Use of 3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyl Tetrazolium Bromide for Rapid Detection of Rifampin-Resistant Mycobacterium tuberculosis

We describe a test which uses the ability of viable cells to reduce 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) to detect resistance to a bactericidal drug, rifampin, in in vitro-cultured Mycobacterium tuberculosis. The assay shows a linear relationship between the number of viable bacteria and the ability to reduce MTT. Dead mycobacteria were unable to reduce MTT. Rifampin-sensitive M. bovis (BCG) and M. tuberculosis exposed to rifampin showed a rifampin concentration-dependent inhibition of the ability to reduce MTT, while the resistant strains were unaffected. The inhibition of MTT reduction after treatment with rifampin paralleled the reduction in the number of CFU. By using mixing experiments in which the population percentages of rifampin-sensitive and -resistant strains were varied, the assay could detect the presence of rifampin resistance in the mixture when at least 1% of the bacterial population was composed of drug-resistant strains. The assay is cheap, can be visually read, and requires less than 3 days to obtain susceptibility results. The total time required to obtain results, from the time sputum is received in the laboratory, is, in most cases, less than 4 to 5 weeks, which is the time required for primary culture of the bacteria. The MTT assay could, in combination with a test to detect resistance to isoniazid, be a cheap and rapid screening method for multidrug-resistant M. tuberculosis that is affordable even by low-income countries where tuberculosis is a major public health problem.     

Medical and social consequences of tuberculosis in rural Ethiopia

An institution based cross sectional study was conducted in June 1996 at a rural health centre in South Gonder Administrative Zone of Northern Ethiopia. Interview and respective review of medical records of patients were done. A total of 211 tuberculosis (TB) patients were included in the study, 133 (63%) males and 78 (37%) females. Majority (79.6%) of patients had pulmonary disease. Clinical observation based on symptoms and signs suggestive of tuberculosis was used as a sole criteria to prescribe anti-tuberculosis treatment in 54.1% (114/211) of all the patients. Acid fast staining of sputum was done for 51.2% (86/168) of the pulmonary patients out of whom only 31.4% were smear positive. Social ostracism was observed to have been affecting tuberculosis patients and their families to a great extent. Divorce rate due to tuberculosis among patients was 29.1% (37/127). Patients have reported loss or threat to lose their job. Dietary misconceptions were rampant. The TB control activities were ineffective and poorly organised. Starting anti-tuberculosis treatment without proper diagnosis was observed to have negative consequences on the patients and the control programme. Appropriate counselling service along with the medical treatment was provided to patients with eventual family reunions. Patients were also organised into local 'TB clubs' by their residential locations so as to improve treatment adherence and the level of TB awareness among the patients and the community. It is strongly recommended that the political system and health authorities of Ethiopia should have to give much more attention and commitment to the TB control activities in Ethiopia.     

Mitochondrial free calcium levels (Rhod-2 fluorescence) and ultrastructural alterations in neuronally differentiated PC12 cells during ceramide-dependent cell death

Mitochondrial free calcium levels measured by Rhod-2 fluorescence and ultrastructure were examined during cell death in nerve growth factor (NGF)-differentiated PC12 cells that were 1) exposed to C2-ceramide, 2) deprived of serum to induce endogenous ceramide production, or 3) treated with calcium ionophore A23187. Rhod-2 fluorescence in mitochondria and also in the nucleolus increased to a maximum within 3 hours after C2-ceramide treatment or serum withdrawal. In A23187-treated cells, Rhod-2 fluorescence remained at baseline levels. In all three models, enlargement of the endoplasmic reticulum was the first ultrastructural alteration, followed by mitochondrial shrinkage in ionophore-treated cells, but by mitochondrial swelling in the ceramide-dependent models, in which rupture of the outer mitochondrial membrane and unfolding of the inner membrane were frequently seen. Dihydro-C2-ceramide, which did not cause cell death, had no effect on cellular ultrastructure. NGF, which inhibits ceramide-dependent cell death, prevented the effects of serum deprivation on mitochondrial ultrastructure but not on endoplasmic reticulum morphology or Rhod-2 fluorescence. Nuclear shrinkage with loss of nuclear membrane integrity, characterized by nuclear pores, free or surrounded by electron-dense filaments, was a late event in ceramide-dependent cell death. Chromatin condensation and other morphological features associated with apoptosis were seen in only a few atypical cells. Ceramide-mediated cell death, therefore, did not involve classical apoptosis but was mediated by a reproducible series of events beginning in the endoplasmic reticulum, followed by the mitochondria, and then the nucleus. NGF-dependent cell death inhibition intervenes at the mitochondrial level, not by blocking the increase in Rhod-2 fluorescence but by preventing the ultrastructural changes that follow.     

High bisphenol A (BPA) concentration in the maternal,but not fetal,compartment increases the risk of spontaneous preterm delivery

Objective: The objective of this study is to determine if BPA exposure, as measured by maternal plasma (MP) and amniotic fluid (AF) BPA concentrations is associated with an increased risk of spontaneous preterm birth (PTB) and preterm premature rupture of membranes (pPROM).

Methods: In this nested case–control study, MP samples from women in term labor (n?=?30), preterm labor that ended with preterm delivery (n?=?25), or who had pPROM (n?=?30) and amniotic fluid samples from term labor (n=?45), preterm labor (n?=?60), and pPROM (n?=?35) were assayed for BPA by enzyme immunoassay.

Results: BPA was detectible in 100% of MP and AF samples. Women with MP BPA concentrations in the fourth quartile were at increased risk of PTB (cOR?=?4.12, 95% CI?=?1.32–12.87; aOR?=?4.78, 95% CI?=?1.14–20) but not pPROM. High (fourth quartile) AF BPA values also tended to increase the risk of pPROM (cOR?=?2.47, 95% CI?=?0.96–6.37) but results were not statistically significant.

Conclusions: Increased BPA concentration is associated with an increased risk for PTB or pPROM depending on the maternal–fetal compartment(s) affected. High MP plasma BPA concentrations are associated with PTB with intact membranes but high AF BPA concentrations may weakly be associated with pPROM.     

Mycobacterium-Specific γ9δ2 T Cells Mediate Both Pathogen-Inhibitory and CD40 Ligand-Dependent Antigen Presentation Effects Important for Tuberculosis Immunity

Numerous pathogens, including Mycobacterium tuberculosis, can activate human γ₉δ₂ T cells to proliferate and express effector mechanisms. γ₉δ₂ T cells can directly inhibit the growth of intracellular mycobacteria and may also act as antigen-presenting cells (APC). Despite evidence for γδ T cells having the capacity to function as APC, the mechanisms involved and importance of these effects on overall tuberculosis (TB) immunity are unknown. We prepared M. tuberculosis-specific γ₉δ₂ T cell lines to study their direct protective effects and APC functions for M. tuberculosis-specific αβ T cells. The direct inhibitory effects on intracellular mycobacteria were measured, and the enhancing effects on proliferative and effector responses of αβ T cells assessed. Furthermore, the importance of cell-to-cell contact and soluble products for γ₉δ₂ T cell effector responses and APC functions were investigated. We demonstrate, in addition to direct inhibitory effects on intracellular mycobacteria, the following: (i) γ₉δ₂ T cells enhance the expansion of M. tuberculosis-specific αβ T cells and increase the ability of αβ T cells to inhibit intracellular mycobacteria; (ii) although soluble mediators are critical for the direct inhibitory effects of γ₉δ₂ T cells, their APC functions do not require soluble mediators; (iii) the APC functions of γ₉δ₂ T cells involve cell-to-cell contact that is dependent on CD40-CD40 ligand (CD40L) interactions; and (iv) fully activated CD4⁺ αβ T cells and γ₉δ₂ T cells provide similar immune enhancing/APC functions for M. tuberculosis-specific T cells. These effector and helper effects of γ₉δ₂ T cells further indicate that these T cells should be considered important new targets for new TB vaccines.     

Disposition of glucosinolates and sulforaphane in humans after ingestion of steamed and fresh broccoli

The cancer-chemopreventive effects of broccoli may be attributed, in part, to isothiocyanates (ITCs), hydrolysis products of glucosinolates. Glucosinolates are hydrolyzed to their respective ITCs by the enzyme myrosinase, which is inactivated by heat. In this study, the metabolic fate of glucosinolates after ingestion of steamed and fresh broccoli was compared in 12 male subjects in a crossover design. During each 48-hour baseline period, no foods containing glucosinolates or ITCs were allowed. The subjects then consumed 200 g of fresh or steamed broccoli; all other dietary sources of ITCs were excluded. Blood and urine samples were collected during the 24-hour period after broccoli consumption. Total ITC equivalents in broccoli and total ITC equivalents in plasma and urine were assayed by high-performance liquid chromatography as the cyclocondensation product of 1,2-benzenedithiol. The content of ITCs in fresh and steamed broccoli after myrosinase treatment was found to be virtually identical (1.1 vs. 1.0 micromol/g wet wt). The average 24-hour urinary excretion of ITC equivalents amounted to 32.3 +/- 12.7% and 10.2 +/- 5.9% of the amounts ingested for fresh and steamed broccoli, respectively. Approximately 40% of total ITC equivalents in urine, 25.8 +/- 13.9 and 6.9 +/- 2.5 micromol for fresh and steamed broccoli, respectively, occurred as the N-acetyl-L-cysteine conjugate of sulforaphane (SFN-NAC). Total ITC metabolites in plasma peaked between 0 and 8 hours, whereas urinary excretion of total ITC equivalents and SFN-NAC occurred primarily between 2 and 12 hours. Results of this study indicate that the bioavailability of ITCs from fresh broccoli is approximately three times greater than that from cooked broccoli, in which myrosinase is inactivated. Considering the cancer-chemopreventive potential of ITCs, cooking broccoli may markedly reduce its beneficial effects on health.     

ABO blood groups,grass pea preparation,and neurolathyrism in Ethiopia

An exploratory study was conducted in the rural Estie district of Ethiopia in 1997 to identify the role of ABO blood group, rhesus factor, and type of grass pea (Lathyrus sativus) diet in the susceptibility to neurolathyrism. Five-hundred study subjects (250 cases and 250 controls) were examined and interviewed, and had their ABO and rhesus blood groups determined. The majority (86%) of the cases were males. Blood group O was the most common in the patients and controls followed by groups A, B, and AB. The vast majority of the study subjects were rhesus-positive. The gravy (Shiro) grass pea preparation was consumed by 91.6% of the study population, boiled (Nifiro) by 86%, and roasted (Kollo) by 56.4%. Almost half (48%) of the cases had consumed grass pea for > 4 months compared to 8% of controls (P < 0.001). There was a significant association between the risk for neurolathyrism and the consumption of boiled (adjusted odds ratio [AOR] = 98.4) and roasted (AOR = 55.62) forms of grass pea. There was no risk of paralysis associated with consumption of the gravy form of grass pea (AOR = 0.40, 95% confidence interval 0.1-2.0). Blood group O remained significantly associated with the disease after adjusting for age, type of grass pea preparation consumed, and duration of consumption (AOR = 2.90).     

Praziquantel efficacy against schistosomiasis mansoni in schoolchildren in north-west Ethiopia

The efficacy of praziquantel against schistosomiasis mansoni has been measured in 10-14 years old schoolchildren in a highly endemic area of north-west Ethiopia. The egg reduction rate was 97% and the cure rate was 94%, as assessed by a single Kato smear. No evidence for praziquantel resistance was detected.     

Pattern and associated factors of the neurolathyrism epidemic in Ethiopia

OBJECTIVES: To describe the neurolathyrism epidemic in Ethiopia and to identify associated household factors. METHODS: We interviewed 589 randomly selected heads of household in Debre Sina district of Ethiopia, the area afflicted by the recent neurolathyrism epidemic. Disease information was obtained for 2987 family members. RESULTS: Neurolathyrism patients were detected in 56 (9.5%) households (prevalence rate 2.38%). The mean number of affected family members per household was 1.27 (SD 0.65, range 1-3). Most (77.5%) patients developed the disability during the epidemic (1995-1999). The median age at onset of paralysis was 11 years with a range of 41 (range 3-44). Younger people were more affected during the epidemic than during the non-epidemic period (P=0.01). The presence of a neurolathyrism patient in the family was associated with illiteracy [adjusted OR (95% CI)=2.23 (1.07-5.10)] of the head of household, with owning a grass pea farm [adjusted OR (95% CI)=2.01 (1.04-3.88)] and with the exclusive cooking of grass pea foods using handmade traditional clay pots [adjusted OR (95% CI=2.06 (1.08-3.90)]. CONCLUSION: Males aged 10-14 years were most affected by neurolathyrism. Increased household risk was associated with illiteracy of the head of the household and exclusive cooking of grass pea foods with handmade traditional clay pots.