A protein microarray immunoassay for the serological evaluation of the antibody response in vertically transmitted infections.

The detection of specific serum antibodies is mainly achieved by enzyme-linked immunosorbent assay (ELISA). Here, we describe the setting up of a microarray-based serological assay to screen for IgG and IgM against vertically transmitted pathogens (Toxoplasma gondii, rubella virus, cytomegalovirus, herpes simplex virus types 1 and 2, varicella zoster virus, Chlamydia trachomatis). The test, accommodated onto a restricted area of a microscope slide, consists of: (a) the immobilization of antigens and human IgG and IgM antibody dilution curves, laid down in an orderly manner; (b) addition of serum samples; (c) detection of antigen-serum antibodies complexes by indirect immunofluorescence. The IgG and IgM curves provide an internal calibration system for the interpolation of the signals from the single antigens. The test was optimized in terms of spotting conditions and processing protocol. The detection limit was 400 fg for the IgG assay and 40 fg for the IgM assay; the analytical specificity was >98%. The clinical sensitivity returned an average value of 78%, the clinical specificity was >96%, the predictive values were >73%, and the efficiency was >88%. The results obtained make this test a promising tool, suitable for introduction in the clinical diagnostic routine of vertically transmitted infections, in parallel (and in future as an alternative) to ELISA.

Relation between psychoneuroendocrine profile in stressful conditions and antibodies to herpesvirus 6 and 7.

The aim of the present study is to evaluate whether the individual susceptibility to infectious disease is influenced by the psychological profile of cadets exposed to stressful events associated with military lifestyle in academy and if the neuroendocrine responses to stressful events is related with humoral immunity estimated by measuring antibody titres to human herpesvirus (HHV-6)7 (HHV-7) and to what extent it is influenced by personality traits. It has been observed that cadets with lower psychoaptitude scores (1-2) have a significant higher susceptibility to infectious disease (x2=7.95; p=0.019) compared to subjects with higher scores. A positive relationship between cortisol and antibody titers to HHV-6 (r=0.304; p=0.024) it has been found. It can be interesting to observe that antibody titers on HHV-6 are also related to psychoaptitude profile (r=0.239; p=0.044). The antibody titers to HHV-7 are negatively related to the 5 scales of BFQ and in particular with subdimension Co (cordiality) of BFQ (r=0.401; p=0.002). The survey carried out on over 1,500 cadets of the Military Academy of Modena shows that the susceptibility to infectious diseases during the first six months of admission to the Academy seem to be influenced by the psychoaptitudinal profile. The finding of a positive relationship between serum cortisol and antibody vs HHV-6 suggests that the impairment of the immune system linked to circulatory cortisol levels may induce a reactivation of a latent herpesvirus 6 with related increase of antibody titers.

Human herpesvirus-6B active infection associated with relapsing bilateral anterior optic neuritis.

BACKGROUND AND OBJECTIVES: Human herpesvirus-6 (HHV-6) is the causative agent of exanthem subitum. Both HHV-6 variants, A and B, have been associated with central nervous system (CNS) diseases, suggesting a wide neuropathogenic potential. We describe a case of recurrent bilateral anterior optic neuritis with HHV-6 active infection associated with clinical relapses. CASE REPORT: A 23-year old woman presented with progressive visual impairment, bilateral papillitis and painful ocular movements. Nested polymerase chain reaction (PCR) for DNA viruses, HHV-6 variant specific real time quantitative PCR, serological analysis and retrotranscription PCR (RT-PCR) for HHV-6 mRNA transcripts were performed. Nested PCR in PBMC and CSF samples was negative for all viruses but positive for HHV-6 DNA, subtyped as HHV-6B. The disease had a relapsing/remitting course. During relapses PBMC samples remained positive for HHV-6 DNA, and HHV-6 active infection was confirmed by the presence of anti-HHV-6 IgM and of HHV-6 U27 mRNA transcript. High viremia levels and relapses were overlapping. After the last relapse, the patient was successfully treated with gancyclovir. CONCLUSIONS: The case reported here suggests a possible association of HHV-6 in bilateral optic neuritis. HHV-6 could be monitored when bilateral optic neuritis is identified, in order to establish an appropriate antiviral therapy.

Growth of human herpesvirus 6 in HEPG2 cells.

HepG2 cells, a well differentiated liver cell line, were shown to be permissive for both human herpesvirus 6 (HHV-6) A and B strains by three independent methods of analysis: detection of viral antigens, viral DNA sequences and infectious virus. HepG2 cell infection with HHV-6 resulted in functional damage as shown by the increased release in the culture medium of some hepatocyte markers. Cells surviving the acute infection were serially passaged without showing cytopathic effect, but, some months later, HHV-6 DNA was still present in the cells and virus induction with a phorbol ester was successful. A possible pathogenetic role of HHV-6 in liver diseases is discussed. Experiments of HepG2 infection with human herpesvirus 7 (HHV-7) were also carried out. The lack of an efficient virus replication suggested a difficulty for HHV-7 to infect hepatic cells.

Viruses and multiple sclerosis.

Multiple sclerosis (MS) is a chronic demyelinating disease of the central nervous system whose cause is still unknown. Many findings suggest an infectious etiology or, at least, that infectious agents in association with host genetic factors may play a role in the pathogenesis of this disease. Accumulating data, including animal models, human models of virus induced demyelination, epidemiologic, and laboratory findings, demonstrate that viruses and host genetic factors can interact to cause immune-mediated demyelination. While many viruses have been postulated as a possible cause of MS, to date, no "MS virus" has been definitively shown to be associated with this disease. Alternatively, ubiquitous viruses are being considered as the environmental "triggers" that have been postulated to be involved in the MS disease process. We will focus on recent studies with human herpesvirus 6 and MS as how a common virus may be associated with this disorder in a subset of infected individuals.

Quantitation of HIV-1 p24 antibody expressed as relative binding capacity p24 antigen (p24 RBC) in infants born to HIV-1 seropositive mothers: correlation of this serological marker with the HIV-1 maternal antibody course and p24 antigenemia detection.

The antibody content to HIV-1 p24 Ag expressed as relative binding capacity to the target antigen (p24 RBC) was retrospectively quantified in serum samples from 20 HIV-1-uninfected infants born to HIV-1 seropositive mothers. p24 RBC values quantified at birth were included either in a low (0-20%) or high (80-100%) range of values, classified as group A (11 infants) and group B (9 infants), respectively. The course of maternal antibodies to HIV-1 antigens p17, p24, p31, gp41, p51, p66, gp120, and gp160 was studied in each group. A substantial difference in the amount and subsequently in the decline of maternal antibodies to gag proteins p17, p24, and p55 and to pol proteins p51 and p66 was observed in the two infant groups in contrast with a similar content and decline of the remaining antibodies. In 7 HIV-1-infected infants of whom 4 resembled infant group A and 3 infant group B for p24 RBC values, a relationship appeared between p24 antibody decline and p24 antigenemia detection.

Herpesvirus DNA is frequently detected in liver tissue from hepatitis C patients.

BACKGROUND AND AIMS: Herpesviruses infect the liver and cause minor hepatitis. Our aim is to verify the presence of herpesviruses in the liver from hepatitis C patients and the possible influence of these viruses in the liver disease. METHODS: We searched for herpesvirus DNA in liver biopsies from patients with hepatitis C and from a control group without hepatitis by means of nested polymerase chain reaction. Serological investigations were carried out as well. RESULTS: Thirty-four liver specimens from hepatitis C patients were examined, 12 of which (35.3%) were positive for at least one herpesvirus DNA, whereas among the 19 control specimens only two were positive (10.5%; P = 0.049). Liver biopsies from seven patients, three with acute hepatitis of unknown origin, three with non-alcoholic steatohepatitis and one with autoimmune hepatitis were also investigated and three positive samples were found. CONCLUSIONS: The prevalence of herpesvirus DNA was found higher in patients with hepatitis C than in individuals without hepatitis. The influence of herpesviruses on the clinical course of hepatitis C is considered.

Suppression of BK virus replication and cytopathic effect by inhibitors of prokaryotic DNA gyrase.

Nalidixic acid and oxolinic acid, two antibacterial agents known to inhibit bacterial DNA gyrase, are shown to suppress the replication, as well as the cytopathic effect, of BK virus in Vero cell cultures. The inhibition of virus replication was detectable at day 4 post infection in cultures which had been continuously exposed to drugs at concentrations as low as 0.02 to 0.04 mM of nalidixic acid and 0.2 mM of oxolinic acid. These active concentrations are inferior to plasma levels attained in the course of clinical use of the drugs for antibacterial chemotherapy. Also, under these circumstances, no cytotoxicity occurred. The inhibition of development of cytopathology and of virus-induced cell death was demonstrable in cultures treated for 12 days with the drugs. Under these circumstances of prolonged action, oxolinic acid proved to be slightly cytotoxic in that virus inhibitory doses reduced the viability of normal cells. No alterations in the topological conformation of the viral genome or accumulation of end products of viral DNA replication were detected. However, accumulation of viral DNA form I at 48 h post infection suggests that the drugs act through a mechanism involving DNA topoisomerase.

HPV typing of cervical squamous lesions by in situ HPV DNA hybridization: influence of HPV type and therapy on the follow-up of low-grade squamous cervical disease.

Papanicolaou (Pap)-stained cervical specimens from 160 squamous lesions were processed for the detection of human papillomavirus (HPV) DNA by an in situ hybridization (ISH) assay. Three biotinylated HPV DNA probes were employed, each containing HPV genotypes 6/11, HPV genotypes 16/18, or HPV genotypes 31/35/51. The HPV etiology of 86 lesions was ascertained (53.8%). In 74 out of 135 (58.8%) HPV-typed low-grade squamous intraepithelial lesions (SILs), HPV 6/11 was found in nine (6.6%), HPV 16/18 in 46 (34.2%), and HPV 31/35/51 in 19 lesions (14.1%); in 11 out of 18 HPV-typed high-grade SILs (61.1%), seven lesions (38.9%) were typed for HPV 16/18 and four (22.2%) for HPV 31/35/51. Of seven invasive carcinomas, only one (14.3%) reacted with the HPV 16/18 DNA probe. A cohort of 124 low-grade SILs were followed cytologically for a year. The results of this study are discussed in light of HPV type association and therapy.

Evaluation of a vero cell toxicity test to detect EHEC infection.

An epidemiologic investigation was carried out in Modena (Italy) to evaluate the prevalence of faecal VEROtoxin (FVT) in diarrhoeal stool specimens. One thousand and sixty-six stool specimens, submitted to the Clinical Microbiology Laboratory of the University Hospital of Modena, were collected and faecal filtrates tested for neutralizable cytotoxin by a toxicity test on VERO cells. Cytopathic effect on VERO cells was produced by 301 stool specimens (28%); neutralizable VT was detected in 40 (13%) out of 301 positive samples (3.7% of 1066 specimens). The prevalent FVT type was VT2 (50%), followed by VT1 (32.5%) and VT1+2 (17.5%). We evaluated an assay that detects both VTs directly from stool specimens to demonstrate that enterohemorrhagic strains (EHEC) should be considerated a causative agent of sporadic non-bloody diarrhoea. Our results suggest that toxin neutralization assay is a sensitive and specific technique and may be used as an alternative method to diagnose diarrhoeal infections caused by EHEC.

Comparison of culture and immunoassay for detection of Escherichia coli O157 in raw minced meat and hamburger.

Verocytotoxin-producing Escherichia coli O157 (VTEC) is an important food-borne pathogen of humans. The serious complications of VTEC infection and the established reservoir of VTEC in cattle used for mass food production are a public health concern. In this study 500 samples of hamburger and minced meat were examined for presence of E. coli O157. For E. coli detection, Tryptic Soy Broth supplemented (with novobiocin and bile salts) and Sorbitol Mc Conkey agar were used; an automated rapid enzyme linked fluorescent immunoassay (VIDAS E. coli O157) was also evaluated. E. coli O157 was found in 5 samples of hamburger, 2 strains were found to be positive for verocytotoxin production on Vero cells.

Antigenic outline of HHV-6 variants from infants with febrile illness.

The surface antigenic make-up of 10 HHV-6 isolates from cases of infantile disease was analyzed by immunofluorescence assay with 5 monoclonal antibodies (Mabs). Three Mabs were directed to glycoproteins (gps) expressed in cells infected with the HHV-6 variant A prototype GS, and 2 to gps expressed in cells infected with the HHV-6 variant B prototype Z29. Of the 10 viral isolates, all belonging to HHV-6 variant B, 9 showed a similar Mab reactivity, while 1, from a case of gastroenteric illness, differed widely from the others. The HHV-6 isolates studied also differed from the variant B prototype Z29 for the absence of reactivity to one of the Mabs. The choice of the HHV-6 new isolates as variant B prototypes is recommended.

Comparison between immunofluorescence and enzyme-linked-immunosorbent assay in the determination of serum HHV-6 IgG.

546 human sera from healthy subjects, subjects with cytomegalovirus or Epstein-Barr virus infection and from cordal blood were tested for the human herpesvirus-6 (HHV-6) IgG content in an enzyme-linked immunosorbent assay (ELISA) to verify results previously obtained with an immunofluorescence assay (IFA). The HHV-6 isolate CV, obtained from a child with exanthem subitum, employed as antigen in both tests, belonged to HHV-6 variant B. The ELISA results were similar to those obtained with IFA. 445/546 (81.5%) sera had the same type of reactivity with the two serological procedures. To establish whether different HHV-6 strains may give different serological results, 117 out of the 546 sera tested with IFA against CV infected cells were also tested against cells infected with the HHV-6 isolate U1102 belonging to variant A. All the sera showed a similar reactivity.

Prevalence of antibodies to human herpesviruses 6 and 7 in early infancy and age at primary infection.

Sera from a sample of children aged 3 months to 6 years and from cord blood were tested in an indirect immunofluorescence assay (IFA) for reactivity to human herpesviruses 6 (HHV-6) and 7 (HHV-7). HHV-6 seropositivity values rose from 19% to 79.3% in the first 18 months of life, while HHV-7 seroprevalence reached a similar value (75.9%) in children aged 3-6 years. These results show that HHV-7, like HHV-6, is a prevalent virus in infancy. In cord blood sera, assayed to study infant humoral situation at birth, similar values for the two viruses (78.9% for HHV-6 and 76.3% for HHV-7) were found. HHV-6 and HHV-7 IgG antibody affinity to the corresponding antigens was assessed by the end point antibody titration in the presence and absence of urea 8M. This test distinguishes antibodies of recent (low affinity) or past (high affinity) production. Together, the data on seroprevalence and antibody affinity suggest that HHV-6 primary infection generally precedes that by HHV-7. These results are discussed in the light of a different pathogenetic role of the two viruses.

IgG antibodies to papillomavirus genus-antigens in adult men with cancer of the urinary bladder.

Forty-five sera from men with bladder cancer were examined in a micro solid-phase enzyme-linked immunosorbent assay (ELISA) and in a Western-blotting (WB) assay for the presence of IgG antibodies to papillomavirus (PV) genus-antigens of bovine origin. The ELISA detected PV antibodies in 75.6% of cancer patients. This antibody frequency was significantly higher than that found in both healthy males (22.7%) and patients with urological disorders (24%). A similar correlation among the PV antibody frequencies of the three groups was found with WB assay: 60% of the neoplastic group showed PV antibodies versus 17.3% in healthy males and 32.6% in non-neoplastic patients. Within the same group, 78% to 87% sera showed the same reactivity to both assays. Of these concordant sera, PV positive sera were 55.6% in cancer patients, 13.3% in healthy adults and 19.6% in patients with urological disorders. ELISA PV antibody level in the cancer group was higher than in each of the two control groups. The meaning of the humoral response to PV genus-antigens in men with bladder cancer is discussed.

Detection of Epstein-Barr virus DNA in cerebrospinal fluid from immunocompetent individuals with brain disorders.

Fifty four cerebrospinal fluid samples obtained from as many immunocomponent patients with disorders of the central nervous system were investigated for the presence of herpesvirus DNA by nested polymerase chain reaction in order to determine an etiological diagnosis. Four of these samples proved positive for the presence of Epstein-Barr virus DNA (7.4%). The result of this diagnostic study is reported to draw insiders' attention to the possible presence of EBV in cerebrospinal fluid from patients with central nervous system diseases.

SupT-1: a cell system suitable for an efficient propagation of both HHV-7 and HHV-6 variants A and B.

HHV-7 growth on Sup-T1, an immature T-cell line, was studied using different HHV-7 isolates obtained in our laboratory. Titration of viral yields showed that all the virus isolates propagate on this cell line more efficiently than in cord blood lymphocytes, the cells usually recommended for HHV-7 growth. The permissivity of Sup-T1 to HHV-6, whose ability to replicate in these cells was still unknown, was also investigated using two virus isolates representative of variants A and B respectively. Both isolates were able to propagate on Sup-T1 and viral titres were similar to those obtained in cord blood lymphocytes. As the efficient propagation of both HHV-7 and HHV-6 isolates in Sup-T1 cultures, these cells may replace more time consuming and expensive cord blood lymphocyte preparations for the propagation of both the viruses.

Primary infection by HHV-6 variant B associated with a fatal case of hemophagocytic syndrome.

The association of a human herpesvirus 6 primary infection with a fatal case of hemophagocytic syndrome in a 3 month old baby is described. The trigger mechanism of the virus for the clinical syndrome is discussed.

Epstein-barr virus DNA in the cerebrospinal fluid of patients with human immunodeficiency virus infection and central nervous system disorders.

Routine search for herpesvirus types 1-5 by nested polymerase chain reaction revealed Epstein-Barr virus (EBV) DNA in the cerebrospinal fluid (CSF) of ten out of seventy-nine patients with human immunodeficiency virus (HIV) infection and central nervous system (CNS) disorders not associated with the presence of primary CNS lymphomas. One out of the ten CSF samples was positive for EBV DNA only, six were also positive for microbial agents of recognised neurological pathogenicity while the remaining three samples had a high content of HIV p24 Ag. When six available CSF samples out of the ten EBV DNA positive specimens were investigated for an intrathecal EBV antibody response, all six samples proved EBV antibody-free. The concurrent detection of neurotropic infectious agents and the absence of EBV antibodies in the CSF contribute to the uncertainty on the role of EBV in the neurological illness of the patients studied. One hypothesis considered is that the presence of EBV DNA in the CSF of a large fraction of the ten patients under study is an incidental event associated with EBV reactivation in the host's peripheral blood monocytes, but not related to the genesis of neurological disorders.

New heterocyclic structures. thiazolo[3,2-a][1,2,5] thiadiazolo[3,4-d]pyrimidine and [1,2,5]thiadiazolo[3',4':4,5] pyrimido[2,1-b][1,3]thiazine. biological assays.

6,7-Dihydro-9H-thiazolo[3,2-a][1,2,5]thiadiazolo [3,4-d]pyrimidin-9-one, 7,8-dihydro-6H,10H-[1,2,5]thiadiazolo[3',4':4,5]pyrimido [2,1-b][1,3]thiazin-10-one and its 3-methyl derivative were prepared by reacting 6,7-diamino-2,3-dihydro-5H-thiazolo[3,2-a]pyrimidin-5-one, 7,8-diamino-3,4-dihydro-2H,6H-pyrimido[2,1-b][1,3]thiazin-6-one or its 3-methyl derivative with N-thionylaniline. A reaction mechanism is proposed. The compounds and the sodium salts of (7-amino-2,3-dihydro-5H-thiazolo[3,2-a]pyrimidin-5-on-6-yl)sulfamic acid, (8-amino-3,4-dihydro-2H,6H-pyrimido[2,1-b][1,3]thiazin-6-on-7-yl) sulfamic acid and its 3-methyl derivative were tested for antimicrobial and antimycotic activity on a number of strains, namely: E. Coli, Proteus mirabilis, P. vulgaris, Pseudomonas aeruginosa, Salmonella spp, Staphylococcus spp, Streptococcus faecalis, Bacillus subtilis, Sarcina lutea, Candida albicans, and for antiviral activity on Herpes simplex virus type 1 and Vescicular stomatitis virus. None of the compounds showed antiviral activity or exhibited biological activity against gram-negative, gram-positive bacteria or against mycetes.