Eshetu Lemma

Detection and Molecular Characterization of 9000-Year-Old Mycobacterium tuberculosis from a Neolithic Settlement in the Eastern Mediterranean

Background Mycobacterium tuberculosis is the principal etiologic agent of human tuberculosis. It has no environmental reservoir and is believed to have co-evolved with its host over millennia. This is supported by skeletal evidence of the disease in early humans, and inferred from M. tuberculosis genomic analysis. Direct examination of ancient human remains for M. tuberculosis biomarkers should aid our understanding of the nature of prehistoric tuberculosis and the host/pathogen relationship. Methodology/Principal Findings We used conventional PCR to examine bone samples with typical tuberculosis lesions from a woman and infant, who were buried together in the now submerged site of Atlit-Yam in the Eastern Mediterranean, dating from 9250-8160 years ago. Rigorous precautions were taken to prevent contamination, and independent centers were used to confirm authenticity of findings. DNA from five M tuberculosis genetic loci was detected and had characteristics consistent with extant genetic lineages. High performance liquid chromatography was used as an independent method of verification and it directly detected mycolic acid lipid biomarkers, specific for the M. tuberculosis complex. Conclusions/Significance Human tuberculosis was confirmed by morphological and molecular methods in a population living in one of the first villages with evidence of agriculture and animal domestication. The widespread use of animals was not a source of infection but may have supported a denser human population that facilitated transmission of the tubercle bacillus. The similarity of the M. tuberculosis genetic signature with those of today gives support to the theory of a long-term co-existence of host and pathogen.

Intensified tuberculosis case finding among HIV-Infected persons from a voluntary counseling and testing center in Addis Ababa, Ethiopia.

Objective:To evaluate commonly available screening tests for pulmonary tuberculosis (TB), using sputum bacteriology as a gold standard, in HIV-infected persons attending an urban voluntary counseling and testing clinic in Addis Ababa, Ethiopia. Design:Prospective enrollment of HIV-infected persons, all of whom underwent TB screening, regardless of symptoms, with: (1) symptom screening and physical examination, (2) 3 sputum specimens for smear microscopy, and (3) chest radiograph. One sputum was also sent for concentrated smear microscopy and mycobacterial culture. Chest radiographs were reviewed by 2 independent radiologists. A confirmed TB diagnosis was defined as 1 positive sputum smear and/or 1 positive sputum culture. Results:We enrolled 438 HIV-infected persons: 265 (61%) females, median age 34 years (range: 18-65), median CD4 cell count 181 cells per cubic millimeter (range: 2-1185). Overall, 32 (7%) persons were diagnosed with TB, of whom 5 (16%) were asymptomatic but culture-confirmed TB cases. Screening for cough >2 weeks would have detected only 12 (38%) confirmed TB cases; screening for cough or fever, of any duration, would have detected 24 (75%) cases, with specificity of 64%. Negative predictive value of screening for these 2 symptoms was 97%. Simulation of the current Ethiopian national guidelines had a sensitivity of 63% and specificity of 83% for diagnosing TB disease among study patients. Conclusions:Traditional symptom screening is insufficient for detecting TB disease among HIV-infected persons but may serve to exclude TB disease. More sensitive, rapid, and low-cost diagnostic tests are needed to meet the demand of resource-limited settings.

Evaluation of Microscopic Observation Drug Susceptibility Assay for Detection of Multidrug-Resistant Mycobacterium tuberculosis

ABSTRACT Early detection of multidrug-resistant Mycobacterium tuberculosis (MDR-TB) is of primary importance for both patient management and infection control. Optimal methods for identifying drug-resistant Mycobacterium tuberculosis in a timely and affordable way in resource-limited settings are not yet available. This study prospectively evaluated a low-technology but rapid drug susceptibility testing method, the microscopic observation drug susceptibility assay (MODS), in the concurrent detection of M. tuberculosis and its susceptibilities to isoniazid and rifampin (two drugs defining multidrug-resistant M. tuberculosis) directly from sputum specimens. Sputum samples were collected from 262 smear-positive TB patients in Addis Ababa, Ethiopia. To undertake MODS, 100 μl of decontaminated samples was inoculated into a 24-well plate containing 1 ml of 7H9 broth with and without appropriate drugs. The assay uses an inverted-light microscope to detect characteristic mycobacterial growth in liquid culture. Of 262 smear-positive patients, MODS detected 254 (96.9%) and culture in Löwenstein-Jensen medium detected 247 (94.3%) (P = 0.016). For the 247 cultures, the sensitivity, specificity, and accuracy of MODS for detecting MDR-TB were 92.0, 99.5, and 98.8%, respectively, using the method of proportion as a reference (concordance, 98.8%; kappa value, 0.932). Results for MODS were obtained in a median time of 9 days. MODS is an optimal alternative method for identifying MDR-TB in a timely and affordable way in resource-limited settings.

The first population-based national tuberculosis prevalence survey in Ethiopia, 2010-2011

SETTING Tuberculosis (TB) remains a major public health problem in Ethiopia. OBJECTIVE To determine the prevalence of pulmonary TB among the general adult population aged ≥15 years in 2010-2011. METHOD A nationwide, cluster-sampled, stratified (urban/rural/pastoralist), cross-sectional survey was conducted in 85 selected clusters. All consenting participants were screened for TB using: 1) chest X-ray (CXR) and 2) an interview to screen for symptoms suggestive of TB disease. RESULT Of 51,667 eligible individuals, 46,697 (90%) participated in the survey and completed at least the screening interview. CXR was performed among 46,548 (99.7%) participants. A total of 6080 (13%) participants were eligible for sputum examination. From the survey, it was estimated that in the national adult population 1) the prevalence of smear-positive TB was 108/100,000 (95%CI 73-143), and 2) that of bacteriologically confirmed TB was 277/100,000 (95%CI 208-347). CONCLUSION We found that the TB burden was lower than previously thought, which may indicate better programme performance. However, a high proportion of TB among young persons suggests that TB is circulating in the community and that there is a need for more efforts to limit the spread of TB disease.

Sensitivity to Anti-tuberculosis Drugs in HIV-positive and -negative Patients in Addis Ababa

A cross-sectional survey was conducted between 1 August and 31 December, 1998 in Addis Ababa, Ethiopia to determine the rate of primary drug resistance to anti-tuberculosis drugs and to investigate its possible association with HIV infection. Sputum culture, sensitivity to first-line anti-tuberculosis drugs and HIV testing were done for 236 sputum smear-positive pulmonary tuberculosis patients. Primary drug resistance level (single or multidrug resistance) had not changed significantly since 1994. Primary resistance occurred more often among HIV-positive than among HIV-negative patients. The association between drug resistance and HIV will have a serious impact in the control of tuberculosis because in recent years the prevalence of HIV has increased dramatically in Ethiopia. Therefore, further studies on drug resistance and HIV infection and the establishment of drug resistance surveillance are recommended.A cross-sectional survey was conducted between 1 August and 31 December, 1998 in Addis Ababa, Ethiopia to determine the rate of primary drug resistance to anti-tuberculosis drugs and to investigate its possible association with HIV infection. Sputum culture, sensitivity to first-line anti-tuberculosis drugs and HIV testing were done for 236 sputum smear-positive pulmonary tuberculosis patients. Primary drug resistance level (single or multidrug resistance) had not changed significantly since 1994. Primary resistance occurred more often among HIV-positive than among HIV-negative patients. The association between drug resistance and HIV will have a serious impact in the control of tuberculosis because in recent years the prevalence of HIV has increased dramatically in Ethiopia. Therefore, further studies on drug resistance and HIV infection and the establishment of drug resistance surveillance are recommended.

Colorimetric Phage-Based Assay for Detection of Rifampin-Resistant Mycobacterium tuberculosis

ABSTRACT Tests based on bacteriophage replication enable rapid screening of Mycobacterium tuberculosis for drug resistance. We describe a novel broth-based colorimetric method for detecting phage replication. When clinical isolates were tested by this novel method, high concordance was observed with both the traditional phage assay and gene mutation analysis for detection of resistance to rifampin.

Attempts to revive Mycobacterium tuberculosis from 300-year-old human mummies

Environmental persistence of Mycobacterium tuberculosis is subject to speculation. However, the reality that infected postmortem tissues can be a danger to pathologists and embalmers has worrisome implications. A few experimental studies have demonstrated the organisms ability to withstand exposure to embalming fluid and formalin. Recently, a failure was reported in an attempt to resuscitate an original isolate of Robert Koch to determine the lifetime of the tubercle bacillus. The present study also considers a historical approach to determine persistence under favorable environmental conditions. It asks whether acid-fast forms observed in tissues of 300-year-old Hungarian mummies can be resuscitated. Finding organisms before the advent of antibiotics and pasteurization may yield valuable genetic information. Using various media modifications, as well as guinea pig inoculation, an attempt was made to culture these tissues for M. tuberculosis. In addition, a resuscitation-promoting factor, known to increase colony counts in high G+C bacteria, was applied to the cultures. Although an occasional PCR-positive sample was detected, no colonies of M. tuberculosis were obtained. Our results may indicate that the life span of the tubercle bacillus is less than a few hundred years, even though in the short run it can survive harsh chemical treatment.