Valerie L. Ng

Diagnosis of Pneumocystis carinii Pneumonia: Improved Detection in Sputum with Use of Monoclonal Antibodies

With the dramatic increase in the frequency of Pneumocystis carinii pneumonia associated with human immunodeficiency virus infection, there has been a need for more rapid and less invasive diagnostic techniques. Recent studies have shown that examination of induced sputum can establish the diagnosis of P. carinii pneumonia in about 55 percent of cases. To assess whether a recently developed indirect immunofluorescent stain using monoclonal antibodies was more sensitive than Giemsa or toluidine blue O stains in detecting P. carinii in sputum, we undertook two prospective studies. Of 63 patients at one institution from whom sputum specimens were obtained, 49 were ultimately given a diagnosis of P. carinii pneumonia, 46 of them by staining of sputum. The sensitivity of the three stains in detecting P. carinii was 45 of 49 (92 percent) for immunofluorescence; 37 of 49 (76 percent) for Diff-Quik (a Giemsa-type stain); and 39 of 49 (80 percent) for toluidine blue O. There were no false positive immunofluorescent stains. In a similar study of a series of 25 patients at another institution, a diagnosis of P. carinii pneumonia was made in 23 of 25 patients by staining of induced sputum. We conclude that examination of induced sputum is a rapid, sensitive, and inexpensive method for diagnosing P. carinii pneumonia and that indirect immunofluorescence is a practical and highly sensitive staining technique for establishing this diagnosis.

The natural history and molecular heterogeneity of HIV-associated primary malignant lymphomatous effusions

Primary malignant lymphomatous effusions arising in individuals infected with the human immunodeficiency virus, type 1 (HIV-1) represent a rare subset of HIV-associated lymphomas. Previous studies have demonstrated that the malignant cells are monoclonal (as defined by rearrangement of the immunoglobulin gene), express cell surface CD38, and are infected with Epstein-Barr virus (EBV) and human herpes virus, type 8 (HHV-8). Despite these detailed molecular and immunophenotypic studies, clinical information on this disease entity is scant, prompting us to review the clinical features of eight cases seen at our institutions. All eight patients had total peripheral CD4+ lymphocytes < 200/microliter and presented with complaints related to body cavity distension. Routine laboratory values were nondiagnostic and yielded no prognostic information. Only two patients could tolerate and thus received chemotherapy with no obvious impact on their clinical course. The mean overall survival after diagnosis was 60 days (range 6-166 days). Four patients were examined at autopsy. The primary malignant lymphomatous effusion either was the immediate cause of death or contributed significantly to the death of only two. All four patients examined post mortem, however, had lymphomatous infiltration of serosal surfaces adjacent to the site of the primary malignant effusion. Molecular and immunologic studies performed on the malignant cells and effusion fluids revealed universal expression of cell surface CD38 and the presence of HHV-8 gene sequences, but in contrast with previous studies, only four had rearranged immunoglobulin genes or EBV present: IL-6 and IL-10 levels in the malignant effusion fluids were markedly elevated. In summary, this rare subset of HIV-associated lymphomas in our eight patients arose late in the course of HIV-associated disease, had a rapid clinical course, and was molecularly heterogeneous. A pathogenetic role for HHV-8 alone in this disease process is strengthened by our observation of four cases lacking EBV but containing HHV-8.

Classifying Laboratory Incident Reports to Identify Problems That Jeopardize Patient Safety

We developed a laboratory incident report classification system that can guide reduction of actual and potential adverse events. The system was applied retrospectively to 129 incident reports occurring during a 16-month period. Incidents were classified by type of adverse event (actual or potential), specific and potential patient impact, nature of laboratory involvement, testing phase, and preventability. Of 129 incidents, 95% were potential adverse events. The most common specific impact was delay in receiving test results (85%). The average potential impact was 2.9 (SD, 1.0; median, 3; scale, 1-5). The laboratory alone was responsible for 60% of the incidents; 21% were due solely to problems outside the laboratorys authority. The laboratory function most frequently implicated in incidents was specimen processing (31%). The preanalytic testing phase was involved in 71% of incidents, the analytic in 18%, and the postanalytic in 11%. The most common preanalytic problem was specimen transportation (16%). The average preventability score was 4.0 (range, 1-5; median, 4; scale, 1-5), and 94 incidents (73%) were preventable (score, 3 or more). Of the 94 preventable incidents, 30% involved cognitive errors, defined as incorrect choices caused by insufficient knowledge, and 73% involved noncognitive errors, defined as inadvertent or unconscious lapses in expected automatic behavior.

Reducing unnecessary inpatient laboratory testing in a teaching hospital

After an inpatient phlebotomy-laboratory test request audit for 2 general inpatient wards identified 5 tests commonly ordered on a recurring basis, a multidisciplinary committee developed a proposal to minimize unnecessary phlebotomies and laboratory tests by reconfiguring the electronic order function to limit phlebotomy-laboratory test requests to occur singly or to recur within one 24-hour window. The proposal was implemented in June 2003. Comparison of fiscal year volume data from before (2002-2003) and after (2003-2004) implementation revealed 72,639 (12.0%) fewer inpatient tests, of which 41,765 (57.5%) were related directly to decreases in the 5 tests frequently ordered on a recurring basis. Because the electronic order function changes did not completely eliminate unnecessary testing, we concluded that the decrease in inpatient testing represented a minimum amount of unnecessary inpatient laboratory tests. We also observed 17,207 (21.4%) fewer inpatient phlebotomies, a decrease sustained in fiscal year 20042005. Labor savings allowed us to redirect phlebotomists to our understaffed outpatient phlebotomy service.

Multicenter Evaluation of the Performance Characteristics of the Bayer VERSANT HCV RNA 3.0 Assay (bDNA)

ABSTRACT In this multicenter evaluation, the VERSANT HCV RNA 3.0 Assay (bDNA) (Bayer Diagnostics, Tarrytown, N.Y.) was shown to have excellent reproducibility, linearity, and analytical sensitivity across specimen collection matrices (serum, EDTA, ACD-A), and hepatitis C virus (HCV) genotypes 1 to 6. The VERSANT HCV bDNA Assay has a reportable range of 615 to 7,690,000 (7.69 × 106) IU/ml. The total coefficient of variation (CV) ranged from 32.4% at 615 IU/ml to 17% at 6.8 × 106 IU/ml. The assay was linear across the reportable range. Analytical specificity of 98.8% was determined by testing 999 specimens from volunteer blood donors. Evaluation of HCV genotypes using RNA transcripts of representative clones of 1a, 1b, 2a, 2b, 2c, 3a, 4a, 5a, and 6a and patient specimens showed that the largest difference between genotype 1, upon which the assay is standardized, and non-1 genotypes was within 1.5-fold. Testing of potentially interfering endogenous substances and exogenous substances and conditions found no interference in HCV-positive or HCV-negative specimens except for unconjugated bilirubin at concentrations of ≥20 mg/dl and protein at concentrations of ≥9 g/dl. Biological variability was estimated from 29 clinically stable individuals not on HCV therapy who were tested weekly over an 8-week period. The combined estimate of total (biologic plus assay) variability was 0.15 log10 standard deviation (CV, 36.1%), a fold change of 2.6. Thus, the observed fold change between any two consecutive HCV RNA measures is expected to be less than 2.6-fold (equivalent to 0.41 log10 IU/ml) 95% of the time in clinically stable individuals.

Reversal of Human Immunodeficiency Virus Type 1-Associated Hematosuppression by Effective Antiretroviral Therapy

The immunodeficiency of human immunodeficiency virus type 1 (HIV-1) disease may be due to accelerated destruction of mature CD4+ T cells and/or impaired differentiation of progenitors of CD4+ T cells. HIV-1 infection may also inhibit the production of other hematopoietic lineages, by directly or indirectly suppressing the maturation of multilineage and/or lineage-restricted hematopoietic progenitor cells. To test this hypothesis, the effects of durable viral suppression on multilineage hematopoiesis in 66 HIV-1-seropositive patients were evaluated. Administration of effective antiretroviral therapy resulted in an increase in circulating CD4+ T cell counts and statistically significant increases in circulating levels of other hematopoietic lineages, including total white blood cells, lymphocytes, polymorphonuclear leukocytes, and platelets. These results suggest that a significant lesion in untreated HIV-1 disease may lie at the level of cell production from hematopoietic progenitors.

Comparative analysis of HIV-1 viral load assays on subtype quantification: Bayer Versant HIV-1 RNA 3.0 versus Roche Amplicor HIV-1 Monitor version 1.5

Summary: Quantification of HIV‐1 subtypes is essential for appropriate clinical management. Whereas viral load assays were initially developed to accurately quantify subtype B, the recent worldwide spread of non‐B subtypes and the introduction of treatment programs in regions with non‐B subtypes have prompted adaptations of these assays. The Bayer Versant HIV‐1 RNA 3.0 Assay (branched DNA [bDNA] 3.0) and the Roche Amplicor HIV‐1 Monitor version 1.5 (Amplicor 1.5) assays are reported to quantify all subtypes in group M; however, evaluation of performance characteristics remains limited. In this study, we evaluated the accuracy and reliability of bDNA 3.0 and Amplicor 1.5 on multiple serially diluted viral isolates from HIV‐1 group M, subtypes A through F. Testing was conducted on both assay systems in two independent laboratories. Comparative pansubtype quantification from regression analysis showed that quantification by bDNA 3.0 was approximately 0.3 log‐fold lower than that by Amplicor 1.5. Comparative pansubtype accuracy analysis showed data points more closely distributed about their respective regression lines and thus showing greater reliability by bDNA 3.0 than by Amplicor 1.5.

Food, shelter and safety needs motivating homeless persons' visits to an urban emergency department.

STUDY OBJECTIVES We determine whether homeless persons present to the emergency department (ED) for food, shelter, and safety and whether the availability of alternative sites for provision of these needs might decrease their ED presentations. METHODS In July to August 2006 and February to March 2007, adult homeless and control (not homeless) patients, who self-presented (nonambulance) to an urban county ED, were interviewed with a structured instrument. RESULTS One hundred ninety-one homeless and 63 control subjects were enrolled. Homeless persons spent a mean (standard deviation [SD]) of 3.5 (3.0) nights/week sleeping without shelter and ate a mean (SD) of 2.1 (1.1) meals per day; 51% stated they had been assaulted on the street. On an analog scale, in which 0=no problem and 10=worst possible problem in their daily lives, the mean (SD) homeless subject responses for hunger, lack of shelter, and safety were 4.8 (3.7), 6.1 (4.2), and 5.1 (4.0), respectively. More homeless (29% [55/189]) than not homeless (10% [6/63]) persons replied that hunger, safety concerns, and lack of shelter were reasons they came to the ED (Delta=20%; 95% confidence interval 10% to 29%). If offered a place that would provide food, shelter, and safety at all times, 24% of homeless subjects stated they would not have come to the ED. CONCLUSION Homeless persons commonly come to the ED for food, shelter, and safety. Provision of these subsistence needs at all times at another site may decrease their ED presentations.

Simultaneous Runs of the Bayer VERSANT HIV-1 Version 3.0 and HCV bDNA Version 3.0 Quantitative Assays on the System 340 Platform Provide Reliable Quantitation and Improved Work Flow

ABSTRACT Branched DNA (bDNA) assays to quantify human immunodeficiency virus type 1 (HIV-1) and hepatitis C virus (HCV) consist of three distinct steps, including sample processing, hybridization, and detection, and utilize the System 340 platform for plate incubation and washing. Sample processing differs: HIV-1 from 1 ml of plasma is concentrated by high-speed centrifugation, whereas HCV plasma or serum samples are used without concentration. The first step of hybridization involves viral lysis at 63°C: HIV-1 is performed in a heat block, whereas HCV is performed in System 340. The remaining hybridization and detection steps are similar for HIV-1 and HCV and executed on System 340. In the present study, the HIV-1 bDNA assay was adapted for viral lysis in the System 340 platform. The adaptation, test method 2, includes a 20-s vortex of concentrated viral pellet and lysis working solution, transfer of viral lysate to the 96-well capture plate, and transfer to System 340 programmed for HCV assay specifications. With test method 2, specificity and quantification were within assay specifications. HCV bDNA methodology remains unchanged. Hence, an HIV-1 and an HCV bDNA can be run simultaneously on System 340. With simultaneous testing, laboratories can run full plates, as well as combinations of full and partial plates. Also, simultaneous HIV-1 and HCV bDNA permits labor consolidation and improved workflow while maintaining multitasking and rapid patient result turnaround.

Comparative genomic analyses of primary effusion lymphoma.

BACKGROUND A rare subset of human immunodeficiency virus (HIV) lymphomas, known as primary effusion lymphomas (PELs), are high-grade tumors carrying human herpes virus 8. Mechanisms postulated to contribute to lymphomagenesis include impaired immune surveillance, alterations in hemopoietic regulatory pathways due to expressed viral genes, and acquisition of genomic alterations in regions of the genome that contain regulatory genes. In PEL, limited information exists about the nature of genome-wide aberrations in these rare lymphomas. METHODS We used comparative genomic hybridization to detect regions of sequence gain and loss throughout the genome of 8 PEL cases. Regions of DNA sequence loss or gain were confirmed using forward and reverse hybridization and t-statistic analyses. RESULTS Genomic aberrations were identified in 6 of 8 cases, including recurrent gain of sequence in chromosomes 12 [ish enh (12q22;12q23, 12q12;12q23)] in 3 of 8 cases and X [ish enh (X, Xp)] in 2 of 8 cases. CONCLUSIONS DNA copy number changes occurred in a majority of PEL cases and are consistent with changes observed in other HIV lymphomas. These observations suggest that common genetic events may occur in HIV-associated lymphoid malignancies, but they probably do not contribute to the unique markers and morphology of PEL. Although individual genetic loci have been evaluated previously in a few PEL cases, to our knowledge this study represents the first reported genome-wide scan of copy number changes in these rare HIV-associated tumors.