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.     

Survival and life table parameters of soybean pod borer Maruca vitrata (Geyer) (Lepidoptera: Crambidae) on leguminous crop cultivars

The soybean pod borer, Maruca vitrata is one of the key insect pests of tropical legumes. It damages tender leaf axils, flower buds, flowers and pods by webbing and boring clusters of flowers and pods. In this study, we investigated the survival and life table parameters of M. vitrata on several leguminous crops; soybean (cvs. Daewon, Poongsannamool and Socheongja), azuki bean (cv. Hongeon), mung bean (cv. Sanpo), and cowpea (cv. Jangchae), compared to artificial diet to assess the antibiosis resistance to M. vitrata. The life‐variables of M. vitrata were significantly affected by the tested legume cultivars. None of the larvae fed cowpea cultivar Jangchae survived. The azuki bean cultivar Hongeon and mung bean cultivar Sanpo were found susceptible to M. vitrata, whereas cowpea cultivar Jangchae and soybean cultivar Daewon showed antibiosis resistance to M. vitrata. Further studies should examine the chemicals associated with leguminous crop cultivars and its mechanism to develop a control method against M. vitrata.     

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.     

Global metabolite analysis: the influence of extraction methodology on metabolome profiles of Escherichia coli

The global pool of all metabolites in a cell, or metabolome, is a reflection of all the metabolic functions of an organism under any particular growth condition. In the absence of in situ methods capable of universally measuring metabolite pools, intracellular metabolite measurements need to be performed in vitro after extraction. In the past, a variety of cell lysis methods were adopted for assays of individual metabolites or groups of intermediates in pathways. In this study, metabolites were extracted from Escherichia coli using six different commonly used procedures including acid or alkaline treatments, permeabilization by freezing with methanol, high-temperature extraction in the presence of ethanol or methanol, and by lysis with chloroform-methanol. Metabolites were extracted by the six methods from cells grown under identical conditions and labeled with [14C]glucose. The metabolomes were compared after 2-dimensional thin-layer chromatography of labeled compounds. For global analysis, extraction with cold (-40 degrees C) methanol showed the greatest promise, allowing simultaneous resolution of more than 95 metabolite spots. In contrast, 80 or less spots were obtained with other extraction methods. Extraction also influenced quantitative analysis of particular compounds. Metabolites such as adenosine exhibited up to 20-fold higher abundance after cold methanol extraction than after extraction with acid, alkali, or chloroform. The simplicity, rapidity, and universality of cold methanol extraction offer great promise if a single method of lysis is to be adopted in metabolome analysis.     

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.