Molecular analysis of diversity within the genus Pseudomonas in the lungs of cystic fibrosis patients

The genus Pseudomonas contains species that can act as human pathogens and are important in many infections such as those occurring in the lungs of many cystic fibrosis (CF) patients. Current methodologies that rely on the cultivation of bacteria are too cumbersome and often lack sufficient discriminatory power to define the diversity of Pseudomonas spp. As a result, molecular-based approaches have many advantages when attempting to differentiate between species of this genus. This study assessed the ability of terminal restriction fragment length polymorphism (T-RFLP) profiling of the 16S–23S rRNA ITS1 gene region to differentiate species of the genus Pseudomonas. Before application to clinical samples, this approach was validated on a panel of 10 different Pseudomonas spp. T-RFLP profiling of the 16S–23S rRNA ITS1 gene region amplified from these strains differentiated all Pseudomonas spp. tested. The presence of Pseudomonas spp. in CF sputum was assessed through the detection of this ITS1 gene region as amplified from DNA extracted from 40 samples of CF sputum. The ITS1 gene region was detected in 75% of these samples, including 5 from which no Pseudomonas spp. had been identified using culture-based methods. In silico analysis showed that all sequences amplified had a high homology with the ITS1 region of Pseudomonas aeruginosa. T-RFLP gave data that were consistent with the band being generated from P. aeruginosa for each patient. No correlation between Pseudomonas diversity and severity of lung disease was observed in this group of CF patients. This study has however shown that molecular analysis of the ITS1 region is effective in resolving diversity within the genus Pseudomonas. The wider use of this application is discussed.     

Allergy‐related diseases and early gut fungal and bacterial microbiota abundances in children

BackgroundThe early gut microbiota has been proposed as an important link between environmental exposures and development of allergy‐related diseases. Beyond the widely investigated associations between the gut bacterial microbiota, we investigated the involvement of early gut mycobiota and gut permeability in the pathogenesis of asthma, allergic rhinoconjunctivitis (AR) and eczema.MethodsIn the Probiotics in the Prevention of Allergy among Children in Trondheim trial with maternal probiotic supplementation, we collected faecal samples at four timepoints between 0 and 2 years from a cohort of 278 children. Clinical information on allergy‐related diseases was collected in a paediatric examination at 2 years and questionnaires at 6 weeks and 1, 2 and 6 years. By quantitative PCR and 16S/ITS1 MiSeq rRNA gene sequencing, we analysed the gut bacterial and fungal microbiota abundance and bacterial diversity and explored associations with allergy‐related diseases. We also measured gut permeability markers (lipopolysaccharide‐binding protein [LBP] and fatty acid‐binding protein 2 [FABP2]).ResultsChildren with higher fungal abundance at 2 years were more likely to develop asthma and AR by 6 years, odds ratios 1.70 (95% CI: 1.06–2.75) and 1.41 (1.03–1.93), respectively. We explored causal connections, and children with eczema at 1–2 years appeared to have more mature bacterial microbiota, as well as being depleted of Enterococcus genus. Although LBP and FABP2 did not correlate with eczema, increased bacterial abundance was associated with increased serum FABP2.ConclusionsWe observed positive associations between gut fungal abundance and allergy‐related disease, but increased gut permeability does not appear to be involved in the underlying mechanisms for this association. Our findings should be confirmed in future microbiota studies.     

Predictors of necessity for endoscopic balloon dilatation in patients with Crohn’s disease-related small bowel stenosis

Background and AimIn patients with Crohn’s disease (CD) and small bowel stenosis, endoscopic balloon dilation (EBD) is considered to be useful in improving stenotic symptoms and avoiding surgery. However, it carries risks such as bleeding and perforation. The aim of this study was to identify the indications for endoscopic intervention in patients with CD and small bowel stenosis.MethodsFrom November 2007 to March 2020, 143 CD patients with small bowel stenosis were enrolled in this study. We identified the factors associated with not requiring endoscopic intervention during long-term follow-up of these patients.ResultsForty of the 143 patients had abdominal symptoms of stenosis and had undergone EBD, whereas the remaining 103 were asymptomatic and had not undergone endoscopic intervention. During long-term follow-up, 95 of those 103 patients never required endoscopic or surgical intervention. Multivariate logistic regression analysis revealed that not consuming an elemental diet (OR 3.18, 95% CI 1.48–6.82; p < .01) and ileocecal valve (ICV) stenosis (OR 0.30, 95% CI 0.11–0.83; p = .02) were independently associated with not requiring EBD. The cumulative emergency hospitalisation-free rate also tended to be higher in patients not consuming an elemental diet or with ICV stenosis.ConclusionsTwo factors, namely not consuming an elemental diet and ICV stenosis, predict a long-term intervention-free prognosis in CD patients with small bowel stenosis.

Key messages

• When an endoscopically impassable small bowel stenosis is found in a CD patient, long-term follow-up without endoscopic intervention may be possible if the patient is asymptomatic, is not using an elemental diet, and the stenosis is ICV.

     

经腹肠道超声对克罗恩病的诊断价值及其表现

目的 探讨经腹肠道超声诊断克罗恩病(CD)的价值。方法 对33例CD患者行经腹肠道超声检查,分析超声图像,并与内镜和(或)消化道造影结果相对照;对肠壁血流进行分级,并检测C反应蛋白(CRP)。结果 CD超声主要表现为受累肠壁不同程度增厚,呈"靶环征"、"三明治征";增厚肠壁回声层次多消失,内膜面呈平板状或"鹅卵石样改变";超声可显示深裂隙状溃疡及较大的黏膜溃疡、并能发现肠管蠕动异常。CD易并发肠管狭窄、炎性包块、肠瘘、穿孔及脓肿;受累肠壁周围出现"爬行脂肪征"。病变类型以小肠-结肠型最多见(16/33,48.48%),其次为小肠型(11/33,33.33%),结肠型占15.15%(5/33),胃-回肠型占3.03%(1/33)。受累肠壁血流分级与CRP存在显著相关(r=0.59,P<0.01),一致性中等(Kappa=0.58,P<0.01)。结论 经腹肠道超声检查在CD的诊断和随访中具有独特优势。     

酿酒酵母菌抗原在炎症性肠病结肠组织中的表达及意义

目的研究酿酒酵母菌抗原在炎症性肠病(IBD)结肠黏膜组织中的表达,评价酿酒酵母菌抗原在IBD诊断中的作用,初步探讨其在IBD发病中的意义。方法随机抽取航天中心医院2003年1月至2009年4月47例有完整临床资料的IBD患者的蜡块标本,其中克罗恩病(CD)22例、溃疡性结肠炎(UC)25例,另取非IBD结肠炎患者的蜡块标本20例作对照组。采用免疫组织化学法对标本石蜡切片进行染色,检测酿酒酵母菌抗原在肠黏膜组织中的表达,采用χ2检验分析酿酒酵母菌抗原在不同肠病中表达的差异性。结果免疫组织化学分析表明酿酒酵母菌抗原在CD阳性表达率为86.4%(19/22),在UC中为80.0%(20/25),在非IBD结肠炎中为70.0%(14/20),三组之间的阳性表达率差异无统计学意义(χ2=1.716,P=0.424)。结论酿酒酵母菌抗原在IBD中的表达无疾病特异性,暂不能作为诊断及鉴别诊断IBD的指标;酿酒酵母菌抗原在CD、UC患者及非IBD结肠炎患者结肠黏膜内的较广泛表达,也提示此类抗原蛋白在IBD的发生发展中发挥着不确定的作用。     

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.     

The biodiversity and composition of the dominant fecal microbiota in patients with inflammatory bowel disease

Clinical and experimental observations in animal models indicate that intestinal commensal bacteria are involved in the initiation and amplification of inflammatory bowel disease (IBD). As the majority of colonic bacteria cannot be identified by culture techniques, the aim of this study was to use sequence-based methods to investigate and characterize the composition of the dominant fecal microbiota in both patients with inflammatory bowel disease and healthy subjects. Fecal microbiota was isolated and quantified using real-time quantitative polymerase chain reaction. Denaturing gradient gel electrophoresis (DGGE) of 16S rDNA was used to evaluate the diversity of the dominant species. Analysis of individual bacterial groups showed a greater change in the fecal microbiota of patients with IBD, especially in those with active ulcerative colitis and active Crohn's disease. DGGE demonstrated the diversity of microbial flora in ulcerative colitis and Crohn's disease was less than in healthy subjects. Our results provide a better understanding of changes in fecal microbiota among patients with inflammatory bowel disease.     

Rapid diagnosis of acute bacterial meningitis: role of a broad range 16S rRNA polymerase chain reaction

Background: Acute bacterial meningitis is a significant cause of morbidity and mortality throughout the world. It can be difficult to diagnose, as the symptoms and signs are often non-specific. Study Objective: To evaluate the performance of an in-house semi-nested polymerase chain reaction (PCR) assay targeting the 16S rRNA gene of Eubacteria for the rapid diagnosis of acute bacterial meningitis using cerebrospinal fluid (CSF) specimens. Methods: A total of 112 CSF samples from 112 patients were used in the study. Among these, 32 samples were obtained from confirmed cases of Streptococcus pneumoniae, six samples were obtained from confirmed cases of Haemophilus influenzae, one sample from a confirmed case of Neisseria meningitidis, and 10 cases of clinically suspected acute bacterial meningitis. The remaining 63 CSF samples were obtained from patients with non-infectious illnesses (n = 47) of the central nervous system (CNS) and autopsy-confirmed tuberculous meningitis (n = 16). Results: The assay had an overall sensitivity of 93% (95% confidence interval [CI] 0.81–0.98, negative predictive value = 95%) and a specificity of 98% (95% CI 0.92–1.0, positive predictive value = 98%). Conclusion: These preliminary findings suggest that the semi-nested PCR assay targeting the 16S rRNA gene may be used as a rapid test for the diagnosis of acute bacterial meningitis.     

Impaired Autophagy of an Intracellular Pathogen Induced by a Crohn's Disease Associated ATG16L1 Variant

The genetic risk factors predisposing individuals to the development of inflammatory bowel disease are beginning to be deciphered by genome-wide association studies. Surprisingly, these new data point towards a critical role of autophagy in the pathogenesis of Crohn's disease. A single common coding variant in the autophagy protein ATG16L1 predisposes individuals to the development of Crohn's disease: while ATG16L1 encoding threonine at amino acid position 300 (ATG16L1*300T) confers protection, ATG16L1 encoding for alanine instead of threonine (ATG16L1*300A, also known as T300A) mediates risk towards the development of Crohn's disease. Here we report that, in human epithelial cells, the Crohn's disease-associated ATG16L1 coding variant shows impairment in the capture of internalized Salmonella within autophagosomes. Thus, we propose that the association of ATG16L1*300A with increased risk of Crohn's disease is due to impaired bacterial handling and lowered rates of bacterial capture by autophagy.     

Use of quantitative 16S rRNA PCR to determine bacterial load does not augment conventional cerebrospinal fluid (CSF) cultures among children undergoing treatment for CSF shunt infection

The aim of this study was to develop a quantitative 16S rRNA assay for determination of bacterial nucleic acid load in cerebrospinal fluid (CSF) shunt infection and to compare quantitative 16S rRNA polymerase chain reaction (PCR) findings to those of conventional bacterial culture in patients treated for CSF shunt infection. We developed a quantitative 16S rRNA PCR assay that detected bacterial load across a range of 2.5 × 10⁹ down to 2.5 × 10⁴ 16S copies/mL CSF under experimental conditions for numerous Gram-positive and Gram-negative organisms. However, when applied to archived CSF samples from 25 shunt infection episodes, correlations between positive bacterial culture and 16S rRNA levels were seen in only half of infections, and 16S rRNA levels dropped precipitously after an initial peak on the first day of sample collection. Bacterial load measured using 16S rRNA PCR does not provide sufficient information beyond bacterial culture to inform CSF shunt infection treatment.     

Adult Crohn disease: can ileoscopy replace small bowel radiology?

Background: This study aimed to document the radiological features and distribution of small bowel Crohn disease (CD) in adults by using a barium follow-through (BaFT) technique and to determine whether disease would be missed or its distribution underestimated if only colonoscopy with ileoscopy were performed. Methods: The BaFT examinations of 121 adults with proven CD were reviewed retrospectively with respect to the stage and distribution of disease. Colonoscopy with attempted ileoscopy was performed in 37 of these subjects, and the results were compared with radiological findings. Results: A normal villous pattern was visualized in 89 studies (74%). BaFT showed small bowel CD in 71 (59%) of 121 patients studied. The terminal ileum (TI) was the most common site of disease, affecting 62 (87%) of patients with small bowel CD. Forty-six patients (65%) had more proximal small bowel disease, including nine (13%) with a normal TI. BaFT showed early mucosal changes of CD in 52 subjects (73%), which was the sole manifestation in 15 (21%). Ileoscopy was possible in the majority of patients colonoscoped but was not achieved in 14 (38%), nine of whom had CD on BaFT. Of the 23 patients in whom ileoscopy was performed, findings agreed with BaFT assessment of the TI in 22. Conclusion: BaFT adequately demonstrates the stage and extent of small bowel CD. The majority of patients with small bowel CD have disease proximal to the TI, which cannot be diagnosed by ileoscopy. Received: 27 August 1996/Accepted: 16 October 1996     

Mathematical Modeling of the Interrelationship of CD4 Lymphocyte Count and Viral Load Changes Induced by the Protease Inhibitor Indinavir

While CD4 cell counts are widely used to predict disease progression in human immunodeficiency virus (HIV)-infected patients, they are poorly explanatory of the progression to AIDS or death after the introduction of chemotherapy. Changes in HIV load (as measured by RNA PCR) have been shown to be a much better predictor of the risk of disease progression. Since the interrelationship of these markers is of great clinical interest, we modeled the time-averaged return of CD4 cell count and change in viral load subsequent to therapy with the HIV protease inhibitor indinavir. We found that CD4 cell return was significantly related to both the baseline CD4 count (r² = 0.86, P < 0.001) and the decline in HIV RNA PCR-determined viral load (also referred to in this work as the HIV RNA PCR decline) (r² = 0.60, P < 0.01). Simultaneously modeling both influences in a linked nonlinear model (r² = 0.93, P < 0.001) demonstrated that (i) the starting number of CD4 cells accounted for the majority of the change in CD4 cell return and (ii) the return of CD4 cells attributable to viral load decrease was 50% of maximal with only a decrease of approximately 0.2 log of HIV RNA as modeled from the first 12 weeks of therapy. Much greater viral inhibition beyond that necessary for maximal CD4 cell return is possible. Given that HIV RNA PCR decline is more strongly linked to ultimate clinical course in HIV disease, our findings indicate that CD4 return is potentially misleading as an indicator of antiviral effect, since it is determined more by the starting CD4 value than by viral load decline and since near-maximal changes occur with minimal antiviral effect.     

Real-time PCR for detection of Streptococcus suis serotype 2 in cerebrospinal fluid of human patients with meningitis

Streptococcus suis serotype 2 is an emerging zoonotic pathogen and is the main cause of acute bacterial meningitis in adult patients in Vietnam. We developed an internally controlled real-time PCR for detection of S. suis serotype 2 in cerebrospinal fluid (CSF) samples targeted at the cps2J gene. Sensitivity and specificity in culture-confirmed clinical samples were 100%. The PCR detected S. suis serotype 2 infection in 101 of 238 (42.4%) prospectively collected CSF samples, of which 55 (23%) were culture positive. Culture-negative but PCR-positive CSF samples were significantly associated with the use of antimicrobial agents before admission. S. suis serotype 2 infection was more common than infections with Streptococcus pneumoniae and Neisseria meningitidis combined. Our results strikingly illustrate the additional diagnostic value of PCR in patients who are pretreated with antimicrobial agents and demonstrate the extremely high prevalence of S. suis infections among Vietnamese adult patients with bacterial meningitis.     

CD4+ T Cells Reactive to Enteric Bacterial Antigens in Spontaneously Colitic C3H/HeJBir Mice: Increased T Helper Cell Type 1 Response and Ability to Transfer Disease

C3H/HeJBir mice are a new substrain that spontaneously develop colitis early in life. This study was done to determine the T cell reactivity of C3H/HeJBir mice to candidate antigens that might be involved in their disease. C3H/HeJBir CD4⁺ T cells were strongly reactive to antigens of the enteric bacterial flora, but not to epithelial or food antigens. The stimulatory material in the enteric bacteria was trypsin sensitive and restricted by class II major histocompatibility complex molecules, but did not have the properties of a superantigen. The precursor frequency of interleuken (IL)-2–producing, bacterial-reactive CD4⁺ T cells in colitic mice was 1 out of 2,000 compared to 1 out of 20,000–25,000 in noncolitic control mice. These T cells produced predominately IL-2 and interferon γ, consistent with a T helper type 1 cell response and were present at 3–4 wk, the age of onset of the colitis. Adoptive transfer of bacterial-antigen–activated CD4⁺ T cells from colitic C3H/HeJBir but not from control C3H/HeJ mice into C3H/HeSnJ scid/scid recipients induced colitis. These data represent a direct demonstration that T cells reactive with conventional antigens of the enteric bacterial flora can mediate chronic inflammatory bowel disease.The inflammatory bowel diseases (IBD)¹, encompassing Crohn''s disease and ulcerative colitis, are complex chronic inflammatory diseases of the intestine whose etiology and pathogenesis remain unknown. There are multiple etiologic theories, one of which is that a dysregulated CD4 T cell response to the abundant antigens in the lumen may be responsible (1, 2). This hypothesis is based on theoretical grounds and there is only limited supporting data in humans as yet (3, 4). However, support for this hypothesis has come from the results of studies done in a number of recently developed experimental models of IBD, some of which have been the unexpected result of gene deletions by selective gene targeting (5–9). In a number of such models, CD4⁺ T cells have been found to mediate colitis, and most commonly this has involved an exaggerated Th1 response manifested by excessive IFN-γ production in the lesions (10–12). The localization of inflammatory disease to the colon of mice that have global deficiencies of an immune molecule suggests that the bacterial flora is the major immune stimulant leading to chronic intestinal inflammation. Indeed, in some models, animals that are raised germ-free no longer develop colitis (5, 13), and in others rederivation with a defined flora (6, 12) or antibiotic treatment (14) ameliorates the disease. Moreover, reconstitution of intestinal bacteria into germ-free animals can restore intestinal inflammation (15). However, it has remained unclear how the bacterial flora generates chronic intestinal inflammation.Humans with IBD do not have absolute deficiencies of the immune molecules whose deletion in mice has resulted in colitis. For this reason, we have derived and studied a new strain of mice which develop colitis spontaneously, namely the C3H/HeJBir strain (16). C3H/HeJBir mice develop a predominantly right-sided colitis early in life that largely resolves by 3 mo of age. Previous studies on the immunopathogenesis of disease in this mouse strain have found that C3H/HeJBir mice, but not mice of the parenteral C3H/HeJ strain, have high titer serum IgG antibodies to a selected subset of antigens of the enteric bacterial flora (17). These antibodies are mainly of the IgG2a subclass, compatible with a predominant Th1 response to these bacterial antigens. This study was undertaken to define the CD4⁺ T cell response of C3H/HeJBir mice to enteric bacterial antigens. Compatible with our earlier results analyzing antibody responses, strong CD4⁺ Th1 T cell reactivity to protein antigens of the enteric bacterial flora was identified. Moreover, disease could be induced by transfer of enteric bacterial antigen-activated C3H/HeJBir CD4⁺ T cells, but not by control C3H/HeJ T cells, into C3H/ HeSnJ scid/scid recipients. These results demonstrate for the first time that CD4⁺ T cells reactive with conventional antigens of the bacterial flora are able to mediate chronic intestinal inflammation.     

Molecular diagnosis of Arcobacter and Campylobacter in diarrhoeal samples among Portuguese patients

The present study was conducted to investigate the prevalence and diversity of Arcobacter and Campylobacter spp. in 298 stool samples of patients with diarrhoea, collected from 22 Portuguese hospitals, between September and November 2012. Detection of Arcobacter and Campylobacter spp. was performed using molecular-based detection techniques, such as real-time fluorescence resonance energy transfer PCR, species-specific PCR, and sequencing of amplified PCR products. Overall, 1.3% of the samples were positive for Arcobacter butzleri and 0.3% for Arcobacter cryaerophilus. Campylobacter spp. were found in 31.9% of diarrhoeic faeces. Campylobacter jejuni and Campylobacter concisus were the most prevalent species (13.7% and 8.0%, respectively). The prevalence of Arcobacter and Campylobacter spp. was significantly different between children and adults (39.7% versus 22.8%, P = 0.003). We underline the high prevalence of these pathogens in diarrhoeal samples among Portuguese patients, with particular relevance in the paediatric age group.     

An rpoD-based PCR procedure for the identification of Pseudomonas species and for their detection in environmental samples

A polymerase chain reaction-based approach was developed for species identification of Pseudomonas strains and for the direct detection of Pseudomonas populations in their natural environment. A highly selective set of primers (PsEG30F and PsEG790R), giving an amplicon of 760 nucleotides in length, was designed based on the internal conserved sequences of 33 selected rpoD gene sequences (the sigma 70 factor subunit of the DNA polymerase) of Pseudomonas type strains, representing the entire intrageneric phylogenetic clusters described in the genus. The utility of the primer set was verified on 96 Pseudomonas type strains and on another 112 recognised Pseudomonas strains. The specificity of the primer set was also tested against strains from species not belonging to the genus Pseudomonas. These primers were also shown to be useful for the direct detection of Pseudomonas species in environmental DNA after a cloning procedure. These results were compared in parallel with other cloning procedures described previously, based on the analysis of other genes (16S rDNA and ITS1) and also by using primers designed for rpoD on sequences from gamma-proteobacteria. All of the cultured Pseudomonas strains tested could be amplified with these novel primers, indicating that this method is also a useful tool for the specific analysis of Pseudomonas populations from environmental samples without the need for cultivation.     

Persistent Enhancement on Contrast-Enhanced Ultrasound Studies of Severe Crohn's Disease: Stuck Bubbles?

A small population of patients with severe Crohn's disease (CD) exhibit atypical lack of intensity decline on intestinal contrast-enhanced ultrasound. From a retrospective CD cohort examined with contrast-enhanced ultrasound, 104 patients were identified. Twenty study patients with severe active disease exhibited high peak enhancement (>23 dB) and minimal decline. From the same cohort, 84 control patients also exhibited high peak enhancement >23dB, but with typical intensity decline. Patient outcomes were assessed. Time–intensity curve analysis revealed a significantly higher (p < 0.0001) area under the curve (44.7 ± 1.5 dB·s), washout time and intensities at 60s and 120s in the study population compared with controls (40.0 ± 1.1 dB·s). Study patients had a worse overall outcome with surgery in 30% versus 10% (p?=?0.027) during follow-up. Heightened enhancement with lack of decline on contrast-enhanced ultrasound suggests microbubbles are stuck within the inflamed bowel wall for an extended period. This observation occurs in patients with severe disease and a bad outcome.     

Comparison of rpoB gene sequencing, 16S rRNA gene sequencing, gyrB multiplex PCR,and the VITEK2 system for identification of Acinetobacter clinical isolates

Since accurate identification of species is necessary for proper treatment of Acinetobacter infections, we compared the performances of 4 bacterial identification methods using 167 Acinetobacter clinical isolates to identify the best identification method. To secure more non-baumannii Acinetobacter (NBA) strains as target strains, we first identified Acinetobacter baumannii in a total of 495 Acinetobacter clinical isolates identified using the VITEK 2 system. Because 371 of 495 strains were identified as A. baumannii using gyrB multiplex 1 PCR and bla_OXA51-like PCR, we performed rpoB gene sequencing and 16S rRNA gene sequencing on remaining 124 strains belonging to NBA and 52 strains of A. baumannii. For identification of Acinetobacter at the species level, the accuracy rates of rpoB gene sequencing, 16S rRNA gene sequencing, gyrB multiplex PCR, and the VITEK 2 were 98.2%, 93.4%, 77.2%, and 35.9%, respectively. The gyrB multiplex PCR seems to be very useful for the detection of ACB complex because its concordance rates to the final identification of strains of ACB complex were 100%. Both the rpoB gene sequencing and the 16S rRNA gene sequencing may be useful in identifying Acinetobacter.     

Analysis of Mycobacterium tuberculosis-Specific CD8 T-Cells in Patients with Active Tuberculosis and in Individuals with Latent Infection

CD8 T-cells contribute to control of Mycobacterium tuberculosis infection, but little is known about the quality of the CD8 T-cell response in subjects with latent infection and in patients with active tuberculosis disease. CD8 T-cells recognizing epitopes from 6 different proteins of Mycobacterium tuberculosis were detected by tetramer staining. Intracellular cytokines staining for specific production of IFN-γ and IL-2 was performed, complemented by phenotyping of memory markers on antigen-specific CD8 T-cells. The ex-vivo frequencies of tetramer-specific CD8 T-cells in tuberculous patients before therapy were lower than in subjects with latent infection, but increased at four months after therapy to comparable percentages detected in subjects with latent infection. The majority of CD8 T-cells from subjects with latent infection expressed a terminally-differentiated phenotype (CD45RA⁺CCR7⁻). In contrast, tuberculous patients had only 35% of antigen-specific CD8 T-cells expressing this phenotype, while containing higher proportions of cells with an effector memory- and a central memory-like phenotype, and which did not change significantly after therapy. CD8 T-cells from subjects with latent infection showed a codominance of IL-2⁺/IFN-γ⁺ and IL-2⁻/IFN-γ⁺ T-cell populations; interestingly, only the IL-2⁺/IFN-γ⁺ population was reduced or absent in tuberculous patients, highly suggestive of a restricted functional profile of Mycobacterium tuberculosis-specific CD8 T-cells during active disease. These results suggest distinct Mycobacterium tuberculosis specific CD8 T-cell phenotypic and functional signatures between subjects which control infection (subjects with latent infection) and those who do not (patients with active disease).