Two nasal swabs may not be enough to exclude SARS-CoV-2 infection in symptomatic patients.

Abstracts: The spread of COVID-19 (COronaVIrus Disease 2019), due to SARS-CoV-2 (Severe Acute Respiratory Syndrome CoronaVirus 2) has taken on dramatic pandemic proportions, affecting over 100 countries in a matter of weeks. Italy has had 237,828 confirmed cases according to the Istituto Superiore di Sanità as of May 13, and 34,448 deaths (1).

Detection of hepatitis C virus-RNA in saliva from chronically HCV-infected patients.

The possibility of the non-parenteral Hepatitis C Virus (HCV) transmission is supported by the demonstration that the actual virus is present in several body fluids, including saliva. From a review of the literature many investigators have found the presence of HCV-RNA in saliva, however, widely contrasting results emerge, with detection rates ranging from 0-100%. To further examine HCV salivary shedding, saliva samples were collected from 46 chronically HCV-infected patients and tested for HCV-RNA and occult blood. Quantification and genotyping of serum HCV-RNA were also carried out for each patient. HCV-RNA was detected in 39.13% of the saliva samples. The viral salivary shedding was significantly related to viraemia levels, serum viral genotype and the presence of salivary occult blood. Our findings indicate that the HCV salivary shedding occurs in about one third of HCV infected patients, but seem to suggest that it is unlikely when the serum viral genotype is 3a. Moreover, blood leakage into the oral cavity is possibly the main source of the salivary HCV-RNA. Although the occurrence of the viral salivary shedding does not necessarily mean that HCV transmission occurs by saliva, our results suggest the need for further investigations into the biological factors possibly involved in HCV mucosal transmission related to both the source and the exposed subjects.

Correlation between seroreactivity to HIV-1 V3 loop peptides and male-to-female heterosexual transmission.

OBJECTIVE: To evaluate the correlation between seroreactivity to peptides corresponding to the V3 loop of the major envelope glycoprotein from different HIV-1 strains and the risk of heterosexual HIV-1 transmission. METHODS: Sera from 39 infected individuals (16 transmitters and 23 non-transmitters) were tested for reactivity against synthetic peptides representing sequences of the V3 loop apex from HIV-1 strains MN, SC, WMJ2, RF and IIIB. RESULTS: A skewed distribution in seroreactivity to RF and IIIB peptides was observed between the two groups: reactivity was more prevalent in sera from non-transmitting than from transmitting individuals. Reactivity to the MN, SC and WMJ2 peptides was very frequent and there were no differences between the two groups. CONCLUSION: These data suggest that antibodies reactive with a larger set of V3 apex peptides (i.e., cross-reactive antibodies) could play a role in the prevention of heterosexual transmission.

Biological correlates of HIV-1 heterosexual transmission.

OBJECTIVES: To study the role of HIV-1 biological phenotype, viral load and neutralizing antibodies in male-to-female heterosexual transmission of HIV-1. METHODS: Seven transmitting and seven non-transmitting HIV-1-seropositive heterosexual male index cases were included in the present study. All couples had engaged in unprotected sex for a period of over 1 year. Transmission was defined by the seroconversion of the female sexual partner. Virus isolates were tested in MT-2 cells for replication and syncytia induction. HIV-1 RNA plasma load was measured by the branched DNA technique. Serum neutralizing activity to primary HIV-1 isolates was tested by using peripheral blood mononuclear cells (PBMC) as target cells. RESULTS: Non-transmitting index cases had a lower HIV-1 RNA concentration in plasma than transmitting index cases. Non-transmitting index cases also tended to have serum neutralizing activity with broad specificity and to have viruses with low replicative capacity, as characterized by 50% infectious dose titres in PBMC and by the lack of MT-2 tropism. CONCLUSIONS: The results indicate that plasma viral-RNA load is a marker for transmission. Moreover, an interplay between the host immune response and viral replication may modulate the level of viral load and thereby influence HIV-1 transmission.

Measurement of viral sequences in cerebrospinal fluid of AIDS patients with cerebral white-matter lesions using polymerase chain reaction.

OBJECTIVES: To optimize the use of polymerase chain reaction (PCR) on cerebrospinal fluid (CSF) for the evaluation of central nervous system (CNS) white-matter lesions that along with clinical findings and magnetic resonance imaging (MRI) can allow a definite diagnosis to be made; also to evaluate treatment with zidovudine plus foscarnet. DESIGN AND METHODS: Fifteen AIDS patients with uncertain CNS white-matter lesions were identified. HIV-1 RNA, cytomegalovirus (CMV) and JC virus (JCV) DNA were measured in a total of 29 CSF samples. The results were correlated with clinical and MRI findings and treatment with zidovudine plus foscarnet was evaluated. RESULTS: Four and five out of 15 patients with CMV DNA > or = 1 : 625 and JCV DNA > or = 10(3) copies/microl detected in the CSF were diagnosed with CMV and progressive multifocal leukoencephalopathy (PML), respectively. Six patients who were CMV/JCV-negative with the highest levels of HIV RNA (median, 6.87 log10 copies/ml) in CSF were considered as having HIV-1 encephalitis. Neurological symptoms were non-supportive for diagnosis as was MRI in 11 out of 15 patients. Nine patients completed a 21-day course of zidovudine plus foscarnet. HIV RNA decreased irrespective of neurological diagnosis. All three HIV-1 encephalitis patients and two out of three patients with CMV leukoencephalopathy improved. In these two latter patients, relief of clinical symptoms coincided with decreased CMV DNA. JCV DNA remained unchanged and all three PML patients deteriorated. CONCLUSIONS: Measurement of CSF viral sequences supports the diagnosis of CNS white-matter lesions in AIDS patients. While effective therapy for PML remains elusive, treatment including zidovudine plus foscarnet may be a promising option for HIV-1 and CMV-related manifestations.

Neurological disorders during HIV-1 infection correlate with viral load in cerebrospinal fluid but not with virus phenotype.

OBJECTIVES: To verify the compartmentalization of HIV-1 within the central nervous system (CNS) and to define whether viral phenotype of HIV-1 isolates from cerebrospinal fluid (CSF) samples and CSF viral load correlate with the presence and type of neurological disorders. METHODS: A total of 33 HIV-1-infected patients with and without neurological disorders were included in the study. HIV-1 isolation from paired CSF and peripheral blood mononuclear cell (PBMC) samples was attempted by a standard cocultivation technique; the biological phenotype of HIV-1 isolates was assessed by the MT-2 cell assay. CSF and plasma HIV-RNA levels were measured by a quantitative reverse transcripase-polymerase chain reaction. RESULTS: The rate of HIV-1 isolation from CSF and PBMC was 66% (22 isolates) and 85% (28 isolates), respectively. Seventeen out of 22 (77%) CSF HIV-1 isolates were characterized as non-syncytium-inducing, and 15 out of 28 (68%) isolates from PBMC were typed as syncytium-inducing (SI). The presence of SI isolates in CSF was limited to patients with HIV-1-, cytomegalovirus- or JC virus-related disorders and was often associated with high levels of HIV-1 RNA in the CSF. DISCUSSION: Our results demonstrate a correlation between high levels of HIV RNA in CSF and the presence of neurological disorders thus indicating a possible role for HIV-1 RNA in the CSF as a biological marker of neurological disease. The finding of viruses with a different phenotype in paired CSF and PBMC indicates that HIV-1 may evolve differently in the brain and in the blood. This suggests compartmentalization of HIV-1 within the CNS.

The role of HIV type 1 phenotype and genotype in long-term responders to zidovudine therapy.

We performed a cross-sectional and partly retrospective virological evaluation of 31 long-term responders (LTRs) to zidovudine (ZDV) (persistent increase in the CD4+ cell counts without progression of HIV infection throughout a period of ZDV therapy > 3 years) and 17 well-matched controls who developed a marked immunological deterioration over a 24-month period of ZDV therapy. The biological phenotype of HIV-1 was assessed by testing the capacity of the isolates to replicate in the MT-2, HUT-78, C-8166, and U-937 T cell lines, and mutations at codons 215 and 41 of RT were checked in proviral DNA from uncultured PBMCs. Show/low non-syncytium-inducing (S/L-NSI) and rapid/high syncytium-inducing (R/H-SI) variants were detected in 25 (81%) and 2 (6%) LTRs, respectively. HIV-1 could not be isolated in the remaining four LTRs (13%). Conversely, 12 of 17 (71%) controls yielded R/H-SI variants. Conversion from the S/L-NSI to R/H to R/H-SI phenotype occurred in 5 controls but in none of the 18 LTRs tested. Mutant sequences in proviral DNA from control PBMCs were consistently detected (94%), while a wild-type sequence of the residues investigated was found in the majority of LTRs (77%). In our series, patients who received immunological and clinical benefits even after prolonged ZDV treatment had S/L-NSI viruses and a low risk to develop ZDV resistance. Conversely, subjects who demonstrated an immunological and clinical deterioration yielded R/H-SI variants or shifted from S/L-NSI to R/H-SI phenotypes and were at higher risk to develop mutations indicating ZDV resistance.

Immunological markers in HIV-infected pregnant and non-pregnant women.

OBJECTIVE: To assess the influence of pregnancy on the course of HIV infection by comparing the behaviour of total lymphocyte counts and lymphocyte subsets (CD4(+) and CD8(+) and their ratio) in a cohort of infected pregnant women. SETTING: Tertiary referral centre for high risk obstetrics and infectious diseases in pregnancy. PATIENTS AND METHODS: A prospective study was designed, HIV infected women being enrolled at the beginning of pregnancy and sampled each trimester and in the puerperium. As controls, a group of non-pregnant HIV-infected women, cross-matched for age, risk factors and stage of disease were included and similarly evaluated in the same period. RESULTS: All the parameters, when longitudinally evaluated, were stable during gestation. Compared with non-pregnant subjects, patients had higher CD4(+) counts at the beginning and increased values of total lymphocytes count and subsets during the puerperium. Antepartum and postpartum risk factors such as drug abuse, smoking, antiretroviral therapy, length of gestation, maternal complications and HIV status of the neonate were not influential on the total lymphocytes counts and subsets. DISCUSSION: According to this data, pregnancy per se seems to have a negligible influence over the course of HIV infection, at least as far as immune parameters are concerned.

Frequent detection of HIV-1 RNA but low rates of HIV-1 isolation in cervicovaginal secretions from infected women.

Information regarding the presence of HIV-1 in the female genital tract is necessary to gain insight into the mechanism of HIV-1 heterosexual transmission. Herein, we present the results of a study on virus isolation and HIV-1 RNA detection from cervicovaginal lavage (CVL) samples from 25 HIV-1 seropositive women. Despite detectable levels of HIV-1 RNA in 88% of CVL samples, HIV-1 was isolated in only four (19%) samples. Although HIV-1 shedding in cervicovaginal secretions is a common event at all disease stages, the recovery of infectious virus in cell cultures appears to be rare; this renders viral isolation in studies aimed to evaluate the infectivity of cervicovaginal secretions relatively useless.

In vitro productive infection of non polarised cervical and rectal biopsies by syncytium-inducing and non syncytium inducing primary HIV-1 isolates.

An in vitro model was used to study the transmission of HIV-1 primary isolates with different biological phenotype to cervical and rectal non polarised bioptic fragments. The method described allowed the productive infection of both cervical and rectal tissues and the virus produced could be propagated onto peripheral blood mononuclear cell cultures. Syncytium-inducing and non-syncytium inducing viral isolates were equally able to produce infection and replication.

Preliminary data on the establishment of permanent cell lines continuously producing HIV-1: factors affecting the transmissibility and adaptability of "wild" HIV-1 isolates.

This study examines some technical aspects of the transmission to and permanent adaptability in continuous cell lines of wild HIV-1 isolates. Three cell systems (the lymphocytic cell lines Molt-3 and H-9 and the monocytoid cell line U-937) and two transmission protocols (cell to cell and cell-free) were used. Two different replicative behaviours were observed among isolates efficiently transmitted: a) transmissibility but not adaptability (consisting in a limited length of viral replication); aa) transmissibility and adaptability (consisting in a stable and long term virus production). The second type of replication was confined to viruses from patients with severe immunodeficiency. Technical and viral factors can affect the rate of transmissibility and adaptability: the modality of infection (cell to cell transmission appeared to be the most efficient) and the tropism of the virus (some viruses could infect only one T cell line).

The use of serum-free medium delays, but does not prevent, the cytotoxic effects of seminal plasma in lymphocyte cultures: implications for studies on HIV infection.

Evidence indicates that seminal plasma is cytotoxic when used in lymphocyte cultures. In fact, an amine oxidase which is normally present in Fetal Calf Serum (FCS) causes spermine oxidation and, finally, cytotoxic substances are released. This seminal quality might obviously hamper studies aiming to evaluate the role of semen in HIV sexual transmission, since lymphocytes are generally used as target or effector cells in virological or immunological studies on HIV. We evaluated the efficacy of FCS free-medium, heat inactivated seminal plasma and/or the washing-out of cultures after a three hour incubation period in preventing the cytotoxicity to lymphocytes. Results show that when cultures are carried out in the absence of FCS and/or after seminal plasma removal, cytotoxicity is not prevented, although delayed. This finding indicates that, along with still unrecognized additional factors, spermine oxidation is still an important factor for semen cytotoxicity, and this considerably hampers any immunological and virological study, requiring the use of lymphocytes, concerning seminal fluid.

Phenotypic patterns of HIV-1 clonal populations during highly active antiretroviral therapy (HAART).

Several studies have demonstrated that during HIV-1 infection many different viral clones may co-exist in the same individual. These clones may differ regarding their biological phenotype and may influence both the natural history of infection and the clinical response to antiretroviral therapy. The aim of the present study was to investigate the influences of combination therapies including protease inhibitors (HAART) on the phenotypical pattern of HIV-1 biological clones in peripheral blood mononuclear cells. Viral isolation and biological characterisation of bulk isolates and clonal viral isolates were performed on two AIDS patients, before and after three months of antiretroviral therapy. A decrease of viral load in plasma specimens in association with a change of clonal composition during antiretroviral therapy was observed in both patients during treatment. Before therapy both of the patients had a syncytium inducing (SI) bulk isolate and the majority of the biological clones were characterised as SI. After treatment, the bulk isolates were still SI in both cases, but the majority of biological clones were characterised as non-syncytium inducing (NSI). These results suggest that HIV clonal composition and relative phenotypic pattern undergo different changes not only during the natural course of HIV infection but also while patients are on antiretroviral combination therapy.

Limited secretory-IgA response in cervicovaginal secretions from HIV-1 infected, but not high risk seronegative women: lack of correlation to genital viral shedding.

The presence and antigen specificity of IgG and secretory-IgA (s-IgA) to HIV-1 were evaluated in cervicovaginal lavages (CVL) from 26 infected and 10 high-risk seronegative women. All the seropositive women had detectable IgG recognizing several viral antigens, while a smaller percentage of women demonstrated s-IgA to the virus. In addition, s-IgA were of limited specificity and provided weak reactivities on Immunoblot bands; an almost constant absence of s-IgA to gp120 was also observed. Neither the presence nor the specificity of either IgG or s-IgA to the virus in CVL prevented the shedding of HIV-1 in this body fluid; in fact, viral RNA was detected in all the women studied and the amounts of viral shedding was unrelated to the genital antibody response. On the other hand, none of the high-risk seronegative women had detectable antibodies to HIV-1 in CVL of either the IgG or s-IgA isotype. Our results a) confirm an impairment of mucosal antibody response during HIV-1 infection and suggest that mucosal immunity is not able to prevent viral shedding in the female genital tract and thus cannot modulate the infectivity of genital secretions; aa) do not provide evidence for a mucosal "memory/protective" antibody response in the genital tract of high-risk seronegative women.

A simple method to detect HIV-1 protein specificity of IgG intrathecal synthesis in HIV-1 infection.

There is currently no simple method to detect the antigen specificity of anti-HIV-1 IgG intrathecal synthesis (IS). Fifty-seven pairs of serum and corresponding CSF from 29 HIV-1 seropositive patients were adjusted to an identical concentration of total IgG and tested by a commercial HIV-1 Western Blot (WB) assay. IgG IS to a given HIV-1 protein was demonstrated when the corresponding band was present in CSF but absent or significantly less represented in serum. A total anti-HIV-1 IS was defined as the presence of an IS to one or more HIV-1 antigens. Our WB analysis of CSF and serum, compared with conventional mathematical formulas, showed a higher sensitivity in demonstrating anti-HIV-1 IgG IS. Moreover, the method disclosed which HIV-1 proteins represent the target of IgG IS. This procedure is easy to perform and therefore may represent a valuable tool to study central nervous system (CNS) involvement by HIV-1 during different stages of infection.

"In vitro" spontaneous production of B-chemokines by endocervical and endometrial short-term bioptic cultures.

Several studies indicate that HIV-1 is present in the cervico-vaginal tissues and secretions of infected women representing an important determinant of both sexual and mother-to-child transmission. HIV-1 genital shedding is influenced by various factors; among these, proinflammatory cytokines, in particular the beta/C-C chemokine group (RANTES, MIP-1alpha and MIP-1beta), are known to suppress HIV-1 replication and thus might affect both sexual and vertical transmission. This study aimed to standardize a procedure to measure "in vitro" uterine spontaneous chemokine production by means of short-term cultures of endocervical and endometrial bioptic fragments. In most cases, "in vitro" chemokine production was observed in both fragment cultures. These results further confirm that beta/C-C chemokines exist in the female genital tract and that uterine mucosa actively produces basal levels of these immuno-active substances. This method constitutes a useful approach to evaluate cytokine production and expression in the female genital tract, their influence on HIV-1 expression and infectivity in this site, and their possible role in viral transmission.

[Maternal viral and gestational risk factors in vertical transmission of HIV-1 infection]. / Fattori di rischio materni, virologici e gestazionali nella trasmissione verticale del virus HIV-1.

BACKGROUND: Several risk factors are related to vertical transfer of HIV-1 infection, but the influence of each one separately is controversial. Various risk factors have been evaluated altogether and separately, in order to find a single condition, that is, early in pregnancy predictive of fetal infection. METHODS: A prospective, observational study has been carried out during the period January 1992-January 1997, at the Centre for infectious diseases in pregnancy, University Hospital of Bari, a tertiary reference setting. Forty-four HIV-1 infected mothers have been recruited in early pregnancy, to be followed-up until delivery. Neonates have been evaluated in the Pediatric Department of the same hospital until 18 months of age. Maternal risk factors, such as age, infection in the partner, mode of acquisition of HIV-1, drug addition, life-style and risky behaviour as well as stage of disease, immunological status and antiviral treatment were considered. Obstetrical factors, too, (parity, preterm delivery, rupture of membranes, duration of labour and mode of delivery) were taken into account. Data were analyzed according to Fisher exact test and Draper and Smith multivariate analysis. RESULTS: Thirty cases (9 inadequate for follow-up, 4 ongoing pregnancies, 1 perinatal death before ascertainment of infection) were evaluated for final results. The overall transmission rate was 13.3%. Single factors significantly related to transmission were stage of disease (p < 0.01), mode of maternal infection (p < 0.03) and maternal HCV infection (p < 0.04). Multivariate analysis has shown the stage of disease to be the only positively-related risk-factor. CONCLUSIONS: The data identify a sub-group among HIV-1 infected women, where the more sustained rate of vertical transmission is associated with risky behaviour, as determined both by drug addiction and positivity for another sexually transmitted disease. Maternal compromise and immunological status are, however, the highest-risk conditions for delivering a HIV-1 infected baby.