Anti-lipid antibody in M. tuberculosis infection--basis for a new biomarker-based test to monitor treatment response

Tuberculosis (TB) faces difficult challenges for its treatment and control. Both the diagnosis of TB and Mycobacterium tuberculosis (M. tuberculosis) drug susceptibility testing take weeks and clinicians often do not know if the patient is taking an appropriate set of drugs until complications or even death occur. Consequently, early determination of a successful drug therapy response in individuals infected with M. tuberculosis is urgently needed. Since the M. tuberculosis cell wall is comprised of a diverse repertoire of lipids, we examined the possible role of these lipids as antigens for serologic response during M. tuberculosis infection. This dissertation is focused on the examination of lipid-antibody response as a potential biomarker used to monitor treatment response in M. tuberculosis infected hosts. Briefly, Chapter 1 describes the current pitfalls of monitoring tuberculosis treatment with current methods, including acid-fast bacilli (AFB) smear and culture conversion. Chapter 1 also covers the definition of biomarkers and the rationale to use M. tuberculosis cell wall lipids to develop an anti-lipid-antibody based test. Evidence from a similar biomarker-based test for syphilis is presented. The chapter also discusses the biological basis which guided the lipid-antibody biomarker search and discovery. Chapter 2 describes the use of a serum bank from patients with pulmonary TB provided by the World Health Organization-Tropical Diseases Research consortium (WHO-TDR) to identify M. tuberculosis lipid candidates as targets of antibody response. These samples were used to look for an antibody response to multiple mycobacterial lipids resolved by thin-layer chromatography immunoblot (TLC-I). This approach allowed us to identify M. tuberculosis cardiolipin by mass spectrometry and we determined that the IgM antibody response to cardiolipin can be used as a biomarker of infection. In Chapter 3, we investigate the biological evidence behind the production of anti-phospholipid IgM antibody during TB infection and anti-TB treatment. For this purpose, we used the Cornell mouse model of infection to monitor the change in IgM antibody response against four phospholipids including cardiolipin (CL), phosphatidyl choline (PTC), phosphatidyl ethanolamine (PE) and phosphatidyl inositol (PI) over the course of M. tuberculosis infection and treatment. We separated BALB/c mice into three groups including acute infection (AI), chronic infection (CI), and healthy control (HC). Both AI and CI groups were infected via the aerosol route with M. tuberculosis strain H37Rv at day 0. The AI group was treated from 4 to 12 weeks post-infection, while the CI group was treated from 20 to 28 weeks post-infection. We also measured the levels of pro-inflammatory cytokines, IL-5 and MCP-1. We observed that in treated AI mice, anti-phospholipid IgM antibody levels decreased compared to those of healthy mice at all time points. Anti-PTC IgM antibodies remained significantly higher in CI mice than in AI mice at all time points post-infection. The anti-PTC IgM antibody levels in CI mice decreased to levels similar to those of AI and HC mice at 32 weeks post-infection. The anti-phospholipid IgM antibody levels correlated with the bacterial load in the lungs, with treated mice showing fewer M. tuberculosis colony-forming units (CFU) after eight weeks of treatment. Furthermore, IL-5 was mainly produced by the site of infection in the lung and decreased with anti-tuberculosis treatment within the CI mice group. Finally, in Chapter 4, we examine the use of anti-phospholipid IgM antibody changes as a biomarker for treatment response in patients with smear positive pulmonary TB. Serum samples were obtained from pulmonary TB patients at the start and end of the intensive phase of treatment (40 doses of anti-TB combination therapy) enrolled from Kampala, Uganda in a CDC-TB Trials Consortium randomized clinical trial. The samples were screened for IgM antibody levels against five commercially available phospholipids by an in-house ELISA assay. The lipid antigens included CL, PI, PE, PTC, and sphingolipid (SL). IgM antibody levels to CL, PE, PI, PTC and SL significantly decreased following anti-TB drug treatment in patients without lung cavities on their baseline chest radiograph. In contrast, patients with cavitary TB showed an overall increase in the anti-phospholipid IgM antibody response following anti-TB drug treatment, notably with a significant increase in anti-PE antibody levels. Thus, anti-lipid IgM response appears to be a useful biomarker for treatment response, especially in those with non-cavitary disease. Chapter 5 summarizes the conclusions in support of using the anti-phospholipid IgM antibody response as a useful biomarker for monitoring TB treatment response. This novel biomarker test would greatly facilitate TB management in resource-poor settings. The development of a point of care (POC) test based on anti-phospholipid IgM antibody will be an affordable and highly sensitive alternative to microscopy or culture testing for monitoring treatment response in individuals with TB. Chapter 5 also gives examples of three platforms that might be used for the development of such a POC test. However, we note that it is necessary to explore the specificity of this assay further by testing patients with HIV infection, latent TB infection, and non-TB pulmonary diseases.Tuberculosis (TB) faces difficult challenges for its treatment and control. Both the diagnosis of TB and Mycobacterium tuberculosis (M. tuberculosis) drug susceptibility testing take weeks and clinicians often do not know if the patient is taking an appropriate set of drugs until complications or even death occur. Consequently, early determination of a successful drug therapy response in individuals infected with M. tuberculosis is urgently needed. Since the M. tuberculosis cell wall is comprised of a diverse repertoire of lipids, we examined the possible role of these lipids as antigens for serologic response during M. tuberculosis infection. This dissertation is focused on the examination of lipid-antibody response as a potential biomarker used to monitor treatment response in M. tuberculosis infected hosts. Briefly, Chapter 1 describes the current pitfalls of monitoring tuberculosis treatment with current methods, including acid-fast bacilli (AFB) smear and culture conversion. Chapter 1 also covers the definition of biomarkers and the rationale to use M. tuberculosis cell wall lipids to develop an anti-lipid-antibody based test. Evidence from a similar biomarker-based test for syphilis is presented. The chapter also discusses the biological basis which guided the lipid-antibody biomarker search and discovery. Chapter 2 describes the use of a serum bank from patients with pulmonary TB provided by the World Health Organization-Tropical Diseases Research consortium (WHO-TDR) to identify M. tuberculosis lipid candidates as targets of antibody response. These samples were used to look for an antibody response to multiple mycobacterial lipids resolved by thin-layer chromatography immunoblot (TLC-I). This approach allowed us to identify M. tuberculosis cardiolipin by mass spectrometry and we determined that the IgM antibody response to cardiolipin can be used as a biomarker of infection. In Chapter 3, we investigate the biological evidence behind the production of anti-phospholipid IgM antibody during TB infection and anti-TB treatment. For this purpose, we used the Cornell mouse model of infection to monitor the change in IgM antibody response against four phospholipids including cardiolipin (CL), phosphatidyl choline (PTC), phosphatidyl ethanolamine (PE) and phosphatidyl inositol (PI) over the course of M. tuberculosis infection and treatment. We separated BALB/c mice into three groups including acute infection (AI), chronic infection (CI), and healthy control (HC). Both AI and CI groups were infected via the aerosol route with M. tuberculosis strain H37Rv at day 0. The AI group was treated from 4 to 12 weeks post-infection, while the CI group was treated from 20 to 28 weeks post-infection. We also measured the levels of pro-inflammatory cytokines, IL-5 and MCP-1. We observed that in treated AI mice, anti-phospholipid IgM antibody levels decreased compared to those of healthy mice at all time points. Anti-PTC IgM antibodies remained significantly higher in CI mice than in AI mice at all time points post-infection. The anti-PTC IgM antibody levels in CI mice decreased to levels similar to those of AI and HC mice at 32 weeks post-infection. The anti-phospholipid IgM antibody levels correlated with the bacterial load in the lungs, with treated mice showing fewer M. tuberculosis colony-forming units (CFU) after eight weeks of treatment. Furthermore, IL-5 was mainly produced by the site of infection in the lung and decreased with anti-tuberculosis treatment within the CI mice group. Finally, in Chapter 4, we examine the use of anti-phospholipid IgM antibody changes as a biomarker for treatment response in patients with smear positive pulmonary TB. Serum samples were obtained from pulmonary TB patients at the start and end of the intensive phase of treatment (40 doses of anti-TB combination therapy) enrolled from Kampala, Uganda in a CDC-TB Trials Consortium randomized clinical trial. The samples were screened for IgM antibody levels against five commercially available phospholipids by an in-house ELISA assay. The lipid antigens included CL, PI, PE, PTC, and sphingolipid (SL). IgM antibody levels to CL, PE, PI, PTC and SL significantly decreased following anti-TB drug treatment in patients without lung cavities on their baseline chest radiograph. In contrast, patients with cavitary TB showed an overall increase in the anti-phospholipid IgM antibody response following anti-TB drug treatment, notably with a significant increase in anti-PE antibody levels. Thus, anti-lipid IgM response appears to be a useful biomarker for treatment response, especially in those with non-cavitary disease. Chapter 5 summarizes the conclusions in support of using the anti-phospholipid IgM antibody response as a useful biomarker for monitoring TB treatment response. This novel biomarker test would greatly facilitate TB management in resource-poor settings. The development of a point of care (POC) test based on anti-phospholipid IgM antibody will be an affordable and highly sensitive alternative to microscopy or culture testing for monitoring treatment response in individuals with TB. Chapter 5 also gives examples of three platforms that might be used for the development of such a POC test. However, we note that it is necessary to explore the specificity of this assay further by testing patients with HIV infection, latent TB infection, and non-TB pulmonary diseases

Water quality effects of intermittent water supply in Arraiján, Panama

Intermittent drinking water supply is common in low- and middle-income countries throughout the world and can cause waterquality to degrade in the distribution system.In this study,we characterized waterquality in one study zone with continuous supply and three zoneswith intermittentsupply in the drinking waterdistribution network in Arraija!n,Panama.Low orzero pressuresoccurred in allzones,and negative pressuresoccurred in the continuouszone and two ofthe intermittentzones.Despite hydraulic conditions that created risks for backflow and contaminant intrusion, only four of 423 (0.9%) grab samplescollected atrandom timeswere positive fortotalcoliform bacteria and only one waspositive for E.coli.Only nine of 496 (1.8%) samples had turbidity >1.0 NTU and all samples had !0.2 mg/L free chlorine residual.In contrast,water quality was often degraded during the first-flush period (when supply first returned after an outage).Still, routine and first-flush water quality under intermittent supply was much better in Arraija!n than that reported in a previous study conducted in India.Better waterquality in Arraija!n could be due to betterwaterquality leaving the treatmentplant,shortersupply outages,highersupply pressures,a more consistentand higherchlorine residual,and fewercontaminant sourcesnearpipes.The resultsillustrate thatintermittentsupply and itseffectson waterquality can vary greatly between and within distribution networks.The study also demonstrated thatmonitoring techniques designed specifically for intermittentsupply,such as continuous pressure monitoring and sampling the firstflush,can detectwaterquality threats and degradation thatwould notlikely be detected with conventionalmonitoring.Intermittent drinking water supply is common in low- and middle-income countries throughout the world and can cause waterquality to degrade in the distribution system.In this study,we characterized waterquality in one study zone with continuous supply and three zoneswith intermittentsupply in the drinking waterdistribution network in Arraija!n,Panama.Low orzero pressuresoccurred in allzones,and negative pressuresoccurred in the continuouszone and two ofthe intermittentzones.Despite hydraulic conditions that created risks for backflow and contaminant intrusion, only four of 423 (0.9%) grab samplescollected atrandom timeswere positive fortotalcoliform bacteria and only one waspositive for E.coli.Only nine of 496 (1.8%) samples had turbidity >1.0 NTU and all samples had !0.2 mg/L free chlorine residual.In contrast,water quality was often degraded during the first-flush period (when supply first returned after an outage).Still, routine and first-flush water quality under intermittent supply was much better in Arraija!n than that reported in a previous study conducted in India.Better waterquality in Arraija!n could be due to betterwaterquality leaving the treatmentplant,shortersupply outages,highersupply pressures,a more consistentand higherchlorine residual,and fewercontaminant sourcesnearpipes.The resultsillustrate thatintermittentsupply and itseffectson waterquality can vary greatly between and within distribution networks.The study also demonstrated thatmonitoring techniques designed specifically for intermittentsupply,such as continuous pressure monitoring and sampling the firstflush,can detectwaterquality threats and degradation thatwould notlikely be detected with conventionalmonitoring

Mycobacterial Lipids Induce Calcium Mobilization and Degranulation of Mast Cells

Cells and culture conditions. Murine MCs from line C1.MC/C57.1 (C57 MC) were kindly provided by Dr. Stephen Galli (Stanford University) and cultured in Dulbecco’s modified Eagle’s medium according to standard methods (9). M. tuberculosis lipid extracts. The bacterial lipid fractions extracted from H37Rv M. tuberculosis, including total lipids (T-L, NR-14837), insoluble lipids (I-L, NR-14843), soluble lipids (S-L, NR-14842), M. leprae, phenolic glycolipid I (PGL-I, NR-19342), and M. tuberculosis phosphatidylinositol mannoside 6 (PIM6, NR-14847), were obtained from BEI Resources. Purified bovine heart L-a-phosphatidylcholine (PC, 840052C) and cardiolipin (CL, 840012C) were obtained from Avanti Polar Lipids. All M. tuberculosis lipid extracts were resuspended according to the manufacturer’s recommendations.Cells and culture conditions. Murine MCs from line C1.MC/C57.1 (C57 MC) were kindly provided by Dr. Stephen Galli (Stanford University) and cultured in Dulbecco’s modified Eagle’s medium according to standard methods (9). M. tuberculosis lipid extracts. The bacterial lipid fractions extracted from H37Rv M. tuberculosis, including total lipids (T-L, NR-14837), insoluble lipids (I-L, NR-14843), soluble lipids (S-L, NR-14842), M. leprae, phenolic glycolipid I (PGL-I, NR-19342), and M. tuberculosis phosphatidylinositol mannoside 6 (PIM6, NR-14847), were obtained from BEI Resources. Purified bovine heart L-a-phosphatidylcholine (PC, 840052C) and cardiolipin (CL, 840012C) were obtained from Avanti Polar Lipids. All M. tuberculosis lipid extracts were resuspended according to the manufacturer’s recommendations

Mycobacterial Lipids Induce Calcium Mobilization and Degranulation of Mast Cells

Tuberculosis (TB) remains the leading cause of death from a single infectious agent, followed by HIV/AIDS (1). Emerging countries, such as Panama, have not been able to curb TB infection rates. The development of novel biomarkers for diagnosis, treatment monitoring, and vaccine development is urgently needed. The innate immune response during TB infection is a source of biomarkers. When a susceptible host is exposed to Mycobacterium tuberculosis, the bacteria translocate to the mucosal barrier, interacting with a variety of immune cells, including mast cells (MCs). The initial interaction between M. tuberculosis and first-line defense cells induces the accumulation and subsequent activation of these immune cells within lung tissue, and the release of proinflammatory mediators (2). Mycobacterial lipids appear to define the clinical fate of the infection by modulating the immune response during TB (3). However, the precise role of M. tuberculosis lipids in the immunopathogenic and molecular mechanisms of MC activation is still unknown. MCs are abundant in the lung tissue, where they act as sentinels for a wide variety of invading pathogens, as well as regulatory cells during the course of acute inflammation (4). Besides specific antigen-driven IgE crosslink stimulation, MCs can be activated by complement components, IgG, neuropeptides, pathogen-associated molecular patterns, mainly peptides, and whole M. tuberculosis interaction (5). MC activation promotes the secretion of stored proinflammatory mediators as well as de novo synthesis of leukotrienes, platelet-activating factor, and prostaglandins. Together with the transcription and release of cytokines and chemokines by MCs, the host begins to make an adaptive immune response (6). Recently, Garcia-Rodriguez and colleagues revised our perspective of the MC strategies used in bacterial defense and reported interactions occurring between M. tuberculosis and MCs (7). Shortly after exposure, MCs recognize M. tuberculosis via the TLR2 and CD48 receptors. During this initial stage, ESAT-6 and MPT-63 induce MC degranulation, cytokine release, and the secretion of antimicrobial peptides such as β-hexosaminidase and LL-37. We hypothesized that M. tuberculosis lipid extracts are able to activate MCs. In this preliminary study, we aimed to elucidate the role of single phospholipids and whole-lipid extracts in MC activation. Some results from these studies have been previously reported in the form of an abstract (8).Tuberculosis (TB) remains the leading cause of death from a single infectious agent, followed by HIV/AIDS (1). Emerging countries, such as Panama, have not been able to curb TB infection rates. The development of novel biomarkers for diagnosis, treatment monitoring, and vaccine development is urgently needed. The innate immune response during TB infection is a source of biomarkers. When a susceptible host is exposed to Mycobacterium tuberculosis, the bacteria translocate to the mucosal barrier, interacting with a variety of immune cells, including mast cells (MCs). The initial interaction between M. tuberculosis and first-line defense cells induces the accumulation and subsequent activation of these immune cells within lung tissue, and the release of proinflammatory mediators (2). Mycobacterial lipids appear to define the clinical fate of the infection by modulating the immune response during TB (3). However, the precise role of M. tuberculosis lipids in the immunopathogenic and molecular mechanisms of MC activation is still unknown. MCs are abundant in the lung tissue, where they act as sentinels for a wide variety of invading pathogens, as well as regulatory cells during the course of acute inflammation (4). Besides specific antigen-driven IgE crosslink stimulation, MCs can be activated by complement components, IgG, neuropeptides, pathogen-associated molecular patterns, mainly peptides, and whole M. tuberculosis interaction (5). MC activation promotes the secretion of stored proinflammatory mediators as well as de novo synthesis of leukotrienes, platelet-activating factor, and prostaglandins. Together with the transcription and release of cytokines and chemokines by MCs, the host begins to make an adaptive immune response (6). Recently, Garcia-Rodriguez and colleagues revised our perspective of the MC strategies used in bacterial defense and reported interactions occurring between M. tuberculosis and MCs (7). Shortly after exposure, MCs recognize M. tuberculosis via the TLR2 and CD48 receptors. During this initial stage, ESAT-6 and MPT-63 induce MC degranulation, cytokine release, and the secretion of antimicrobial peptides such as β-hexosaminidase and LL-37. We hypothesized that M. tuberculosis lipid extracts are able to activate MCs. In this preliminary study, we aimed to elucidate the role of single phospholipids and whole-lipid extracts in MC activation. Some results from these studies have been previously reported in the form of an abstract (8)

Serum samples can be substituted by plasma samples for the diagnosis of paratuberculosis

Employing plasma samples rather than serum samples for serological paratuberculosis diagnosis is practical, especially when bovine TB is assessed in the same cattle herd with the gamma interferon bovine avian (IFN- BA) test. We demonstrate that antibody titers in serum and plasma samples, utilizing the PARACHECK® ELISA kit, are highly comparable (Cohen’s kappa test, k = 0.955). We conclude that serum can be replaced with plasma in this commercially available antibody detection assay resulting in working hour savings for sampling and blood sample work-up and cost reductions for materials and sample storage.Employing plasma samples rather than serum samples for serological paratuberculosis diagnosis is practical, especially when bovine TB is assessed in the same cattle herd with the gamma interferon bovine avian (IFN- BA) test. We demonstrate that antibody titers in serum and plasma samples, utilizing the PARACHECK® ELISA kit, are highly comparable (Cohen’s kappa test, k = 0.955). We conclude that serum can be replaced with plasma in this commercially available antibody detection assay resulting in working hour savings for sampling and blood sample work-up and cost reductions for materials and sample storage

Mycobacterium tuberculosis Isolates from Single Outpatient Clinic in Panama City Exhibit Wide Genetic Diversity

Understanding Mycobacterium tuberculosis biodiversity and transmission is significant for tuberculosis control. This short report aimed to determine the genetic diversity of M. tuberculosis isolates from an outpatient clinic in Panama City. A total of 62 M. tuberculosis isolates were genotyped by 12 loci mycobacterial interspersed repetitive units-variable number of tandem repeats (MIRU-VNTR) and Spoligotyping. Forty-five (72.6%) of the isolates showed unique MIRU-VNTR genotypes, and 13 (21%) of the isolates were grouped into four clusters. Four isolates showed polyclonal MIRU-VNTR genotypes. The MIRU-VNTR Hunter-Gaston discriminatory index reached 0.988. The Spoligotyping analysis revealed 16 M. tuberculosis families, including Latin American-Mediterranean, Harlem, and Beijing. These findings suggest a wide genetic diversity of M. tuberculosis isolates at one outpatient clinic. A detailed molecular epidemiology survey is now warranted, especially following second massive immigration for local Panama Canal expansion activities.Understanding Mycobacterium tuberculosis biodiversity and transmission is significant for tuberculosis control. This short report aimed to determine the genetic diversity of M. tuberculosis isolates from an outpatient clinic in Panama City. A total of 62 M. tuberculosis isolates were genotyped by 12 loci mycobacterial interspersed repetitive units-variable number of tandem repeats (MIRU-VNTR) and Spoligotyping. Forty-five (72.6%) of the isolates showed unique MIRU-VNTR genotypes, and 13 (21%) of the isolates were grouped into four clusters. Four isolates showed polyclonal MIRU-VNTR genotypes. The MIRU-VNTR Hunter-Gaston discriminatory index reached 0.988. The Spoligotyping analysis revealed 16 M. tuberculosis families, including Latin American-Mediterranean, Harlem, and Beijing. These findings suggest a wide genetic diversity of M. tuberculosis isolates at one outpatient clinic. A detailed molecular epidemiology survey is now warranted, especially following second massive immigration for local Panama Canal expansion activities

Household stored water quality in an intermittent water supply network in Panama

Safe water storage is critical to preserve water quality, especially when intermittent piped drinking water supply creates a need for household storage. This study characterized household storage practices and stored water quality in 94 households (N = 94) among four peri-urban neighborhoods in Arraiján, Panama with varying degrees of supply intermittency. We found that 18 (19.1%) households stored drinking water in unsafe containers. Forty-four (47%) samples of household stored drinking water had residual chlorine levels 10 most probable number (MPN)/100 mL total coliform bacteria. Eight (44%) samples were positive for Escherichia coli, whereas only one (1.3%) sample from the safe containers was positive. Twenty-nine (30.9%) samples had >500 MPN/mL heterotrophic plate count bacteria. These findings suggest that longer supply interruptions were associated with longer storage times and lower chlorine residual, which were associated with higher concentrations of indicator bacteria. This is one of the first studies in the Central-American region to show an association between the lack of turnover (replacement with fresh water) and greater contamination during household water storage. Thus, when drinking water supply is not completely continuous and household storage is required, decreasing the time between supply periods can facilitate safer water storage. Public awareness and education are also recommended to increase hygiene practices during water collection and storage.Safe water storage is critical to preserve water quality, especially when intermittent piped drinking water supply creates a need for household storage. This study characterized household storage practices and stored water quality in 94 households (N = 94) among four peri-urban neighborhoods in Arraiján, Panama with varying degrees of supply intermittency. We found that 18 (19.1%) households stored drinking water in unsafe containers. Forty-four (47%) samples of household stored drinking water had residual chlorine levels 10 most probable number (MPN)/100 mL total coliform bacteria. Eight (44%) samples were positive for Escherichia coli, whereas only one (1.3%) sample from the safe containers was positive. Twenty-nine (30.9%) samples had >500 MPN/mL heterotrophic plate count bacteria. These findings suggest that longer supply interruptions were associated with longer storage times and lower chlorine residual, which were associated with higher concentrations of indicator bacteria. This is one of the first studies in the Central-American region to show an association between the lack of turnover (replacement with fresh water) and greater contamination during household water storage. Thus, when drinking water supply is not completely continuous and household storage is required, decreasing the time between supply periods can facilitate safer water storage. Public awareness and education are also recommended to increase hygiene practices during water collection and storage

Compositions and methods for detecting mycobacterium

The present disclosure provides methods of detecting mycobacterium in an individual, generally involving detecting antibody to a mycobacterial lipid in a biological sample obtained from the individual. The present disclosure further provides compositions and kits for carrying out the methodsThe present disclosure provides methods of detecting mycobacterium in an individual, generally involving detecting antibody to a mycobacterial lipid in a biological sample obtained from the individual. The present disclosure further provides compositions and kits for carrying out the method

High clustering rates of multidrug-resistant Mycobacterium tuberculosisgenotypes in Panama

Background: Tuberculosis continues to be one of the leading causes of death worldwide and in the American region. Although multidrug-resistant tuberculosis (MDR-TB) remains a threat to TB control in Panama, few studies have focused in typing MDR-TB strains. The aim of our study was to characterize MDR Mycobacterium tuberculosis clinical isolates using PCR-based genetic markers. Methods: From 2002 to 2004, a total of 231 Mycobacterium tuberculosis isolates from TB cases country-wide were screened for antibiotic resistance, and MDR-TB isolates were further genotyped by double repetitive element PCR (DRE-PCR), (GTG)5-PCR and spoligotyping. Results: A total of 37 isolates (0.85%) were resistant to both isoniazid (INH) and rifampicin (RIF). Among these 37 isolates, only two (5.4%) were resistant to all five drugs tested. Dual genotyping using DRE-PCR and (GTG)5-PCR of MDR Mycobacterium tuberculosis isolates revealed eight clusters comprising 82.9% of the MDR-TB strain collection, and six isolates (17.1%) showed unique fingerprints. The spoligotyping of MDR-TB clinical isolates identified 68% as members of the 42 (LAM9) family genotype.Background: Tuberculosis continues to be one of the leading causes of death worldwide and in the American region. Although multidrug-resistant tuberculosis (MDR-TB) remains a threat to TB control in Panama, few studies have focused in typing MDR-TB strains. The aim of our study was to characterize MDR Mycobacterium tuberculosis clinical isolates using PCR-based genetic markers. Methods: From 2002 to 2004, a total of 231 Mycobacterium tuberculosis isolates from TB cases country-wide were screened for antibiotic resistance, and MDR-TB isolates were further genotyped by double repetitive element PCR (DRE-PCR), (GTG)5-PCR and spoligotyping. Results: A total of 37 isolates (0.85%) were resistant to both isoniazid (INH) and rifampicin (RIF). Among these 37 isolates, only two (5.4%) were resistant to all five drugs tested. Dual genotyping using DRE-PCR and (GTG)5-PCR of MDR Mycobacterium tuberculosis isolates revealed eight clusters comprising 82.9% of the MDR-TB strain collection, and six isolates (17.1%) showed unique fingerprints. The spoligotyping of MDR-TB clinical isolates identified 68% as members of the 42 (LAM9) family genotype