Polymerase chain reaction on cerebrospinal fluid for diagnosis of virus-associated opportunistic diseases of the central nervous system in HIV-infected patients

OBJECTIVE: To assess the diagnostic reliability of polymerase chain reaction (PCR) on cerebrospinal fluid (CSF) for virus-associated opportunistic diseases of the central nervous system (CNS) in HIV-infected patients. DESIGN: CSF samples from 500 patients with HIV infection and CNS symptoms were examined by PCR. In 219 patients the PCR results were compared with CNS histological findings. METHODS: Nested PCR for detection of herpes simplex virus (HSV) type 1 or 2, varicella zoster virus (VZV), cytomegalovirus (CMV), Epstein-Barr virus (EBV), human herpesvirus 6 (HHV-6), and JC virus (JCV) DNA. Histopathological examination of CNS tissue obtained at autopsy or on brain biopsy. RESULTS: DNA of one or more viruses was found in CSF in 181 out of 500 patients (36%; HSV-1 2%, HSV-2 1%, VZV 3%, CMV 16%, EBV 12%, HHV-6 2%, and JCV 9%). Among the 219 patients with histological CNS examination, HSV-1 or 2 was detected in CSF in all six patients (100%) with HSV infection of the CNS, CMV in 37 out of 45 (82%) with CMV infection of the CNS, EBV in 35 out of 36 (97%) with primary CNS lymphoma, JCV in 28 out of 39 (72%) with progressive multifocal leukoencephalopathy. Furthermore, HSV-1 was found in one, VZV in four, CMV in three, EBV in three, HHV-6 in seven, and JCV in one patient without histological evidence of the corresponding CNS disease. CONCLUSIONS: CSF PCR has great relevance for diagnosis of virus-related opportunistic CNS diseases in HIV-infected patients as demonstrated by its high sensitivity, specificity, and the frequency of positive findings.     

Vancomycin cerebrospinal fluid concentrations after intravenous administration in premature infants

OBJECTIVE: Staphylococcal species are the most common cause of nosocomial infections in the neonate. Because of staphylococcal resistance patterns, vancomycin has become the drug of choice for treatment. Although the blood stream is the usual site of infection, premature infants are at increased risk for the development of meningitis. The aim of this study was to determine vancomycin cerebrospinal fluid (CSF) concentration and penetration following intravenous (IV) administration in critically ill premature infants. STUDY DESIGN: A multiple-dose, open-label, case series was performed at a level III neonatal intensive care unit in a university teaching hospital. Three critically ill premature infants, 26 to 31 weeks of gestation requiring a course of IV vancomycin for suspected or proved sepsis were studied. Vancomycin was administered intravenously at 20 mg/kg, every 18 to 24 hours over 60 minutes. Serum and CSF vancomycin concentrations were obtained and pharmacokinetic analysis and CSF penetration was calculated. RESULTS: Serum vancomycin pharmacokinetics were consistent with those previously reported. CSF vancomycin concentrations ranged from 2.2 to 5.6 micrograms/ml and the calculated vancomycin CSF penetration ranged from 26% to 68%. CONCLUSIONS: CSF penetration of vancomycin after IV administration was much higher than that reported in older infants and children. This higher penetration may improve clinical outcomes in neonates with central nervous system infections. These data should be encouraging to clinicians who choose to use IV vancomycin for neonatal meningitis.     

Detection of viral DNA in neonatal herpes encephalitis autopsy tissues by solution-phase PCR: comparison with pathology and immunohistochemistry

To detect DNA sequences of herpes simplex virus (HSV) in neural and non-neural tissue sections in disseminated human neonatal HSV infection, a solution polymerase chain reaction (PCR) protocol was developed which amplified HSV thymidine kinase and host genomic DNA sequences that were hybridized with sequence-specific probes in Southern blots. Serial sections of formalin-fixed, paraffin embedded autopsy tissues were tested by PCR and compared to histology and HSV antigen detection. The sensitivity, specificity and reproducibility of this PCR protocol were determined on uninfected and HSV-infected mouse tissues and on HSV DNA from infected tissue culture cells. Samples estimated to contain as few as 60 copies of preserved HSV DNA target sequence gave a positive PCR result. In nine neonates that died during acute HSV infection, all non-neural tissues and a minority of neural tissues with histological lesions had HSV antigen; when DNA could be amplified, HSV DNA sequences were detected by PCR. Together, these findings indicate a direct role for virus in the pathogenesis of these lesions. In the same cases, some or all brain samples were negative for HSV antigen, but nevertheless had HSV DNA sequences detected by PCR. The possible explanations for this finding are discussed. In one neonate dying seven weeks after birth, HSV sequences were found in brain lesions in the absence of HSV antigen; neither HSV DNA nor antigen were found in non-neural tissues, suggesting a latent HSV infection in brain. It is practical to apply PCR methods to detect minute quantities of viral DNA in formalin-fixed, paraffin embedded autopsy tissues.(ABSTRACT TRUNCATED AT 250 WORDS)     

Legal rights of veterinarians under veterinary Good Samaritan statutes and equine liability statutes

OBJECTIVE: To determine the prevalence and clinical features of Candida species in hospital-acquired urinary tract infections (UTI) in a neonatal intensive care unit. DESIGN: A retrospective study was conducted of hospital-acquired UTI occurring in infants admitted to a neonatal intensive care unit between January 1, 1989, and June 30, 1995. Hospital-acquired infection was defined as one occurring in an infant who was at least 7 days of age and hospitalized since birth. Urinary tract infection was defined by a urine culture yielding a single organism with > 1000 colony-forming units/ml from a suprapubic aspiration or > 10,000 colony-forming units/ml via urethral catheterization. RESULTS: Fifty-seven infants had 60 UTI during the study period. Candida spp. were responsible for 25 of 60 (42%) UTI. The median gestational age of infants with candidal UTI was 26 weeks (range, 23 to 37) which was significantly less than that for infants with bacterial UTI, 28 weeks (range, 23 to 40) (P = 0.04). Candidemia was present in 13 of 25 (52%) candidal UTI which was significantly more often than bacteremia with bacterial UTI, 3 of 35 (8%) (odds ratio, 11.6; 95% confidence interval, 2.8 to 47.8). The median age of infection for candidal UTI was 34 days (range, 9 to 228), which was significantly earlier than for bacterial UTI, 79 days (range, 7 to 247) (P = 0.003). Renal pelvis fungus balls were present in 7 of 20 (35%) infants with candidal UTI who had renal ultrasound studies. CONCLUSIONS: Candida spp. were the pathogens identified in 42% of hospital-acquired urinary tract infections in a neonatal intensive care unit. Candidemia was associated with 52% of candidal UTI and bacteremia with 8% of bacterial UTI. Candidal UTI occurred significantly earlier than bacterial UTI. Renal fungus balls were present in 35% of infants with candidal UTI.     

A case of chronic encephalitis due to double infection with herpes simplex and measles viruses

In a healthy 49-year-old man, a decrease in job efficiency was noticed along with bizarre behavior. On admission, he was euphoric, childish, superficial and had increased libido. Neurological findings were normal. There were no abnormal findings on routine blood tests, hematochemistry or urine analysis. MRI showed no abnormal findings. However, single photon emission CT (SPECT) showed diffuse hypoaccummulation of tracer from the temporal to frontal regions. Lumbar puncture showed clear cerebrospinal fluid (CSF) with pleocytosis and an elevated protein level. Moreover, antibody IgG titers to herpes simplex virus (HSV) and measles virus were elevated, according to EIA [serum HSV -1,202.2x, measles virus 47.1x: CSF HSV-116.1x, measles virus 9.9x]. The ratio of serum to CSF antibody titers of HSV and measles virus were 12.5 and 4.75, respectively. The antibody index values of HSV and measles virus IgG titers were 8.42 and 22.22. The ratio of albumin was 105.7. Chronic, progressive HSV encephalitis is rare, and there have been very few reports of encephalitis due to double infection by HSV and another virus. Our patient was diagnosed as having encephalitis due to double infection with HSV and measles virus, because the ratio of serum to CSF antibody titers was less than 20 and the antibody index values were over 1.91. Moreover, since the IgG index was elevated and the ratio of albumin was not low, it was suggested that the blood-brain-barrier had not been disrupted, and antibodies were being produced chronically in the medullary cavity. Hyperaccummulation of tracer on SPECT studies has been reported in the early stages of HSV encephalitis. In our case, while CT and MRI showed no abnormal findings, SPECT showed diffuse hypoaccummulation. SPECT appears to be a useful tool in the diagnosis of this disorder. In case of chronic, progressive personality change in middle-aged adults, we must be aware of double virus infection of the brain as a possible causal factor.     

Recurrent ascending myelitis: an unusual presentation of herpes simplex virus type 1 infection

We report on a healthy female with a unique relapsing transverse myelitis accompanied by herpes simplex virus type 1 (HSV-1) infection. Magnetic resonance imaging showed cord enlargement and increased signal intensity on T1-weighted image with gadolinium enhancement from T-4 to T-10 during the first attack and from C-1 to C-2 during the second episode. She was not diagnosed during the first attack. During the second episode, laboratory studies disclosed IgM and IgG antibodies to HSV at the outset with greater than fourfold increases in antibody levels in the serum and cerebrospinal fluid (CSF). Cells cultured from the CSF were positive for HSV-1 according to the immunofluorescence method. The presence of HVS-1 DNA in CSF was documented by polymerase chain reaction (PCR) technique. Acyclovir was given with a partial recovery. We anticipate that PCR assay of CSF will assist early diagnosis of herpetic central nervous system disorders.     

Major histocompatibility complex class II DR alleles DRB1*1501 and those encoding HLA-DR13 are preferentially associated with a diminution in maternally transmitted human immunodeficiency virus 1 infection in different ethnic groups: determination by an automated sequence-based typing method

Transmission of human immunodeficiency virus 1 (HIV-1) from an infected women to her offspring during gestation and delivery was found to be influenced by the infant's major histocompatibility complex class II DRB1 alleles. Forty-six HIV-infected infants and 63 seroreverting infants, born with passively acquired anti-HIV antibodies but not becoming detectably infected, were typed by an automated nucleotide-sequence-based technique that uses low-resolution PCR to select either the simpler Taq or the more demanding T7 sequencing chemistry. One or more DR13 alleles, including DRB1*1301, 1302, and 1303, were found in 31.7% of seroreverting infants and 15.2% of those becoming HIV-infected [OR (odds ratio) = 2.6 (95% confidence interval 1.0-6.8); P = 0.048]. This association was influenced by ethnicity, being seen more strongly among the 80 Black and Hispanic children [OR = 4.3 (1.2-16.4); P = 0.023], with the most pronounced effect among Black infants where 7 of 24 seroreverters inherited these alleles with none among 12 HIV-infected infants (Haldane OR = 12.3; P = 0.037). The previously recognized association of DR13 alleles with some situations of long-term nonprogression of HIV suggests that similar mechanisms may regulate both the occurrence of infection and disease progression after infection. Upon examining for residual associations, only only the DR2 allele DRB1*1501 was associated with seroreversion in Caucasoid infants (OR = 24; P = 0.004). Among Caucasoids the DRB1*03011 allele was positively associated with the occurrence of HIV infection (P = 0.03).     

Herpes simplex virus type 2 infections presenting as brainstem encephalitis and recurrent myelitis

We describe here 3 patients with central nervous system infection caused by herpes simplex virus type 2 (HSV-2); one patient with brainstem encephalitis and two with recurrent transverse thoracic myelitis. All three patients showed increased IgG antibodies to HSV in the cerebrospinal fluid (CSF). HSV-2 DNA was demonstrated in the CSF by polymerase chain reaction (PCR) amplification. Upon treatment with acyclovir, one patient with myelitis partially recovered and the others completely recovered. It is important to recognize the wide spectrum of clinical manifestations of HSV-2 infection in the central nervous system (CNS).     

Severe myelotoxicity of oral etoposide in heavily pretreated patients with non-Hodgkin''s lymphoma or chronic lymphatic leukemia

In this study we aimed to determine the incidence of herpes simplex virus (HSV) in the lungs of burns patients, and its association with the presence of adult respiratory distress syndrome (ARDS) and pneumonia. Haematoxylin and eosin (H&E), and immunohistochemical (IHC) staining for HSV was performed on lung tissue from 54 patients who had died following burn injury and from nine control cases. Polymerase chain reaction (PCR) for HSV deoxyribonucleic acid (DNA) was performed on a subset both of burns cases and controls. No viral inclusions were detected in H&E sections, but 50% of the burns cases were positive for HSV by IHC staining; no control cases were positive. Nuclear and cytoplasmic immunopositivity for HSV was seen in macrophages and epithelial lining cells. HSV was strongly associated with ARDS (p=0.007), but not with pneumonia (p=0.577). The relative risk of HSV infection was higher for cases with ARDS (2.21) than for those with pneumonia (1.26). PCR for HSV DNA was positive in three out of five burns cases, and in one out of five control cases. Immunohistochemical staining is more sensitive for the detection of herpes simplex virus than haematoxylin and eosin staining for detection of viral inclusions. Burns cases have a high incidence of pulmonary herpes simplex virus infection. Polymerase chain reaction results may not be fully representative due to problems of tissue necrosis postmortem. Pulmonary herpes simplex virus is strongly associated with adult respiratory distress syndrome and the two may be causally linked. Early detection and treatment of pulmonary herpes simplex virus in burns patients may reduce pulmonary complications and mortality.     

Effect of rHuEpo therapy in dialysis patients on endogenous erythropoietin synthesis after renal transplantation

BACKGROUND: Pertussis is a highly contagious respiratory disease and the most serious effects occur in young infants. Recently it has been shown that rapid and highly specific PCR can be a useful diagnostic tool for detection of pertussis infection. To our knowledge there are no previous studies concerning the disappearance of Bordetella pertussis DNA from the nasopharynx during antimicrobial treatment. METHODS: We studied prospectively how rapidly live B. pertussis organisms and DNA of these bacteria disappear from the nasopharynx during erythromycin therapy in unvaccinated infants. Eighty-five nasopharyngeal swabs obtained from nine erythromycin-treated infants with pertussis on consecutive days during hospitalization were tested by PCR and culture. The PCR products were further analyzed by Southern hybridization. RESULTS: On the fourth day of treatment 56% of the samples were positive by culture and 89% by PCR, whereas after 7 days the rates were 0 and 56%, respectively. In seven of nine patients PCR remained positive for 1 to 7 days longer than culture. The follow-up study also showed the semiquantitative nature of the PCR assay. The intensity of the PCR products in agarose gel usually weakened with time during erythromycin therapy. CONCLUSIONS: The results of this study show that PCR assay can achieve the specific diagnosis of pertussis infection in a large proportion of infants even when antimicrobial treatment has killed the organisms and culture is no longer positive.     

The prevalence of herpes family virus DNA in the conjunctiva of patients positive and negative for human immunodeficiency virus using the polymerase chain reaction

OBJECTIVE: To help understand the pathogenesis of herpes family virus ocular infection among patients positive for HIV, the authors compared the rates of detection of herpes family virus DNA from the conjunctiva of patients who are positive and negative for human immunodeficiency virus (HIV) using the polymerase chain reaction (PCR). DESIGN: Cross-sectional study. PARTICIPANTS: The conjunctival scrapings of 30 patients positive for HIV and 30 patients negative for HIV were examined. INTERVENTION: PCR was used to assay for the presence of herpes simplex virus type 1 (HSV), varicella-zoster virus (VZV), cytomegalovirus (CMV), and Epstein-Barr virus (EBV) DNA (n = 240 samples). MAIN OUTCOME MEASURE: The rate of detection of virus DNA in the two groups, controlling for age, gender, and race, was measured. RESULTS: HSV and VZV DNA were not detected in any of the HIV-positive or HIV-negative samples. CMV DNA was detected in 20% (6 of 30) of patients positive for HIV and was undetected in control subjects negative for HIV (P = 0.01). EBV DNA was detected in 40% (12 of 30) of patients positive for HIV and in 47% (14 of 30) of control subjects negative for HIV (P = 0.58). CONCLUSIONS: There was no difference in the frequency of detection of HSV, VZV, or EBV DNA from the conjunctiva of patients positive or negative for HIV. Only CMV DNA was detected at a significantly higher rate in the conjunctiva of patients positive for HIV compared with control subjects negative for HIV. These different rates of peripheral virus shedding may be one possible explanation for the different rates of clinical infection among the herpes family viruses among patients positive for HIV.     

Diagnosis of meningoencephalitis caused by herpes simplex virus type 1 using nested PCR in CSF samples

BACKGROUND: The aim of this study was to achieve the early diagnosis of the neurologic alteration caused by the Herpes Simplex virus type 1 (HSV-1) with the nested PCR technique in CSF. PATIENTS AND METHODS: From January, 1994 to October, 1995, 140 CSF from 140 patients were studied in our laboratory. Ninety-five were diagnosed with viral meningoencephalitis (Group A) and 45 with other neurologic diseases (Group B). Nested PCR of HSV-1 and conventional viral cultures were carried out in all the cases. RESULTS: Laboratory diagnosis was achieved in 13 (13.68%) of Group A patients: in 12 (12.63%) HSV-1 genome was detected by nested PCR and in one patient adenovirus was isolated. In Group B, the HSV-1 was detected by nested PCR in 2 patients (4.44%). CONCLUSIONS: The results obtained demonstrate the usefulness of nested PCR in HSV-1 infection for the diagnosis of herpetic meningoencephalitis in initial stages of the disease, from a single CSF sample.     

Quantitation of human cytomegalovirus (HCMV) DNA in cerebrospinal fluid by competitive PCR in AIDS patients with different HCMV central nervous system diseases

The current study was designed to quantitate human cytomegalovirus (HCMV) DNA in cerebrospinal fluid (CSF) of persons with AIDS with specific HCMV-related CNS disease. DNA present in CSF obtained from AIDS patients was initially detected by a qualitative PCR procedure and then quantitated using a competitive PCR assay. In a group of 21 AIDS patients with HCMV-related CNS disease, 12 patients with HCMV polyradiculopathy had a mean +/- SEM of 11,982 +/- 4,480 copies/microliters in their CSF compared to 1,747 +/- 929 for 9 patients with HCMV encephalitis p = 0.017). Of the 14 patients with > 1,000 copies/microliters of HCMV DNA in CSF, 11(79%) had HCMV polyradiculopathy including all 3 with > 10,000 copies/microliters. Ganciclovir treatment of 3 patients with HCMV-related CNS disease was associated with a decline in HCMV DNA detectable within CSF. These data indicate that quantities of HCMV DNA in CSF are higher in persons with HCMV-related polyradiculopathy than encephalitis, and that quantitation of HCMV DNA can be useful in monitoring antiviral therapy.     

Herpes simplex virus type 1 in brain and risk of Alzheimer's disease

BACKGROUND: The apolipoprotein E epsilon 4 (APOE-epsilon 4) allele is a risk factor for Alzheimer's disease (AD), but it is neither essential nor sufficient for development of the disease. Other factors-genetic or environmental-must therefore have a role. By means of a PCR we have detected herpes simplex virus type 1 (HSV1) in latent form in brains of elderly people with and without AD. We have postulated that limited reactivation of the virus causes more damage in AD patients than in elderly people without AD because of a difference in the hosts. We now report the APOE genotypes of AD patients and non-AD sufferers with and without HSV1 in brain. METHODS: DNA was extracted from 84 samples of brain from 46 AD patients (39 temporal lobe, 39 frontal lobe, three hippocampus) and from 75 samples of brain from 44 non-AD elderly people (33 temporal lobe, 36 frontal lobe, six hippocampus). PCR amplification was used to detect HSV1 thymidine kinase gene and the host APOE gene. FINDINGS: By multiple logistic regression, the APOE-epsilon 4 allele frequency was significantly higher in the patients positive for HSV1 in brain than in the HSV1-negative AD group, the HSV1-positive non-AD group, or the HSV1-negative non-AD group (52.8% vs 10.0%, 3.6%, and 6.3%, respectively). The odds ratio for APOE-epsilon 4 in the HSV1-positive AD group compared with HSV1-negative non-AD group was 16.8 (95% CI 3.61-77.8) and in the HSV1-negative AD group, 1.67 (0.21-13.4). We also compared APOE genotypes of 40 people who had recurrent cold sores and 33 non-sufferers; the APOE-epsilon 4 allele frequencies were 36% and 9%, respectively (p < 0.0001). INTERPRETATION: These findings suggest that the combination of HSV1 in brain and carriage of an APOE-epsilon 4 allele is a strong risk factor for AD, whereas either of these features alone does not increase the risk of AD. The findings in people with cold sores support our hypothesis that APOE-epsilon 4 and HSV1 together are damaging in the nervous system.     

Central nervous system infection with Listeria monocytogenes. 33 years' experience at a general hospital and review of 776 episodes from the literature

We reviewed 776 previously reported and 44 new cases of CNS listeriosis outside of pregnancy and the neonatal period, and evaluated the epidemiologic, diagnostic, and therapeutic characteristics of this infection. Among patients with Listeria meningitis/meningoencephalitis, hematologic malignancy and kidney transplantation were the leading predisposing factors, but 36% of patients had no underlying diseases recognized. The infection occurred throughout life, with a higher incidence before the age of 3 and after the age of 45-50 years. Fever, altered sensorium, and headache were the most common symptoms, but 42% of patients had no meningeal signs on admission. Compared with patients with acute meningitis due to other bacterial pathogens, patients with Listeria infection had a significantly lower incidence of meningeal signs, and the CSF profile was significantly less likely to have a high WBC count or a high protein concentration. Gram stain of CSF was negative in two-thirds of cases of CNS listeriosis. One-third of patients had focal neurologic findings, and approximately one-fourth developed seizures over their course. Mortality was 26% overall, and was higher among patients with seizures and those older than 65 years of age. Relapse occurred in 7% of episodes. Ampicillin for a minimum of 15-21 days (with an aminoglycoside for at least the first 7-10 days) remains the treatment of choice. Cerebritis/abscess due to L. monocytogenes, without meningeal involvement, is less common but may be diagnosed by blood cultures and CNS imaging, or by stereotactic biopsy. Longer antibiotic therapy (at least 5-6 weeks) is needed in the presence of localized CNS involvement.     

Association of herpesvirus infection with the development of genital cancer

Clinical observations and epidemiological studies on genital cancer have revealed an association with sexual behavior, thus motivating research into sexually transmitted agents which may be responsible for the neoplasia. In this study, we used the PCR technique to examine the presence of CMV, HSV and EBV viruses in 187 cases of human genital lesions and found that infection with CMV or HSV was associated with cervical cancer. When we stratified according to HPV status this association was found only for HPV-DNA-negative cases. These findings indicate that past infection with CMV or HSV could be interpreted as a surrogate marker of HPV infection. However, these viruses may play an important role themselves in cervical cancer.     

Atopic keratoconjunctivitis

Rapid, non-culture, serogroup determination of meningococcal infection is important in contact management where vaccination may be possible. The impending availability of polysaccharide-protein conjugate vaccines for serogroup C disease requires maximal case ascertainment, with serogroup determination, at a time when the number of culture confirmed meningococcal infections is decreasing. A polymerase chain reaction assay (PCR), based on a restriction fragment length polymorphism (RFLP) in the meningococcal serogroup B and C sialytransferase (siaD) gene, was developed to combine the non-culture diagnosis of meningococcal infection from CSF, whole blood and serum with serogroup (B and C) identification. The PCR assay was adapted to an ELISA format incorporating hybridization with serogroup-specific B and C oligonucleotide probes. Specificity for CSFs was 100% and sensitivities were respectively 81, 63 and 30% for CSFs, whole blood and sera. The serogroup-specific PCR ELISA is a significant addition to currently available tests for non-culture diagnosis of meningococcal infection and outbreak investigation.     

Alloimmune neonatal neutropenia in Australian aboriginals: an unrecognized disorder?

Alloimmune neonatal neutropenia (ANN) is reported for the first time in two Australian aboriginals. Both infants displayed the typical clinical features of ANN with profound neutropenia which persisted for 7 weeks and only minor infectious episodes. However, management strategies differed for the two infants because in one case (complicated by uncertain paternity) serological confirmation of ANN was not obtained until after recovery of the infant's neutrophil count. The maternal antibodies could not be assigned to known neutrophil antigen specificities and a new antigen may be involved. The antibodies were reactive with > 99% of neutrophils in a Caucasian population. Aboriginals comprise 1% of the total population of Australia and 1-2% of the obstetric population at our institution. Thus, ANN may be an unrecognized disorder in this ethnic group and a possible cause of neonatal infection and mortality.