Gregory J. Buffone

Rapid diagnosis of herpes simplex virus encephalitis by using the polymerase chain reaction

To determine the diagnostic value of the polymerase chain reaction (PCR) in establishing the rapid diagnosis of herpes simplex virus encephalitis (HSE) in the pediatric age group, we performed PCR to detect herpes simplex virus (HSV) in the cerebrospinal fluid of 8 neonates with HSV infection (4 with central nervous system involvement), 11 infants and children with suspected HSE (4 proved, 1 presumed, 6 excluded), and 105 control patients who had cerebrospinal fluid obtained as part of the evaluation for other diagnoses. The HSV DNA was amplified and typed by using primers specific for the DNA polymerase gene of HSV types 1 and 2. Herpes simplex virus DNA was detected in the cerebrospinal fluid of 3 of 4 neonates with CNS involvement (all with HSV type 2) and 3 of the 4 patients with proved HSE (all with HSV type 1). No HSV DNA was detected in the 4 neonates without CNS disease, the 1 patient with presumed HSE, the 6 patients who had HSE excluded from the diagnosis, and the 105 control patients. Overall, HSV PCR had a sensitivity of 75%, a specificity of 100%, a positive predictive value of 100%, and a negative predictive value of 98%. These results indicate that PCR is a useful noninvasive test in establishing the diagnosis of acute HSE, but a negative result did not exclude the diagnosis.

Growth and hepatospecific gene expression of human hepatoma cells in a defined medium.

SummaryThe production of albumin, α-fetoprotein (AFP), and α-1 antitrypsin has been compared among human hepatoma cells cultured in medium containing serum, medium containing hormones and growth factors, and a basal medium containing selenium as the only supplement. Growth is sustained in all three media, and the expression of all three proteins was maintained for over 4 mo. in the various media. However, the quantitative production of albumin and AFP were dramatically different in the three media. Two hormones, insulin and triiodothyronine, influenced the level of secreted proteins. Triiodothyronine increases the amount of secreted albumin whereas insulin at 10 μg/ml reduced the level of total secreted protein.

High-performance liquid chromatography of human hemoglobins on a new cation exchanger

We have investigated the use of a high-performance liquid chromatographic (HPLC) column packed with a unique weak cation exchanger prepared by coating silica with poly(aspartic acid) for hemoglobin analysis. The complete separation of hemoglobin Barts F, A0, A2, S, C, D, E, G, SG, Winnipeg and Sealy was achieved by gradient elution within 30 min. The high resolution made it possible to distinguish hemoglobin variants such as Barts, AC, AD, AE, AG, AS, ASG, CC, SC, SS, Winnipeg, Sealy and beta-chain variants with thalassemia such as S/beta +, S/beta 0 and S(C)-beta + thalassemia. Comparison of DEAE-cellulose column chromatography and our HPLC method for the quantitation of hemoglobin A2 yielded a good correlation. Hemoglobins A2, C and E are completely resolved on PolyCAT A columns in contrast to both cellulose acetate electrophoresis and DEAE-cellulose column chromatography. The high resolution of the system and the accuracy of the method combined with complete automation make this procedure useful for diagnosis of hemoglobin disorders in both a research and clinical laboratory environment.

Demonstration of Epstein-Barr virus in primary central nervous system lymphomas by the polymerase chain reaction and in situ hybridization☆

Primary lymphomas of the central nervous system (CNS) account for 0.3% to 1.5% of all intracranial neoplasms. Several reports have noted a coincidence between this neoplasm and serologic evidence of Epstein-Barr virus (EBV) infection, but in only a few instances has the EBV genome been demonstrated in these tumors. To further evaluate the frequency of this occurrence, we analyzed primary CNS lymphomas using nucleic acid hybridization methods and the polymerase chain reaction (PCR). In situ hybridization was used in selected cases. Sequences of EBV were found in two of nine cases by PCR and in situ hybridization. Southern blot hybridization of genomic DNA from these samples was negative for EBV. Both tumors arose in patients with conditions shown to produce secondary immunodeficiency, namely, chronic alcohol abuse and diabetes mellitus. We conclude that the association of EBV and CNS lymphoma is not restricted to patients with severe primary immune deficiency, and that PCR can be applied successfully to paraffin-embedded tissue for the detection of low-abundance viral sequences.

Deficient classical complement pathway activity in newborn sera.

Summary: The bactericidal activity of 20 maternal-neonatal paired sera was assessed employing a clinical type la, group B streptococcal isolate, strain 515, known to be opsonized by the classical complement pathway in a non-antibody dependent fashion. Twelve neonatal sera had efficient bactericidal activity for this isolate (mean 96%, range 91-99%) whereas eight had significantly lower bactericidal indices (mean 29%, range 0-54%) (p < 0.001). The mean bactericidal index for 20 maternal sera (88%) did not differ from that of 20 adult control sera (91%). Bactericidal activity was not influenced by the concentration of antibody to the capsular polysaccharide antigen of type Ia, group B Streptococcus present in these sera. When the classical pathway of selected neonatal sera with high or low bactericidal activity was inactivated by MgEGTA chelation of C1, bacterial growth was observed uniformly when concentrations of specific antibody were low. The bactericidal index of neonatal sera correlated significantly with the total hemolytic complement titer (r = 0.611, P < 0.01). The mean levels of each complement component or control protein assessed by radial immunodiffusion (C1q, C4, C3, B, H, and I) were lower in neonatal sera with low than in those with efficient bactericidal activity, and levels of C1q and H were depressed significantly (P < 0.025 and <0.001, respectively). Hemolytic titers of C4 but not C1 or C2 were also significantly lower for low than for selected high-killing neonatal sera (P < 0.05). Bactericidal activity in neonatal serum with a bactericidal index of zero was restored in a dose-dependent fashion by the addition of fresh frozen plasma.

Impaired Chemotaxigenesis by Type III Group B Streptococci in Neonatal Sera: Relationship to Diminished Concentration of Specific Anticapsular Antibody and Abnormalities of Serum Complement

Summary: A chemotaxigenesis (CTG) assay employing adult or neonatal sera, type III group B streptococci (GBS) and polymorphonuclear leukocytes (PMNs) was designed to evaluate the role of PMN mobilization in the pathogenesis of type III GBS infection in neonates. Generation of C5a in healthy adult sera with moderate-high (3–40 μg/ml) or low (≤2 μg/ml) levels of specific anticapsular antibody was confirmed by PMN aggregometry and by the neutralization of CTG by goat anti-human C5. CTG was significantly (P < 0.001) greater in high as compared to low specific antibody-containing adult sera; stepwise increases in CTG occurred when specific IgG was added to untreated, but not heat-inactivated, hypogammaglobutinemic serum. Immunospecificity of CTG was shown by a failure of type III GBS to generate C5a in heterologous (type Ia) high antibody sera. Mean CTG values in three high and 16 low antibody-containing sera from healthy term neonates were 24% and 62% of high (P < 0.001) and low (P < 0.01) antibody adult sera, respectively. The addition of both complement and specific IgG to low antibody-containing neonatal sera was required to enhance their CTG activity to high antibody adult values. CTG by type III GBS in neonatal sera-neonatal PMN mixtures was only 25% (high antibody sera) and 14% (low antibody sera) of values for paired maternal sera mixtures reacted with adult PMNs (P < 0.001). These studies demonstrate that CTG by type III GBS in neonatal sera is markedly diminished and that low concentrations of specific anticapsular antibody and abnormalities of complement function contribute to impaired PMN mobilization in human neonates.

The diagnosis of CMV pneumonitis in lung and heart/lung transplant patients by PCR compared with traditional laboratory criteria

Polymerase chain reaction (PCR) amplification of CMV DNA recovered from bronchial alveolar lavage (BAL) and peripheral blood samples was compared with tissue culture, cytology, and/or histology for the earlier detection of CMV pneumonitis in 12 recipients of singlelung or heart/lung transplants. In patients with confirmed CMV pneumonitis, cytological evidence of CMV disease in BAL samples was detected 38±14 days posttransplantation, while tissue culture and PCR-positive results were noted as early as 30±4.0 days and 18±4.6 days, respectively. While PCR was positive earlier than culture in a number of cases, culture-positive results were subsequently obtained in each case, consistent with earlier detection of viral replication by PCR as opposed to detection of latent virus. CMV was detected by PCR in 6 of 24 blood samples from patients with confirmed or suspected CMV pneumonitis, while results of all 24 blood samples were negative when assayed by tissue culture. PCR-based testing was more sensitive than traditional tests, allowing detection of viral replication earlier than tissue culture in the posttransplant period. PCR could provide a powerful means of monitoring the immunocompromised patients in whom preemptive therapeutic intervention for CMV disease is desirable.

Performance of a Rapid Influenza Test in Children During the H1N1 2009 Influenza A Outbreak

OBJECTIVE: To evaluate the performance of a rapid influenza diagnostic test (RIDT) in detecting H1N1 2009 influenza A virus in respiratory samples from pediatric patients in comparison to that of real-time reverse-transcriptase polymerase chain reaction (rRT-PCR) and viral culture. Methodology. This was a cross-sectional diagnostic-accuracy study conducted at a tertiary care childrens hospital. Patients for whom the RIDT (BinaxNOW [Binax, Inc, Portland, ME]), viral culture, and rRT-PCR results were known were included. Sensitivity, specificity, and likelihood ratios (LRs) were calculated. RESULTS: A total of 3030 specimens had RIDT results paired with both rRT-PCR and viral culture results. With rRT-PCR as the reference, overall test sensitivity was 45% (95% confidence interval [CI]: 43.3%–46.3%) and specificity was 98.6% (95% CI: 98.1%–99%). Positive and negative LRs were 32.9 (95% CI: 22.9–45.4) and 0.56 (95% CI: 0.54–0.58), respectively. RIDT sensitivity was significantly higher in young infants and children younger than 2 years than in older children. Using viral culture as the reference standard, RIDT sensitivity was 55.5% (95% CI: 51.9%–95.6%) and specificity was 95.6% (95% CI: 95%–96.1%). The positive and negative LRs were 12.6 and 0.47, respectively. CONCLUSIONS: The RIDT had relatively poor sensitivity but excellent specificity in this consecutive series of respiratory specimens obtained from pediatric patients. Although a positive RIDT result was highly accurate in predicting infection with influenza type A H1N1 2009 in children, a negative RIDT result did not preclude a child having H1N1. Therefore, for children at high risk with influenza-like illnesses during high-prevalence periods of influenza, empiric initiation of antiviral therapy should be considered for patients with a negative RIDT result.

Clinical Applications of DNA Probes in the Diagnosis of Genetic Diseases

Currently, advances in molecular technology involving recombinant DNA have led to dramatic breakthroughs in genetic diseases, cancer research, and identification of foreign DNA. Of particular interest is the impact these tools have made and will make on the clinical laboratory. We describe the techniques and their effects on clinical testing in the chemistry laboratory by using selected examples of available applications. Specific examples include carrier detections and prenatal diagnosis in cystic fibrosis and hemophilia, and sickle cell anemia.

Detection of mRNA from the immediate early gene of human cytomegalovirus in infected cells by in vitro amplification

It would be desirable to have a facile means of detecting the presence of viral DNA or mRNA in any given biological sample, especially in view of the growing immunocompromised population in which the diagnosis of viral disease is a common problem. In vitro amplification of DNA using methods such as the polymerase chain reaction, offers a sensitive means of detecting both DNA and mRNA. We have used the polymerase chain reaction to detect DNA and mRNA from human cytomegalovirus infected human fibroblasts. Viral mRNA was differentiated from DNA using primers which flank a splice junction, resulting in a smaller product for the mRNA template. A cDNA was prepared from total RNA using a primer specific for the gene of interest, in this case the major immediate early transcript of human cytomegalovirus. The cDNA was then amplified using a modified polymerase chain reaction protocol. mRNA from the major immediate early gene was detected in cultured fibroblasts as early as 6 h after infection, and continued to be expressed for at least 96 h post infection. Sensitive and facile detection of viral mRNA should facilitate diagnostic and basic studies of viral pathogenesis.