p = 0.033) were predictors of indeterminate results. One patient with the QFT-GIT indeterminate status and high interferon-γ level of negative control result developed active TB with a TB IR of 18.5 per 1000 person-years and an IRR of 0.1 (95% confidence interval, 0.01-0.71) when compared with positive QFT-GIT patients without prophylaxis treatment. CONCLUSION: Abnormal ranges of WBC and lymphocyte differential count percentage were independent predictors useful to determine the optimal timing of implementing QFT-GIT test in patients with HMs.

Dynamics of the lung microbiome in intensive care patients with chronic obstructive pulmonary disease and community-acquired pneumonia.

Little is known about the composition and clinical implications of lung microbiome in patients with chronic obstructive pulmonary disease (COPD) and community-acquired pneumonia requiring invasive mechanical ventilation and intensive care unit admission. Therefore, this study aimed to explore the longitudinal changes in microbial airway composition and its variations between COPD patients with different weaning outcomes. Fifty-one endotracheal aspirate samples from 21 participants and 5 saline samples were collected as the patient and control group, respectively. Sequence analysis revealed significant increases and upward trends in the relative abundance of the Acinetobacter genus and Acinetobacter baumannii complex species in paired comparisons of sampling points and over time, respectively, in patients with failed weaning (p for trend = 0.012 and 0.012, respectively) but not in those with successful weaning (p for trend = 0.335 and 0.426, respectively). Furthermore, significant changes in the composition of the bacterial community were observed in paired comparisons of sampling points in patients with failed weaning compared with those with successful weaning. The alpha diversity did not differ between the patients with different weaning outcomes. These results further the understanding of longitudinal airway microbiome structure analysis and its clinical implications when managing critically ill patients with and without COPD.

Association of Carbonic Anhydrase 9 Polymorphism and the Epithelial Growth Factor Receptor Mutations in Lung Adenocarcinoma Patients.

Carbonic anhydrase 9 (CA9) plays a vital role in lung cancer progression. The current study explored the effect of CA9 gene polymorphisms and the epidermal growth factor receptor (EGFR) mutations on the clinicopathological characters of lung adenocarcinoma. In this study, three loci of CA9 single nucleotide polymorphism (SNP) (rs2071676 A>G, rs3829078 A>G, and rs1048638 C>A) were genotyped using the TaqMan allelic discrimination method in 193 EGFR wild type individuals and 281 EGFR mutation subjects. After adjusting for age, gender, and cigarette smoking status in logistic regression, all three CA9 SNPs illustrated a non-significant difference for the distribution between the EGFR wild type group and EGFR mutation group. Nevertheless, a significantly lower rate of CA9 SNP rs2071676 AG (adjusted odds ratio (AOR): 0.40, 95% confidence interval (CI): 0.16-0.95, p = 0.039) and AG+GG (AOR: 0.43, 95% CI: 0.18-0.98, p = 0.046) were found in the male population with L858R EGFR mutation compared to men with EGFR wild type. In addition, the CA9 SNP rs2071676 AG+GG genotype were significantly correlated to the lower tumor stage of lung adenocarcinoma in the whole study population (p = 0.044) and EGFR wild type individuals (p = 0.033). For the male population, the presence of CA9 SNP rs2071676 AG+GG genotype was also correlated to a lower tumor stage (p = 0.037) and fewer lymph node invasion (p = 0.003) in those with EGFR wild type. In conclusion, the existence of CA9 SNP rs2071676 is associated with the rate of EGFR L858R mutation in males. Furthermore, the CA9 SNP rs2071676 is correlated to lower tumor stage and lower risk for developing lymph node metastasis in lung adenocarcinoma, mainly in the EGFR wild type.

Aqueous Ocimum gratissimum extract induces cell apoptosis in human hepatocellular carcinoma cells.

Treatment of advanced hepatocellular carcinoma (HCC) has exhibited a poor overall survival rate of only six to ten months, and the urgency of the development of more effective novel agents is ever present. In this line of research, we aimed to investigate the effects and inhibitive mechanisms of aqueous Ocimum gratissimum leaf extract (OGE), the extract of Ocimum gratissimum, which is commonly used as a therapeutic herb for its numerous pharmacological properties, on malignant HCC cells. Our results showed that OGE decreased the cell viability of HCC SK-Hep1 and HA22T cells in a dose-dependent manner (from 400 to 800 µg/mL), while there is little effect on Chang liver cells. Moreover, cell-cycle analysis shows increased Sub-G1 cell count in SK-Hep1 and HA22T cells which is not observed in Chang liver cells. These findings raise suspicion that the OGE-induced cell death may be mediated through proteins that regulate cell cycle and apoptosis in SK-Hep1 and HA22T cells, and further experimentation revealed that OGE treatment resulted in a dose-dependent decrease in caspase 3 and PARP expressions and in CDK4and p-ERK1/2expressions. Moreover, animal tests also exhibited decreased HCC tumor growth by OGE treatment. We therefore suggest that the inhibition of cell viability and tumor growth induced by OGE may be correlated to the alteration of apoptosis-related proteins.

The association between airflow limitation and blood eosinophil levels with treatment outcomes in patients with chronic obstructive pulmonary disease and prolonged mechanical ventilation.

The clinical implications of airflow limitation severity and blood eosinophil level in patients with chronic obstructive pulmonary disease (COPD) and prolonged mechanical ventilation (PMV) are unknown. Thus, this study aimed to identify whether or not these two indicators were significantly associated with short-term in-respiratory care center (RCC) treatment outcomes in this population. Of all participants (n = 181) in this retrospective cross-sectional study, 41.4%, 40.9%, 8.3%, and 52.5% had prolonged RCC admission (RCC length of stay >21 days), failed weaning, death, and any adverse outcomes of interest, respectively. Compared to participants without any adverse outcomes of interest, moderate (the Global Initiative for Chronic Obstructive Lung Disease (GOLD) II) and/or severe (GOLD III) airflow limitation were significantly associated with short-term in-RCC adverse outcomes in terms of failed weaning (for III versus I, OR = 15.06, p = 0.003) and having any adverse outcomes of interest (for II versus I, OR = 17.66, p = 0.002; for III versus I, OR = 37.07, p = 0.000) though the severity of airflow limitation did not have associations with prolonged RCC admission and death after adjustment. Meanwhile, blood eosinophilia defined by various cut-off values was not associated with any adverse outcomes. The findings have significant clinical implications and are useful in the management of patients with COPD and PMV.

Clinical features, bacteriology of endotracheal aspirates and treatment outcomes of patients with chronic obstructive pulmonary disease and community-acquired pneumonia in an intensive care unit in Taiwan with an emphasis on eosinophilia versus non-eosinophilia: a retrospective case-control study.

OBJECTIVES: The clinical implications of blood eosinophil level in patients with chronic obstructive pulmonary disease (COPD) and community-acquired pneumonia (CAP) requiring invasive mechanical ventilation (IMV) and intensive care unit (ICU) admission are still unknown. Thus, this study aimed to compare the features of such patients with and without blood eosinophilia. DESIGN: This was a retrospective case-control study. SETTING: An ICU of a medical centre in central Taiwan. PARTICIPANTS: A total of 262 patients with COPD and CAP requiring IMV and ICU admission. RESULTS: Of all participants (n=262), 32 (12.2%) had an eosinophil percentage (EP) >2% and 169 (64.5%) had an absolute eosinophil count (AEC) >300 cells/µL. Regardless of whether 2% or 300 cells/µL was used as a cut-off value, the eosinophilia group were slightly older (years) (82.9±5.4 vs 78.1±9.1, p=0.000 and 79.2±8.4 vs 77.6±9.6, p=0.246, respectively), and had a higher forced expiratory volume in 1 s/forced vital capacity (%) (56.0±8.0 vs 51.3±11.6, p=0.005 and 53.1±11.2 vs 49.5±11.2, p=0.013, respectively), less severe spirometric classification (p=0.008 and p=0.001, respectively), and lower white cell count 109/L (8.8±3.2 vs 11.1±4.9, p=0.009 and 10.3±4.4 vs 11.8±5.3, p=0.017, respectively) than the non-eosinophilia group. The bacteriology of endotracheal aspirates showed that Pseudomonas aeruginosa and other gram-negative bacilli were the most common organisms in all study groups. Participants with an EP >2% had a shorter ICU length of stay (OR=12.13, p=0.001) than those with an EP ≤2%, while an AEC >300 cells/µL was not associated with any in-ICUoutcomes. CONCLUSIONS: The results of this study have significant clinical implications and should be considered when making treatment decisions for the management of patients with COPD and CAP requiring IMV and ICU admission.

In vitro activity of aminoglycosides, clofazimine, d-cycloserine and dapsone against 83 Mycobacterium avium complex clinical isolates.

BACKGROUND/PURPOSE: Treatment success rates for Mycobacterium avium complex (MAC) diseases range from 50% to 55% only. To explore effective antimicrobials against either Mycobacterium intracellulare or M. avium, we determined in vitro activities of five aminoglycosides, clofazimine, dapsone and d-cycloserine compared with primary (clarithromycin) and secondary (moxifloxacin and linezolid) antimycobacterial agents. METHODS: 83 non-duplicate clinical MAC isolates were collected from sputum and identified at the species level by PCR and restriction enzyme analysis of the 65 kDa hsp and rpoB genes. Drug susceptibility testing was performed using broth microdilution method. The fractional inhibitory concentration was calculated to determine synergy between isepamicin and clofazimine. RESULTS: High susceptibility rates of five aminoglycosides (isepamicin, amikacin, kanamycin, streptomycin, capreomycin, 82.7-88%), d-cycloserine (82.7%), clofazimine (97.3%) and clarithromycin (92%) against M. intracellulare, and 2 aminoglycosides (isepamicin, streptomycin, 87.5%), d-cycloserine (100%) and clarithromycin (100%) against M. avium were found. Dapsone had no inhibitory activity and moxifloxacin had little effect against both M. intracellulare and M. avium. Linezolid had modest activity whereas clofazimine had little effect against M. avium. Most MAC isolates with non-susceptibility to isepamicin were also non-susceptible to the other four aminoglycosides. Most streptomycin-susceptible MAC isolates were also susceptible to amikacin. Synergistic effect of combination of isepamicin and clofazimine was demonstrated in all (100%) M. intracellulare isolates whereas in only 50% M. avium isolates. CONCLUSION: When treating MAC diseases, species identification plays an important role in choosing treatment regimens. Combination of isepamicin and clofazimine may be a promising regimen in M. intracellulare-associated disease.

Evaluation of double locus (clfB and spa) sequence typing for studying molecular epidemiology of methicillin-resistant Staphylococcus aureus in Taiwan.

BACKGROUND: Pulsed-field gel electrophoresis (PFGE) is the "gold standard" for epidemiological investigation of methicillin-resistant Staphylococcus aureus (MRSA), but several DNA sequence-based methods have been developed in MRSA typing because of the unambiguous results. METHODS: Ninety-one MRSA isolates were collected from the blood cultures of different patients from July 2008 to December 2008 in central Taiwan. The molecular characteristics of each isolate, including double locus sequence typing (DLST; spa and clfB typing), Staphylococcus cassette chromosome mec (SCCmec), and PFGE were determined for comparison. RESULTS: Five major clfB types (types A-E), 18 spa types, 33 DLST genotypes, five SCCmec types, 17 pulsotypes have been observed. Three major DLST genotypes (A1-t002, C0-t037, and B1-t437) and two major pulsotypes (6 and 8) were identified. Most clfB type A isolates (97.1%) were SCCmec type II and all clfB type C isolates (100%) were SCCmec type III. Most clfB type B isolates (88.9%) were SCCmec type IV (59.3%) and VT (29.6%). All (100%) clfB subtypes A1, A2, and C isolates and 70.4% of clfB type B isolates belonged to healthcare-associated-MRSA. The average congruence was 57.7% between DLST and PFGE, and 96.6% between clfB and SCCmec type. The index of discrimination of SCCmec, clfB, spa, PFGE, and DLST was 0.72, 0.79, 0.80, 0.81, and 0.87, respectively. CONCLUSION: ClfB type has high congruence with SCCmec type. The DLST method in this study yielded a higher discriminatory power than PFGE in local investigation of molecular epidemiology of MRSA and a promising alternative to PFGE.

Isolated tuberculous liver abscess in an immunocompetent adult patient: A case report and literature review.

Tuberculous liver abscess is a rare disease entity even in endemic areas of Mycobacterium tuberculosis. It is usually accompanied by pulmonary tuberculosis or enteric tuberculosis. Further, an isolated tuberculous liver abscess is extremely rare. The disease is diagnosed by laparotomy or postmortem autopsy in most cases, and some authors adopted a 9-month antituberculosis regimen. We herein report a case of an isolated tuberculous liver abscess that initially manifested as persistent fever and general malaise, which was diagnosed by liver biopsy and treated successfully with a 6-month antituberculosis regimen and percutaneous abscess drainage.

Quantiferon TB-Gold conversion can predict active tuberculosis development in elderly nursing home residents.

AIM: The study was carried out on elderly nursing home residents in Taiwan. We assessed whether the serial QuantiFERON-TB Gold (QFT-G) assay and serial tuberculin skin test (TST) were reliable tools to predict or exclude the development of active tuberculosis (TB). METHODS: This prospective observational cohort study involved non-bacillus Calmette-Guérin-vaccinated 259 elderly nursing home residents free of active TB at baseline. Of these, 147 were eligible for follow up. Participants underwent serial QFT-G and TST at baseline and 2-year follow up, and were monitored for active TB over 5 years. Agreement between QFT-G and TST, incidence rate ratio, positive predictive value, and negative predictive value for progression to active TB were measured. RESULTS: During 5-year follow up, three participants developed active TB. The agreement between these two tests was 54.13% (ĸ = 0.167, P = 0.001). The incidence rate ratio was 15.8 (P = 0.016) for the QFT-G-conversion group compared with the TST-positive group at baseline. Positive predictive value for QFT-G conversion groups was 25%. Negative predictive value was 100% for the TST-negative group at baseline. CONCLUSION: In the elderly nursing home residents, QFT-G conversion is a more reliable tool to predict the development of active TB. Meanwhile, TST is a valuable tool for predicting the chance of not developing active TB.

A reduction in anti-tuberculosis drug resistance after the implementation of the national "STOP TB" program in central Taiwan, 2003-2007.

The aim of this study was to determine the performance of the national "STOP TB" program in central Taiwan during 2003-2007 by examining trends in the combined drug resistance to first-line anti-tuberculosis (TB) drugs among clinical Mycobacterium tuberculosis isolates. Using 4,819 clinical M. tuberculosis isolates obtained from two mycobacteriology referral laboratories, the resistance to drugs was measured and analyzed along with the treatment outcomes in notified TB patients. The proportion of isolates showing total resistance and multidrug-resistant tuberculosis (MDR-TB) isolates were 17.7% and 3.67%, respectively. More number of MDR-TB isolates showed high-level resistance to isoniazid (84.18%) and streptomycin (SM) (30.51%); low-level resistance to ethambutol (EMB) (61.58%), SM (41.81%), and pyrazinamide (66.1%); and resistance to ofloxacin (30.4%). However, fewer isolates showed high-level resistance to EMB (19.77%), levofloxacin (17.9%), moxifloxacin (19.6%), kanamycin (8.9%), amikacin (8.9%), and capreomycin (8.9%). Of these MDR-TB isolates, 7.1% were extensively drug-resistant. Trends in combined drug resistance to all the first-line anti-TB drugs and the incidence of MDR-TB were stable during the 2 years (2003-2004) before the implementation of the national "STOP TB" program. After the "STOP TB" program, there were significant declines in the incidence of MDR-TB during 2005-2007 in central Taiwan as well as improved TB-treatment outcomes. Thus, the national "STOP TB" program had a significant positive impact on TB control in central Taiwan.

Synergistic activities of tigecycline with clarithromycin or amikacin against rapidly growing mycobacteria in Taiwan.

The occurrence of diseases caused by rapidly growing mycobacteria (RGM) is increasing in Taiwan. In this study, the in vitro antimicrobial activities of tigecycline, minocycline, tetracycline and doxycycline were evaluated against 160 clinical RGM isolates, including 34 Mycobacterium abscessus sensu stricto (s.s.), 44 Mycobacterium massiliense, 1 Mycobacterium bolletii, 58 Mycobacterium fortuitum and 23 Mycobacterium chelonae. Clarithromycin and amikacin were tested alone as well as for synergistic effect with tigecycline. Both amikacin and tigecycline showed excellent activities against the RGM. More than 85% of each of the five RGM species isolates showed susceptibility to the two drugs. The MIC50 and MIC90 values (drug concentrations at which 50% and 90%, respectively, of the tested isolates did not show any visible growth) of amikacin were 1-4 mg/L and 2-8 mg/L, respectively, whilst those of tigecycline were 0.125-1 mg/L and 0.5-2.0 mg/L. Clarithromycin had only moderate activity, with ≥42.9% but ≤87.5% of each RGM species isolates showing susceptibility. The other three drugs had limited or no antimicrobial activity, with <40% of each RGM species isolates showing susceptibility. Combined with clarithromycin, tigecycline had synergistic activity against 92.9%, 68.8%, 100%, 35.7% and 46.2% of M. abscessus s.s., M. massiliense, M. bolletii, M. fortuitum and M. chelonae isolates, respectively. However, tigecycline combined with amikacin had synergistic activity against <25% but antagonistic activity against >18% of each RGM species. Thus, tigecycline alone may be an alternative for treating RGM diseases in patients who are intolerant to cefoxitin, imipenem or amikacin. However, it should be used with caution or not used in combination with amikacin for RGM diseases.

Suitable restriction enzyme for standardization of pulsed-field gel electrophoresis protocol and interlaboratory comparison of Acinetobacter baumannii.

BACKGROUND/PURPOSE: Interlaboratory comparison of pulsed-field gel electrophoresis (PFGE) patterns is difficult. A key reference of standardized PFGE protocol for Acinetobacter baumannii may address this issue. This study aimed to determine restriction enzymes with rare cutting sites on A baumannii genomes and evaluate their cost-effectiveness, discriminatory power, and interlaboratory consistence of band assignments. METHODS: There were 42 A baumannii isolates collected, including nine from three hospital outbreaks and 33 sporadic isolates. The numbers of cutting sites for the restriction enzymes were explored using the "Restriction Digest and PFGE" program. The cost-effectiveness for PFGE analysis was evaluated for the tested restriction enzymes, while its discriminatory ability was expressed through a discriminatory index and 95% confidence interval. The interlaboratory consistence of band assignments was evaluated for the 42 A baumannii isolates. RESULTS: ApaI was the most cost-effective restriction enzyme for a PFGE protocol for A baumannii. Both AscI and AsiSI were reasonable in terms of costs. ApaI, AscI, and AsiSI exhibited similar discriminatory indices. ApaI generated more than 40 fragments that were close and not easy to resolve, resulting in less consistence of band assignments. AscI and AsiSI generated 10-20 fragments that were clearly resolved, resulting in higher consistence of band assignments. AscI exhibited a close discriminatory power to that of AsiSI and at half of the cost of AsiSI for PFGE analysis. CONCLUSION: We recommend AscI as the primary enzyme and AsiSI as the secondary enzyme for standardizing the PFGE protocol and interlaboratory comparisons of A baumannii.

Combined rpoB duplex PCR and hsp65 PCR restriction fragment length polymorphism with capillary electrophoresis as an effective algorithm for identification of mycobacterial species from clinical isolates.

BACKGROUND: Mycobacteria can be quickly and simply identified by PCR restriction-enzyme analysis (PRA), but misidentification can occur because of similarities in band sizes that are critical for discriminating among species. Capillary electrophoresis can provide computer-aided band discrimination. The aim of this research was to develop an algorithm for identifying mycobacteria by combined rpoB duplex PRA (DPRA) and hsp65 PRA with capillary electrophoresis. RESULTS: Three hundred and seventy-six acid-fast bacillus smear-positive BACTEC cultures, including 200 Mycobacterium tuberculosis complexes (MTC) and 176 non-tuberculous mycobacteria (NTM) were analyzed. With combined hsp65 and rpoB DPRA, the accuracy rate was 100% (200 isolates) for the MTC and 91.4% (161 isolates) for the NTM. Among the discordant results (8.6%) for the NTM, one isolate of Mycobacterial species and an isolate of M. flavescens were found as new sub-types in hsp65 PRA. CONCLUSIONS: This effective and novel identification algorithm using combined rpoB DPRA and hsp65 PRA with capillary electrophoresis can rapidly identify mycobacteria and find new sub-types in hsp65 PRA. In addition, it is complementary to 16 S rDNA sequencing.

SoRT2: a tool for sorting genomes and reconstructing phylogenetic trees by reversals, generalized transpositions and translocations.

SoRT(2) is a web server that allows the user to perform genome rearrangement analysis involving reversals, generalized transpositions and translocations (including fusions and fissions), and infer phylogenetic trees of genomes being considered based on their pairwise genome rearrangement distances. It takes as input two or more linear/circular multi-chromosomal gene (or synteny block) orders in FASTA-like format. When the input is two genomes, SoRT(2) will quickly calculate their rearrangement distance, as well as a corresponding optimal scenario by highlighting the genes involved in each rearrangement operation. In the case of multiple genomes, SoRT(2) will also construct phylogenetic trees of these genomes based on a matrix of their pairwise rearrangement distances using distance-based approaches, such as neighbor-joining (NJ), unweighted pair group method with arithmetic mean (UPGMA) and Fitch-Margoliash (FM) methods. In addition, if the function of computing jackknife support values is selected, SoRT(2) will further perform the jackknife analysis to evaluate statistical reliability of the constructed NJ, UPGMA and FM trees. SoRT(2) is available online at http://bioalgorithm.life.nctu.edu.tw/SORT2/.