Comparison of real-time and nested PCR assays for detection of herpes simplex virus DNA

We performed a real-time PCR assay to detect herpes simplex virus (HSV) DNA, and compared it prospectively with a nested PCR assay in 164 clinical samples (109 cerebrospinal fluid and 55 sera) from patients suspected of having neonatal HSV infection or HSV encephalitis. In 25 of 164 samples, HSV DNA was detected by the nested PCR assay. All samples positive for HSV DNA in the nested PCR assay were also positive in the real-time PCR assay, and all but two samples negative for HSV DNA in the nested assay were negative in the real-time assay. The real-time PCR assay thus had a sensitivity of 100% and a specificity of 99%, when compared with the nested assay. Sequential assays in a case of disseminated HSV showed that a decrease in HSV DNA paralleled clinical improvement. Quantification of HSV DNA by real-time PCR was useful for diagnosing and monitoring patients with HSV encephalitis and neonatal HSV infection.     

Quantitation of herpes simplex virus DNA in cerebrospinal fluid of patients with herpes simplex encephalitis by the polymerase chain reaction

Background: Previous studies have shown the diagnostic utility of qualitative detection of herpes simplex virus (HSV) DNA by the polymerase chain reaction (PCR) in cerebrospinal fluid samples (CSF) from patients with herpes simplex encephalitis (HSE).Objectives: To determine whether quantitation of HSV DNA in CSF could be useful for monitoring efficacy of antiviral therapy and provide prognostic indications.Study design: A quantitative PCR assay using an internal control for evaluation of PCR efficiency and detection of PCR inhibitors was developed and used for retrospective testing of 98 CSF samples from 26 patients with serologically diagnosed HSE during the period 1980–1995.Results: HSV DNA was detected in 36 CSF samples from 23 patients. PCR positivity was 100% for CSF samples collected within 10 days after onset, and 30.4 and 18.7% for samples collected 11–20 and 21–40 days later, respectively. The 3 PCR-negative patients had their first CSF collected 14, 16, and 28 days after onset, respectively. Three of 98 (3.1%) CSF samples were completely or partially inhibitory to PCR. Initial DNA levels were not significantly different in patients with HSE due to either primary or recurrent HSV infection. In addition, they were not related to severity of clinical symptoms nor were predictive of the outcome. A progressive decrease in viral DNA levels was observed both in patients who received acyclovir therapy and in a small number of untreated patients.Conclusions: This study: (i) confirms the high sensitivity of PCR for the diagnosis of HSE; (ii) emphasizes the need for an internal control of amplification to achieve maximal sensitivity and perform reliable quantitation of viral DNA; and (iii) suggests that CSF might not be the best specimen to investigate in studies of the natural history of HSE.     

Serum and cerebrospinal fluid herpes simplex virus type 1 immunoglobulin G and M titers in four cases of herpes simplex encephalitis

Herpes simplex virus type 1 (HSV-1) IgG and IgM ELISA titers were serially determined in serum and cerebrospinal fluid (CSF) samples from 4 patients with HSV-1 encephalitis during a follow-up period of 1-26 months. In 3 out of 4 patients HSV-1 IgM titers raised in CSF during the acute phase of disease, thus allowing differentiation between primary and reactivated forms of HSV-1 encephalitis. HSV-1 IgG titers showed a sharp elevation earlier in serum than in CSF. Specific IgG index documented a large intrathecal production of HSV-1 IgG and their persistence 2 years following clinical onset. The initial trend of serum and CSF specific IgG titer represents a reliable tool for a retrospective diagnosis of HSV-1 encephalitis.     

A simple method for obtaining cerebrospinal fluid from a pig model of herpes encephalitis

A procedure is described for obtaining cerebrospinal fluid (CSF) from anesthetized pigs by puncturing the cisterna magna. CSF samples of at least 5 ml. can be obtained. The punctures are well tolerated and can be repeated with intervals of approximately 4 days.     

Direct PCR assay for detection ofNeisseria meningitidis in human cerebrospinal fluid

A PCR amplification was performed to detectNeisseria meningitidis insertion sequence1106 (IS-1106) in the humancerebrospinalfluid (CSF) in cases of meningitis. The study included 27 CSF samples from suspected meningitis patients. Although the inflammatory response in most of the samples was slightly increased, the results showed that 7 (26%) and 8 (30%) CSF samples were diagnosed as meningococcal meningitis by Gram staining and by culture, respectively. The primers of theIS-1106 were used for direct diagnosis ofN. meningitidis in the human spinal fluid after a minor treatment of the CSF samples. The sample was diagnosed as meningococcal meningitis, if a DNA band of about 600 bp was detected in the ethidium bromide-stained agarose gel. The 27 CSF samples were analyzed in a random manner. Of these, 18 samples including the Gram staining- and culture-positive samples were also positive in PCR amplification. However, a CSF sample, which was diagnosed to be meningococcal meningitis in culture was negative in both Gram staining and PCR analysis. The specificity of theIS-1106 primers was determined to be 95%, with 100% sensitivity in comparison to Gram staining and culture. The primers were sensitive to 10 pg or more of meningococcal DNA. In addition, the PCR amplification showed high predictive values (89 and 100%) in diagnosing meningitis in patients that were negative and positive responders when tested by culture and by Gram staining. In conclusion, the PCR amplification ofIS-1106 ofN. meningitidis is specific and sensitive to both culture-positive and-negative meningococcal meningitis. Hence, PCR assay is highly recommended for use in a rapid diagnosis of suspected meningitis patients.     

Detection of cytomegalovirus in infant cerebrospinal fluid by conventional PCR, nested PCR and PCR-Digene

The possibility of amplification of human cytomegalovirus (HCMV) DNA in cerebrospinal fluid (CSF) for the diagnosis of HCMV central nervous system (CNS) infection in infants was studied. Single-step PCR, nested PCR and PCR-Digene were used to assay CSF specimens from 37 patients. Criteria for patient inclusion in the study were: 1. clinical manifestations suggesting CMV neuroinfection such as seizures, hypertonia, hypotonia, intracranial calcification, microcephaly, chorioretinitis; 2. any of the following symptoms: anaemia, hepetomegaly, prolonged cholestatic jaundice, or hepatitis, splenomegaly, thrombocytopenia, intrauterine hypotrophy; 3. serologic presentation, and/or positive results for CMV infection obtained by single-step PCR and PCR-Digene in urine and/or blood. PCR-Digene results were positive in 6 CSF samples. Four CSF samples were positive by nested PCR and 1 CSF sample by single step PCR. We found that the double PCR was about ten or more times more sensitive than single PCR and the PCR-Digene was only three times more sensitive than nested-PCR. The results were correlated with serology. Thirty-three out of 37 examined patients were seropositive (ELISA IgG); ELISA IgM gave positive results in 9 patients. In control studies, cells infected with other members of the herpes virus family were negative with these methods, which suggest that amplification combined with primers from the IE and the L-region of CMV is specific. In conclusion, nested-PCR seems to be the best method for early diagnosis of CMV infection in CSF due to an absence of false positive results and its high specificity and sensitivity.     

Mother-child separation: a study in a remote health care setting.

The literature indicates that the birth of a sibling and the consequent temporary separation from the mother is usually a stressful experience for a child. It was hypothesized that this stress would result in an increased number of visits by the child to health care facilities because of new health problems. In a controlled study of 89 matched pairs of Indian families in a remote region of northwestern Ontario this hypothesis was not supported. During the intervals studied - the anticipatory period before delivery, the separation itself and the period immediately following the mother''s return home - the number of diagnoses of new medical problems was significantly less for the children who were separated from their mothers for the birth of a sibling. As well, the number of diagnoses of new medical problems in the children separated form their mothers decreased over the three intervals. The fathers'' reluctance to seek health care probably played a major role in this decrease.     

Comprehensive mental health care in a pediatric dialysis-transplantation program.

The dialysis-transplantation (D-T) program at The Hospital for Sick Children, Toronto has a mental health component directed by a psychiatrist and a social worker. As of Jan. 1, 1975, 53 kidney transplants had been carried out on 44 children. Patients and their families are counselled continuously by the psychiatrist and the social worker before, during and after transplantation. Members of the multidisciplinary team meet regularly to plan treatment for the children. Mental health issues are an integral part of team discussions and help determine D-T program policy. Psychological preparation, mental health consultation, therapeutic intervention and continuous counselling prevent many of the mental health problems that plague a D-T program.     

Advance in herpes simplex viruses for cancer therapy

Oncolytic virotherapy is an attractive approach that uses live viruses to selectively kill cancer cells. Oncolytic viruses can be genetically engineered to induce cell lyses through virus replication and cytotoxic protein expression. Herpes simplex virus (HSV) has become one of the most widely clinically used oncolytic agent. Various types of HSV have been studied in basic or clinical research. Combining oncolytic virotherapy with chemotherapy or radiotherapy generally produces synergic action with unclear molecular mechanisms. Arming HSV with therapeutic transgenes is a promising strategy and can be used to complement conventional therapies. As an efficient gene delivery system, HSV has been successfully used to deliver various immunomodulatory molecules. Arming HSV with therapeutic genes merits further investigation for potential clinical application.     

Development and assessment of sensitive immuno‐PCR assays for the quantification of cerebrospinal fluid three‐ and four‐repeat tau isoforms in tauopathies

Characteristic tau isoform composition of the insoluble fibrillar tau inclusions define tauopathies, including Alzheimer's disease (AD), progressive supranuclear palsy (PSP) and frontotemporal dementia with parkinsonism linked to chromosome 17/frontotemporal lobar degeneration‐tau (FTDP‐17/FTLD‐tau). Exon 10 splicing mutations in the tau gene, MAPT, in familial FTDP‐17 cause elevation of tau isoforms with four microtubule‐binding repeat domains (4R‐tau) compared to those with three repeats (3R‐tau). On the basis of two well‐characterised monoclonal antibodies against 3R‐ and 4R‐tau, we developed novel, sensitive immuno‐PCR assays for measuring the trace amounts of these isoforms in CSF. This was with the aim of assessing if CSF tau isoform changes reflect the pathological changes in tau isoform homeostasis in the degenerative brain and if these would be relevant for differential clinical diagnosis. Initial analysis of clinical CSF samples of PSP (n = 46), corticobasal syndrome (CBS; n = 22), AD (n = 11), Parkinson's disease with dementia (PDD; n = 16) and 35 controls revealed selective decreases of immunoreactive 4R‐tau in CSF of PSP and AD patients compared with controls, and lower 4R‐tau levels in AD compared with PDD. These decreases could be related to the disease‐specific conformational masking of the RD4‐binding epitope because of abnormal folding and/or aggregation of the 4R‐tau isoforms in tauopathies or increased sequestration of the 4R‐tau isoforms in brain tau pathology.     

Detection of herpes simplex and varicella zoster viruses in clinical specimens using direct immunofluorescence and cell culture assays

Patients (33 in toto) with a clinical diagnosis of herpes infections (simplex, zoster or chickenpox) were investigated for the presence of herpes simplex virus (HSV) and varicella zoster virus (VZV) in skin samples, using direct immunofluorescence and cell culture assays. Five patients with nonherpetic vesiculobullous disorders were included as negative controls. Of the 33 patients, nineteen (57.6%) were positive for HSV or VZV and fourteen (42.4%) were negative. Five controls were all negative for HSV or VZV. Of the nineteen positive patients, HSV was isolated from eight (42.1%) patients, by both direct immunofluorescence and cell culture assays. VZV was isolated from eleven (57.9%) patients, eleven (100%) by direct immunofluorescence assay, and six (54.5%) by cell culture assays. HSV was isolated from one patient clinically diagnosed as chickenpox (VZV), but otherwise the positive laboratory results were concordant with the clinical diagnosis. For epidemiological studies, atypical cases and immunocompromised patients the clinical diagnosis should be confirmed in the laboratory.     

Determination of 4-methylpropranolol in cerebrospinal fluid,serum, and urine by nonprotected fluid room-temperature phosphorescence using simplex optimization

A direct and simple procedure for the determination of 4-methylpropranolol, a specific beta-adrenergic receptor blocking agent, in biological fluids was developed. The method was based on the measurement of the nonprotected fluid room-temperature phosphorescence of the drug. This technique enables us to determine analytes in complex matrices without the need for a tedious prior separation process. The appropriate experimental conditions to obtain suitable reproducibility and maximum phosphorescence signal, when sodium sulfite is used to eliminate the oxygen from the solution and when potassium iodide is used as heavy atom, were studied. The optimum concentration of KI was 3.2 M. The optimization of Na(2)SO(3) (7.0 x 10(-3) M) and the accurate value of pH (10.88) were determined using a simplex as the method of optimization. A sodium carbonate-hydrogen carbonate buffer solution (5.0 x 10(-2) M) was used to adjust the value of pH. The delay time (124 micros), gate time (206 micros), and time between flashes (5 ms) were also optimized using a simplex. Under the above conditions, the maximum signal of phosphorescence appears instantly once the sample has been prepared, and the intensity was measured at lambda(ex) = 300 nm and lambda(em) = 537 nm, in the concentration range 25-500 ng/ml. Overall least-squares regression was used to find the straight line that fit the experimental data. The detection limit according to the error propagation theory was 6.2 ng/ml and the detection limit calculated as proposed by C. A. Clayton et al. (1987, Anal. Chem. 59, 2506) was 11.7 ng/ml. The repeatability was studied using 10 solutions of 200 ng/ml 4-methylpropranolol; if error propagation theory was assumed, the relative error was 1.78% and the standard deviation for replicate samples was 3.5 ng/ml. This method was successfully applied to the determination of 4-methylpropranolol in urine, serum, and cerebrospinal fluid, with recoveries of 99.3 +/- 0.5% in the case of urine, 99.8 +/- 0.2% for serum, and 101.5 +/- 1.5% for cerebrospinal fluid.     

Proteomic profiling of cerebrospinal fluid identifies prostaglandin D2 synthase as a putative biomarker for pediatric medulloblastoma: A pediatric brain tumor consortium study

The aims of this study were to demonstrate the feasibility of centrally collecting and processing high-quality cerebrospinal fluid (CSF) samples for proteomic studies within a multi-center consortium and to identify putative biomarkers for medulloblastoma in CSF. We used 2-DE to investigate the CSF proteome from 33 children with medulloblastoma and compared it against the CSF proteome from 25 age-matched controls. Protein spots were subsequently identified by a combination of in-gel tryptic digestion and MALDI-TOF TOF MS analysis. On average, 160 protein spots were detected by 2-DE and 76 protein spots corresponding to 25 unique proteins were identified using MALDI-TOF. Levels of prostaglandin D2 synthase (PGD2S) were found to be six-fold decreased in the tumor samples versus control samples (p<0.00001). These data were further validated using ELISA. Close examination of PGD2S spots revealed the presence of complex sialylated carbohydrates at residues Asn(78) and Asn(87) . Total PGD2S levels are reduced six-fold in the CSF of children with medulloblastoma most likely representing a host response to the presence of the tumor. In addition, our results demonstrate the feasibility of performing proteomic studies on CSF samples collected from patients at multiple institutions within the consortium setting.     

Urinary schistosomiasis on Pemba Island: low-cost diagnosis for control in a primary health care setting

In the 1970s and early 1980s indirect diagnosis of urinary schistosomiasis, using urinalysis reagent strips for proteinuria and haematuria, was proposed as a possible alternative to the more accurate but very time-consuming parasitological methods. The recent experience o f the Schistosomiasis Control Programme for Pemba Island, which used a combination of (1) observations o f grossly bloody urine specimens, (2) results from reagent strips for measuring haematuria, and (3) treatment with praziquantel, is the first large-scale example o f a simple, inexpensive and promising alternative for controlling the morbidity caused by this parasite.     

Challenges and progress in setting a standard for PCR for invasive aspergillosis

Despite extensive experience in the past decade using polymerase chain reaction for diagnosing aspergillosis, the technique was not included as mycologic evidence in either the first consensus definitions of invasive fungal disease or in recent revisions because there was no standardized or validated method. Consequently, several research groups decided to join forces and form a working party of the International Society of Human and Animal Mycology to establish a standard for Aspergillus polymerase chain reaction testing. This group opted for screening tests on blood because this is most likely to find a place in integrated care pathways for managing invasive aspergillosis. Progress is being made such that a standard will be proposed in the near future. This standard will then be formally validated in a prospective trial.