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.

Changes in mRNA levels of poly(ADP-ribose) polymerase during activation of human lymphocytes.

The level of mRNA encoding the nuclear enzyme poly(ADP-ribose) polymerase (ADP-ribosyltransferase, EC 2.4.2.30) was found to be very low in quiescent human lymphocytes and to increase at least 10-fold between 1 and 2 dyas after stimulation with the mitogen phytohaemagglutinin, staying high for several days thereafter. This increase was inhibited by 3-methoxybenzamide (a competitive inhibitor of poly(ADP-ribose) polymerase) but was not affected significantly by aphidicolin. Incubation of activated cells with cycloheximide for 2 h increased the expression slightly. These data demonstrate that, during lymphocyte activation, the level of mRNA of the poly(ADP-ribose) polymerase gene correlates with, and hence is presumably responsible for, the increase in poly(ADP-ribose) polymerase protein detectable by enzyme assay or immunochemistry.

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.

Cytotoxic effects of amiodarone and desethylamiodarone on human thyrocytes.

Since recent in vivo evidence suggests that the benzofuran antiarrhythmic drug amiodarone has a direct toxic effect on the human thyroid gland, we have investigated the effects of both amiodarone and its metabolite desethylamiodarone on a novel immortalized functional human thyrocyte line (SGHTL-34 cells). Desethylamiodarone markedly reduced cell number as assessed from both DNA and protein content. Few cells were left after 24 hr exposure to 12.5 micrograms/ml; the concentration producing death of 50% of cells (EC50) was 6.8 +/- 1.1 micrograms/ml (mean +/- SE, N = 15). Amiodarone was much less potent, producing a maximum decrease in cell number of approximately 25% at concentrations up to 50 micrograms/ml. The effect of desethylamiodarone was seen within 24 hr of culture. T3 in concentrations up to 0.75 micrograms/ml had no effect on the action of amiodarone or desethylamiodarone on SGHTL-34 cells. Light microscopy demonstrated vacuolation of SGHTL-34 cells after 4-day culture with either the drug or its metabolite. Studies using primary cultures of human retroorbital fibroblasts demonstrated that the greater cytotoxicity of desethylamiodarone was not confined to thyrocytes. When SGHTL-34 cells were incubated with 2.5 micrograms/ml desethylamiodarone for 4 days, 71.7 +/- 0.9% was taken up by the cells; there was no detectable conversion to amiodarone. Incubation of thyrocytes with 50 micrograms/ml amiodarone for 4 days resulted in the uptake of 80.1 +/- 2.1% by the cells. In addition, 5.0 +/- 0.1% of the amiodarone was converted to material with the same retention time as desethylamiodarone standard; of this material, 72.9 +/- 2.8% was taken up by the cells. We conclude that desethylamiodarone, at concentrations near those found in the plasma of patients on long-term amiodarone therapy, exerts a direct cytotoxic effect on human thyroid cells in short-term culture. This effect may play a role in the aetiology of clinical thyroid disease during amiodarone therapy. We suggest that, since the effect is not restricted to thyrocytes, desethylamiodarone may play a role in the aetiology of amiodarone toxicity which occurs clinically in many tissues.

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.

Effect of dietary protein on glomerular filtration rate in pregnancy.

High dietary protein intake is known to increase glomerular filtration rate in the nonpregnant state. The effects of dietary protein on renal function in human pregnancy are not well known. We studied 392 patients with singleton uncomplicated pregnancies at gestational ages of 7-40 weeks. Creatinine clearance was correlated with protein intake estimated from 24-hour dietary recall during the same period. Creatinine clearances were significantly higher when dietary protein was more than 50 g than when it was less than 50 g (P less than .005). The short- or long-term significance of this finding is unclear.

Cellular immunity in preeclampsia: alterations in T-lymphocyte subpopulations during early pregnancy.

T-lymphocyte subpopulations were evaluated longitudinally in 62 primigravidas. Beginning early in the second trimester, women who later developed preeclampsia showed a significantly lower proportion of T-helper cells compared with those who remained normotensive (27.0 +/- 7.1 versus 34.7 +/- 7.9%; P less than or equal to .001). The decrease in the T-helper cell count occurred several weeks before the development of preeclampsia and reverted to normal 6-10 weeks postpartum.

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.

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.

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.

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.

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.

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.

Changes in topoisomerase I activity after irradiation of lymphoid cells.

The activity of topoisomerase I in nuclear extracts increased about three-fold 5 min after gamma-irradiation (840-2500 rads) of human peripheral blood lymphocytes or cultured lymphoblastoid cells. The change may reflect modification of the enzyme by nuclear ADP-ribosyl transferase, which is known to be activated by DNA breaks.

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.