Restriction fragment mass polymorphism (RFMP) analysis based on MALDI-TOF mass spectrometry for detecting antiretroviral resistance in HIV-1 infected patients

AbstractViral genotype assessment is important for effective clinical management of HIV-1 infected patients, especially when access and/or adherence to antiretroviral treatment is reduced. In this study, we describe development of a matrix-assisted laser desorption/ionization-time of flight mass spectrometry-based viral genotyping assay, termed restriction fragment mass polymorphism (RFMP). This assay is suitable for sensitive, specific and high-throughput detection of multiple drug-resistant HIV-1 variants. One hundred serum samples from 60 HIV-1-infected patients previously exposed to nucleoside reverse transcriptase inhibitors (NRTIs), non-nucleoside reverse transcriptase inhibitors (NNRTIs) and protease inhibitors (PIs) were analysed for the presence of drug-resistant viruses using the RFMP and direct sequencing assays. Probit analysis predicted a detection limit of 223.02 copies/mL for the RFMP assay and 1268.11 copies/mL for the direct sequencing assays using HIV-1 RNA Positive Quality Control Series. The concordance rates between the RFMP and direct sequencing assays for the examined codons were 97% (K65R), 97% (T69Ins/D), 97% (L74VI), 97% (K103N), 96% (V106AM), 97% (Q151M), 97% (Y181C), 97% (M184VI) and 94% (T215YF) in the reverse transcriptase coding region, and 100% (D30N), 100% (M46I), 100% (G48V), 100% (I50V), 100% (I54LS), 99% (V82A), 99% (I84V) and 100% (L90M) in the protease coding region. Defined mixtures were consistently and accurately identified by RFMP at 5% relative concentration of mutant to wild-type virus while at 20% or greater by direct sequencing. The RFMP assay based on mass spectrometry proved to be sensitive, accurate and reliable for monitoring the emergence and early detection of HIV-1 genotypic variants that lead to drug resistance

Response of a Lake Michigan coastal lake to anthropogenic catchment disturbance

A paleolimnological investigation of post-European sediments in a Lake Michigan coastal lake was used to examine the response of Lower Herring Lake to anthropogenic impacts and its role as a processor of watershed inputs. We also compare the timing of this response with that of Lake Michigan to examine the role of marginal lakes as ‘early warning’ indicators of potential changes in the larger connected system and their role in buffering Lake Michigan against anthropogenic changes through biotic interactions and material trapping. Sediment geochemistry, siliceous microfossils and nutrient-related morphological changes in diatoms, identified three major trophic periods in the recent history of the lake. During deforestation and early settlement (pre-1845–1920), lake response to catchment disturbances results in localized increases in diatom abundances with minor changes in existing communities. In this early phase of disturbance, Lower Herring Lake acts as a sediment sink and a biological processor of nutrient inputs. During low-lake levels of the 1930s, the lake goes through a transitional period characterized by increased primary productivity and a major shift in diatom communities. Post-World War II (late 1940s–1989) anthropogenic disturbances push Lower Herring Lake to a new state and a permanent change in diatom community structure dominated by Cyclotella comensis . The dominance of planktonic summer diatom species associated with the deep chlorophyll maximum (DCM) is attributed to epilimnetic nutrient depletion. Declining Si:P ratios are inferred from increased sediment storage of biogenic silica and morphological changes in the silica content of Aulacoseira ambigua and Stephanodiscus niagarae . Beginning in the late 1940s, Lower Herring Lake functions as a biogeochemical processor of catchment inputs and a carbon, nutrient and silica sink. Microfossil response to increased nutrients and increased storage of biogenic silica in Lower Herring Lake and other regional embayments occur approximately 20–25 years earlier than in a nearby Lake Michigan site. Results from this study provide evidence for the role of marginal lakes and bays as nutrient buffering systems, delaying the impact of anthropogenic activities on the larger Lake Michigan system.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/43091/1/10933_2004_Article_1688.pd