Reduced Mycobacterium tuberculosis association with monocytes from diabetes patients that have poor glucose control.

The re-emerging importance of type 2 diabetes mellitus (DM) to tuberculosis (TB) control is of growing concern, but the basis for this relationship is poorly understood. Given the importance of mononuclear phagocytes for TB control and the reported alterations in monocytes of DM patients, we evaluated whether the initial interaction between both was affected in diabetics. Mycobacterium tuberculosis-naïve individuals with and without DM were group matched by age and gender and the efficiency of M. tuberculosis association (attachment and ingestion) with their monocytes was assessed in the presence of autologous serum. The association of M. tuberculosis with monocytes was significantly lower in diabetics (19.2 ± 6.1) than non-diabetics (27.5 ± 7.9; p = 0.02). Multivariate analysis controlling for host socio demographics, DM characteristics and serum lipids indicated that male gender (p = 0.04) and poorly-controlled DM (high HbA1c and hyperglycemia; p = 0.01) were significantly associated with the lower interaction of M. tuberculosis with monocytes. Serum heat-inactivation reduced the association of M. tuberculosis to similar levels in both study groups (p = 0.69) suggesting alterations in the complement pathway of DM patients. These findings suggest an altered route of entry of the pathogen in DM patients that may influence the downstream activation of signaling pathways in the monocyte and the survival of mycobacteria.

Rapid DNA extraction for specific detection and quantitation of Mycobacterium tuberculosis DNA in sputum specimens using Taqman assays.

Rapid tuberculosis (TB) detection is critical for disease control, and further quantitation of Mycobacterium tuberculosis (Mtb) in sputum is valuable for epidemiological and clinical studies. We evaluated a simple, robust and cost-efficient in-house DNA extraction and downstream Taqman approach for detection and quantitation of Mtb genomes from sputum of newly-diagnosed TB patients and non-TB controls. DNA was extracted using guanidine isothiocyanate and silica-based spin columns in less than 2 h, stored frozen, and Taqman assays were used to detect Mtb with IS6110 and quantify it targeting RD1 and IS1081. The Taqmans had a sensitivity >95% in 108 culture-confirmed TB patients and specificity of 100% in 43 non-TB controls. Genome counts were correlated with the Mycobacterial Growth Indicator Tubes' (MGIT) time-to-detection values (1/TTD × 1000; rho = 0.66; p < 0.001) in 91 TB patients (33 excluded with MGIT contamination). This linear relationship was nearly identical between mycobacteria isolated from sputum and H37Rv Mtb grown in-vitro to its log phase. TB treatment between 3 and 7 days was associated with lower 1/TTD × 1000 values but not with genome counts. Together, our protocol provides rapid, specific, inexpensive and quantitative detection of Mtb DNA in fresh or stored sputa making it a robust tool for prompt TB diagnosis, and with potential use for clinical and epidemiologic studies.

Systematic interpretation of molecular beacon polymerase chain reaction for identifying rpoB mutations in Mycobacterium tuberculosis isolates with mixed resistant and susceptible bacteria.

Detection of multidrug-resistant tuberculosis (MDR-TB), a frequent cause of treatment failure, takes 2 or more weeks to identify by culture. Rifampicin (RIF) resistance is a hallmark of MDR-TB, and detection of mutations in the rpoB gene of Mycobacterium tuberculosis using molecular beacon probes with real-time quantitative polymerase chain reaction (qPCR) is a novel approach that takes

Selective enrichment and detection of mycobacterial DNA in paucibacillary specimens.

A major challenge for tuberculosis control is mycobacterial detection in paucibacillary disease, particularly in pediatric, extrapulmonary and smear-negative pulmonary infections. We developed a simple and efficient DNA extraction and real-time quantitative PCR (qPCR) protocol for mycobacterial detection and quantification in paucibacillary specimens. The method was refined using an in vitro model mimicking blood specimens which are characterized by the presence of numerous qPCR inhibitors. Mycobacterial DNA detection in blood is of interest given the high sensitivity we previously reported using conventional PCR in blood of patients with tuberculosis lymphadenitis. Mechanical lysis of mycobacteria in the presence of an organic solvent provided the highest sensitivity. Mycobacterial DNA amplification was compromised when the human:bacterial genome ratio was at least 190:1. Separation of the specimen into bacterial- and host-rich fractions prior to DNA extraction improved mycobacterial DNA detection by 30%. Preliminary testing of our protocol in smear-negative, culture-positive specimens (gastric and lymph node aspirates, pleural and cerebrospinal fluid, and blood) confirmed the applicability of our technique to a range of paucibacillary specimens for the detection, quantification and speciation (M. tuberculosis versus M. avium) of mycobacteria, several weeks before culture results were available. Our protocol provides a novel, efficient and simple strategy to improve the performance of qPCR in paucibacillary specimens, including those with excess human DNA background. This tool is useful to study the pathophysiology of early pulmonary or occult tuberculosis, and for more rapid and accurate diagnosis in difficult to diagnose infections.

In situ detection of antigenic glycoproteins in Taenia solium metacestodes.

Taenia solium has a complex life cycle. Its cysticercus can lodge in the brain, causing neurocysticercosis (NCC), and the adult tapeworm's survival in the intestine results in taeniasis. In this study, the in situ detection of previously described glycoprotein antigens used for serological diagnosis of NCC and the detection of other glycoconjugates was explored in cysticerci and the surrounding porcine tissue to understand their potential role in pathogenesis. Immunohistochemistry with an antiserum specific for glycoprotein antigens rich in N-linked carbohydrates and in situ histochemistry with a battery of lectins that have affinity to a variety of glycoconjugates were performed. The glycoconjugates rich in N-linked carbohydrates were detected in the vesicular fluid and tegument of the vesicular membrane and scolex, where the parasite has direct contact with the host tissues during cysticercosis and taeniasis, respectively. Additionally, as the inflammatory response progressed, the parasite's antigenic glycoproteins were also detected in the cytoplasm of inflammatory cells in the surrounding granuloma. In contrast, the spiral canal tegument, which will be exposed to intestinal enzymes in taeniasis, had N-acetyl-galactosamine-rich mucins. Thus, the differential saccharidic composition in T. solium metacestode structures may be important for the survival of the parasite in different host sites.

Similar diagnostic performance for neurocysticercosis of three glycoprotein preparations from Taenia solium metacestodes.

The detection of antibodies to Taenia solium metacestodes is very important in the differential diagnosis of neurocysticercosis (NCC). In this study, an electroimmunotransfer blot (EITB) assay that uses an elaborate protocol with metacestode glycoproteins as antigens was compared with two other Western blots that use glycoproteins obtained using simpler methods, including an eluate from a lectin column, or the vesicular fluid (VF) of the parasite. The concordance between the three assays was 91% in patients with active NCC and 100% in patients with suspected NCC and previous documentation of negative serology. The specificities for the Western blots and the EITB assay were 98% and 100%, respectively (98% concordance). These data suggest that the simplest of these immunoassays, the one that uses the VF of T. solium metacestodes in a Western blot format, can be reliably used for the serologic diagnosis of NCC in developing countries where access to the EITB assay is difficult.

El papel de los carbohidratos en la antigenicidad de las glicoproteínas del cisticerco de la Taenia solium / Hydrocarbon role in Taenia solium cysticercus glycoproteins antigenicity

La neurocisticercosis es una infección causada por el cisticerco de la T. Solium y puede confundirse con otras afecciones del sistema nervioso central. Las glicoproteínas de 12-28 kD de este parásitos son útiles para el diagnóstico serológico de la neurocisticercosis. Estas glicoproteínas contiene abundantes carbohidratos asociados vía asparagina (tipo N). Objetivo: Determinar la contribución de los carbahidratos tipo N en la antigenicidad de las glicoproteínas. Materiales y Métodos: se purificaron las glicoproteínas de 12, 16 y 18 kD de los cisticercos utilizando un gel preparativo de poliacrilamida y se sometieron a deglicosilación enzimática con PNGase F. Luego se evaluaron los cambios en antigenicidad entre las proteínas nativas y deglicosiladas por Western blot. Resultados: los antígenos deglicosilados redujeron su peso molecular a 7 kD y perdieron parte de su antigenicidad. Esta reducción fue más notoria para la proteína de 18 kD. La cual tiene mayor contenido de carbohidratos que la de 12 y 16 kD. Conclusión: estos resultados sugieren que los carbohidratos no sólo contribuyen a la antigenicidad, sino que además causan un bloqueo estérico que inhibe que el sistema inmune detecte otros epítopes no expuesto. Estos datos sugieren que la antigenicidad de las glicoproteínas de T. Solium se debe a una combinación de epítopes sacarídicos y probablemente proteicos