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August 2011

Editor:
Michael Bissell, MD, PhD, MPH

PCA3 as a prostate biomarker PCA3 as a prostate biomarker

Prostate cancer is the most common noncutaneous cancer in males in Sweden, with an incidence of nearly 9,000 cases per year. The standard diagnostic tools, total prostate-specific antigen in serum or plasma (tPSA) and digital rectal examination (DRE), have low specificity, leading to unnecessary prostate biopsies. TPSA has been shown to be a very strong predictor of risk when obtained during early middle age. However, specificity decreases with age owing to nonmalignant conditions, such as inflammation and benign prostatic hyperplasia (BPH), that increase tPSA to levels at or above the recommended biopsy cutoffs. Attempts have been made to increase the specificity of the tPSA test, the most validated of such methods being percentage free PSA, PSA kinetics, and PSA in relation to gland volume. Except for percentage free PSA, which to some degree increases specificity, these methods have fallen short after being tested in more robust settings or have been hampered by operator-dependent factors. Therefore, new diagnostic biomarkers are needed. Prostate cancer antigen 3 (PCA3) is a gene located on chromosome 9. However, no immunohistochemical studies of PCA3 are available because the PCA3 gene transcript is not translated into a protein. Urine tests for PCA3 have, in several studies, been suggested to perform better than tPSA in predicting the outcome of first or repeat biopsy in patients with mild to moderately elevated tPSA levels. The Progensa uPCA3 assay (Gen-Probe) is a urine test that has shown good analytical properties and is available commercially. The test analyzes PCA3 and PSA messenger RNA in the first portion of voided urine after a standardized DRE and gives the result as a uPCA3 score. The authors conducted a study to evaluate the performance of uPCA3 as a prebiopsy diagnostic marker for prostate cancer and to compare the performance with variables such as tPSA and prostate volume. The study included 62 men scheduled for prostate biopsy at Skane University Hospital, Malmo, Sweden. Urine samples were obtained according to the Progensa uPCA3 assay. Logistic regression and receiver operating characteristic curves were used to test associations between levels of biomarkers and prostate cancer. According to pathological examination of core needle biopsies, prostate cancer was found in 18 of 62 patients. A one-step increase in uPCA3 was associated with an increase in the odds of cancer of 1.026 (P=0.005). Differences in the odds ratio between uPCA3 and tPSA were not statistically significant. A model using both markers did not increase prediction of event. Areas under the curve for uPCA3, tPSA, and a model combining uPCA3 and tPSA did not differ significantly. No significant correlation was found between uPCA3 and tPSA or prostate volume. The authors concluded that in this small set of mixed patients, uPCA3 alone and tPSA performed equally well as diagnostic markers for prostate cancer. A combination of the two markers did not improve diagnostic performance. This study does not support adding the uPCA3 urine test to tPSA for detecting prostate cancer or using the uPCA3 urine test as a replacement for tPSA.

Nyberg M, Ulmert D, Lindgren A, et al. PCA3 as a diagnostic marker for prostate cancer: a validation study on a Swedish patient population. Scand J Urol Nephrol. 2010;44:378–383.

Correspondence: A. Bjartell at anders.bjartell@med.lu.se

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Use of mass spectrometry to identify enteric pathogens Use of mass spectrometry to identify enteric pathogens

Infectious diarrhea continues to be a problem worldwide and accounts for more than 2 million deaths annually. In addition to Salmonella, Shigella, and enterohemorrhagic Escherichia coli, the list of potential enteric bacterial pathogens has been expanded to include such microorganisms as Campylobacter jejuni, Aeromonas hydrophila, Yersinia enterocolitica, and Vibrio species. Bacterial stool cultures remain the mainstay laboratory diagnostic method for evaluating diarrheal illnesses. Identifying enteric bacterial pathogens rapidly, accurately, and specifically is essential for directing the antimicrobial therapy of diarrheal illnesses. Selective agars are used in isolating suspicious enteric bacterial pathogens. Pure cultures obtained subsequently allow the microorganisms to be characterized more fully and identified by morphological and biochemical characteristics. However, these conventional methods are time consuming and labor intensive, and they require experienced clinical microbiologists. Multiplex polymerase chain-reaction–based molecular methods have the potential to provide rapid results, but detecting and identifying a panel of pathogens has been difficult and costly. In recent years, matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) has emerged as a new technology for species identification. After detection signals are processed and interpreted in the mass spectra, the characteristic mass peaks are used to characterize and eventually identify the microorganisms. By measuring the exact sizes of peptides and small proteins, which are assumed to be characteristic for each bacterial species, it is possible to determine the species within a few minutes of when the analysis is started with whole cells, cell lysates, or crude bacterial extracts. A MALDI-TOF MS-based Biotyper system (Bruker Daltonics) has been developed for rapidly identifying and characterizing microorganisms based on the characterization of biomarker molecules. The authors conducted a preliminary study in which they evaluated the performance and cost-effectiveness of the Biotyper system for routinely identifying common enteric bacterial pathogens seen in middle Tennessee from suspicious colonies grown on selective stool culture media. A total of 304 suspicious colonies were selected and further identified from 605 stool specimens. The suspicious colonies were analyzed by the Biotyper system, and the results were compared with those from routine phenotypic methods, which identified 22 Salmonella species, 39 Shigella species, three enterohemorrhagic E. coli isolates, two Yersinia enterocolitica isolates, two Campylobacter species, and 236 gastrointestinal normal flora isolates. The Biotyper system identified the Salmonella species, Yersinia enterocolitica, and Campylobacter species but failed to distinguish the Shigella species and enterohemorrhagic E. coli isolates from E. coli. Among the 236 normal flora isolates, 233 (98.7 percent) and 228 (96.6 percent) agreed at the genus and species levels, respectively, between the phenotypic and Biotyper methods. Organism identification scores showed no significant difference between colonies directly from selective media and subsequently from pure subculture. The entire Biotyper identification procedure, from smear preparation to final result reporting, can be completed within 30 minutes. The authors concluded that the Biotyper system provides a rapid and simple screening tool for identifying many, but not all, suspicious colonies grown on selective media within 24 hours of inoculation, which shortens test turnaround time by two to three days.

He Y, Li H, Lu X, et al. Mass spectrometry Biotyper system identifies enteric bacterial pathogens directly from colonies grown on selective stool culture media. J Clin Microbiol. 2010;48:3888–3892.

Correspondence: Yi-Wei Tang at yiwei.tang@vanderbilt.edu

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Decisionmaking in newborn screening Decisionmaking in newborn screening

Newborn screening programs have grown rapidly as a result of new technologies and the work of advocacy groups. Historically, diseases that fit defined criteria, such as having a well-understood natural history, being identifiable at a presymptomatic stage, and benefitting from early treatment, were considered candidates for population-wide screening. In recent years, suggestions have been made to expand the criteria to consider benefits to other family members, facilitation of new research, and prevention of diagnostic odysseys. However, little is known about the newborn screening decisions parents would make if given a choice. Previous studies of parents’ attitudes toward newborn screening involved families affected by the targeted disease or considered specific and relatively well-known disorders. Those studies generally demonstrated support for newborn screening for specific conditions, but the specificity of the disease limited the studies’ ability to be generalized. The studies provided little information about general attitudes toward newborn screening for unrelated disorders. Other studies focused on psychological aspects of the newborn screening process, such as waiting for confirmatory testing, receiving false-positive results, and identifying carrier status, rather than parents’ preferences. Findings related to newborn and prenatal screening have shown that parents may be influenced by previous experiences with diseases and understanding of the clinical outcomes associated with a specific disease. A deeper understanding of parents’ potentially diverse views could assist policymakers in shaping future newborn screening policies. The authors conducted a study to determine the factors parents would consider when making a decision about newborn screening and to determine what they perceive to be the risks and benefits of such screening. They conducted focus groups with parents from primary care clinics and interviewed parents from a genetics clinic. The 45 participants discussed seven vignettes about newborn screening that the authors developed and refined with assistance from an expert panel. Two coders coded the data independently, compared coding, and resolved disagreements through discussion. Using framework analysis, the authors analyzed the data and identified how parents’ preferences varied according to disease characteristics, test characteristics, and perceptions of the associated risks and benefits. The authors found that study participants strongly supported population-wide screening for disorders with well-defined, effective treatments, even if the treatment—for example, a bone marrow transplant—had significant morbidity. However, particularly among primary care clinic participants, there were more varied preferences and greater difficulty making decisions about disorders associated with older age at onset, less accurate screening tests, or less-effective treatment. In those cases, many participants suggested that screening be optional. For all disorders, participants expressed a desire for more information to facilitate decisionmaking. The authors concluded that participants supported newborn screening for treatable disorders but suggested that screening be optional for other disorders. The variable influences on parents’ decisionmaking suggest that parents with diverse experiences, if they were included in decisionmaking regarding screening policies, could provide critical perspectives and help screening programs address parents’ preferences and meet parents’ needs for accurate information.

Lipstein EA, Nabi E, Perrin JM, et al. Parents’ decision-making in newborn screening: opinions, choices, and information needs. Pediatrics. 2010;126:696–704.

Correspondence: Dr. Ellen A. Lipstein at ellen.lipstein@cchmc.org

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RET genetic screening for medullary thyroid cancer RET genetic screening for medullary thyroid cancer

Medullary thyroid carcinoma is a rare tumor arising from parafollicular thyroid C cells and can occur sporadically (70 to 80 percent of patients) or as part of the multiple endocrine neoplasia (MEN) type 2 syndromes (20 to 30 percent of patients). In these patients, other endocrine organs—that is, parathyroid and adrenal glands—may be involved, and three different syndromes (MEN 2A, MEN 2B, and familial medullary thyroid carcinoma) can be distinguished. Clinical classification of MEN 2 has been established based on the spectrum of clinical manifestations in the index case and in other affected family members. When a pheochromocytoma or primary hyperparathyroidism, or both, is present in a patient with medullary thyroid carcinoma (MTC) or in relatives, MEN 2A syndrome can be diagnosed. The clinical feature of a marfanoid habitus with mucosal neurinomas with or without associated pheochromocytoma in a subject with MTC is suggestive of MEN 2B syndrome. When several members of the same family have a history of MTC with no evidence of involvement in any other endocrine gland, a diagnosis of a familial medullary thyroid carcinoma syndrome can be advocated. According to the literature, the clinical prevalence of the three hereditary syndromes is about 60 percent for MEN 2A, 10 percent for MEN 2B, and 30 percent for familial medullary thyroid carcinoma. Conversely, the sporadic form of MTC is defined by the absence of a familial history of MTC and the other MEN 2-related tumors. In 1993, RET germline mutations were recognized as the causative molecular alterations in MEN 2 syndromes. American Thyroid Association guidelines for managing patients with MTC recommend that all patients with clinical suspicion of hereditary MTC be screened for a germline RET mutation and, if they are positive, all first-degree family members be tested to identify the gene carriers. A few years after the pathogenetic roles of RET germline mutations in MEN 2 syndromes were recognized, several authors demonstrated that about 10 percent of patients with apparently sporadic MTC also have identifiable RET mutations, which allows these cases to be reclassified as hereditary forms. The authors conducted a study to demonstrate the clinical benefits of RET genetic screening in patients with apparently sporadic MTC not only to identify the hereditary nature of the disease in the index case but also to discover family members harboring the same germline mutations but who are unaware of their condition. The authors performed RET genetic screening for 729 apparently sporadic MTC patients by direct sequencing RET exons 5, 8, 10, 11, and 13–16. They also performed clinical and biochemical evaluation of gene carriers. The authors discovered an unsuspected germline RET mutation in 47 of 729 (6.5 percent) apparently sporadic MTCs that were reclassified as hereditary. They found 60 of 146 (4.1 percent) gene carriers, 35 of whom had biochemical or clinical evidence of MTC. Thirty gene carriers underwent total thyroidectomy, and 27 of 30 (90 percent) were persistently cured after a mean followup of six years. The authors also observed a significantly higher prevalence of familial medullary thyroid cancer in their series with respect to the largest series of the International RET Consortium (P=0.0002). The authors concluded that RET genetic screening of patients with apparently sporadic MTC represents a major tool for the preclinical diagnosis and early treatment of unsuspected affected family members and allows the identification of a relevant percentage of hidden familial medullary thyroid cancer.

Romei C, Cosci B, Renzini G, et al. RET genetic screening of sporadic medullary thyroid cancer (MTC) allows the preclinical diagnosis of unsuspected gene carriers and the identification of a relevant percentage of hidden familial MTC (FMTC). Clin Endocrinol. 2011;74:241–247.

Correspondence: R. Elisei at relisei@endoc.med.unipi.it

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Detection of West Nile and Japanese encephalitis viruses Detection of West Nile and Japanese encephalitis viruses

Species within the Flavivirus genus can cause public health problems worldwide. For example, the increasing number of Dengue and Japanese encephalitis virus (JEV) infections in Asia, frequent outbreaks of yellow fever in Africa and South America, and the ongoing spread of West Nile virus throughout the Americas exemplify the geographical burden of flavivirus diseases. Global flavivirus incidence has grown in the past 25 years. This increase is due primarily to unprecedented population growth and migration, uncontrolled urbanization, and lack of effective mosquito control. West Nile virus and JEV are arthropod-borne flaviviruses, termed arborviruses, that can emerge or re-emerge in many regions due to climate change and increased travel to such regions. They are associated with human and equine encephalitis worldwide. If a human pathogen such as West Nile virus emerges or re-emerges in a country, very strict epidemiological regulations need to be implemented immediately. To monitor infection and prevent rapid viral spread, methods are required for rapid viral detection in suspect cases and potential vectors. In areas where JEV is endemic, such as Korea and Japan, distinguishing between West Nile virus and JEV is critical to detecting West Nile virus invasion. JEV is a mosquito-borne flavivirus from the JEV serocomplex that causes encephalitis in humans and horses. It is widespread throughout most of Asia. However, Japanese encephalitis serocomplex flaviviruses cross-react antigenically with West Nile virus and, therefore, are not readily differentiated by serology. For this reason, molecular diagnostic methods are preferred, and reverse transcriptase-polymerase chain reaction (RT-PCR) methods have been used to develop sensitive and specific assays for identifying West Nile virus. Recently, more sensitive assays, such as fluorogenic real-time (TaqMan) PCR, SYBR green-based real-time PCR, and loop-mediated isothermal amplification (LAMP), have been developed to detect West Nile virus genes. However, these diagnostic methods were designed only to detect strains of West Nile virus, isolated in the United States, that are closely related to lineage one. They may not detect lineage two strains found in Africa and, more recently, Europe. In areas where JEV is endemic and West Nile virus is absent, it is possible that other West Nile virus lineages can emerge, such as lineage two. Therefore, it is necessary to design assays that recognize all lineages of the virus and can distinguish between JEV and West Nile virus for purposes of making a definitive diagnosis. The authors conducted a study to develop a more rapid molecular diagnostic method that could detect and distinguish between West Nile virus and JEV using a conventional RT-PCR format in a single-tube duplex platform with a primer mixture specific to JEV strains and all of the lineages of West Nile virus. The bioinformatic analysis of published sequences of West Nile virus and JEV revealed conserved regions not targeted by previously reported primers. Thirteen primers were designed based on these regions to detect all of the West Nile virus and JEV lineages and to discriminate between the two viruses by the generation of 482- and 241-bp cDNA products, respectively. The results indicate that single-tube duplex PCR using these primers is a useful technique for detecting and differentiating West Nile virus and JEV in plasma or brain tissue. The novel duplex RT-PCR described in this study enables the early diagnosis of these two encephalitic flaviviruses. In addition, this technique may be useful as part of a testing regimen for humans, horses, and other susceptible animal species because it is rapid (less than 3.5 hours from RNA extraction), sensitive, and specific.

Yeh JY, Lee JH, Seo HJ, et al. Fast duplex one-step reverse transcriptase PCR for rapid differential detection of West Nile and Japanese encephalitis viruses. J Clin Microbiol. 2010;48:4010–4014.

Correspondence: Jung-Yong Yeh at yeh02@nvrqs.go.kr

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Cerebrospinal fluid reference ranges in febrile infants Cerebrospinal fluid reference ranges in febrile infants

Fever in infants one to 90 days of age is a common clinical problem. Medical evaluation of these infants often includes an analysis of cerebrospinal fluid (CSF) to exclude bacterial meningitis. Controversy surrounds the interpretation of CSF profiles from young infants. The authors identified 18 publications that analyzed the CSF profiles of presumptively uninfected infants. Many of the publications did not include the precise age of the infants, and most did not document all bacterial and viral testing performed. Most studies included data from no more than 50 infants, and only three studies included data on 100 or more infants, making it difficult to determine normative values for the CSF profile of young infants. The normal values referenced in U.S. pediatric textbooks for infant CSF profile are based on four studies with small numbers of infants. Three studies were performed before the availability of polymerase chain-reaction (PCR) testing for enteroviruses, the most common cause of aseptic meningitis in this age group. A recent study describing normative CSF white blood cell (WBC) counts in infants younger than 56 days old reported negative enterovirus PCR test results for approximately 20 percent of the cohort and no enterovirus PCR testing for the remainder. Further, although controversial, recent literature has associated CSF pleocytosis with urinary tract infection, the most common cause of serious bacterial infection in this age group. Two of the commonly referenced studies describing normal infant CSF WBC values did not report the infants’ status for urinary tract infection. Finally, the presence of red blood cells (RBC) in CSF may alter the number of WBCs observed. Traumatic lumbar punctures are common in pediatric practice. Several authors have evaluated the contribution of RBCs in older infants, children, and adults. However, information about the effect of CSF RBCs on the CSF WBC counts in infants one to 90 days of age is limited. The authors conducted a study to describe the CSF profiles of febrile infants with negative bacterial culture test results of blood, urine, and CSF and negative test results for enterovirus with PCR and to determine the contribution of CSF RBCs to the CSF WBCs in these infants. They analyzed statistically a retrospective cohort of CSF profiles from 823 infants with negative test results for infection. For 677 infants with atraumatic lumbar punctures (RBC count, less than 1,000/mm3), the mean and median CSF WBC counts were 4.3/mm3 and 3.0/mm3, respectively, with a range from zero to 12/mm3. Mean CSF WBC counts (6.1/mm3 versus 3.1/mm3 and 3.0/mm3) and protein levels (75.4 mg/dL versus 58.9 mg/dL and 39.2 mg/dL) were higher in the first month compared with months two and three, respectively (P<0.001 for all). CSF glucose levels were lower in the first month compared with month three (45.3 mg/dL versus 48.0 mg/dL and 57.7 mg/dL; P<0.001). Increasing RBC counts were statistically associated with increasing WBC counts (P<0.001). However, the contribution of RBC counts of less than 10,000/mm3 was small, and the reference range for WBC counts in uninfected infants with traumatic lumbar punctures was zero to 16/mm3. The authors concluded that CSF WBC counts in febrile infants without evidence of bacterial or enteroviral infection, even in those with traumatic lumbar puncture, are lower than reported in pediatric references.

Byington CL, Kendrick J, Sheng X. Normative cerebrospinal fluid profiles in febrile infants. J Pediatr. 2011;158:33–37.

Correspondence: Carrie L. Byington at carrie.byington@hsc.utah.edu

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Clinical pathology abstracts editor: Michael Bissell, MD, PhD, MPH, professor, Department of Pathology, Ohio State University, Columbus.