Mycobacterium kansasii infection in squirrel monkeys (Saimiri sciureus sciureus)

Abstract: This report documents asymptomatic infections of Mycobacterium kansasii in four of five tuberculin positive squirrel monkeys (Saimiri sciureus sciureus). The mycobacterial DNA amplified by polymerase chain reaction (PCR) from a bronchial lymph node had no affinity for the species specific probes of M. tuberculosis, M. avium, and M. intracellular, thus allowing the presumptive diagnosis of an atypical mycobacterial infection. Infection by Mycobacterium kansasii was confirmed by culture of bronchial lymph nodes from three monkeys. The source of the infection was never identified.     

Establishment of an effective TLC bioautographic method for the detection of Mycobacterium tuberculosis H37Ra phosphoglucose isomerase inhibition by phosphoenolpyruvate

A bioautographic assay based on thin layer chromatography was developed for phosphoenolpyruvate (PEP) detecting as a known but rarely studied inhibitor of phosphoglucose isomerase (PGI). The protocol with NADP⁺/NBT/PMS (β-nicotinamide adenine dinucleotide phosphate/nitrotetrazolium blue chloride/phenazine methosulfate) staining was capable of detecting Mycobacterium tuberculosis H₃₇Ra PGI inhibition using PEP. According to this method, visibly brighter spots (zones) against purple background are observed in the area of inhibition of the above-mentioned enzyme activity. The detection limit for PEP as an inhibitor of Mycobacterium tuberculosis H₃₇Ra PGI was 226?μg per spot/zone. Noteworthy is that we are the first authors to have successfully used a bioautographic assay to detect Mycobacterium tuberculosis H₃₇Ra PGI inhibition by PEP.     

Molecular basis of bacterial virulence and survival within infected hosts and the environment

The EMBO–FEBS lecture course on the ‘Molecular Basis of Bacterial Virulence and Survival Within Infected Hosts and in the Environment’, organized by Pascale Cossart, Efstathios Gonos and Roberto Kolter, was held on the island of Spetsai, Greece, September 3–13, 2002.     

Fine needle aspiration cytology in HIV‐related lymphadenopathy: experience at a single centre in north India

P. K. Sarma, A. K. Chowhan, V. Agrawal and V. Agarwal
Fine needle aspiration cytology in HIV‐related lymphadenopathy: experience at a single centre in north India Objective: Fine needle aspiration (FNA) is emerging as a rapid and minimally invasive tool in evaluating lymphadenopathy associated with human immunodeficiency virus (HIV). We evaluated the role of FNA in differentiating various causes of lymphadenopathy in patients with HIV and correlated the cytological diagnosis with CD4 counts. Methods: Seventy‐nine HIV‐positive patients (median age 35 years, 68 male) underwent ultrasound‐guided (n = 16) and unguided (n = 63) FNA from 1999 to 2006. Smears were stained with May–Grünwald–Giemsa, haematoxylin & eosin and Papanicolaou stains. Ziehl–Neelsen (ZN) staining for acid‐fast bacilli (AFB) was performed in all cases. Staining for fungus was performed whenever required. Results: The aspirates were adequate in 75 cases (95%). Non‐specific reactive hyperplasia was the most common FNA diagnosis (39, 52%) followed by granulomatous necrotizing lymphadenitis (15, 20%), necrotizing lymphadenitis (13, 17.3%) and granulomatous lymphadenitis (4, 5.2%). Fungal infection and non‐Hodgkin lymphoma (NHL) were seen in two patients each. ZN staining was positive for AFB in 25 (33.3%) cases. One of these was morphologically interpreted as reactive hyperplasia, 12 as necrotizing lymphadenitis and 12 as granulomatous necrotizing lymphadenitis. Both patients with NHL had CD4 counts below 100/dl. Necrotizing lymphadenitis and granulomatous lymphadenitis were significantly associated with CD4 counts below and above 200/dl, respectively (P = 0.0002). Conclusions: FNA is an important tool for assessing the cause of lymphadenopathy in HIV patients. Necrotizing inflammation is more often seen in patients with low CD4 counts. AFB are commonly found in necrotic aspirates with or without granulomas. However, a stain for AFB should be performed in all aspirates from HIV‐related lymphadenopathy including reactive hyperplasia.     

Sensing of mycobacterial arabinogalactan by galectin‐9 exacerbates mycobacterial infection

Mycobacterial arabinogalactan (AG) is an essential cell wall component of mycobacteria and a frequent structural and bio‐synthetical target for anti‐tuberculosis (TB) drug development. Here, we report that mycobacterial AG is recognized by galectin‐9 and exacerbates mycobacterial infection. Administration of AG‐specific aptamers inhibits cellular infiltration caused by Mycobacterium tuberculosis (Mtb) or Mycobacterium bovis BCG, and moderately increases survival of Mtb‐infected mice or Mycobacterium marinum‐infected zebrafish. AG interacts with carbohydrate recognition domain (CRD) 2 of galectin‐9 with high affinity, and galectin‐9 associates with transforming growth factor β‐activated kinase 1 (TAK1) via CRD2 to trigger subsequent activation of extracellular signal‐regulated kinase (ERK) as well as induction of the expression of matrix metalloproteinases (MMPs). Moreover, deletion of galectin‐9 or inhibition of MMPs blocks AG‐induced pathological impairments in the lung, and the AG‐galectin‐9 axis aggravates the process of Mtb infection in mice. These results demonstrate that AG is an important virulence factor of mycobacteria and galectin‐9 is a novel receptor for Mtb and other mycobacteria, paving the way for the development of novel effective TB immune modulators.     

Association of Mycobacterium infections in patients with Mendelian susceptibility to mycobacterial disease with venous thromboembolism

An association between a hypercoagulable state and Mendelian susceptibility to mycobacterial disease (MSMD) has been established in a few studies; resultant thrombosis is considered rare. In a case‐control study, the prevalence of factor V Leiden, prothrombin G20210A and methylenetetrahydrofolate reductase (MTHFR) C677T, A1298C mutations were investigated in mycobacterium‐infected patients. The study comprised 30 patients with mycobacterial infections (invasive, disseminated and/or recurrent infections with Bacille Calmette–Guerin or non‐tuberculosis mycobacteria and Mycobacterium Tuberculosis with positive results for acid‐fast bacilli and tuberculin skin tests) and 30 normal healthy controls. Forty female (66.7%) and 20 male subjects (33.3%) aged from 3 to 70 years were recruited into this study. Genotyping of targeted genes was performed by RT‐PCR and cytokine TNF‐α concentrations were quantified using a commercially available ELISA kit. Significant associations between mycobacterial infection and TNF‐α production after stimulating peripheral blood mononuclear cells with LPS alone and with IFN‐γ plus LPS were identified. Moreover, genotyping analysis in the studied population revealed a significant association between MTHFR c.677C>T (OR, 3.28; 95% CI, 1.35–7.92; P < 0.05), MTHFR c.1298A>C (OR, 2.33; 95% CI, 1.10–4.93; P < 0.05) and mycobacterial infection in affected patients, indicating susceptibility to venous thromboembolism according to previous studies. Additionally, mycobacterium‐infected patients had a significantly greater prevalence of MTHFR C677T and A1298C mutations than controls.     

The utility of fine needle aspiration in HIV positive children

Objective:  To determine the spectrum of disease, diagnostic accuracy and adequacy of fine needle aspirates (FNA) in human immunodeficiency virus (HIV) positive children who present with mass lesions.
Methods:  Between January 1997 and December 2002, 95 FNAs were performed in 91 children aged 15 years and younger who were known to be infected with the human immunodeficiency virus (HIV).
Results:  Head and neck masses including salivary gland swellings were the most common presentation (58.9%) followed by axillary masses (25.3%). Groin masses were aspirated in six children, flank and abdominal masses in four children, buttock masses in three children, a chest wall mass in one child and a sonar guided FNA of a lung mass in one child. Eight FNAs (8.4%) proved inadequate. Reactive lymphadenopathy was diagnosed in 42 cases, mycobacterial infection in 22, four children were diagnosed with abscess, one child had a fungal infection and five were found to have non-Hodgkin's lymphoma. There were four cases each of lymphoepithelial lesion and Kaposi sarcoma. There was one case each of nephroblastoma, rhabdomyosarcoma, myeloma, melanotic progonoma and spindle cells, not otherwise specified.
Conclusion:  Fine needle aspiration in HIV positive children is a worthwhile procedure and in most instances allows a rapid diagnosis obviating the need for surgery and enabling swift treatment to be undertaken where necessary. Ancillary studies form an important diagnostic component. Universal safety precautions must be strictly adhered to.     

Structure-Function Analysis of the Acyl Carrier Protein Synthase (AcpS) from Mycobacterium tuberculosis

We have solved the crystal structure of the acyl carrier protein synthase (AcpS) from Mycobacterium tuberculosis (Mtb) at 1.95 Å resolution. AcpS, a 4-phosphopantetheinyl transferase, activates two distinct acyl carrier proteins (ACPs) that are present in fatty acid synthase (FAS) systems FAS-I and FAS-II, the ACP-I domain and the mycobacterial ACP-II protein (ACPM), respectively. Mtb, the causal agent of tuberculosis (TB), and all other members of the Corynebacterineae family are unique in possessing both FAS systems to produce and to elongate fatty acids to mycolic acids, the hallmark of mycobacterial cell wall. Various steps in this process are prime targets for first-line anti-TB agents. A comparison of the Mtb AcpS structure determined here with those of other AcpS proteins revealed unique structural features in Mtb AcpS, namely, the presence of an elongated helix followed by a flexible loop and a moderately electronegative surface unlike the positive surface common to other AcpSs. A structure-based sequence comparison between AcpS and its ACP substrates from various species demonstrated that the proteins of the Corynebacterineae family display high sequence conservation, forming a segregated subgroup of AcpS and ACPs. Analysis of the putative interactions between AcpS and ACPM from Mtb, based on a comparison with the complex structure from Bacillus subtilis, showed that the Mtb AcpS and ACPM lack the electrostatic complementarity observed in B. subtilis. Taken together, the common characteristic of the Corynebacterineae family is likely reflected in the participation of different residues and interactions used for binding the Mtb AcpS to ACP-I and ACPM. The distinct features and essentiality of AcpS, as well as the mode of interaction with ACPM and ACP-I in Mtb, could be exploited for the design of AcpS inhibitors, which, similarly to other inhibitors of fatty acid synthesis, are expected to be effective anti-TB-specific drugs.