Evaluation of cytokine responses against novel Mtb antigens as diagnostic markers for TB disease.

OBJECTIVE: We investigated the accuracy of host markers detected in Mtb antigen-stimulated whole blood culture supernatant in the diagnosis of TB. METHODS: Prospectively, blood from 322 individuals with presumed TB disease from six African sites was stimulated with four different Mtb antigens (Rv0081, Rv1284, ESAT-6/CFP-10, and Rv2034) in a 24 h whole blood stimulation assay (WBA). The concentrations of 42 host markers in the supernatants were measured using the Luminex multiplex platform. Diagnostic biosignatures were investigated through the use of multivariate analysis techniques. RESULTS: 17% of the participants were HIV infected, 106 had active TB disease and in 216 TB was excluded. Unstimulated concentrations of CRP, SAA, ferritin and IP-10 had better discriminating ability than markers from stimulated samples. Accuracy of marker combinations by general discriminant analysis (GDA) identified a six analyte model with 77% accuracy for TB cases and 84% for non TB cases, with a better performance in HIV uninfected patients. CONCLUSIONS: A biosignature of 6 cytokines obtained after stimulation with four Mtb antigens has moderate potential as a diagnostic tool for pulmonary TB disease individuals and stimulated marker expression had no added value to unstimulated marker performance

Limited efficacy of repeated praziquantel treatment in Schistosoma mansoni infections as revealed by highly accurate diagnostics, PCR and UCP-LF CAA (RePST trial)

BACKGROUND: Most studies assessing praziquantel (PZQ) efficacy have used relatively insensitive diagnostic methods, thereby overestimating cure rate (CR) and intensity reduction rate (IRR). To determine accurately PZQ efficacy, we employed more sensitive DNA and circulating antigen detection methods. METHODOLOGY: A sub-analysis was performed based on a previously published trial conducted in children from Cote d'Ivoire with a confirmed Schistosoma mansoni infection, who were randomly assigned to a standard (single dose of PZQ) or intense treatment group (4 repeated doses of PZQ at 2-week intervals). CR and IRR were estimated based on PCR detecting DNA in a single stool sample and the up-converting particle lateral flow (UCP-LF) test detecting circulating anodic antigen (CAA) in a single urine sample, and compared with traditional Kato-Katz (KK) and point-of-care circulating cathodic antigen (POC-CCA). PRINCIPAL FINDINGS: Individuals positive by all diagnostic methods (i.e., KK, POC-CCA, PCR, and UCP-LF CAA) at baseline were included in the statistical analysis (n = 125). PCR showed a CR of 45% (95% confidence interval (CI) 32-59%) in the standard and 78% (95% CI 66-87%) in the intense treatment group, which is lower compared to the KK results (64%, 95% CI 52-75%) and 88%, 95% CI 78-93%). UCP-LF CAA showed a significantly lower CR in both groups, 16% (95% CI 11-24%) and 18% (95% CI 12-26%), even lower than observed by POC-CCA (31%, 95% CI 17-35% and 36%, 95% CI 26-47%). A substantial reduction in DNA and CAA-levels was observed after the first treatment, with no further decrease after additional treatment and no significant difference in IRR between treatment groups. CONCLUSION/SIGNIFICANCE: The efficacy of (repeated) PZQ treatment was overestimated when using egg-based diagnostics. Quantitative worm-based diagnostics revealed that active Schistosoma infections are still present despite multiple treatments. These results stress the need for using accurate diagnostic tools to monitor different PZQ treatment strategies, in particular when moving toward elimination of schistosomiasis. CLINICAL TRIAL REGISTRATION: www.clinicaltrials.gov, NCT02868385

Association between cervical dysplasia and female genital schistosomiasis diagnosed by genital PCR in Zambian women.

BACKGROUND: Female genital schistosomiasis (FGS) is a neglected tropical gynaecological disease that affects millions of women in sub-Saharan Africa (SSA). FGS is caused by Schistosoma haematobium, a parasitic carcinogen involved in the pathogenesis of squamous cell carcinoma of the bladder. Cervical cancer incidence and mortality are highest in SSA, where pre-cancerous cervical dysplasia is often detected on screening with visual inspection with acetic acid (VIA). There are no studies evaluating the association between VIA positivity and FGS diagnosed by genital PCR. METHODS: Women were recruited from the Bilharzia and HIV (BILHIV) study in Zambia a community-based study comparing genital self-sampling to provider obtained cervicovaginal-lavage for the diagnosis of FGS in women aged 18-31. FGS was defined as positive Schistosoma DNA from any genital PCR. Urogenital schistosomiasis diagnostics included urine circulating anodic antigen, urine microscopy and portable colposcopy. Participants were offered cervical cancer screening using VIA at Livingstone Central Hospital. Associations of PCR confirmed FGS and other diagnostics with VIA positivity were assessed using multivariable logistic regression. RESULTS: VIA results were available from 237 BILHIV participants. A positive Schistosoma PCR in any genital specimen was detected in 14 women (5.9%), 28.6% (4/14) of these women had positive VIA compared to 9.0% without PCR evidence of schistosome infection (20/223). Schistosoma PCR positivity in any genital specimen was strongly associated with VIA positivity (OR: 6.08, 95% CI: 1.58-23.37, P = 0.02). CONCLUSIONS: This is the first study to find an association between FGS and positive VIA, a relationship that may be causal. Further longitudinal studies are needed

How can schistosome circulating antigen assays be best applied for diagnosing male genital schistosomiasis (MGS): an appraisal using exemplar MGS cases from a longitudinal cohort study among fishermen on the south shoreline of Lake Malawi

We provide an update on diagnostic methods for the detection of urogenital schistosomiasis (UGS) in men and highlight that satisfactory urine-antigen diagnostics for UGS lag much behind that for intestinal schistosomiasis, where application of a urine-based point-of-care strip assay, the circulating cathodic antigen (CCA) test, is now advocated. Making specific reference to male genital schistosomiasis (MGS), we place greater emphasis on parasitological detection methods and clinical assessment of internal genitalia with ultrasonography. Unlike the advances made in defining a clinical standard protocol for female genital schistosomiasis, MGS remains inadequately defined. Whilst urine filtration with microscopic examination for ova of Schistosoma haematobium is a convenient but error-prone proxy of MGS, we describe a novel low-cost sampling and direct visualization method for the enumeration of ova in semen. Using exemplar clinical cases of MGS from our longitudinal cohort study among fishermen along the shoreline of Lake Malawi, the portfolio of diagnostic needs is appraised including: the use of symptomatology questionnaires, urine analysis (egg count and CCA measurement), semen analysis (egg count, circulating anodic antigen measurement and real-time polymerase chain reaction analysis) alongside clinical assessment with portable ultrasonography

An integrated, self-contained microfluidic cassette for isolation, amplification, and detection of nucleic acids

A self-contained, integrated, disposable, sample-to-answer, polycarbonate microfluidic cassette for nucleic acid-based detection of pathogens at the point of care was designed, constructed, and tested. The cassette comprises on-chip sample lysis, nucleic acid isolation, enzymatic amplification (polymerase chain reaction and, when needed, reverse transcription), amplicon labeling, and detection. On-chip pouches and valves facilitate fluid flow control. All the liquids and dry reagents needed for the various reactions are pre-stored in the cassette. The liquid reagents are stored in flexible pouches formed on the chip surface. Dry (RT-)PCR reagents are pre-stored in the thermal cycling, reaction chamber. The process operations include sample introduction; lysis of cells and viruses; solid-phase extraction, concentration, and purification of nucleic acids from the lysate; elution of the nucleic acids into a thermal cycling chamber and mixing with pre-stored (RT-)PCR dry reagents; thermal cycling; and detection. The PCR amplicons are labeled with digoxigenin and biotin and transmitted onto a lateral flow strip, where the target analytes bind to a test line consisting of immobilized avidin-D. The immobilized nucleic acids are labeled with up-converting phosphor (UCP) reporter particles. The operation of the cassette is automatically controlled by an analyzer that provides pouch and valve actuation with electrical motors and heating for the thermal cycling. The functionality of the device is demonstrated by detecting the presence of bacterial B.Cereus, viral armored RNA HIV, and HIV I virus in saliva samples. The cassette and actuator described here can be used to detect other diseases as well as the presence of bacterial and viral pathogens in the water supply and other fluids

Towards a target label-free suboptimum oligonucleotide displacement-based detection system

A novel method for the future development of label-free DNA sensors is proposed here. The approach is based on the displacement of a labelled suboptimum mutated oligonucleotide hybridised with the immobilised biotin-capture probe. The target fully complementary to the biotin-capture probe can displace the labelled oligonucleotide causing a subsequent decrease of the signal that verifies the presence of the target. The decrease of signal was demonstrated to be proportional to the target concentration. A study of the hybridisation of mutated and complementary labelled oligonucleotides with an immobilised biotin-capture probe was carried out. Different kinetic and thermodynamic behaviour was observed for heterogeneous hybridisation of biotin-capture probe with complementary or suboptimum oligonucleotides. The displacement method evaluated colourimetrically achieved the objective of decreasing the response time from 1 h for direct hybridisation of 19-mer oligonucleotides in the direct enzyme-linked oligonucleotide assay (ELONA) to 5 min in the case of displacement detection in the micromolar concentration range

The uronic acid content of coccolith-associated polysaccharides provides insight into coccolithogenesis and past climate

Unicellular phytoplanktonic algae (coccolithophores) are among the most prolific producers of calcium carbonate on the planet, with a production of ∼1026 coccoliths per year. During their lith formation, coccolithophores mainly employ coccolith-associated polysaccharides (CAPs) for the regulation of crystal nucleation and growth. These macromolecules interact with the intracellular calcifying compartment (coccolith vesicle) through the charged carboxyl groups of their uronic acid residues. Here we report the isolation of CAPs from modern day coccolithophores and their prehistoric predecessors and we demonstrate that their uronic acid content (UAC) offers a species-specific signature. We also show that there is a correlation between the UAC of CAPs and the internal saturation state of the coccolith vesicle that, for most geologically abundant species, is inextricably linked to carbon availability. These findings suggest that the UAC of CAPs reports on the adaptation of coccolithogenesis to environmental changes and can be used for the estimation of past CO2 concentrations

What's New Is Old: Resolving the Identity of Leptothrix ochracea Using Single Cell Genomics, Pyrosequencing and FISH

Leptothrix ochracea is a common inhabitant of freshwater iron seeps and iron-rich wetlands. Its defining characteristic is copious production of extracellular sheaths encrusted with iron oxyhydroxides. Surprisingly, over 90% of these sheaths are empty, hence, what appears to be an abundant population of iron-oxidizing bacteria, consists of relatively few cells. Because L. ochracea has proven difficult to cultivate, its identification is based solely on habitat preference and morphology. We utilized cultivation-independent techniques to resolve this long-standing enigma. By selecting the actively growing edge of a Leptothrix-containing iron mat, a conventional SSU rRNA gene clone library was obtained that had 29 clones (42% of the total library) related to the Leptothrix/Sphaerotilus group (≤96% identical to cultured representatives). A pyrotagged library of the V4 hypervariable region constructed from the bulk mat showed that 7.2% of the total sequences also belonged to the Leptothrix/Sphaerotilus group. Sorting of individual L. ochracea sheaths, followed by whole genome amplification (WGA) and PCR identified a SSU rRNA sequence that clustered closely with the putative Leptothrix clones and pyrotags. Using these data, a fluorescence in-situ hybridization (FISH) probe, Lepto175, was designed that bound to ensheathed cells. Quantitative use of this probe demonstrated that up to 35% of microbial cells in an actively accreting iron mat were L. ochracea. The SSU rRNA gene of L. ochracea shares 96% homology with its closet cultivated relative, L. cholodnii, This establishes that L. ochracea is indeed related to this group of morphologically similar, filamentous, sheathed microorganisms

Transcriptomic response of the red tide dinoflagellate, Karenia brevis, to nitrogen and phosphorus depletion and addition

<p>Abstract</p> <p>Background</p> <p>The role of coastal nutrient sources in the persistence of <it>Karenia brevis </it>red tides in coastal waters of Florida is a contentious issue that warrants investigation into the regulation of nutrient responses in this dinoflagellate. In other phytoplankton studied, nutrient status is reflected by the expression levels of N- and P-responsive gene transcripts. In dinoflagellates, however, many processes are regulated post-transcriptionally. All nuclear encoded gene transcripts studied to date possess a 5' <it>trans</it>-spliced leader (SL) sequence suggestive, based on the trypanosome model, of post-transcriptional regulation. The current study therefore sought to determine if the transcriptome of <it>K. brevis </it>is responsive to nitrogen and phosphorus and is informative of nutrient status.</p> <p>Results</p> <p>Microarray analysis of N-depleted <it>K. brevis </it>cultures revealed an increase in the expression of transcripts involved in N-assimilation (nitrate and ammonium transporters, glutamine synthetases) relative to nutrient replete cells. In contrast, a transcriptional signal of P-starvation was not apparent despite evidence of P-starvation based on their rapid growth response to P-addition. To study transcriptome responses to nutrient addition, the limiting nutrient was added to depleted cells and changes in global gene expression were assessed over the first 48 hours following nutrient addition. Both N- and P-addition resulted in significant changes in approximately 4% of genes on the microarray, using a significance cutoff of 1.7-fold and p ≤ 10^-4. By far, the earliest responding genes were dominated in both nutrient treatments by pentatricopeptide repeat (PPR) proteins, which increased in expression up to 3-fold by 1 h following nutrient addition. PPR proteins are nuclear encoded proteins involved in chloroplast and mitochondria RNA processing. Correspondingly, other functions enriched in response to both nutrients were photosystem and ribosomal genes.</p> <p>Conclusions</p> <p>Microarray analysis provided transcriptomic evidence for N- but not P-limitation in <it>K. brevis</it>. Transcriptomic responses to the addition of either N or P suggest a concerted program leading to the reactivation of chloroplast functions. Even the earliest responding PPR protein transcripts possess a 5' SL sequence that suggests post-transcriptional control. Given the current state of knowledge of dinoflagellate gene regulation, it is currently unclear how these rapid changes in such transcript levels are achieved.</p