Commercial products to preserve specimens for tuberculosis diagnosis: a systematic review.

SETTING: Eliminating tuberculosis in high-burden settings requires improved diagnostic capacity. Important tests such as Xpert® MTB/RIF and culture are often performed at centralised laboratories that are geographically distant from the point of specimen collection. Preserving specimen integrity during transportation, which could affect test performance, is challenging. OBJECTIVE: To conduct a systematic review of commercial products for specimen preservation for a World Health Organization technical consultation. DESIGN: Databases were searched up to January 2018. Methodological quality was assessed using Quality Assessment of Technical Studies, a new technical study quality-appraisal tool, and Quality Assessment of Diagnostic Accuracy Studies-2. Studies were analysed descriptively in terms of the different products, study designs and diagnostic strategies used. RESULTS: Four products were identified from 16 studies: PrimeStore-Molecular-Transport-Medium (PS-MTM), FTA card, GENO•CARD (all for nucleic acid amplification tests [NAATs]) and OMNIgene•SPUTUM (OMS; culture, NAATs). PS-MTM, but not FTA card or GENO•CARD, rendered Mycobacterium tuberculosis non-culturable. OMS reduced Löwenstein-Jensen but not MGIT™ 960™ contamination, led to delayed MGIT time-to-positivity, resulted in Xpert performance similar to cold chain-transported untreated specimens, and obviated the need for N-acetyl-L-cysteine-sodium hydroxide decontamination. Data from paucibacillary specimens were limited. Evidence that a cold chain improves culture was mixed and absent for Xpert. The effect of the product alone could be discerned in only four studies. CONCLUSION: Limited evidence suggests that transport products result in test performance comparable to that seen in cold chain-transported specimens.

An investigation of fingerstick blood collection for point-of-care HIV-1 viral load monitoring in South Africa.

BACKGROUND: Viral load (VL) quantification is an important tool in determining newly developed drug resistance or problems with adherence to antiretroviral therapy (ART) in HIV-positive patients. VL monitoring is becoming the standard of care in many resource-limited settings. Testing in resource-limited settings may require sampling by fingerstick because of general shortages of skilled phlebotomists and the expense of venepuncture supplies and problems with their distribution. OBJECTIVE: To assess the feasibility and ease of collecting 150 µL capillary blood needed for the use of a novel collection device following a classic fingerstick puncture. METHODS: Patients were recruited by the study nurse upon arrival for routine ART monitoring at the Themba Lethu Clinic in Johannesburg, South Africa. Each step of the fingerstick and blood collection protocol was observed, and their completion or omission was recorded. RESULTS: One hundred and three patients consented to the study, of whom three were excluded owing to the presence of callouses. From a total of 100 patients who consented and were enrolled, 98% of collection attempts were successful and 86% of participants required only one fingerstick to successfully collect 150 µL capillary blood. Study nurse adherence to the fingerstick protocol revealed omissions in several steps that may lower the success rate of capillary blood collection and reduce the performance of a subsequent VL assay. CONCLUSION: The findings of this study support the feasibility of collecting 150 µL of capillary blood via fingerstick for point-of-care HIV-1 VL testing in a resource-limited setting.

Transcriptional activation of the chlorocatechol degradative genes of Ralstonia eutropha NH9.

Ralstonia eutropha (formerly Alcaligenes eutrophus) NH9 degrades 3-chlorobenzoate via the modified ortho-cleavage pathway. A ca. 5.7-kb six-gene cluster is responsible for chlorocatechol degradation: the cbnABCD operon encoding the degradative enzymes (including orfX of unknown function) and the divergently transcribed cbnR gene encoding the LysR-type transcriptional regulator of the cbn operon. The cbnRAB orfXCD gene cluster is nearly identical to the chlorocatechol genes (tcbRCD orfXEF) of the 1,2, 4-trichlorobenzene-degrading bacterium Pseudomonas sp. strain P51. Transcriptional fusion studies demonstrated that cbnR regulates the expression of cbnABCD positively in the presence of either 3-chlorobenzoate or benzoate, which are catabolized via 3-chlorocatechol and catechol, respectively. In vitro transcription assays confirmed that 2-chloro-cis,cis-muconate (2-CM) and cis, cis-muconate (CCM), intermediate products from 3-chlorocatechol and catechol, respectively, were inducers of this operon. This inducer-recognizing specificity is different from those of the homologous catechol (catBCA) and chlorocatechol (clcABD) operons of Pseudomonas putida, in which only the intermediates of the regulated pathway, CCM for catBCA and 2-CM for clcABD, act as significant inducers. Specific binding of CbnR protein to the cbnA promoter region was demonstrated by gel shift and DNase I footprinting analysis. In the absence of inducer, a region of ca. 60 bp from position -20 to position -80 upstream of the cbnA transcriptional start point was protected from DNase I cleavage by CbnR, with a region of hypersensitivity to DNase I cleavage clustered at position -50. Circular permutation gel shift assays demonstrated that CbnR bent the cbnA promoter region to an angle of 78 degrees and that this angle was relaxed to 54 degrees upon the addition of inducer. While a similar relaxation of bending angles upon the addition of inducer molecules observed with the catBCA and clcABD promoters may indicate a conserved transcriptional activation mechanism of ortho-cleavage pathway genes, CbnR is unique in having a different specificity of inducer recognition and the extended footprint as opposed to the restricted footprint of CatR without CCM.

Transcriptional activation of the catechol and chlorocatechol operons: variations on a theme.

The ortho-cleavage pathways of catechol and 3-chlorocatechol are central catabolic pathways of Pseudomonas putida that convert aromatic and chloroaromatic compounds to tricarboxylic acid (TCA)-cycle intermediates. They are encoded by the evolutionarily related catBCA and clcABD operons, respectively. Expression of the cat and clc operons requires the LysR-type transcriptional activators CatR and ClcR, and the inducer molecules cis,cis-muconate and 2-chloro-cis,cis-muconate. In addition to sequence similarities, CatR and ClcR share functional similarities which allow catR to complement clcR mutants. DNase-I footprinting, DNA bending and in vitro transcription analyses with RNA polymerase mutants indicate that CatR and ClcR activate transcription via a similar mechanism which involves interaction with the C-terminal domain of the alpha-subunit (alpha-CTD) of RNA polymerase. In vitro transcription assays with different regions of the clc promoter indicate that the ClcR dimer bound to the promoter proximal site (the activation binding site) interacts with the alpha-CTD. Gel shift assays and DNase-I footprinting have demonstrated that CatR occupies two adjacent sites proximal to the catBCA promoter in the presence of inducer and an additional binding site within the catB structural gene called the internal binding site (IBS). CatR binds the IBS with low intrinsic affinity that is increased by cooperativity in presence of the two promoter binding sites. Site-directed mutations in the IBS indicate a probable cis-acting repressor function for the IBS. The location of the IBS within the catB structural gene, the cooperativity observed in footprinting studies and phasing studies suggest that the IBS participates in the interaction of CatR with the upstream binding sites by looping out the intervening DNA. Although the core transcriptional activation mechanisms of CatR and ClcR have been conserved, nature has provided some flexibility to respond to different environmental signals in addition to the presence of inducer. Transcriptional fusion studies demonstrate that the expression from the clc promoter is repressed when the cells are grown on succinate, citrate or fumarate and that this repression is ClcR-dependent and occurs at the transcriptional level. The presence of these organic acids did not affect the expression from the cat promoter. In vitro transcription assays demonstrate that the TCA-cycle intermediate, fumarate, directly and specifically inhibits the formation of the clcA transcript. No such inhibition was observed when CatR was used as activator on either the cat or clc template. Since both the catechol and the chlorocatechol pathways feed into the TCA cycle, but only the chlorocatechol pathway is inhibited by fumarate, there is a subtle difference in the regulation of these two pathways where intracellular sensing of a TCA-cycle intermediate leads to a reduction of chloroaromatic degradation.

A tricarboxylic acid cycle intermediate regulating transcription of a chloroaromatic biodegradative pathway: fumarate-mediated repression of the clcABD operon.

The ortho-cleavage pathways of catechol and 3-chlorocatechol are central catabolic pathways of Pseudomonas putida that convert aromatic and chloroaromatic compounds to tricarboxylic acid (TCA) cycle intermediates. They are encoded by the evolutionarily related catBCA and clcABD operons, respectively. Expression of the cat and clc operons requires the LysR-type transcriptional activators CatR and ClcR, respectively, and the inducer molecules cis,cis-muconate and 2-chloro-cis,cis-muconate, respectively. The regulation of the cat and clc promoters has been well studied, but the extent to which these operons are repressed by growth in TCA cycle intermediates has not been explored. We demonstrate by transcriptional fusion studies that the expression from the clc promoter is repressed when the cells are grown on succinate, citrate, or fumarate and that this repression is ClcR dependent and occurs at the transcriptional level. The presence of these organic acids did not affect the expression from the cat promoter. In vitro transcription assays demonstrate that the TCA cycle intermediate fumarate directly and specifically inhibits the formation of the clcA transcript. No such inhibition was observed when CatR was used as the activator on either the cat or clc template. Titration studies of fumarate and 2-chloromuconate show that the fumarate effect is concentration dependent and reversible, indicating that fumarate and 2-chloromuconate most probably compete for the same binding site on ClcR. This is an interesting example of the transcriptional regulation of a biodegradative pathway by the intracellular sensing of the state of the TCA cycle.

2-chloromuconate and ClcR-mediated activation of the clcABD operon: in vitro transcriptional and DNase I footprint analyses.

In Pseudomonas putida, the plasmid-borne clcABD operon encodes enzymes involved in 3-chlorocatechol degradation. Previous studies have demonstrated that these enzymes are induced when P. putida is grown in the presence of 3-chlorobenzoate, which is converted to 3-chlorocatechol, and that ClcR, a LysR-type regulator, is required for this induction. The clcABD operon is believed to have evolved from the chromosomal catBCA operon, which encodes enzymes that utilize catechol and is regulated by CatR. The inducer for the catBCA operon is an intermediate of the catechol pathway, cis,cis-muconate. In this study, we demonstrate by the use of in vitro transcription assays and lacZ transcription fusions in vivo that the analogous intermediate of the 3-chlorocatechol pathway, 2-chloromuconate, is the inducer of the clcABD operon. The DNase I footprints of ClcR with and without 2-chloromuconate were also determined. An extended region of the promoter from -79 to -25 was occupied in the absence of inducer, but the -35 region was unprotected. When 2-chloromuconate was added to the binding assays, the footprint contracted approximately 4 bp at the proximal end of the promoter, and the -35 region was contacted. It is interesting to note that CatR actually extends its footprint 14 bp on the catBCA promoter in response to its inducer. Although CatR and ClcR change their nucleotide protection patterns in different manners when exposed to their respective inducers, their final footprints resemble each other. Therefore, it is possible that their transcriptional activation mechanisms may be evolutionarily conserved.

DNase I footprinting, DNA bending and in vitro transcription analyses of ClcR and CatR interactions with the clcABD promoter: evidence of a conserved transcriptional activation mechanism.

In Pseudomonas putida, benzoate and 3-chlorobenzoate are converted to catechol and 3-chlorocatechol, respectively, which are then catabolized to tricarboxylic acid cycle intermediates via the catBCA and clcABD pathways. The catBCA and clcABD operons are regulated by homologous transcriptional activators CatR and ClcR. Previous studies have demonstrated that in addition to sequence similarities, CatR and ClcR share functional similarities which allow catR to complement clcR. In this study, we demonstrate that CatR activates the clcABD promoter in vitro without inducer, but more transcript is produced when inducer is added. DNase I footprinting and DNA-bending analyses demonstrate that CatR binds to and bends the clcABD promoter to the same angle as does ClcR plus its inducer, 2-chloromuconate. This implies that CatR binds to the clc promoter in its active conformation. Transcription of the clcABD promoter by the alpha-subunit truncation mutant (alpha-235) of RNA polymerase was sharply reduced, indicating that the alpha-subunit C-terminal domain is important. However, a small amount of transcript was produced under these conditions, indicating that other contact sites on the RNA polymerase may play a role in activation.

Interaction of two LysR-type regulatory proteins CatR and ClcR with heterologous promoters: functional and evolutionary implications.

The soil bacteria Pseudomonas putida can use benzoate or 3-chlorobenzoate as a sole carbon source. Benzoate and 3-chlorobenzoate are converted into catechol and 3-chlorocatechol, respectively, which are in turn converted into tricarboxylic acid cycle intermediates. The catabolic pathways of both compounds proceed through similar intermediates, have similar genetic organization, and have homologous enzymes responsible for different catabolic steps. This has led to suggestions that the plasmid-borne 3-chlorocatechol degradation genes evolved from the chromosomal catechol degradation genes. Both catechol and 3-chlorocatechol pathways are positively regulated by the homologous regulatory proteins CatR and ClcR, respectively. These proteins belong to the LysR family of DNA binding proteins and bind to highly conserved target sequences. We examined the ability of CatR and ClcR to cross-regulate the two pathways. CatR was shown in vitro by DNase I footprinting and gel-shift assays to interact with the clcABD promoter region. Likewise, ClcR was shown to interact in vitro with the catBC promoter region. In in vivo experiments, CatR complemented a ClcR- P. putida strain harboring the clcABD operon for growth on 3-chlorobenzoate. However, ClcR was not capable of complementing a CatR- P. putida strain for growth on benzoate. These observations were confirmed by lacZ-transcriptional fusion expression experiments. Differences in the CatR and ClcR binding sites and their in vitro binding characteristics may explain the ability of CatR and not ClcR to cross-activate. These differences may provide insight about the evolution of regulatory systems in P. putida.