Failure or relapse predictors for the STREAM Stage 1 short regimen for RR-TB.

BACKGROUND: STREAM (Standardised Treatment Regimens of Anti-tuberculosis drugs for Multidrug-Resistant Tuberculosis) Stage 1 demonstrated non-inferior efficacy of a short regimen for rifampicin-resistant TB (RR-TB) compared to a long regimen as recommended by the WHO. The present paper analyses factors associated with a definite or probable failure or relapse (FoR) event in participants receiving the Short regimen.METHODS: This analysis is restricted to 253 participants allocated to the Short regimen and is based on the protocol-defined modified intention to treat (mITT) population. Multivariable Cox regression models were built using backwards elimination with an exit probability of P = 0.157, equivalent to the Akaike Information Criterion, to identify factors independently associated with a definite or probable FoR event.RESULTS: Four baseline factors were identified as being significantly associated with the risk of definite or probable FoR (male sex, a heavily positive baseline smear grade, HIV co-infection and the presence of costophrenic obliteration). There was evidence of association of culture positivity at Week 8 and FoR in a second model and Week 16 smear positivity, presence of diabetes and of smoking in a third model.CONCLUSION: The factors associated with FoR outcomes identified in this analysis should be considered when determining the optimal shortened treatment regimen.

Effectiveness of GenoType MTBDRsl in excluding TB drug resistance in a clinical trial.

OBJECTIVES: To assess the performance of the GenoType MTBDRsl v1, a line-probe assay (LPA), to exclude baseline resistance to fluoroquinolones (FQs) and second-line injectables (SLIs) in the Standard Treatment Regimen of Anti-tuberculosis Drugs for Patients With MDR-TB 1 (STREAM 1) trial.METHODS: Direct sputum MTBDRsl results in the site laboratories were compared to indirect phenotypic drug susceptibility testing (pDST) results in the central laboratory, with DNA sequencing as a reference standard.RESULTS: Of 413 multidrug-resistant TB (MDR-TB) patients tested using MTBDRsl and pDST, 389 (94.2%) were FQ-susceptible and 7 (1.7%) FQ-resistant, while 17 (4.1%) had an inconclusive MTBDRsl result. For SLI, 372 (90.1%) were susceptible, 5 (1.2%) resistant and 36 (8.7%) inconclusive. There were 9 (2.3%) FQ discordant pDST/MTBDRsl results, of which 3 revealed a mutation and 5 (1.3%) SLI discordant pDST/MTBDRsl results, none of which were mutants on sequencing. Among the 17 FQ- and SLI MTBDRsl-inconclusive samples, sequencing showed 1 FQ- and zero SLI-resistant results, similar to frequencies among the conclusive MTBDRsl. The majority of inconclusive MTBDRsl results were associated with low bacillary load samples (acid-fast bacilli smear-negative or scantily positive) compared to conclusive results (P < 0.001).CONCLUSION: MTBDRsl can facilitate the rapid exclusion of FQ and SLI resistances for enrolment in clinical trials.

Reduction of diagnostic and treatment delays reduces rifampicin-resistant tuberculosis mortality in Rwanda.

SETTING: In 2005, in response to the increasing prevalence of rifampicin-resistant tuberculosis (RR-TB) and poor treatment outcomes, Rwanda initiated the programmatic management of RR-TB, including expanded access to systematic rifampicin drug susceptibility testing (DST) and standardised treatment.OBJECTIVE: To describe trends in diagnostic and treatment delays and estimate their effect on RR-TB mortality.DESIGN: Retrospective analysis of individual-level data including 748 (85.4%) of 876 patients diagnosed with RR-TB notified to the World Health Organization between 1 July 2005 and 31 December 2016 in Rwanda. Logistic regression was used to estimate the effect of diagnostic and therapeutic delays on RR-TB mortality.RESULTS: Between 2006 and 2016, the median diagnostic delay significantly decreased from 88 days to 1 day, and the therapeutic delay from 76 days to 3 days. Simultaneously, RR-TB mortality significantly decreased from 30.8% in 2006 to 6.9% in 2016. Total delay in starting multidrug-resistant TB (MDR-TB) treatment of more than 100 days was associated with more than two-fold higher odds for dying. When delays were long, empirical RR-TB treatment initiation was associated with a lower mortality.CONCLUSION: The reduction of diagnostic and treatment delays reduced RR-TB mortality. We anticipate that universal testing for RR-TB, short diagnostic and therapeutic delays and effective standardised MDR-TB treatment will further decrease RR-TB mortality in Rwanda.

Two vs. three sputum samples for microscopic detection of tuberculosis in a high HIV prevalence population.

SETTING: A busy urban hospital in Cameroon. OBJECTIVES: To compare the yield in bacteriologically proven tuberculosis (TB) cases examining two morning vs. three spot-morning-spot sputum specimens (MM vs. SMS) by direct microscopy for acid-fast bacilli (AFB). DESIGN: Repeated temporal cross-over between MM and SMS sampling for successive TB suspects, using culture as gold standard. RESULTS: A total of 799 suspects were screened using the MM strategy, identifying 223 smear-positives, and 808 suspects with the SMS strategy, yielding 236 smear-positives. Of the MM, 256 were culture-positive, of whom 195 (76%) were smear-positive. For SMS, these figures were respectively 281 and 206 (73%), a non-significant difference. The MM and SMS strategies also detected respectively 28 and 30 smear-positive cases not confirmed by culture. No cases were lost to treatment with either strategy. CONCLUSIONS: In this population with a high prevalence of human immunodeficiency virus (HIV) with late case presentation, smear microscopy of two morning specimens detected at least as many positive cases as the classical strategy, and no cases were lost before treatment. Two specimens for initial TB suspect screening can thus be recommended, also without excessive workload. Comparative studies in populations presenting with paucibacillary sputum are needed to determine the equivalent quality and yield of an alternative strategy with two spot specimens at consultation.

Evaluation of the FluoreslenS and fluorescence microscopy blinded rechecking trial, Nairobi, Kenya.

SETTING: Three busy laboratories in Nairobi, Kenya. OBJECTIVES: To determine the performance of an affordable fluorescence system (FluoreslenS) for tuberculosis microscopy, and to test an auramine-smear rechecking system. DESIGN: Alternating routine use of Ziehl-Neelsen (ZN) and fluorescence microscopy (FM) was performed to compare detection and errors found while rechecking. RESULTS: Overall, 19.5% of 25,250 ZN and 23% of 21,104 FM smears were positive (P < 10(-3)). The proportional increment of FM over ZN was 18% (range -6%-29%), with one centre detecting fewer positives (non-significant, NS). The average error frequencies were comparable (1.8% vs. 2.6% false-negative and 0.2% vs. 0.4% high false-positive for ZN and FM, respectively, NS). The superior performance of controllers and the overall equal ZN/FM quality in the laboratories could be demonstrated only after converting error percentages to relative sensitivity (RS). CONCLUSIONS: FM with the FluoreslenS system considerably improved sensitivity without loss of specificity in two of the busy routine laboratories, but the system is not sufficiently practical or user-friendly. Rechecking by FM can be done using guidelines for ZN smears, provided that routine ZN confirmation of positives is omitted. Calculation of RS allows an objective comparison of microscopy quality, independent of the variable prevalence of positives and sample size.

Yield of fluorescence microscopy versus culture for tuberculosis at a middle-income country referral hospital.

The aim of this study was to determine the usefulness of fluorescence microscopy (FM) at a referral centre in a middle-income country. Direct Ziehl-Neelsen (ZN) and direct, as well as concentrated, smear FM were performed on 2179 suspect sputa, with Löwenstein-Jensen (LJ) culture as the gold standard. ZN, direct FM and concentration FM detected 36.0, 38.6 and 37.0%, respectively, of 272 culture-positive specimens. Patient-wise, there were 8.1% (126/1553) positives on any smear compared with 12.0% (187/1553) on any culture. ZN, direct FM and concentrated FM smear were positive in 43.3, 46.5 and 45.5%, respectively, of culture-proven cases. All differences between microscopy and culture were significant (P<0.001), but not those between microscopy techniques. Acid-fast bacilli (AFB) were not rare in 60% of 48 duplicate smears, positive in ZN or FM only. Simple LJ culture, but not FM on direct or concentrated smears, was thus significantly more sensitive than ZN smears. The considerable numbers of AFB found in positive direct smears from discordant microscopy sets suggest that repeating smears can improve microscopy sensitivity more than variations of correctly executed technique, provided that overload is avoided. Thus FM could be particularly useful, as it is time-saving and could protect against the sensitivity loss associated with high workload.

Evaluation of a novel line probe assay to detect resistance to pyrazinamide, a key drug used for tuberculosis treatment.

OBJECTIVES: The development of rapid molecular diagnostic assays for pyrazinamide (PZA) resistance is considered technically challenging as mutations are highly diverse, scattered along the full length of the pncA gene and not all are associated with PZA resistance. We evaluated the performance of the novel Genoscholar PZA-TB II line probe assay (PZA-LPA2; NIPRO Corporation, Japan). METHODS: To evaluate the applicability of the PZA-LPA2 in clinical settings, we compared the performance of the PZA-LPA2 to a composite reference standard pncA Sanger and Illumina sequencing plus phenotypic susceptibility testing on a panel of 87 Mycobacterium tuberculosis isolates from World Health Organization (WHO) drug resistance surveys, harbouring mutations previously classified as associated or not associated with resistance according to data from peer-reviewed literature. In addition, the PZA-LPA2 was challenged against a selection of isolates with lineage-specific and non-resistance-associated mutations, for which the frequency among clinical isolates is unknown, and tested directly on 59 sputum extracts. RESULTS: For the survey isolates, the PZA-LPA2 reached an overall agreement with the composite reference of 97.6% (80/82) or 94.3% (82/87) excluding or including heteroresistance, respectively. The PZA-LPA2 failed on 8.5% (5/59) of clinical samples; among valid results, 100% (14/14) sensitivity and 100% (7/7) specificity was reached relative to pncA Sanger sequencing. CONCLUSIONS: The PZA-LPA2 represents a valid and rapid alternative for indirect PZA susceptibility testing. Preliminary findings on clinical samples show promise for direct testing. Further studies are needed to assess the clinical risk of missing heteroresistance and falsely detecting lineage-specific, silent and nonassociated mutations.

3 IS-RFLP: a powerful tool for geographical clustering of global isolates of Yersinia pestis.

Multiple copies of several classes of insertion sequences (IS) are found in the genome of Yersinia pestis, the causative agent of bubonic and pneumonic plague. We used the genetic instability generated by these IS to develop a method (designated 3IS-RFLP) based on the restriction fragment length polymorphism of the IS100, IS285 and IS1541 elements for studying Y. pestis strains of worldwide origin. We show that 31S-RFLP is a powerful tool to group Y. pestis isolates according to their geographical origin, and therefore that this method may be valuable for investigating the origin of new or re-emerging plague foci or for addressing forensic issues.

Detection of circulating antibodies to purified protein derivative by enzyme-linked immunosorbent assay: its potential for the rapid diagnosis of tuberculosis.

An enzyme-linked immunosorbent assay for detecting antibodies to purified protein derivative was evaluated as a rapid method for serodiagnosis of childhood tuberculosis. Its specificity for IgG antibodies was 0.98 as determined in 55 sera from nontuberculous children who showed no significant effect of previous Bacillus Calmette-Guérin vaccination on the production of specific antibodies. Results were negative in 29 of 33 (87.9%) tuberculin-positive children and in 18 of 20 (90.0%) contacts, none of whom had evidence of tuberculosis. The sensitivity of this test was 0.51 as determined in 49 sera from bacteriologically confirmed cases; 17 of 27 smear positive cases and 8 of 22 children with positive cultures were detected. Results were positive in 32 of 114 (28.1%) patients with a diagnosis of tuberculosis not confirmed by microbiology. Consequently whereas a negative result does not rule out tuberculosis, a positive result is a strong indication of the disease. The IgM antibody determination yielded much less discriminative results.

[Evaluation of an enzyme immunoassay for the rapid diagnosis of paucibacillary tuberculosis in adults]. / Evaluación del enzimoinmunoensayo para el diagnóstico rápido de la tuberculosis paucibacilar del adulto.

The ELISA has been extensively evaluated as a serodiagnostic method for tuberculosis. However, there is scarce information about its application to cases that cannot be diagnosed by microscopic examination: those with closed lesions or undergoing early stages of the disease. Since a reliable serological test might substantially contribute to their prompt detection, the objective of the present study was to determine the diagnostic value of an ELISA applied to adult smear-negative cases of tuberculosis. Sera from 235 patients with active tuberculosis--176 pulmonary and 59 extrapulmonary cases--and 181 control subjects were tested for IgG antibodies to PPD by ELISA. Eleven cases of non tuberculous mycobacterial (MOTT) disease and 33 cases of mycosis were also included in this group. With the adopted cut-off value, 73.9% (105/142) of smear positive and 52.7% (49/93) of smear negative tuberculosis cases, were correctly classified. Particularly in the latter, the test was positive in 55.2% (32/58) of patients with positive cultures for Mycobacterium tuberculosis and in 48.6% (17/35) of patients diagnosed by clinical, radiological and or histopathological findings. No antibody activity was demonstrated in 92.7% of sera from the control population which included 92 healthy volunteers, 32 non tuberculous diseased subjects and 13 household contacts of smear-positive cases. Among those control subjects who were skin tested, ELISA results were not related to the tuberculin reactivity: 93.7% (30/32) of tuberculin negative and 95.2% (40/42) of tuberculin positive healthy individuals had no detectable antibodies.(ABSTRACT TRUNCATED AT 250 WORDS)

[Evaluation of four antigens for the detection of anti-Mycobacterium bovis antibodies by enzyme immunoassay]. / Evaluación de cuatro antígenos para la detección de anticuerpos anti-Mycobacterium bovis por enzimoinmunoensayo.

An enzyme-linked immunosorbent assay (ELISA) for the diagnosis of bovine tuberculosis through the detection of specific seric antibodies has recently been developed in our laboratory. In order to assess its reproducibility and select the most adequate antigen, four bovine PPDs from different sources were evaluated in parallel: PPD M. bovis strain AN5, CEPANZO standard (CPZ), PPD M. bovis strain AN5, European Economic Community standard (EEC), PPD M. bovis strain AN5, prepared from non heated bacilli, killed by phenol (P) and PPD. M. bovis BCG strain prepared at the Pasteur Institute, Paris (BCG). Sera from 22 healthy cattle from tuberculosis free area and 20 bacteriologically confirmed tuberculous animals were employed in simultaneous assays. Antibody mean and standard deviations from healthy cattle expressed as optical density (OD) values were 45 +/- 22 when CPZ was used as antigen, 24 +/- 10 with EEC, 103 +/- 56 with P and 56 +/- 20 with BCG. Mean O.D. from tuberculous cattle were 588 +/- 158, 510 +/- 234, 782 +/- 138 and 441 +/- 189 with antigens CPZ, EEC, P and BCG respectively. A close correlation was observed when results obtained with EEC and P were compared with that of CPZ (r: 0.97 and 0.94 respectively). A lower specificity was achieved when BCG was used as antigen being also lower its correlation with the results obtained with CPZ (r: 0.87). It is concluded that our ELISA would achieve similar sensitivity and specificity if CPZ, EEC and P were used as antigens. On the other hand, BCG would not be suitable for this assay.

Evaluation of the Genotype® MTBDRplus assay as a tool for drug resistance surveys.

SETTING: A national tuberculosis (TB) drug resistance survey in Tanzania. OBJECTIVE: To compare the performance of the Genotype® MTBDRplus line-probe assay (LPA) on smear-positive sputum specimens with conventional culture and isoniazid (INH) plus rifampicin (RMP) drug susceptibility testing (DST). DESIGN: Mycobacterium tuberculosis isolates tested at the Tanzanian Central TB Reference Laboratory (CTRL) were submitted for quality assurance of phenotypic DST to its supranational reference laboratory (SRL), together with ethanol-preserved sputum specimens for LPA DST. RESULTS: Only 321 samples could be tested using LPA; of these, three were identified as being non-tuberculous mycobacteria using CTRL DST. Both tests had 269 sets with interpretable results. CTRL DST yielded almost the same number of interpretable results as LPA, with 90% concordance (κ = 0.612, P < 0.001). Five (1.9%) multidrug-resistant (MDR) strains, 46 (17.1%) resistant to INH only and 0 RMP only, were found by CTRL DST. For the LPA, these results were respectively 5 (1.9%), 26 (9.7%) and 2 (0.7%). With SRL DST as the gold standard, LPA was more accurate than CTRL DST for RMP, but missed almost half the INH-resistant samples. CONCLUSION: LPA applied directly on ethanol-preserved sputum specimens was similar to phenotypic DST in terms of yield of interpretable results. Although probably more accurate for RMP and MDR-TB, it appears to seriously underestimate INH resistance. Considering speed, easy and safe specimen transportation and low infrastructure requirements, LPA DST from sputum can be recommended for surveys in resource-poor settings.

Comparison of two LED fluorescence microscopy build-on modules for acid-fast smear microscopy.

SETTING: National Reference Laboratory, Benin. OBJECTIVES: To compare the performance of Fraen FluoLED and LW Lumin light-emitting diode (LED) fluorescence microscopy modules. DESIGN: Acid-fast bacilli (AFB) smears, routinely examined with a classical fluorescence microscope, were blindly re-read with both LED systems at 200x magnification. Smears with discordant results were rechecked on all systems at 200x, and 100 randomly chosen smears were read again at 400x. Confirmed presence of AFB with any system was accepted as a true positive. RESULTS: A total of 1937 smears were examined by all systems. The Fraen and LW detected 895 (46.2%) and 817 (42.2%) positive and scanty positive smears. After rechecking 201 smears, 15 false-positive and 61 false-negative results were declared for Fraen, against 11 and 135 for LW. The systems had similar false-positive rates (1.7% for Fraen and 1.4% for LW), but differed significantly regarding detection of confirmed microscopy positives (93.5% and 85.6% respectively, P < 0.00001). A high correlation between both LED systems was found at 400x magnification. CONCLUSIONS: The Fraen LED fluorescence microscopy module performed significantly better than the LW LED at the most efficient 200x magnification. It was also more appreciated by all users. The LW module may perform equally well at higher magnification.

HIV-related incremental yield of bleach sputum concentration and fluorescence technique for the microscopic detection of tuberculosis.

Bleach sputum concentration and fluorescence microscopy (FM) are reportedly more sensitive than direct Ziehl-Neelsen (ZN) sputum smears for tuberculosis detection, and might be particularly valuable for human immunodeficiency virus (HIV)-positive patients excreting fewer bacilli. This study, implemented in Yaoundé, Cameroon, determined the yield from both direct and bleach-concentrated FM and ZN duplicate smears against culture on Löwenstein-Jensen medium, with HIV testing from the sputa. From 418 HIV-positive and 518 HIV-negative tuberculosis suspects, 185 (44.3%) and 243 (46.9%) cultures, respectively, grew Mycobacterium tuberculosis. Direct ZN was positive for, respectively, 87 (47.0%) and 202 (83.1%) of the culture-positive cases. Proportional incremental yield over direct ZN from ZN and FM bleach smears was 14.9% (P < 10(-3)) and 17.2% (P < 10(-4)) for HIV-positive versus 4.9% (P < 10(-2)) and 2.0% (non-significant) for HIV-negative cases. There was no gain from direct FM. Bleach FM showed 2% excess false positives. The bleach concentration, therefore, increases the yield of ZN and FM, particularly from HIV-positive patients, but with a higher risk for false positives with bleach FM. With excellent baseline direct ZN, the gain remains modest. Field studies under real-life conditions are needed to determine whether it is worth the risks and operational challenges in HIV high-prevalence populations. FM was not more sensitive than ZN in this study, probably because of sub-optimal objective power and background staining. Culture on solid media with sparing laurylsulfate decontamination was clearly superior for HIV-positives, but it remains to be seen if culture also leads to more cases started on treatment routinely.

First molecular epidemiology study of Mycobacterium tuberculosis in Burkina Faso.

We conducted a molecular epidemiology study on 120 Mycobacterium tuberculosis isolates from patients presenting pulmonary tuberculosis (TB) in Burkina Faso. Classical antibiogram studies and genetic characterization, using mycobacterial interspersed repetitive-unit-variable-number tandem-repeat (MIRU-VNTR) typing and spoligotyping, were applied after culture. Molecular analysis of specific signatures showed that all TB cases reported in this study were caused by M. tuberculosis and identified no Mycobacterium bovis or Mycobacterium africanum isolates. This result is unexpected, as M. africanum strains were reportedly the etiologic agent in 20% of TB cases 2 decades ago. The comparison of spoligotypes from Burkina Faso with an international spoligotype database (SpolDB4) showed that the majority of isolates belong to major clades of M. tuberculosis (Haarlem, 9%; Latin American-Mediterranean, 30%; and T, 20%). The predominant group of isolates (30%) corresponds to spoligotype 61, described in Cameroon as the "Cameroon family." In Burkina Faso, as in Cameroon, this family could be associated with recent transmission of TB, suggesting a recent expansion in West Africa. Our data suggest a low level of primary drug resistance that may be a positive result of the Directly Observed Therapy Shortcourse program. Besides, based on spoligotyping plus MIRU-VNTR, data showed a high number of clusters in our sample, suggesting a high level of recent TB transmission in Burkina Faso. Nevertheless, an important genetic polymorphism was observed in this country, reflecting an endemicity situation where the control of TB would have less impact in the main towns.

Chromosomal DNA fingerprinting analysis using the insertion sequence IS6110 and the repetitive element DR as strain-specific markers for epidemiological study of tuberculosis in French Polynesia.

The polymorphism of Mycobacterium tuberculosis strains was evaluated in French Polynesia, an area with a low incidence of tuberculosis and a population which has been geographically stable during recent decades. Nonrepetitive strains isolated from 64 patients during 1991 and 1992 were subjected to DNA restriction fragment length polymorphism (RFLP) analysis, using the insertion sequence IS6110 and the repetitive element DR as probes. Thirty-eight different IS6110 RFLP types were identified. They could be clustered in 11 groups. All the members of each group are identical or differ by one to three bands. All the other strains are gathered in the miscellaneous group. In some cases, transmission of strains with identical RFLP types between patients of the same family or between patients living in the same area was identified. Strains exhibiting similar IS6110 RFLP types also exhibited identical DR RFLP patterns, confirming that strains with similar types were genetically linked. Strains belonging to two different IS6110 clusters exhibited the same DR RFLP type. These data may also indicate a common origin for these strains and evolution to new IS6110 types. The results obtained in this study suggest that not only reactivation of latent tuberculous infections but also active transmissions are still occurring in French Polynesia.

Evaluation of tuberculosis transmission in a community by 1 year of systematic typing of Mycobacterium tuberculosis clinical isolates.

Interhuman transmission of Mycobacterium tuberculosis was investigated by using molecular typing, including restriction fragment length polymorphism with probes IS6110, DR (direct repeat) and PGRS (polymorphic GC-rich sequence) and a PCR method using the inverted repeat sequences of IS6110 as primers. From 105 patients hospitalized for tuberculosis during a 1-year survey in three hospitals in Paris, France, 111 isolates were collected and analyzed. Eighty-eight patients were infected with genetically different isolates, demonstrating the clonal heterogeneity of M. tuberculosis in these patients originating from various geographical areas. Fourteen patients were infected by strains clustered with identical fingerprints. An epidemiological relatedness was demonstrated for isolates from only seven of these patients. Thus, the typing of isolates from all tuberculous patients in hospitals during 1 year allows the detection of transmission in the general community. This would improve the case findings, thereby further improving the detection of outbreaks.

Yersinia pestis, the cause of plague, is a recently emerged clone of Yersinia pseudotuberculosis.

Plague, one of the most devastating diseases of human history, is caused by Yersinia pestis. In this study, we analyzed the population genetic structure of Y. pestis and the two other pathogenic Yersinia species, Y. pseudotuberculosis and Y. enterocolitica. Fragments of five housekeeping genes and a gene involved in the synthesis of lipopolysaccharide were sequenced from 36 strains representing the global diversity of Y. pestis and from 12-13 strains from each of the other species. No sequence diversity was found in any Y. pestis gene, and these alleles were identical or nearly identical to alleles from Y. pseudotuberculosis. Thus, Y. pestis is a clone that evolved from Y. pseudotuberculosis 1,500-20,000 years ago, shortly before the first known pandemics of human plague. Three biovars (Antiqua, Medievalis, and Orientalis) have been distinguished by microbiologists within the Y. pestis clone. These biovars form distinct branches of a phylogenetic tree based on restriction fragment length polymorphisms of the locations of the IS100 insertion element. These data are consistent with previous inferences that Antiqua caused a plague pandemic in the sixth century, Medievalis caused the Black Death and subsequent epidemics during the second pandemic wave, and Orientalis caused the current plague pandemic.

Identification of mycobacterium tuberculosis DNA sequences encoding exported proteins by using phoA gene fusions.

The activity of bacterial alkaline phosphatase (PhoA) is dependent on it being exported across the plasma membrane. A plasmid vector (pJEM11) allowing fusions between phoA and genes encoding exported proteins was constructed to study protein export in mycobacteria. Introduction of the Mycobacterium fortuitum beta-lactamase gene (blaF*) into this vector led to the production in M. smegmatis of protein fusions with PhoA activity. A genomic library from M. tuberculosis was constructed in pJEM11 and screened in M. smegmatis for clones with PhoA activity. Sequences of the M. tuberculosis inserts directing the production of protein fusions in these PhoA-positive clones were determined. They include part of the already-known exported 19-kDa lipoprotein, a sequence with similarities to the exported 28-kDa antigen from M. leprae, a sequence encoding a protein sharing conserved amino acid motifs with stearoyl-acyl-carrier-protein desaturases, and unknown sequences. This approach thus appears to identify sequences directing protein export, and we expect that more extensive screening of such libraries will lead to a better understanding of protein export in M. tuberculosis.