Investigating the quality of expectorated sputum for tuberculosis diagnosis in Bolivia.

A low-power microscope-based cytological system to assess the quality of expectorated sputum provided for tuberculosis (TB) diagnosis was piloted in Bolivia. A total of 3688 samples were subjected to visual and cytological examination in nine laboratories: of these, 591 (16%) were misclassified by visual examination and 294 (8%) were found to be degraded. The degree of discordance varied between locations, and laboratories received a higher number of degraded specimens from isolated health clinics. Cytological assessment of sputum was found to be feasible and identified areas for improvement in the Bolivian diagnostic system for TB.

Large-scale whole genome sequencing of M. tuberculosis provides insights into transmission in a high prevalence area.

To improve understanding of the factors influencing tuberculosis transmission and the role of pathogen variation, we sequenced all available specimens from patients diagnosed over 15 years in a whole district in Malawi. Mycobacterium tuberculosis lineages were assigned and transmission networks constructed, allowing ≤10 single nucleotide polymorphisms (SNPs) difference. We defined disease as due to recent infection if the network-determined source was within 5 years, and assessed transmissibility from forward transmissions resulting in disease. High-quality sequences were available for 1687 disease episodes (72% of all culture-positive episodes): 66% of patients linked to at least one other patient. The between-patient mutation rate was 0.26 SNPs/year (95% CI 0.21-0.31). We showed striking differences by lineage in the proportion of disease due to recent transmission and in transmissibility (highest for lineage-2 and lowest for lineage-1) that were not confounded by immigration, HIV status or drug resistance. Transmissions resulting in disease decreased markedly over time.

Microscopy compared to culture for the diagnosis of tuberculosis in induced sputum samples: a systematic review.

BACKGROUND: Resource-limited settings rely on sputum examination using microscopy to diagnose tuberculosis (TB); however, the sensitivity of the test is poor and case detection rates are low. Sputum induction is proposed as a way to improve sample collection and enhance test sensitivity. OBJECTIVE: To undertake a systematic review of studies comparing microscopy and culture sensitivity in induced sputum samples. METHODS: We ran duplicate searches of databases (up to August 2011) and searchable websites of major human immunodeficiency virus (HIV) and TB conferences (up to November 2010) to identify studies comparing the performance of microscopy compared to culture on induced sputum samples, with culture as the reference standard. RESULTS: A total of 23 studies met our inclusion criteria. The overall success of the induction was high, ranging from 76.4% (95%CI 68.5-83.2) to 100% (95%CI 98.5-100), while adverse events associated with sputum induction were infrequent and mild. The sensitivity of microscopy compared to culture ranged from 0% to 100%; only eight studies reported on the species of mycobacterium isolated in culture. Yield was generally higher for sputum induction compared to nasopharyngeal aspiration and gastric lavage, and compared equally well to bronchoalveolar lavage and physiotherapy. DISCUSSION: Sputum induction increases TB case detection and is useful for people who are negative on spontaneous smear microscopy or unable to expectorate spontaneously. It is well-tolerated by children and adults, irrespective of HIV status, and can be used where culture is not available. The use of induced sputum samples with molecular tests, such as Xpert(®) MTB/RIF, warrants further investigation.

Challenges and perspectives for improved management of HIV/Mycobacterium tuberculosis co-infection.

HIV and Mycobacterium tuberculosis (MTB) are two widespread and highly successful microbes whose synergy in pathogenesis has created a significant threat for human health globally. In acknowledgement of this fact, the European Union (EU) has funded a multinational support action, the European Network for global cooperation in the field of AIDS and TB (EUCO-Net), that brings together experts from Europe and those regions that bear the highest burden of HIV/MTB co-infection. Here, we summarise the main outcome of the EUCO-Net project derived from an expert group meeting that took place in Stellenbosch (South Africa) (AIDS/TB Workshop on Research Challenges and Opportunities for Future Collaboration) and the subsequent discussions, and propose priority areas for research and concerted actions that will have impact on future EU calls.

Costs and cost-effectiveness of tuberculosis cultures using solid and liquid media in a developing country.

SETTING: The expansion of culture has been proposed to aid tuberculosis (TB) control in developing countries. OBJECTIVES: To examine the cost and cost-effectiveness at the Zambian National TB Reference Laboratory of homemade and commercially produced Löwenstein-Jensen culture (HLJ and CLJ) as well as automated and manually read liquid culture (AMGIT and MMGIT). DESIGN: Costs were estimated from the provider's perspective and based on the average monthly throughput. Cost-effectiveness estimates were based on yield during the study period. RESULTS: All techniques show comparable costs per culture (between US$28 and $32). Costs per Mycobacterium tuberculosis specimen detected were respectively US$197, $202, $312 and $340 for MMGIT, AMGIT, CLJ and HLJ. When modelled for the maximum throughput, costs were above US$95 per M. tuberculosis specimen detected for all techniques. When only performed among smear-negative specimens, costs per additionally identified M. tuberculosis would be US$487 for MMGIT and higher for other methods. CONCLUSION: Based on cost-effectiveness grounds, liquid media compare well with conventional solid media, especially where yield of MGIT is substantially higher than that of LJ media. The results indicate high overall costs per culture; the expansion of culture to decentralised levels with lower throughputs may result in even higher costs.

Quality assessment of sputum smear microscopy for detection of acid fast bacilli in peripheral health care facilities in Dar es Salaam, Tanzania.

BACKGROUND: There is no published information regarding the quality of sputum smear microscopy in Tanzania. OBJECTIVE: To evaluate technical quality and results of smear microscopy for acid-fast bacilli (AFB) in peripheral health care facilities in Kinondoni and Ilala Districts in Dar es Salaam, Tanzania. DESIGN: Cross-sectional study. SETTING: All tuberculosis diagnostic centres in Dar es Salaam, Tanzania. RESULTS: The proportion of well prepared smears was 86.2% and that of well stained smears was 81.2%. The overall average agreement in reading was (89.2%). The overall sensitivity was 88.5% and specificity was 100%. High false negatives (HFN) were the major errors found in this study and Low false negative (LFN) and quantification errors (QE) were the minor errors found. There were no false positive errors. Minor errors occurred more frequently in hospitals than dispensaries, while major errors occurred more frequently in dispensaries than in hospitals. CONCLUSIONS: The types of errors found in this survey, HFN, LFN and QE, suggest a systematic under-reading of smears in all the surveyed health facilities, probably due to a number of technical factors (quality of smears, poor stains, bad microscopes, or inadequate training) and other factors such as overwork and lack of motivation which need to be addressed. RECOMMENDATIONS: Regular supervision using the new WHO quality assurance guidelines should be conducted countrywide. We do recommend that blind re-checking as the most efficient means of making the first broad assessment of sputum smear microscopy in Tanzania.

Evaluation of Dot-Blot and Phage Replication Techniques for Detection of Drug Resistant Mycobacterium Tuberculosis

We have assessed the utility of two new methods; dot-blot and bacteriophage replication techniques; for use in a routine diagnosis laboratory in poor resource settings in the screening of drug resistant Mycobacteria tuberculosis by comparing with the conventional proportion method. A total of 145 M. tuberculosis clinical isolates were tested for resistance to rifampicin; isoniazid; streptomycin and ethambutol. The dot blot had sensitivities of 91.7; 100; 93.5and 85.7 and specificities of 99.2; 99.2; 99.1and 99.2 for rifampicin; streptomycin; isoniazid and ethambutol; respectively. The phage technique had sensitivities of 92and 84.6and specificities of 99.2and 99.2for rifampicin and streptomycin; respectively. Both techniques yielded results within 48 hours of receipt of the culture on solid media.The high sensitivity and specificity coupled with rapidity of results indicate that these methods are potentially useful tools for screening resistance to anti-tuberculosis drugs in our setting. However; the phage replication technique; which is simpler and technically less demanding; seems the most suitable for routine screening of drug resistant mycobacteria in resource deprived countries such as Tanzania. We are recommending further field evaluation of the phage replication method so that it can complement; and possibly replace; the conventional proportion method in drug susceptibility testing

Comparison of two bacteriophage tests and nucleic acid amplification for the diagnosis of pulmonary tuberculosis in sub-Saharan Africa.

SETTING: National reference laboratory in Zambia, a high-incidence setting with a high prevalence of HIV infection. OBJECTIVE: To compare the performance of a commercial bacteriophage kit with a nucleic acid amplification kit and an 'in-house' bacteriophage method for rapid diagnosis of pulmonary tuberculosis (TB). METHODS: Sputum specimens from suspected pulmonary TB cases were examined by direct fluorescence microscopy and culture on Löwenstein Jensen (LJ). In a blinded study, remaining samples were tested by AMTD and FASTPlaqueTB or an in-house bacteriophage assay. Two specimen decontamination protocols were investigated. RESULTS: Microbial contamination of 40.4% was observed when using the FASTPlaqueTB kit specimen preparation protocol. When compared to culture on LJ, the sensitivity of the FASTPlaqueTB test was 20.7%. Implementation of a modified Petroff's decontamination protocol reduced contamination to 5.8% and the FASTPlaqueTB test detected 8/25 (32%) of culture-positive specimens. The sensitivity of AMTD and smear microscopy for these specimens were 64% and 48%, respectively. In a separate experiment the sensitivity of an in-house bacteriophage assay was 45.3% compared to 64.2% for AMTD and 45.3% for direct smear microscopy. CONCLUSIONS: Additional analysis of sputum specimens by bacteriophage assay provided no advantage in this setting. For the rapid diagnosis of TB, AMTD offered improved sensitivity over direct smear microscopy.

Guidelines for establishing trials of new tests to diagnose tuberculosis in endemic countries.

Efforts to control tuberculosis are currently hampered by the lack of effective tools for the detection of infected individuals. Although new diagnostic tests are being developed and marketed, there is a paucity of data on the value of new technology for the diagnosis of tuberculosis in endemic regions of the world. Trials of new tools need to be undertaken in appropriate settings and to a high standard. The value of previous studies has frequently been compromised by inadequate study design and poor execution. We offer guidelines to assist in planning trials for new tests for the diagnosis of tuberculosis.

Utility of nucleic acid amplification techniques for the diagnosis of pulmonary tuberculosis in sub-Saharan Africa.

SETTING: Lusaka, Zambia. OBJECTIVES: To investigate the utility of nucleic amplification tests for the diagnosis of pulmonary tuberculosis in a resource-poor setting with a high incidence of human immunodeficiency virus (HIV). DESIGN: Sputum specimens from suspects attending a referral chest clinic were examined by low-cost 'in-house' one-tube nested polymerase chain reaction (PCR), the enhanced Gen-Probe Amplified Mycobacterium Direct Test (AMTD), auramine smear and Lowenstein-Jensen culture. RESULTS: PCR and AMTD detected respectively 80% and 92% of smear-positive specimens and 40% and 60% of smear-negative, culture-positive specimens. AMTD was positive for 18 culture-negative suspects; subsequent investigation indicated these to be six confirmed tuberculosis patients, nine judged from radiological data and clinical follow-up studies to have pulmonary tuberculosis, and three non-tuberculosis patients. Sensitivity for smear, culture, PCR and AMTD, when compared to a gold standard incorporating both microbiological and clinical data, was respectively 29%, 69%, 55% and 81%. CONCLUSION: In this setting, the sensitivity of the low-cost PCR proved insufficient for its effective use as a tool for diagnosing pulmonary tuberculosis, while AMTD performed considerably better than the current laboratory methods for diagnosis of pulmonary tuberculosis. However, the high cost of this technology may limit its application in the public sector of low-income countries.

Phage replication technology for diagnosis and drug susceptibility testing.

Of the world's infectious diseases, tuberculosis remains the leading cause of mortality and it has been estimated that of the eight million new cases that occur each year 95% are found in the less developed countries (1,2). Diagnostic methods that involve culture of Mycobacterium tuberculosis are necessarily slow due to the protracted growth times required by these bacteria. Rapid molecular tests have been developed for both diagnosis (3) and drug resistance testing (4). However, the high costs and requirement for specialized equipment prohibits the routine application of this technology in low-income countries, and there is an urgent need for sensitive, rapid tests that are affordable and appropriate for use in these areas of the world (5).

Micro-well phage replication assay for screening mycobacteria for resistance to rifampin and streptomycin.

Phenotypic methods for screening Mycobacterium tuberculosis or Mycobacterium Ulcerans for susceptibility to therapeutic drugs are necessarily slow due to the protracted growth times of these bacteria. Rapid testing is now possible for the potent antituberculosis drug rifampicin using molecular methods to detect those mutations that confer resistance (1, 2). However, the high cost and requirement for specialized equipment may prohibit the application of this technology in resource-poor settings and there is a need for low-cost, rapid tests that are appropriate for use in low-income countries.

An incremental cost-effectiveness analysis of the first, second and third sputum examination in the diagnosis of pulmonary tuberculosis.

SETTING: St. Francis Hospital in Katete District, Eastern Province, Zambia. OBJECTIVE: To compare the incremental cost-effectiveness of examining serial sputum smears for screening suspects for pulmonary tuberculosis at a rural district hospital in Zambia. DESIGN: An incremental cost-effectiveness analysis of serial sputum smear examinations for diagnosing pulmonary tuberculosis based on laboratory results collected during 1997 and 1998 in a rural district hospital in Zambia. The cost analysis took a health service provider perspective, and used the ingredients approach. The cost-effectiveness is expressed in terms of the incremental cost per tuberculosis case diagnosed. Relevant information was obtained from various sources, including administrative records, interviews and direct observation. RESULTS: Of a total of 166 acid-fast bacilli positive suspects who had three sputum smears examined sequentially, 128 (77.1%) were found on the first smear, a further 25 (15%) on the second smear and 13 (7.9%) additional cases were identified on the third smear. The economic analysis shows that the incremental cost of performing a third test, having already done two, increases rapidly with only a small gain in terms of additional cases of tuberculosis identified. CONCLUSION: A policy of examining two samples should be considered in resource-poor settings, if the remaining steps of the national diagnostic algorithm can be adhered to with respect to smear-negative suspects.

Rapid screening of Mycobacterium tuberculosis for susceptibility to rifampicin and streptomycin.

OBJECTIVE: To investigate rapid detection of drug-resistant tuberculosis using the genotypic Inno-LiPA Rif TB assay and a novel, low-cost, bacteriophage-based susceptibility assay. DESIGN: The performance of the microwell phage replication assay (MPRA) on 18 isolates from suspected multidrug-resistant tuberculosis patients was compared to the LiPA assay performed directly on sputum specimens. Mutations in the rpoB gene identified by LiPA that confer resistance to rifampicin (RMP) were confirmed by DNA sequencing, while susceptibilities were confirmed by the proportion method and BACTEC. A further 19 isolates undergoing routine screening for both RMP and streptomycin susceptibility were included for comparison. RESULTS: Susceptibility to RMP was determined for 17/18 (94.4%) sputum specimens tested by LiPA. Correlation between MPRA, molecular and conventional methods was 100% for the detection of RMP susceptibility. However, for susceptibility to streptomycin one discrepant result was found: an isolate susceptible to streptomycin by the proportion method was found resistant by MPRA to 2 microg/ml of streptomycin. Similarly, an isolate initially resistant by MPRA upon re-testing was found susceptible in agreement with the conventional method. CONCLUSION: LiPA enables rapid detection of drug-resistant infection, while MPRA offers simple, low-tech testing of drug susceptibilities that may be appropriate for application in low-income countries.

TB: the return of the phage. A review of fifty years of mycobacteriophage research.

The first mycobacteriophage was isolated in 1947, and since that time over 250 of these viruses have been identified. Phages have made a significant contribution to our knowledge of mycobacteria over the past fifty years, and following the development of typing techniques in the 1960s and 1970s they were widely used in epidemiological studies of tuberculosis. Unfortunately, attempts to use lytic phages therapeutically during tuberculosis infection have so far failed to elicit cure in experimentally infected animals. During the past decade phages have become important in molecular studies of mycobacteria, both in terms of studying phage biology and as tools in recombinant DNA technology, thus facilitating the investigation of mycobacterial pathogenesis. Today their potential as diagnostics reagents is also being realised with the development of exciting new techniques for rapid bacterial detection and drug susceptibility testing. This review outlines the history of these remarkable organisms, from their discovery fifty years ago to the current developments in rapid diagnostic techniques.

Inactivation of mycobacteriophage D29 using ferrous ammonium sulphate as a tool for the detection of viable Mycobacterium smegmatis and M. tuberculosis.

There is still an urgent requirement for more sensitive, cost-effective methods for detection and susceptibility testing of mycobacteria in clinical samples. We have been investigating a simple bacteriophage-based system which could be used for both purposes. As this depends upon the detection of phages which have successfully infected cells, a key step is the efficient removal or inactivation of phages remaining free in the culture medium. We demonstrate here the use of ferrous ammonium sulphate as an effective agent for the inactivation of mycobacteriophage D29 without impairing phage replication in previously infected host bacteria. Using this property, we report the detection of viable Mycobacterium smegmatis, M. bovis BCG and M. tuberculosis using simple low-cost technology. The method is highly sensitive, since it is able to detect 10 colony-forming units of M. smegmatis. It is also rapid, with the detection of M. tuberculosis in sputum specimens within 48 h.

An improved colorimetric PCR-based method for detection and differentiation of Entamoeba histolytica and Entamoeba dispar in feces.

The epidemiological implications of the recent separation of "Entamoeba histolytica" into two separate species, pathogenic E. histolytica sensu stricto and commensal E. dispar, will not become apparent without methods of distinguishing between them which are applicable to large numbers of specimens. We have modified a PCR-based method to produce such a technique which may be completed in 1 day while still identifying 10(-1) E. histolytica and 1 to 10 E. dispar trophozoites per g of feces when present separately and 10 E. histolytica and 100 E. dispar trophozoites per g in the presence of 10(6) trophozoites per g of the other species. Applied to fecal specimens from 18 patients from which E. histolytica or E. dispar had been grown and identified to the species level by hexokinase isoenzyme analysis, the method in every case yielded the correct result. Positive and negative results are easily distinguished by eye, and we are now applying this technique to a large-scale epidemiological study of amebiasis in the eastern Mediterranean region.