Accuracy of Tongue Swab Testing Using Xpert MTB-RIF Ultra for Tuberculosis Diagnosis.

Tongue dorsum swabs have shown promise as alternatives to sputum for detecting Mycobacterium tuberculosis (MTB) in patients with pulmonary tuberculosis (TB). Some of the most encouraging results have come from studies that used manual quantitative PCR (qPCR) to analyze swabs. Studies using the automated Cepheid Xpert MTB/RIF Ultra qPCR test (Xpert Ultra) have exhibited less sensitivity with tongue swabs, possibly because Xpert Ultra is optimized for testing sputum, not tongue swab samples. Using two new sample preprocessing methods that demonstrated good sensitivity in preliminary experiments, we assessed diagnostic accuracy and semi-quantitative signals of Xpert Ultra performed on tongue swabs collected from 183 adults with presumed TB in Kampala, Uganda. Relative to a sputum Xpert Ultra reference standard, the sensitivity of tongue swab Xpert Ultra was 77.8% (95% confidence interval [CI] 64.4-88.0) and specificity was 100.0% (95% CI, 97.2-100.0). When compared to a microbiological reference standard (MRS) incorporating both sputum Xpert Ultra and sputum mycobacterial culture, sensitivity was 72.4% (95% CI, 59.1-83.3) and specificity remained the same. Semi-quantitative Xpert Ultra results were generally lower with tongue swabs than with sputum, and cycle threshold values were higher. None of the eight sputum Xpert Ultra "trace" or "very low" results were detected using tongue swabs. Tongue swabs should be considered when sputum cannot be collected for Xpert Ultra testing, or in certain mass-screening settings. Further optimization of tongue swab analysis is needed to achieve parity with sputum-based molecular testing for TB.

Microfluidic one-step synthesis of alginate microspheres immobilized with antibodies.

Micrometre- and submicrometre-size functionalized beads are frequently used to capture targets of interest from a biological sample for biological characterizations and disease diagnosis. The main challenge of the microbead-based assay is in the immobilization of probe molecules onto the microbead surfaces. In this paper, we report a versatile droplet microfluidics method to fabricate alginate microspheres while simultaneously immobilizing anti-Mycobacterium tuberculosis complex IgY and anti-Escherichia coli IgG antibodies primarily on the porous alginate carriers for specific binding and binding affinity tests. The binding affinity of antibodies is directly measured by fluorescence intensity of stained target bacteria on the microspheres. We demonstrate that the functionalized alginate microspheres yield specificity comparable with an enzyme-linked immunosorbent assay. The high surface area-to-volume ratio of the functionalized porous alginate microspheres improves the detection limit. By using the droplet microfluidics, we can easily modify the size and shape of alginate microspheres, and increase the concentration of functionalized alginate microspheres to further enhance binding kinetics and enable multiplexing.

Rapid extraction and preservation of genomic DNA from human samples.

Simple and rapid extraction of human genomic DNA remains a bottleneck for genome analysis and disease diagnosis. Current methods using microfilters require cumbersome, multiple handling steps in part because salt conditions must be controlled for attraction and elution of DNA in porous silica. We report a novel extraction method of human genomic DNA from buccal swab and saliva samples. DNA is attracted onto a gold-coated microchip by an electric field and capillary action while the captured DNA is eluted by thermal heating at 70 °C. A prototype device was designed to handle four microchips, and a compatible protocol was developed. The extracted DNA using microchips was characterized by qPCR for different sample volumes, using different lengths of PCR amplicon, and nuclear and mitochondrial genes. In comparison with a commercial kit, an equivalent yield of DNA extraction was achieved with fewer steps. Room-temperature preservation for 1 month was demonstrated for captured DNA, facilitating straightforward collection, delivery, and handling of genomic DNA in an environment-friendly protocol.

Outbreak of tuberculosis in a homeless population involving multiple sites of transmission.

SETTING: During 2002-2003, a large outbreak of tuberculosis (TB) occurred among persons using multiple homeless facilities in King County, Washington. OBJECTIVE: To control the transmission of TB in multiple settings. DESIGN: In 2002, contacts exposed to patients in homeless facilities were screened using tuberculin skin tests (TSTs) and symptom review. Based on these screening results, sites of transmission were identified and prioritised, and exposed cohorts at these sites were offered intensive screening tests in 2003 (e.g., symptom review, TST, chest radiograph [CXR], sputum examination and culture). Mycobacterium tuberculosis isolates from patients were genotyped using PCR-based methods to identify outbreak-associated patients quickly. RESULTS: During 2002-2003, 48 (15%) of 313 patients diagnosed in King County were outbreak-associated; 47 culture-positive patients had isolates that matched the outbreak strain by genotyping. Three facilities visited by >12 patients in 2002 had a higher prevalence of TST positive results (approximately 30%) among clients compared with the background rate (7%) in the homeless community. Screening contacts with one sputum culture was as sensitive as CXR in detecting TB disease (77% vs. 62%, respectively). CONCLUSIONS: A comprehensive, resource-intensive approach likely helped to control transmission. This outbreak highlights the vulnerability of homeless populations and the need to maintain robust TB programs in urban settings.

The white morphotype of Mycobacterium avium-intracellulare is common in infected humans and virulent in infection models.

Isolates of Mycobacterium avium-intracellulare (MAI) form multiple colony types named red-opaque, white-opaque, red-transparent (RT), and white-transparent (WT). The newly discovered WT morphotype is multidrug resistant relative to other variants in vitro. To determine whether the WT morphotype occurs in humans, 32 MAI-positive clinical samples from 2 sites were plated directly onto indicator agar without prior passage in vitro. WT was the predominant morphotype in 26 (81%) of these samples and was absent in only 2 samples. WT variants grew better than isogenic RT variants in mouse and human macrophage models of infection, and RT clones that passed through such systems underwent rapid shifts to the WT morphotype. The RT morphotype was heterogeneous with regard to infectivity. In summary, the white morphotype was common in humans and was favored in disease models. It may play an important role in the establishment and persistence of MAI infection.

Drug susceptibility testing of Mycobacterium tuberculosis: a neglected problem at the turn of the century.

The prevailing opinion in most of the world is that drug susceptibility testing of Mycobacterium tuberculosis isolates may not be necessary, especially in countries where the diagnosis of tuberculosis is based predominantly on sputum smear examination without culture isolation, and even if it were functionally important it is too expensive to be practical. However, we believe that the spread of primary drug resistance calls for immediate action to prevent the real threat in a number of countries of an epidemic of incurable polyresistant tuberculosis. One step in this direction, among other necessary measures, is implementation of a system for timely detection of drug resistance in new patients. An overview of the currently available methods for drug susceptibility testing, and those under development, is presented in this article. Our aim is to stimulate discussion regarding where, when, and which methods can and should be implemented now, and which of them has the best potential for the future. This overview includes phenotypic approaches based on conventional cultivation and other techniques, as well as perspectives for the genotypic principle. Regardless of the methods chosen for now or to be implemented in the future, they are all laboratory methods, and we contend that dreams of a revolutionary method which can do away with the laboratory are unrealistic, at least for the time being. We argue that it is essential to create a system of direct, centralized laboratory services in order to make drug susceptibility testing reliable, practical and affordable. Such centralized laboratories would provide both more timely and trustworthy diagnosis of tuberculosis, and facilitate detection of primary drug resistance. Although such systems would require significant initial investment, we suggest that, over time, they would prove highly cost-effective for many countries.

Detection of stable pre-rRNA in toxigenic Pseudo-nitzschia species.

Nucleotide sequence analysis of ribosomal DNA (rDNA) spacer regions is useful for taxonomic comparisons of closely related microorganisms. These regions have been less useful for routine microbial identification and detection, partly because rRNA precursors (pre-rRNAs) in microbial cells are assumed to be too labile to be detectable by high-throughput probe hybridization methods. We characterized the sequence diversity and physiological stability of pre-rRNA in the toxigenic marine diatoms Pseudo-nitzschia australis, P. multiseries, and P. pungens. As with nucleotide sequences of the first internal transcribed spacer (ITS1) reported previously, sequences of ITS2 and the 5' external transcribed spacer (ETS1) exhibited considerable divergence among these species, including large insertions-deletions detectable by PCR-based spacer length analysis. In slot blot hybridization assays on RNA extracted from lysates of Pseudo-nitzschia cells, oligonucleotide probes directed to pre-rRNA spacers generated much stronger signals than did complementary probes directed to the coding strands of the rDNAs, indicating that the pre-rRNA-targeted probes detected multicopy transcripts. A group of probes directed to a discrete 90-base region within the ITS1 pre-rRNA gave no detectable signal, suggesting that this region is degraded early in the rRNA maturation pathway. Other pre-rRNA regions were always detectable and, in marked contrast to prokaryotic systems analyzed in this manner, were stable and abundant in both actively dividing and nondividing cells. Long, multilabeled RNA probes, which would exhibit considerable cross-reactivity if directed to mature rRNA sequences, detected species-specific pre-rRNA sequences from as few as 1,000 cells. Pre-rRNA is a potentially useful molecular target for detecting and identifying Pseudo-nitzschia species and possibly other unicellular eukaryotes as well.

Depletion of pre-16S rRNA in starved Escherichia coli cells.

Specific hybridization assays for intermediates in rRNA synthesis (pre-rRNA) may become useful for monitoring the growth activity of individual microbial species in complex natural systems. This possibility depends upon the assumption that rRNA processing in microbial cells continues after growth and pre-rRNA synthesis cease, resulting in drainage of the pre-rRNA pool. This is not the case in many eukaryotic cells, but less is known about the situation in bacteria. Therefore, we used DNA probes to measure steady-state cellular pre-16S rRNA pools during growth state transitions in Escherichia coli. Pre-16S rRNA became undetectable when cells entered the stationary phase on rich medium and was replenished upon restoration of favorable growth conditions. These fluctuations were of much greater magnitude than concurrent fluctuations in the mature 16S rRNA pool. The extent of pre-16S rRNA depletion depended upon the circumstances limiting growth. It was significantly more pronounced in carbon-energy-starved cells than in nitrogen-starved cells or in cells treated with energy uncouplers. In the presence of the transcriptional inhibitor rifampin, rates of pre-16S rRNA depletion in carbon-energy-starved cells and nitrogen-starved cells were similar, suggesting that the difference between these conditions resides primarily at the level of pre-rRNA synthesis. Chloramphenicol, which inhibits the final steps in rRNA maturation, halted pre-16S rRNA depletion under all conditions. The data show that E. coli cells continue to process pre-rRNA after growth and rrn operon transcription cease, leading to drainage of the pre-rRNA pool. This supports the feasibility of using pre-rRNA-targeted probes to monitor bacterial growth in natural systems, with the caveat that patterns of pre-rRNA depletion vary with the conditions limiting growth.

Detection of rifampin- and ciprofloxacin-resistant Mycobacterium tuberculosis by using species-specific assays for precursor rRNA.

rRNA precursor (pre-rRNA) molecules carry terminal stems which are removed during rRNA synthesis to form the mature rRNA subunits. Their abundance in bacterial cells can be markedly affected by antibiotics which directly or indirectly inhibit RNA synthesis. We evaluated the feasibility of rapidly detecting antibiotic-resistant Mycobacterium tuberculosis strains by measuring the effects of brief in vitro antibiotic exposure on mycobacterial pre-rRNA. By hybridizing extracted M. tuberculosis nucleic acid with radiolabeled nucleic acid probes specific for pre-16S rRNA stem sequences, we detected clear responses to rifampin and ciprofloxacin within 24 and 48 h, respectively, of exposure of cultured cells to these drugs. Detectable pre-rRNA was depleted in susceptible cells but remained abundant in resistant cells. In contrast, no measurable responses to isoniazid or ethambutol were observed. Probes for pre-rRNA were specific for the M. tuberculosis complex when tested against a panel of eight Mycobacterium species and 48 other bacteria. After 24 h of incubation with rifampin, resistant M. tuberculosis strains were detectable in a reverse transcriptase PCR assay for pre-rRNA with a calculated lower limit of sensitivity of approximately 10(2) cells. Susceptible cells were negative in this assay at over 500 times the calculated lower limit of sensitivity. This general approach may prove useful for rapidly testing the susceptibility of slowly growing Mycobacterium species to the rifamycin and fluoroquinolone drugs and, with possible modifications, to other drugs as well.

Detection of rifampin-resistant bacteria using DNA probes for precursor rRNA.

Ribosomal RNA precursor (pre-rRNA) molecules have terminal domains (tails) which are removed during late steps in rRNA processing, to yield the mature rRNA subunits. Transcriptional inhibitors such as rifampin can deplete pre-rRNA in sensitive cells by inhibiting de novo pre-rRNA synthesis while allowing maturation to proceed. We developed direct DNA probe assays for pre-rRNA tail sequences of Escherichia coli, and evaluated their ability to rapidly distinguish rifampin-resistant from rifampin-sensitive strains in cultures treated with the drug. Pre-rRNA became undetectable in sensitive cells less than a generation time after rifampin exposure, but remained abundant in resistant cells. Resistant cells were detectable by this method against a 100-fold excess of sensitive cells, showing that this method can detect resistant mutants even when present as a small percentage of a pathogen population. Our data indicate that the response of pre-rRNA to antibiotic treatment is sufficient in rate and magnitude to make it a useful metabolic marker for antibiotic sensitivity.

Oligonucleotide probes for mutans streptococci.

Oligodeoxyribonucleotide probes complementary to hypervariable regions of the 16S ribosomal RNA were designed for specificity toward Streptococcus mutans and Streptococcus sobrinus. The probes were tested for specificity and sensitivity by hybridization with nucleic acid from over 100 mutans and non-mutans oral streptococci and other common oropharyngeal bacteria. Probes designated SM002 and SM010 were 100% sensitive and > 99% specific for S. mutans. Probe SSP001, designed to detect both S. mutans and S. sobrinus, was 88% sensitive and > 99% specific. The probes were able to detect nucleic acid extracted from 3 x 10(4)-1 x 10(5) homologous bacteria. Sensitivity did not vary significantly with the growth state of the cells, except for diminished signals when using nucleic acid extracted from very old cultures. The probes correctly identified 72 S. mutans colonies isolated from 10 volunteer saliva samples, using sugar utilization patterns as a reference standard. Ten isolates resembling S. mutans by colony morphology but not by sugar utilization patterns were correctly distinguished from mutans streptococci by the probes. These results demonstrate that oligonucleotide probes can accurately identify S. mutans in saliva samples.

Sugars induce the Agrobacterium virulence genes through a periplasmic binding protein and a transmembrane signal protein.

Phenolic plant metabolites such as acetosyringone induce transcription of the virulence (vir) genes of Agrobacterium tumefaciens through the transmembrane VirA protein. We report here that certain sugars induce the vir genes synergistically with phenolic inducers by way of a distinct regulatory pathway that includes VirA and a chromosomally encoded virulence protein, ChvE. Sequence comparison showed that ChvE is a periplasmic galactose-binding protein corresponding to the GBP1 protein isolated from Agrobacterium radiobacter. Like homologous sugar-binding proteins in Escherichia coli, ChvE was required for chemotaxis toward galactose and several other sugars. These sugars strongly induced vir gene expression in wild-type cells when acetosyringone was absent or present in low concentrations. Mutations in chvE abolished vir gene induction by sugars and resulted in a limited host range for infection but did not affect vir gene induction by acetosyringone. A mutant lacking the periplasmic domain of VirA exhibited the same regulatory phenotype and limited host range as chvE mutants. These data show that the vir genes are regulated by two separate classes of plant-derived inducers by way of distinct regulatory pathways that can be separated by mutation. Induction by sugars is essential for infection of some but not all plant hosts.

Osmosensitivity phenotypes of Agrobacterium tumefaciens mutants that lack periplasmic beta-1,2-glucan.

The periplasmic cyclic beta-1,2-glucan of Agrobacterium tumefaciens is believed to maintain high osmolarity in the periplasm during growth of the bacteria on low-osmotic-strength media. Strains with mutations in the chvA or chvB gene do not accumulate beta-1,2-glucan in their periplasm and exhibit pleiotropic phenotypes, including inability to form crown gall tumors on plants. We examined the effects of medium osmolarity to determine whether some or all of these phenotypes result from suboptimal periplasmic osmolarity. The mutants grew more slowly than wild-type cells and exhibited altered periplasmic and cytoplasmic protein content when cultured in low-osmotic-strength media, but not when cultured in high-osmotic-strength media. These observations support a role for periplasmic glucan in osmoadaptation. However, the mutants were avirulent and exhibited reduced motility regardless of the osmolarity of the medium. Therefore, beta-1,2-glucan may play roles in virulence and motility that are unrelated to its role in osmoadaptation.

A chromosomal Agrobacterium tumefaciens gene required for effective plant signal transduction.

The vir gene products of Agrobacterium tumefaciens carry out the transfer of T-DNA to the plant genome. Effective transcriptional induction of the vir genes by plant signal molecules is controlled by two vir gene products, VirA and VirG. In this study we have identified and cloned a chromosomal region which is also required for vir gene induction. Transposon insertions within this region reduce induction significantly and strongly attenuate virulence, resulting in a restricted host range for infection. The reduction in vir gene transcription can be partially overcome by high concentrations of the inducer molecule acetosyringone. Expression of virG at low pH and low phosphate concentrations, which is independent of plant signals, is not affected by these mutations. Sequence analysis of the region revealed two divergent open reading frames, which we have designated chvE and ORF1. Several transposon insertions mapped in chvE; this resulted in attenuated virulence. chvE codes for a putative protein which is homologous to two periplasmic receptor proteins involved in chemotaxis and uptake of sugars. Whether ORF1 is required for virulence is uncertain. One transposon insertion resulting in avirulence maps in or near the 5' end of ORF1, and several which do not affect virulence map in its 3' end. ORF1 codes for a putative protein which is homologous to a family of transcriptional activator proteins.

Biochemical characterization of avirulent exoC mutants of Agrobacterium tumefaciens.

The synthesis of periplasmic beta(1-2)glucan is required for crown gall tumor formation by Agrobacterium tumefaciens and for effective nodulation of alfalfa by Rhizobium meliloti. The exoC (pscA) gene is required for this synthesis by both bacteria as well as for the synthesis of capsular polysaccharide and normal lipopolysaccharide. We tested the possibility that the pleiotropic ExoC phenotype is due to a defect in the synthesis of an intermediate common to several polysaccharide biosynthetic pathways. Cytoplasmic extracts from wild-type A. tumefaciens and from exoC mutants of A. tumefaciens containing a cloned wild-type exoC gene synthesized in vitro UDP-glucose from glucose, glucose 1-phosphate, and glucose 6-phosphate. Extracts from exoC mutants synthesized UDP-glucose from glucose 1-phosphate but not from glucose or glucose 6-phosphate. Membranes from exoC mutant cells synthesized beta(1-2)glucan in vitro when exogenous UDP-glucose was added and contained the 235-kilodalton protein, which has been shown to carry out this synthesis in wild-type cells. We conclude that the inability of exoC mutants to synthesize beta(1-2)glucan is due to a deficiency in the activity of the enzyme phosphoglucomutase (EC 2.7.5.1), which in wild-type bacteria converts glucose 6-phosphate to glucose 1-phosphate, an intermediate in the synthesis of UDP-glucose. This interpretation can account for all of the deficiencies in polysaccharide synthesis which have been observed in these mutants.

Role for [corrected] Agrobacterium tumefaciens ChvA protein in export of beta-1,2-glucan.

Functional chvA and chvB genes are required for attachment of Agrobacterium tumefaciens to plant cells, an early step in crown gall tumor formation. Strains defective in these loci do not secrete normal amounts of cyclic beta-1,2-glucan. Whereas chvB is required for beta-1,2-glucan synthesis, the role of chvA in glucan synthesis or export has not been clearly defined. We found that cultures of chvA mutants contained as much neutral beta-1,2-glucan in the cell pellets as did the wild type, with no detectable accumulation of glucan in the culture supernatant. The cytoplasm of chvA mutant cells contained over three times more soluble beta-1,2-glucan than did the cytoplasm of the wild-type parent. Unlike the wild type, chvA mutants contained no detectable periplasmic glucan. The amino acid sequence of chvA is highly homologous to the sequences of bacterial and eucaryotic export proteins, as observed previously in the case of ndvA, a rhizobial homolog of chvA. Strong sequence homology within this family of export proteins is concentrated in the carboxy-terminal portions of the proteins, but placement of consensus ATP-binding sites, internal signal sequences, and hydrophobic domains are conserved over their entire lengths. These data suggest a model for beta-1,2-glucan synthesis in A. tumefaciens in which glucan is synthesized inside the inner membrane with the participation of ChvB and transported across the inner membrane with the participation of ChvA.

Identification of the product of an Agrobacterium tumefaciens chromosomal virulence gene.

The chvB operon of Agrobacterium tumefaciens is required for bacterial attachment to plant cells and for efficient crown gall tumor formation. As defined by the virulence phenotypes of mutants with transposon insertions mapping in the region, the operon was previously mapped to a 5-kilobase (kb) stretch of chromosomal DNA. We report here that the operon is actually about 8.5 kb long and that it contains a 7-kb gene coding for a large membrane protein involved in the synthesis of cyclic beta-1,2-glucan. Mutants with transposon insertions within the 5-kb phenotypically defined operon do not synthesize this functional protein, do not synthesize beta-1,2-glucan, and do not form tumors. However, mutants with insertions that map up to 3.5 kb downstream of the phenotypically defined operon synthesize truncated proteins that are active in beta-1,2-glucan synthesis. These mutants form tumors. The truncated proteins correspond closely in size with the map positions of the insertions, suggesting that the insertions truncate the proteins by translational termination. A plasmid that contains only the phenotypically defined chvB operon also codes for a truncated protein. A fusion product between the protein and beta-galactosidase carried on a Tn3-HoHo1 insertion was observed in one mutant. Partial trypsin digestion of wild-type inner membranes generated truncated proteins that were active in beta-1,2-glucan synthesis, demonstrating that a large portion of the protein is not required for beta-1,2-glucan synthesis. The correlation between beta-1,2-glucan synthesis by the truncated proteins and tumorigenesis strongly implicates the polysaccharide product of this protein in tumor formation.

Common loci for Agrobacterium tumefaciens and Rhizobium meliloti exopolysaccharide synthesis and their roles in plant interactions.

Mutants of Rhizobium meliloti have been isolated which are deficient in exopolysaccharide (EPS) production and effective nodulation of alfalfa (J. A. Leigh, E. R. Signer, and G. C. Walker, Proc. Natl. Acad. Sci. USA 82:6231-6235, 1985). We isolated approximately 100 analogous EPS-deficient (Exo) mutants of the closely related plant pathogen Agrobacterium tumefaciens, including strains whose EPS deficiencies were specifically complemented by each of five cloned R. meliloti exo loci. We also cloned A. tumefaciens genes which complemented EPS defects in three of the R. meliloti Exo mutants. In two of these cases, symbiotic defects were also complemented. All of the A. tumefaciens Exo mutants formed normal crown gall tumors on four different plant hosts, except ExoC mutants, which were nontumorigenic and unable to attach to plant cells in vitro. Like their R. meliloti counterparts, A. tumefaciens Exo mutants were deficient in production of succinoglycan, the major acidic EPS species produced by both genera. A. tumefaciens ExoC mutants also produced extremely low levels of another major EPS, cyclic 1,2-beta-D-glucan. This deficiency has been noted previously in a different set of nontumorigenic, attachment-defective A. tumefaciens mutants.