BACKGROUND

There is limited available data on the strain diversity of M tuberculosis in Peru, though there may be interesting lessons to learn from a setting where multidrug resistant TB has emerged as a major problem despite an apparently well-functioning DOTS control programme.

METHODS

Spoligotyping was undertaken on 794 strains of M tuberculosis collected between 1999 and 2005 from 553 community-based patients and 241 hospital-based HIV co-infected patients with pulmonary tuberculosis in Lima, Peru. Phylogenetic and epidemiologic analyses permitted identification of clusters and exploration of spoligotype associations with drug resistance.

RESULTS

Mean patient age was 31.9 years, 63% were male and 30.4% were known to be HIV+. Rifampicin mono-resistance, isoniazid mono-resistance and multidrug resistance (MDR) were identified in 4.7%, 8.7% and 17.3% of strains respectively. Of 794 strains from 794 patients there were 149 different spoligotypes. Of these there were 27 strains (3.4%) with novel, unique orphan spoligotypes. 498 strains (62.7%) were clustered in the nine most common spoligotypes: 16.4% SIT 50 (clade H3), 12.3% SIT 53 (clade T1), 8.3% SIT 33 (LAMLAMLAM), and 2.8% SIT 92 (X3). Amongst HIV-negative community-based TB patients no associations were seen between drug resistance and specific spoligotypes; in contrast HIV-associated MDRTB, but not isoniazid or rifampicin mono-resistance, was associated with SIT42 and SIT53 strains.

CONCLUSIONS

Two spoligotypes were associated with MDR particularly amongst patients with HIV. The MDR-HIV association was significantly reduced after controlling for SIT42 and SIT53 status; residual confounding may explain the remaining apparent association. These data are suggestive of a prolonged, clonal, hospital-based outbreak of MDR disease amongst HIV patients but do not support a hypothesis of strain-specific propensity for the acquisition of resistance-conferring mutations.

The anti-tuberculosis drug, ethambutol (Emb), was previously shown to inhibit the synthesis of arabinans of both the cell wall arabinogalactan (AG) and lipoarabinomannan (LAM) of Mycobacterium tuberculosis and other mycobacteria. However, an Emb-resistant mutant, isolated by consecutive passage of the Mycobacterium smegmatis parent strain in media containing increasing concentrations of Emb, while synthesizing a normal version of AG, produced truncated forms of LAM when maintained on 10 microg/ml Emb (Mikusová, K., Slayden, R. A., Besra, G. S., and Brennan, P. J. (1995) Antimicrob. Agents Chemother. 39, 2482-2489). We have now isolated and characterized the truncated LAMs made by both the resistant mutant and a recombinant strain transfected with a plasmid containing the emb region from Mycobacterium avium which encodes for Emb resistance. By chemical analysis, endoarabinanase digestion, high pH anion exchange chromatography, and mass spectrometry analyses, truncation was demonstrated as primarily a consequence of selective and partial inhibition of the synthesis of the linear arabinan terminal motif, which constitutes a substantial portion of the arabinan termini in LAM but not of AG. However, at higher concentrations, Emb also affected the general biosynthesis of arabinan destined for both AG and LAM, resulting in severely truncated LAM as well as AG with a reduced Ara:Gal ratio. The results suggested that Emb exerts its antimycobacterial effect by inhibiting an array of arabinosyltransferases involved in the biosynthesis of arabinans unique to the mycobacterial cell wall. It was further concluded that the uniquely branched terminal Ara6 motif common to both AG and LAM is an essential structural entity for a functional cell wall and, consequently, that the biosynthetic machinery responsible for its synthesis is the effective target of Emb in its role as a potent anti-tuberculosis drug.

BACKGROUND

Reducing mortality from HIV-associated tuberculosis (TB) requires diagnostic tools that are rapid and have high sensitivity among patients with poor prognosis. We determined the relationship between disease severity and the sensitivity of new sputum-based and urine-based diagnostic assays.

METHODS

Consecutive ambulatory patients enrolling for antiretroviral treatment in South Africa were screened for TB regardless of symptoms using diagnostic assays prospectively applied to sputum (fluorescence smear microscopy, Xpert MTB/RIF and liquid culture (reference standard)) and retrospectively applied to stored urine samples (Determine TB-LAM and Xpert MTB/RIF). Assay sensitivities were calculated stratified according to pre-defined indices of disease severity: CD4 count, symptom intensity, serum C-reactive protein (CRP), hemoglobin concentration and vital status at 90 days.

RESULTS

Sputum culture-positive TB was diagnosed in 15% (89/602) of patients screened and data from 86 patients were analyzed (median CD4 count, 131 cells/μL) including 6 (7%) who died. The sensitivity of sputum microscopy was 26.7% overall and varied relatively little with disease severity. In marked contrast, the sensitivities of urine-based and sputum-based diagnosis using Determine TB-LAM and Xpert MTB/RIF assays were substantially greater in sub-groups with poorer prognosis. Rapid diagnosis from sputum and/or urine samples was possible in >80% of patients in sub-groups with poor prognosis as defined by either CD4 counts <100 cells/μL, advanced symptoms, CRP concentrations >200 mg/L or hemoglobin <8.0 g/dl. Retrospective testing of urine samples with Determine TB-LAM correctly identified all those with TB who died.

CONCLUSIONS

The sensitivities of Xpert MTB/RIF and Determine TB-LAM for HIV-associated TB were highest among HIV-infected patients with the most advanced disease and poorest prognostic characteristics. These data provide strong justification for large-scale intervention studies that assess the impact on survival of screening using these new sputum-based and urine-based diagnostic approaches.

BACKGROUND

The HIV pandemic raised the potential for facultative-pathogenic mycobacterial species like, Mycobacterium kansasii, to cause disseminating disease in humans with immune deficiencies. In contrast, non-pathogenic mycobacterial species, like M. smegmatis, are not known to cause disseminating disease even in immunocompromised individuals. We hypothesized that this difference in phenotype could be explained by the strong induction of an innate immune response by the non-pathogenic mycobacterial species.

RESULTS

A comparison of two rapid-growing, non-pathogenic species (M. smegmatis and M. fortuitum) with two facultative-pathogenic species (M. kansasii and M. bovis BCG) demonstrated that only the non-pathogenic bacteria induced strong apoptosis in human THP-1 cells and murine bone marrow-derived macrophages (BMDM) and dendritic cells (BMDD). The phospho-myo-inositol modification of lipoarabinomannan (PI-LAM) isolated from non-pathogenic species may be one of the cell wall components responsible for the pro-inflammatory activity of the whole bacteria. Indeed, PI-LAM induces high levels of apoptosis and IL-12 expression compared to the mannosyl modification of LAM isolated from facultative-pathogenic mycobacteria. The apoptosis induced by non-pathogenic M. smegmatis was dependent upon caspase-3 activation and TNF secretion. Consistently, BALB/c BMDM responded by secreting large amounts of TNF upon infection with non-pathogenic but not facultative-pathogenic mycobacteria. Interestingly, C57Bl/6 BMDM do not undergo apoptosis upon infection with non-pathogenic mycobacteria despite the fact that they still induce an increase in TNF secretion. This suggests that the host cell signaling pathways are different between these two mouse genotypes and that TNF is necessary but not sufficient to induce host cell apoptosis.

CONCLUSIONS

These results demonstrate a much stronger induction of the innate immune response by non-pathogenic versus facultative-pathogenic mycobacteria as measured by host cell apoptosis, IL-12 and TNF cytokine induction. These observations lend support to the hypothesis that the strong induction of the innate immune response is a major reason for the lack of pathogenicity in fast-growing mycobacteria.

OBJECTIVE

Severe alcoholic hepatitis occurs mainly in patients with cirrhosis, and has a high death rate. Corticosteroid therapy has been particularly advocated as reducing mortality in patients with severe histologic lesions. However, identification of these patients is difficult, requiring transvenous liver biopsy. Extracellular matrix serum markers have been proposed as non-invasive diagnostic tools in alcoholic liver disease. The aim of this study was to determine the accuracy of 5 extracellular matrix serum markers, i.e. laminin (Lam), N-terminal peptide of type III procollagen (PIIINP), type I (CI), type III (CIII) and type IV (CIV) collagens in identifying patients with severe histologic alcoholic hepatitis from among those with cirrhosis and suspected alcoholic hepatitis.

METHODS

We studied 80 consecutive patients with alcoholic cirrhosis and clinical suspicion of alcoholic hepatitis referred for transvenous liver biopsy. Clinical severity of alcoholic hepatitis was assessed according to Maddrey's score. Histological severity was scored using the sum of the 3 following items: polynuclear infiltration (0-3); hepatocytes alterations (0-3); Mallory bodies (0-2). According to this score, patients were divided into 3 groups: mild (1-3), moderate (4-6), and severe (7-8) alcoholic hepatitis. Serum levels of the 5 extracellular matrix serum markers were measured at the time of biopsy using radioimmunoassays. Diagnostic value for histologically severe alcoholic hepatitis of the 5 extracellular matrix serum markers was assessed using receiver operating characteristic curves.

RESULTS

Histological alcoholic hepatitis was present in 67 patients (mean alcoholic hepatitis score: 3.4+/-2.3). Maddrey's score was 66% sensitive and 69% specific for the diagnosis of severe histologic alcoholic hepatitis. The serum Lam and CIV concentrations were the most accurate in identifying correctly patients with severe histologic alcoholic hepatitis. At a cut-off of 4.1 UI/ml, Lam was 90% sensitive and 77% specific, whereas at a cut-off of 150 ng/ml, CIV was 89% sensitive and 77% specific. Combination of markers did not result in improved diagnostic value.

CONCLUSIONS

In patients with cirrhosis, determination of serum Lam or CIV could represent a simple and accurate non-invasive method for identification of patients with histologically severe alcoholic hepatitis eligible for corticosteroid treatment.

Immunoglobulin G subclass responses to lipoarabinomannan (LAM) of Mycobacterium tuberculosis were determined by ELISA in both HIV-1 antibody positive (n = 31) and negative (n = 43) patients with tuberculosis (TB). Responses were also studied in a group of healthy controls (n = 16) and HIV-1 antibody positive (n = 60) individuals without TB. IgG2 antibodies were the predominant subclass, being present in 25 of 43 non-HIV-infected TB patients (58%) and in 11 of 31 HIV-infected TB patients (35%). However, HIV+ TB patients also showed IgG4 (n = 16; 52%), and IgG1 (n = 4, 13%) responses to LAM, whereas these subclasses were absent in sera from HIV-TB patients. Individuals in both non-tuberculous control groups showed no antibody responses to LAM. The influence of HIV infection on B cell responses to LAM, and possible mechanisms for antibody-mediated regulation of immunity to TB, are explored.

BACKGROUND

Renal dysfunction and Fanconi's syndrome associated with hypophosphatemia caused by long-term administration of low-dose adefovir dipivoxil (ADV) has been reported in recent years. The aim of this retrospective study was to determine the incidence and factors associated with renal dysfunction and hypophosphatemia in patients with hepatitis B infection on long-term treatment with ADV and lamivudine (LAM).

METHODS

The study subjects were 292 patients treated with 10 mg/day ADV and 100 mg/day LAM for more than 6 months. We evaluated estimated glomerular filtration rate (eGFR), serum creatinine and serum phosphate level at the start of ADV and every 6 months.

RESULTS

During a median treatment duration of 64 months, 28 (9.6 %) patients developed renal impairment (defined as eGFR < 50 ml/min/1.73 m(2)), and 73 (27.1 %) developed hypophosphatemia, including 14 with persistent hypophosphatemia. The cumulative incidences of renal impairment at 1, 3, and 5 years were 1.4, 7.5, 10.5 %, respectively, and those of hypophosphatemia were 6.8, 20.6, 26.7 %, respectively. Multivariate analysis identified old age, liver cirrhosis and hypertension as determinants of renal impairment, and male sex, HCC, low baseline serum phosphate as determinants of hypophosphatemia. Three of the 14 patients with persistent hypophosphatemia developed Fanconi's syndrome; their serum creatinine level remained normal, but eGFR was lower than at baseline.

CONCLUSIONS

Long-term treatment of hepatitis B with low-dose (10 mg/day) ADV and LAM can potentially cause renal impairment and hypophosphatemia. We advocate regular monitoring of serum phosphate and evaluation of eGFR, in addition to serum creatinine, in such patients.

Lymphangioleiomyomatosis (LAM) is a female-predominant interstitial lung disease that can lead to respiratory failure. LAM cells typically have inactivating TSC2 mutations, leading to mTORC1 activation. The gender specificity of LAM suggests that estradiol contributes to disease development, yet the underlying pathogenic mechanisms are not completely understood. Using metabolomic profiling, we identified an estradiol-enhanced pentose phosphate pathway signature in Tsc2-deficient cells. Estradiol increased levels of cellular NADPH, decreased levels of reactive oxygen species, and enhanced cell survival under oxidative stress. Mechanistically, estradiol reactivated Akt in TSC2-deficient cells in vitro and in vivo, induced membrane translocation of glucose transporters (GLUT1 or GLUT4), and increased glucose uptake in an Akt-dependent manner. (18)F-FDG-PET imaging demonstrated enhanced glucose uptake in xenograft tumors of Tsc2-deficient cells from estradiol-treated mice. Expression array study identified estradiol-enhanced transcript levels of glucose-6-phosphate dehydrogenase (G6PD), the rate-limiting enzyme of the pentose phosphate pathway. Consistent with this, G6PD was abundant in xenograft tumors and lung metastatic lesions of Tsc2-deficient cells from estradiol-treated mice. Molecular depletion of G6PD attenuated estradiol-enhanced survival in vitro, and treatment with 6-aminonicotinamide, a competitive inhibitor of G6PD, reduced lung colonization of Tsc2-deficient cells. Collectively, these data indicate that estradiol promotes glucose metabolism in mTORC1 hyperactive cells through the pentose phosphate pathway via Akt reactivation and G6PD upregulation, thereby enhancing cell survival under oxidative stress. Interestingly, a strong correlation between estrogen exposure and G6PD was also found in breast cancer cells. Targeting the pentose phosphate pathway may have therapeutic benefit for LAM and possibly other hormonally dependent neoplasms.

OBJECTIVE

To determine the gender-specific prevalence of pulmonary cysts typical for lymphangioleiomyomatosis (LAM) in adult patients with known tuberous sclerosis complex (TSC).

METHODS

A retrospective, cross-sectional study in a cohort of 206 adult TSC patients was performed. Institutional review board approval was obtained, and patient informed consent was waived. Patients had routinely undergone abdominal CT scanning between 1996 and 2006. All 186 patients (mean age 38 years; range 19-72 years; 91 (49%) male patients) in whom at least the lung bases were depicted on computed tomography (CT) were included. Images were reviewed for the presence of pulmonary thin-walled cysts. Descriptive statistics, two sample t-test to compare means, and χ(2)-test to compare proportions were applied.

RESULTS

CT demonstrated pulmonary thin-walled cysts in the lung bases in 52 (28%) of 186 patients. Size varied from 2mm in diameter to more than 2 cm. Pulmonary cysts were detected in 40 (42%) of 95 female patients and in 12 (13%) of 91 male patients (p<0.001). In general, cysts were larger and more numerous in women than in men. Only minimal cystic changes were found in four women and two men, moderate cystic changes were seen in three women and seven men, but considerable cystic changes were seen almost exclusively in women (33 women versus three men).

CONCLUSIONS

CT demonstrated thin-walled pulmonary cysts in the lung bases in 28% of 186 included patients with tuberous sclerosis complex. Female patients were more affected than male patients.

BACKGROUND

Lymphangioleiomyomatosis (LAM) is a prominent finding in the setting of tuberous sclerosis complex (TSC).

OBJECTIVE

The present study was designed to compare cystic lung changes consistent with LAM in patients with a TSC1 disease-causing mutation, TSC2 disease-causing mutation, or no mutation identified (NMI).

RESULTS

We conducted a retrospective review of the chest computed tomography (CT) of 45 female and 20 male patients with TSC and found cysts consistent with LAM in 22 (49%) women and two (10%) men. In the female population, changes consistent with LAM were observed in six of 15 (40%) patients with TSC1, 11 of 23 (48%) with TSC2, and five of seven (71%) with NMI. While the predominant size of cysts did not differ across these three groups, TSC2 women with LAM had a significantly greater number of cysts than did TSC1 patients (p = 0.010).

CONCLUSIONS

These findings suggest a higher rate of LAM in TSC1 than previously recognised, as well as a fundamental difference in CT presentation between TSC1 and TSC2.

BACKGROUND

The relationship between anemia and undiagnosed tuberculosis (TB) in patients living with HIV in sub-Saharan Africa is incompletely defined. We assessed the prevalence of TB among those with HIV-related anemia and evaluated new means of rapid TB diagnosis.

METHODS

Blood hemoglobin levels were measured in unselected antiretroviral treatment-naive patients in Cape Town, South Africa, and anemia was classified according to World Health Organization criteria. All patients were screened for TB by testing paired sputum samples using liquid culture (reference standard), fluorescence microscopy, and Xpert MTB/RIF. Urine samples were tested for lipoarabinomannan (LAM) using the Determine TB-LAM diagnostic assay.

RESULTS

Of 602 adults screened, 485 had complete results. Normal hemoglobin levels were found in 44.5% (n = 216) of patients, and mild, moderate, or severe anemia were present in 24.9% (n = 121), 25.4% (n = 123) and 5.2% (n = 25) of patients, respectively. Culture-confirmed pulmonary TB was diagnosed in 8.8% (19/216) of those without anemia compared with 16.5% (20/121), 26.0% (32/123), and 40.0% (10/25) among those with mild, moderate, or severe anemia, respectively (P < 0.001). Anemia was a strong independent predictor of TB. The sensitivities of diagnostic assays were much higher among those with moderate/severe anemia compared with those with no/mild anemia using sputum microscopy (42.9% vs 15.4%), urine LAM (54.8% vs 0%), sputum microscopy plus urine LAM (71.4% vs 15.4%), and sputum Xpert (73.8% vs 41.0%) (P < 0.01 for all).

CONCLUSIONS

A very high prevalence of undiagnosed TB was found in patients with moderate or severe anemia. Such patients should be prioritized for routine microbiological investigation using rapid diagnostic assays.

BACKGROUND

Tuberculosis (TB) is one of the major public health problems in Zambia. However, information about lineages of M. tuberculosis complex (MTBC) isolates useful for epidemiology investigations is unknown. In this study, we investigated the diversity of MTBC isolates from Ndola, a typical Zambian urbanized city with a documented high HIV prevalence.

METHODS

This was part of a prospective cohort study in subjects with sputum smear-positive pulmonary TB. Spoligotyping was used to genotype the MTBC isolates and establish the circulating lineages. The 15-locus Mycobacterial Interspersed Repetitive Units - Variable Number Tandem Repeats (MIRU-VNTR) typing was used to study recent transmission.

RESULTS

A total of 98 different spoligotypes were identified among 273 MTBC isolates. The majority (64.8%) of the isolates belonged to 9 known families, while 96 (35.2%) of the isolates were orphans. While <em>LAM</em> (41.8%) was the largest spoligotype family observed, most of the isolates (87.7%) belonging to the SAF1 family, with a significant portion coming from the T (13.6%), and X (5.9%) families. A few isolates (3.6%) belonged to the CAS, EAI, H, S, X1-<em>LAM</em>9 or U families. MIRU-VNTR typing was highly discriminatory (h = 0.988) among the 156 isolates tested in our sample, and increased the discrimination among 82 SAF1 isolates from 6 to 46 distinct patterns. In addition, 3.2% (5/156) of cases with available MIRU-VNTR results harbored more than one MTBC strain.

CONCLUSIONS

Our findings show a limited diversity of MTBC in Ndola with a high clustering rate (37.7%), which indicates that recent transmission plays an appreciable role in the dynamics of TB disease in this setting. This conclusion emphasizes the importance of early diagnosis and timely treatment. The results also confirm that MIRU-VNTR typing is suitable for studying the molecular epidemiology of TB in Ndola.

BACKGROUND

Understanding the true prevalence of lymphangioleiomyomatosis (LAM) is important in estimating disease burden and targeting specific interventions. As with all rare diseases, obtaining reliable epidemiological data is difficult and requires innovative approaches.

OBJECTIVE

To determine the prevalence and incidence of LAM using data from patient organizations in seven countries, and to use the extent to which the prevalence of LAM varies regionally and nationally to determine whether prevalence estimates are related to health-care provision.

METHODS

Numbers of women with LAM were obtained from patient groups and national databases from seven countries (n = 1001). Prevalence was calculated for regions within countries using female population figures from census data. Incidence estimates were calculated for the USA, UK and Switzerland. Regional variation in prevalence and changes in incidence over time were analysed using Poisson regression and linear regression.

RESULTS

Prevalence of LAM in the seven countries ranged from 3.4 to 7.8/million women with significant variation, both between countries and between states in the USA. This variation did not relate to the number of pulmonary specialists in the region nor the percentage of population with health insurance, but suggests a large number of patients remain undiagnosed. The incidence of LAM from 2004 to 2008 ranged from 0.23 to 0.31/million women/per year in the USA, UK and Switzerland.

CONCLUSIONS

Using this method, we have found that the prevalence of LAM is higher than that previously recorded and that many patients with LAM are undiagnosed.

In Burkina Faso, most people in particular, in rural areas, use traditional medicine and medicinal plants to treat usual diseases. In the course of new antimalarial compounds, an ethnobotanical survey has been conducted in different regions. Seven plants, often cited by traditional practitioners and not chemically investigated, have been selected for an antiplasmodial screening: Pavetta crassipes (K. Schum), Acanthospermum hispidum (DC), Terminalia macroptera (Guill. et Perr), Cassia siamea (<em>Lam</em>), Ficus sycomorus (L), Fadogia agrestis (Schweinf. Ex Hiern) and Crossopteryx febrifuga (AFZ. Ex G. Don) Benth. Basic, chloroform, methanol, water-methanol and aqueous crude extracts have been prepared and tested on Plasmodium falciparum chloroquine-resistant W2 strain. A significant activity has been observed with alkaloid extract of P. crassipes (IC(50)<4 microg/ml), of A. hispidum, C. febrifuga, and F. agrestis (4<IC(50)<10 microg/ml). The best result is obtained with aqueous extract of T. macroptera with an IC(50)=1 microg/ml. These results confirm the traditional use of these plants.

Intravesical administration of Mycobacterium bovis bacillus Calmette-Guérin (BCG) continues to be a successful immunotherapy for superficial bladder cancer. Recently, workers in our laboratory observed expression of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) on neutrophils in voided urine following BCG therapy. Neutrophils released a soluble and functional form of TRAIL when they were stimulated in vitro with BCG, and the activity was localized predominantly to the cell wall fraction. In this study, we examined the ability of individual mycobacterial components to stimulate TRAIL release from neutrophils. Our results demonstrated that cell wall-derived lipoarabinomannan (LAM), mycolyl arabinogalactan-peptidoglycan complex, and a Triton X-114 (Tx114)-solubilized protein pool were effective agonists of TRAIL release from neutrophils. Mycobacterial DNA was also an agonist of TRAIL release from neutrophils. Furthermore, purified antigen 85 ABC complex and alpha-crystallin (HspX), two major cell wall antigens present in the Tx114 pool, induced TRAIL release from neutrophils. The Tx114 pool stimulated HEK-293 cells expressing either Toll-like receptor 2/1 (TLR2/1) or TLR2/6, but only HspX was able to stimulate TLR2/6-expressing cells. TLR4/MD2/CD14-expressing cells responded only to LAM. Collectively, these results suggested that TRAIL release from neutrophils was induced through the recognition of multiple mycobacterial components by TLR2 and TLR4.

Pathogenic mycobacteria are highly adapted for survival within host mononuclear phagocytes. This is largely due to the organism's capacity to prevent macrophage activation, block phagosome acidification and maturation, and attenuate presentation of antigens to the immune system. Mycobacterium avium subsp. paratuberculosis (MAP) is one such organism that modulates the ruminant innate immune response. It is the causative agent in paratuberculosis, a chronic progressive granulomatous enteritis in ruminants. MAP initially interacts with cell membrane receptors on bovine mononuclear phagocytes and initiates cell signaling responses and phagocytosis. Mannosylated liparabinomannan (Man-LAM) is a major component of the MAP cell wall that interacts with the cell membrane of mononuclear phagocytes and may be a major virulence factor. Toll-like receptor 2 (TLR2) has been incriminated as major signaling receptor that binds to MAP and initiates signaling though the mitogen-activated protein kinase (MAPK)-p38 pathway. This pathway induces transcription of interleukin (IL)-10. Early production of IL-10 suppresses proinflammatory cytokines, chemokines, IL-12, and major histocompatability factor class-II expression. Both IL-10 dependent and IL-10 independent mechanisms appear to be involved in attenuation of phagosome acidification and phagolysosome fusion. Many of the suppressive effects of MAP on bovine mononuclear phagocytes can be reproduced by exposure of bovine monocytes to Man-LAM. Therefore, MAP Man-LAM-induced TLR2-MAPK-p38 signaling with resultant excessive IL-10 expression has emerged as one of the mechanisms by which MAP organisms suppress inflammatory, immune, and antimicrobial responses and promote their survival within host mononuclear phagocytes.

This investigation provides a novel method of endophyte-aided removal of polycyclic aromatic hydrocarbons (PAHs) from plant bodies. A phenanthrene-degrading endophytic bacterium Pseudomonas sp. Ph6 was isolated from clover (Trifolium pratense L.) grown in a PAH-contaminated site. After being marked with the GFP gene, the colonization and distribution of strain Ph6-gfp was directly visualized in plant roots, stems, and leaves for the first time. After ryegrass (Lolium multiflorum Lam.) roots inoculation, strain Ph6-gfp actively and internally colonized plant roots and transferred vertically to the shoots. Ph6-gfp had a natural capacity to cope with phenanthrene in vitro and in planta. Ph6-gfp degraded 81.1% of phenanthrene (50 mg · L(-1)) in a culture solution within 15 days. The inoculation of plants with Ph6-gfp reduced the risks associated with plant phenanthrene contamination based on observations of decreased concentration, accumulation, and translocation factors of phenanthrene in ryegrass. Our results will have important ramifications in the assessment of the environmental risks of PAHs and in finding ways to circumvent plant PAH contamination.

The study was aimed at evaluating the antioxidant activity of alcoholic extract of Cassia siamea Lam. (Fabaceae) flowers. The extract was found to contain a large amount of polyphenols and also exhibited an immense reducing ability. At a concentration of 250 microg/ml, 96% of DPPH radicals and at 500 microg/ml, 42.7, 32.7 and 64.5% of O2-, H2O2 and NO respectively could be scavenged by C. siamea flower extract. The extract also inhibited OH radical induced oxidation of protein (BSA) and LPO in murine hepatic microsomes. The determination of metal chelating capacity of the extract indicated chelating of metal ions (Fe2+) to be a putative mechanism implicated in the inhibition of OH radical-induced BSA oxidation and LPO. C. siamea flower extract also exhibited a significant antioxidant activity in acute oxidative tissue injury animal model constituted by CCl4 induced hepatotoxicity. Oral administration of the extract at a dose of 50-150 mg/kg of body weight significantly protected from CCl4 induced elevation in AST and ALT in the serum, elevation in hepatic LPO, depletion of hepatic GSH and decrease in the activities of hepatic antioxidant enzymes: SOD, CAT and GPX. The extract also protected against histopathological changes produced by CCl4 such as necrosis, fatty changes, ballooning degeneration, etc. The data obtained in the present study suggests that the alcoholic extract of C. siamea flowers have potent antioxidant activity against free radicals, prevent oxidative damage to major biomolecules and afford significant protection against oxidative damage in the liver.

Arabinan polymers are major components of the cell wall in Mycobacterium tuberculosis and are involved in maintaining its structure, as well as playing a role in host-pathogen interactions. In particular, lipoarabinomannan (LAM) has multiple immunomodulatory effects. In the nonpathogenic species Mycobacterium smegmatis, EmbC has been identified as a key arabinosyltransferase involved in the incorporation of arabinose into LAM, and an embC mutant is viable but lacks LAM. In contrast, we demonstrate here that in M. tuberculosis, embC is an essential gene under normal growth conditions, suggesting a more crucial role for LAM in the pathogenic mycobacteria. M. tuberculosis EmbC has an activity similar to that of M. smegmatis EmbC, since we were able to complement an embC mutant of M. smegmatis with embC(Mtb), confirming that it encodes a functional arabinosyltransferase. In addition, we observed that the size of LAM produced in M. smegmatis was dependent on the level of expression of embC(Mtb). Northern analysis revealed that embC is expressed as part of a polycistronic message encompassing embC and three upstream genes. The promoter region for this transcript was identified and found to be up-regulated in stationary phase but down-regulated during hypoxia-induced nonreplicating persistence. In conclusion, we have identified one of the key genes involved in LAM biosynthesis in M. tuberculosis and confirmed its essential role in this species.

Regular consumption of fruit and vegetables is associated with a lower risk of some chronic diseases including various forms of cancer and cardiovascular diseases. The health-promoting potential of these foods may be due, in part, to the phytochemical bioactive compounds present in the plants. Fruit of Euphoria longana Lam. (longan) are consumed throughout Asia and are a major crop in Thailand. In the present study phytochemicals were extracted with 70% methanol from peel, pulp, and seed tissues of longan fruit, and the major components were identified as gallic acid, corilagin (an ellagitannin), and ellagic acid. A high-through-put reversed phase HPLC method was developed to determine the content of these three compounds in different parts of the longan fruit and among different cultivars. The analyses showed that there was a large variation in the contents of gallic acid, corilagin, and ellagic acid in different plant tissues and cultivars. Seed contained the highest levels of the three phenolics, and pulp contained the lowest. Among commercial cultivars, Biewkiew and Edor contained the highest levels of gallic and ellagic acid while Srichompoo contained the highest content of corilagin. These three cultivars may be used in directed breeding and cultivation programs and to develop concentrated longan seed extracts to promote good health. Utilization of this byproduct material will support the use of thousands of tons of waste longan seeds after the production of canned longan pulp.

BACKGROUND

The detection of Mycobacteriumtuberculosis (MTB)-specific human antibodies has been an important diagnostic aid in the diagnosis of TB, especially for the bacterium-negative TB. The humoral antibody responses to different antigens of M.tuberculosis (MTB) are heterogeneous in active TB patients. Hence, detection of antibody responses to several MTB antigens may improve the sensitivity and specificity of serological diagnosis of active TB.

METHODS

Seventeen MTB antigens (38kD, 16kD, Ag85A, Ag85B, MPT32, MPT63, MPT64, Mtb39, MTB48, Mtb81, MTC28, Rv1009, ESAT6, CFP10, CFP10-ESAT6, katG, and LAM) were prepared by cloning, expression, and purification from E. coli, and their antigenicities were evaluated in the antibody responses of 210 active TB patients (103 sera from smear- or culture-positive patients, and 107 from smear- or culture-negative patients) and 192 healthy control (95 sera from purified protein derivative-negative healthy donors, and 97 sera from BCG-vaccinated individuals) by an enzyme-linked immunosorbent assay (ELISA).

RESULTS

The levels of antibodies against these antigens in bacterium-negative TB patients were significantly higher than that in healthy controls (p<0.001). The sensitivity with individual antigens to detect antibody responses ranged from 55.7 to 82.9%, with the specificity from 62.0 to 92.2%. Importantly, the sensitivity with five antigens (LAM, 38kD, katG, 16kD, and MPT63 or Mtb39) to detect antibody responses reached 69.5% (146/210), with a specificity of 91.1% (17/192), and the sensitivity with another five antigens (LAM, katG, 16kD, Mtb39 and Mtb81) to detect antibody responses reached 67.1% (141/210), with a specificity of 92.7% (14/192).

CONCLUSIONS

The combination of optimal multiple antigens to detect anti-MTB antibody responses increased the sensitivity and specificity. Therefore, detection of anti-MTB antibody responses with multiple antigens may be valuable in the clinical diagnosis of TB patients.

BACKGROUND

Multidrug- (MDR) and extensively drug resistant (XDR) tuberculosis (TB) presents a challenge to disease control and elimination goals. In Lisbon, Portugal, specific and successful XDR-TB strains have been found in circulation for almost two decades.

RESULTS

In the present study we have genotyped and sequenced the genomes of 56 Mycobacterium tuberculosis isolates recovered mostly from Lisbon. The genotyping data revealed three major clusters associated with MDR-TB, two of which are associated with XDR-TB. Whilst the genomic data contributed to elucidate the phylogenetic positioning of circulating MDR-TB strains, showing a high predominance of a single SNP cluster group 5. Furthermore, a genome-wide phylogeny analysis from these strains, together with 19 publicly available genomes of Mycobacterium tuberculosis clinical isolates, revealed two major clades responsible for M/XDR-TB in the region: Lisboa3 and Q1 (LAM).The data presented by this study yielded insights on microevolution and identification of novel compensatory mutations associated with rifampicin resistance in rpoB and rpoC. The screening for other structural variations revealed putative clade-defining variants. One deletion in PPE41, found among Lisboa3 isolates, is proposed to contribute to immune evasion and as a selective advantage. Insertion sequence (IS) mapping has also demonstrated the role of IS6110 as a major driver in mycobacterial evolution by affecting gene integrity and regulation.

CONCLUSIONS

Globally, this study contributes with novel genome-wide phylogenetic data and has led to the identification of new genomic variants that support the notion of a growing genomic diversity facing both setting and host adaptation.

Early diagnosis of active tuberculosis (TB) remains an elusive challenge, especially in individuals with disseminated TB and HIV co-infection. Recent studies have shown a promise for the direct detection of pathogen-specific biomarkers such as lipoarabinomannan (LAM) for the diagnosis of TB in HIV-positive individuals. Currently, traditional immunoassay platforms that suffer from poor sensitivity and high non-specific interactions are used for the detection of such biomarkers. In this manuscript, we demonstrate the development of sandwich immunoassays for the direct detection of three TB-specific biomarkers, namely LAM, early secretory antigenic target 6 (ESAT6) and antigen 85 complex (Ag85), using a waveguide-based optical biosensor platform. Combining detection within the evanescent field of a planar optical waveguide with functional surfaces that reduce non-specific interactions allows for the ultra-sensitive and quantitative detection of biomarkers (an order of magnitude enhanced sensitivity, as compared to plate-based ELISA) in complex patient samples (urine, serum) within a short time. We also demonstrate the detection of LAM in urine from a small sample of subjects being treated for TB using this approach with excellent sensitivity and 100% corroboration with disease status. These results suggest that pathogen-specific biomarkers can be applied for the rapid and effective diagnosis of disease. It is likely that detection of a combination of biomarkers offers greater reliability of diagnosis, rather than detection of any single pathogen biomarker. NCT00341601.

BACKGROUND

Molecular genotyping methods have shown infection with more than one Mycobacterium tuberculosis strain genotype in a single sputum culture, indicating mixed infection.

OBJECTIVE

This study aimed to develop a PCR-based genotyping tool to determine the population structure of M. tuberculosis strain genotypes in primary Mycobacterial Growth Indicator Tubes (MGIT) and Löwenstein-Jensen (LJ) cultures to identify mixed infections and to establish whether the growth media influenced the recovery of certain strain genotypes.

METHODS

A convenience sample of 206 paired MGIT and LJ M. tuberculosis cultures from pulmonary tuberculosis patients resident in Khayelitsha, South Africa were genotyped using an in-house PCR-based method to detect defined M. tuberculosis strain genotypes.

RESULTS

The sensitivity and specificity of the PCR-based method for detecting Beijing, Haarlem, S-family, and LAM genotypes was 100%, and 75% and 50% for detecting the Low Copy Clade, respectively. Thirty-one (15%) of the 206 cases showed the presence of more than one M. tuberculosis strain genotype. Strains of the Beijing and Haarlem genotypes were significantly more associated with a mixed infection (on both media) when compared to infections with a single strain (Beijing MGIT p = 0.02; LJ, p<0.01) and (Haarlem: MGIT p<0.01; LJ, p = 0.01). Strains with the Beijing genotype were less likely to be with "other genotype" strains (p<0.01) while LAM, Haarlem, S-family and LCC occurred independently with the Beijing genotype.

CONCLUSIONS

The PCR-based method was able to identify mixed infection in at least 15% of the cases. LJ media was more sensitive in detecting mixed infections than MGIT media, implying that the growth characteristics of M. tuberculosis on different media may influence our ability to detect mixed infections. The Beijing and Haarlem genotypes were more likely to occur in a mixed infection than any of the other genotypes tested suggesting pathogen-pathogen compatibility.