Development and Evaluation of a Blood Culture PCR Assay for Rapid Detection of Salmonella Paratyphi A in Clinical Samples

Background

Enteric fever remains an important cause of morbidity in many low-income countries and Salmonella Paratyphi A has emerged as the aetiological agent in an increasing proportion of cases. Lack of adequate diagnostics hinders early diagnosis and prompt treatment of both typhoid and paratyphoid but development of assays to identify paratyphoid has been particularly neglected. Here we describe the development of a rapid and sensitive blood culture PCR method for detection of Salmonella Paratyphi A from blood, potentially allowing for appropriate diagnosis and antimicrobial treatment to be initiated on the same day.

Methods

Venous blood samples from volunteers experimentally challenged orally with Salmonella Paratyphi A, who subsequently developed paratyphoid, were taken on the day of diagnosis; 10 ml for quantitative blood culture and automated blood culture, and 5 ml for blood culture PCR. In the latter assay, bacteria were grown in tryptone soy broth containing 2.4% ox bile and micrococcal nuclease for 5 hours (37°C) before bacterial DNA was isolated for PCR detection targeting the fliC-a gene of Salmonella Paratyphi A.

Results

An optimized broth containing 2.4% ox bile and micrococcal nuclease, as well as a PCR test was developed for a blood culture PCR assay of Salmonella Paratyphi A. The volunteers diagnosed with paratyphoid had a median bacterial burden of 1 (range 0.1–6.9) CFU/ml blood. All the blood culture PCR positive cases where a positive bacterial growth was shown by quantitative blood culture had a bacterial burden of ≥ 0.3 CFU/ ml blood. The blood culture PCR assay identified an equal number of positive cases as automated blood culture at higher bacterial loads (≥0.3 CFU/ml blood), but utilized only half the volume of specimens.

Conclusions

The blood culture PCR method for detection of Salmonella Paratyphi A can be completed within 9 hours and offers the potential for same-day diagnosis of enteric fever. Using 5 ml blood, it exhibited a lower limit of detection equal to 0.3 CFU/ml blood, and it performed at least as well as automated blood culture at higher bacterial loads (≥0.3 CFU/ml blood) of clinical specimens despite using half the volume of blood. The findings warrant its further study in endemic populations with a potential use as a novel diagnostic which fills the present gap of paratyphoid diagnostics.     

Platelet sparing effect of COX II inhibition used with pegylated interferon alfa-2a for the treatment of chronic hepatitis C: a short term pilot study

The role of a cyclooxygenase (COX) II inhibitor in reducing microvascular inflammation and the platelet count associated with interferon (IFN) plus ribavirin therapy of chronic hepatitis C (HCV) was assessed. Three plasma mediators (biomarkers) associated with platelet activation, inflammation and fibrosis were measured. Eighteen IFN na?ve patients were studied. Nine were treated with pegylated IFN alfa-2a (PEG-IFN alpha-2a) plus ribavirin and rofecoxib; nine were treated with PEG-IFN alpha-2a plus ribavirin. A complete blood count, liver panel and HCV-RNA were assayed weekly. Human soluble P-selectin (hs-P-selectin), human interleukin-8 (IL-8), human interleukin-13 (IL-13) and human thrombopoietin (TPO) were assayed at 4 week intervals. The COX II inhibitor reduced the platelet reduction experienced with PEG-IFN alpha-2a treatment of HCV despite a reduction in the plasma TPO level. Hs-P-selectin was increased in both groups. In contrast, human IL-8 levels declined to undetectable levels in virologic responders. Similarly, human IL-13 levels declined with therapy (P < 0.001). These data suggest that: (1) a COX II inhibition is associated with an increase in the platelet count despite a reduction in the TPO level; (2) human IL-8 and human IL-13 but not hs-P-selectin levels decline in those who experience an early virologic response.     

In Vitro Culture of Functionally Active Buffalo Hepatocytes Isolated by Using a Simplified Manual Perfusion Method

Background

In farm animals, there is no suitable cell line available to understand liver-specific functions. This has limited our understanding of liver function and metabolism in farm animals. Culturing and maintenance of functionally active hepatocytes is difficult, since they survive no more than few days. Establishing primary culture of hepatocytes can help in studying cellular metabolism, drug toxicity, hepatocyte specific gene function and regulation. Here we provide a simple in vitro method for isolation and short-term culture of functionally active buffalo hepatocytes.

Results

Buffalo hepatocytes were isolated from caudate lobes by using manual enzymatic perfusion and mechanical disruption of liver tissue. Hepatocyte yield was (5.3±0.66)×10⁷ cells per gram of liver tissue with a viability of 82.3±3.5%. Freshly isolated hepatocytes were spherical with well contrasted border. After 24 hours of seeding onto fibroblast feeder layer and different extracellular matrices like dry collagen, matrigel and sandwich collagen coated plates, hepatocytes formed confluent monolayer with frequent clusters. Cultured hepatocytes exhibited typical cuboidal and polygonal shape with restored cellular polarity. Cells expressed hepatocyte-specific marker genes or proteins like albumin, hepatocyte nuclear factor 4α, glucose-6-phosphatase, tyrosine aminotransferase, cytochromes, cytokeratin and α1-antitrypsin. Hepatocytes could be immunostained with anti-cytokeratins, anti-albumin and anti α1-antitrypsin antibodies. Abundant lipid droplets were detected in the cytosol of hepatocytes using oil red stain. In vitro cultured hepatocytes could be grown for five days and maintained for up to nine days on buffalo skin fibroblast feeder layer. Cultured hepatocytes were viable for functional studies.

Conclusion

We developed a convenient and cost effective technique for hepatocytes isolation for short-term culture that exhibited morphological and functional characteristics of active hepatocytes for studying gene expression, regulation, hepatic genomics and proteomics in farm animals.     

Network pharmacological evaluation of strigolactones efficacy as potential inhibitors against therapeutic targets of hepatocellular carcinoma

Biotechnology Letters - Hepatocellular carcinoma (HCC) is the uncontrolled growth of hepatocytes which results in nearly 5 million deaths worldwide. Specific strategies have been developed to treat...     

Increasing Anaerobic Acetate Consumption and Ethanol Yields in Saccharomyces cerevisiae with NADPH-Specific Alcohol Dehydrogenase

Saccharomyces cerevisiae has recently been engineered to use acetate, a primary inhibitor in lignocellulosic hydrolysates, as a cosubstrate during anaerobic ethanolic fermentation. However, the original metabolic pathway devised to convert acetate to ethanol uses NADH-specific acetylating acetaldehyde dehydrogenase and alcohol dehydrogenase and quickly becomes constrained by limited NADH availability, even when glycerol formation is abolished. We present alcohol dehydrogenase as a novel target for anaerobic redox engineering of S. cerevisiae. Introduction of an NADPH-specific alcohol dehydrogenase (NADPH-ADH) not only reduces the NADH demand of the acetate-to-ethanol pathway but also allows the cell to effectively exchange NADPH for NADH during sugar fermentation. Unlike NADH, NADPH can be freely generated under anoxic conditions, via the oxidative pentose phosphate pathway. We show that an industrial bioethanol strain engineered with the original pathway (expressing acetylating acetaldehyde dehydrogenase from Bifidobacterium adolescentis and with deletions of glycerol-3-phosphate dehydrogenase genes GPD1 and GPD2) consumed 1.9 g liter⁻¹ acetate during fermentation of 114 g liter⁻¹ glucose. Combined with a decrease in glycerol production from 4.0 to 0.1 g liter⁻¹, this increased the ethanol yield by 4% over that for the wild type. We provide evidence that acetate consumption in this strain is indeed limited by NADH availability. By introducing an NADPH-ADH from Entamoeba histolytica and with overexpression of ACS2 and ZWF1, we increased acetate consumption to 5.3 g liter⁻¹ and raised the ethanol yield to 7% above the wild-type level.     

Post-pandemic seroprevalence of pandemic influenza A (H1N1) 2009 infection (swine flu) among children <18 years in Germany

Background

We determined antibodies to the pandemic influenza A (H1N1) 2009 virus in children to assess: the incidence of (H1N1) 2009 infections in the 2009/2010 season in Germany, the proportion of subclinical infections and to compare titers in vaccinated and infected children.

Methodology/Principal Findings

Eight pediatric hospitals distributed over Germany prospectively provided sera from in- or outpatients aged 1 to 17 years from April 1^st to July 31^st 2010. Vaccination history, recall of infections and sociodemographic factors were ascertained. Antibody titers were measured with a sensitive and specific in-house hemagglutination inhibition test (HIT) and compared to age-matched sera collected during 6 months before the onset of the pandemic in Germany. We analyzed 1420 post-pandemic and 300 pre-pandemic sera. Among unvaccinated children aged 1–4 and 5–17 years the prevalence of HI titers (≥1∶10) was 27.1% (95% CI: 23.5–31.3) and 53.5% (95% CI: 50.9–56.2) compared to 1.7% and 5.5%, respectively, for pre-pandemic sera, accounting for a serologically determined incidence of influenza A (H1N1) 2009 during the season 2009/2010 of 25,4% (95% CI : 19.3–30.5) in children aged 1–4 years and 48.0% (95% CI: 42.6–52.0) in 5–17 year old children. Of children with HI titers ≥1∶10, 25.5% (95% CI: 22.5–28.8) reported no history of any infectious disease since June 2009. Among vaccinated children, 92% (95%-CI: 87.0–96.6) of the 5–17 year old but only 47.8% (95%-CI: 33.5–66.5) of the 1–4 year old children exhibited HI titers against influenza A virus (H1N1) 2009.

Conclusion

Serologically determined incidence of influenza A (H1N1) 2009 infections in children indicates high infection rates with older children (5–17 years) infected twice as often as younger children. In about a quarter of the children with HI titers after the season 2009/2010 subclinical infections must be assumed. Low HI titers in young children after vaccination with the AS03_B-adjuvanted split virion vaccine need further scrutiny.