Evaluation of real time polymerase chain reaction and enzyme immunoassay in diagnosis of clostridium difficile infection

Background: Clostridium difficile infection [CDI] is the most common cause of antibiotic associated diarrhea [AAD]. Rapid diagnosis of CDI is essential to prevent hospital spread of infection

Objectives: The aim of this work were to determine the prevalence of CDI among cases of AAD in Zagazig University Hospitals, identify risk factors, and evaluate real-time polymerase chain reaction [PCR] and enzyme immunoassay [EIA], against toxigenic culture [TC]

Methodology: Stools were collected from 150 patients with AAD

Results: They were tested for TC, toxin A/B EIA, and C. difficile tcdA/tcdB genes. Thirty four toxigenic C. difficile isolates were obtained [22.7%] out of the 150 patients and those patients were considered positive for CDI. On the other hand, 6 non-toxigenic C. difficile isolates were obtained [4%], while culture of the remaining 110 patients [73.3%] did not yield C. difficile. The later 116 patients [77.3%] were considered negative for CDI. Analysis of risk factors revealed that advanced age, prolonged hospitalization, long duration of antibiotic intake, potentiated penicillins, 3rd generation cephalosporins, antibiotic combined therapy, liver cirrhosis, malignancy, proton pump inhibitors, enteral tube feeding, and cancer chemotherapy were significantly associated with CDI. Sensitivitiy, specificitiy, positive predictive value, negative predictive value, and accuracy of real-time PCR against TC were all 100%, however, values of EIA were 79.4%, 100%, 100%, 94.3%, 95.3%, respectively

Conclusions: CDI is an underappreciated nosocomial infection predisposed by many risk factors. Real-time PCR proved superior diagnostic performance to toxin A/B EIA

Comparative study between conventional blood culture method versus polymerase chain reaction assay for diagnosis of neonatal septicemia

Neonatal septicemia represents a major clinical problem in neonatology with high morbidity and mortality rates despite the progress in neonatal intensive care and antibiotics. Clinical diagnosis of septicemia in newborn infants is not easy and there is no laboratory test with 100% specificity and sensitivity with the exception of blood culture, the results of which are not available for at least 48-72 hours. The purpose of this study was to compare the utility of a 16S rRNA PCR assay to that of the conventional blood culture for detecting bacteria in blood obtained from neonates suspected of having septicemia. The present work included 50 neonates with provisional diagnosis of neonatal septicemia and 25 non infected neonates as control group. For each neonate the following was done: Full history taking, full clinical diagnosis, routine investigations including complete blood cell count and C-reactive protein [CRP], conventional blood culture for isolation of the causative organism and its identification and lastly the polymerase chain reaction for detection of the 16S rRNA gene from blood of septicemic neonates. The results of this study showed that: Only thirty nine neonates [78%] of those diagnosed as having or suspected to have septicemia on clinical backgrounds had given positive blood culture results. The most common isolated organisms were Klebsiella pneumonia [41.02%], coagulase negative staphylococci [CoNS] [20.51%], E. coli [10.26%] and S. aureus [10.26%]. K. pneumonia was the commonest isolated organism in early onset infection while CoNS was the commonest in late onset infection. Thirty eight of cases with positive blood culture [97.44%], two cases with negative blood culture [18.18%] and one of the control group [4%] were positive by 16S rRNA PCR. The sensitivity, specificity, positive and negative predictive values for 16S rRNA PCR in relation to blood culture were 97.4, 91.7, 92.7 and 97.1%, respectively and the accuracy was 94.7%. These values were superior to that of CRP [89.7, 83.3, 85.4 and 88.2%, respectively with accuracy of 86.7%]. Our results suggested that 16S rRNA PCR is a rapid, sensitive and specific diagnostic test for ruling out neonatal septicemia

Diagnosis of acute bacterial endophthalmitis by nested polymerase chain reaction

Microbiological investigations of vitreous fluid [VF] and aqueous humour [AH] samples have often failed to detect the infecting agent in infectious endophthalmitis, resulting in a clinical dilemma regarding therapy. So in this study we aim to assess the usefulness of polymerase chain reaction in the diagnosis of acute bacterial endophthalmitis. This study included forty intraocular samples [25 vitreous fluids [VF] from 25 cases of endophthalmitis and 15 samples; 7 VF and 8 AH as control from non infective patients during vitreous and cataract surgery respectively. The samples were processed for microbiological investigation [Gram stain and periodic acid schiff and culture for aerobic, anaerobic bacteria and fungi]. Nested PCR directed at the 16S rDNA using universal primers for eubacterial genome was done. The 25 cases were divided into four groups according to the initial ophthalmic procedure. PCR for eubacterial genome showed 100% correlation with 12 [48%] bacterial culture positive samples. Two samples were positive by both direct smear and PCR but negative culture. Eubacterial genome was detected in 5 [38.4%] of 13 bacterial culture negative samples. Among the 8 eubacterial PCR negative samples, one was positive for fungus infection. Only one of 15 control samples was positive for eubacterial genome. The sensitivity and specificity of PCR in relation to culture results were 100% and 93.75%. respectively. The most common organisms recovered from patients with culture proven endophthalmitis were Coagulase Negative Staphylococci [CoNS] [41.7%], S.aureus [16.7] S. viridans [16.7%], Pneumococci [8.3%], P. aeruginosa [8.3%] and E. coli [8.3%]. The most common factors predisposing to acute bacterial endophthalmitis were intraoperative complications, use of anti-metabolite [mitomycin C] and diabetes mellitus. PCR performed on aqueous humour [AH] and vitreous fluid [VF] is a reliable tool for the diagnosis of acute bacterial endophthalmitis particularly in smear and culture negative samples

Assessment of T helper 1 and T helper 2 cytokines and their correlations with viral load in HCV patients co-infected with S. mansoni

The aim of this study was to investigate the effect of infection with S. mansoni on the balance between Th-1 and Th-2 cytokines in patients with chronic hepatitis C virus [HCV] infection and to show the relations between these two groups of cytokines to the degrees of viral load in these patients. 44 individuals were classified into 4 groups: group I of control subjects [n=10], group II of patients with S. mansoni mono-infection [n=9], group III of patients with chronic HCV mono-infection [n=13] and group IV of patients with both S. mansoni and HCV co-infection [n=12]. For individuals of all studied groups, interferon-gamma and IL-2 [cytokines of Th-1 cells] and IL-4 and IL-10 [cytokines of Th-2 cells] were measured. Viral load was measured for patients of group III [HCV mono-infection] and group IV [co-infected patients]. The results showed that Th-1 cytokines [IFN-gamma and IL-2] were significantly higher in HCV mono-infection patients and significantly lower in patients co-infected with HCV and S. mansoni compared to normal subjects group. In S. mansoni mono-infection group, IFN-gamma was decreased while IL-2 was normal compared to normal control group. Th-2 cytokines [IL-4 and IL-10] were significantly higher in the three patients groups compared to the control group but the degree of increase of IL-4 showed no significant difference between S. mansoni mono-infection patients and HCV mono-infection patients while the degree of increase of IL-10 was higher in S. mansoni mono-infection patients compared to HCV monoinfection patients. The degree of increase of both IL-4 and IL-10 was significantly higher in the coinfection patients compared to the HCV mono-infection patients. Viral load was significantly higher in the co-infected group compared to HCV mono-infection group and in both groups, the viral load was positively correlated with Th-2 cytokines and negatively correlated with Th-1 cytokines [except IL-2 in the group of HCV mono-infection patients]. In conclusion, these results suggest that HCV patients co-infected with S. mansoni suffer from strong activation of Th-2 cells and so increase of Th-2 cytokines that suppress Th-1 cells and related cytokines which is the type of immune response needed in face of HCV. This Th-1/Th-2 imbalance allows more viral replication and higher viral load in these patients compared to HCV patients who are not co-infected with S. mansoni and this may give an explanation to the rapid hepatic deterioration of these co-infected patients

Soluble L-selectin, vascular endothelial growth factor and endothelin-1 in patients with ulcerative colitis

Recent evidence increasingly suggests that ulcerative colitis [UC] is the result of dysfunctional immunoregulation manifested by an inappropriate production of mucosal cytokines. An abnormal microcirculatory system has also been implicated in its pathogenesis. The objective of this study was to assessed serum concentrations of soluble L-selectin [sL-selectin] and vascular endothelial growth factor [VEGF] and the plasma level of endothelin-1[ET-1] in the patients with UC, compared with healthy controls, and to analyze the results depending on the stage of the disease. This study was conducted on two groups of subjects, the patient group including 30 patients with active ulcerative colitis [UC], and the control group which included 15 healthy volunteers. We assessed serum sL-selectin, VEGF and Plasma ET-1 Level at the beginning of the study in patients and controls then measured again in patients after remission. The levels of sL-selectin, VEGF, and ET-1 were significantly higher in active UC than those in the controls [p < 0.001]. But in remission there was no significant difference between UC patients and controls in VEGF and ET-1 levels. We also found that serum Level of sL-selectin, VEGF, and Plasma Level of ET-1 were significantly higher in patients with active UC compared with patients in remission [p < 0.001]. In addition, it is shown that UC patients in remission have significantly lower levels of sL-selectin than controls. There was a significant positive correlation among the serum levels of VEGF and the plasma level of ET-1; that is, elevated VEGF, and ET-1 levels correlated well with each other in active UC patients [r= 0.631, p < 0.001]. The most common form of the disease observed in our patient population was of mild to moderate severity. Pro-inflammatory cytokines, including sL-selectin, VEGF, and ET-1 appear to play a significant role in the pathogenesis of ulcerative colitis [UC]. Their levels were higher during exacerbation while it is low in periods of remission in UC patients

Association of chlamydia pneumoniae infection with coronary heart disease

C. pneumoniae is an obligatory intracellular bacterium responsible for upper and lower respiratory tract infections. This work was carried out to study the association between C. pneumoniae infection and coronary heart disease [CHD]. This study included 70 patients, divided into two groups[Group I ,included 55 patients with acute coronary syndromes: 32 patients with acute myocardial infarction and 23 patients with unstable angina Group II, It included 15 patients with previously diagnosed chronic coronary heart disease]and healthy Control Group [Group III],It included 22 healthy subjects as control. Venous blood samples were collected from all patients and controls for: determination of total blood cholesterol level, detection of C. pneumoniae-specific IgG by ELISA and detection of C. pneumoniae DNA in the peripheral blood mononuclear cells by PCR The results of this study showed that: C. pneumoniae-specific IgG was detected by ELISA in 83.6% of the acute patients, 73.3% of the chronic patients, and 68.2% of the healthy control subjects.There was no statistically significant difference among the different studied groups as regarding the prevalence of C. pneumoniae-specific IgG antibodies.C. pneumoniae IgG seropositivity among the CHD patients was correlated with smoking but not with age, male sex, hypertension, diabetes mellitus or hypercholesterolemia. C. pneumoniae-specific DNA was detected by PCR in the PBMCs of 61.8% of the acute patients, 26.7% of the chronic patients, and 18.2% of the healthy control subjects. Positive PCR results were significantly higher among the whole studied CHD patients [acute plus chronic] compared to the control subjects. Also, positive PCR results were significantly higher among the acute patients than both the chronic patients and the healthy controls. In contrast, the difference between the chronic patients and healthy controls as regarding the prevalence of C. pneumoniae DNA in the PBMCs was not statistically significant. C. pneumoniae DNA positivity among the patients [either the whole patients or acute patients only] was not significantly correlated with age, sex or any of the studied classic coronary risk factors [smoking, hypertension, diabetes mellitus and hypercholesterolemia]. After adjustment for the classic coronary risk factors and demographic characteristics in the multiple logistic regression analysis, C. pneumoniae infection [as indicated by PCR positivity] was associated with the CHD.No statistically significant difference was found between C. pneumonia DNA positive and C. pneumoniae DNA negative patients [either the whole patients or acute patients only] as regarding the prevalence of C. pneumoniae-specific IgG. This study concluded that: C. pneumoniae DNA detection in the PBMCs was found to be an independent predictor for CHD, particularly acute coronary events.The prevalence of C. pneumoniae DNA in the peripheral blood was found to be higher among the acute CHD patients with recurrent attacks than those with first attacks.C. pneumoniae IgG seropositivity was unreliable predictor for the presence of C. pneumoniae DNA in the CHD patients

Evaluation of screening methods for detection of methicillin resistatance in coagulase-negative staphylococci

In the present study, 100 strains of coagulase-negative staphylococci were isolated from 57 males and 43 females, their ages ranged from eighteen years to fifty years. Cases were admitted in General Surgery, Haemodialysis Unit, Gynaecology and Obstetric Departments and Outpatient Clinics of Zagazig University Hospitals. The objective of the present study was to evaluate the most commonly used susceptibility test methods for detection of methicillin resistance among coagulase negative staphylococci [CoNS] using oxacillin disk diffusion and oxacillin salt agar screen methods and to compare the results of these methods to PCR detection of the mec A gene for 100 clinical isolates of CoNS. Typing, of isolated CoNS into methicillin resistant coagulase-negative staphylococci [MRCoNS] strains and methicillin sensitive coagulase-negative staphylococci [MS CoNS] strains was done using the disk diffusion method: seventy two [72%] strains of the total CoNS isolates were susceptible to oxacillin while the remaining twenty eight [28%] strains were resistant. On using oxacillin salt agar screen it was revealed that 69 [69%] strains of the total 100 CoNS isolates were susceptiblandto oxacillin while the remaining 31 [31%] strains were resistant. The DNA extracted from 28 oxacillin resistant CoNS strains and 12 oxacillin susceptible CoNS were amplified by polymerase chain reaction. Then, analysis of the amplified products by agarose gel electrophoresis for detection of the mecA gene. The gel electrophoresis revealed that 4 strains of MRCoNS strains as detected by disk diffusion were negative by PCR and out of MSCoNS we found that 3 strains were PCR positive. The sensitivities of the disk diffusion method and oxacillin salt agar screen method at 48 hr in relation to PCR results were 88.9% and 100% respectively. While, the specificities of the disk diffusion method and oxacillin salt agar screen method at 48 hr in relation to PCR results were 69.2% and 92.3% respectively. We concluded that oxacillin salt agar screen method at 48 hr by the current National Committee for Clinical Laboratory Standards [NCCLS] methodology for determination of methicillin susceptibility to CoNS can be considered reliable and cost-effective alternative to PCR assay [for detection of mec A gene] for identification of MRCoNS isolates