Rapid molecular detection of tuberculosis and rifampicin drug resistance: retrospective analysis of a national U.K. molecular service over the last decade.

BACKGROUND: Fast and reliable detection of Mycobacterium tuberculosis complex (MTBC) and drug resistance is crucial in establishing effective treatment and enforcing timely public health measures. METHODS: The authors analysed the performance of a national U.K. molecular diagnostic service over a decade, based on the use of a line probe assay (Innolipa, LiPA) compared with conventional liquid and solid cultures with rapid molecular identification and culture-based drug resistance testing. FINDINGS: Data were available for 7836 consecutive patient samples using LiPA and the reference microbiological technique (conventional liquid and solid cultures with rapid molecular identification and culture-based drug resistance testing). For all sputum specimens (n=3382) the sensitivity, specificity, positive predictive value, negative predictive value and accuracy for MTBC detection were 93.4%, 85.6%, 92.7%, 86.9% and 90.7%; the equivalent values for smear-positive sputum specimens (n=2606) were 94.7%, 80.9%, 93.9%, 83.3% and 91.3%. The sensitivity, specificity, positive predictive value, negative predictive value and accuracy for detection of rifampicin resistance in all sputum samples (n=1667) were 92.1%, 99.3%, 89.4%, 99.5% and 98.9%, respectively; the equivalent values for smear-positive sputum specimens (n=1477) were 93.3%, 99.3%, 87.5%, 99.6% and 99%. Between January 2006 and December 2008, LiPA saved 25.3 and 32.2 days for TB diagnosis and rifampicin resistance of smear-positive samples, respectively. INTERPRETATION: A molecular diagnostic service, using a non-automated line probe assay approach, provides a rapid and reliable national service for diagnosing MTBC and rifampicin resistance.

Human Mycobacterium bovis infections in London and Southeast England.

Variable-number tandem repeat (VNTR) and spoligotyping analyses were used to assess transmission of Mycobacterium bovis between humans. VNTR was more discriminatory than spoligotyping. Low case numbers, despite a substantial animal reservoir, and resolution of all isolates provided no evidence of recent human-to-human transmission or recent significant infection from animals.

The added value of a European Union tuberculosis reference laboratory network--analysis of the national reference laboratory activities.

National reference laboratories (NRL) and other laboratories are the cornerstones of well-functioning tuberculosis programmes and surveillance activities. However, the scope and activity of NRL services for mycobacterial identification and drug susceptibility testing (DST) has not been examined in detail across the European Union (EU), nor has the added value of cooperation and networking at the European level been explored with regard to strengthening laboratory services. Therefore, the European Centre for Disease Prevention and Control (ECDC) has commissioned a survey to explore these issues and to identify areas of work that could bring added value by supporting networking activities of tuberculosis (TB) reference laboratories in the EU. Structured questionnaires were sent to TB reference laboratory experts in the EU and European Economic Area (EEA) countries, and in three additional countries selected on the basis of their networking activities with EU projects and other initiatives (Switzerland, Croatia and Israel). The compiled results describe the activities and structure of 32 NRLs (29 countries replied, a response rate of 91%). The analysis of the survey led to the following recommendations for strengthening TB laboratory services: (1) implementing of the published European standards for TB laboratory services with respect to infrastructure, national reference functions, biosafety, human resources, quality assurance, operational research (including evaluation of new medical diagnostics), accuracy and speed, appropriately trained staff; (2) ensuring that laboratories only perform activities for which they have demonstrated proficiency; (3) implement validated and standardised second-line drug susceptibility testing (DST), including drugs used to define extensively drug-resistant tuberculosis (XDR TB); (4) aiming to identify Mycobacterium tuberculosis complex (MTBC) and rifampicin (RIF) resistance in over 90% of cultures and cases from smear-positive sputum directly within one to two working days. To realise some of the above recommendations and to strengthen links of TB surveillance and microbiology activities in the EU, a list of suggested generic areas of activities for an EU network of reference laboratories is presented. Such a network would build on and link to existing networks and initiatives at the European and global level.

Molecular epidemiology and prevalence of mutations conferring rifampicin and isoniazid resistance in Mycobacterium tuberculosis strains from the southern Ukraine.

Understanding the molecular epidemiology of tuberculosis (TB) and mutations in genes associated with drug resistance may contribute to the development of appropriate interventions to improve tuberculosis control. A structured questionnaire was used to collect basic epidemiological data from 589 patients with radiologically confirmed TB in the Odessa and Nikolaev regions of the Ukraine in 2003-2004. A non-commercial reverse hybridisation assay and DNA sequencing were used to detect mutations associated with rifampicin and isoniazid resistance. Genotyping was performed using multilocus variable number tandem repeat (VNTR) typing and spoligotyping. Mutations conferring rifampicin and isoniazid resistance were detected in 32.9% and 44.0%, respectively, of 225 Mycobacterium tuberculosis isolates from individual consecutive patients. Mutations in codon 531 and codon 315 of the rpoB and katG genes, respectively, were predominant among drug-resistant isolates. Multidrug (MDR) resistance rates were significantly higher among former prison inmates compared with non-prisoners (54.8% vs. 27.3%; RR 2.01; 95% CI 1.35-2.97) and the prevalence of mutations was higher in Beijing strains sharing the VNTR signature 223325173533424 than in other Beijing strains (71.4% vs. 45.7%; RR 1.74; 95% CI 1.17-2.57), suggesting that this group may be responsible for rapid transmission of MDR TB in the southern Ukraine.

The use of macroarrays for the identification of MDR Mycobacterium tuberculosis.

The emergence of Mycobacterium tuberculosis (Mtb), resistant to both isoniazid (INH) and rifampicin (RIF) (MDR-TB), is an increasing threat to tuberculosis control programs. Susceptibility testing of Mtb complex isolates by phenotypic methods requires a minimum of 14 days from a primary specimen. This can be reduced significantly if molecular analysis is used. Low density oligonucleotide arrays (macroarrays) have been used successfully for the detection of RIF resistance in Mtb. We describe the use of macroarray technology to identify Mtb complex isolates resistant to INH and/or RIF. The macroarray MDR-Mtb screen has been designed to detect mutations in the RIF resistance determining region (RRDR) of Mtb rpoB and loci in katG and mabA-inhA associated with INH resistance. A panel of Mtb isolates containing 38 different RRDR genotypes, 4 different genotypes within codon 315 of katG and 2 genotypes at mabA-inhA was used to validate the macroarray. The wild type (WT) genotype was correctly identified at all three loci. Of the 37 mutant rpoB genotypes, 36 were correctly detected; the single mutant not detected contained a 9 base insertion. All mutations within katG and mabA-inhA were correctly identified. We conclude that this low cost, rapid system can usefully detect the mutations associated with the vast majority of MDR-Mtb.

[Molecular typing of the Mycobacterium tuberculosis strains circulating in central Russia: efficiency of spoligotyping and VNTR-MIRU].

Current problems of molecular epidemiology of the Mycobacterium tuberculosis strains circulating in Samara Region, Russia are discussed. A total of 190 isolates of Mycobacterium tuberculosis were typed using two PCR-based molecular methods. The cultures were isolated from civil and prison patients with pulmonary tuberculosis recruited from different tuberculosis institutions across the Samara region. The usefulness of spoligotyping and 15-locii VNTR-MIRU was assessed for genotyping of Mycobacterium in population with high prevalence of Beijing strains (67.9%) using statistical analyses that included calculation of Hunter-Gaston index. The VNTR-MIRU method was demonstrated to be more efficient and was characterized by higher discrimination (index 0.747) compare to spoligotyping (index 0.572). VNTR-MIRU loci 10, 26, 31, 39, 40 and ETR-A were mostly polymorphic and therefore recommended for use in screening. It could be performed by manual electrophoresid, provided that automated sequencing is not available.

[Mutations linked with antimicrobial resistance in Mycobacterium tuberculosis isolated from tuberculosis patients in the Samara region].

A total of 234 M. tuberculosis isolates were used to demonstrate the leading role of mutations in, respectively, codon 531 of gene rpoB (90.0%) and codon 315 of gene katG (92.9%), in the development of resistance to rifampicin and isoniazid by the methods of reverse hybridization with oligonucleotide probes and the sequencing of gene stretches. The levels of primary resistance of M. tuberculosis to rifampicin, isoniazid and multiresistance, according to the molecular-genetic analysis, were 41.0%, 57.7% and 37.2% respectively. The coincidence of the results of the bacteriological and molecular-genetic analyses of the antimicrobial resistance of the isolates was 90.4% and 95.3% for isoniazid and rifampicin respectively. The prevalence of individual types of mutations, linked with antimicrobial resistance, in the presence of a considerable spread of strains of the family Beijing in the region may be indicative of the limited number of M. tuberculosis clones circulating in the region.

Modern laboratory diagnosis of tuberculosis.

One-third of the global population is believed to be infected with bacteria of the Mycobacterium tuberculosis complex, the causative agent of tuberculosis. More than 8 million new cases of tuberculosis occur annually leading to 2 million deaths. Mortality is particularly high in those coinfected with HIV and where the bacteria are multiple-drug-resistant strains--ie, strains resistant to at least isoniazid and rifampicin. Early diagnosis of tuberculosis and drug resistance improves survival and by identifying infectious cases promotes contact tracing, implementation of institutional cross-infection procedures, and other public-health actions. This review addresses significant advances made in the diagnosis of infection, clinical disease, and drug resistance over the past decade. It proposes operational criteria for a modern diagnostic service in the UK (as a model of a low-incidence country) and explores some of the economic issues surrounding the use of these techniques.

Modern laboratory diagnosis of mycobacterial infections.

This review summarises recent advances made in microscopic techniques (fluorescence and peptide nucleic acids) and culture techniques (solid, liquid, radiometric, and non-radiometric systems) and in the development of rapid methods for the identification of mycobacterial cultures (high performance liquid chromatography, thin layer chromatography, RNA sequencing, and polymerase chain reaction restriction enzyme assays). The role of molecular amplification systems in identifying Mycobacterium tuberculosis is described. Most methods record high specificity and sensitivity for smear positive sputum but have variable sensitivity for sputum smear negative and extrapulmonary specimens. Specimen quality will affect the performance of these assays and organisational delays, such as the batching of specimens, can reduce the time saved. In house assays can be as effective as commercial systems as long as appropriate controls are used.

A national audit of the laboratory diagnosis of tuberculosis and other mycobacterial diseases within the United Kingdom.

In order to audit United Kingdom laboratory diagnostic and reference services including novel molecular methods for tuberculosis, a questionnaire was sent to laboratories submitting specimens to the PHLS Mycobacterium Reference Unit (MRU) and regional centres and to the Scottish Mycobacteria Reference Laboratory (SMRL) in 1996-7. Nationally, 67.2% of laboratories responded. Most UK laboratories were fully or conditionally CPA accredited and take part in the NEQAS proficiency scheme. On average only 3.3% of primary samples submitted for mycobacterial diagnosis in 1995 produced a mycobacterial culture from approximately half as many patients (that is, a mean of 1488 specimens producing 49 isolates from 23 patients). Potentially over 380,000 specimens are processed for mycobacteria in the UK each year. The majority of laboratories use 4% NaOH +/- NALC for specimen decontamination. Culture on solid media was used by most laboratories and 62.9% also use liquid media. Most laboratories incubated cultures for eight weeks. Few laboratories use molecular diagnostic methods. Laboratories were most likely to use molecular methods for diagnosing tuberculous meningitis and for specimens from immunocompromised patients, although usage was strongly influenced by cost. Within England and Wales 43.9% (47/107) and 56% (61/109) of laboratories wanted a rapid service for rifampicin resistance detection in M tuberculosis from immunocompetent and immunocompromised patients, respectively. In regard to a tuberculous meningitis service, 80.5% (43/112) and 84.3% (102/121) of laboratories wanted this service for immunocompetent and immunocompromised patients, respectively. The quality of reference services was rated as "very good"/"good" by 85.6% of respondents nationally. Rapid molecular amplification diagnostic services were established at the PHLS MRU for rifampicin drug resistance detection nationally and for tuberculous meningitis at the MRU.

Molecular techniques in the diagnosis of Mycobacterium tuberculosis and the detection of drug resistance.

Early diagnosis of Mycobacterium tuberculosis disease is crucial in initiating treatment and interrupting the train of transmission. The increasing incidence of MDR TB worldwide has also placed emphasis on the need for early detection of drug resistance, particularly to isoniazid and rifampicin. Molecular diagnostic techniques and automated culture systems have reduced turnaround times in the modern mycobacteriology laboratory, and the continuing evaluation and development of such techniques is increasing the use of molecular technology in developed nations. Simple phenotypic methods for the detection of resistance to first-line drugs and genotypic kit-form assays for detection of rifampicin resistance have been developed that have become key tools in the containment of MDR TB.

The rapid diagnosis of isoniazid and rifampicin resistance in Mycobacterium tuberculosis--a molecular story.

Isoniazid and rifampicin resistance are assayed phenotypically by the resistance ratio, absolute concentration or proportion methods. Assay methods are often difficult to standardise and the World Health Organization (WHO) Global Programme on Drug Resistance is attempting to produce standardised drug resistance data worldwide. Broth-based methods are faster than solid media systems, and a commercial radiometric system, the Bactec 460, is arguably the fastest method and permits testing to be completed within 7-14 days; however, this method is expensive and requires disposal of radioactive material. Novel phenotypic methods that utilise mycobacteriophages have shown promise. Other molecular detection systems require knowledge of the genes encoding the drug target (the inhA/mabA, katG, oxyR and ahpC genes for isoniazid; rpoB for rifampicin) and the mutations producing resistance. These genotypic methods are limited in that not all resistance mechanisms are known, but advanced assays for rifampicin resistance that use gene sequencing, heteroduplex analysis, solid-phase hybridisation or single-strand conformation polymorphism analysis are becoming available.

Tuberculosis and AIDS.

Since the mid-1980s, the rate of decline in reported cases of tuberculosis (TB) has reached a plateau or reversed because of a combination of poverty and increased homelessness, immigration and displacement, poorly managed and supplied TB control programmes and, particularly in the developing world, the emergence of human immunodeficiency virus (HIV) infection. TB in HIV-positive patients may present atypically, both clinically and radiologically, with a lower probability of sputum positivity, greater difficulty in diagnosis, and a more rapid clinical deterioration than TB in HIV-seronegative patients. The emergence of multiple-drug-resistant strains of Mycobacterium tuberculosis, particularly in patients infected by HIV, carries a high mortality and has been associated with outbreaks in Europe and the USA. Microscopy and culture form the basis of diagnosis, but there is a need for more rapid diagnostic techniques and novel methods of drug susceptibility testing. Prolonged supervised treatment programmes and the development of new chemotherapeutic agents and regimens are essential prerequisites for successful TB therapy in AIDS patients. This review examines the clinical, microbiological and epidemiological issues associated with TB in HIV-infected individuals.

A clinical, microbiological and economic analysis of a national service for the rapid molecular diagnosis of tuberculosis and rifampicin resistance in Mycobacterium tuberculosis.

A clinical, microbiological and economic study of a national rapid molecular service for the identification of Mycobacterium tuberculosis and the determination of rifampicin resistance in smear-positive sputum samples (and other primary specimens) was performed. Ninety-one primary specimens, of which 55 were smear-positive sputum, were examined by molecular and conventional assays. Concordance of molecular results from smear-positive sputum specimens with tuberculosis diagnosis and rifampicin resistance by conventional analysis was 52 (94.5%) of 55 and 44 (91.7%) of 48, respectively. Concordance of molecular analysis on all primary specimens was 81 (89.0%) of 91 (diagnosis) and 55 (90.2%) of 61 (rifampicin resistance). Approximately 28 days were saved in the time to diagnosis by using the molecular assay. Hospitals can reduce the cost of inappropriate isolation of patients with risk factors for multiple drug-resistant tuberculosis (MDRTB) who subsequently are shown to have drug-sensitive tuberculosis. At one hospital potential annual savings were between pound sterling 50000 and pound sterling 150000. Of the nine MDRTB cases identified, all had a previous diagnosis of tuberculosis, 78% were born overseas, 44% were known to be non-compliant with therapy, but only one case (12.5%) was HIV positive. HIV status was not significantly different between MDRTB and drug-sensitive tuberculosis cases. Over 75% of specimens were taken while the patient was on therapy. Isolates from >50% of the MDRTB cases were resistant to three or more drugs and one was resistant to seven drugs. All patients were placed on additional therapy once the molecular result was known; this was subsequently modified based on the results of in-vitro drug susceptibility testing. All survived at least 6 months of follow-up. There was no difference in the proportion of successful cultures from smear-positive samples from patients with drug-sensitive tuberculosis or MDRTB who were on therapy. Molecular rifampicin resistance assays are reliable for diagnosis in cases with smear-positive disease.

Tuberculosis and the role of war in the modern era.

OBJECTIVE: Tuberculosis (TB) remains a major global health problem; historically, major wars have increased TB notifications. This study evaluated whether modern conflicts worldwide affected TB notifications between 1975 and 1995. DESIGN: Dates of conflicts were obtained and matched with national TB notification data reported to the World Health Organization. Overall notification rates were calculated pre and post conflict. Poisson regression analysis was applied to all conflicts with sufficient data for detailed trend analysis. RESULTS: Thirty-six conflicts were identified, for which 3-year population and notification data were obtained. Overall crude TB notification rates were 81.9 and 105.1/100,000 pre and post start of conflict in these countries. Sufficient data existed in 16 countries to apply Poisson regression analysis to model 5-year pre and post start of conflict trends. This analysis indicated that the risk of presenting with TB in any country 2.5 years after the outbreak of conflict relative to 2.5 years before the outbreak was 1.016 (95%CI 0.9435-1.095). CONCLUSION: The modelling suggested that in the modern era war may not significantly damage efforts to control TB in the long term. This might be due to the limited scale of most of these conflicts compared to the large-scale civilian disruption associated with 'world wars'. The management of TB should be considered in planning post-conflict refugee and reconstruction programmes.

Surveillance of drug-resistant tuberculosis and molecular evaluation of transmission of resistant strains in refugee and non-refugee populations in North-Eastern Kenya.

SETTING: Three refugee camp complex clinics and an adjacent non-refugee treatment centre in North-Eastern Kenya. OBJECTIVES: To use conventional and molecular epidemiology tools to determine: 1) the prevalence of drug resistance in newly diagnosed patients with smear-positive pulmonary tuberculosis in refugee and non-refugee populations; 2) risk factors for resistance in the two populations; and 3) whether IS6110 restriction fragment length polymorphism (RFLP) and spoligotyping showed similarities in DNA fingerprinting patterns of drug-resistant isolates that could infer transmission within and between the two populations. RESULTS: Of 241 isolates from the camps, 44 (18.3%) were resistant to one or more drugs, seven of which (2.9%) were multidrug-resistant TB (MDR-TB). Of 88 isolates from the non-refugees, five (5.7%) were resistant to one or more drugs without MDR-TB. Drug resistance was higher in the camps than in the non-refugee population (OR = 3.7; 95%CI 1.42-9.68; P < 0.007). Resistance was significantly higher in one camp compared with the other two, despite a comparable ethnic distribution. Unusually, females were more associated with drug resistance than their male counterparts in both populations (OR = 2.3; 95%CI 1.2-4.8; P = 0.008). There was evidence of transmission of streptomycin-resistant strains in the refugee population. DNA fingerprints of resistant strains from the non-refugee population were unique and different from those in the refugee camps. CONCLUSION: The observed high levels of drug resistance and MDR-TB, combined with evidence of transmission of strains resistant to streptomycin in the refugee population, suggest a need for strengthened TB control programmes in settings with a high risk of developing drug-resistant strains.

Molecular biology in the diagnosis and epidemiology of tuberculosis.

Tuberculosis is the predominant infectious cause of mortality today, killing 3 million people annually. The cornerstones of diagnosis rest on microscopy of specimens using auramine and Ziehl-Neelsen stains followed by culture on Lowenstein-Jensen or alternative media. The long generation time of Mycobacterium tuberculosis means 2-8 weeks usually elapse before a result is available to the clinician. This has stimulated research into the use of molecular diagnostic techniques. This article reviews the use and limitations of DNA hybridization, restriction fragment length polymorphism, pulsed-field gel electrophoresis and the polymerase chain reaction in the diagnosis and epidemiology of tuberculosis. The applicability of molecular biology to determine drug resistance is also addressed.

Restriction fragment length polymorphism analysis rules out cross-infection among renal patients with tuberculosis.

A cluster of five cases of tuberculosis occurred on a renal unit in 1993. The initial impression was that this was an outbreak, and cross-infection was suspected. Restriction fragment length polymorphism analysis was carried out on the strains of Mycobacterium tuberculosis isolated from these cases, using a DNA probe directed against the insertion sequence IS6110. DNA fingerprints obtained by this method differed for all the strains tested, ruling out cross-infection as a cause of the outbreak. This technique is a useful adjunct to standard epidemiological investigations in outbreaks of tuberculosis.

An outbreak of multi-drug-resistant tuberculosis in a London teaching hospital.

We describe the epidemiology and control of a hospital outbreak of multi-drug-resistant tuberculosis (MDR-TB). A human immunodeficiency virus (HIV)-negative patient with drug-sensitive tuberculosis developed MDR-TB during a period of unsupervised therapy. She was admitted to an isolation room in a ward with HIV-positive patients, but the room, unbeknown to hospital staff, was at positive-pressure relative to the main ward. Seven HIV-positive contacts developed MDR-TB. The diagnosis in the second patient was delayed, partly because acid-fast bacilli in his sputum were assumed to be Mycobacterium avium-intracellulare. All the available Mycobacterium tuberculosis isolates were indistinguishable by molecular typing. Nearly 1400 staff and patient contacts were offered screening, but the screening programme detected only one of the cases. Despite therapy, the index patient and two of the contacts died. HIV-positive patients are more likely than others to develop tuberculosis after exposure, and the disease may progress more rapidly. In these patients the possibility that acid-fast bacilli may represent M. tuberculosis must always be considered. Patients with tuberculosis (suspected or proven) should not be nursed in the same wards as immunosuppressed patients, and should be isolated. MDR-TB cases must be isolated in negative-pressure rooms. Hospital side-rooms may be positive-pressure as a fire safety measure; infection control teams must be aware of the airflows in all isolation rooms, and must be consulted during the design of hospital buildings. Good communication between infection control teams and clinicians is important, and all medical and nursing staff must be aware of the principles of management of patients with proven or suspected tuberculosis and MDR-TB.