Mycobacterium

Detection of Tuberculosis in HIV-Infected and -Uninfected African Adults Using Whole Blood RNA Expression Signatures: A Case-Control Study.

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Abstract

Background

A major impediment to tuberculosis control in Africa is the difficulty in diagnosing active tuberculosis (TB), particularly in the context of HIV infection. We hypothesized that a unique host blood RNA transcriptional signature would distinguish TB from other diseases (OD) in HIV-infected and -uninfected patients, and that this could be the basis of a simple diagnostic test.

Methods and Findings

Adult case-control cohorts were established in South Africa and Malawi of HIV-infected or -uninfected individuals consisting of 584 patients with either TB (confirmed by culture ofMycobacterium tuberculosis [M.TB] from sputum or tissue sample in a patient under investigation for TB), OD (i.e., TB was considered in the differential diagnosis but then excluded), or healthy individuals with latent TB infection (LTBI). Individuals were randomized into training (80%) and test (20%) cohorts. Blood transcriptional profiles were assessed and minimal sets of significantly differentially expressed transcripts distinguishing TB from LTBI and OD were identified in the training cohort. A 27 transcript signature distinguished TB from LTBI and a 44 transcript signature distinguished TB from OD. To evaluate our signatures, we used a novel computational method to calculate a disease risk score (DRS) for each patient. The classification based on this score was first evaluated in the test cohort, and then validated in an independent publically available dataset (GSE19491).

In our test cohort, the DRS classified TB from LTBI (sensitivity 95%, 95% CI [87–100]; specificity 90%, 95% CI [80–97]) and TB from OD (sensitivity 93%, 95% CI [83–100]; specificity 88%, 95% CI [74–97]). In the independent validation cohort, TB patients were distinguished both from LTBI individuals (sensitivity 95%, 95% CI [85–100]; specificity 94%, 95% CI [84–100]) and OD patients (sensitivity 100%, 95% CI [100–100]; specificity 96%, 95% CI [93–100]).

Limitations of our study include the use of only culture confirmed TB patients, and the potential that TB may have been misdiagnosed in a small proportion of OD patients despite the extensive clinical investigation used to assign each patient to their diagnostic group.

Conclusions

In our study, blood transcriptional signatures distinguished TB from other conditions prevalent in HIV-infected and -uninfected African adults. Our DRS, based on these signatures, could be developed as a test for TB suitable for use in HIV endemic countries. Further evaluation of the performance of the signatures and DRS in prospective populations of patients with symptoms consistent with TB will be needed to define their clinical value under operational conditions.

Discussion

We have identified a host blood transcriptomic signature that distinguishes TB from a wide range of OD prevalent in HIV-infected and -uninfected African patients. We found that patients with TB can be distinguished from LTBI with only 27 transcripts and from OD with 44 transcripts. Our findings appear robust as the results are reproducible in both HIV-infected and -uninfected cohorts, in different geographic locations, and in an independent TB patient dataset. The high sensitivity and specificity of the signatures in distinguishing TB from OD, even in the HIV-infected patients that have differing levels of T cell depletion and a wide spectrum of opportunistic infections as well as HIV-related complications, suggests that the signatures are promising biomarkers of TB. The relatively small number of transcripts in our signatures may increase the potential for using transcriptional profiling as a clinical diagnostic tool from a single peripheral blood sample (i.e., using a multiplex assay [35],[36]).

The major challenge for diagnosis of TB in Africa is how to distinguish this disease from the range of other conditions that show similar symptoms in countries where TB and HIV are co-endemic. Previous TB biomarker studies have focused on distinguishing patients with TB from healthy controls, or from LTBI [21],[22],[24], or have used other disease controls that may not represent the “real world” disease spectra from which TB should be clinically differentiated [19],[25]. Furthermore, these TB biomarker studies have also excluded HIV co-infected patients who are the group that most need new diagnostics. Our study design should ensure that our signatures are applicable in TB/HIV endemic countries as we recruited patients with TB concurrently with patients with a range of conditions that present with similar clinical features to TB, as well as recruiting both HIV-infected and -uninfected individuals.

We have identified separate signatures for distinguishing TB/OD and TB/LTBI, which only overlap in three transcripts. In practice the clinical applications of these signatures might be distinct as the TB/LTBI signature would be of value in contact screening, where the concern is distinguishing active disease from previous exposure in minimally symptomatic individuals. The TB/OD signature would be of most value in evaluating symptomatic patients presenting to medical services with symptoms of TB. We have also explored whether a single signature might be used to distinguish TB from both LTBI and OD. The combined signature showed lower performance to the separate TB/LTBI and TB/OD signatures. Further exploration of the operational performance of a combined signature or separate signatures is needed to establish the best strategy.

Although our signatures and DRS distinguished the majority of patients with TB from those with LTBI or OD, a proportion of patients were not correctly classified. There is increasing recognition that TB and LTBI may represent a dynamically evolving continuum, particularly in HIV-infected patients and thus failure to culture M.TB is not absolute proof that TB is not present. Some false assignment by our current “gold standard” is to be expected as noted by post mortem studies at which undiagnosed TB is confirmed [14],[15]. All patients in the OD group presented with symptoms for which TB was included in the differential diagnosis, and it is possible that TB may have been misdiagnosed in a small proportion of OD patients despite the extensive clinical investigation used to assign each patient to each diagnostic group. Some improvement in sensitivity and specificity of our DRS may also be achieved by weighting the signal from the most discriminatory transcripts, and this could be explored in subsequent refinements of the method.

A major concern in using transcriptional signatures as a clinical diagnostic tool in resource poor settings is the complexity, as well as cost, of the current methodologies. Our results have shown that transcriptional signatures can be used to distinguish TB from OD in an African setting. We explored the feasibility of a simplified method for disease categorization that may facilitate development of a diagnostic test based on our signatures. Our DRS provides a new approach that enables the use of multi-transcript signatures for individual disease risk assignment without the requirement for complex analysis. Our method could be used to develop a simple test in which the transcripts comprising the diagnostic signature (separated into those that are either up- or down-regulated in TB relative to controls) are each measured using a suitable detection system [35], and the combined signature used to identify each patient’s risk of TB. For example, a simple test using the TB/OD signature probes that show increased transcript expression in TB relative to OD could be located in a single well or tube, and those probes that show reduced transcript expression in TB located in a second well or tube. Binding of RNA from a patient’s blood to these probes could be detected as a combined signal from each tube using one of the aforementioned detection systems. To allow normalization, expression of up- or down-regulated transcripts in an individual patient could be compared with that of housekeeping genes, which do not show variation between healthy and disease states. There are methods for rapid detection of multi-transcript signatures including lateral flow reverse transcription (RT)-PCR based systems, nano-pore technology [37], nano-particle enzyme linked detection [38],[39], and detection using nano-wires and electrical impedance [40]. Some of these may be suitable for direct analysis of multiple transcript signatures in blood and at a relatively low cost.

While this study provides a proof of principle that relatively small numbers of RNA transcripts can be used to discriminate active TB from latent TB infection and OD in Africa, limitations remain that need to be addressed in order to translate these results into a clinical test. One such limitation is that our study has not assessed performance of our DRS in patients treated for TB solely on the basis of clinical suspicion, without any microbiological confirmation. Amongst these “probable/possible” patients with TB, there is no gold standard to evaluate any new biomarker. Exclusion of probable/possible patients with TB may have produced better estimates of sensitivity and specificity than would be achieved in a prospective “all comers” study including the entire cohort of patients in whom TB is included in the differential diagnosis. Thus, further evaluation using a prospective population based study in which the decision whether and when to initiate TB treatment is evaluated against the new biomarker is required. Future studies will also be required to refine the use of these biomarkers in a clinical decision process either as an initial screening tool, or in conjunction with more detailed culture based diagnostics.

From a clinical perspective a simple transcriptome-based test that reliably diagnoses or excludes TB in the majority of patients undergoing investigation for suspected TB, using a single blood sample, would be of great value, allowing scarce hospital resources to be focused on the small proportion of patients where the result was indeterminate. The challenge for the academic research community and for industry is to develop innovative methods to translate multi-transcript signatures into simple, cheap tests for TB suitable for use in African health facilities.

SOURCE: PLOS

Rifampicin and moxifloxacin for tuberculous meningitis.

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Ravina Ruslami and colleagues presented a study assessing pharmacokinetics, safety, and survival benefit of different treatment regimens containing high-dose rifampicin and moxifloxacin in patients with tuberculous meningitis in a hospital setting.1

Their findings that a treatment regimen containing a higher dose of rifampicin and standard-dose or high-dose moxifloxacin during the first 2 weeks is safe in patients with tuberculous meningitis, and that high-dose intravenous rifampicin could be associated with a survival benefit in patients with severe disease are important to note.

We agree with the authors that, on the basis of the small number of patients per group, clinical results should be interpreted carefully. To compensate for small group sizes, one could consider a different strategy with drug exposure as a continuous variable. Additionally, isoniazid concentrations should also be measured since isoniazid contributes to rapid culture conversion and penetrates well in cerebrospinal fluid. Receiver operating characteristic analysis could show the extent to which cumulative drug exposures of rifampicin, moxifloxacin, and isoniazid relate to outcome. Resultant potentially crucial values for positive treatment outcome could be detected and related to the antagonistic effect on cell kill as observed after co-administration of rifampicin and moxifloxacin in in vitro and in vivo studies.2, 3

Another consideration is the potential benefit of a higher oral dosage to reach similar drug exposure as achieved with intravenous dosing. The proposed alternative strategy to analyse the data would also compensate for the difference in drug exposure due to intravenous administration compared with oral dosing, especially in the presence of predisposing factors for poor drug absorption like HIV co-infection.

We therefore would like to encourage the authors to do further analyses of their data to generate additional hypotheses.

References

1 Ruslami R, Ganiem AR, Dian S, et al. Intensified regimen containing rifampicin and moxifloxacin for tuberculous meningitis: an open-label, randomised controlled phase 2 trial. Lancet infect Dis 2013; 13: 27-35. Summary | Full Text | PDF(279KB) |CrossRef | PubMed

2 Drusano GL, Sgambati N, Eichas A, Brown DL, Kulawy R, Louie A. The combination of rifampin plus moxifloxacin is synergistic for suppression of resistance but antagonistic for cell kill of Mycobacterium tuberculosis as determined in a hollow-fiber infection model. mBio 2010; 1: e00139. e00110 PubMed

3 Balasubramanian V, Solapure S, Gaonkar S, et al. Effect of coadministration of moxifloxacin and rifampin onMycobacterium tuberculosis in a murine aerosol infection model. Antimicrob Agents Chemother 2012; 56: 3054-3057. PubMed

Source:Lancet

 

 

 

Bacterial Gene Transfer Gets Sexier.

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Mycobacterium smegmatis can donate larger portions of its genome to other bacteria than previously thought, approaching the level of gene shuffling seen in sexual reproduction.

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n what appears to be a novel form of bacterial gene transfer, or conjugation, the microbeMycobacterium smegmatis can share multiple segments of DNA at once to fellow members of its species, according to a study published today (July 9) in PLOS Biology. The result: the generation of genetic diversity at a pace once believed to be reserved for sexual organisms.

“It is a very nice study providing clear evidence that, in Mycobacterium smegmatis at least, conjugation underlies much of species diversity,” said Richard Meyer, who studies conjugation at The University of Texas at Austin, in an email toThe Scientist.

Traditionally, transfer of genetic material through conjugation has been considered an incremental process. Plasmids mediate the transfer of short segments of DNA, one at a time, between pairs of touching bacterial cells, often conferring such traits as antibiotic resistance.

But M. smegmatis, a harmless bacterium related to the pathogen M. tuberculosis, appears to use a more extensive method of gene shuffling, endowing each recipient cell with a different combination of new genes. The researchers dubbed this form of conjugation “distributive conjugal transfer.” “We can generate a million [hybrid bacteria] overnight, and each of those million will be different than each other,” said coauthor Todd Gray, a geneticist at the New York State Department of Health’s Wadsworth Center.

Coauthor Keith Derbyshire, also a geneticist at the Wadsworth Center, and colleagues had previously published data indicating that M. smegmatis used a novel form of conjugation, but the new study confirms and expands on their suspicions using genetic data. The researchers compared the whole genome sequences of donor and recipient bacteria before and after the massive gene transfers.

The researchers found that, after the transfers, up to a quarter of the recipient bacteria’s genomes were made up of donated DNA, scattered through the chromosomes in segments of varying lengths.

According to the authors, the diversity resulting from distributive conjugal transfer approaches that achieved by meiosis, the process of cell division that underlies sexual reproduction. “The progeny were like meiotic blends,” said Derbyshire. “The genomes are totally mosaic.”

The genes and machinery behind distributive conjugal transfer remain largely unknown, but Gray, Derbyshire, and colleagues have zeroed in on a region of the genome that may determine whether a bacterium becomes a DNA donor or recipient. The region encodes the ESX-1 family of proteins, which are also involved in secreting molecules from M. tuberculosis that give the bacterium its pathogenicity.

The researchers suspect distributive conjugal transfer is important in multiple species of Mycobacteria. Earlier this year, Roland Brosch, a tuberculosis researcher at the Pasteur Institute in France, and colleagues sequenced various strains of the pathogenic M. canettii, which is closely related to M. tuberculosis, and found they were genetically variable—possible evidence of distributive conjugal transfer, according to Gray and Derbyshire. Brosch said he had not yet been able to demonstrate distributive conjugal transfer in M. canettii, however, and he noted that such large-scale gene transfer is unlikely to be occurring in M. tuberculosis, which is a highly genetically homogenous species that shows little sign of recent horizontal gene transfer.

Brosch agreed with Derbyshire and Gray that distributive conjugal transfer could have been important in the evolutionary history of the Mycobacteria genus as a whole, however.  Gray pointed out that understanding the prevalence of distributive conjugal transfer could change views on the time scale of mycobacterial evolution. “I think it’s really going to open some eyes about how quickly things can change,” he said.

Asked whether distributive conjugal transfer could be happening in bacteria outside of theMycobacterium genus, Derbyshire said it remained a mystery, but added: “It’s likely to be more prevalent than currently is known.”

T.A. Gray, “Distributive conjugal transfer in Mycobacteria generates progeny with meiotic-like genome-wide mosaicism, allowing mapping of a mating identity locus,” PLOS Biology, 11: e1001602, 2013.

Source: the-scientist.com

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sexual reproduction , mycobacteria, DNA sequencing, diversity, conjugation, bacterial evolution andbacteria

Steroids control paradoxical worsening ofMycobacterium ulcerans infection following initiation of antibiotic therapy.

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A 19-year-old man with a 3-month history of a non-healing ulcer of the right knee and indurated oedema involving the entire lower leg (Box 1) presented after not responding to multiple courses of antibiotics. A punch biopsy showed necrosis of the dermis and subcutaneous tissues, and extensive infiltration with extracellular acid-fast bacilli on auramine–rhodamine staining. Polymerase chain reaction (PCR) testing for the IS2404 insertion sequence of Mycobacterium ulcerans was positive,1 andM. ulcerans was isolated from culture at 6 weeks. The patient reported holidaying on the Bellarine Peninsula, an endemic area for M. ulcerans, 6 months before the development of the lesion. Surgical intervention was deferred due to the extensive area involved and World Health Organization guidelines suggesting antibiotics as first-line treatment for extensive oedematous disease.2

He commenced oral daily rifampicin (600 mg) and moxifloxacin (400 mg) and, within a week, noted a marked decrease in exudate and improvement in the painful oedematous disease of the lower leg. Therapy was well tolerated and the ulcer remained stable with only one small area of new skin loss lateral to the primary lesion.

Four weeks later, there was a rapid deterioration in clinical condition, with fevers, sweats, anorexia and a recurrence of lower limb oedema and pain. Significant skin breakdown occurred around the ulcer and distally on the leg, with copious serous exudate (Box 2). Full blood examination showed a neutrophilia (neutrophil count, 14.9 × 109/L [reference interval, 1.9–8.0 × 109/L]) and a raised C-reactive protein level (69.8 mg/L [reference interval, < 3 mg/L]). Prednisolone 50 mg daily and

intravenous piperacillin–tazobactam 4.5 g three times daily was commenced. An ultrasound of the limb showed extensive liquefaction of the subcutaneous fat, and swabs were PCR-positive for M. ulcerans but culture-negative following prolonged incubation.

Within 12 hours of commencing prednisolone therapy (combined with broad-spectrum antibiotics), the patient became afebrile, with neutrophil and C-reactive protein levels within reference intervals on full blood examination and an improvement in ulcer appearance. He was discharged on a 25 mg maintenance dose of prednisolone while continuing rifampicin and moxifloxacin. Increasing areas of skin loss and exudate developed when prednisolone was decreased to 12.5 mg daily, and thus he underwent a limited debridement of the lateral knee and negative-pressure wound therapy (VAC GranuFoam dressing, KCI Medical), followed by successful split skin grafting. Heat therapy (Bair Hugger, 3M Arizant Healthcare) was used to maintain skin temperature at 39°C.

Prednisolone was continued for 2 weeks postoperatively (15 mg daily) and then stopped without further deterioration in the patient’s condition. The patient completed a 3-month course of antimycobacterial antibiotics, at which time there was no sign of ongoing infection. Two skin sites subsequently discharged a small amount of culture-negative material but, at 12 months, there was no evidence of microbiological relapse.

Mycobacterium ulcerans infection is a geographically restricted infection often referred to by its local name as Daintree, Buruli or Bairnsdale ulcer (BU). The disease occurs in sub-Saharan Africa and within discrete regions of Australia, predominantly coastal Victoria and northern Queensland. Significant diagnostic delays, which place patients at risk of more extensive disease, are common. A recent review identified a mean duration of 42 days of symptoms before diagnosis (range, 2–270 days).3 These delays often occur when patients present outside endemic areas, a factor affected by the long incubation period of 4–7 months.4

BU is characterised by slowly progressive skin lesions with local necrosis, destruction of lipocytes and surprisingly little systemic inflammation. This unusual pattern of infection is the result of its unique virulence factor mycolactone, an immunomodulatory toxin that induces necrosis of host tissues and can limit initiation of immune responses and the recruitment of inflammatory cells.5 Following the increased use of antibiotics in BU, there have been descriptions of worsening clinical condition after an initial response to therapy.6 Such deteriorations (referred to as paradoxical reactions) are most common in patients with extensive disease and can take a variety of forms including increased ulcer size, ulceration of previously non-ulcerative papules or the development of new lesions not detectable before antibiotics.7 It is particularly important that paradoxical reactions are not misinterpreted as antibiotic failure, as the vast majority of lesions will resolve without a change in antibiotic regimen.

Paradoxical reactions may be the result of antibiotic-induced suppression of mycolactone synthesis, leading to decreased mycolactone concentrations and thus a reversal of macrophage and neutrophil dysfunction with renewed immune surveillance and response.7 There are many parallels to the paradoxical reactions described with Mycobacterium tuberculosis in HIV co-infected patients who initiate antiretroviral therapy. In that setting, the reaction likely represents an increased inflammatory response secondary to antiretroviral-induced immune reconstitution. As with M. tuberculosis,paradoxical reactions to BU commonly occur 4–8 weeks after therapy commences.7

In our case, the deterioration 4 weeks into therapy at the time that mycobacterial cultures became sterile indicates that this was not a failure of antibiotics but an unmasked immune phenomenon. Although our patient did receive a short course of intravenous antibiotics at the time of initiation of steroid therapy, the rapidity of his response and lack of identification of a new bacterial pathogen suggests that the steroid therapy itself was responsible for his improvement. This is supported by the fact that his condition deteriorated when his prednisolone dose was weaned.

It has been shown in animal models that corticosteroids do not adversely affect the outcome of antibiotic therapy in BU,8 and Friedman and colleagues reported a marked improvement in the appearance of lesions when steroids were used for paradoxical reactions in this setting.9Established dosing guidelines are available for the management of paradoxical reactions to M. tuberculosis (most popularly, 1 mg/kg/day until improvement, followed by a 2-week wean),10 yet the degree of applicability of these guidelines to BU management is unknown.

As treatment evolves from a purely surgical approach to an adjunctive or primary antibiotic focus, optimal management remains controversial. In patients with extensive and oedematous disease, pre-emptive steroid therapy may have a role in preventing paradoxical deteriorations, although to date there is no randomised evidence to support its use in this way.

  • Mycobacterium ulcerans should be considered in patients who present with non-healing chronic ulcers or atypical cellulitis that do not respond to standard treatment.
  • It is common for M. ulcerans infections to worsen following the initiation of antibiotics; these paradoxical reactions are likely due to an enhanced immune response directed at dead and dying bacteria and do not reflect a failure of antibiotic therapy.
  • Antibiotics should be continued unchanged when a paradoxical reaction occurs.
  • Steroids may play a role in diminishing skin loss and systemic symptoms associated with paradoxical reactions.

Source: MJA