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Culturing before DNA extraction represents a major time-consuming step in whole-genome sequencing of slow-growing bacteria, such as Mycobacterium tuberculosis. We report a workflow to extract DNA from frozen isolates without reculturing. Prepared libraries and sequence data were comparable with results from recultured aliquots of the same stocks., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

Nutritional supplementation to tuberculosis (TB) patients has been associated with increased weight and reduced mortality, but its effect on the pharmacokinetics of first-line anti-TB drugs is unknown. A cohort of 100 TB patients (58 men; median age, 35 [interquartile range {IQR}, 29 to 40] years, and median body mass index [BMI], 18.8 [17.3 to 19.9] kg/m(2)) were randomized to receive nutritional supplementation during the intensive phase of TB treatment. Rifampin plasma concentrations were determined after 1 week and 2 months of treatment. The effects of nutritional supplementation, HIV, time on treatment, body weight, and SLCO1B1 rs4149032 genotype were examined using a population pharmacokinetic model. The model adjusted for body size via allometric scaling, accounted for clearance autoinduction, and detected an increase in bioavailability (+14%) for the patients in the continuation phase. HIV coinfection in patients not receiving the supplementation was found to decrease bioavailability by 21.8%, with a median maximum concentration of drug in serum (Cmax) and area under the concentration-time curve from 0 to 24 h (AUC0-24) of 5.6 μg/ml and 28.6 μg · h/ml, respectively. HIV-coinfected patients on nutritional supplementation achieved higher Cmax and AUC0-24 values of 6.4 μg/ml and 31.6 μg · h/ml, respectively, and only 13.3% bioavailability reduction. No effect of the SLCO1B1 rs4149032 genotype was observed. In conclusion, nutritional supplementation during the first 2 months of TB treatment reduces the decrease in rifampin exposure observed in HIV-coinfected patients but does not affect exposure in HIV-uninfected patients. If confirmed in other studies, the use of defined nutritional supplementation in HIV-coinfected TB patients should be considered in TB control programs. (This study has the controlled trial registration number ISRCTN 16552219.)., (Copyright © 2014, American Society for Microbiology. All Rights Reserved.)

Transmission of Mycobacterium tuberculosis continues at high rates among Greenland-born persons in Greenland and Denmark, with 203 and 450 notified cases per 10(5) population, respectively, in the year 2010. Here, we document that the predominant M. tuberculosis outbreak strain C2/1112-15 of Danish origin has been transmitted to Greenland-born persons in Denmark and subsequently to Greenland, where it is spreading at worrying rates and adding to the already heavy tuberculosis burden in this population group. It is now clear that the C2/1112-15 strain is able to gain new territories using a new population group as the "vehicle." Thus, it might have the ability to spread even further, considering the potential clinical consequences of strain diversity such as that seen in the widely spread Beijing genotype. The introduction of the predominant M. tuberculosis outbreak strain C2/1112-15 into the Arctic circumpolar region is a worrying tendency which deserves attention. We need to monitor whether this strain already has, or will, spread to other countries.

Molecular genotyping of Mycobacterium tuberculosis has proved to be a powerful tool in tuberculosis surveillance, epidemiology, and control. Based on results obtained through 15 years of nationwide IS6110 restriction fragment length polymorphism (RFLP) genotyping of M. tuberculosis cases in Denmark, a country on the way toward tuberculosis elimination, we discuss M. tuberculosis transmission dynamics and point to areas for control interventions. Cases with 100% identical genotypes (RFLP patterns) were defined as clustered, and a cluster was defined as cases with an identical genotype. Of 4,601 included cases, corresponding to 76% of reported and 97% of culture-verified tuberculosis cases in the country, 56% were clustered, of which 69% were Danes. Generally, Danes were more often in large clusters (≥ 50 persons), older (mean age, 45 years), and male (male/female ratio, 2.5). Also, Danes had a higher cluster frequency within a 2-year observation window (60.8%), and higher clustering rate of new patterns over time, compared to immigrants. A dominant genotype, cluster 2, constituted 44% of all clustered and 35% of all genotyped cases. This cluster was primarily found among Danish males, 30 to 59 years of age, often socially marginalized, and with records of alcohol abuse. In Danes, cluster 2 alone was responsible for the high cluster frequency level. Immigrants had a higher incidence of clustered tuberculosis at a younger age (0 to 39 years). To achieve tuberculosis elimination in Denmark, high-risk transmission environments, like the cluster 2 environment in Danes, and specific transmission chains in immigrants in the capital area, e.g., homeless/socially marginalized Somalis/Greenlanders, often with alcohol abuse, must be targeted, including groups with a high risk of reactivation.

A new immunodiagnostic test based on the Mycobacterium tuberculosis-specific antigens CFP-10/ESAT-6(QFT-RD1) has been launched as an aid in the diagnosis of latent tuberculosis (TB) infection (LTBI). The aim of this study was to evaluate this test for the diagnosis of active TB. Eighty-two patients with suspicion of TB and 39 healthy BCG-vaccinated persons were enrolled. Forty-eight had active TB, 25 did not, and 9 were excluded. Sensitivity and specificity of the test for active TB were evaluated in a prospective blinded manner in patients suspected of TB. The sensitivity of the QFT-RD1 was 85% (40/48; confidence interval [CI], 75 to 96), and it was higher than the sensitivity of microscopy, 42% (20/48; CI, 27 to 56; P = 0.001), and culture, 59% (27/46; CI, 44 to 73; P = 0.009). Of patients with extrapulmonary TB, 92% (12/13) were QFT-RD1 positive, whereas only 31% (4/13) were positive by microscopy and 42% (5/12) by culture (P < 0.05), and 87% (13/15) of those who were negative by both microscopy and culture were QFT-RD1 positive. By combining microscopy and culture with the QFT-RD1 test, sensitivity increased to 96% (CI, 90 to 102). Ten of 25 (40%) non-TB patients were QFT-RD1 positive, resulting in a specificity of 60%. However, 80% (8/10) of these had risk-factors for TB, indicating latent infection in this group. In healthy controls, only 3% (1/39) were QFT-RD1 positive. In conclusion, the QFT-RD1 test is sensitive for diagnosis of TB, especially in patients with negative microscopy and culture. The accuracy of the QFT-RD1 test will vary with the prevalence of LTBI. We suggest that the QFT-RD1 test could be a very useful supplementary tool for the diagnosis of TB.

Does immigration from a high-prevalence area contribute to an increased risk of tuberculosis in a low-incidence country? The tuberculosis incidence in Somalia is among the highest ever registered. Due to civil war and starvation, nearly half of all Somalis have been forced from their homes, causing significant migration to low-incidence countries. In Denmark, two-thirds of all tuberculosis patients are immigrants, half from Somalia. To determine the magnitude of Mycobacterium tuberculosis transmission between Somalis and Danes, we analyzed DNA fingerprint patterns of isolates collected in Denmark from 1992 to 1999, comprising >97% of all culture-positive patients (n = 3,320). Of these, 763 were Somalian immigrants, 55.2% of whom shared identical DNA fingerprint patterns; 74.9% of these were most likely infected before their arrival in Denmark, 23.3% were most likely infected in Denmark by other Somalis, and 1.8% were most likely infected by Danes. In the same period, only 0.9% of all Danish tuberculosis patients were most likely infected by Somalis. The Somalian immigrants in Denmark could be distributed into 35 different clusters with possible active transmission, of which 18 were retrieved among Somalis in the Netherlands. This indicated the existence of some internationally predominant Somalian strains causing clustering less likely to represent recent transmission. In conclusion, M. tuberculosis transmission among Somalis in Denmark is limited, and transmission between Somalis and Danes is nearly nonexistent. The higher transmission rates between nationalities found in the Netherlands do not apply to the situation in Denmark and not necessarily elsewhere, since many different factors may influence the magnitude of active transmission.

Mycobacterium tuberculosis complex strains cultured in Denmark have been analyzed by IS6110 restriction fragment length polymorphism (RFLP) on a routine basis from 1992 and onwards. Due to the influx of immigrants with tuberculosis, the number of strains harboring only one to five copies of IS6110 has increased steadily. Since the discriminatory power of IS6110 fingerprinting for such strains is poor, we have performed additional genotyping of all low-copy-number strains by the recently described PCR-based method known as spoligotyping. A total of 311 clinical strains were typed: 14 Mycobacterium bovis BCG, 48 M. bovis, and 249 M. tuberculosis strains. Spoligotyping correctly differentiated M. bovis and M. bovis BCG from M. tuberculosis strains, but it did not differentiate M. bovis from M. bovis BCG. All M. bovis BCG strains exhibited identical spoligotype patterns. The discriminatory power of spoligotyping of low-copy-number M. tuberculosis strains was higher than that of IS6110 fingerprinting. Based on RFLP typing solely, 83% of the low-copy-number M. tuberculosis strains were found to form part of a cluster, and 75% were found to form a cluster on the basis of spoligotyping. When the two techniques were combined, the amount of clustering decreased to 55%. The combination of these two techniques might be valuable in studying the epidemiology of M. tuberculosis strains harboring few copies of the IS6110 element.

In the present study restriction fragment length polymorphism analyses with the recently described insertion sequence IS1245 as a probe was performed with clinical Mycobacterium avium complex strains cultured in Denmark during a 2-year period. The overall aim of the study was to disclose potential routes of transmission of these microorganisms. As a first step, the genetic diversity among isolates from AIDS patients and non-human immunodeficiency virus (HIV)-infected patients was described. In addition, a number of isolates from nonhuman sources cultured during the same period were analyzed and compared to the human isolates. A total of 203 isolates from AIDS patients (n = 90), non-HIV-infected patients (n = 91), and nonhuman sources (n = 22) were analyzed. The presence of IS1245 was restricted to Mycobacterium avium subsp. avium isolates. The majority of human isolates had large numbers of IS1245 copies, while nonhuman isolates could be divided into a high-copy-number group and a low-copy-number group. Groups of identical strains were found to be geographically widespread, comprising strains from AIDS patients as well as strains from non-HIV-infected patients. Samples of peat (to be used as potting soil) and veterinary samples were found to contain viable M. avium isolates belonging to genotypes also found in humans.

Recently a novel insertion element, IS1245, has been described and suggested for use as a probe in restriction fragment length polymorphism studies of Mycobacterium avium strains. An important issue in this context is the stability of the insertion element. We analyzed single colonies of M. avium cultures and found frequent small one- to two-band changes. However, following repeated in vitro passages over 1 year, similar one- to two-band changes were observed in the IS1245 patterns of only six M. avium strains investigated.

Results from DNA fingerprint analyses of Mycobacterium tuberculosis complex isolates from tuberculosis (TB) patients diagnosed during 5 years in Denmark are presented. The lack of success in eradicating TB in this low-incidence country may be explained by an unrecognized high frequency of active TB transmission (57%) among native Danes. Only two strains of M. tuberculosis are responsible for 40% of all clustered cases of TB among Danes.

Culture fluids after growth of Mycobacterium bovis BCG on Sauton medium contain actively secreted proteins and proteins released by bacterial lysis. BCG culture fluids and sonicates of Mycobacterium tuberculosis and Mycobacterium paratuberculosis were tested after separation by gel filtration and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The localization of marker proteins was determined by enzyme-linked immunosorbent assay and Western blotting with selected monoclonal antibodies of known specificities. Soluble secreted proteins (MPB64 and proteins of the antigen 85 complex) and three heat shock proteins (DnaK, GroEL, and GroES) were recovered in a single peak after gel filtration, indicating their occurrence as a free monomer in the culture fluid and cytosol, respectively. Other constituents eluted in two distinct peaks during gel filtration. The first peak corresponded to the void volume, indicating complex formation between several proteins or attachment to lipids in the surface layer or the cytoplasmic membrane; the second peak corresponded to the expected monomer size indicated by SDS-PAGE under conditions that separate proteins from each other during sample preparation. The two-peak group contained constituents with known lipid contents, the 19- and 38-kDa lipoproteins and lipoarabinomannan. The 26-kDa form of MPB83 behaved similarly. After extraction with Triton X-114, these constituents entered into the detergent phase, confirming the lipoprotein nature of 26-kDa MPB83. The MPB83 molecule was shown to be available on the surface of BCG Tokyo bacilli for reaction with monoclonal antibody MBS43 by flow cytometry.

During 1994, a cross-contamination problem leading to false-positive cultures of Mycobacterium tuberculosis was revealed in a mycobacteriology laboratory processing 30,000 to 35,000 samples per year. Molecular strain typing based on restriction fragment length polymorphism confirmed the contaminations. Out of 1,439 positive cultures of Mycobacterium tuberculosis, 49 samples from 48 patients were suspected to be cross-contaminated. In 37 cases, growth was observed both in BACTEC vials and on solid media, indicating that the contamination took place during the processing of the samples. The majority of the contaminated samples had been handled by the same technician.

The gene encoding the immunogenic protein MPT64 found in culture filtrates of Mycobacterium tuberculosis H37Rv was expressed in Escherichia coli K-12 and purified as a recombinant protein. The purified recombinant MPT64 elicited delayed-type hypersensitivity (DTH) in outbred guinea pigs sensitized with Mycobacterium bovis BCG Tokyo. The skin reactions were comparable to those obtained with native MPT64. No skin reactions were observed when either recombinant MPT64 or native MPT64 was used in guinea pigs sensitized with M. bovis BCG Danish 1331. Amino- and carboxy-terminal deletion mutants of MPT64 were purified as fusion proteins for the mapping of DTH-inducing epitopes on recombinant MPT64 by use of the guinea pig skin test model. The part of the molecule responsible for the biological activity was located at the carboxy-terminal end. Further studies with overlapping synthetic peptides have pinpointed the biological activity at a single DTH-inducing epitope consisting of 15 residues between amino acids Gly-173 and Ala-187. Screening by PCR of 56 clinical isolates of M. tuberculosis from Danish and Tanzanian patients demonstrated the presence of mpt64 in all of the strains. These results point to MPT64 as a possible candidate for a skin test reagent specific for diagnosis of human tuberculosis.

The incidence of tuberculosis (TB) is increasing all over the world, including in countries with a high standard of living and good social security. Denmark represents such a region. Furthermore, it is a small country (5 million inhabitants) with a long tradition in TB control, including a centralization of the bacteriological diagnostic facility. The present study was intended to analyze the transmission of Mycobacterium tuberculosis in a country in which TB has low endemicity by a combination of conventional epidemiological approaches and DNA fingerprinting techniques, whereby individual bacterial strains can be traced. M. tuberculosis isolates from 92% of all new cases of bacteriologically verified TB in Denmark during 1992 were subjected to IS6110 DNA fingerprinting to visualize the DNA restriction fragment length polymorphism (RFLP) patterns of the isolated strains. The data obtained from the RFLP analyses were interpreted by using demographic data, such as age, sex, ethnicity, and residence, for the patients. The risk factors among the patients for being part of an active chain of transmission, as opposed to demonstrating reactivation of a previously acquired latent infection, were estimated by statistical analyses. The magnitude of TB transmission in 1992 in Denmark was determined, and transmitted infections were shown to comprise at least one quarter of the total number of cases. Almost half of the TB cases involved patients of foreign origin. However, most of these isolates showed unique DNA fingerprint patterns and were rarely part of an active chain of transmission. The major chains of recent transmission were localized to distinct geographical regions in the country. TB is frequent among immigrants, especially from Asia and Africa, but it is apparently readily suspected, diagnosed, and treated by the health care system. Danish patients with pulmonary symptoms are not primarily suspected to have TB and, therefore, play an important role in recent TB transmission in Denmark.

A novel immunogenic antigen, the 6-kDa early secretory antigenic target (ESAT-6), from short-term culture filtrates of Mycobacterium tuberculosis was purified by hydrophobic interaction chromatography and anion-exchange chromatography by use of fast protein liquid chromatography. The antigen focused at two different pIs of 4.0 and 4.5 during isoelectric focusing, and each of these components separated into three spots ranging from 4 to 6 kDa during two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The apparent differences in molecular masses or pIs of these isoforms were not due to posttranslational glycosylation. The molecular weight of the purified native protein was determined by applying gel filtration and nondenaturing polyacrylamide gel electrophoresis and found to be 24 kDa. ESAT-6 is recognized by the murine monoclonal antibody HYB 76-8, which was used to screen a recombinant lambda gt11 M. tuberculosis DNA library. A phage expressing a gene product recognized by HYB 76-8 was isolated, and a 1.7-kbp fragment of the mycobacterial DNA insert was sequenced. The structural gene of ESAT-6 was identified as the sequence encoding a polypeptide of 95 amino acids. The N terminus of the deduced sequence could be aligned with the 10 amino-terminal amino acids derived from sequence analyses of the native protein. N-terminal sequence analysis showed that the purified antigen was essentially free from contaminants, and the amino acid analysis of the antigen was in good agreement with the DNA sequence-deduced amino acid composition. Thus, the heterogeneities observed in the pI and molecular weight of the purified antigen do not derive from contaminating proteins but are most likely due to heterogeneity of the antigen itself. Native and recombinant ESAT-6 are immunologically active in that both elicited a high release of gamma interferon from T cells isolated from memory-immune mice challenged with M. tuberculosis. Analyses of subcellular fractions of M. tuberculosis showed the presence of ESAT-6 in cytosol- and cell wall-containing fractions. Interspecies analyses showed the presence of ESAT-6 in filtrates from M. tuberculosis complex species. Among filtrates from mycobacteria not belonging to the M. tuberculosis complex, reactivity was observed in Mycobacterium kansasii, Mycobacterium szulgai, and Mycobacterium marinum.

In order to describe the transmission of tuberculosis (TB) at the clonal level in a defined geographic region during a certain period of time, all isolates of Mycobacterium tuberculosis collected during 1992 from Greenland were subjected to analyses of DNA restriction fragment length polymorphism (RFLP). The RFLP patterns obtained by probing the genomic DNA with the repetitive insertion segment IS6110 revealed a high degree of similarity among the isolates, indicating a relatively high transmission rate and a close relationship between the individual M. tuberculosis clones. This was further confirmed by reprobing the Southern blots with two more-stable genetic markers, IS1081 and the DR sequence. The RFLP patterns were compared with those of 245 M. tuberculosis strains collected from Denmark during the same period (representing 91% of all new, bacteriologically verified cases of TB in Denmark in 1992). One of the three prevalent IS6110-defined clusters was traced to a group of immigrants from Greenland living in a small, defined geographical region in Denmark and to a group of Danish citizens either with known contact with these immigrants or, in other cases, with a record of previous travel or working activities in Greenland. The study showed that the present technique is extremely helpful in monitoring the spread of TB and thereby also contributing to improved disease control.

The gene of the immunogenic protein MPT64 found in culture filtrates of Mycobacterium tuberculosis H37Rv was cloned and sequenced. A comparison showed mpt64 and the gene encoding MPB64 from Mycobacterium bovis BCG Tokyo to be identical except for one silent mutation. The regions encoding the promoter and the signal peptide were also well conserved for the two sequences. Southern blot experiments on genomic mycobacterial DNA showed the presence of mpt64 in the M. tuberculosis substrains H37Rv, H37Ra, and Erdman and in the M. bovis BCG substrains Tokyo, Moreau, and Russian, whereas the M. bovis BCG substrains Glaxo, Pasteur, Canadian, Tice, and Danish 1331 and Mycobacterium leprae lack the gene. Southern blot analyses revealed differences in the restriction enzyme patterns within the M. tuberculosis substrains as well as within the M. bovis BCG substrains, indicating either different chromosomal localization of mpt64 or that mutations have occurred at different locations on the chromosomes. N-terminal and C-terminal deletion mutants were constructed for the mapping of B-cell epitopes on MPT64 with five monoclonal antibodies, C24b1, C24b2, C24b3, L24b4, and L24b5. Western blot (immunoblot) analysis revealed that the murine antibodies bind to one linear and three conformational epitopes.

An international workshop was sponsored by the World Health organization to screen new antimycobacterial monoclonal antibodies and to identify antibodies which could be recommended as standard reagents giving consistent results under differing assay conditions. Fifty-eight antibodies were submitted to the workshop by eight independent laboratories. Nineteen of the antibodies recognized antigens distinct from those identified in earlier workshops, defining at least 10 new protein antigens. Monoclonal antibodies characterized in the workshop provide a set of convenient reagents for further characterization of mycobacterial antigens.

A gene encoding a protein antigen from Mycobacterium tuberculosis with a molecular weight of 40,000 has been sequenced. On the basis of sequence homology and functional analyses, we demonstrated that the protein is an L-alanine dehydrogenase (EC 1.4.1.1). The enzyme was demonstrated in M. tuberculosis and Mycobacterium marinum but not in Mycobacterium bovis BCG. The enzyme may play a role in cell wall synthesis because L-alanine is an important constituent of the peptidoglycan layer. Although no consensus signal sequence was identified, we found evidence which suggests that the enzyme is secreted across the cell membrane. The enzyme was characterized and purified by chromatography, thus enabling further studies of its role in virulence and interaction with the immune system of M. tuberculosis-infected individuals.

A polymerase chain reaction for the specific detection of mycobacteria belonging to the Mycobacterium tuberculosis complex was developed. Using a single primer pair derived from the nucleotide sequence of protein antigen b of M. tuberculosis, we achieved specific amplification of a 419-base-pair DNA fragment in M. tuberculosis and M. bovis. After DNA was extracted from mycobacteria by using a simple, safe lysis procedure, we detected the 419-base-pair sequence in samples containing few mycobacteria. Preliminary data suggested that this technique could be applied to clinical specimens for early and specific diagnosis of tuberculosis.

Only a limited number of proteins from Mycobacterium tuberculosis have so far been shown to possess species-specific epitopes as defined by monoclonal antibodies. One such protein is protein antigen b (Pab) of molecular weight 38,000, which binds the monoclonal antibodies HYT 28, HAT 2, HBT 12, HGT 3, TB 71, and TB 72. The gene encoding this protein was isolated from a lambda gt11 M. tuberculosis DNA library. The nucleotide sequence of the recombinant mycobacterial insert was determined, and an open reading frame of 374 amino acids was identified. The amino acid sequence exhibited 30% homology to a phosphate-binding protein, PstS, from Escherichia coli. The pab gene was subcloned into pBR322 in conjunction with the lacZ gene, and deletions were obtained from the 3' end. The anti-Pab monoclonal antibodies were used to probe crude protein lysates of E. coli transformed with the deletion plasmids. The monoclonal antibodies showed two reactivity patterns; one group of antibodies were dependent on the presence of the ultimate 91 amino acids of the protein, whereas another group of antibodies recognized an antigenic domain located on the middle portion of the molecule. None of the antibodies bound to the N-terminal 117-amino-acid peptide.

Five different murine monoclonal antibodies (MAbs) to Mycobacterium tuberculosis were examined for degree of cross-reactivity with other mycobacterial species by enzyme-linked immunosorbent assay and immunoblotting. One MAb reacted solely with M. tuberculosis and M. bovis BCG. Two of the MAbs reacted with all mycobacterial species examined, whereas two MAbs demonstrated a limited reactivity pattern. The epitopes are located on molecules susceptible to protease treatment, and two of these molecules possess concanavalin A-binding moieties. Two of the antigens defined by these five MAbs are present in tuberculin purified protein derivative.

A genomic lambda gt11 DNA library of Mycobacterium tuberculosis was screened for expression of mycobacterial protein antigens with murine monoclonal antibodies. The reactivity patterns of the monoclonal antibodies ranged from those showing a limited interspecies reactivity to antibodies widely cross-reactive among different mycobacterial species. Twelve recombinant bacteriophages were isolated, containing eight mycobacterial genes (paa, pab, pac, pad, paeA, paeB, pafA, and pafB) encoding protein antigens. Physical maps of the phages were generated and the products of the recombinant genes were analyzed by immunoblotting techniques. PaeA and PaeB are distinct proteins but were shown to share an epitope. A similar condition was observed between PafA and PafB. Among the phages isolated, two groups expressed epitopes specific for M. tuberculosis and Mycobacterium bovis BCG. One group of phages produced an antigenic determinant which is found in M. tuberculosis and Mycobacterium marinum but not in M. bovis BCG.