Blood Of HIV-positive Patients Research Articles

Evaluation of a New Histoplasma spp. Quantitative RT-PCR Assay

Laboratory diagnosis of histoplasmosis is based on various methods, including microscopy, culture, antigen, and DNA detection of Histoplasma capsulatum var. capsulatum or Histoplasma capsulatum var. duboisii. To improve sensitivity of existing real-time quantitative PCR (qPCR) assays, we developed a new RT-qPCR assay that allows amplification of whole nucleic acids of Histoplasma spp. validated on suspected cases. The limit of detection was 20 copies, and the specificity against 114 fungal isolates/species was restricted to Histoplasma spp. Whole nucleic acids of 1319 prospectively collected consecutive samples from 907 patients suspected of having histoplasmosis were tested routinely between May 2015 and May 2019 in parallel with standard diagnostic procedures performed in parallel. Forty-four had proven histoplasmosis attributable to H. capsulatum var. capsulatum (n = 40) or H.capsulatum var. duboisii (n = 4) infections. The results of RT-qPCR were positive in 43 of 44 patients (97.7% sensitivity) in at least one specimen. Nine of 863 cases (99% specificity) were RT-qPCR positive and therefore classified as possible cases. RT-qPCR was positive in 13 of 30 (43.3%) blood samples tested in proven cases. A positive RT-qPCR result in blood was significantly associated with H. capsulatum var. capsulatum progressively disseminated histoplasmosis with a positive RT-qPCR result in 92.3% of the immunocompromised patients with disseminated disease. This new Histoplasma RT-qPCR assay enabling amplification of H. capsulatum var. capsulatum and H. capsulatum var. duboisii is highly sensitive and allows the diagnosis of histoplasmosis advantageously from blood and bronchoalveolar lavage fluid.

Antibiotic Susceptibility and Genotyping of Mycobacterium avium Strains That Cause Pulmonary and Disseminated Infection.

Mycobacterium avium subsp. hominissuis mainly causes disseminated infection in immunocompromised hosts, such as individuals with human immunodeficiency virus (HIV) infection, and pulmonary infection in immunocompetent hosts. However, many aspects of the different types of M. avium subsp. hominissuis infection remain unclear. We examined the antibiotic susceptibilities and genotypes of M. avium subsp. hominissuis isolates from different hosts by performing drug susceptibility testing using eight antibiotics (clarithromycin, rifampin, ethambutol, streptomycin, kanamycin, amikacin, ethionamide, and levofloxacin) and variable-number tandem-repeat (VNTR) typing analysis for 46 isolates from the sputa of HIV-negative patients with pulmonary M. avium subsp. hominissuis disease without previous antibiotic treatment and 30 isolates from the blood of HIV-positive patients with disseminated M. avium subsp. hominissuis disease. Interestingly, isolates from pulmonary M. avium subsp. hominissuis disease patients were more resistant to seven of the eight drugs, with the exception being rifampin, than isolates from HIV-positive patients. Moreover, VNTR typing analysis showed that the strains examined in this study were roughly classified into three clusters, and the genetic distance from reference strain 104 for isolates from pulmonary M. avium subsp. hominissuis disease patients was statistically significantly different from that for isolates from HIV-positive patients (P = 0.0018), suggesting that M. avium subsp. hominissuis strains that cause pulmonary and disseminated disease have genetically distinct features. Significant differences in susceptibility to seven of the eight drugs, with the exception being ethambutol, were noted among the three clusters. Collectively, these results suggest that an association between the type of M. avium subsp. hominissuis infection, drug susceptibility, and the VNTR genotype and the properties of M. avium subsp. hominissuis strains associated with the development of pulmonary disease are involved in higher levels of antibiotic resistance.

The Distribution of HIV DNA and RNA in Cell Subsets Differs in Gut and Blood of HIV-Positive Patients on ART: Implications for Viral Persistence

Even with optimal antiretroviral therapy, human immunodeficiency virus (HIV) persists in plasma, blood cells, and tissues. To develop new therapies, it is essential to know what cell types harbor residual HIV. We measured levels of HIV DNA, RNA, and RNA/DNA ratios in sorted subsets of CD4+ T cells (CCR7+, transitional memory, and effector memory) and non-CD4+ T leukocytes from blood, ileum, and rectum of 8 ART-suppressed HIV-positive subjects. Levels of HIV DNA/million cells in CCR7+ and effector memory cells were higher in the ileum than blood. When normalized by cell frequencies, most HIV DNA and RNA in the blood were found in CCR7+ cells, whereas in both gut sites, most HIV DNA and RNA were found in effector memory cells. HIV DNA and RNA were observed in non-CD4+ T leukocytes at low levels, particularly in gut tissues. Compared to the blood, the ileum had higher levels of HIV DNA and RNA in both CD4+ T cells and non-CD4+ T leukocytes, whereas the rectum had higher HIV DNA levels in both cell types but lower RNA levels in CD4+ T cells. Future studies should determine whether different mechanisms allow HIV to persist in these distinct reservoirs, and the degree to which different therapies can affect each reservoir.

The use of nested Polymerase Chain Reaction (nested PCR) for the early diagnosis of Histoplasma capsulatum infection in serum and whole blood of HIV-positive patients*

The aim of the study was to detect the rDNA sequences and their regions in Histoplasma capsulatum, which could be considered species-specific and used as a molecular method for this diagnosis by the technique of nested polymerase chain reaction (nested PCR), employing specific sequences (primers) for H. capsulatum: 18S rDNA region (HC18), 100 kDa (HC100) and the sequence 5.8 S-ITS rDNA (HC5.8). The PCR sequences HC18, HC100 and HC5.8 resulted in a specificity of 100%. The molecular assays may increase the specificity, sensitivity and speed in the diagnosis of Histoplasmosis.

Loss of IL-7Rα is associated with CD4 T-cell depletion, high interleukin-7 levels and CD28 down-regulation in HIV infected patients

Elevated levels of interleukin (IL)-7 are present in the blood of HIV-positive patients and it is known that IL-7 receptor (IL-7R)alpha expression decreases on T cells during HIV infection. The subset(s) of T cells with low IL-7Ralpha and the consequence of low IL-7Ralpha expression for T-cell survival are poorly characterized. The frequency of IL-7Ralpha-negative T cells in HIV-positive patients was studied in relation to CD4 T-cell counts, IL-7 concentration and survival in culture. We analysed IL-7Ralpha expression in different T-cell populations and in relation to Bcl-2 expression. Specimens from 38 HIV-1 patients and 17 controls were examined. IL-7Ralpha and Bcl-2 expression in different T-cell populations was studied by flow cytometry. The influence of IL-7Ralpha expression on T-cell survival was studied by culturing T cells in the presence of IL-7. Down-regulation of IL-7Ralpha on T cells correlated with depletion of CD4 T cells (P < 0.001) and also with increased concentration of serum IL-7 (P < 0.05). The decreased IL-7Ralpha expression was associated with low Bcl-2 expression and with the reduced survival capacity of T cells in the presence of IL-7 in vitro. Particularly, T cells with memory phenotype showed a decreased IL-7Ralpha expression in association with CD28 down-regulation. The positive effects of IL-7 on survival and homeostatic proliferation of T cells might be severely impaired in HIV-infected individuals due to IL-7Ralpha down-regulation. Differentiation towards a CD28-negative memory phenotype in response to chronic activation may lead to an overall decrease of IL-7 mediated survival within the peripheral T-cell pool.

Detection of JC virus DNA in the peripheral blood leukocytes of HIV-infected patients.

To assess whether JC virus (JCV) DNA is frequently harboured by peripheral blood leukocytes (PBL) in HIV-positive patients, before the onset of progressive multifocal leukoencephalopathy (PML). The polyomavirus JCV induces PML in immunocompromised persons and particularly AIDS patients. Leukocytes may play a central part in the onset of PML, but their precise role in JCV latency and reactivation still remains hypothetical. The controversial presence of JCV DNA in PBL has been, until now, investigated only among small groups of patients. We therefore studied 157 HIV-positive persons and compared them with 65 HIV-negative immunocompromised patients. DNA was extracted from PBL. The presence of JCV DNA was demonstrated by the polymerase chain reaction (PCR) alone or combined with a molecular hybridization assay. RESULTS. The presence of JCV DNA was ascertained by PCR and hybridization in 28.9% of 135 HIV-infected persons at all stages of HIV infection and only 16.4% of 61 HIV-negative immunocompromised patients. No correlation could be drawn between the detection of JCV DNA and the clinical or biological status of the HIV-positive patients. JCV DNA is detectable in the PBL of 28.9% of HIV-infected persons, even in the early stages of infection. JCV is more seldomly amplified in HIV-negative immunocompromised patients. Further work is in progress to determine the prognostic value of the presence of JCV DNA in the blood of HIV-positive patients.