Direct matrix-assisted laser desorption ionisation time-of-flight mass spectrometry identification of mycobacteria from colonies

Matrix-assisted laser desorption ionisation time-of-flight mass spectrometry (MALDI-TOF-MS) identification of mycobacteria requires a standard acetonitrile/formic acid pre-MALDI-TOF-MS. We prospectively compared this standard protocol with direct deposit with matrix for the identification of mycobacteria cultured on solid media. We first verified that Mycobacterium tuberculosis was killed after it was mixed with matrix. Then, 111 Mycobacterium isolates previously identified by partial rpoB gene sequencing were tested in parallel by the two protocols. An identification score?>1.7 was obtained in 86/111 (77.5 %) isolates after protein extraction versus 97/111 (87.4 %) isolates after direct deposit (p?=?0.039, Chi-squared test). In a third step, we determined that direct deposit achieved identification for as few as 2.10⁴ M. tuberculosis organisms. In a fourth step, we evaluated direct deposit of one colony for 116 solid medium-cultured clinical isolates finally identified as representative of 12 species (63.8 %?M. tuberculosis). For 114/116 (98.3 %) isolates with an identification score >1.2, the MALDI-TOF-MS identification was in complete agreement with the reference rpoB gene sequencing identification. One isolate with a MALDI-TOF-MS identification score of 1.22 for M. fortuitum was identified as M. avium by partial rpoB gene sequencing. One other isolate with a MALDI-TOF-MS identification score of 1.22 for M. tuberculosis was identified as M. tuberculosis by genotyping. All the original MALDI-TOF-MS spectra reported here have been deposited in a public database. Direct deposit of one colony on a MALDI-TOF-MS plate allows for an accurate identification of mycobacteria for an identification score?>1.3.     

The correlation between <Emphasis Type="Italic">Toxoplasma gondii</Emphasis> infection and prenatal depression in pregnant women

Previous studies have demonstrated that latent toxoplasmosis is associated with neuropsychiatric disorders. We evaluated the correlation between Toxoplasma gondii infection and prenatal depression. In this case–control study, we enrolled 116 depressed pregnant women and 244 healthy controls. The Edinburgh Postpartum Depression Scale (EPDS) was used to evaluate the depression symptom severity in study participants. All participants were screened for the anti-Toxoplasma IgG by enzyme-linked immunosorbent assay. Seroprevalence of T. gondii did not significantly differ between the depressed pregnant women and healthy controls (OR?=?1.4; 95 % CI?=?0.9–2.19; P?=?0.142). T. gondii IgG titer was significantly higher in depressed women (18.6?±?10.9 IUs) than those in the control group (13.6?±?8.1 IUs) (z?= ?5.36, P?<?0.001). The T. gondii–positive depressed women showed a positive correlation of T. gondii IgG titer with the EPDS scores (r?=?0.52; P?<?0.01). The mean EPDS score was also significantly higher in the T. gondii–positive depressed women (20.7?±?2.7) compared with the controls (18.36?±?2.7) (P?<?0.001). The results obtained from the current study revealed that T. gondii infection might affect susceptibility to depression and severity of depressive symptoms in pregnant women, particularly in those patients who have high antibody titers. Further study is required to fully elucidate the characteristics and mechanisms of this association.     

Clinical significance of <Emphasis Type="Italic">Aspergillus</Emphasis> species isolated from respiratory specimens in patients with <Emphasis Type="Italic">Mycobacterium avium</Emphasis> complex lung disease

Chronic pulmonary aspergillosis (CPA) is associated with mortality in patients with Mycobacterium avium complex lung disease (MAC-LD). An Aspergillus-positive respiratory specimen often reflects colonization, and thus the clinical significance of Aspergillus isolation in MAC-LD patients is not well understood. The objective of this study was to investigate the clinical characteristics and outcomes of MAC-LD patients in whom Aspergillus was isolated from respiratory specimens. We performed a retrospective review of the medical records of 329 MAC-LD patients. We compared the characteristics and mortality rates between patients with Aspergillus isolation and those without. All Aspergillus species detected from respiratory specimens within the follow-up period were reviewed. Aspergillus was detected in 40 (12.2%) of the 329 patients. There were no significant differences in the clinical characteristics and mortality rates between patients with and without Aspergillus isolation. Among the 40 patients with Aspergillus isolation, 9 (22.5%) developed CPA. CPA was most often caused by A. fumigatus. In the 40 Aspergillus-positive patients, patients with A. fumigatus isolation had a significantly higher mortality rate than those without (P < 0.001). The multivariate Cox proportional hazards model showed older age (P = 0.050), presence of respiratory comorbidities (P = 0.008), hypoalbuminemia (P < 0.001), and isolation of A. fumigatus (P = 0.005) to be prognostic factors for mortality in MAC-LD patients. There was no significant difference in the mortality rates between patients with Aspergillus isolation and those without. However, isolation of A. fumigatus may be associated with poor prognosis in MAC-LD patients.     

A novel inhibition ELISA for the detection and monitoring of <Emphasis Type="Italic">Penicillium marneffei</Emphasis> antigen in human serum

The thermally dimorphic fungus Penicillium marneffei is a causative agent of penicilliosis marneffei, a disease considered to be an acquired immune deficiency syndrome (AIDS)-defining illness in Southeast Asia and southern China. We have developed an inhibition enzyme-linked immunosorbent assay (inh-ELISA) incorporating the yeast phase specific mannoprotein-binding monoclonal antibody 4D1 for the detection of P. marneffei infection. In our sample set, the test detected antigenemia in all 45 (100 %) patients with P. marneffei, with a mean antigen concentration of 4.32 μg/ml. No cross-reactivity in this assay was found using serum from 44 additional patients with other fungal infections, such as Aspergillus fumigatus, Cryptococcus neoformans, and Candida albicans, as well as 44 patients with bacterial infections, such as Mycobacterium tuberculosis and Streptococcus suis. Additionally, no reactivity occurred using serum from 31 human immunodeficiency virus (HIV)-infected patients without a history of fungal infections and 113 healthy controls residing in endemic areas. To investigate the potential of the inh-ELISA for disease monitoring, we followed the reduction in antigenemia in six patients who clinically responded to itraconazole and P. marneffei was no longer isolated from their blood or tissues. In contrast, we correlated increased concentrations of antigenemia in patients with relapsed P. marneffei infection with the progression of their clinical symptoms and the isolation of P. marneffei from their clinical specimens. In summary, the P. marneffei inh-ELISA is a promising new assay for the rapid diagnosis of P. marneffei, as well as a tool for evaluating clinical response and clearance of the fungus during treatment.     

The Phenomenon of the Formation of Biofilms by <Emphasis Type="Italic">Brevibacillus</Emphasis> spp. and <Emphasis Type="Italic">Bacillus</Emphasis> spp. in the Presence of Clinical Strains of <Emphasis Type="Italic">Mycobacterium tuberculosis</Emphasis>

The formation of biofilms by M. tuberculosis on Shkolnikova’s medium (synthetic medium, analogue of Sauton’s medium) has been researched. We studied 150 clinical and 20 laboratory strains of M. tuberculosis. None of the 150 strains isolated from human beings produced biofilms (pellicle), but all yielded abundant planktonic growth. Twenty reference strains of M. tuberculosis produced both biofilms (pellicle) and planktonic growth. The phenomenon of biofilm formation by mixed cultures was observed when inoculating sputum treated with NALC-NaOH from patients with tuberculosis. We obtained 63 mixed biofilms. In 30.2% (19/63) of cases, biofilms contained the DNA of the causative agent of tuberculosis. The RV-PCR method was used to select six samples with the highest concentration of mycobacterial DNA. Molecular cloning and sequencing of a fragment of the 16S rRNA gene from one of the biofilms was carried out. The nucleotide sequence had 99% homology with the Bacillus thermoamylovorans species. From the mixed biofilms obtained, three strains of spore-forming bacilli were isolated. Strains are identified by Sanger’s sequencing of the 16S rRNA gene, one as Bacillus licheniformis, and the other two as Brevibacillus spp. A study of the resistance of isolated strains of spore bacilli against 12 antituberculosis drugs of the first and second series was carried out. All three strains were resistant to maximum concentrations of isoniazid, streptomycin, ethionamide, and ethambutol. Strains of Brevibacillus spp. were additionally resistant to para-aminosalicylic acid (PAS) and kanamycin. In a model experiment, the possibility of cogrowth of clinical strains of M. tuberculosis and B. licheniformis was demonstrated with prolonged co-incubation in Shkolnikova’s medium. In the first few days of growth, B. licheniformis produced a biofilm that remained stable for the entire observation period of 45 days. The hypothesis suggesting the possibility of a short-term persistence of some “saprophytic” species of bacilli in the caseous contents of necrosis foci in the late stages of pulmonary tuberculosis has been postulated.     

Preliminary antimycobacterial study on selected Turkish plants (Lamiaceae) against <Emphasis Type="Italic">Mycobacterium tuberculosis</Emphasis> and search for some phenolic constituents

Background

The global resurgence of tuberculosis is a significant threat. Lamiaceae members have been used in folk remedies for centuries. This study was designed to assess the in-vitro antimycobacterial activity of eighteen crude extracts from six plants (Lamiaceae) and to characterize their phenolic and flavonoid compounds.

Methods

Six Turkish medicinal plants of the family Lamiaceae (Stachys tmolea Boiss., Stachys thirkei C. Koch, Ballota acetabulosa (L.) Benth., Thymus sipthorpii Benth., Satureja aintabensis P.H. Davis, and Micromeria juliana (L.) Benth. ex Reich.) were collected in 2009 – 2010. Dried and crushed plant samples were subjected to sequential extraction with petroleum ether, ethyl acetate, and methanol in order of increasing polarity. A broth microdilution method was employed to screen extracts against four mycobacterial strains of Mycobacterium tuberculosis. Phenolic and flavonoid compounds were characterized by liquid chromatography–mass spectrometry.

Results

S. aintabensis, T. sibthorpii, and M. juliana were found to develop considerable activity against the four strains of M. tuberculosis with the minimal inhibitory concentrations value of 12.5-100 μg/ml. S. aintabensis and T. sibthorpii extracts killed M. tuberculosis with the minimum bactericidal concentration value of 50–800 μg/ml. On the basis of these prominent antimycobacterial activity, we suggest that they could be a source of natural anti-tuberculosis agents.

Conclusion

S. aintabensis and T. sibthorpii showed activity by killing Mycobacteria strains. The major phenolic compound was rosmarinic for T. sibthorpii and S. aintabensis. Flavonoids might be “a modal” for the drug design.

     

Involvement of methylated HBHA expressed from <Emphasis Type="Italic">Mycobacterium smegmatis</Emphasis> in an IFN-γ release assay to aid discrimination between latent infection and active tuberculosis in BCG-vaccinated populations

IFN-γ release assays (IGRAs) based on region of difference 1 (RD1) antigens have improved diagnosis of Mycobacterium tuberculosis (M. tb) infection. However, IGRAs with these antigens cannot discriminate between active tuberculosis (ATB) and latent tuberculosis infection (LTBI). M. tb heparin-binding-hemagglutinin (HBHA) induces relatively high IFN-γ responses in LTBI individuals and low responses in ATB patients, but purification of the native methylated HBHA from cultures of M. tb for immunological tests is complex and time-consuming. To overcome these cumbersome procedures, we constructed a recombinant Mycobacterium smegmatis strain that over-expressed HBHA under control of a strong furA promoter. The methylated activity of purified protein was verified by hybridization with anti-methylated Lys antibody, and the methylated HBHA (mHBHA) was further evaluated for antigen-specific IFN-γ responses in BCG-vaccinated Chinese population. A total of 138 individuals including 86 active TB (ATB) patients, 15 latent TB infection (LTBI) cases, and 37 healthy controls (HC) were tested by using an IFN-γ enzyme-linked immunospot (ELISPOT) assay. The results showed that T-cell responses against mHBHA were always lower in ATB patients than in LTBI individuals, regardless of the site of infection or the results of bacteriological tests. This allowed for a good discrimination between these two groups of M. tb-infected individuals, even in the BCG-vaccinated and high TB-incidence setting that is China. Additionally, combination of mHBHA and RD1 antigens in an IFN-γ release assay enhanced diagnostic efficacy for active TB cases. Taken together, inclusion of the immune response to mHBHA can discriminate healthy LTBI cases from ATB patients.     

Polymorphisms and expression of inflammasome genes are associated with the development and severity of rheumatoid arthritis in Brazilian patients

Objective

In the present study, we analyzed the possible association of inflammasome gene variants and expression to rheumatoid arthritis (RA)’s development and severity in the Brazilian population.

Materials and methods

Thirteen single nucleotide polymorphisms within six inflammasome genes (NLRP1, NLRP3, NLRC4, AIM2, CARD8, CASP1) as well as IL1B and IL18 genes in two different Brazilian populations (from Northeast and Southeast Brazil) were analyzed. We also evaluated inflammasome gene expression profile in resting and LPS?+?ATP-treated monocytes from RA patients and healthy individuals. For genetic association study, 218 patients and 307 healthy controls were genotyped. For gene expression study, inflammasome genes mRNA levels of 12 patients and ten healthy individuals were assessed by qPCR.

Results

Our results showed that rs10754558 NLRP3 and rs2043211 CARD8 polymorphisms are associated with RA development (p value?=?0.044, OR?=?1.77, statistical power?=?0.999) and severity measured by Health Assessment Questionnaire (HAQ) (p value?=?0.03), respectively. Gene expression analyses showed that RA patients display activation of CASP1, IL1B and IL1R genes independently of LPS + ATP activation. In LPS?+?ATP-treated monocytes, NLRP3 and NLRC4 expressions were also significantly higher in patients compared with controls.

Conclusions

The first reported results in Brazilian populations support the role of inflammasome in the development of RA.

     

Asthma and Hypogammaglobulinemia: an Asthma Phenotype with Low Type 2 Inflammation

Purpose

Little is known about hypogammaglobulinemia (HGG) in asthma patients. No data are available on the characteristics of adult patients with asthma and HGG.

Methods

We conducted a retrospective monocentric study between January 2006 and December 2012. Asthma patients with a serum immunoglobulin (Ig) quantitative analysis were included and classified into two groups depending on their serum IgG concentration: presence or absence of HGG. Clinical, biological, functional, and radiologic characteristics were compared in univariate and multivariate analysis, using a logistic regression model.

Results

In univariate analysis, asthma patients with HGG (n?=?25) were older (58 years old?±?18 vs 49?±?18, p?=?0.04) and more frequently active or former smokers as compared to patients with normoglobulinemia (n?=?80) (56.0 vs 35.0 %, p?=?0.01). Total IgE?<?30 kUI/L was more frequently observed in patients with HGG (53.0 vs 18.3 %, p?=?0.01). HGG asthma patients had lower fraction of exhaled nitric oxide (p?=?0.02), blood eosinophilia (p?=?0.0009), and presented with more severe composite score for bronchiectasis (p?=?0.01). In multivariate analysis, asthma patients with HGG had increased risk of being smokers [OR?=?6.11 (IC 95 %?=?1.16–32.04)], having total IgE concentration?<?30 kUI/L [OR?=?12.87 (IC 95 %?=?2.30–72.15)], and a more severe composite score of bronchiectasis [OR?=?20.65 (IC 95 %?=?2.13–199.74)].

Conclusion

Asthma patients with HGG are older and more often tobacco smoker than asthma patients without HGG. These patients have low type-2 inflammation markers.

     

Impact of serum soluble programed death ligand 1 on end of treatment metabolic response of diffuse large B cell lymphoma patients

Programmed death ligand-1 (PD-L1) plays an important role in the immune evasion of cancer cells and, in turn, can influence the outcome of many malignancies. The serum soluble PD-L1 (sPD-L1) levels were measured in diffuse large B cell lymphoma (DLBCL) patients at diagnosis and at end of treatment. Their impact on end of treatment metabolic response was analyzed. Serum sPD-L1 level was significantly elevated in DLBCL patients at diagnosis than in controls (P?<?0.001). Also, serum sPD-L1 level at diagnosis was significantly higher than that at end of treatment (P?<?0.001). Patients who achieved partial response (PR) had significantly higher serum sPD-L1 level at end of treatment than controls (P?<?0.001). In contrast, all patients especially those who achieved complete response (CR) had insignificantly different serum sPD-L1 level at end of treatment than controls (P?=?0.354 and P?=?0.090, respectively). There was a significant difference between serum sPD-L1 level at diagnosis and that at end of treatment in patients who achieved PR and CR (P?=?0.023 and P?<?0.001, respectively). On univariate analysis, presence of comorbidities, Ann Arbor stage IV, high serum sPD-L1 level at diagnosis and high serum sPD-L1 level at end of treatment were significantly associated with achievement of PR (P?=?0.018 and P?=?0.043, P?=?0.045 and P?<?0.001, respectively). On multivariate analysis, serum sPD-L1 levels at diagnosis and at end of treatment were still influencing metabolic response significantly (P?=?0.014 and P?=?0.007, respectively). Serum sPD-L1 is a predictor for metabolic response to immunochemotherapy in DLBCL patients.     

RD7 genotyping of <Emphasis Type="Italic">Mycobacterium tuberculosis</Emphasis> strains isolated from patients with lung tuberculosis in various areas of Kazakhstan

A three-primer PCR assay has been designed for detecting possible deletions in the RD7 chromosomal region of the Mycobacterium tuberculosis complex. The assay gives rise to amplicons of different sizes depending on the presence or absence of deletions. The PCR assay was applied to 176 isolates from lung tuberculosis cases collected in various areas of Kazakhstan in the summer of 2004. Prior to assay, the isolates were characterized by culture and biochemical tests. The RD7 genotyping showed neither polymorphism nor deletions in the RD7 genome region. Some strains were additionally characterized by a PCR-RFLP analysis of the gyrB and hsp64 genes. The RFLP patterns corresponded to M. tuberculosis. The results of the study were consistent with certain previous studies, which indicates the population stability of RD7 in M. tuberculosis strains. Species identification of the isolates showed that M. tuberculosis sensu stricto was the principal causative agent of human lung tuberculosis in Kazakhstan.     

Rheumatoid factor is correlated with disease activity and inflammatory markers in antineutrophil cytoplasmic antibody-associated vasculitis

Background

Some patients with antineutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV) also have positivity of rheumatoid factor (RF). However, the clinical significance of this occurrence remains unknown in AAV patients. The aim of this study was to clarify an association between the presence of RF and clinical features in patients with AAV.

Results

Forty-seven patients diagnosed with AAV who were not complicated with RA were enrolled in this study. We compared clinical manifestations of AAV between an RF-positive subset (n?=?29) and an RF-negative subset (n?=?18). The Birmingham Vasculitis Activity Score (BVAS) was higher (P?=?0.026) in the RF-positive subset than in the RF-negative subset. The levels of CRP and ESR were higher in the RF-positive patients (P?=?0.020 and P?=?0.007, respectively) compared to the RF-negative subset. IgM-RF titers were significantly correlated with the BVAS (r?=?0.50, P?=?0.0004). In addition, the IgM-RF titers had significant correlations with the levels of CRP (r?=?0.41, P?=?0.004), ESR (r?=?0.39, P?=?0.016), IgM (r?=?0.36, P?=?0.016) and IgG (r?=?0.37, P?=?0.015). The frequency of commencement of dialysis therapy, usage of mechanical ventilation and mortality were higher in the RF-positive subset than in the RF-negative subset.

Conclusions

In patients with AAV, RF titers were significantly correlated with disease activity and the levels of inflammatory markers. The presence of RF could be a poor prognostic factor in patients with AAV.

     

Passive administration of purified secretory IgA from human colostrum induces protection against <Emphasis Type="Italic">Mycobacterium tuberculosis</Emphasis> in a murine model of progressive pulmonary infection

Background

Immunoglobulin A is the most abundant isotype in secretions from mucosal surfaces of the gastrointestinal, respiratory and genitourinary tracts and in external secretions such as colostrum, breast milk, tears and saliva. The high concentration of human secretory IgA (hsIgA) in human colostrum strongly suggests that it should play an important role in the passive immune protection against gastrointestinal and respiratory infections.

Materials and methods

Human secretory IgA was purified from colostrum. The reactivity of hsIgA against mycobacterial antigens and its protective capacity against mycobacterial infection was evaluated.

Results

The passive administration of hsIgA reduces the pneumonic area before challenge with M. tuberculosis. The intratracheal administration of M. tuberculosis preincubated with hsIgA to mice greatly reduced the bacterial load in the lungs and diminished lung tissue injury.

Conclusions

HsIgA purified from colostrum protects against M. tuberculosis infection in an experimental mouse model.

     

Humoral immune consequences of <Emphasis Type="Italic">Staphylococcus aureus</Emphasis> ST239-associated bacteremia

The humoral immune responses against 46 different staphylococcal antigens in 27 bacteremia patients infected by clonally related methicillin-resistant Staphylococcus aureus (MRSA) strains of a single sequence type (ST) 239 were investigated. A group of non-infected patients (n = 31) hospitalized for different reasons served as controls. All strains were confirmed as ST 239 by S. aureus and mecA-specific PCR, spa, and multi-locus sequence typing (MLST). In each bacteremia patient, a unique pattern of S. aureus antigen-specific immune responses after infection was observed. Antibody levels among bacteremia patients were significantly higher than controls for HlgB (P = 0.001), LukD (P = 0.009), LukF (P = 0.0001), SEA (P = 0.0001), SEB (P = 0.011), SEC (P = 0.010), SEQ (P = 0.049), IsaA (P = 0.043), IsdA (P = 0.038), IsdH (P = 0.01), SdrD (P = 0.001), SdrE (P = 0.046), EsxA (P = 0.0001), and SA0104 (P = 0.0001). On the other hand, the antibody levels were significantly higher among controls for SSL3 (P = 0.009), SSL9 (P = 0.002), and SSL10 (P = 0.007) when the IgG level on the day of infection was compared with that measured on the day of admission. Diversity was observed in the immune response against the antigens. However, a set of antigens (IsaA, IsdA, IsdH, SdrD, and HlgB) triggered a similar type of immune response in different individuals. We suggest that these antigens could be considered when developing a multi-component (passive) vaccine. SEA and/or its specific antibodies seem to play a critical role during ST239 MRSA bacteremia and SEA-targeted therapy may be a strategy to be considered.     

Differences in humoral immune response between patients with or without nasal carriage of <Emphasis Type="Italic">Staphylococcus aureus</Emphasis>

The humoral immune response against 43 staphylococcal antigens was compared among hospitalized patients where none of them had any staphylococcal infection on the day of admission with or without nasal Staphylococcus aureus carriage. Fifty-nine carriers and 59 matched non-carriers were studied. The carriers harbored S. aureus of 35 different spa types, including three t037/ST239 methicillin-resistant S. aureus (MRSA) (5.1%). Among the 118 patients, 31 acquired S. aureus during hospitalization. In colonized and non-colonized patients, unique patterns of S. aureus-specific immune responses were observed. The mean fluorescence indices (MFIs) of antibodies against 36/43 (83.7%) antigens were seen to be elevated among carriers. The MFI among carriers with acquisition was significantly higher for staphylococcal superantigen-like protein 5 (SSL5, p?=?0.028) when compared to carriers without acquisition. High antibody levels against staphylococcal enterotoxin A (SEA) among carriers illustrate its role as a superantigen in both infection and colonization. We also report a dynamic immune response in S. aureus-carrying patients against the recently reported formyl peptide receptor-like inhibitory (FLIPr)-like protein. In the current study, the dynamics of antibodies against staphylococcal antigens among carrier patients seem quite similar to non-carrier patients. To better understand the dynamic immunogenicity during S. aureus infection and colonization, artificial colonization studies and investigation of the changes in the levels of antibodies against other staphylococcal antigens are recommended.     

A first insight into the genotypic diversity of <Emphasis Type="Italic">Mycobacterium tuberculosis</Emphasis> from Rwanda

Background

Mycobacterium tuberculosis complex (MTC) is the causative agent of tuberculosis (TB). Globally, increasing evidence shows that in M. tuberculosis, transmission varies from strain to strain and that different strains exhibit a range of geographical and host specificities, pathogenicity, and drug susceptibility. Therefore rapid and accurate differentiation of the members of MTC is critical in guiding treatment and public health decisions. We carried out a study at different health units and the National Reference Laboratory in Rwanda identify Mycobacterium tuberculosis complex species prevalent in TB patients in Rwanda. We further characterized the isolates using spoligotyping in order to gain an insight into the strain diversity of drug resistant and susceptible isolates of M. tuberculosis in this setting.

Methods

A total of 151 isolates from culture positive sputum samples were harvested, heat killed at 80°C for two hours, and then shipped to Makerere University College of Health Sciences, Uganda, for speciation and typing. Species identification was achieved by regions of difference (RD) analysis, while Spoligotyping was done to identify strain types.

Results

Region of difference analysis identified all the 151 isolates as M. tuberculosis. Spoligotyping revealed predominance of the T2 family (58.3%, 88/151), with SIT 52 being the most prevalent strain (31.8%, 48/151). Among the 151 isolates, 64 (42.4%) were multidrug resistant (MDR) with 3 cases on mono-resistance. Of 94 retreatment cases, 48 (51.1%) were MDR and of 46 newly presenting cases 14 (30.4%) were MDR. There was a significant difference (p=0.01) in anti-TB drug resistance between new and retreatment cases in the sample. However, there was no significant relationship between HIV serostatus and the two major strain types SIT 52 (p =0.15and SIT 152 (p = 0.41).

Conclusion

Mycobacterium tuberculosis is the most prevalent species of Mycobacterium tuberculosis complex in Rwanda, and SIT 52 (T2) the predominant strain. There is significantly more MDR in the retreatment cases but no significant difference was observed by HIV status in relation to any spoligotypes.

     

The relevance of Tim-3 polymorphisms and F protein to the outcomes of HCV infection

Hepatitis C virus (HCV) is one of the major causes of liver inflammation. The aim of this study was to investigate the associations of T-cell immunoglobulin and mucin domain-3 (Tim-3) polymorphisms and the alternate reading frame protein (F protein) with the outcomes of HCV infection. Three single-nucleotide polymorphisms (SNPs; rs10053538, rs12186731, and rs13170556) of Tim-3 were genotyped in this study, which included 203 healthy controls, 558 hepatitis C anti-F-positive patients, and 163 hepatitis C anti-F-negative patients. The results revealed that the rs12186731 CT and rs13170556 TC and CC genotypes were significantly less frequent in the anti-F-positive patients [odds ratio (OR)?=?0.54, 95 % confidence interval (CI)?=?0.35–0.83, p?=?0.005; OR?=?0.26, 95 % CI?=?0.18–0.39, p?<?0.001; and OR?=?0.19, 95 % CI?=?0.10–0.35, p?<?0.001, respectively), and the rs13170556 TC genotype was more frequent in the chronic HCV (CHC) patients (OR?=?1.70, 95 % CI?=?1.20–2.40, p?=?0.002). The combined analysis of the rs12186731 CT and rs13170556 TC/CC genotypes revealed a locus-dosage protective effect in the anti-F-positive patients (OR?=?0.22, 95 % CI?=?0.14–0.33, p _trend?<?0.001). Stratified analyses revealed that the frequencies of the rs12186731 (CT?+?TT) genotypes were significantly lower in the older (OR?=?0.31, 95 % CI?=?0.15–0.65, p?=?0.002) and female (OR?=?0.30, 95 % CI?=?0.17–0.52, p?<?0.001) subgroups, and rs13170556 (TC?+?CC) genotypes exhibited the same effect in all subgroups (all p?<?0.001) in the anti-F antibody generations. Moreover, the rs13170556 (TC?+?CC) genotypes were significantly more frequent in the younger (OR?=?1.86, 95 % CI?=?1.18–2.94, p?=?0.007) and female (OR?=?2.38, 95 % CI?=?1.48–3.83, p?<?0.001) subgroups of CHC patients. These findings suggest that the rs12186731 CT and rs13170556 TC/CC genotypes of Tim-3 provide potential protective effects with the F protein in the outcomes of HCV infection and that these effects are related to sex and age.     

Fecal calprotectin concentrations in cancer patients with <Emphasis Type="Italic">Clostridium difficile</Emphasis> infection

Fecal calprotectin (fCPT) has been used as a surrogate marker for assessment of intestinal inflammation. We explore the utility of fCPT values as a diagnostic aid in cancer patients with suspected Clostridium difficile infection (CDI). A total of 232 stool specimens submitted for GeneXpert C. difficile PCR testing were included in the study. All specimens were tested for fCPT and toxin/GDH antigens. Clinical severity of CDI cases was determined by the IDSA/SHEA criteria. Significant differences of median fCPT values between CDI (n?=?117, Median 183.6 μg/g) and non-CDI (n?=?115, 145.6 μg/g, p?=?0.006) patients were seen. In CDI patents, significantly lower fCPT values were found in patients with mild to moderate (n?=?95, 182.1 μg/g) than those with severe and severe to complicated (n?=?22, 218.5 μg/g, p?=?0.014) scores, and among those that were toxin positive (n?=?24, 200.2 μg/g) vs. toxin negative (n?=?86, 182.8 μg/g, p?=?0.044). Despite this overall trend, wide variations in fCPT values were found in all categories examined. A logistic regression analysis revealed that the fCPT values correlated independently with the severity of clinical manifestations (OR?=?2.021, 95%CI?=?1.132–3.608); however, it did not correlate with other clinical outcomes. Our study findings show that high fecal calprotectin levels correlate with toxin-positive and clinically severe CDI; however, wide variations in individual measurements preclude establishment of reliable cut-offs for routine diagnostic use in cancer patients.     

Phage display of functional αβ single-chain T-cell receptor molecules specific for CD1b:Ac<Subscript>2</Subscript>SGL complexes from <Emphasis Type="Italic">Mycobacterium tuberculosis</Emphasis>-infected cells

The development of molecules specific for M. tuberculosis-infected cells has important implications, as these tools may facilitate understanding of the mechanisms regulating host pathogen interactions in vivo. In addition, development of new tools capable to targeting M. tuberculosis-infected cells may have potential applications to diagnosis, treatment, and prevention of tuberculosis (TB). Due to the lack of CD1b polymorphism, M. tuberculosis lipid-CD1b complexes could be considered as universal tuberculosis infection markers. The aim of the present study was to display on the PIII surface protein of m13 phage, a human αβ single-chain T-cell receptor molecule specific for CD1b:2-stearoyl-3-hydroxyphthioceranoyl-2´-sulfate-α-α´-D-trehalose (Ac₂SGL) which is a complex presented by human cells infected with M. tuberculosis. The results showed the pIII fusion particle was successfully displayed on the phage surface. The study of the recognition of the recombinant phage in ELISA and immunohistochemistry showed the recognition of CD1b:Ac₂SGL complexes and cells in human lung tissue from a tuberculosis patient respectively, suggesting the specific recognition of the lipid-CD1b complex.