Identification of a potent and selective LAPTc inhibitor by RapidFire-Mass Spectrometry, with antichagasic activity.

BACKGROUND: Chagas disease is caused by the protozoan parasite Trypanosoma cruzi and leads to ~10,000 deaths each year. Nifurtimox and benznidazole are the only two drugs available but have significant adverse effects and limited efficacy. New chemotherapeutic agents are urgently required. Here we identified inhibitors of the acidic M17 leucyl-aminopeptidase from T. cruzi (LAPTc) that show promise as novel starting points for Chagas disease drug discovery. METHODOLOGY/PRINCIPAL FINDINGS: A RapidFire-MS screen with a protease-focused compound library identified novel LAPTc inhibitors. Twenty-eight hits were progressed to the dose-response studies, from which 12 molecules inhibited LAPTc with IC50 < 34 µM. Of these, compound 4 was the most potent hit and mode of inhibition studies indicate that compound 4 is a competitive LAPTc inhibitor, with Ki 0.27 µM. Compound 4 is selective with respect to human LAP3, showing a selectivity index of >500. Compound 4 exhibited sub-micromolar activity against intracellular T. cruzi amastigotes, and while the selectivity-window against the host cells was narrow, no toxicity was observed for un-infected HepG2 cells. In silico modelling of the LAPTc-compound 4 interaction is consistent with the competitive mode of inhibition. Molecular dynamics simulations reproduce the experimental binding strength (-8.95 kcal/mol), and indicate a binding mode based mainly on hydrophobic interactions with active site residues without metal cation coordination. CONCLUSIONS/SIGNIFICANCE: Our data indicates that these new LAPTc inhibitors should be considered for further development as antiparasitic agents for the treatment of Chagas disease.

Correction to "Bespoke Drug Discovery Training for Low-Middle Income Countries".

[This corrects the article DOI: 10.1021/acsmedchemlett.3c00215.].

Bespoke Drug Discovery Training for Low-Middle Income Countries.

Working in drug discovery is difficult for many institutions due to the need for resources, funding, and in-country expertise. The Wellcome Centre for Anti-Infective Research (WCAIR) is responding to the unmet training needs for individuals/institutions working in drug discovery in low-middle income countries. Through their training program, individuals can undertake a practical placement, either online or at the center, with access to a dedicated trainer from their field of research. Practical placements are tailored to the needs of the individual/institute to enable capability building on return to their home institute. In addition to training placements, the center is focused on building partnerships by supporting institutes to work in drug discovery. Here we highlight WCAIR's training program and the partnerships that have developed from this.

Preparation, biological evaluation and QSAR analysis of urea substituted 2,4-diamino-pyrimidine anti-malarials.

The synthesis and evaluation of twenty six new phenylurea substituted 2,4-diamino-pyrimidines against Plasmodium falciparum (Pf) 3D7 are reported. Compounds were prepared to improve both anti-malarial activity and selectivity of the series previously reported by our group. Additional properties have been determined to assess their potential as anti-malarial leads including; HepG2 cytotoxicity, solubility, permeability, and lipophilicity, as well as in vitro stability in human and rat microsomes. We also assess their inhibition profile against a diverse set of 10 human kinases. Molecular docking, cheminformatics and bioinformatics analyses were also undertaken. Compounds 40 demonstrated the best anti-malarial activity at Pf 3D7 (0.09 µM), good selectivity with respect to mammalian cytotoxicity (SI = 54) and low microsomal clearance. Quantitative structure activity relationship (QSAR) analyses point to lipophilicity being a key driver of improved anti-malarial activity. The most active compounds in the series suffered from high lipophilicity, poor aqueous solubility and low permeability. The results provide useful information to guide further chemistry iterations.

Preparation, biological & cheminformatics-based assessment of N2,N4-diphenylpyrimidine-2,4-diamine as potential Kinase-targeted antimalarials.

Twenty eight new N2,N4-diphenylpyrimidine-2,4-diamines have been prepared in order to expand our understanding of the anti-malarial SAR of the scaffold. The aim of the study was to make structural modifications to improve the overall potency, selectivity and solubility of the series by varying the anilino groups attached to the 2- and 4-position. We evaluated the activity of the compounds against Plasmodium falciparum (Pf) 3D7, cytotoxicity against HepG2, % inhibition at a panel of 10 human kinases, solubility, permeability and lipophilicity, and human and rat in vitro clearance. 11 was identified as a potent anti-malarial with an IC50 of 0.66 µM at the 3D7 strain and a selectivity (SI) of ~ 40 in terms of cytotoxicity against the HepG2 cell line. It also displayed low experimental logD7.4 (2.27), reasonable solubility (124 µg/ml), good metabolic stability, but low permeability. A proteo-chemometric workflow was employed to identify putative Pf targets of the most promising compounds. Ligand-based similarity searching of the ChEMBL database led to the identification of most probable human targets. These were then used as input for sequence-based searching of the Pf proteome. Homology modelling and molecular docking were used to evaluate whether compounds could indeed bind to these targets with valid binding modes. In vitro biological testing against close human analogs of these targets was subsequently undertaken. This allowed us to identify potential Pf targets and human anti-targets that could be exploited in future development.

Development of a High-Throughput Screening Assay to Identify Inhibitors of the Major M17-Leucyl Aminopeptidase from Trypanosoma cruzi Using RapidFire Mass Spectrometry.

Leucyl aminopeptidases (LAPs) are involved in multiple cellular functions, which, in the case of infectious diseases, includes participation in the pathogen-host cell interface and pathogenesis. Thus, LAPs are considered good candidate drug targets, and the major M17-LAP from Trypanosoma cruzi (LAPTc) in particular is a promising target for Chagas disease. To exploit LAPTc as a potential target, it is essential to develop potent and selective inhibitors. To achieve this, we report a high-throughput screening method for LAPTc. Two methods were developed and optimized: a Leu-7-amido-4-methylcoumarin-based fluorogenic assay and a RapidFire mass spectrometry (RapidFire MS)-based assay using the LSTVIVR peptide as substrate. Compared with a fluorescence assay, the major advantages of the RapidFire MS assay are a greater signal-to-noise ratio as well as decreased consumption of enzyme. RapidFire MS was validated with the broad-spectrum LAP inhibitors bestatin (IC50 = 0.35 µM) and arphamenine A (IC50 = 15.75 µM). We suggest that RapidFire MS is highly suitable for screening for specific LAPTc inhibitors.

Research Techniques Made Simple: An Introduction to Drug Discovery for Dermatology.

This article aims to provide an overview of drug discovery with a focus on application within dermatology. The term "drug" can be used to describe a wide variety of agents, including small molecules, cell therapies, and antibodies, which may be dosed intravenously, orally, topically, or by other routes of administration. We summarize the economics and risks involved in drug discovery. Understanding the needs of patients and clinicians through use of a target product profile before initiating drug discovery can reduce time and effort spent developing a poor or unneeded drug. For small molecule drug discovery, a chemical starting point is then required. We present four options for finding a chemical starting point for drug discovery projects: screening libraries of compounds or modifying, reformulating, or repositioning a known drug. Examples of each technique's use in dermatology are provided. We also describe the subsequent steps involved in discovery of a new drug. To help interested readers, we provide information on how to engage with academic drug discovery centers or industrial partners.

Development of Chemical Proteomics for the Folateome and Analysis of the Kinetoplastid Folateome.

The folate pathway has been extensively studied in a number of organisms, with its essentiality exploited by a number of drugs. However, there has been little success in developing drugs that target folate metabolism in the kinetoplastids. Despite compounds being identified which show significant inhibition of the parasite enzymes, this activity does not translate well into cellular and animal models of disease. Understanding to which enzymes antifolates bind under physiological conditions and how this corresponds to the phenotypic response could provide insight on how to target the folate pathway in these organisms. To facilitate this, we have adopted a chemical proteomics approach to study binding of compounds to enzymes of folate metabolism. Clinical and literature antifolate compounds were immobilized onto resins to allow for "pull down" of the proteins in the "folateome". Using competition studies, proteins, which bind the beads specifically and nonspecifically, were identified in parasite lysate ( Trypanosoma brucei and Leishmania major) for each antifolate compound. Proteins were identified through tryptic digest, tandem mass tag (TMT) labeling of peptides followed by LC-MS/MS. This approach was further exploited by creating a combined folate resin (folate beads). The resin could pull down up to 9 proteins from the folateome. This information could be exploited in gaining a better understanding of folate metabolism in kinetoplastids and other organisms.

Identifying diabetes cases from administrative data: a population-based validation study.

BACKGROUND: Health care data allow for the study and surveillance of chronic diseases such as diabetes. The objective of this study was to identify and validate optimal algorithms for diabetes cases within health care administrative databases for different research purposes, populations, and data sources. METHODS: We linked health care administrative databases from Ontario, Canada to a reference standard of primary care electronic medical records (EMRs). We then identified and calculated the performance characteristics of multiple adult diabetes case definitions, using combinations of data sources and time windows. RESULTS: The best algorithm to identify diabetes cases was the presence at any time of one hospitalization or physician claim for diabetes AND either one prescription for an anti-diabetic medication or one physician claim with a diabetes-specific fee code [sensitivity 84.2%, specificity 99.2%, positive predictive value (PPV) 92.5%]. Use of physician claims alone performed almost as well: three physician claims for diabetes within one year was highly specific (sensitivity 79.9%, specificity 99.1%, PPV 91.4%) and one physician claim at any time was highly sensitive (sensitivity 93.6%, specificity 91.9%, PPV 58.5%). CONCLUSIONS: This study identifies validated algorithms to capture diabetes cases within health care administrative databases for a range of purposes, populations and data availability. These findings are useful to study trends and outcomes of diabetes using routinely-collected health care data.

Effect of Electrophysiology Assessment on Mortality and Hospitalizations in Patients With New-Onset Atrial Fibrillation.

Few patients with atrial fibrillation (AF) receive care by cardiac electrophysiologists. Although previous work has highlighted differential care for patients with AF treated by electrophysiologists, it is unclear whether this is associated with improved clinical outcomes. This retrospective population-level propensity score-matched cohort study included patients aged 20 to 80 years with new-onset AF presenting to an emergency department (ED) in Ontario, Canada, between 2010 and 2012. Patients were followed until March 31, 2015. Patients who saw an electrophysiologist within 1 year of the index ED visit were matched to patients who did not see an electrophysiologist. Linked administrative databases were used for cohort construction and allow 1-year follow-up to assess for the clinical end points of all-cause mortality and hospitalization for AF, heart failure, bleeding, and stroke. A total of 5,221 unique pairs of patients were matched. One hundred seventeen patients (2.2%) in the electrophysiologist cohort underwent an AF ablation procedure during the 1-year follow-up period. All-cause mortality (hazard ratio [HR] = 1.1, p = 0.17) and stroke (HR = 1.4, p = 0.09) were not significantly different between the 2 groups. Hospitalization for AF (HR = 1.4, p <0.001), bleeding (HR = 1.5, p = 0.0001), and congestive heart failure (HR = 1.5, p <0.0001) was increased in the group that saw an electrophysiologist. In conclusion, electrophysiologist care was not associated with improved clinical outcomes in patients with new-onset AF.

The Relationship Between Cardiologist Care and Clinical Outcomes in Patients With New-Onset Atrial Fibrillation.

BACKGROUND: Increased mortality is well described in patients with atrial fibrillation (AF), primarily related to death from cardiovascular causes. One may hypothesize that cardiology care may be associated with a reduction in cardiovascular deaths in patients with AF, thereby improving their overall survival. The aim of this study was to assess the association between cardiologist care and clinical outcomes, including all-cause mortality, in patients with new-onset AF. METHODS: This was a retrospective population-level, propensity score-matched cohort study of patients aged 20-80 years with new-onset AF presenting to an emergency department in Ontario, Canada between 2010 and 2012. Patients who saw a cardiologist within 1 year of the initial diagnosis were matched to patients who did not see a cardiologist. Linked administrative databases were used for cohort construction and allow for 1-year follow-up to assess for the clinical end points of death, hospitalization for AF, stroke syndromes, bleeding, and heart failure. RESULTS: Cardiologist care was associated with a lower 1-year rate of death (5.3% vs 7.7%; hazard ratio, 0.68; 95% confidence interval, 0.55-0.84), despite an increased rate of hospitalizations for AF (17.9% vs 8.2%), stroke syndromes (1.7% vs 0.5%), bleeding (3.1% vs 2.0%), and heart failure (3.2% vs 1.4%). CONCLUSIONS: Cardiologist care was associated with a reduction in death in patients with new-onset AF. Further study to obtain a greater understanding of the processes of care associated with the observed survival improvement is warranted.

Factors Associated With Cardiac Electrophysiologist Assessment and Catheter Ablation Procedures in Patients With Atrial Fibrillation.

OBJECTIVES: This study sought to determine factors associated with cardiac electrophysiologist assessment and atrial fibrillation (AF) ablation in patients with new-onset AF. BACKGROUND: Factors driving variation in the use of AF ablation have not been well described. METHODS: All individuals with new-onset AF in Ontario, Canada, between January 1, 2010, and December 31, 2012, were identified. Survival analysis accounting for the competing risk of death was used to evaluate the association between clinical and nonclinical factors and receipt of an electrophysiologist assessment. Factors associated with AF ablation were then determined in the subgroup of patients who received an electrophysiologist assessment. RESULTS: A total of 22,032 patients with new-onset AF were identified, 8,161 (37%) of whom received an electrophysiology assessment. Prior cardiologist care was associated with electrophysiologist assessment (hazard ratio [HR]: 1.57; p < 0.0001). Rural residence was associated with a decreased incidence of electrophysiology assessment (HR: 0.80; p < 0.0001). A total of 424 (5.2%) patients receiving an electrophysiologist assessment had an AF ablation. Recurrent AF emergency department (ED) visits between the index ED visit and the initial electrophysiologist assessment (HR for ≥2 ED visits: 4.22; p < 0.0001) and rural residence (HR: 1.50; p = 0.002) were both associated with AF ablation. Cardiovascular comorbidities were associated with a decreased incidence of AF ablation. CONCLUSIONS: Rural patients with AF have a lower incidence of electrophysiologist assessment but paradoxically a higher incidence of AF ablation compared with their urban counterparts. Clinical factors such as recurrent ED visits for AF and cardiovascular comorbidities are the most important factors associated with of AF ablation.

Power Mobility Training for Young Children with Multiple, Severe Impairments: A Case Series.

AIMS: Young children with neurodevelopmental conditions are often limited in their ability to explore and learn from their environment. The purposes of this case series were to (1) describe the outcomes of using an alternative power mobility device with young children who had multiple, severe impairments; (2) develop power mobility training methods for use with these children; and (3) determine the feasibility of using various outcome measures. METHODS: Three children with cerebral palsy (Gross Motor Function Classification System Levels IV, V, and V) ages 17 months to 3.5 years participated in the case series. Examination included the Pediatric Evaluation of Disability Inventory-Computer Adaptive Test (PEDI-CAT) and the Dimensions of Mastery Questionnaire (DMQ). An individualized, engaging power mobility training environment was created for each participant. Intervention was provided for 60 minutes per week over 12 weeks. RESULTS: All participants exhibited improvements in power mobility skills. Post-intervention PEDI-CAT scores increased in various domains for all participants. Post-intervention DMQ scores improved in Participants 1 and 2. DISCUSSION: The participants appeared to make improvements in their beginning power mobility skills. Additional research is planned to further explore the impact of power mobility training in this unique population.

Validation of Algorithms to Identify Invasive Electrophysiology Procedures Using Administrative Data in Ontario, Canada.

BACKGROUND: Administrative database research can provide insight into the real-world effectiveness of invasive electrophysiology procedures. However, no validated algorithm to identify these procedures within administrative data currently exists. OBJECTIVE: To develop and validate algorithms to identify atrial fibrillation (AF), atrial flutter (AFL), supraventricular tachycardia (SVT) catheter ablation procedures, and diagnostic electrophysiology studies (EPS) within administrative data. METHODS: Algorithms consisting of physician procedural billing codes and their associated most responsible hospital diagnosis codes were used to identify potential AF, AFL, SVT catheter ablation procedures and diagnostic EPS within large administrative databases in Ontario, Canada. The potential procedures were then limited to those performed between October 1, 2011 and March 31, 2013 at a single large regional cardiac center (Sunnybrook Health Sciences Center) in Ontario, Canada. These procedures were compared with a gold-standard cohort of patients known to have undergone invasive electrophysiology procedures during the same time period at the same institution. The sensitivity, specificity, positive and negative predictive values of each algorithm was determined. RESULTS: Algorithms specific to each of AF, AFL, and SVT ablation were associated with a moderate sensitivity (75%-86%), high specificity (95%-98%), positive (95%-98%), and negative (99%) predictive values. The best algorithm to identify diagnostic EPS was less optimal with a sensitivity of 61% and positive predictive value of 88%. CONCLUSIONS: Algorithms using a combination of physician procedural billing codes and accompanying most responsible hospital diagnosis may identify catheter ablation procedures within administrative data with a high degree of accuracy. Diagnostic EPS may be identified with reduced accuracy.

Cholesterol testing among men and women with disability: the role of morbidity.

PURPOSE: Despite more frequent use of health services by people living with disability, the quality of preventive care received may be suboptimal. In this retrospective cohort study, we used administrative data to examine the relationship between cholesterol testing and levels of disability and morbidity among women and men in Ontario, Canada. METHODS: We linked multiple provincial-level databases in this study. In stratified analyses for women and men, we used multivariable logistic regression to examine differences in cholesterol testing, and we tested for an interaction effect between disability and morbidity. In a secondary analysis, we tested for a three-way interaction between sex, disability, and morbidity on the entire cohort. RESULTS: There was an interaction between morbidity and disability for both women and men. Women and men with no chronic conditions appeared to be least likely to be up-to-date on cholesterol testing, and among this group, those with moderate disability were more likely to be up-to-date on cholesterol testing than those with no disability (adjusted odds ratio [AOR] =1.51; 95% confidence interval [CI] 1.20-1.90 for women; AOR =1.16; 95% CI 1.00-1.34 for men). Among women and men who had one chronic condition, having severe disability put them at significant disadvantage versus those with no disability. Only 58.5% of men with no disability and no chronic conditions were up-to-date on cholesterol testing. CONCLUSION: An intermediate level of health care need (reflected in this study as level of disability and level of morbidity) may provide a benefit for cholesterol testing, and conversely, health care needs that are too few or too great may negatively affect testing. Public health and practice-based interventions need to be explored to address these findings.

Inpatient mortality of HIV-infected adults in sub-Saharan Africa and possible interventions: a mixed methods review.

BACKGROUND: Despite the increased availability of anti-retroviral therapy, in-hospital HIV mortality remains high in sub-Saharan Africa. Reports from Senegal, Malawi, and Tanzania show rates of in-hospital, HIV-related mortality ranging from 24.2% to 44%. This mixed methods review explored the potential causes of preventable in-hospital mortality associated with HIV infections in sub-Saharan Africa in the anti-retroviral era. RESULTS: Based on our experience as healthcare providers in Africa and a review of the literature we identified 5 health systems failures which may cause preventable in-hospital mortality, including: 1) late presentation of HIV cases, 2) low rates of in-hospital HIV testing, 3) poor laboratory capacity which limits CD4 T-cell testing and the diagnosis of opportunistic infections, 4) delay in initiation of anti-retroviral therapy in-hospital, and 5) problems associated with loss to follow-up upon discharge from hospital. CONCLUSION: Our findings, together with the current available literature, should be used to develop practical interventions that can be implemented to reduce in-hospital mortality.

Superior outcomes and lower outpatient costs with scale-up of antiretroviral therapy at the GHESKIO clinic in Port-au-Prince, Haiti.

BACKGROUND: Treatment protocols and prices of antiretroviral therapy (ART) have changed over time. Yet, limited data exist to evaluate the impact of these changes on patient outcomes and treatment costs in resource-poor settings. METHODS: We compared patient-level data on outcomes, utilization, and cost for the first 2 years of ART for a cohort of adult patients initiating ART in 2003-2004 and a cohort initiating ART in 2006-2008 at the Haitian Group for the Study of Kaposi's Sarcoma and Opportunistic Infections clinic (GHESKIO) in Port-au-Prince, Haiti. Costs were measured from the health center perspective. Multivariate analyses were conducted to account for the potential impact of differences in disease severity at baseline. RESULTS: With the exclusion of patients who transferred care, 92% (167/181) of patients in the 2006-2008 cohort and 75% (150/200) in the 2003-2004 cohort were alive and in care at the end of the study period. The mean cost per patient for the 2-year study period was US$723 for the 2006-2008 cohort vs. US$1191 for the 2003-2004 cohort, a cost difference of US$468 (P < 0.0001). The mean cost per patient alive and in care at the end of the 2-year study period was US$744 for the 2006-2008 cohort vs. US$1489 for the 2003-2004 cohort (P < 0.0001). CONCLUSIONS: HIV treatment outcomes in Haiti have improved over time while treatment costs declined by over 50% per patient alive and in care at the end of the 2-year study period. The major drivers in the reduction of treatment costs were the lower price of ART, lower costs for laboratory testing, and lower overhead costs.

The co-use of tobacco and cannabis among adolescents over a 30-year period.

BACKGROUND: This study explores the patterns of use and co-use of tobacco and cannabis among Ontario adolescents over 3 decades and if characteristics of co-users and single substance users have changed. METHODS: Co-use trends for 1981-2011 were analyzed using the Centre for Addiction and Mental Health Ontario Student Drug Use and Health Survey, which includes 38,331 students in grades 7, 9, and 11. A co-user was defined as someone reporting daily tobacco and/or cannabis use in the past month. Trends over time (by gender and academic performance) were analyzed with logistic regression. RESULTS: The prevalence of tobacco-only use, cannabis-only use, and co-use fluctuated considerably. During 1981-1993, there were more tobacco-only users than co-users and cannabis-only users; since 1993 the prevalence of tobacco use has decreased dramatically. Co-use prevalence peaked at 12% (95% confidence interval: 9, 15) in 1999, when prevalence of overall use of both substances was highest. In 2011, 92% of tobacco users also used cannabis, up from 16% in 1991. CONCLUSIONS: In 2011 nearly all students who smoke tobacco daily also use cannabis. Non-regular use of either substance is highest now compared with the past 3 decades. Contemporary tobacco and cannabis co-users are significantly different than past users. Youth prevention programs should understand the changing context of cannabis and tobacco among youth.

Epidemiology and genetic diversity of multidrug-resistant tuberculosis in East Africa.

Multidrug-resistant tuberculosis (MDR-TB) is an emerging problem in many parts of the world, and levels of MDR-TB among new TB patients are increasing in sub-Saharan Africa. We reviewed the prevalence and molecular epidemiology of MDR-TB in East Africa, including Burundi, Kenya, Rwanda, Tanzania, and Uganda. In 16 epidemiologic surveys, the prevalence of MDR among new cases ranges from 0.4% in Tanzania to 4.4% in Uganda, and among recurrent cases ranges from 3.9% in Tanzania to 17.7% in Uganda. There is a gap of 5948 cases between the estimated number of MDR-TB cases in East Africa and the number actually diagnosed. The only confirmed risk factors for MDR-TB are prior treatment for TB and refugee status. HIV has not been reported as a risk factor, and there are no reports of statistical association between spoligotype and drug resistance pattern. Increased capacity for diagnosis and treatment of MDR-TB is needed, with an emphasis on recurrent TB cases and refugees.

Early and efficient detection of Mycobacterium tuberculosis in sputum by microscopic observation of broth cultures.

Early, efficient and inexpensive methods for the detection of pulmonary tuberculosis are urgently needed for effective patient management as well as to interrupt transmission. These methods to detect M. tuberculosis in a timely and affordable way are not yet widely available in resource-limited settings. In a developing-country setting, we prospectively evaluated two methods for culturing and detecting M. tuberculosis in sputum. Sputum samples were cultured in liquid assay (micro broth culture) in microplate wells and growth was detected by microscopic observation, or in Löwenstein-Jensen (LJ) solid media where growth was detected by visual inspection for colonies. Sputum samples were collected from 321 tuberculosis (TB) suspects attending Bugando Medical Centre, in Mwanza, Tanzania, and were cultured in parallel. Pulmonary tuberculosis cases were diagnosed using the American Thoracic Society diagnostic standards. There were a total of 200 (62.3%) pulmonary tuberculosis cases. Liquid assay with microscopic detection detected a significantly higher proportion of cases than LJ solid culture: 89.0% (95% confidence interval [CI], 84.7% to 93.3%) versus 77.0% (95% CI, 71.2% to 82.8%) (p = 0.0007). The median turn around time to diagnose tuberculosis was significantly shorter for micro broth culture than for the LJ solid culture, 9 days (interquartile range [IQR] 7-13), versus 21 days (IQR 14-28) (p<0.0001). The cost for micro broth culture (labor inclusive) in our study was US $4.56 per sample, versus US $11.35 per sample for the LJ solid culture. The liquid assay (micro broth culture) is an early, feasible, and inexpensive method for detection of pulmonary tuberculosis in resource limited settings.