Isoniazid-resistant Tuberculosis Research Articles

Colorimetric redox-indicator methods for the rapid detection of multidrug resistance in Mycobacterium tuberculosis: a systematic review and meta-analysis

With the spread of multidrug-resistant tuberculosis (MDR-TB) there is increasing demand for new accurate and cost-effective tools for rapid drug susceptibility testing (DST), particularly for developing countries. The reference standard method used today for DST is very slow and cumbersome. Colorimetric assays using redox indicators have been proposed to be used in low-resource countries as rapid alternative culture methods for the detection of resistance especially to rifampicin and isoniazid. These methods appear as promising new tools but their accuracy has not been systematically evaluated. We did a meta-analysis to evaluate the accuracy of the colorimetric assays for the detection of rifampicin and isoniazid-resistant tuberculosis among clinical isolates. We searched Medline, PubMed (NCBI), Global health-CAB, EJS-E (EbscoHost), ISI Web, Web of Science and IFCC databases and contacted authors if additional information was needed. Eighteen studies met our inclusion criteria for rifampicin resistance detection and 16 for isoniazid. We used a summary receiver operating characteristic (SROC) curve to perform meta-analysis and summarize diagnostic accuracy. For both drugs, all studies had a sensitivity and specificity that ranged between 89% and 100%. There is evidence that colorimetric methods are highly sensitive and specific for the rapid detection of MDR-TB. These new tools could offer affordable technologies for TB laboratories especially in places where resources are limited and where the prevalence of MDR-TB is important and make TB control efforts more effective. Additional studies are needed in high MDR prevalence countries and cost-effectiveness analysis to have more evidence on the utility of these methods. Future developments to detect resistance directly from smear-positive sputum specimens should be taken into consideration to speed up the process.

Cerebellar abscess and syringomyelia due to isoniazid-resistant Mycobacterium tuberculosis

A 19-year-old immunocompetent man was admitted to hospital with diplopia, nausea, vomiting and change in mental status. The patient had a history of tuberculous meningitis that was diagnosed at another hospital 6 months before the present admission, and at that time anti-tuberculosis treatment was initiated using a first-line drug combination. A computed tomography (CT) scan of the brain revealed non-communicating hydrocephalus. A ventriculo-peritoneal shunt was inserted surgically. Two months later, the patient was hospitalized again for fever, dysphagia and left hemiparesis. At that time, his cranial CT findings were within normal limits; however, magnetic resonance imaging (MRI) revealed an irregular multilocular peripheral contrast-enhancing lesion in the posterior fossa. The abscess was surgically drained. The presence of acid-fast bacilli in the abscess material was demonstrated by Ziehl-Neelsen staining. Mycobacterium tuberculosis grew on Lowenstein-Jensen culture medium, and the strain was found to be resistant to isoniazid. One month after the operation, the patient became quadriparetic. Cervical MRI revealed a cervico-thoracic syringomyelitic cavity, after which a syringoperitoneal shunt was placed. Treatment with four drugs was continued for 10 months, and then treatment with three drugs for a total period of 18 months. The patient recovered, with residual quadriparesis. Even though very rare, isoniazid-resistant M. tuberculosis may be the causative agent of progressive tuberculosis.

Public health impact of isoniazid-resistant Mycobacterium tuberculosis strains with a mutation at amino-acid position 315 of katG: a decade of experience in The Netherlands

A previous limited study demonstrated that Mycobacterium tuberculosis isolates with a mutation at amino-acid position 315 of katG (Delta315) exhibited high-level resistance to isoniazid and were more frequently resistant to streptomycin. In the present study, isoniazid-resistant M. tuberculosis isolates from 8,332 patients in The Netherlands (1993-2002) were screened for the Delta315 mutation. Isoniazid resistance was found in 592 (7%) isolates, of which 323 (55%) carried Delta315. IS6110 restriction fragment length polymorphism analysis showed that Delta315 isolates occurred in clusters, suggesting recent transmission, at the same frequency as isoniazid-susceptible isolates. In contrast, other isoniazid-resistant isolates clustered significantly less frequently. Delta315 isolates were high-level isoniazid-resistant, streptomycin-resistant and multidrug-resistant significantly more often, and may have a greater impact on public health, than other isoniazid-resistant isolates.

Impact of Bacterial Genetics on the Transmission of Isoniazid-Resistant Mycobacterium tuberculosis

Understanding the ecology of drug-resistant pathogens is essential for devising rational programs to preserve the effective lifespan of antimicrobial agents and to abrogate epidemics of drug-resistant organisms. Mathematical models predict that strain fitness is an important determinant of multidrug-resistant Mycobacterium tuberculosis transmission, but the effects of strain diversity have been largely overlooked. Here we compared the impact of resistance mutations on the transmission of isoniazid-resistant M. tuberculosis in San Francisco during a 9-y period. Strains with a KatG S315T or inhA promoter mutation were more likely to spread than strains with other mutations. The impact of these mutations on the transmission of isoniazid-resistant strains was comparable to the effect of other clinical determinants of transmission. Associations were apparent between specific drug resistance mutations and the main M. tuberculosis lineages. Our results show that in addition to host and environmental factors, strain genetic diversity can influence the transmission dynamics of drug-resistant bacteria.

Isoniazid preventive therapy and risk for resistant tuberculosis.

In the context of tuberculosis (TB) resurgence, isoniazid preventive therapy (IPT) is increasingly promoted, but concerns about the risk for development of isoniazid-resistant tuberculosis may hinder its widespread implementation. We conducted a systematic review of data published since 1951 to assess the effect of primary IPT on the risk for isoniazid-resistant TB. Different definitions of isoniazid resistance were used, which affected summary effect estimates; we report the most consistent results. When all 13 studies (N = 18,095 persons in isoniazid groups and N = 17,985 persons in control groups) were combined, the summary relative risk for resistance was 1.45 (95% confidence interval 0.85-2.47). Results were similar when studies of HIV-uninfected and HIV-infected persons were considered separately. Analyses were limited by small numbers and incomplete testing of isolates, but findings do not exclude an increased risk for isoniazid-resistant TB after IPT. The diagnosis of active TB should be excluded before IPT. Continued surveillance for isoniazid resistance is essential.

Molecular Dynamics Simulation Studies of the Wild-Type, I21V, and I16T Mutants of Isoniazid-Resistant Mycobacterium tuberculosis Enoyl Reductase (InhA) in Complex with NADH: Toward the Understanding of NADH-InhA Different Affinities

The increasing prevalence of tuberculosis in many areas of the world, associated with the rise in drug-resistant Mycobacterium tuberculosis (MTB) strains, presents a major threat to global health. InhA, the enoyl-ACP reductase from MTB, catalyzes the nicotinamide adenine dinucleotide (NADH)-dependent reduction of long-chain trans-2-enoyl-ACP fatty acids, an intermediate in mycolic acid biosynthesis. Mutations in the structural gene for InhA are associated with isoniazid resistance in vivo due to a reduced affinity for NADH, suggesting that the mechanism of drug resistance may be related to specific interactions between enzyme and cofactor within the NADH binding site. To compare the molecular events underlying ligand affinity in the wild-type, I21V, and I16T mutant enzymes and to identify the molecular aspects related to resistance, molecular dynamics simulations of fully solvated NADH-InhA (wild-type and mutants) were performed. Although very flexible, in the wild-type InhA-NADH complex, the NADH molecule keeps its extended conformation firmly bound to the enzyme's binding site. In the mutant complexes, the NADH pyrophosphate moiety undergoes considerable conformational changes, reducing its interactions with its binding site and probably indicating the initial phase of ligand expulsion from the cavity. This study should contribute to our understanding of specific molecular mechanisms of drug resistance, which is central to the design of more potent antimycobacterial agents for controlling tuberculosis.

An action plan for tuberculosis in England

Since the mid-1980s the world has seen a steady increase in tuberculosis of about 1% a year, but in the UK the increase has been nearer 2% a year.1,2 In the Americas and most of Western (though not Eastern) Europe, tuberculosis case rates are falling; sub-Saharan Africa is seeing huge rises, because of the immunocompromising effects of HIV. In the UK the increase in cases has effectively been confined to London, where a doubling of numbers in the past decade is attributable to immigration from countries with a high prevalence. Particularly affected is the London Borough of Newham, where the incidence now exceeds 80/100 000 per year. Poor housing and a deficient infrastructure make matters worse. In October 2004, the Department of Health launched a report with the bold title Stopping Tuberculosis in England.3 The Chief Medical Officer's action plan centres on ten broad principles—increased awareness, strong commitment and leadership, a high quality of service, excellence of clinical care, well organized and well coordinated patient services, first-class laboratory services, highly effective disease control at population level, an expert workforce, leading edge research, and international partnership. Nobody could quarrel with these, but how is this plan to be implemented? On 9 March 2005 the RSM held a meeting to debate the action plan and some key points are recorded here. As usual, resources loomed large. The Government's report admiringly uses the word 'can-do' to describe the strategy adopted in New York in the early 1990s, when an increase in tuberculosis similar to that in London was successfully reversed. What it omits to mention is the considerable expense of the New York programme; each case of tuberculosis prevented cost around $10 000.4 One plus-point for the UK, in dealing with the epidemic, is the high quality of the Mycobacterial Laboratory Reference system. New molecular techniques (such as the polymerase chain reaction and gene probe identification) have brought the times for identification of species and drug resistance down to a few days. Unfortunately, there has been little improvement in sensitivity; so we still find ourselves treating 20–40% of patients empirically, without confirmation of the diagnosis. One very promising development for early diagnosis is a blood test for T cells specific for antigens of Mycobacterium tuberculosis. This is more accurate than the tuberculin skin test, now a hundred years old, and should be particularly useful for detection of latent infection.5 On 'excellence of clinical care', a difficulty in England is the patchiness of tuberculosis, so that some clinicians gain much experience and others very little. Practice and control have until now been overseen by the British Thoracic Society's Tuberculosis Committee, with occasional pronouncements on best practice.6,7 From next February, everyone who manages tuberculosis will need to consult guidelines from the National Institute for Clinical Excellence (NICE). Meanwhile, some promising new treatment agents are entering phase 2 trials;8 and, on the vaccine front, the leading contender is a vaccinia-based mycobacterial antigen that boosts immunity to BCG.9 Two important areas of controversy were highlighted at the RSM meeting. Contrary to popular opinion (and to much political opinion), no good argument can be made for routine screening of new immigrants for active disease. Where screening is in operation, it identifies only one person with active tuberculosis for every thousand immigrants examined, and few of these individuals are infectious. Most immigrants who present with tuberculosis acquire their symptoms and signs between one and five years after arrival, so the disease is unlikely to be picked up on entry. Moreover, the numbers of specialist nurses, whose principal role is to provide support for patients taking medication and identifying contacts for tracing, are insufficient to provide a comprehensive immigrant screening service. There is a better case for screening of young immigrants who might benefit from preventive therapy. Also, some uninfected children should receive BCG. This is not true of UK children in general: the rates of tuberculosis in white teenagers and young adults are now so low that about 4500 routine vaccinations at the age of thirteen are being given to prevent a single case; the existing vaccination programme is far from being cost-effective.10 No new monies, we are told, are to be made available for implementing the Government's action plan, so can the old monies be applied more effectively? A recent outbreak of isoniazid-resistant tuberculosis in North London might have been curtailed if resources had been better targeted.11,12 Discontinuation of the school BCG programme could release funds for employment of more specialist nurses to provide patient support and directly observed therapy. Screening of immigrants, where necessary, might best be done in primary care. Routine screening of symptom-free adult immigrants could be suspended, with redirection of resources to prevention of disease in the young.

Genotypic Diversity among Isoniazid-Resistant Isolates of Mycobacterium tuberculosis from Rashid Hospital in Dubai, United Arab Emirates

Objective: To perform molecular fingerprinting for strain relatedness among isoniazid-resistant Mycobacterium tuberculosis isolates recovered from tuberculosis (TB) patients in the United Arab Emirates (UAE). Materials and Methods: Thirty-two consecutive isoniazid-resistant M. tuberculosis strains isolated from TB patients (4 natives and 28 expatriates) at Rashid Hospital, Dubai, UAE were analyzed. The typing was carried out by touchdown double-repetitive-element PCR (DRE-PCR). The status of R463 or L463 polymorphism and the presence of S315T mutation in the katG gene were also determined for isolates exhibiting cluster pattern in DRE-PCR. Results: All the 32 isolates (28 from expatriate patients and 4 from UAE nationals) exhibited 21 distinct patterns in DRE-PCR with 20 of 32 isolates exhibiting unique patterns and the remaining 12 exhibiting cluster ‘A’ pattern. All the isolates (19 of 19) yielding two or more DNA fragments in DRE-PCR were unique strains. The genotypic heterogeneity among 10 of the 12 cluster isolates was suggested by the varying susceptibility of these isolates to anti-TB drugs, presence of R463 or L463 polymorphism in the katG gene and the presence or absence of S315T mutation in the katG gene. Conclusion: The genotypic diversity among isoniazid-resistant M. tuberculosis isolates mostly recovered from expatriate patients indicates that most expatriates were infected with a unique strain imported, most likely, from their country of origin and that their latent infection was reactivated in UAE.

Molecular characterization of isoniazid-resistant Mycobacterium tuberculosis clinical strains isolated in the Philippines

The prevalence of mutations in the katG, inhA and oxyR- ahpC genes of isoniazid (INH)-resistant Mycobacterium tuberculosis isolates in the Philippines were determined. Of 306 M. tuberculosis isolates studied, 81 (26.5%) exhibited INH-resistance. Forty-four strains (54.3%) had mutations in the katG gene, eighteen strains (22.2%) had mutations in the putative inhA locus region, seven had mutations in both regions and five strains had mutations in the oxyR- ahpC operon. Only seven strains had no mutations. A total of 71 of the 81 (87.6%) resistant strains and 65 of the 72 (90.3%) INH sensitive randomly selected strains showed amino acid substitution in codon 463 (Arg to Leu) (88.9%). This fact supports the hypothesis that mutations at codon 463 are independent of INH-resistance and are linked to the geographical origins of the strains.

Molecular characterization of isoniazid-resistant Mycobacterium tuberculosis clinical strains isolated in the Philippines

The prevalence of mutations in the kat G, inh A and oxy R- ahp C genes of isoniazid (INH)-resistant Mycobacterium tuberculosis isolates in the Philippines were determined. Of 306 M. tuberculosis isolates studied, 81 (26.5%) exhibited INH-resistance. Forty-four strains (54.3%) had mutations in the kat G gene, eighteen strains (22.2%) had mutations in the putative inh A locus region, seven had mutations in both regions and five strains had mutations in the oxy R- ahp C operon. Only seven strains had no mutations. A total of 71 of the 81 (87.6%) resistant strains and 65 of the 72 (90.3%) INH sensitive randomly selected strains showed amino acid substitution in codon 463 (Arg to Leu) (88.9%). This fact supports the hypothesis that mutations at codon 463 are independent of INH-resistance and are linked to the geographical origins of the strains.

Poncet's disease—a novel cause of non-compliance with anti-tuberculous drugs

We present a patient with isoniazid resistant pulmonary tuberculosis (Tb) associated with large joint polyarthropathy. A diagnosis of Poncet’s disease (PD) was made. This case highlights the problems of non-compliance with drug therapy, directly observed therapy (DOT) and difficulty in diagnosing PD. The mechanisms for PD are discussed and the links between mycobacterial infection and rheumatoid arthritis (Rh.A) are reviewed.

Contribution of AGC to ACC and other mutations at codon 315 of the katG gene in isoniazid-resistant Mycobacterium tuberculosis isolates from the Middle East

The presence of ACC and other mutations at codon 315 in the katG gene was detected by PCR amplification followed by restriction fragment length polymorphism (PCR-RFLP) generated with restriction enzymes Msp I and MspA1 I in 37 isoniazid-resistant and 22-susceptible Mycobacterium tuberculosis isolates from Kuwait obtained in 2001. The mutation AGC to ACC was detected in 22 (60%) isolates while any mutation at codon 315 of the katG gene was present in 24 (65%) of 37 isoniazid-resistant isolates. The typing studies showed that majority of the isolates carrying mutations at codon 315 exhibited unique DNA banding patterns. The results were extended by additional analysis of 67, 28 and 17 isoniazid-resistant and 18, seven and six-susceptible M. tuberculosis isolates from Kuwait, Dubai and Beirut, respectively, that were analyzed previously for ACC mutation alone. These studies showed that one of 21, one of 10 and two of 11 isolates (all recovered from patients of Middle Eastern origin) with no AGC to ACC mutation from Kuwait, Dubai and Beirut, respectively, contained other mutations at codon 315 of the katG gene. None of the susceptible strains contained any mutation at codon 315. The PCR-RFLP with MspA1 I that detects all mutations at codon 315, compared with Msp I that detects only ACC mutation, identified more isoniazid-resistant strains with mutations at codon 315 in the katG gene. The data also showed that mutations other than AGC to ACC at codon 315 in the katG gene occur frequently in M. tuberculosis isolates recovered from Middle Eastern patients and should be incorporated in a rapid screen for the detection of mutations for isoniazid-resistance in the katG gene from this ethnic group.

Detection of katG Ser315Thr substitution in respiratory specimens from patients with isoniazid-resistant Mycobacterium tuberculosis using PCR-RFLP.

Mutations in the katG locus of catalase peroxidase in Mycobacterium tuberculosis (MTB) account for major isoniazid (INH) resistance. In the South China region, a collection of 906 respiratory specimens and 142 MTB isolates was used to evaluate the sensitivity and specificity of a PCR-RFLP method for the detection of INH resistance-associated mutations. Except for four catalase-negative MTB isolates, katG PCR for a 620-bp amplicon was successful for all purified MTB isolates. For respiratory specimens, diagnostic sensitivity and specificity of katG PCR was 85 and 100 %. Subsequent RFLP of the katG amplicons by MspI digestion identified that 51 % of INH-resistant MTB were associated with the Thr315 phenotype, and that codon 463 was a polymorphic site with no linkage to INH resistance. The Arg463 wild-type MTB isolates predominant in the Western world were replaced by isolates carrying Leu463 in the South China region. RFLP patterns of katG amplicons from respiratory specimens were identical to those of the corresponding MTB cultured colonies. This method has potential application for rapid diagnosis of INH resistance due to katG Ser315Thr mutation.

Frequency of mutations in rpoB and codons 315 and 463 of katG in rifampin- and/or isoniazid-resistant Mycobacterium tuberculosis isolates from northeast Mexico.

A total of 48 isoniazid (INH)- and rifampin (RIF)-resistant Mycobacterium tuberculosis isolates, 19 INH-resistant isolates, and 9 RIF-resistant isolates were randomly selected and tested for detecting mutations at codons 315 and 463 of katG by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP), and/or for detecting mutations at a 69-bp region of the rpoB gene by the INNO-LiPA Rif TB assay. Of the 67 INH-resistant isolates tested, 36 (53.7%) showed the mutation at codon 315 of katG; however, none of them showed the mutation at codon 463. The majority of the RIF-resistant samples analyzed (49 of 57, 86.0%) reacted positive with one of the four R-type probes. The R5-pattern (S531L mutation) was the most frequently observed (31 of 57, 54.4%), followed by R4a-pattern (H526Y mutation) 13 isolates (22.8%), R4b-pattern (H526D mutation) 4 isolates (7.0%), and R2-pattern (D516V mutation) 1 isolate (1.8%). Overall, there was agreement between the line probe kit and phenotypic RIF-susceptibility test for 56 (98.2%) of 57 RIF-resistant isolates tested. These results show that the mutation analysis at codon 315 of katG could be used as a screening assay prior to standard susceptibility testing, whereas mutations in the rpoB gene could be used successfully as genetic markers to rapidly detect RIF-resistant M. tuberculosis clinical isolates from northeast Mexico.

Treatment of Isoniazid Resistant Mycobacterium Tuberculosis(INHr-TB), a Retrospective Study of Biweekly Versus Daily Therap

PURPOSE: To compare isoniazid resistant mycobacterium tuberculosis (INHr-TB) treatment outcomes using a biweekly versus daily therapy.METHOD: We retrospectively reviewed all cases of INH resistant and INH-SM resistant TB at a county health department from Dec 1991 to Jan 2003. We included cases with 6 or more months of drug therapy consisting primarily of rifampin(R), pyrazinamide(Z), and ethambutol(E) and active follow up after completion of treatment for 6 months or greater. Fisher exact tests and unpaired t tests were used to assess for statistically significant differences between the biweekly and daily therapy cohorts.

Molecular fingerprinting of isoniazid-resistant Mycobacterium tuberculosis isolates from chest diseases hospital in Kuwait.

Touchdown double-repetitive-element-PCR (DRE-PCR) was carried out for typing 38 consecutive isoniazid-resistant Mycobacterium tuberculosis strains isolated at Chest Diseases Hospital, Kuwait, during 1998-2000. The polymorphism at codon 463 in the katG gene was also determined and correlated with genotypic relationships among the isolates. The isolates exhibited 21 distinct patterns in DRE-PCR. Nearly half of the isolates (18 of 38) exhibited unique patterns. Majority of isolates (16 of 20) yielding multiple DNA fragments in DRE-PCR were unique strains while most of the isolates (16 of 18) yielding a single DNA fragment in DRE-PCR clustered together. The prevalence of L463 in the katG gene was much higher in isolates from Middle Eastern (mostly Kuwaiti) patients than is reported for this ethnic group. The data indicate the possibility of some strains of South Asian/Southeast Asian origin spreading among local populations.

Detection of isoniazid-resistant Mycobacterium tuberculosis strains by a multiplex allele-specific PCR assay targeting katG codon 315 variation.

We describe a simple multiplex allele-specific (MAS)-PCR assay to detect mutations in the second base of the katG gene codon 315, including AGC-->ACC and ACA (Ser-->Thr) substitutions that confer resistance to isoniazid (INH) in Mycobacterium tuberculosis clinical isolates. The 315 ACC allele is found in the majority of Inh(r) strains worldwide, especially in areas with a high incidence of tuberculosis. The 315 ACA allele is characteristic of the New York City multidrug-resistant (MDR) strain W and its progenies in the United States. The mutations in katG315 are revealed depending on the presence or absence of an indicative fragment amplified from the wild-type allele of this codon. Initially optimized on the purified DNA samples, the assay was then tested on crude cell lysates and auramine-stained sputum slide preparations with the same reproducibility and interpretability of profiles generated by agarose gel electrophoresis. The MAS-PCR assay can be used for the detection of resistance to INH in clinical laboratories in regions with a high prevalence of MDR M. tuberculosis strains.

Rapid screening of katG gene mutation in isoniazid-resistant Mycobacterium tuberculosis

OBJECTIVE: To evaluate the relationship between katG gene mutation and isoniazid (INH) resistance and to develop a rapid screening method of point mutation in the katG gene associated with MTB resistance. METHODS: Twenty-four clinical isolates of MTB with 8 INH resigtance isolates and 16 INH-sensitive isolates were analyzed by PCR-RFLP, with the H(37)Rv reference strain as the control. RESULTS: G-->C point mutations were detected in 7 of 8 isoniazid-resistant strains and no gene mutation was shown in 16 isoniazid-sensitive isolates. The sensitivity and specificity were 87.5 % and 100 % respectively. No katG gene sequence deletion was observed in any specimen. CONCLUSION: Our results suggest katG gene mutation is one of the most important mechanisms of INH-resistant TB. PCR-RFLP may be useful in detection of katG gene mutation.

A continuing outbreak of isoniazid resistant tuberculosis in North London

A continuing outbreak of isoniazid resistant tuberculosis in North London

Nightclubs said to be cause of serious TB outbreak

Last month the Daily Mail reported that young people in crowded nightclubs were spreading tuberculosis (TB) and causing a serious “outbreak.” The London Evening Standard also stated that, according to experts, “the hot, sweaty conditions of nightclubs and busy bars are ideal places for bacteria to breed. The disease can be spread by the saliva of a TB sufferer, either through kissing or sharing a drink.” A spokesperson from the Department of Health confirmed that around 60 cases of the isoniazid-resistant TB had been seen over the past six years in Greater London. However, …