Diagnosis and quantitative detection of HSV DNA in samples from patients with suspected herpes simplex encephalitis

Diagnosis of herpes simplex encephalitis (HSE) is based on the detection of herpes simplex virus (HSV) DNA in patients' CSF samples. HSV DNA quantitation has the potential for estimating the effects of antiviral therapy. The aim of this study was to diagnose HSV DNA in HSE suspected patients and the quantitative analysis of its genome using real-time PCR to assess the value of the viral load in the course of antiviral treatment. The CSF samples were collected from 236 consecutive HSE suspected patients from November 2004 to May 2008. Upon DNA extraction, the samples were analyzed by Real-Time PCR assay. A set of primers amplified a common sequence of HSV glycoprotein B gene. The copy numbers of unknown samples were expressed via a standard curve drawn with a known amount of amplified cloned plasmid. Of the 236 samples, 137 (58 percent) came from males and 99 (42 percent) from females. The HSV genome was detected in 22 (9.3 percent) patients by PCR, 13 males/ 9 females. Serial CSF samples were available from 10 of the 22 patients. The range of the HSV DNA copy numbers in the clinical samples ranged from 2.5 × 10² to 1.7 × 10(6) copies/mL of CSF. Quantitative PCR results can be helpful in evaluating the efficacy of antiviral therapy in the above-mentioned patients. There is an association between the initial viral load and the duration of treatment course.

An improvement in isolation and preservation of clinical strains of Helicobacter pylori.

Background and Aim: Isolation of H. pylori from gastric mucosal biopsy specimens is a prerequisite for further studies addressing drug susceptibility testing, analysis and characterization of virulence factors, molecular epidemiology studying or other comparative studies. In this study, we used a modified enriched culture medium with short incubation time to improve the isolation rate of H. pylori from the clinical specimens. Methods: Between October 2008 and October 2009, 266 dyspeptic patients attending the endoscopy ward of Motahhary Clinic of Shiraz University of Medical Sciences, were investigated. The biopsy samples were cultured on two selective media called M1, which we used in our previous studies, and a modified medium called M2. The cultures were kept in a microaerophilic atmosphere at 37 °C. The plates were inspected first on day 1, and then on daily basis for a total of 10 days. The isolates were confirmed as H. pylori by colony morphology and positive oxidase, catalase and rapid urease tests. We used the same media and culture conditions to subculture the isolates for several times. Specimens were considered to be H. pylori positive if either the culture or two of the three diagnostic methods yielded positive results. . Results: The isolation rate of H. pylori strains from the samples was significantly higher on M2 in comparison with M1 medium (p<0.05). The bacterial growth on M2 was observed after a significantly shorter time (p<0.05), i.e., after incubation for about 24 hrs. Following these procedures, the preservation time could be extended beyond 6 months without a significant loss of viability. Conclusion: The modified culture technique enabled a shorter incubation time and a higher isolation rate for H.pylori obtained from clinical samples .

Antibacterial susceptibility patterns and cross-resistance of methicillin resistant and sensitive Staphyloccus aureus isolated from the hospitalized patients in Shiraz, Iran

Nosocomial infections caused by methicillin-resistant staphylococci (MRSA) pose a serious problem in many countries. This study aimed to determine the antibacterial susceptibility patterns of methicillin sensitive and resistant Staphylococcus aureus isolates from the hospitalized patients. Totally 356 isolates of Staphylococcus aureus (S. aureus) including 200, 137 and 19 corresponding to MSSA, MRSA, and intermediate MRSA strains, respectively were isolated. Antibacterial susceptibility patterns of the isolates to 14 antibiotics were examined using Kirby-Bauer method. MICs of 15 antibiotics to 156 MRSA isolates were determined by E test method. Cross-resistances of MRSA isolates (137+19) to the other tested antibiotics were also determined. S.aureus with high frequencies were isolated from the blood, sputum and deep wound samples. All of 200 MSSA isolates were sensitive to oxacillin, vancomycin, tecoplanin, rifampin, linezolid, quinupristin/dalfopristin, mupirocin and fusidic acid. A gradient of reduced susceptibility of MSSA to cephalexin, co-trimoxazole, ciprofloxacin, clindamycin, tetracycline, erythromycin and gentamicin were evident. MRSA isolates were sensitive to vancomycin, tecoplanin, linezolid, quinupristin/dalfopristin, mupirocin and fusidic acid, while reduced susceptibility of them to rifampin, co-trimoxazole, clindamycin, cephalexin, tetracycline, ciprofloxacin, erythromycin and gentamicin were observed. MRSA isolates exhibited a high range of cross-resistance to the eight tested antibiotics. Overall, co-trimoxazole, ciprofloxacin, clindamycin, tetracycline, erythromycin and gentamicin showed low activity against MSSA and MRSA isolates which may indicate they are not suitable to be used in clinical practices. To preserve the effectiveness of antibiotics, rational prescription and concomitant application of preventive measures against the spread of MRSA are recommended.

Multidrug-resistant bacteria isolated from intensive-care-unit patient samples

We examined epidemiological aspects and bacterial resistance patterns of bacteria isolated from intensive care unit (ICU) patient samples. During a 10 month period (from June 2006 to March 2007), 812 samples of blood, urine and cerebral spinal fluid (CSF) from 553 hospitalized patients, in ICU wards, including pediatric surgical, neonatal, adult surgical I, adult surgical II, general pediatrics, neurosurgical I, neurosurgical II, and internal medical, were collected. Minimum inhibitory concentration (MIC) of antibiotics for bacteria isolates was determined by the E-test method. The internal medicine ICU with 28.7 percent admissions gave the largest contribution. Coagulase negative staphylococci at frequencies of 66.7 percent and 36.5 percent and E. coli at 20.9 percent were the bacteria most frequently isolated from the blood, CSF and urine samples, respectively. Samples taken from patients 20-40 years old were the most frequent (32.2 percent), while the group of patients over sixty years contributed least (18.5 percent). Both Gram-positive and - negative isolates expressed resistance to most of the penicillins and cephalosporins tested. Combined therapy with vancomycin and meropenem or imipenem gave the most effective treatment against Gram-positive and Gram-negative isolates based on empirical therapy. High frequencies of multiresistant bacteria in ICUs warn us to administer a few effective antibiotics in our hospitals more wisely in order to reduce selective pressure on sensitive strains. This could help save the life of ICU patients and prevent of spread of resistant isolates in these critical wards. Due to continuous changes in antibacterial susceptibility patterns, periodical antibacterial sensitivity assessment in ICUs should be mandatory.