Fluoromycobacteriophages for rapid, specific, and sensitive antibiotic susceptibility testing of Mycobacterium tuberculosis

Rapid antibiotic susceptibility testing of Mycobacterium tuberculosis is of paramount importance as multiple- and extensively- drug resistant strains of M. tuberculosis emerge and spread. We describe here a virus-based assay in which fluoromycobacteriophages are used to deliver a GFP or ZsYellow fluorescent marker gene to M. tuberculosis, which can then be monitored by fluorescent detection approaches including fluorescent microscopy and flow cytometry. Pre-clinical evaluations show that addition of either Rifampicin or Streptomycin at the time of phage addition obliterates fluorescence in susceptible cells but not in isogenic resistant bacteria enabling drug sensitivity determination in less than 24 hours. Detection requires no substrate addition, fewer than 100 cells can be identified, and resistant bacteria can be detected within mixed populations. Fluorescence withstands fixation by paraformaldehyde providing enhanced biosafety for testing MDR-TB and XDR-TB infections. © 2009 Piuri et al

Prevalence of head lice and hygiene practices among women over twelve years of age in Sindh, Balochistan, and North West Frontier Province: National Health Survey of Pakistan, 1990-1994

Background: Head lice infestation is an infection of the scalp and skin which causes blood loss, discomfort, and social and psychological distress with the possibility of secondary bacterial infections occurring at scratch sites. In Pakistan, although some small scale studies have been conducted to investigate prevalence of head lice in school children and the general population, no population based estimates have been reported. The National Health Survey of Pakistan (NHSP 1990 - 94) was a nationally representative health examination survey of the Pakistani population. The NHSP is the first population based household survey to collect data on the prevalence of head lice in adult women in Pakistan. In this paper we use data from the NHSP to present an epidemiological profile of personal hygiene practices and head lice infestation among women aged 12 years or older in three provinces of Pakistan, Balochistan, Sindh and North West Frontier Province (NWFP). Results: Overall about 7% women aged 12 years and older suffered from head lice infestation. Multivariable logistic regression analysis identified factors independently associated with presence of head lice. Age less than 16 years and crowding at home were associated with higher infestation-rates. The impact of household socio-economic status on infestation rates among women was different in urban and rural settings, urban women with low socio-economic status were more vulnerable than similar women in rural settings. Bathing infrequently in summer was associated with higher prevalence rates only in Sindh, possibly due to the fact that among the three provinces Sindh has a hotter and more humid summer. Conclusions: The results of our analysis of NHSP indicate high levels of head lice infestation among girls and women in the three Provinces. The epidemiological profile of hygienic practices of women indicated that NWFP and Balochistan as compared to Sindh, and rural as compared to urban areas were less developed with respect to access to water supply and soap for maintaining personal hygiene. Simple and cost-effective measures such as provision of water and soap, and improving awareness regarding maintaining personal hygiene can contribute significantly towards improving public health status of the women in Pakistan

Multiple Colonization with S. pneumoniae before and after Introduction of the Seven-Valent Conjugated Pneumococcal Polysaccharide Vaccine

Simultaneous carriage of more than one strain of Streptococcus pneumoniae promotes horizontal gene transfer events and may lead to capsule switch and acquisition of antibiotic resistance. We studied the epidemiology of cocolonization with S. pneumoniae before and after introduction of the seven-valent conjugated pneumococcal vaccine (PCV7)

Isolation of Non-Tuberculous Mycobacteria in Children Investigated for Pulmonary Tuberculosis

OBJECTIVE: To evaluate the frequency and clinical significance of non-tuberculous mycobacteria (NTM) isolates among children investigated for pulmonary tuberculosis in a rural South African community. METHODS: Children were investigated for pulmonary tuberculosis as part of a tuberculosis vaccine surveillance program (2001–2005). The clinical features of children in whom NTM were isolated, from induced sputum or gastric lavage, were compared to those with culture-proven M. tuberculosis. RESULTS: Mycobacterial culture demonstrated 114 NTM isolates from 109 of the 1,732 children investigated, a crude yield of 6% (95% CI 5–7). The comparative yield of positive NTM cultures from gastric lavage was 40% (95% CI 31–50), compared to 67% (95% CI 58–76) from induced sputum. 95% of children with NTM isolates were symptomatic. Two children were HIV-infected. By contrast, M. tuberculosis was isolated in 187 children, a crude yield of 11% (95% CI 9–12). Compared to those with culture-proven M. tuberculosis, children with NTM isolates were less likely to demonstrate acid-fast bacilli on direct smear microscopy (OR 0.19; 95% 0.0–0.76). Children with NTM were older (p<0.0001), and more likely to demonstrate constitutional symptoms (p = 0.001), including fever (p = 0.003) and loss of weight or failure to gain weight (p = 0.04), but less likely to demonstrate a strongly positive tuberculin skin test (p<0.0001) or radiological features consistent with pulmonary tuberculosis (p = 0.04). DISCUSSION: NTM were isolated in 6% of all children investigated for pulmonary tuberculosis and in more than one third of those with a positive mycobacterial culture. NTM may complicate the diagnosis of PTB in regions that lack capacity for mycobacterial species identification. The association of NTM isolates with constitutional symptoms suggestive of host recognition requires further investigation

Early-infantile onset epilepsy and developmental delay caused by bi-allelic GAD1 variants

Gamma-aminobutyric acid (GABA) and glutamate are the most abundant amino acid neurotransmitters in the brain. GABA, an inhibitory neurotransmitter, is synthesized by glutamic acid decarboxylase (GAD). Its predominant isoform GAD67, contributes up to ∼90% of base-level GABA in the CNS, and is encoded by the GAD1 gene. Disruption of GAD1 results in an imbalance of inhibitory and excitatory neurotransmitters, and as Gad1−/− mice die neonatally of severe cleft palate, it has not been possible to determine any potential neurological dysfunction. Furthermore, little is known about the consequence of GAD1 disruption in humans. Here we present six affected individuals from six unrelated families, carrying bi-allelic GAD1 variants, presenting with developmental and epileptic encephalopathy, characterized by early-infantile onset epilepsy and hypotonia with additional variable non-CNS manifestations such as skeletal abnormalities, dysmorphic features and cleft palate. Our findings highlight an important role for GAD1 in seizure induction, neuronal and extraneuronal development, and introduce GAD1 as a new gene associated with developmental and epileptic encephalopathy

D-Cbl Binding to Drk Leads to Dose-Dependent Down-Regulation of EGFR Signaling and Increases Receptor-Ligand Endocytosis

Proper control of Epidermal Growth Factor Receptor (EGFR) signaling is critical for normal development and regulated cell behaviors. Abnormal EGFR signaling is associated with tumorigenic process of various cancers. Complicated feedback networks control EGFR signaling through ligand production, and internalization-mediated destruction of ligand-receptor complexes. Previously, we found that two isoforms of D-Cbl, D-CblS and D-CblL, regulate EGFR signaling through distinct mechanisms. While D-CblL plays a crucial role in dose-dependent down-regulation of EGFR signaling, D-CblS acts in normal restriction of EGFR signaling and does not display dosage effect. Here, we determined the underlying molecular mechanism, and found that Drk facilitates the dose-dependent regulation of EGFR signaling through binding to the proline-rich motif of D-CblL, PR. Furthermore, the RING finger domain of D-CblL is essential for promoting endocytosis of the ligand-receptor complex. Interestingly, a fusion protein of the two essential domains of D-CblL, RING- PR, is sufficient to down-regulate EGFR signal in a dose-dependent manner by promoting internalization of the ligand, Gurken. Besides, RING-SH2Drk, a fusion protein of the RING finger domain of D-Cbl and the SH2 domain of Drk, also effectively down-regulates EGFR signaling in Drosophila follicle cells, and suppresses the effects of constitutively activated EGFR. The RING-SH2Drk suppresses EGFR signaling by promoting the endosomal trafficking of ligand-receptor complexes, suggesting that Drk plays a negative role in EGFR signaling by enhancing receptor endocytosis through cooperating with the RING domain of D-Cbl. Interfering the recruitment of signal transducer, Drk, to the receptor by the RING-SH2Drk might further reduces EGFR signaling. The fusion proteins we developed may provide alternative strategies for therapy of cancers caused by hyper-activation of EGFR signaling

Force-Controlled Balance Perturbations Associated with Falls in Older People: A Prospective Cohort Study

Balance recovery from an unpredictable postural perturbation can be a challenging task for many older people and poor recovery could contribute to their risk of falls. This study examined associations between responses to unpredictable perturbations and fall risk in older people. 242 older adults (80.064.4 years) underwent assessments of stepping responses to multi-directional force-controlled waist-pull perturbations. Participants returned monthly falls calendars for the subsequent 12 months. Future falls were associated with lower force thresholds for stepping in the posterior and lateral but not anterior directions. Those with lower posterior force thresholds for stepping were 68% more likely to fall at home than those with higher force thresholds for stepping. These results suggest that amount of force that can be withstood following an unpredictable balance perturbation predicts future falls in community-dwelling older adults. Perturbations in the posterior direction best discriminated between future fallers and non-fallers

Drug Metabolism in Human Brain: High Levels of Cytochrome P4503A43 in Brain and Metabolism of Anti-Anxiety Drug Alprazolam to Its Active Metabolite

Cytochrome P450 (P450) is a super-family of drug metabolizing enzymes. P450 enzymes have dual function; they can metabolize drugs to pharmacologically inactive metabolites facilitating their excretion or biotransform them to pharmacologically active metabolites which may have longer half-life than the parent drug. The variable pharmacological response to psychoactive drugs typically seen in population groups is often not accountable by considering dissimilarities in hepatic metabolism. Metabolism in brain specific nuclei may play a role in pharmacological modulation of drugs acting on the CNS and help explain some of the diverse response to these drugs seen in patient population. P450 enzymes are also present in brain where drug metabolism can take place and modify therapeutic action of drugs at the site of action. We have earlier demonstrated an intrinsic difference in the biotransformation of alprazolam (ALP) in brain and liver, relatively more α-hydroxy alprazolam (α-OHALP) is formed in brain as compared to liver. In the present study we show that recombinant CYP3A43 metabolizes ALP to both α-OHALP and 4-hydroxy alprazolam (4-OHALP) while CYP3A4 metabolizes ALP predominantly to its inactive metabolite, 4-OHALP. The expression of CYP3A43 mRNA in human brain samples correlates with formation of relatively higher levels of α-OH ALP indicating that individuals who express higher levels of CYP3A43 in the brain would generate larger amounts of α-OHALP. Further, the expression of CYP3A43 was relatively higher in brain as compared to liver across different ethnic populations. Since CYP3A enzymes play a prominent role in the metabolism of drugs, the higher expression of CYP3A43 would generate metabolite profile of drugs differentially in human brain and thus impact the pharmacodynamics of psychoactive drugs at the site of action

Diagnostic Accuracy of Adenosine Deaminase and Lymphocyte Proportion in Pleural Fluid for Tuberculous Pleurisy in Different Prevalence Scenarios

BACKGROUND: Tuberculous pleural effusion (TPE) is a paucibacillary manifestation of tuberculosis, so isolation of Mycobacterium tuberculosis is difficult, biomarkers being an alternative for diagnosis. Adenosine deaminase (ADA) is the most cost-effective pleural fluid marker and is routinely used in high prevalence settings, whereas its value is questioned in areas with low prevalence. The lymphocyte proportion (LP) is known to increase the specificity of ADA for this diagnosis. We analyse the diagnostic usefulness of ADA alone and the combination of ADA ≥ 40 U/l (ADA(40)) and LP ≥ 50% (LP(50)) in three different prevalence scenarios over 11 years in our area. MATERIALS AND METHODS: Biochemistry, cytology and microbiology studies from 472 consecutive pleural fluid samples were retrospectively analyzed. ADA and differential cell count were determined in all samples. We established three different prevalence periods, based on percentage of pleural effusion cases diagnosed as tuberculosis: 1998-2000 (31.3%), 2001-2004 (11.8%), and 2005-2008 (7.4%). ROC curves, dispersion diagrams and pre/post-test probability graphs were produced. TPE accounted for 73 episodes (mean prevalence: 15.5%). The sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) for ADA(40) were 89%, 92.7%, 69.2% and 97.9%, respectively. For ADA(40)+LP(50) the specificity and PPV increased (98.3% and 90%) with hardly any decrease in the sensitivity or NPV (86.3% and 97.5%). No relevant differences were observed between the three study periods. CONCLUSIONS/SIGNIFICANCE: ADA remains useful for the diagnosis of TPE even in low-to-intermediate prevalence scenarios when combined with the lymphocyte proportion