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CAP Home > CAP Reference Resources and Publications > CAP TODAY > CAP TODAY 2005 Archive > Clinical Abstracts - May 2005
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  Clinical Abstracts





cap today

May 2005

Michael Bissell, MD, PhD, MPH, Professor and Director of Clinical Services and Vice Chair, Department of Pathology, Ohio State University Medical Center, Columbus
Ronald Domen, MD, Professor of Pathology, Medicine, and Humanities, Penn State University College of Medicine, Hershey, Pennsylvania

Prothrombin fragments associated with carotid wall thickness
Use of rush venom immunotherapy
DNA sequencing of gram-positive rods
Avian influenza A in Vietnam
Logistics of cord blood collection and banking
Multiplex real-time PCR for viral infections
Laboratory-acquired SARS
An outcomes study of plasma coagulation
Experiences with in vivo blood gas monitoring

Prothrombin fragments associated with carotid wall thickness

Thrombin, a central enzyme of the hemostatic system, is considered a key mechanism in the pathophysiology of cardiovascular diseases. The incidence of cardiovascular disease (CVD) and the recurrence of coronary events have been positively associated with several coagulation factors, including fibrinogen, factor VII, and factor VIII. The conversion of prothrombin to thrombin is a central event in the coagulation cascade. Prothrombin fragment 1+2 (F1+2) is a polypeptide released from prothrombin during its activation to thrombin by the prothrombinase complex. Measurement of circulating levels of F1+2 has been considered a specific marker of thrombin generation in vivo. Elevated F1+2 has been found in patients with peripheral arterial disease, coronary atherosclerosis, and in relation to the presence of conventional coronary artery disease (CAD) risk factors, such as age, smoking, and dyslipidemia. Whether markers of thrombin generation are associated only with the thrombotic component of CVD or also with the atherosclerotic process is still being debated. A relationship between F1+2 and the thickness of the arterial wall would be important because this might make it possible to identify asymptomatic subjects who might benefit from anti-thrombotic strategies. The aim of this study was, therefore, to examine the relationship between F1+2 and carotid intima-media thickness (IMT) in middle-aged adults free of clinically overt cardiovascular disease. The authors examined 181 asymptomatic middle-aged subjects (mean age, 55.6 years, 76.7 percent men) who were free of overt clinical atherosclerotic disease. F1+2 was measured by enzyme-linked immunosorbent assay and IMT by duplex ultrasonography of carotid artery. Multiple linear regression analysis was used to assess the relationship between the two parameters. Compared with individuals in the lowest tertile of F1+2, those in the upper tertile (greater than 0.55 nmol/L) showed significantly higher IMT (P<0.01). In correlation analysis, a positive relationship was found between plasma F1+2 and carotid IMT. F1+2 also correlated positively with total cholesterol (P<0.008) and low-density lipoprotein cholesterol (P<0.005) but not with blood pressure or body mass index. In multivariate analysis, the association of F1+2 with carotid IMT remained significant (P<0.001) after adjusting for age, gender, body mass index, systolic blood pressure, cholesterol, diabetes, and smoking. The authors concluded that in a population sample of adults without clinically overt atherosclerotic disease, the plasma levels of F1+2 were significantly associated with carotid IMT, suggesting a relationship between thrombin generation and the development of atherosclerosis.

Páramo JA, Orbe J, Beloqui O, et al. Prothrombin fragment 1+2 is associated with carotid intima-media thickness in subjects free of clinical cardiovascular disease. Stroke. 2004;35:1085-1089.

Use of rush venom immunotherapy

Insect venom immunotherapy is considered protective against future insect stings only after the maintenance dose has been achieved. However, an unknown number of venom-allergic patients fail to reach the maintenance dose during the build-up of conventional venom immunotherapy (VIT) due to recurrent systemic reactions. The incidence of systemic reactions that results from administration of VIT varies from 5.8 percent to 58.8 percent, according to different studies. The incidence is higher during the build-up period than during maintenance treatment. Patients occasionally experience recurrent systemic reactions immediately after venom injections. In most of these patients, lowering the dose after the systemic reaction and subsequent minute gradual increments of the dose will allow the patient to eventually reach the full maintenance dose. In a few patients, recurrent trials of decreasing and slowly increasing the dose are repeatedly associated with systemic reactions. The build-up period of conventional VIT is composed of gradual increments of the venom dosage at weekly intervals for three to four months until the maintenance dose is reached. Previous studies with various protocols have demonstrated the efficacy and safety of rapid VIT. In rush VIT, the build-up period is significantly shortened by administering many venom injections at 15-min. intervals for several days until the maintenance dose has been reached. The underlying mechanism responsible for long-term protection may be similar in both procedures. However, the early protection achieved within several days by rush VIT probably implies a different mechanism compared with the one responsible for long-term protection. Therefore, the authors hypothesized that rush VIT might be a possible alternative for patients who develop systemic reactions during the build-up period of conventional VIT, thus enabling them to achieve the full maintenance dose and gain maximal protection. For this study, the authors attempted to establish an alternative VIT protocol that will enable these patients to reach a full protective maintenance dose. Venom-allergic patients who had experienced recurrent systemic reactions during the build-up period of conventional VIT underwent rush VIT. The authors found that of the nine patients who participated in this study, the six who underwent eight treatment courses tolerated the rush VIT well and reached the maintenance dose within three days. In three of these patients, mild cutaneous systemic reactions were overcome with loratadine. In two patients who experienced recurrent and more severe systemic reactions, the original three-day rush VIT had to be modified and extended to five days until the maintenance dose was reached. In a single patient who experienced an anaphylactic reaction, VIT was discontinued. The authors concluded that rush VIT is an appropriate therapeutic alternative that allows most patients with recurrent systemic reactions throughout the build-up period of conventional VIT to reach full protective maintenance dose.

Goldberg A, Confino-Cohen R. Rush venom immunotherapy in patients experiencing recurrent systemic reactions to conventional venom immunotherapy. Ann Allergy Asthma Immunol. 2003;91:405-410.

Reprints: Dr. Aaron Goldberg, Allergy and Clinical Immunology Unit, Meir General Hospital, 44281 Kfar Saba, Israel; arnong

DNA sequencing of gram-positive rods

It is important to accurately and rapidly identify isolated microorganisms in the clinical microbiology laboratory. Genotypic identification techniques involve the amplification of a phylogenetically informative target, such as the small-subunit (16S) rRNA gene. Broad-range primers that recognize 16S ribosomal DNA (rDNA) sequences conserved among a wide variety of bacteria are used to amplify species-specific variable regions of interest. Sequence determination and comparative database searches allow the unknown isolate to be assigned to a group of bacteria. 16S rDNA sequencing has been used extensively for bacterial phylogeny to identify uncultivated bacterial pathogens and cultural isolates. However, few studies have reported on the use of rDNA sequencing to identify bacterial isolates in a more systematic fashion. In this study, the authors evaluated the suitability of 16S rDNA sequencing for identifying aerobic gram-positive rods under routine conditions in a clinical microbiology laboratory. They investigated 136 clinical isolates for which conventional phenotypic identification did not result in species identification. In addition, 37 randomly selected clinical isolates that had been well-identified by standard microbiological procedures were used to compare the accuracy of molecular identification procedures with those of conventional identification methods. Over a period of 18 months, the authors evaluated the use of 16S rDNA sequence analysis as a means of identifying aerobic gram-positive rods in the clinical laboratory. Two collections of strains were studied: 37 clinical strains of gram-positive rods well-identified by phenotypic tests and 136 clinical isolates difficult to identify by standard microbiological investigations—that is, identification at the species level was impossible. Results of molecular analyses were compared with those of conventional phenotypic identification procedures. Good overall agreement between phenotypic and molecular identification procedures was found for the collection of 37 clinical strains well identified by conventional means. For the 136 clinical strains which were difficult to identify by standard microbiological investigations, phenotypic characterization identified 71 of 136 (52.2 percent) isolates at the genus level; 65 of 136 (47.8 percent) isolates could not be discriminated at any taxonomic levels. In comparison, 16S rDNA sequencing identified 89 of 136 (65.4 percent) isolates at the species level, 43 of 136 (31.6 percent) isolates at the genus level, and four of 136 (2.9 percent) isolates at the family level. The authors concluded that rDNA sequencing is an effective means for identifying aerobic gram-positive rods that are difficult to identify by conventional techniques and that molecular identification procedures are not required for isolates well-identified by phenotypic investigations.

Bosshard PP, Abels S, Zbinden R, et al. Ribosomal DNA sequencing for identification of aerobic gram-positive rods in the clinical laboratory (an 18-month evaluation). J Clin Microbiol. 2003;41:4134-4140.

Reprints: P.P. Bosshard, Institute of Medical Microbiology, University of Zürich, Gloriastrasse 30, CH-8028 Zürich, Switzerland; philboss

Avian influenza A in Vietnam

Although the natural reservoir for all known subtypes of influenza A (hemagglutinins H1 through H15 and neuraminidases N1 through N9) is wild waterfowl, only three subtypes are currently circulating among humans—H1N1, H1N2, and H3N2. During the past few years, however, several subtypes of avian influenza A have been shown to cross the species barrier and infect humans. During an outbreak of a highly pathogenic influenza A (H5N1) virus among poultry in Hong Kong in 1997, six of 18 people with confirmed infection died. In February 2003, two additional cases were reported, one of which resulted in death. Possibly as a result of heightened surveillance, avian influenza A (H9N2) viruses were also isolated from children in Hong Kong in 1999, but this infection resulted in only mild, self-limiting illnesses. A total of 89 human infections with influenza A (H7N7), including one resulting in the death of a Dutch veterinarian, occurred during the extensive outbreak in 2003 that decimated the Dutch poultry industry. During late 2003 and early 2004, there were reports of large outbreaks of H5N1 among poultry throughout Asia, including South Korea, Japan, Indonesia, Vietnam, Thailand, Laos, Cambodia, and China. In January 2004, there was confirmation that influenza A (H5N1) virus had been isolated from patients who had died of a respiratory illness in Hanoi and Ho Chi Minh City, Vietnam. The authors reported on the epidemiologic, clinical, and radiographic features in 10 patients with confirmed influenza A (H5N1) infection. In all 10 cases, the diagnosis of influenza A (H5N1) was confirmed by means of a viral culture or reverse transcriptase-polymerase chain reaction with primers specific for H5 and N1. None of the 10 patients (mean age, 13.7 years) had pre-existing medical conditions. Nine of them had a clear history of direct contact with poultry (median time before onset of illness, three days). All patients presented with fever (temperature, 38.5°C to 40°C), respiratory symptoms, and clinically significant lymphopenia (median lymphocyte count, 700/mm3). The median platelet count was 75,500/mm3. Seven patients had diarrhea. In all patients, there were marked abnormalities on chest radiography. There was no definitive evidence of human-to-human transmission. Eight patients died, one patient recovered, and one is recovering. The authors concluded that influenza A (H5N1) infection, characterized by fever, respiratory symptoms, and lymphopenia, carries a high risk of death. In all 10 cases, the infection appeared to have been acquired directly from infected poultry, but the potential exists for genetic reassortment with human influenza viruses and the evolution of human-to-human transmission. Therefore, containment of influenza A (H5N1) in poultry throughout Asia is urgently necessary.

Hien TT, Liem NT, Dung NT, et al. Avian influenza A (H5N1) in 10 patients in Vietnam. N Engl J Med. 2004;350:1179-1188.

Reprints: Dr. Jeremy Farrar, Hospital for Tropical Diseases, 190 Ben Ham Tu, Quan 5, Ho Chi Minh City, Vietnam; jeremyjf @hcm.vnn.v

Logistics of cord blood collection and banking

The collection and banking of cord blood for transplantation has grown considerably in recent years, with more than 110,000 units stored worldwide. Cord blood may be collected at any time and shipped from a collection site to a central processing facility where it is processed (typically red blood cell depleted) and cryopreserved. As with blood banking, cord blood collection is typically performed at multiple sites—usually hospitals—nationwide. The sample must be stored in liquid phase and shipped before cryopreservation. As international systems to coordinate hematopoietic progenitor cell transplantation were developed, issues surrounding the shipping of cryopreserved cells were examined. The authors previously examined the duration of and conditions for optimal liquid storage of cord blood and the influence of the liquid storage period on the post-thaw viability of the blood. An implicit objective of the current study was to determine shipping conditions that would permit the authors to maintain the desired thermal history for the sample. This information will be essential in developing and validating shipping protocols during liquid storage of hematopoietic progenitor cells. For this study, anticoagulated cord blood was divided, with one sample diluted 1:1 using the storage solution STM-sav and the other left undiluted. Units were shipped from Minneapolis to Memphis and returned RBC depleted, cryopreserved, stored for 14 days, and thawed. Mononuclear cell (MNC) counts, percent viable cells, quantity of CD34+ cells, and frequency of CFU-GM were measured. Temperature during shipment was continuously monitored. Preliminary studies showed that the packing and processing protocol influenced the temperatures attained during shipping. Samples achieved temperatures below 10°C within four to eight hours, with a few units dropping near or below 1°C with cold ambient temperatures. The MNC recovery, CD34+45+ recovery, and frequency of CFU-GM for samples that were shipped were comparable to those observed using static liquid storage. The post-thaw viable cell recovery was greatest for storage and shipping times of 24 hours and decreased when the storage and shipping times were longer. The authors concluded that ambient conditions and the packing and processing protocol influenced the temperature history of the sample. Samples stored beyond 24 hours in liquid storage and shipping exhibited a decreased post-thaw recovery.

Hubel A, Carlquist D, Clay M, et al. Liquid storage, shipment, and cryopreservation of cord blood. Transfusion. 2004;44:518-525.

Reprints: Dr. Allison Hubel, Dept. of Mechanical Engineering, University of Minnesota, 1100 Mechanical Engineering, 111 Church St. SE, Minneapolis, MN 55455; hubel001

Multiplex real-time PCR for viral infections

Respiratory infections caused by influenza virus type A, influenza virus type B, respiratory syncytial virus, parainfluenza virus (PIV) type 1, PIV2, and PIV3 are major causes of upper and lower respiratory tract diseases in infants and young children and cause croup, bronchiolitis, and pneumonia. Viral culture, usually in combination with immunofluorescence, is the gold standard for laboratory diagnosis. Multiplex PCR for clinical diagnosis has a significant advantage because it permits simultaneous amplification of several viruses in a single reaction mixture, facilitating cost-effective diagnosis. The real-time PCR method can perform multiplex amplification and detection. In some real-time PCR platforms, four different amplification products can be distinguished in a single tube. Published real-time PCR formats for detecting respiratory viruses have only included one or two organisms. In this study, two multiplex RNA PCRs were designed for detecting the pathogenic respiratory RNA viruses, influenza A and influenza B viruses, RSV, PIV1, PIV2, PIV3, and PIV4. The assays were compared to viral culture, retrospectively, and their potential application in routine diagnosis was assessed. Laboratory diagnosis of viral respiratory infections is generally performed by virus isolation in cell culture and immunofluorescent assays. Reverse transcriptase PCR is recognized as a sensitive and specific alternative for detecting respiratory RNA viruses. A rapid, real-time multiplex PCR assay was developed to detect influenza A and influenza B viruses, RSV, PIV1, PIV2, PIV3, and PIV4 in a two-tube multiplex reaction that used molecular beacons to discriminate between the pathogens. A total of 358 respiratory samples taken over a one-year period were analyzed by the multiplex assay. The incidence of respiratory viruses detected in these samples was 67 of 358 (19 percent) and 87 of 358 (24 percent) by culture and real-time PCR, respectively. Culture detected three influenza A virus, two influenza B virus, 57 RSV, two PIV1, and two PIV3 infections. All of these culture-positive samples and an additional five influenza A virus, six RSV, two PIV1, one PIV2, one PIV3, and three PIV4 infections were detected by multiplex real-time PCR. The authors concluded that applying real-time PCR to clinical samples increases the sensitivity for respiratory viral diagnosis. In addition, results can be obtained within six hours, which increases clinical relevance. Therefore, use of this real-time PCR assay would improve patient management and infection control.

Templeton KE, Scheltinga SA, Beersma MFC, et al. Rapid and sensitive method using multiplex real-time PCR for diagnosis of infections by influenza A and influenza B viruses, respiratory syncytial virus, and parainfluenza viruses 1, 2, 3, and 4. J Clin Microbiol. 2004;42:1564-1569.

Reprints: Eric C.J. Claas, Dept. of Medical Microbiology, Leiden University Medical Center, P.O. Box 9600, 2300 RC Leiden, Netherlands;

Laboratory-acquired SARS

An outbreak of severe acute respiratory syndrome in Singapore ended in late May 2003. The Centers for Disease Control and Prevention subsequently removed its travel alerts for Toronto, Hong Kong, China, and Taiwan. The authors reported on the first case of severe acute respiratory syndrome (SARS) to occur in Singapore after the initial worldwide outbreak ended. They documented the transmission of SARS in a laboratory setting. A 27-year-old graduate student in microbiology at a local university was admitted to Singapore General Hospital with a fever on Sept. 3, 2003. In July and August 2003, he had worked with a nonattenuated strain of West Nile Virus in a biosafety level three (BSL-3) laboratory at Institute A, where research was also conducted on SARS-associated coronavirus (SARS-CoV), dengue virus, and Kunjin virus. The patient's illness had begun on Aug. 26, 2003, with fever, headache, and polyarthralgia. Three days later, evaluation at the emergency department at Singapore General Hospital revealed apyrexia and a normal complete blood count and chest radiograph, so the patient was discharged. He returned with persistent fever on Sept. 3 and was admitted. A review of systems was notable only for a dry cough of two days' duration. The patient reported no history of exposure to SARS and no clinically significant travel history. On admission, the patient had a temperature of 37.6ºC and oxygen saturation of 98 percent while breathing ambient air. Physical examination was unremarkable. Laboratory evaluation showed mild leukopenia, thrombocytopenia, transaminitis, and an elevated lactate dehydrogenase level of 709 U/L (normal range, 180-380). The patient's chest radiograph was normal. Investigations included routine blood cultures and testing for various pathogens. Sputum, blood, stool, and conjunctival swab specimens obtained on Sept. 3, 4, and 8 were sent for SARS-CoV testing. The patient was initially admitted to a general ward. The following day, however, he was transferred to an isolation room because of concern regarding the remote possibility of exposure to SARS. On Sept. 8, a polymerase-chain-reaction assay of a sputum sample and a serologic test of a blood sample were reported to be positive for SARS-CoV. The Singapore Ministry of Health was notified, and the patient was transferred to the designated isolation facility at Tan Tock Seng Hospital. Chest radiographs on Sept. 11 showed a left mid-zone pulmonary infiltrate. Computed tomography of the thorax two days later confirmed the presence of the pulmonary infiltrate in the apical segment of the left lower lobe. The patient's respiratory status remained stable, requiring no supplemental oxygen. His fever resolved 12 days after onset, and his dry cough persisted for another week. He was discharged on Sept. 16 and spent 14 days in home quarantine.

Lim PL, Kurup A, Gopalakrishna G, et al. Laboratory-acquired severe acute respiratory syndrome. N Engl J Med. 2004;350: 1740-1745.

Reprints: Dr. Poh Lian Lim, Dept. of Infectious Diseases, Tan Tock Seng Hospital, 11 Jalan Tan Tock Seng, Singapore, 308433; pllim@post.

An outcomes study of plasma coagulation

The incidence of venous thromboembolism in surgical patients is well known. The clinical benefit and cost-effectiveness of thromboprophylaxis in this patient population is well established, as it is for certain categories of medical patients, such as those who suffer from stroke or myocardial infarction. The situation is less clear in most medical populations because of the heterogeneity in the studied patient populations and in the design of the trials, as well as the lack of uniformity in the evaluated endpoint. The MEDENOX study was a controlled, multi-center, double-blind, randomized study to evaluate the outcome of patients older than 40 years who were newly admitted for an acute medical condition and thought to be at moderate risk of developing venous thromboembolism (VTE). The objectives of the study were to establish the incidence of VTE in this population using the most accurate evaluation technique possible and to determine the efficacy of two different regimens of the low-molecular-weight heparin (LMWH) enoxaparin for preventing deep vein thrombosis (DVT) and pulmonary embolism. Similar to all LMWHs, enoxaparin has a high anti-Xa/anti-IIa ratio. The authors conducted a sub-study of the MEDENOX study to determine whether a correlation existed between plasma coagulation parameters and patient characteristics, thromboprophylaxis, and the incidence of VTE in the context of the MEDENOX study. The authors investigated the relationship between plasma coagulation parameters and patient characteristics, including renal function, thromboprophylaxis, and incidence of VTE, in a controlled, multi-center, double-blind, randomized study of the MEDENOX study population. Two hundred and twenty-four acutely ill patients were given 20 mg or 40 mg of enoxaparin administered subcutaneously or a placebo once daily for 10 (±4) days. The main outcome measures were VTE and plasma anti-Xa and anti-IIa activities, D-dimer, and thrombin-antithrombin levels in blood collected before prophylaxis was given and after the last injection of the study drug. The authors concluded that anti-Xa activity correlated with the dose of enoxaparin. In patients with mild or moderate renal impairment, no significant relationship was noted between anti-Xa activity and the creatinine clearance rate. D-dimer concentrations were lower at day 10 in the 40-mg group, which had a 63 percent lower VTE incidence, than at day zero. No venographically confirmed thromboses were found in patients with a normal D-dimer concentration (<0.5 µg/mL [0.5 mg/L]). D-dimer levels were higher in patients with VTE than in those without VTE, but no predictive value could be demonstrated for individual patients.

Desjardins L, Bara L, Boutitie F, et al. Correlation of plasma coagulation parameters with thromboprophylaxis, patient characteristics, and outcome in the MEDENOX study. Arch Pathol Lab Med. 2004; 128: 519-526.

Reprints: Dr. Louis Desjardins, Centre Hosptalier de l'Université Laval, 2705, Boulevard Laurier, Ste-Foy, Quebec, G1V 4G2, Canada;

Experiences with in vivo blood gas monitoring

Management of critically ill patients in an intensive care unit requires frequent arterial blood gas analyses to assess respiratory and hemodynamic function. Intermittent arterial blood gas analyses (IABG), however, provide only snapshots of information about the dynamically changing pulmonary and circulatory function. Moreover, blood gas analyses are often performed only after a deleterious event occurs. IABG, therefore, provides no adequate "alarm function" for unexpected events. Continuous intra-arterial blood gas monitoring (CABG) can circumvent some of the problems related to IABG. A CABG sensor with a new sensing elements architecture recently became available for clinical use. The former electrochemical Clark electrode for measuring PO2 by a self-consuming process of the electrode chemicals was replaced by a pure optode system. These technical changes led to the length of the sensor tip being reduced to less than 2 cm. The precision and drift of this new sensor have not yet been investigated in clinical or experimental settings. The authors conducted a prospective explorative study to test the new CABG sensor in patients requiring arterial blood gas monitoring for an estimated minimum of four days. Simultaneous measurements of intermittent blood gas analyses (ABL 610, Radiometer, Copenhagen) and CABG (Diametrics Medical, High Wycombe, Bucks, United Kingdom) were compared using Bland-Altman analysis. The study involved 25 patients admitted to the ICU who required mechanical ventilation for an expected minimum of approximately 96 hours. The mean monitoring time was 106.1 (range, 15-231) hours. Bias and precision for PO2 were -0.2 kPa (one percent) ±1.8 kPa (9.5 percent); PCO2: 0.03 kPa (0.6 percent) ± 0.44 kPa (9.3 percent); pH: -0.001 (0.01 percent) ± 0.04 (0.45 percent). The sensor showed no change in measurement characteristics during four days of measurement. However, continuous monitoring was interrupted (reversible sudden drops of PO2 measurement) in 69 cases, possibly caused by thrombotic deposition or sensor bending and accidental sensor retraction, or both. The authors concluded that the precision and bias of the PCO2 and pH-sensing elements were in line with the finding of the older sensor technology. The possibility that the PO2 optode could offer greater accuracy than the older technology is suggested by comparisons with results reported in previous studies. No sensor drift occurred during long-term measurement over more than four days.

Menzel M, Soukup J, Henze D, et al. Experiences with continuous intra-arterial blood gas monitoring: precision and drift of a pure optode-system. Intensive Care Med. 2003; 29: 2180-2186.

Reprints: Matthias Menzel, Dept. of Anesthesiology and Intensive Care Medicine, Martin-Luther University, Magdeburger Strasse 16, 06097 Halle, Germany; matthias. menzel




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