Sulphadimidine Acetylation Test for Classification of Patients as Slow or Rapid Inactivators of Isoniazid

Sulphadimidine acetylation studies were undertaken in 103 patients, 52 of whom had been classified as slow and 51 as rapid inactivators of isoniazid by a standard microbiological assay method. Each patient received sulphadimidine by mouth in a dose of 44 mg./kg. body weight, and free and total sulphadimidine were estimated in blood and urine collected at six hours. The findings suggest that patients may be classified as slow inactivators of isoniazid if the proportion of acetylated sulphadimidine (total minus free) is (a) less than 25% in blood or (b) less than 70% in urine. The sulphadimidine test is easy to perform and the result is available the same day; urine specimens for the test can be stored at room temperature for over a week without any loss of drug.     

Specificity of the Isoniazid Drop Test for Control of Domiciliary Treatment of Tuberculosis

A method for determining N-acetyl isoniazid in urine was found to be specific in a double-blind experiment on 1673 urine specimens of which 328 were positive and 1345 were negative. The urine specimens were obtained from patients in a sanatorium ((a) Tuberculosis Division, (b) Mental Health Division) and a general hospital. The patients in the Tuberculosis Division were receiving isoniazid and other antituberculosis drugs alone or in combination. The patients in the Mental Health Division and in the general hospital were receiving a wide variety of other drugs. After a single dose of 300 mg. of isoniazid, N-acetyl isoniazid could be detected in urines of 36 patients for at least 12 hours. In three 24-hour urine specimens trace amounts could still be demonstrated.This test provides evidence that isoniazid is being ingested by home-treated tuberculous patients, and it can be performed by a doctor or nurse during routine visits to the home or clinic.     

The determination of isoniazid and its metabolites acetylisoniazid, monoacetylhydrazine, diacetylhydrazine, isonicotinic acid and isonicotinylglycine in serum and urine

1. A solvent system was devised for the extraction of isoniazid and its metabolites acetylisoniazid, monoacetylhydrazine, diacetylhydrazine, isonicotinic acid and isonicotinylglycine from serum and urine. 2. Specific chemical and fluorimetric methods were developed for the determination of the extracted isoniazid and acetylisoniazid, and chemical methods for the determination of monoacetylhydrazine, diacetylhydrazine, isonicotinic acid and isonicotinylglycine. 3. When applied to serum, these methods were capable of measuring concentrations of down to about 0.005mug of isoniazid/ml, 0.05mug of acetylisoniazid/ml, 0.2mug of monoacetylhydrazine/ml, 0.2mug of diacetylhydrazine/ml, 0.02mug of isonicotinic acid/ml and 0.1mug of isonicotinylglycine/ml. 4. In urine, these methods were capable of measuring concentrations of down to about 0.05mug of isoniazid/ml, 0.2mug of acetylisoniazid/ml, 1mug of diacetylhydrazine/ml, 0.1mug of isonicotinic acid/ml and 0.2mug of isonicotinylglycine/ml. 5. The stability of these compounds was studied in serum and urine and a method devised to decrease their decomposition in serum.     

Bacteriological conversion in twenty urinary tuberculosis patients treated with ofloxacin, rifampin and isoniazid: a 10-year follow-up study

Twenty patients with urinary tuberculosis were treated with ofloxacin (200 mg/day, 6 months), rifampin (600 mg/day, 3 months) and isoniazid (300 mg/day, 3 months) between 1989 and 1990. All patients were new cases, diagnosed by observation and/or isolation of Mycobacterium tuberculosis in one of the three morning urine samples. Bacteriological culture conversion (negativization) was assessed as a clinical guide of efficacy, comparing it, as the only parameter, against a control group (150 patients) with urinary tuberculosis who received conventional therapy. Bacteriological follow-up studies were performed in both groups monthly for 6 months, then again 6 months later and then every year for 10 years after completion of treatment. In the 20 patients, the initial culture was positive with over 100 colonies per culture (>50%); the smear was positive in 45% of the patients (most were 2+). All strains were susceptible to rifampin, isoniazid and ofloxacin. Two patients discontinued treatment. Beginning with the first month of treatment, the bacteriological conversion was 100%, 89.5% and 100% in the remaining controls. In the control group, which received conventional treatment, the conversion was: 90%, 87%, 93% and 100% in the remaining controls. Treatment with ofloxacin resulted in a bacteriological conversion similar to that following conventional treatment (p>0.05, Fisher's exact test). After 10 years of patient follow-up, we conclude that ofloxacin, in combination with rifampin and isoniazid (both for 3 months only is effective against M. tuberculosis, providing satisfactory bacteriological and clinical efficacy. Electronic Publication     

Indian time for nature? A multi-level approach to American Indian outdoor time in everyday life

Social scientists have utilized daily time use studies as one method of understanding everyday lives. The bulk of this research, usually quantitative, identifies broad racial, ethnic and gender differences. Yet, certain groups and questions are typically excluded in daily time use research. One such group is American Indians. To address this lacuna, we look at the deeply discussed view that American Indians are closer to nature than other US ethnic groups. We use a nationally representative sample of individual daily time use (American Time Use Survey; n?=?136,960) to look at leisure time outdoors. Our results show that American Indians report greater time spent outdoors but that this is only statistically significant for those who identify as exclusively American Indian (not for American Indians that are multi- and bi-racial). This study confirms previous qualitative research that suggests American Indians have a distinct relationship with nature.     

Antituberculous drug susceptibility testing of Mycobacterium bovis BCG strain Montréal

Testing of Mycobacterium bovis BCG strain Montréal for susceptibility to four primary antituberculous drugs (isoniazid, ethambutol, streptomycin, and rifampin) and to one secondary drug (p-aminosalicylic acid) showed the strain to be susceptible to all five substances. Mycobacterium bovis strains ATCC 35735, which is isoniazid sensitive, and ATCC 35747, which is isoniazid resistant, were included in the test; with the exception of their respective susceptibility to isoniazid, both were inhibited by the other four drugs.     

Metabolic Response to Oral Glucose in Healthy South African White,Indian, and African Subjects

The response of serum insulin, growth hormone, plasma free fatty acids, triglycerides, and blood glucose to an oral glucose load was investigated in healthy White, African, and Indian subjects. Serum cholesterol, uric acid, platelet adhesiveness, and urine insulin clearance were also measured. Each racial group responded differently. Most striking were the differences between Africans and Whites; despite similar mean blood glucose values at all times during the test, the Africans had lower serum insulin levels, a lower urine insulin clearance, a much greater rise of growth hormone, a more definite and prolonged suppression of free fatty acid release, lower serum cholesterol and uric acid levels, and a trend towards lower plasma triglyceride values.The Indians tended to resemble Whites rather than Africans with respect to their insulin, growth hormone, cholesterol, and triglyceride levels. Their glucose tolerance was decreased compared with that of the other two groups, but suppression of their free fatty acids was enhanced. Platelet adhesiveness was similar in all three groups.The reasons for these differences are unknown, but must be related to genetic and environmental differences among the three races.     

Polydactyly in the American Indian.

Polydactyly has an incidence in the American Indian twice that of Caucasians. A minimum estimate of this incidence is 2.40 per 1,000 live births. Preaxial type 1 has an incidence three to four times that reported for Caucasians or Negroes. The overall sex ratio in Indians is distorted with more males affected than females. The preaxial type 1 anomaly has a strong predilection for the hands and always is unilateral in contrast to postaxial type B where more than one-half are bilateral. The evidence to date, consisting of varying incidences of specific types of polydactyly among American whites, Negroes, and Indians in varying enviroments, suggests different gene-frequencies for polydactyly in each population. The incidence in Indians with 50% Caucasian admixture suggests that the factors controlling polydactyly are in large part genetically determined. Family studies and twin studies reported elsewhere offer no clear-cut genetic model which explains the highly variable gene frequencies.     

Mutagenicity induced by lyophilization or storage of urine from isoniazid-treated rats.

Urine collected during 24 h after treatment of rats with 90--550 mg/kg isonicotinic acid hydrazide (isoniazid, INH) was after lyophilization, mutagenic for Salmonella typhimurium TA1535. Urine collected directly from bladders of INH-treated rats was not mutagenic, and solutions of INH in water or urine became mutagenic only after lyophilization. In the absence of lyophilization, sterile urine from INH-treated rats became mutagenic after 8--14 days' storage at room temperature.     

A novel genotypic test for rapid detection of multidrug-resistant Mycobacterium tuberculosis isolates by a multiplex probe array

AIMS: To develop and evaluate a novel genotypic test for rapid detection of rifampicin and isoniazid resistance of multidrug-resistant (MDR) Mycobacterium tuberculosis isolates by a multiplex probe array. METHODS AND RESULTS: A multiplex probe array was designed for genotypic test to simultaneously screen the mutations of rpoB, katG, inhA and ahpC genes, associated with rifampin and isoniazid resistance in M. tuberculosis, with a probe detecting one of the recently confirmed genetic markers of isoniazid resistance ahpC-6 and -9 locus added. By using the genotypic test developed, 52 MDR isolates were identified, among which 46 isolates had mutations in rpoB (88.5%) and 45 at codon 315 of katG, regulatory region of inhA and oxyR-ahpC intergenic region (86.5%), whereas all 35 susceptible isolates identified showed a wild-type hybridization pattern. The sensitivity and specificity were 88.5% and 100% for rifampicin resistance, and 86.5% and 100% for isoniazid resistance, respectively. CONCLUSION: A rapid and simultaneous detection of rifampicin and isoniazid resistance caused by the mutations of rpoB, katG, inhA and ahpC genes in M. tuberculosis isolates could be achieved by a multiplex probe array developed. SIGNIFICANCE AND IMPACT OF THE STUDY: This genotypic test protocol has the potential to be developed on clinical application for the rapid detection of drug resistant M. tuberculosis isolates before an efficient chemotherapy is initiated.     

Standardized Treatment of Active Tuberculosis in Patients with Previous Treatment and/or with Mono-resistance to Isoniazid: A Systematic Review and Meta-analysis

Background

A standardized regimen recommended by the World Health Organization for retreatment of active tuberculosis (TB) is widely used, but treatment outcomes are suspected to be poor. We conducted a systematic review of published evidence of treatment of patients with a history of previous treatment or documented isoniazid mono-resistance.

Methods and Findings

PubMed, EMBASE, and the Cochrane Central database for clinical trials were searched for randomized trials in previously treated patients and/or those with with mono-resistance to isoniazid, published in English, French, or Spanish between 1965 and June 2008. The first two sources were also searched for cohort studies evaluating specifically the current retreatment regimen. In studies selected for inclusion, rifampin-containing regimens were used to treat patients with bacteriologically confirmed pulmonary TB, in whom bacteriologically confirmed failure and/or relapse had been reported. Pooled cumulative incidences and 95% CIs of treatment outcomes were computed with random effects meta-analyses and negative binomial regression. No randomized trials of the currently recommended retreatment regimen were identified. Only six cohort studies were identified, in which failure rates were 18%–44% in those with isoniazid resistance. In nine trials, using very different regimens in previously treated patients with mono-resistance to isoniazid, the combined failure and relapse rates ranged from 0% to over 75%. From pooled analysis of 33 trials in 1,907 patients with mono-resistance to isoniazid, lower failure, relapse, and acquired drug resistance rates were associated with longer duration of rifampin, use of streptomycin, daily therapy initially, and treatment with a greater number of effective drugs.

Conclusions

There are few published studies to support use of the current standardized retreatment regimen. Randomized trials of treatment of persons with isoniazid mono-resistance and/or a history of previous TB treatment are urgently needed. Please see later in the article for the Editors'' Summary     

Crystal structure of Mycobacterium tuberculosis catalase-peroxidase

The Mycobacterium tuberculosis catalase-peroxidase is a multifunctional heme-dependent enzyme that activates the core anti-tuberculosis drug isoniazid. Numerous studies have been undertaken to elucidate the enzyme-dependent mechanism of isoniazid activation, and it is well documented that mutations that reduce activity or inactivate the catalase-peroxidase lead to increased levels of isoniazid resistance in M. tuberculosis. Interpretation of the catalytic activities and the effects of mutations upon the action of the enzyme to date have been limited due to the lack of a three-dimensional structure for this enzyme. In order to provide a more accurate model of the three-dimensional structure of the M. tuberculosis catalase-peroxidase, we have crystallized the enzyme and now report its crystal structure refined to 2.4-A resolution. The structure reveals new information about dimer assembly and provides information about the location of residues that may play a role in catalysis including candidates for protein-based radical formation. Modeling and computational studies suggest that the binding site for isoniazid is located near the delta-meso heme edge rather than in a surface loop structure as currently proposed. The availability of a crystal structure for the M. tuberculosis catalase-peroxidase also permits structural and functional effects of mutations implicated in causing elevated levels of isoniazid resistance in clinical isolates to be interpreted with improved confidence.     

Utility of nitrate reductase assay for detection of multidrug-resistant Mycobacterium tuberculosis in a low resource setting

Introduction. The performance of a drug susceptibility test may change when moving from the research stage to implementation on a population level in actual public health practice. Objective. The performance of a rapid drug susceptibility test was described for detecting multidrug-resistant Mycobacterium tuberculosis when implemented in the routine workflow of a low-resource reference laboratory. Materials and methods. A prospective study was done comparing the performance of the nitrate reductase assay with the conventional proportion method for rifampicin and isoniazid on 364 isolates were obtained from multidrug-resistant tuberculosis risk patients referred from diffrent Colombian laboratories. Results. When compared with the proportion method, the nitrate reductase assay sensitivity was 86.8% and 84.9% for rifampicin and isoniazid, respectively, whereas nitrate reductase assay specificity was 100% for isoniazid and rifampicin. Nitrate reductase assay sensitivity was significantly higher when the age of isolate was less than 70 days. A sensitivity of 94.4% dropped to 78.1% for rifampicin resistance for fresh and old isolates, respectively (Fisher exact test, p=0.05). For isoniazid resistance using fresh and old isolates, 94.7% vs.74.3% sensitivities, were achieved (chi square test, p=0.03). The proportion of nitrate reductase assay ambiguous results was significantly higher in multidrug-resistant than in non-multidrug-resistant isolates (17.6% vs. 4.0%, chi square test, p<0.005). Conclusions. The nitrate reductase assay demonstrated provided reliable results for antibiotic resistance. However, using old cultures leds to a higher proportion of false sensitive results; furthermore, the nitrate reductase assay capability to detect multidrug-resistant tuberculosis decreased due to a higher proportion of non-interpretable results.     

Detection of mutations associated with multidrug-resistant Mycobacterium tuberculosis clinical isolates

Antimicrobial resistance was studied in 100 Mycobacterium tuberculosis strains selected randomly from sputum cultures of newly diagnosed tuberculosis patients. Resistance of the isolates to rifampicin, isoniazid, and ethambutol was tested by both drug susceptibility testing (DST) and allele-specific PCR (AS-PCR). A total of 19 (19%) isolates were found resistant to at least one of the antituberculosis drugs investigated by PCR compared with 14 (14%) resistant isolates detected by DST. Eleven mutations were detected by AS-PCR in the rpoB gene (codons 516, 526, and 531), associated with rifampicin resistance, a marker of multidrug-resistant tuberculosis (MDR-TB), 14 mutations in the katG gene codon 315 that confers resistance to isoniazid, and nine mutations in the embB gene codon 306 that confers resistance to ethambutol. Mutations in the six multidrug-resistant isolates were confirmed by DNA sequencing. Results were compared with phenotypic DST data. Nineteen different mutation types to at least one of the drugs were found; six isolates (6%) were classified as MDR-TB, defined as resistance to at least rifampicin and isoniazid. The rates of concordance of the PCR with the phenotypic susceptibility test were 71.4, 54.5, and 44.4 for isoniazid, rifampicin, and ethambutol, respectively. These results highlight the importance of molecular epidemiology studies of tuberculosis in understudied regions with a tuberculosis burden to uncover the true prevalence of the MDR-TB.     

Microalbuminuria in non-insulin-dependent diabetes: its prevalence in Indian compared with Europid patients

Non-insulin-dependent diabetes mellitus is strikingly common in British Indians, but their susceptibility to diabetic complications is unknown. The ratio of albumin to creatinine concentrations was measured in samples of the first urine voided in the morning in 154 Indian and 82 Europid patients with non-insulin-dependent diabetes and in a control group of 129 non-diabetic Indians. The ratio was significantly higher in the Indian patients than in the Europid patients and the Indian controls. There were no significant correlations between the logarithm of the albumin: creatinine ratio and age, known duration of diabetes, haemoglobin A₁ concentration, or body mass index within either diabetic group. Hypertension and raised albumin:creatinine ratio were significantly associated, and significant correlations were seen between the logarithm of the albumin:creatinine ratio and systolic and diastolic blood pressures in the Indian but not the Europid diabetics.Because of the high prevalence of diabetes at a relatively early age in Indians, nephropathy may emerge as an important clinical problem.     

Assessing Accuracy of Genotype Imputation in American Indians

Background

Genotype imputation is commonly used in genetic association studies to test untyped variants using information on linkage disequilibrium (LD) with typed markers. Imputing genotypes requires a suitable reference population in which the LD pattern is known, most often one selected from HapMap. However, some populations, such as American Indians, are not represented in HapMap. In the present study, we assessed accuracy of imputation using HapMap reference populations in a genome-wide association study in Pima Indians.

Results

Data from six randomly selected chromosomes were used. Genotypes in the study population were masked (either 1% or 20% of SNPs available for a given chromosome). The masked genotypes were then imputed using the software Markov Chain Haplotyping Algorithm. Using four HapMap reference populations, average genotype error rates ranged from 7.86% for Mexican Americans to 22.30% for Yoruba. In contrast, use of the original Pima Indian data as a reference resulted in an average error rate of 1.73%.

Conclusions

Our results suggest that the use of HapMap reference populations results in substantial inaccuracy in the imputation of genotypes in American Indians. A possible solution would be to densely genotype or sequence a reference American Indian population.     

In Vitro Chemotherapeutic Combinations Against Isoniazid-Resistant Mycobacterium tuberculosis and Mycobacterium fortuitum

It is an acceptable medical practice to use second-line antimycobacterial drugs in combination with isoniazid in treatment of isoniazid-resistant tuberculosis. Recent investigations have demonstrated the importance of determining chemotherapeutic interaction in instances of multiple antibiotic use. We studied the inhibitory effect of combinations of isoniazid with ethambutol, rifampin, ethionamide, cycloserine, viomycin, and kanamycin against three isoniazid-resistant strains of Mycobacterium tuberculosis and three strains of M. fortuitum. The isobologram technique with drug concentrations of 0.4 to 100 mug/ml was used. With the exception of single instances in which kanamycin plus isoniazid (M. tuberculosis strain 9999) and ethionamide plus isoniazid (M. fortuitum strain 2080) seemed to have a synergistic effect, neither synergy nor antagonism was noted for any of the combinations. These studies show that the combined use of isoniazid and a second line antimycobacterial agent results in vitro in indifferent inhibitory activity.     

Mechanism for the pyridoxal neutralization of isoniazid action of Mycobacterium tuberculosis

In Sauton's synthetic liquid medium, 10 mug of pyridoxal per ml completely protected Mycobacterium tuberculosis (H37R(a)) from the effects of a minimal inhibitory concentration of isoniazid (0.01 mug/ml). (14)C-labeled isoniazid was employed to study the nature of this protective effect. Uptake of the drug by cells in a Sauton environment containing 0.01 mug of (14)C-isoniazid per ml was inhibited 20 to 40% by 10 mug of pyridoxal per ml during the early hours of drug exposure. A stronger inhibition of uptake resulted when labeled isoniazid and pyridoxal were increased to 0.1 mug/ml and 50 to 100 mug/ml, respectively. Further studies revealed that certain Sauton nutrients are required to achieve this effect. When l-asparagine or salts (MgSO(4) and ferric ammonium citrate) or both were deleted from the menstruum, pyridoxal did not inhibit isoniazid incorporation by the tubercle bacilli. Pyridoxal also failed to inhibit uptake when (NH(4))(2)SO(4) was substituted for l-asparagine. Growth experiments in Sauton's medium modified to contain (NH(4))(2)SO(4) instead of l-asparagine were consistent with the latter finding. Pyridoxal did not prevent isoniazid growth inhibition in this medium. It is postulated that a large excess of pyridoxal in Sauton's medium protects tubercle bacilli from the effects of isoniazid through formation of an extracellular complex involving drug, vitamin, and certain medium constituents, thereby reducing the level of isoniazid available to the cells.     

Hydrogen cyanide and phenylthiocarbamide sensitivity, mid-phalangeal hair and color blindness in Yucatán, Mexico

Approximately 1450 persons, 800 of them unrelated, of both sexes from age eigth upward from the town of Ticul, Yucatán, Mexico, formed an American Indian (Maya) sample with apparently little Caucasian admixture for four genetic tests: ability to taste PTC and smell HCN, presence of mid-phalangeal hair, and color blindness. A modified PTC sorting test indicated a nontaster allele (t) frequency of 0.29 in Ticul, a relatively high result for American Indians but compatible with previously reported Mayan data. A color vision deficiency frequency of 3.6% was found in the total male sample, and a subsample of unrelated males had a rate of 2.8%. No color blind females were detected. The Ticuleños exhibited a high rate of midphalangeal hairlessness, characteristic of American Indians, with a notable sex differential: 75.9% for males, 87.1% for females. Previous studies of the inability to smell HCN, most of which suggest an X-linked recessive inheritance with an allele frequency of about 0.2, are reviewed. The Yucatán material, with a nonsmeller prevalence around 50% and no significant difference between the sexes, contradicts the X-linked recessive hypothesis on the basis of both population and family analyses. No simple Mendelian scheme appears to fit the Ticul HCN sensitivity test results.     

Genome-wide analysis in brazilian xavante indians reveals low degree of admixture

Characterization of population genetic variation and structure can be used as tools for research in human genetics and population isolates are of great interest. The aim of the present study was to characterize the genetic structure of Xavante Indians and compare it with other populations. The Xavante, an indigenous population living in Brazilian Central Plateau, is one of the largest native groups in Brazil. A subset of 53 unrelated subjects was selected from the initial sample of 300 Xavante Indians. Using 86,197 markers, Xavante were compared with all populations of HapMap Phase III and HGDP-CEPH projects and with a Southeast Brazilian population sample to establish its population structure. Principal Components Analysis showed that the Xavante Indians are concentrated in the Amerindian axis near other populations of known Amerindian ancestry such as Karitiana, Pima, Surui and Maya and a low degree of genetic admixture was observed. This is consistent with the historical records of bottlenecks experience and cultural isolation. By calculating pair-wise F(st) statistics we characterized the genetic differentiation between Xavante Indians and representative populations of the HapMap and from HGDP-CEPH project. We found that the genetic differentiation between Xavante Indians and populations of Ameridian, Asian, European, and African ancestry increased progressively. Our results indicate that the Xavante is a population that remained genetically isolated over the past decades and can offer advantages for genome-wide mapping studies of inherited disorders.