Evaluation of a multiplex polymerase chain reaction for the diagnosis of infectious diarrhea in intensive care unit patients in Upper Egypt.

The rapid diagnosis of infectious diarrhea is lifesaving for intensive care unit (ICU) patients. This study evaluated a commercially available multiplex polymerase chain reaction (PCR) (BioFire FilmArray) for the diagnosis of parasitic and bacterial infections in ICU patients with secretory diarrhea in comparison to other traditional methods. This cross-sectional study included 50 subjects with infectious diarrhea. Their stool samples were subjected to macroscopic and microscopic examinations, concentration techniques, permanent staining techniques, stool culture, identification of bacterial infection by the Vitek 2 Compact 15 System, and molecular diagnosis of bacterial or parasitic infections by BioFire FilmArray multiplex PCR. Parasitological examination showed that the sensitivity and specificity of BioFire FilmArray multiplex PCR in the diagnosis of Cryptosporidium oocysts were 83.33% and 100.0%, respectively compared with 100% and 92.5% in diagnosis of G. lamblia cysts. Bacteriological examination showed that the sensitivity and specificity of BioFire FilmArray multiplex PCR in the diagnosis of E. coli and salmonella were 100% and 100.0%, respectively. The BioFire FilmArray multiplex PCR gastrointestinal (GI) panel assay was more sensitive and specific in the diagnosis of bacterial infections than parasitic infections. The BioFire FilmArray multiplex PCR GI panel assay was less sensitive in the detection of Cryptosporidium oocysts than traditional methods. In conclusion, the BioFire FilmArray multiplex PCR may be useful for rapid diagnosis of ICU patients with infectious diarrhea.

Evaluation of the accuracy of multiplex polymerase chain reaction in differentiation between bacterial and viral meningitis.

BACKGROUND: Meningitis is one of the most dangerous infection affecting children. The need for rapid and accurate diagnosis is mandatory for improving the outcome. AIM OF THE WORK: To evaluate the role of multiplex polymerase chain reaction (PCR) in diagnosis of meningitis either bacterial or viral and to detect its accuracy. PATIENTS AND METHODS: A cross-sectional study was carried out in University Children Hospital, Faculty of Medicine, between November 2019 and September 2020. The study was approved by the Ethics Review Board of Faculty of Medicine, Assiut University, and informed written consent was obtained. The committee's reference number is 17200161. Clinicaltrails.gov ID: NCT03387969. Forty-eight children aged 2 to 18 years with meningitis were included. Detailed history and examination, blood glucose level at time of admission prior to lumbar puncture, and multiplex PCR in cerebrospinal fluid (CSF) were evaluated. RESULTS: The mean age of children was 3.27 ± 1.27 years. Thirty-five (72.9%) cases were bacterial meningitis while 13 (27.1%) cases were viral meningitis. Multiplex PCR had 94% sensitivity and 100% specificity for diagnosis of bacterial meningitis. CONCLUSION: Multiplex PCR may help in diagnosis and differentiation of bacterial and viral meningitis with accurate and rapid results.

Inetrleukin-6 and C-reactive protein as predictors of mortality among critically ill COVID-19 patients in Assiut university hospitals (ICUs).

COVID-19 represents a serious global threat due to scarcity of definitive cure and its infectious nature. The death rate of COVID-19 patients admitted to hospitals was quite high, and cytokine storms could be the mechanism of severity. Interleukin-6 (IL6) and C-reactive protein (CRP) may predict severity and mortality. We attempted to determine the role of IL6 and CRP as predictors of death in intensive care unit (ICU) patients. This Cross-sectional hospital study included 100 patients admitted to ICUs at Assiut University Hospitals from October 2020 to October 2021. Data including age, sex and comorbidities were recorded, laboratory investigations included CRP, ferritin, and IL6. Data were collected and analyzed. Morality predictors in ICU patients with COVID-19 infection were older age (>60 years), presence of diabetes mellitus, chest diseases, CRP >49, IL-6 >70 pg/ml. In conclusion, early ranking and identification of people, who are at risk of death among ICU patients, by monitoring of CRP, IL6, early treatment of cytokine storm, and good management of pre-existing comorbidities would be a very useful approach to reduce the mortality among ICU patients.

Accuracy of Cerebrospinal Fluid C-Reactive Protein and Multiplex Polymerase Chain Reaction and Serum Procalcitonin in Diagnosis of Bacterial and Viral Meningitis in Children.

BACKGROUND: Meningitis is one of the most dangerous infection affecting children. The need for rapid and accurate diagnosis is mandatory for improving the outcome. AIM OF THE WORK: to evaluate the role of multiplex polymerase chain reaction (PCR), cerebrospinal fluid (CSF)-C-reactive protein (CRP) and serum procalcitonin (PCT) in diagnosis of meningitis and to detect its accuracy. PATIENTS AND METHODS: A cross-sectional study was carried out in University Children hospital, Faculty of Medicine, between November 2019 and September 2020. The study was approved by the Ethics Review Board of Faculty of Medicine, Assiut University, and informed written consent was obtained. The committee's reference number is 17200161. CLINICALTRIALS: gov ID: NCT03387969. 48 Children aged 2 to 18 years with meningitis were included. Detailed history and examination. Blood glucose level at time of admission prior to lumbar puncture, serum CRP level, serum PCT, CSF-CRP level and Multiplex PCR were evaluated. FUNDING: The study was supported by Grant Office of Faculty of Medicine, Assiut University with grant NO. 2018-01-04-006-R2. RESULTS: The mean age of children was 3.27 plus or minus 1.27 years. 35 (72.9%) cases were bacterial meningitis, while 13 (27.1%) cases were viral meningitis. Patients with bacterial meningitis had significantly higher serum CRP, serum PCT and higher CSF-CRP and significantly lower CSF/blood glucose compared to viral meningitis. Multiplex PCR had 94% sensitivity and 100% specificity for diagnosis of bacterial and viral meningitis. CONCLUSION: CSF-CRP, CSF/blood glucose, PCT and Multiplex-PCR may help in diagnosis and differentiation of bacterial and viral meningitis.

Role of procalcitonine, hsCRP and 16SrRNA in diagnosis of culture negative endocarditis.

Infective endocarditis infects the heart's inner surface, mostly the valves. It is a lethal disease with a high rate of morbidity and fatality. It mostly caused by bacteria, but fungi can also cause it. Microbiological diagnosis relies on blood culture. Molecular and biochemical indicators add to diagnosis, particularly in culture-negative patients. This study aimed to decide the role of 16s rRNA, procalcitonin, and high sensitivity C reactive protein (hsCRP) in the diagnosis of culture negative infective endocarditis (CNIE) in Assiut university hospitals. This cross-sectional study included 60 patients who were admitted to cardiac hospitals with suspected infective endocarditis according to modified DUKE criteria. A group of 20 apparently healthy subjects served as a control group for investigation of inflammatory biomarkers. We performed blood culture, biochemical markers and molecular investigations. Of the 60 patients, there were 46 (76.7%) culture positives and 14 (23.3%) culture negatives. Staphylococcus aureus was the most prevalent pathogen in culture positive patients, followed by enterococcus and klebsiella. Linezolid and imipenem were the most sensitive antibiotics for Gram-positive and Gram-negative bacteria, respectively. Amplification of bacterial 16S rRNA gene, after DNA extraction from whole blood samples, was positive in 11/14 cases (78.5%) of culture negative patients and in 42/46 cases (91.3%) of culture positive patients. The range of procalcitonin in culture positive patients was (0.32- 89) ng/ml, significantly higher than in culture negative patients (0.02- 8.8) ng/ml, and in the control group (0.01-0.08) ng/ml. hsCRP showed the same pattern. In conclusion, our data suggest that PCR was the most accurate diagnostic tool for diagnosing CNIE, followed by procalcitonin, and hsCRP, respectively.

Intralesional vitamin D3 versus intralesional purified protein derivative in treatment of multiple warts: A comparative clinical and immunological study.

Intralesional (IL) vitamin D3 is an emerging treatment for cutaneous warts. However, its effectiveness and exact mechanism is not fully evaluated. We aimed to compare the efficacy and safety of IL purified protein derivative (PPD) and IL vitamin D3 in multiple warts and to investigate their systemic effect clinically and immunologically. Forty-five patients with multiple extragenital warts were treated with IL-PPD (22 patients) or IL vitamin D3 injection (23 patients) for a maximum of three sessions at 3 week intervals. Decrease in size and number of warts and adverse effects were evaluated. Serum interleukin-12 (IL-12) and interferon-gamma (IFN-γ) levels were measured before and 3 weeks after the last session. Higher clearance rates for all warts were observed with IL-PPD compared to IL vitamin D (59.1% vs. 21.7% complete clearance, p < .001). Significant increase was found in both serum IL-12 and IFN-γ after PPD treatment (p = .034 and p = .04, respectively), but only IFN-γ after vitamin D3 treatment (p = 0.02). Both IL vitamin D3 and PPD showed positive results in treatment of multiple warts. However, PPD showed higher clinical efficacy and more increase in both IL-12 and IFN-γ levels.

Clinical Value of Presepsin in Comparison to hsCRP as a Monitoring and Early Prognostic Marker for Sepsis in Critically Ill Patients.

Sepsis carries a poor prognosis for critically ill patients, even withintensive management. We aimed to determined early predictors of sepsis-related in-hospital mortality and to monitor levels of presepsin and high sensitivity C reactive protein (hsCRP) during admission relative to the applied treatment and the development of complications.An observational study was conducted on 68 intensive care unit (ICU) patients with sepsis. Blood samples from each patient were collected at admission (day 0) for measuring presepsin, hsCRP, biochemical examination, complete blood picture and microbiological culture and at the third day (day 3) for measuring presepsin and hsCRP. Predictors of sepsis-related in-hospital mortality were assessed using regression analysis. Predictive abilities of presepsin and hsCRP were compared using the area under a receiver operating characteristic curve. The Kaplan⁻Meier method was used to estimate the overall survival rate.Results showed that the sepsis-related in-hospital mortality was 64.6%. The day 0 presepsin and SOFA scores were associated with this mortality. Presepsin levels were significantly higher at days 0 and 3 in non-survivors vs. survivors (p = 0.03 and p < 0.001 respectively) and it decreased over the three days in survivors. Presepsin had a higher prognostic accuracy than hsCRP at all the evaluated times. Overall, in comparison with hsCRP, presepsin was an early predictor of sepsis-related in-hospital mortality in ICU patients. Changes in presepsin concentrations over time may be useful for sepsis monitoring, which in turn could be useful for stratifying high-risk patients on ICU admission that benefit from intensive treatment.

Health care-associated infections in pre-transplant liver intensive care unit: Perspectives and challenges.

BACKGROUND: Health care-associated infections (HAIs) threaten patient's safety worldwide especially in the intensive care units (ICU). In end-stage liver disease (ESLD), the condition is much more complicated. Data regarding HAIs among ESLD patients is lacking. We aimed to assess the incidence of HAIs, risk factors, causative micro-organisms, antimicrobial susceptibilities and mortality rates among patients with end-stage liver disease (ESLD) admitted to pre-transplant liver intensive care unit (LICU). METHOD: This prospective observational study included 337 ESLD patients admitted to LICU, Al-Rajhi liver center, Assiut University Hospital, Assiut, Egypt between January 2016 and June 2016 and they were followed up for the development of HAI manifestations. The medical history, physical examination and severity of underlying disease were determined. Clinical samples were taken from patients who developed HAIs for microbiological diagnosis and antimicrobial susceptibility testing. RESULTS: A total of 57 (16.9%) ESLD patients developed HAIs with the incidence density of 26.8 per 1000 patient-days. Blood stream infection was the most common (49.1%). Escherichia coli (21.1%) followed by methicillin-resistant Staphylococcus aureus (MRSA) (15.8%) were the commonest bacteria. Multidrug resistant organisms were reported in 52.6% of the isolates. Fungal causes were 15.8% with Candida species dominance. Sphingomonas paucimobilis and Achromobacter dentrificans were reported for the first time as pathogens for HAIs in LICU. Prolonged hospital stay, intravenous line duration, prolonged use of proton pump inhibitors and paracentesis were predictors for HAIs. No significant difference between ESLD patients with and without HAIs regarding mortality (36.8% vs. 48.6%, P=0.2). CONCLUSION: High HAI rate among ESLD patients is a matter of worry. Effective surveillance program, active infection control measures and national antibiotic policies are necessary to reduce the burden of HAIs.

Effect of 6-gingerol on AMPK- NF-κB axis in high fat diet fed rats.

OBJECTIVES: Adenosine monophosphate (AMP)-activated protein kinase (AMPK) plays a central role in metabolic homeostasis and regulation of inflammatory responses through attenuation of nuclear factor kappa-B (NF-κB), Thus AMPK may be a promising pharmacologic target for the treatment of various chronic inflammatory diseases. We examined the effect of 6-gingerol, an active ingredient of ginger on AMPK-NF-κB pathway in high fat diet (HFD) rats in comparison to fish oil. METHODS: Protein levels of AMPK-α1 and phosphorylated AMPK-α1 were measured by western blot while Sirtuin 6 (Sirt-6), resistin and P65 were estimated by RT-PCR, TNF-α was determined by ELISA, FFAs were estimated chemically as well as the enzymatic determination of the metabolic parameters. RESULTS: 6-Gingerol substantially enhanced phosphorylated AMPK-α1 more than fish oil and reduced the P65 via upregulation of Sirt-6 and downregulation of resistin, and resulted in attenuation of the inflammatory molecules P65, FFAs and TNF-α more than fish oil treated groups but in an insignificant statistical manner, those effects were accompanied by a substantial hypoglycemic effect. CONCLUSION: Gingerol treatment effectively modulated the state of inflammatory privilege in HFD group and the metabolic disorders via targeting the AMPK-NF-κB pathway, through an increment in the SIRT-6 and substantial decrement in resistin levels.

Toll Like Receptor 3 Polymorphisms in Hepatitis B Virus Infection.

Hepatitis B virus (HBV) is the one of the major causes of chronic liver disease. Individuals exposed to HBV show wide spectrum outcomes including immunized persons, asymptomatic carrier, chronic active hepatitis, cirrhosis and HCC. The outcome of HBV infection and the severity of associated liver diseases are determined by the nature and strength of host immune responses against the virus. There is accumulating evidence that the innate branch of the host immune system plays an important role in the control of HBV infection. Various components including toll-like receptor (TLR) contribute to this nonspecific innate immune response .TLR3 play an important role in innate immune response against viral pathogens. Single nucleotide polymorphisms (SNPs) in the TLR3 could be considered as factors for the susceptibility to viral pathogens including HBV. This study aimed to investigate the distribution of six SNPs of the TLR3 gene in patients infected with HBV and to determine the relation between these SNPs and the clearance of hepatitis B virus. These SNPs were tested by direct sequencing. Three groups were investigated: chronic HBV carrier (25 patients), chronic active HBV carrier (16 patients) and 13 persons who were previously exposed to HBV and became immunized. These 3 groups were examined for six SNPs (rs5743311, rs5743312, rs5743313, rs5743314, rs5743315, and rs78726532). The analysis showed high frequencies of GCTCCA haplotype and CCA haplotype in the immunized group when compared to chronic hepatitis B groups (P < 0.05). These findings indicate that genetic variations in the TLR3 gene could affect the outcome of HBV infection.

Diagnosis of Pulmonary Tuberculosis and Detection of Resistance to Rifampin and Isoniazid through Direct Molecular Methods in Stool Samples.

BACKGROUND: Tuberculosis (TB) is an infectious disease that is caused by Mycobacterium tuberculosis (M.tb). TB has high morbidity and mortality around the world. OBJECTIVE: To evaluate molecular-based methods performed directly on stool samples for the diagnosis of pulmonary tuberculosis (PTB) and to determine the susceptibility to Rifampicin (RMP) and Isoniazid (INH). STUDY DESIGN: This is a descriptive study evaluating the performance of the PCR-based method for direct PTB diagnosis and to determine the susceptibility of RMP and INH using stool samples from PTB patients. The study was conducted between March 2011 and March 2014. METHODS: Three stool samples and three sputum samples (n=300 stool and 300 sputum) were collected from 100 PTB patients (75 pretreatment and 25 follow up). Stool samples (n=60) were also collected from 20 healthy individuals to serve as controls. DNA was extracted from stool samples using Chelex®-100. PTB was diagnosed using a Genekam® kit. RMP and INH susceptibility testing was performed using the Genotype MTBDRplus® assay. The sputum Lowenstein Jensen (LJ) culture and agar proportion method (PM) of drug susceptibility testing for INH and RMP were considered to be the gold standard methods for comparing the results of the molecular methods. RESULTS: The Genekam kit showed 100% sensitivity and 95.24% specificity for diagnosing new patients and showed 100% sensitivity and 80% specificity for follow-up patients. The Genotype MTBDR-plus assay showed 86.4% and 100% sensitivity and 98.1% and 97.8% specificity for determining INH and RMP sensitivity, respectively, in newly diagnosed patients and 85.7% and 94.4% sensitivity and specificity, respectively, for both INH and RMP in follow-up patients. CONCLUSION: Molecular-based methods are promising techniques for the diagnosis and susceptibility testing of PTB when the ease of sample collection and the speed of diagnosis are taken into consideration. However, they are not as useful for assessing follow-up patients.

Fungal infection in patients with end-stage liver disease: low frequency or low index of suspicion.

BACKGROUND: End-stage liver disease (ESLD) is associated with dysregulation of the immune system and increased susceptibility to infections. Although invasive fungal infection (IFI) is a growing public health problem, studies of IFI in ESLD are lacking. The aims of this study were to screen for IFI in ESLD and to assess risk factors and serum interleukin 17 (IL-17) as a marker of the cellular immune response. METHODS: Both blood and ascitic fluid samples were collected from 46 patients with ESLD for fungal culture and PCR. Serum IL-17 levels were determined. RESULTS: Seven patients had isolated IFI (four had spontaneous fungal peritonitis, two had fungemia, and one had a disseminated fungal infection) and five cases had combined fungal and bacterial infections. Spontaneous fungal peritonitis was attributed to Candida species, while fungemia was caused by Aspergillus species. Patients with IFI had higher serum IL-17 levels and increased mortality compared to patients without IFI. A history of antibiotic use (p = 0.002), higher model for end-stage liver disease (MELD) scores (p = 0.04), and hepatorenal syndrome (p = 0.006) were risk factors for IFI. CONCLUSIONS: Patients with ESLD had a low frequency of IFI; however, in patients with these infections, delayed diagnosis and treatment may contribute to a high fatality rate. Thus, clinicians should have a high index of suspicion for this unusual but lethal entity, as prompt detection and appropriate treatment can improve the outcome.