Development of Multiplex PCR Detection of Blood-borne Viruses by Nucleic Acid Hybridization

Polymerase chain reaction (PCR) has been used as a substitute for conventional serological methods in order to provide blood or blood products free from contaminating viruses and recently attempts have focused to detect 2 or 3 viruses by a single multiplex PCR (M-PCR) reaction. We were able to detect human immunodeficiency virus type 1 (HIV-1), hepatitis C virus (HCV), hepatitis B virus (HBV) and human cytomegalovirus (HCMV) simultaneously by a single M-PCR. However detection by gel electrophoresis of the products from M-PCR suffers from drawbacks such as low sensitivity and product sizes. Here we report enhanced detection systems of M-PCR based on nucleic acid hybridization with arrays built on membrane. Membrane array was manufactured by spotting appropriate probe DNAs on nylon membrane. Single or multiplex PCR was performed and the PCR products were labeled with DIG and allowed to hybridize with the membrane array. Results indicate that nonspecific hybridization was not observed for membrane DNA array. Additionally, membrane array method could detect small amount of viruses that were not detectable by conventional gel electrophoresis. At least 25-fold, and in some cases more than 125-fold increases in sensitivity was obtained with DNA array method. Thus, the nucleic acid hybridization with membrane array could be applied for the detection of M-PCR of viruses in blood or blood products.

Detection of Human Parvovirus B19 in Human Blood by Polymerase Chain Reaction

Viruses present in the blood or blood products serve important infection source to transfusion patients or users of blood products. Human parvovirus B19 has been recognized as a new viral pathogen in human mainly transmitted via blood. Thus, detection of human parvovirus B19 has become an urgent problem to be solved. This study was intended to develop methods to detect human parvovirus B19 in the blood or blood products by nucleic acid amplification technique (NAT) or polymerase chain reaction (PCR). Five sets of primer DNAs were tested for the detection of human parvovirus B19 by PCR. A primer set amplifying 258 nucleotides corresponding Vp1 gene of human parvovirus B19 was chosen and further studies were done to determine the optimum condition to detect human parvovirus B19 from human blood or blood products. PCR detection of human parvovirus B19 was almost 1,000 times more sensitive than the receptor-mediated hemagglutination assay developed by the Japanese Red Cross Center. Although direct PCR of B19 virus without DNA extraction could detect B19 virus from PBS buffer, attempts to detect the virus from whole blood or plasma failed. PCR after DNA extraction from blood or plasma samples could detect B19 virus as little as 104 PFU/ml. Our results can further be applied for developing routine methods to identify human parvovirus B19 in human blood or commercial blood products.

Comparison of Meperidine and Meperidine with Ketamine for Postoperative Analgesia after Total Abdominal Hysterectomy / 대한마취과학회지

BACKGROUND: The purpose of this study was to compare meperidine and meperidine with ketamine for postoperative analgesia after total abdominal hysterectomy (TAH) and to establish a correlation between three types of pain: pain at rest, pain with movement and pain with coughing (maximal pain). METHODS: This present study compared the quality of pain during pain management in 65 patients undergoing TAH. Patients received i.v. meperidine as the loading dose in the recovery room and PCA with meperidine 600 mg, droperidol 5 mg, normal saline 35 ml for three days (Group 1, n = 36), or with meperidine 600 mg, ketamine 200 mg, droperidol 5 mg, normal saline 16 ml for three days (Group 2, n = 29). Patients were then interviewed on postoperative day 1, 2 and 3 (POD1, 2 and 3) to assess their pain on a visual analogue scale (VAS) of 0 (none) to 10 (worst imaginable) and to compare meperidine and meperidine with ketamine for postoperative analgesia. RESULTS: The mean VAS of pain at rest was 4.5 on POD and decreased to 1.8 on POD 3 for patients receiving meperidine with ketamine was lower than the VAS scores of patients receiving meperidine 5.4 to 2.5. Patients receiving meperidine with ketamine also had less difficulty with side effects, less headache, nausea and vomiting. CONCLUSIONS: IVPCA ketamine in combination with meperidine provides superior postsurgical pain relief, especially at rest and with movement and has fewer side effects than meperidine alone.

Comparison of Open and Laparoscopic Cholecystectomies / 대한마취과학회지

Laparoscopic cholecystectomy, a surgical technique first performed in France, which has gained widespread acceptance among surgeons in Korea. The advantages of the laparoscopic technique include lesser patient discomfort, shorter hospitalization, and a shorter return interval to full activities after operation. It has been postulated that due to the minimal incisional discomfort of laparoscopic cholecystectomy, the postoperative pulmonary function following this procedure would be improved as compared to open cholecystectomy. Laparoscopic cholecystectomy uses carbon dioxide, a highly diffusable gas, for insufflation. With extended periods of insufflation, a patient's arterial carbon dioxide levels may be adversely altered. We performed 44 laparoscopic cholecystectomy in 1993. There were 22 women and 22 men, with a mean age of 50.0+/-11.9 years. The mean operative time was 86.7+/-24.3 minutes, reflecting a 62 percent decrease in operative time compared to the open cholecystectomy. The mean hospital stay was 4.6+/-2.0 days. To analyze the hemodynamic effects of carbon dioxide during laparoscopic cholecystectomy, the changes of pH, PaCO2, PaO2, and SaO2 were studied. The measurement showed significant increase of arterial carbon dioxide. Finally, during laparoscopic cholecystectomy patients may require careful intraopererative arterial blood gas monitoring of the absorbed carbon dioxide.

The Need of Central Venous Pressure Monitoring in Patients with Severe Preeclampsia before Epidural Anesthesia / 대한마취과학회지

Most obstetric anesthesiologist now consider epidural anesthesia to be the preferred anesthetic technique for patients with preeclampsia. In the severe preeclampsia effective ciirculatory volume is usually low, so appropriate preanesthetic hydration is essential feature of safe management. We performed CVP monitoring before epidural anesthesia in nine severe preeclamptic patients undergoing cesarean section. Mean CVP of them was 2+/-1 mm Hg which was not significantly different from normal term parturient. In four patients whose CVP was less than 2 mmHg, preanesthetic hydration with Hartman's solution 500~700 rrd was performed under the monitoring of CVP until it became to rise 2~3 mmHg. Five patients out of nine whose systolic blood pressure dropped below 100 mmHg during anesthesia were IV injected ephedrine 4 mg immmediately and three of them needed repeat injection of ephedrine 4 mg. With the monitoring of CVP, the operations of all the nine severe preeclamptic patients were completed successfully without any problem during and after anesthesia. lt is important to reaUze the limitation of central venous pressure monitoring : but if used with caution in proper circumstance it can provide clinically useful information with minimal risk to the patient.

Alkalinization on Epidural 2 % Lidocaine Solution for Cesarean Section / 대한마취과학회지

Sixty parturients scheduled for elective cesarean section at term under epidural anesthesia were randomly divided into one of two groups. Group 1 patients were given a prepared 2% lidocaine solution with 1:200,000 epinephrine plus 1ml of normal saline per 10ml of lidocaine; the solution pH was 2.45. Group 2 patients were given a prepared 2% lidocaine solution with 1:200,000 epinephrine plus 1ml(lmEq) NaHCO3 per 10ml of lidocaine; the solution pH was 7.20. Just before injection, either 2ml normal saline(group 1)or 2ml(2mEq) 8.4 % NaHCO3 solution(group 2) was added to 20ml 2% lidocaine hydrochloride with 1:200, 000 epinephrine. The time to onset of the T5 sensory block (time between completion of injection and loss of cold sensation at T5 dermatome) was significantly more rapid in the group 2 that received the pH adjusted solution. The mean onset time was 4.9+/-1.3 min in the group 2 and 6.4+/-2.0 min in the group 1. The time to peak level was also significantly more rapid in the group 2 at 10.5+/-2.7 min and group 1 at 13.3+/-4.0 min. But the peak level was not statistically signifieant between the two groups. The spread of sensory blockade was significantly more rapid in the pH-adjusted group at 5, 10 and 15 min after epi- dural injection. Incidence of maternal hypotension during the operation did not differ signifi- cantly between the two groups. The time from end of injection to Bromage scale 0 did not differ significantly between the two groups.