Viral excretion in cervicovaginal secretions of HIV-1-infected women receiving antiretroviral therapy.

A longitudinal study was conducted to evaluate the viral shedding present in cervicovaginal secretions of HIV-1-seropositive women receiving antiretroviral therapy. A total of 128 paired cervicovaginal and blood samples was obtained from 37 women during a median follow-up period of 21 months. A sensitive, competitive, polymerase chain reaction and a reverse transcription polymerase chain reaction were used for the simultaneous quantitation of HIV-1 proviral DNA and RNA in cervicovaginal cells and cell-free RNA in cervicovaginal secretions, as well as HIV-1 RNA in peripheral blood. The cumulative probability of detecting proviral DNA in genital secretions was significantly higher over time in women with detectable viremia than in women in whom HIV-1 RNA was persistently undetectable in plasma (< 50 copies/ml) (P = 0.028 by log-rank test). The presence and amount of proviral DNA, cell-associated RNA and cell-free RNA in the cervicovaginal secretions were positively correlated with the presence of detectable viremia or the number of HIV-1 RNA copies in plasma (Spearman rank correlation, 0.290, 0.279, and 0.305, respectively; all P < 0.01), but no correlation was found with the CD4+ cell count. In addition, vaginal infections were positively correlated with the detection of proviral DNA in cervicovaginal secretions (odds ratio, 2.60; 95% confidence interval, 1.07-5.70). However, the positive correlation between the presence and amount of HIV in cervicovaginal secretions and the viral load in plasma provides no assurance that HIV shedding does not occur in the genital tract of women with undetectable HIV-RNA in plasma.

Screening of homocysteine from newborn blood spots by high-performance liquid chromatography with coulometric array detection.

Homocystinuria, due to a deficiency of cystationine-beta-synthase, refers to the rare inborn error of the metabolism of homocysteine. The identification and prompt treatment of homocystinuria during the neonatal period can prevent or greatly reduce the severity of the clinical consequences. We report a new method for homocystinuria diagnosis from dried blood spots on newborn screening cards, based on high-performance liquid chromatography with electrochemical coulometric array detection. This method shows an excellent linearity (y=10.36x+0.04; r=0.999), precision (RSDs ranged from 2.7 to 5.8%), recovery (87%) and appears to be a cost-effective approach, being simple, rapid, sensitive and cheap.

Quantification of HIV-1 proviral DNA in patients with undetectable plasma viremia over long-term highly active antiretroviral therapy.

OBJECTIVES: To assess the prognostic role of proviral DNA in peripheral blood mononuclear cells (PBMC) of patients with undetectable viremia over long-term highly active antiretroviral therapy (HAART). METHODS: Eighty-two human immunodeficiency virus (HIV)-1-infected patients, free of acquired immunodeficiency syndrome (AIDS), received zidovudine plus lamivudine plus indinavir. Levels of plasma HIV-RNA, and PBMC proviral DNA and RNA unspliced (US) transcripts were evaluated by using competitive polymerase chain reaction (cPCR) assays, every 3 months over 1 year. RESULTS: Among patients with undetectable viremia at baseline, 13 of 18 with CD4 cell count 350/mm3 or less and 12 of 16 with CD4 between 351 and 700/mm3, constantly maintained undetectable RNA levels; in these patients, a mean proviral DNA decrease of 0.67 6 0.7 and 1.03 6 0.53 log (P < 0.001), respectively, a significant decrease of RNA-US transcripts (P < 0.001), and significant correlations between decreases of proviral DNA and RNA-US transcripts (P = 0.008 and P < 0.001, respectively) were observed. CONCLUSIONS: Proviral DNA quantitation permits the continued monitoring of HAART in patients with undetectable viremia.

Determination of illicit drugs and related substances by high-performance liquid chromatography with an electrochemical coulometric-array detector.

A general method for the simultaneous determination of fifteen common drugs (6-acetylmorphine, 3,4-methylenedioxymetamphetamine, buprenorphin, cocaine, codeine, dihydrocodeine, ethylmorphine, heroin, hydrocodone, lidocaine, methadone, morphine, naloxone, procaine and thebaine) was developed using reversed-phase HPLC and electrochemical detection. The separation of the drugs was achieved by using as the mobile phase 20 mM monobasic sodium phosphate-acetonitrile (90:10) with a gradient to 50% of the organic modifier, on a silica based C18 column (150 x 4.6 mm I.D.) of 3 microns particle size and by the selectivity supplied by an array of eight coulometric electrodes at increasing potential. It was possible to identify and to determine fifteen different drugs in the same chromatographic run in 50 min. The method was tentatively applied to the determination of drugs in extracts of human hair.

HTLV infection among Italian intravenous drug users and North African subjects detected by the polymerase chain reaction and serological methods.

Six hundred intravenous drug users (IVDUs) and two hundred North Africans were screened for human T-cell leukemia virus (HTLV) antibodies using several serological methods. Eighteen of the eighty-two HTLV-seropositive individuals were also tested by the polymerase chain reaction-DNA enzyme immunoassay (PCR-DEIA), a non-isotopic method of immunoenzymatic detection of the amplified DNA. Of these eighteen subjects, eight IVDUs were found to be HTLV-II-positive by the PCR-DEIA, whereas all of the eighteen subjects were negative for HTLV-I. Western blot (WB) confirmed six of the eight HTLV-positive subjects, while the results of the remaining two were indeterminate. The results confirmed the PCR-DEIA as a rapid and an efficient method of discriminating between HTLV-I and HTLV-II infection, whereas serological tests, including the WB, have limitations in terms of specificity and sensitivity. Moreover, this study showed a higher frequency of HTLV seroreactivity in the Italian IVDU population than in previous studies and confirmed that HTLV-II is more frequent than HTLV-I in this population.

HIV type 1 intraperitoneal infection of rabbits permits early detection of serum antibodies to Gag, Pol, and Env proteins, neutralizing antibodies, and proviral DNA from peripheral blood mononuclear cells.

The aim of this study is the development of an animal model useful for studying HIV-1 pathogenesis, candidate vaccines, and antiviral drugs. Aseptic thioglycolate peritonitis was induced in six rabbits. After 4 days, four rabbits were infected with 1 ml of HIV-1 stock containing 100 times the MID50. Blood samples were collected every 2 weeks for 8 months. Serum antibodies were tested by ELISA, using as antigen the recombinant protein p24; synthetic peptides of highly conserved regions of p31, gp41, and gp120; and a synthetic peptide of gp120 at the V3 loop region of HIV-1 strains IIIB and MN. Furthermore, neutralizing antibodies were tested by a microscale neutralization assay. Proviral DNA was detected by PCR, and virus isolation was performed by a cocultivation technique using primary rabbit peripheral blood mononuclear cells (PBMCs). All infected rabbits produced antibodies to HIV-1 proteins within 2 weeks and up to 8 months after virus infection. Serum antibodies were directed against the Env (gp120 and gp41), Gag (p24), and Pol (p31) proteins and against two synthetic peptides whose sequence corresponds to gp120 at the V3 loop region of HIV-1 strains IIIB and MN. Neutralizing antibodies were also detected in the sera of infected animals. Proviral DNA was detected in PBMCs by PCR within 4 weeks and up to 8 months after HIV-1 infection. HIV-1 was also isolated from PBMCs of infected animals at 30, 60, and 120 days after infection. Results obtained indicate that HIV-1 intraperitoneal infection of the rabbit permits the early detection of serum antibodies to Gag, Pol, and Env proteins, neutralizing antibodies, and proviral DNA sequences from PBMCs.

[Hypotension following maximum physical exercise. Evaluation of hemodynamic and humoral mechanisms]. / Ipotensione dopo esercizio fisico massimale. Valutazione dei meccanismi emodinamici ed umorali.

The period after exercise has received little attention although there are rapid and arge changes in the loading conditions of the heart and circulation which may precipitate hypotension or arrhythmias. Little is known of the time course of the recovery of cardiac output and humoral changes occurring during this periods. After a single bout of prolonged muscular exercise, systolic and diastolic blood pressure decrease, sometimes for several hours. In a recent controlled study for the possible effects of the expecting of the exercise, a reduction in diastolic blood pressure was observed particularly in the first 10 min and lasting to 60 min. The mechanisms of the acute hypotensive effect of upright dynamic exercise have not yet been clarified. Little is known of the time course of the recovery of cardiac output, humoral and autonomic changes occurring during this period. Conflicting data are presented by different authors. The aim of the present study was to study the role of the haemodynamic and humoral changes in the modifications in blood pressure occurring in the hour of recovery after maximal exercise in normal subjects. Nine normal male volunteers (age: 28:34 years) have been studied on 2 separate days. Subjects were studied on a non-exercise (control) day (the subjects maintained the upright position for 30 min, followed by 60 min supine) and an exercise day (maximal upright bicycle exercise followed by supine rest for 60 min), in a random order. The following data have been recorded before the test and serially during 60 minute supine: systolic and diastolic blood pressure, heart rate, haemodynamic changes (by suprasternal aortic Doppler), and humoral changes (renin).(ABSTRACT TRUNCATED AT 250 WORDS)

Molecular characterization of two isolates of human T cell leukaemia virus type II from Italian drug abusers and comparison of genome structure with other isolates.

The human T cell leukaemia virus type II (HTLV-II), whose pathogenicity is as yet unclear, was recently found to be associated with intravenous drug abuse in North America and Europe. HTLV-II was isolated from two Italian drug abusers belonging to the same cohort and coinfected with human immunodeficiency virus type 1. Two new isolates, HTLV-II Gu and Va, were established in a culture of BJAB cells, a continuous B cell line (Epstein-Barr virus-negative), and characterized by nucleotide sequence analysis of the long terminal repeat (LTR) and portions of the gag, env and X regions. These sequences were compared to those of the HTLV-II Mo isolate reported in the literature. No major variations were observed in important regulatory elements of LTR nor in the stem-bulge-loop configuration known to be essential for binding of rex protein. The results obtained from the sequence of the 1988 nucleotides examined indicated a 1.6% variability between the Gu and Va isolates and about 6% with respect to Mo. Notable differences were found in the structure of putative open reading frames of the X region when compared to those reported for the Mo isolate. Restriction analysis of proviral DNA of two isolates and comparison with the physical map of the Mo isolate confirmed the existence of genetic heterogeneity in the HTLV-II group and demonstrated that the new isolates Gu and Va belong to the HTLV-IIb subtype. The results of this study show that the new isolates have distinct features with respect to the Mo isolate though all important regulatory elements of the LTR appear to be well conserved.

Seroepidemiology of HIV infection: an early survey in a peripheral area of Uganda.

At the end of 1985, when the AIDS epidemic was in its early stages in Uganda, a survey was carried out in a peripheral area of the country. Sera were collected from groups of people, and examined for the presence of HIV infection. The results show a very limited number of positive cases, present only among sexually active subjects. High specificity and sensitivity in the laboratory tests was shown by the Western blot technique.

Sensitivity and specificity of anti-HIV ELISA employing recombinant (p24, p66, gp120) and synthetic (gp41) viral antigenic peptides.

We have developed two immunoassay systems, one designated HIV (p24, p66, gp41) ELISA that uses as antigens the immunodominant epitopes mixed from each of three major groups of HIV-1 proteins: the core (p24), the pol (p66) and the env (gp41) gene. The other immunoassay system consists of four separate ELISAs for detection of single antibodies to HIV gag gene (p24), HIV pol gene (p66) and HIV env gene (gp41 and gp120). In the present study 200 specimens from patients with AIDS and 200 specimens from patients with ARC were repeatedly positive by HIV (p24, p66, gp41) ELISA. 1425 specimens from HIV drug addicts positive at W.B. were positive at HIV (p24, gp41, p66) ELISA. In addition, 60 samples that were indeterminate by W.B., were repeatedly positive at HIV (p24, p66, gp41) ELISA. The sensitivity and specificity of HIV (p24, p66, gp41) is estimated to be 100%. In this study 1507 specimens from HIV drug addicts, positive at W.B., were all positive (more than one test positive) at HIV p24 ELISA, HIV gp41 ELISA, HIV p66 ELISA and HIV gp120 ELISA used in combination. 135 samples from HIV positive drug addicts, positive at standard ELISA but indeterminate at W.B., were positive by HIV p24 ELISA, HIV gp41 ELISA, HIV p66 ELISA and HIV gp120 ELISA using the same criteria as in W.B. interpretation. The specificity (defined in terms of percentage of non-reacting persons in a low risk population) of HIV p24 ELISA, HIV gp41 ELISA, HIV p66 ELISA, HIV gp120 ELISA is 100%. In this work we demonstrated that: a) HIV (p24, p66, gp41) ELISA could be used as an adjunct or reliable alternative to standard ELISA for detection or confirmation of HIV antibodies in human sera; b) the specificity and sensitivity of antibodies to p24, p66, gp41, gp120 by ELISA used alone and/or in combination, is equal to or greater than W.B.

The association of "standard ELISA" and HIV (env) ELISA for detection and confirmation of HIV antibodies in human sera.

Sera from various population of subjects, including patients with AIDS and ARC, drug-addicts seropositive for HIV and healthy blood donors were screened with "standard ELISA" and HIV (env) ELISA. In the present study all 80 specimens from patients with AIDS and all 60 patients with ARC were positive by HIV (env) ELISA, and 1507 specimens from HIV drug positive by W.B. were detected as positive by HIV (env) ELISA. The specificity of HIV (env) ELISA was defined in terms of percentage of non-reacting persons in a low risk population is 100%. Furthermore the HIV (env) ELISA is highly specific and sensitive and could be used in association with "standard ELISA" for detection and confirmation of HIV antibodies in human sera and plasma.

Detection of antibodies to p24 and gp41 epitopes of HIV by ELISA using the recombinant core protein (p24) and envelope synthetic protein (gp41).

We have developed a system consisting of two separate ELISA, one designed to detect antibodies to HIV gag gene (p24) and the other to detect antibodies to HIV env gene (gp41). The antigen used in these ELISA was produced as recombinant DNA-derived proteins expressed in E. coli for HIV gag gene (p24) and synthetic peptide for the HIV env gene (gp41). These HIV (env-gag) ELISA, that provide independent determinations of the antibody response to the core and envelope proteins, are highly specific and sensitive. In this work we have demonstrated that determinations of antibodies such as those to p24 and gp41 by HIV (env-gag) ELISA are among the criteria for a confirmation procedure, and sensitivity one (gp41) and/or both these determination should be equal or greater than the sensitivity of W.B. In addition, the procedure should be objective and standardized and the antigen source used should be different from that adopted in the "classical" W.B. and screening test. In view of these considerations, this HIV (env-gag) ELISA could be used as a reliable alternative to W.B. for confirmation of antibody detection.

Determination of neurochemicals in biological fluids by using an automated high-performance liquid chromatographic system with a coulometric array detector.

The chromatographic separation of 33 neurochemicals was achieved by using a combined gradient of organic modifier, pH and counter-ion. A secondary separation of unresolved analytes was obtained by using electrochemical detection with a coulometric array of sixteen electrodes. The stability of the analytes was studied and data on analytical performance are reported in addition to a list of neurochemicals detected in a normal plasma sample.

Seroepidemiology of HIV infection: an early survey in a peripheral area of Uganda

At the end of 1985; when the AIDS epidemic was in its early stages in Uganda; a survey was carried out in a peripheral area of the country. Sera were collected from groups of people; and examined for the presence of HIV infection. The results show a very limited number of positive cases; present only among sexually active subjects. High specificity and sensitivity in the laboratory tests was shown by the Western blot technique

Major antigenic domain recognized by monoclonal antibodies maps within the carboxy-terminal moiety of a recombinant human immunodeficiency virus-1 p24 protein.

Antigenicity in mice of a recombinant polypeptide including the complete amino acid sequence of mature human immunodeficiency virus type 1 p24 protein was studied by induction of monoclonal antibodies (MAbs). A panel of nine recloned hybridomas secreting MAbs with anti-p24 reactivity was isolated and further characterized. Competitive inhibition experiments suggested that the MAbs could be grouped into four epitopic classes corresponding to at least two distinct determinants. Analysis of reactivity to recombinant p24 deletion variants indicated that all the recognized epitopes are localized within a carboxy-terminal domain (amino acids 168-208) which should be largely exposed in recombinant as well as authentic antigen. Lack of response to N-terminal and central portions of p24 suggests that the antigenicity of those regions in the natural polypeptide is strongly conformation-dependent.

HTLVI antibodies in multiple sclerosis and other neurological diseases.

The sera and the CSFs from 157 Multiple Sclerosis (MS) patients and 43 other neurological diseases (OND) cases have been evaluated for the presence of HTLVI antibodies. A commercial passive agglutination assay (PPA), an indirect fluorescence assay (IFA), and a Western Immunoblotting (WIB) performed in our laboratories have been employed. No OND samples showed HTLVI antibodies, while 14 sera and 1 CSF from MS patients resulted positive by PPA and 4 sera were positive with the IFA. When tested with WIB 6 MS sera showed a reactivity against one or more HTLVI proteins. Our results lead us to affirm that in a small number of MS patients, when sensitive tests are employed, it is possible to observe an antibody response toward proteins that share one or more epitope with HTLVI antigens.