Acute lymphoblastic leukaemia patients treated with PEGasparaginase develop antibodies to PEG and the succinate linker.

Polyethylene glycol (PEG) conjugated asparaginase (PEGasparaginase) is essential for treatment of paediatric acute lymphoblastic leukaemia. We developed an assay identifying antibodies against the PEG-moiety, the linker and the drug itself in patients experiencing hypersensitivity reactions to PEGasparaginase. Eighteen patients treated according to the DCOG ALL-11 protocol, with a neutralizing hypersensitivity reaction to PEGasparaginase to the first PEGasparaginase doses in induction (12 patients) or during intensification after interruption of several months (6 patients) were included. ELISA was used to measure antibodies, coating with the succinimidyl succinate linker conjugated to BSA, PEGfilgrastim and Escherichia coli asparaginase, and using hydrolysed PEGasparaginase and mPEG5,000 for competition. Anti-PEG antibodies were detected in all patients (IgG 100%; IgM 67%) of whom 39% had anti-PEG antibodies exclusively. Pre-existing anti-PEG antibodies were also detected in patients who not previously received a PEGylated therapeutic (58% IgG; 21% IgM). Antibodies against the SS-linker were predominantly detected during induction (50% IgG; 42% IgM). Anti-asparaginase antibodies were detected in only 11% during induction but 94% during intensification. In conclusion, anti-PEG and anti-SS-linker antibodies predominantly play a role in the immunogenic response to PEGasparaginase during induction. Thus, switching to native E. coli asparaginase would be an option for adequate asparaginase treatment.

Within-Host Selection of Drug Resistance in a Mouse Model Reveals Dose-Dependent Selection of Atovaquone Resistance Mutations.

The evolutionary selection of malaria parasites within an individual host plays a critical role in the emergence of drug resistance. We have compared the selection of atovaquone resistance mutants in mouse models reflecting two different causes of failure of malaria treatment, an inadequate subtherapeutic dose and an incomplete therapeutic dose. The two models are based on cycles of insufficient treatment of Plasmodium berghei-infected mice: repeated inadequate treatment associated with a subtherapeutic dose (RIaT) (0.1 mg kg-1 of body weight) and repeated incomplete treatment with a therapeutic dose (RIcT) (14.4 mg kg-1 of body weight). The number of treatment cycles for the development of a stable resistance phenotype during RIaT was 2.00 ± 0.00 cycles (n = 9), which is not statistically different from that during RIcT (2.57 ± 0.85 cycles; combined n = 14; P = 0.0591). All mutations underlying atovaquone resistance selected by RIaT (M133I, T142N, and L144S) were found to be in the Qo1 (quinone binding 1) domain of the mitochondrial cytochrome b gene, in contrast to those selected by RIcT (Y268N/C, L271V, K272R, and V284F) in the Qo2 domain or its neighboring sixth transmembrane region. Exposure of mixed populations of resistant parasites from RIaT to the higher therapeutic dose of RIcT revealed further insights into the dynamics of within-host selection of resistance to antimalarial drugs. These results suggest that both inadequate subtherapeutic doses and incomplete therapeutic doses in malaria treatment pose similar threats to the emergence of drug resistance. RIcT and RIaT could be developed as useful tools to predict the potential emergence of resistance to newly introduced and less-understood antimalarials.

Within-Host Selection of Drug Resistance in a Mouse Model of Repeated Incomplete Malaria Treatment: Comparison between Atovaquone and Pyrimethamine.

The evolutionary selection of malaria parasites within individual hosts is an important factor in the emergence of drug resistance but is still not well understood. We have examined the selection process for drug resistance in the mouse malaria agent Plasmodium berghei and compared the dynamics of the selection for atovaquone and pyrimethamine. Resistance to these drugs has been shown to be associated with genetic lesions in the dihydrofolate reductase gene in the case of pyrimethamine and in the mitochondrial cytochrome b gene for atovaquone. A mouse malaria model for the selection of drug resistance, based on repeated incomplete treatment (RICT) with a therapeutic dose of antimalarial drugs, was established. The number of treatment cycles for the development of stable resistance to atovaquone (2.47 ± 0.70; n = 19) was found to be significantly lower than for pyrimethamine (5.44 ± 1.46; n = 16; P < 0.0001), even when the parental P. berghei Leiden strain was cloned prior to the resistance selection. Similar results were obtained with P. berghei Edinburgh. Mutational changes underlying the resistance were identified to be S110N in dihydrofolate reductase for pyrimethamine and Y268N, Y268C, Y268S, L271V-K272R, and G280D in cytochrome b for atovaquone. These results are consistent with the rate of mitochondrial DNA mutation being higher than that in the nucleus and suggest that mutation leading to pyrimethamine resistance is not a rare event.

Differences in the reporting rates of serious allergic adverse events from intravenous iron by country and population.

BACKGROUND: Previous studies have compared rates of adverse events between intravenous (IV) iron preparations; however, there has been no comparison of adverse event rates by country and population. OBJECTIVES: To compare rates of adverse events to IV iron products by country and population. METHODS: All adverse events reported from 18 countries from January 1, 2003 to June 30, 2009 were obtained for iron dextran (ID), iron sucrose (IS), IS similars (ISS), and sodium ferric gluconate (FG). Rates of all adverse events and serious adverse events (anaphylaxis plus other serious allergic reactions) were calculated as number of events per gram of iron sold (gFe) per million inhabitants (mil) × 10-3. Odds ratios (ORs) were calculated for the risks of adverse events between products. RESULTS: Iron use ranged from 1 gFe/mil (Poland) to 48,674 gFe/mil (Italy). Rates of all adverse events (reports/gFe/mil × 10-3) varied: for IS, it ranged from 0 (Poland, Austria, Czech Republic) to 1,222 (Ireland); for FG, from 3.3 (Czech Republic) to 183.6 (United States); for ID, from 0.9 (Turkey) to 46,875 (Switzerland). There were no reports of adverse events in ISS. In a subset of countries that used 2 or more iron products and had more than 1 serious adverse event, rates (reports/gFe/mil × 10-3) of all adverse events and serious adverse events were lowest for IS (39.8 and 1.7), intermediate for FG (54.8 and 4.5), and greatest for ID (337.7 and 20.5). IS had lower risks for all adverse events (OR, 0.63; P<.0001) and serious adverse events (OR, 0.31; P=.001) versus FG, and for all adverse events (OR, 0.13; P<.0001) and serious adverse events (OR, 0.07; P<.0001) versus ID. FG had lower risks for all adverse events (OR, 0.20; P<.0001) and serious adverse events (OR, 0.24; P<.0001) versus ID. CONCLUSIONS: Considerable international variation existed in the extent and choice of iron product and adverse event reporting, suggesting under-reporting in some instances. Clinicians should appreciate the differential risks between available products, and should critically review local reporting practices.

Neutralization of Neisseria meningitidis outer membrane vesicles.

INTRODUCTION: We aimed to determine the neutralization of Neisseria meningitidis outer membrane vesicles (blebs) by humoral and cellular elements of whole blood. METHODS: The interaction of FITC-labeled blebs with monocytes was studied by spectrofluorometry. Blebs are able to induce an oxidative burst in neutrophils, and we evaluated the inhibitory effect of plasma on this process. RESULTS: Human plasma reduced the priming activity of blebs containing 1-3 ng/ml lipopolysaccharide (LPS) by 50-60% and bactericidal permeability increasing protein (BPI) reduced priming to background levels. A complete neutralization of LPS and blebs by plasma and BPI was measured using the limulus amebocyte lysate (LAL) assay. Furthermore, only 3% of blebs were cell-associated, while the remainder were in the supernatant. CONCLUSIONS: Plasma and BPI are able to neutralize blebs, with phagocytosis playing only a minor role. As such, we conclude that blebs do not behave like particles but more like free LPS.

Ethnogeographical structure of hepatitis B virus genotype distribution in Indonesia and discovery of a new subgenotype, B9.

The distribution of hepatitis B virus (HBV) in the populations of island Southeast Asia is of medical and anthropological interest and is associated with an unusually high genetic diversity. This study examined the association of this HBV genetic diversity with the ethnogeography of the populations of the Indonesian archipelago. Whole genome analysis of 21 HBV isolates from East Nusa Tenggara and Papua revealed two recently reported HBV/B subgenotypes unique to the former, B7 (7 isolates) and B8 (5 isolates), and uncovered a further novel subgenotype designated B9 (4 isolates). Further isolates were collected from 419 individuals with defined ethnic backgrounds representing 40 populations. HBV/B was predominant in Austronesian-language-speaking populations, whereas HBV/C was the major genotype in Papua and Papua-influenced populations of Moluccas; HBV/B3 was the predominant subgenotype in the western half of the archipelago (speakers of the Western Malayo-Polynesian [WMP] branch of Austronesian languages), whereas B7, B8 and B9 were specific to Nusa Tenggara (Central Malayo-Polynesian (CMP)). The result provides the first direct evidence that the distribution of HBV genotypes/subgenotypes in the Indonesian archipelago is related to the ethnic origin of its populations and suggests that the HBV distribution is associated with the ancient migratory events in the peopling of the archipelago.

Novel multiple-locus variable-number tandem-repeat analysis method for rapid molecular typing of human Staphylococcus aureus.

We evaluated the use of a novel multiple-locus variable-number tandem-repeat analysis (MLVA) method for typing of human Staphylococcus aureus. For a total of 150 clinical isolates, MLVA demonstrated the highest discriminatory power. MLVA correctly assigned isolates to outbreaks or identified isolates as unlinked. MLVA is a rapid and simple method for the epidemiological typing of S. aureus.

Mannose binding lectin plays a crucial role in innate immunity against yeast by enhanced complement activation and enhanced uptake of polymorphonuclear cells.

BACKGROUND: Mannose binding lectin (MBL) is an important host defence protein against opportunistic fungal pathogens. This carbohydrate-binding protein, an opsonin and lectin pathway activator, binds through multiple lectin domains to the repeating sugar arrays displayed on the surface of a wide range of clinically relevant microbial species. We investigated the contribution of MBL to antifungal innate immunity towards C. parapsilosis in vitro. RESULTS: High avidity binding was observed between MBL and C. albicans and C. parapsilosis. Addition of MBL to MBL deficient serum increased the deposition of C4 and C3b and enhanced the uptake of C. albicans, C. parapsilosis and acapsular C. neoformans by polymorphonuclear cells (PMNs). Compared to other microorganisms, such as Escherichia coli, Staphylococcus aureus and Cryptococcus neoformans, C. parapsilosis and Candida albicans were potent activators of the lectin pathway. CONCLUSION: Our results suggest that MBL plays a crucial role in the innate immunity against infections caused by yeast by increasing uptake by PMN.

Quantifying the relationship between Staphylococcus aureus bacteremia and S. aureus bacteriuria: a retrospective analysis in a tertiary care hospital.

In this retrospective cohort study, patients who had Staphylococcus aureus bacteremia but who lacked signs and symptoms of urinary tract infection due to S. aureus and who did not have an indwelling urinary catheter had a likelihood of S. aureus bacteriuria of 2.5% (2 of 79 patients). Therefore, we strongly question the theory that S. aureus bacteremia causes S. aureus bacteriuria.

Surveillance uncovers the smoking gun for resistance emergence.

Today, antibiotic resistance is becoming a major healthcare concern. As global travel increases, more antibiotic-resistant bacteria will be disseminated from one country to another, thereby imposing a problem worldwide. Since the development of resistance is an evolutionary process, constant surveillance is needed to gain insight into the problem and surveillance studies needed to document the spread of antibiotic resistance. The basic objectives of surveillance studies in antimicrobial resistance are: to determine the level of resistance in a particular geographical area; to monitor changes in the level of resistance and make this information available to therapeutic policy-makers, as well as to detect new mechanisms of resistance for use as early warning signs; to study how such resistance develops, persists and spreads, and to monitor interventions. Although, surveillance provides the smoking gun for emergence of antibiotic resistance, improvement of the system is necessary and may be achieved through enhanced information technology and diagnostic tools.

Nosocomial pneumonia : rationalizing the approach to empirical therapy.

Nosocomial pneumonia or hospital-acquired pneumonia (HAP) causes considerable morbidity and mortality. It is the second most common nosocomial infection and the leading cause of death from hospital-acquired infections. In 1996 the American Thoracic Society (ATS) published guidelines for empirical therapy of HAP. This review focuses on the literature that has appeared since the ATS statement. Early diagnosis of HAP and its etiology is crucial in guiding empirical therapy. Since 1996, it has become clear that differentiating mere colonization from etiologic pathogens infecting the lower respiratory tract is best achieved by employing bronchoalveolar lavage (BAL) or protected specimen brush (PSB) in combination with quantitative culture and detection of intracellular microorganisms. Endotracheal aspirate and non-bronchoscopic BAL/PSB in combination with quantitative culture provide a good alternative in patients suspected of ventilator-associated pneumonia. Since culture results take 2-3 days, initial therapy of HAP is by definition empirical. Epidemiologic studies have identified the most frequently involved pathogens: Enterobacteriaceae, Haemophilus influenzae, Streptococcus pneumoniae and Staphylococcus aureus ('core pathogens'). Empirical therapy covering only the 'core pathogens' will suffice in patients without risk factors for resistant microorganisms. Studies that have appeared since the ATS statement issued in 1996, demonstrate several new risk factors for HAP with multiresistant pathogens. In patients with risk factors, empirical therapy should consist of antibacterials with a broader spectrum. The most important risk factors for resistant microorganisms are late onset of HAP (>/=5 days after admission), recent use of antibacterial therapy, and mechanical ventilation. Multiresistant bacteria of specific interest are methicillin-resistant S. aureus (MRSA), Pseudomonas aeruginosa, Acinetobacter calcoaceticus-baumannii, Stenotrophomonas maltophilia and extended-spectrum beta-lactamase (ESBL)-producing Enterobacteriaceae. Each of these organisms has its specific susceptibility pattern, demanding appropriate antibacterial treatment. To further improve outcomes, specific therapeutic options for multiresistant pathogens and pharmacological factors are discussed. Antibacterials developed since 1996 or antibacterials with renewed interest (linezolid, quinupristin/dalfopristin, teicoplanin, meropenem, new fluoroquinolones, and fourth-generation cephalosporins) are discussed in the light of developing resistance.Since the ATS statement, many reports have shown increasing incidences of resistant microorganisms. Therefore, one of the most important conclusions from this review is that empirical therapy for HAP should not be based on general guidelines alone, but that local epidemiology should be taken into account and used in the formulation of local guidelines.

Isolation and structural characterization of epilancin 15X, a novel lantibiotic from a clinical strain of Staphylococcus epidermidis.

The potential application of lantibiotics as food-preserving agents and more recently as antibiotics has strongly increased the interest in these antibacterial peptides. Here, we report the elucidation of the primary and three-dimensional structures of the novel lantibiotic epilancin 15X from Staphylococcus epidermidis using high-resolution nuclear magnetic resonance spectroscopy and tandem mass spectrometry. The molecule contains ten post-translationally modified amino acids, three lanthionine ring structures and a hydroxy-propionyl N-terminal moiety. The primary and tertiary structure and the distribution of positive charges are closely similar to the previously identified lantibiotic epilancin K7, most likely indicative of a common mode of action.

Methicillin-resistant Staphylococcus aureus carriage among patients after hospital discharge.

BACKGROUND AND OBJECTIVE: At the University Medical Center Utrecht (UMCU), follow-up implies an inventory of risk factors and screening for MRSA colonization among all MRSA-positive patients for at least 6 months. If risk factors or positive cultures persist or re-emerge, longer follow-up is indicated and isolation at readmission. This study investigated how long MRSA-positive patients remained colonized after hospital discharge and which risk factors were important. Furthermore, the results of eradication therapy were evaluated. DESIGN: All patients who were positive for MRSA at the UMCU between January 1991 and January 2001 were analyzed regarding carriage state, presence of risk factors for prolonged carriage of Staphylococcus aureus, and eradication treatment. RESULTS: A total of 135 patients were included in the study. The median follow-up time was 1.2 years. Eighteen percent of the patients were dismissed from follow-up 1 year after discharge. Only 5 patients were dismissed after 6 months. Among patients with no risk factors, eradication treatment was effective for 95% within 1 year. Among patients with persistent risk factors, treatment was effective for 89% within 2 years. CONCLUSIONS: Based on these findings, eradication therapy should be prescribed for all MRSA carriers, independent of the presence of risk factors. MRSA-positive patients should be evaluated for 6 months for the presence of risk factors and MRSA carriage. Screening for risk factors is important because intermittent MRSA carriage was found in a significant number of our patients. Patients with negative MRSA cultures and without risk factors for 12 months can be safely dismissed from follow-up.

Integrons in Escherichia coli from food-producing animals in The Netherlands.

The presence and character of class 1 integrons in multidrug-resistant Escherichia coli from slaughter animals and meat was determined by integrase-specific PCR and conserved segment PCR-restriction fragment length polymorphism (RFLP). At least five different class 1 integron types were found and three types were shared between hospitalized patients, humans in the community, meat, and slaughter animals. Common integron types indicate that antibiotic resistance genes are exchanged via the food chain between different reservoirs of both human and animal origin.

Genogeography and Immune Epitope Characteristics of Hepatitis B Virus Genotype C Reveals Two Distinct Types: Asian and Papua-Pacific.

Distribution of hepatitis B virus (HBV) genotypes/subgenotypes is geographically and ethnologically specific. In the Indonesian archipelago, HBV genotype C (HBV/C) is prevalent with high genome variability, reflected by the presence of 13 of currently existing 16 subgenotypes. We investigated the association between HBV/C molecular characteristics with host ethnicity and geographical distribution by examining various subgenotypes of HBV/C isolates from the Asia and Pacific region, with further analysis on the immune epitope characteristics of the core and surface proteins. Phylogenetic tree was constructed based on complete HBV/C genome sequences from Asia and Pacific region, and genetic distance between isolates was also examined. HBV/C surface and core immune epitopes were analyzed and grouped by comparing the amino acid residue characteristics and geographical origins. Based on phylogenetic tree and geographical origins of isolates, two major groups of HBV/C isolates--East-Southeast Asia and Papua-Pacific--were identified. Analysis of core and surface immune epitopes supported these findings with several amino acid substitutions distinguishing the East-Southeast Asia isolates from the Papua-Pacific isolates. A west-to-east gradient of HBsAg subtype distribution was observed with adrq+ prominent in the East and Southeast Asia and adrq- in the Pacific, with several adrq-indeterminate subtypes observed in Papua and Papua New Guinea (PNG). This study indicates that HBV/C isolates can be classified into two types, the Asian and the Papua-Pacific, based on the virus genome diversity, immune epitope characteristics, and geographical distribution, with Papua and PNG as the molecular evolutionary admixture region in the switching from adrq+ to adrq-.

Amyloid-beta-induced chemokine production in primary human macrophages and astrocytes.

In Alzheimer's disease (AD), chemotaxis might be responsible for attracting glial cells towards the neuritic plaque. Using primary monocyte-derived macrophages and primary adult astrocytes as a model, amyloid-beta (Abeta) (1-42) was able to stimulate the production, as measured by RT-PCR, of MIP-1alpha and MIP-1beta mRNA in macrophages and MCP-1 in astrocytes. Cocultures showed in unstimulated as well as in Abeta-stimulated cells an increase in MIP-1alpha, MIP-1beta and MCP-1 mRNA. ELISAs of supernatant samples of stimulated macrophages and astrocytes also showed an increase in MIP-1alpha and MIP-1beta in macrophages and MCP-1 in astrocytes. Stimulated cocultures showed an increase in MIP-1alpha, MIP-1beta and MCP-1 protein levels in contrast to unstimulated cocultures.

Spare CD14 molecules on human monocytes enhance the sensitivity for low LPS concentrations.

Human monocytes express on their plasma membrane relatively large number of CD14 molecules, known to play a crucial role in the lipopolisaccharide (LPS)-mediated cellular activation. Indirect data (J. Biol. Chem. 270 (1995) 9904) suggest that not all of these CD14 molecules participate in LPS-signaling, but the importance of these spare receptors and the exact number of CD14 involved in activation upon different LPS-stimuli is not known. Using different concentrations of a blocking anti-CD14 monoclonal antibody (mAb 60bca) we created monocytes with graded amounts of CD14. The exact number of occupied and free receptors was quantitated by flow cytometry and special mAb-labeled standard beads. The number of free CD14 molecules per monocyte in the presence of 10, 3.33, 0.73, 0.25 and 0.041 microg/ml mAb was 0, 13,100, 49,300, 97,700 and 165,900. Stimulation of these partially blocked monocytes with 0.1, 1, 10 and 100 ng/ml ReLPS in the presence of 3% human serum revealed that already 13,100 and 97,700 CD14 molecules provided a maximal Tumor necrosis factor alpha (TNFalpha) mRNA response using 100 and 10 ng/ml ReLPS, while the activation totally depended on the number of available CD14 molecules in the case of 1 and 0.1 ng/ml ReLPS. Our data imply that the number of CD14 molecules available for LPS-binding influence the cellular response. In the presence of higher concentrations of LPS only fractions of CD14 participate in the cell activation, while the presence of the spare receptors enhance the sensitivity against lower LPS amounts.

Discrimination of Klebsiella pneumoniae and Klebsiella oxytoca phylogenetic groups and other Klebsiella species by use of amplified fragment length polymorphism.

Bacteria of the genus Klebsiella are opportunistic pathogens responsible for an increasing number of multiresistant infections in hospitals. The two clinically and epidemiologically most important species, Klebsiella pneumoniae and K. oxytoca, have recently been shown to be subdivided into three and two phylogenetic groups, respectively. The aim of this study was an in depth evaluation of the amplified fragment length polymorphism (AFLP) genetic characterization method for epidemiological and phylogenic analyzes of Klebsiella isolates. First, we investigated the variability of AFLP patterns for Klebsiella strains within and between different outbreaks. Second, by use of carefully characterized phylogenetically representative strains, we examined whether different Klebsiella species and phylogenetic groups can be discriminated using AFLP. Twenty-four strains originating from seven presumed outbreaks and 31 non-associated strains were investigated. The AFLP fingerprints of all epidemiologically associated strains showed three or fewer fragment differences, whereas unrelated strains differed by at least four fragments. Cluster analysis of the AFLP data revealed a very high concordance with the phylogenetic assignation of strains based on the gyrA sequence and ribotyping data. The species K. pneumoniae, K. oxytoca, K. terrigena and the possibly synonymous pair K. planticola/K. ornithinolytica each formed a separate cluster. Similarly, strains of the phylogenetic groups of K. pneumoniae and K. oxytoca fell into their corresponding clusters, with only two exceptions. This study provides a preliminary cut-off value for distinguishing epidemiologically non-related Klebsiella isolates based on AFLP data; it confirms the sharp delineation of the recently identified phylogenetic groups, and demonstrates that AFLP is suitable for identification of Klebsiella species and phylogenetic groups.

Identification of a new geographically widespread multiresistant Acinetobacter baumannii clone from European hospitals.

The aim of the study was to investigate the genetic diversity of Acinetobacter baumannii clinical strains that had previously been allocated to three major groups based on automated ribotyping. Forty-seven isolates from European hospitals and one isolate from a South African hospital, geographically representative of the three ribogroups (ribogroups 1, 2 and 3 with 10, 23 and 15 isolates, respectively), were analysed using the highly discriminatory fingerprinting methods AFLP and pulsed-field gel electrophoresis (PFGE). Based on AFLP data, the isolates clustered into three main groups, each corresponding to one ribogroup. Inclusion of reference strains of the previously described clones I and II, responsible for outbreaks in northwestern European hospitals, showed that ribogroups 1 and 2 correspond to clones I and II, respectively, whereas ribogroup 3 apparently represents a new clone. This clone III was found in France, The Netherlands, Italy and Spain. Clones I and II were not limited to northwestern European countries, as they were also recovered from Spain, South Africa, Poland and Italy (clone I) and from Spain, Portugal, South Africa, France, Greece and Turkey (clone II). Combined AFLP and PFGE data showed intraclonal diversity and led to the distinction of 23 different genotypes. Three genotypes, two of them belonging to clone II and one to clone III, were found in different hospitals and may correspond to subsets of isolates with a more recent clonal relationship, which emphasizes the epidemic potential of these organisms.

Aspirin-like molecules that inhibit human immunodeficiency virus 1 replication.

Some anti-inflammatory molecules are also known to possess anti-human immunodeficiency virus (HIV) activity. We found that o-(acetoxyphenyl)hept-2-ynyl sulfide (APHS), a recently synthesized non-steroidal anti-inflammatory molecule can inhibit HIV-1 replication. The aim of this study was to clarify the mechanism of action of APHS. When administered during the first steps of the infection, APHS was capable of inhibiting the replication of several HIV-1 strains (macrophage-tropic and/or lymphocytotropic) in a dose-dependent manner in both peripheral blood mononuclear cells (PBMC), monocyte-derived macrophages and peripheral blood lymphocytes with 50% inhibitory concentration values of approximately 10 microM. The 50% toxic concentration of APHS varied between 100 and 200 microM in the different primary cells tested. APHS did not affect HIV-1 replication once the provirus was already inserted into the cellular genome. APHS also did not inhibit HIV-1 entry into the host cells as determined by quantification of gag RNA inside PBMC 2h after infection. However, APHS did inhibit gag DNA synthesis during reverse transcription in primary cells, which indicates that APHS may target the reverse transcription process.