Rapid detection and identification of Aspergillus from lower respiratory tract specimens by use of a combined probe-high-resolution melting analysis.

Diagnosis of invasive aspergillosis (IA) requires increasingly rapid molecular methods that enable sensitive detection and discrimination between species. We designed and evaluated a real-time PCR-based method that combined melting temperature (T(m)) calling analysis of a specific probe with high-resolution melting analysis of the full amplicon. The test correctly identified 78 isolates of Aspergillus section Fumigati and non-Fumigati sections of Aspergillus with a limit of detection of 10(2) conidia/ml (10(2) fg/ml). No cross-reactivity with other fungi was found. The assay was further validated on lower respiratory tract specimens containing Aspergillus or not. It successfully identified Aspergillus to section level in 56 of 59 specimens. With culture as the gold standard, our assay shows 100% sensitivity and specificity and constitutes an efficient alternative for identification of Aspergillus in lower respiratory tract samples.

Seroreversion of HIV antibodies in patients with prolonged suppression of viraemia under HAART.

Prolonged virus suppression in chronically HIV-infected patients could hypothetically lead to antibody seroreversion. Eighty-four HIV-positive individuals with undetectable viraemia for longer than 5 years under HAART were examined. Only one individual, who had initiated HAART shortly after primary HIV infection, showed seroreversion. In contrast, the cure of hepatitis C virus (HCV) with interferon in 25 controls led to the loss of HCV antibodies in most cases. This information indirectly reflects that whereas HCV may be eradicable HIV is not.

A comparison of fluorescent in situ hybridization and multiplex short tandem repeat polymerase chain reaction for quantifying chimerism after stem cell transplantation.

BACKGROUND AND OBJECTIVES: Despite the great utility of chimerism analysis after allogeneic stem cell transplantation, a gold standard method for its quantification has not yet been defined. The objective of the present investigation was to compare the sensitivity (detection limit) and the quantification accuracy of fluorescent in situ hybridization with specific probes for the sex chromosomes (XY-FISH) and multiplex short tandem repeat polymerase chain reaction (STR-PCR) revealed by capillary electrophoresis for the quantification of chimerism after stem cell transplantation. DESIGN AND METHODS: A first experiment was performed on two sets of artificial cell mixtures from two sex-mismatched healthy donors mixed in different proportions (% male: 100, 75, 50, 25, 10, 5, 3, 1, 0.1, 0). In a second experiment, 58 samples obtained from 10 selected patients with different clinical courses and chimerism evolution after sex-mismatched stem cell transplantation, which had been studied by XY-FISH, were retrospectively analyzed by STR-PCR. In a third experiment, 60 unselected prospective samples belonging to 15 patients (5 of whom had also been included in the retrospective study) were analyzed by both XY-FISH and STR-PCR. RESULTS: Both techniques showed high quantification accuracy and were highly reproducible. The sensitivity of both approaches reached 1% under standard conditions. Moreover, the use of long injection times for the capillary electrophoresis (30 and 50s vs. the standard 10s) resulted in an increase of sensitivity of the STR-PCR assay up to 0.1%, which has interesting clinical implications. INTERPRETATION AND CONCLUSIONS: Considering the high sensitivity and quantification accuracy of multiplex STR-PCR and the fact that this assay is sex-independent and can be applied to virtually all patients, STR-PCR could be considered as the method of choice for chimerism quantification after stem cell transplantation when high sensitivity is not a requirement.

An additive effect of protective host genetic factors correlates with HIV nonprogression status.

INTRODUCTION: In HIV-positive individuals, complex multifactorial mechanisms control viral infection. In addition to viral and immunological factors, the host genetic background also plays an important role. Our objective was to evaluate how various genetic factors associated with delayed AIDS onset. METHODS: Thirty HIV+ long-term nonprogressors (LTNPs) and 30 known progressors were analyzed. Host genes were analyzed in peripheral blood mononuclear cells DNA: CCR5 and HLA were polymerase chain reaction typed. HLA-C5', HCP5 polymorphisms, and CCL3L1 copy number were determined using real-time polymerase chain reaction. RESULTS: The CCL3L1high-copy-CCR5 deletion genetic risk groups was overrepresented in LTNPs. However, separately, neither CCL3L1 nor CCR5 were significantly associated with clinical outcome. HLA seemed as a strong nonprogression determinant, mainly HLA-B and the less-studied HLA-C. HLA-Cw0102 and HLA-C5' had an impact on LTNP phenotype along with HLA-B5701 and B2705. The presence of allele combinations like HLA- B*5701-Cw0602, HLA-B*2705-Cw0102, or HLA-B*3801-Cw1203 had the strongest effect in non-progression. As for HCP5, no independent effect was observed. The studied factors had additive effects, and although the number of patients was small, it seemed that carrying a high number of protective alleles associated with progression delay. CONCLUSIONS: We showed the additive load of protective host factors was predictive of nonprogression, and that HLA-associated factors were predominant in this global effect.

Mutation N155H in HIV-2 integrase confers high phenotypic resistance to raltegravir and impairs replication capacity.

BACKGROUND: Raltegravir has been shown to be active against wildtype HIV-2 with a phenotypic susceptibility similar to HIV-1. Due to the recent introduction of these novel inhibitors, information on the selection of resistance mutations and its phenotypic effect in this population is scarce. OBJECTIVES: To explore in vitro the effect of raltegravir resistance in one individual with HIV-2 infection who failed raltegravir-HAART. METHODS: A 20-year-old man with HIV-2 infection received a raltegravir-based HAART regimen. Drug resistance mutations were examined in the integrase gene by sequence analysis. Phenotypic analyses were performed in two HIV-2 isolates from the patient (wildtype isolate: SP-2p2-175 and mutant isolate: SP-2p2-189) and a laboratory reference strain (HIV-2 ROD). Susceptibility to raltegravir was assessed in a PBMC culture assay. Furthermore, a replicative capacity assay was performed. RESULTS: After introduction of raltegravir, patient's HIV-2 viremia dropped 1 log but did not reach undetectability. Genotypic analysis at month 8 with raltegravir, revealed the development of N155H resistant mutation along with other changes in the HIV-2 integrase: V72I, I84V, A153G, N160K and S163S/G. These changes resulted in a 37-fold increase in phenotypic resistance to raltegravir. Wildtype HIV-2 integrase (SP-2p2-175) had an IC(50) of 21.5nM and HIV-2 mutant virus (SP-2p2-189) showed an IC(50) of 789nM. SP-2p2-189 virus presented also lower replicative capacity in the absence of raltegravir than wildtype. CONCLUSION: A continued low HIV-2 viral load seems to be enough to select the N155H mutation, which despite significantly impairing viral replication, shows a level of resistance sufficient to give a selective advantage to the virus that maintains this pathway of resistance to raltegravir overtime.

Molecular and epidemiological characteristics of blood-borne virus infections among recent immigrants in Spain.

The increased immigration from developing regions to Western countries raises public health concerns related to blood-borne viruses. The prevalence of human immunodeficiency virus (HIV), hepatitis B virus (HBV), hepatitis C virus (HCV), and human T-lymphotropic virus (HTLV) infections among recent immigrants attending several Spanish diagnostic centers in years 2002 and 2003 was examined. Genetic characterization of viral subtypes and its relationship with distinct at-risk populations was carried out. A total of 1,303 immigrants were identified. They originated in Latin America (46.9%), Sub-Saharan Africa (23.7%), Eastern Europe (9.4%), and the Maghreb (9.2%). Seroprevalence rates were as follows: HIV-1 4.2%, HBV 4.1%, HCV 2.9%, and HTLV-1 0.8%. All patients with HIV-1 non-B subtypes, HBV genotypes E and A3, and HCV genotype 4 were sub-Saharan Africans, and had been infected mainly through heterosexual contacts. In contrast, Latin American homo/bisexual men carried HIV-1 subtype B most likely acquired after their arrival to Spain. In conclusion, while Sub-Saharan Africans carry wide diverse genetic variants of blood-borne viruses, the absence of high-risk practices in most cases could limit the spread of these variants. In contrast, Latin Americans with high-risk sexual practices may be a particularly vulnerable collective to acquire blood-borne viruses in the receptor country.