| Methods of Testing for HIV Drug Resistance|
Drug resistance can be measured using either genotypic or phenotypic assays. Genotypic assays detect mutations that cause drug resistance. Phenotypic assays are drug susceptibility assays in which a fixed inoculum of HIV-1 is cultured in the presence of serial dilutions of an inhibitory drug. Current commercially available genotypic and phenotypic assays both test HIV-1 extracted from plasma. After extraction, the contiguous protease and reverse transcriptase (RT) genes are reverse transcribed to cDNA and then amplified using polymerase chain reaction (PCR) to generate sufficient DNA for genotypic or phenotypic testing. Because the segment of the HIV-1 genome used for genotypic testing (about 1500-2000 base pairs [bp]) is larger than that used for quantitative assays (about 100 bp), the sensitivity of most genotypic assays is lower (between 100 and 1000 RNA copies/mL depending on the assay) than most quantitative assays, which can typically detect <20 to 50 RNA copies/mL.
Genotypic testing is used more commonly than phenotypic testing because of its lower cost, wider availability, and shorter turnaround time. Genotypic testing provides early evidence of drug resistance within a virus population. Genotypic assays detect mutations present as mixtures even if the mutation is present at a level too low to affect susceptibility in a phenotypic assay, and detect transitional mutations that do not cause drug resistance by themselves but indicate the presence of selective drug pressure. Genotypic assays detect mutations even if the phenotypic effect of the mutation is suppressed by other mutations in the sequence. The clinical usefulness of genotypic testing has been demonstrated in four of five prospective randomized studies;(1,2,3,4,5) in contrast, phenotypic testing has been shown to be clinically useful in just one of four prospective randomized studies.(5,6,7,8)
Phenotypic testing in research settings is essential for establishing genotype-phenotype correlations, which provide the basis for interpreting genotypic tests and for designing new antiviral drugs that are effective against existing drug-resistant strains. Phenotypic testing in clinical settings is expected to be most useful for testing isolates with unusual combinations of drug resistance mutations. Phenotypic testing may also be useful in combination with therapeutic drug monitoring for designing salvage regimens in heavily treated patients whose viruses contain multiple drug resistance mutations.
| Genotypic Resistance Testing|
| Sample Processing and Sequencing|
Because the half-life of HIV-1 in plasma is approximately 6 hours,(9) only actively replicating virus can be isolated from this source; thus the sequence of plasma virus represents the quasispecies most recently selected by antiretroviral drug therapy. The evolution of HIV-1 drug resistance mutations in proviral DNA in peripheral blood mononuclear cells (PMBCs) lags behind that in plasma HIV-1 RNA.(10,11,12-14) In patients with multiple virologic failures, proviral PBMC DNA may contain multiple archived mutations that are not present in plasma. However, the utility of PBMC sequencing has not been evaluated in either prospective or retrospective clinical trials.
Clonal sequencing of individual virus strains is performed in research settings to answer questions about the evolution of HIV-1 drug resistance. Direct PCR, or viral population-based sequencing is done in clinical settings because it is quicker and more affordable than testing multiple clones individually. For both population-based and clonal sequencing, the ability to detect minor variants is related to the proportion of those minor variants within the whole virus population. In direct PCR sequencing, a nucleotide mixture can be detected when the least common nucleotide is present in at least 20% of the sampled virus population.(15-19)
1 shows the distribution of known drug resistance mutations within HIV-1 protease and RT. Mutations that may individually confer resistance to one or more drugs are represented by tall lines. Accessory mutations that confer resistance only when present with other mutations are represented by short lines. Nearly all of the RT mutations are in the fingers and palm of the 5´ polymerase region of the enzyme. Sequencing for clinical purposes should encompass nearly all of the protease and positions 41- 236 of the RT. Mutations at position 318 have been shown to increase nonnucleoside reverse transcriptase inhibitor (NNRTI) resistance when present with other mutations.(20) G333E is a naturally occurring polymorphism that facilitates zidovudine resistance in isolates from some patients receiving zidovudine and lamivudine that also have multiple zidovudine resistance mutations,(21) but dual resistance to these drugs usually emerges without this change.
| Dideoxynucleotide Sequencing|
Dideoxynucleotide sequencing is the standard approach to HIV genotyping; it involves the synthesis of a new DNA chain in the presence of a primer and a carefully created mix of deoxynucleotide triphosphates (dNTPs) and dideoxynucleotide triphosphate chain terminators (ddNTPs). New strand synthesis starts with the primer and continues until a ddNTP is incorporated in place of the appropriate dNTP. This reaction creates a set of DNA strands each differing from one another by the length of one nucleotide. The strands are separated using polyacrylamide gel electrophoresis, and the final nucleotide on each strand is read by an automated sequencer using fluorometric methods that depend upon the labeling of either the primer or the terminators.
Most diagnostic laboratories use their own "home-brew" methods: sequencing is done using reagents obtained from separate vendors, and polyacrylamide gel electrophoresis and sequencing reading is generally done using an automated sequencer. In 2001, a commercial HIV-1 RT and protease genotyping kit (TruGene, Visible Genetics, Toronto, Canada) was approved by the FDA for use in clinical settings.(22) A second kit is under consideration for FDA approval (ViroSeq, Applied Biosystems, Foster City, California).(23) Preliminary data suggest that results obtained with each assay are highly concordant.(24) The kits have stronger quality control and validation profiles than home-brew methods, making them preferable in clinical laboratories. However, they are more expensive and may not provide the versatility of home-brew methods.
| Hybridization Methods|
Sequencing by hybridization can be used to detect specific mutations, and in some situations, to determine the complete sequence of a gene. The Affymetrix GeneChip (Santa Clara, California) was designed to determine the complete sequence of HIV-1 protease and the first 1200 nucleotides of HIV-1 RT. Although this product is no longer available, DNA chip sequencing is likely to become increasingly common in the future and may be again applied to HIV genotyping. The INNO-LiPA HIV-1 line probe assays (Innogenetics, Ghent, Belgium) are point-mutation assays designed to detect specific HIV-1 protease and RT mutations.
The GeneChip was divided into several thousand segments each containing millions of similar probes designed to interrogate every nucleotide position in a test DNA or RNA molecule. Every nucleotide in the test molecule requires at least four sets of oligonucleotide probes to determine whether that nucleotide is an A, C, G, or T. DNA chip sequencing is also called "resequencing" because some prior knowledge of the sequence is required to design probes that can bind to nucleotides adjacent to the position being interrogated.
Because of genetic variability, sequencing HIV-1 by hybridization is challenging. Several studies compared the performance of the GeneChip with dideoxynucleotide sequencing and found that dideoxynucleotide sequencing was more reliable at detecting HIV-1 RT and protease mutations.(16,25-27) DNA chips were less capable of detecting insertions or deletions in viral sequences and of sequencing viral subtypes other than subtype B--the subtype on which the original chip tiling was based.(25,26)
Point mutation assays are inexpensive and have the potential to be highly sensitive for mutations present in only a small proportion of circulating viruses.(28,29) Because they require only simple laboratory equipment, they may be useful in areas that do not have ready access to expensive sequencers. The INNO-LiPA assays have probes for wild-type and mutant alleles of each codon, attached to a nitrocellulose strip.(30-32) Biotin-labeled RT-PCR product from the patient sample is hybridized to the strip. An avidin-enzyme complex and the enzyme substrate produce a color change on the paper strip where the PCR product has hybridized with a probe. This assay is limited because it can only detect a subset of drug resistance mutations and has had a 10% rate of uninterpretable results due to poor hybridization, particularly when uncommon mutations are present at key codons.(29,33)
| Dideoxynucleotide Sequencing Reproducibility|
Two large multicenter comparisons of sequence results obtained from samples containing mixtures of plasmid clones (ENVA-1) and spiked plasma samples (ENVA-2) have identified two important causes of interlaboratory variation: 1) variability in the quality of work done by different laboratories, and 2) the difficulty in consistently detecting minor variants even by laboratories performing high-quality work.(17,34) The variability in laboratory performance--which was independent of the particular sequencing methodology used--suggests a need for ongoing quality assurance testing. Although specific reasons for poor laboratory performance are not cited in the published literature, it is likely that cost-saving shortcuts (such as sequencing just one strand of DNA or using sequence primers that are too far apart to obtain consistent, high-quality electrophoretic peaks) may be responsible. The difficulty in consistently detecting minor variants is a function of the quasispecies nature of HIV-1 and is characteristic of all currently used susceptibility testing methods.
In experienced laboratories, dideoxynucleotide sequencing can be highly reproducible. In one study, 13 research laboratories were shipped cell pellets from cultured HIV-1 isolates.(35) The sequence concordance among laboratories was 99.7% at all nucleotide positions and 97% at positions associated with zidovudine resistance. Sequencing cultured cell pellets is simpler than sequencing plasma because RNA extraction and reverse transcription are not necessary and because cultured virus is more homogeneous than uncultured virus.(36,37) Nonetheless, the high interlaboratory concordance in this study attests to the intrinsic reliability of the dideoxy method for HIV-1 analysis.
In another study, two clinical laboratories also assessed the reproducibility of HIV-1 RT and protease sequencing using plasma aliquots obtained from 46 heavily treated HIV-1-infected individuals.(38) Although both laboratories used sequencing reagents from the same commercial source, each used a different in-house protocol for plasma HIV-1 RNA extraction, reverse transcription, PCR, and sequencing. Overall sequence concordance between the two laboratories was 99.0%. Approximately 90% of the discordances were partial, defined as one laboratory detecting a mixture when the second laboratory detected only one of the mixture's components. Discordance was significantly more likely to occur in plasma samples with lower plasma HIV-1 RNA levels.
Nucleotide mixtures were detected at approximately 1% of the nucleotide positions, and, in every case in which one laboratory detected a mixture, the second laboratory detected either the same mixture or one of the mixture's components. The high concordance in detecting mixtures and the fact that most discordance between the two laboratories was partial suggest that most discordances were due to variation in sampling the HIV-1 quasispecies rather than to technical errors. Figure
2 shows that the few drug resistance mutations detected by one laboratory but missed by the other laboratory were present as minor variants.
| Sequence Quality Control|
Sequence quality control should aim to avoid PCR contamination and sample mix-ups. To prevent sample contamination with DNA from other sources, laboratories should use standard physical precautions,(39) run negative controls with each PCR step, and examine their final sequence results for the possibility of contamination with other samples sequenced at the same time.(40) Uracil N-glycosylase (UNG), which is used in Applied Biosystems' ViroSeq kit, has also been used to diminish the risk of PCR-based laboratory contamination. UNG works by destroying any PCR product from previous reactions at the start of each PCR assay.
Sequence analyses can be extremely useful for detecting both laboratory contamination and sample mix-up and should be incorporated into every laboratory's testing procedure. These analyses should compare each new sequence with other recently generated sequences to look for unexpectedly high levels of similarity. Phylogenetic trees can also be constructed to visually detect unexpectedly similar isolates. The HIV Sequence Database at Los Alamos National Laboratories has a tutorial to assist with sequence analysis for quality control purposes.(41) If previous sequences from a patient are available, then each new sequence should be compared with these earlier sequences. Although one would expect some change because of selective drug pressure, many aspects of the sequence, particularly polymorphic amino acids and silent nucleic acid positions, should undergo minimal change over time. Finally, if a sequence is incompatible with the patient treatment history, sequencing should be repeated on a new clinical sample.
| Intersubtype Variability|
During its spread among humans, group M HIV-1 has evolved into multiple subtypes that differ from one another by 10-30% along their genomes.(42,43) In North America and Europe, most HIV-1 isolates belong to subtype B, and the available antiretroviral drugs have been developed by drug screening and susceptibility testing using subtype B isolates. However, subtype B accounts for only a small proportion of HIV-1 isolates worldwide, and non-B isolates are being identified with increasing frequency, particularly in Europe.
It might be expected that the functional constraints on the protease and RT enzymes would prevent their genes from displaying much intersubtype variation. But several groups have shown that the protease and RT genes of global isolates are sufficiently different from one another to allow subtype grouping.(44,45,46,47) Although most intersubtype variation is caused by synonymous nucleotide substitutions, there are subtype-specific amino acid patterns.(44,45,46,47,48)
Protease intersubtype sequence variation is more likely to occur at positions associated with drug resistance than RT intersubtype variation. A protease substrate cleft mutation, V82I occurs commonly in subtype G isolates. Preliminary data suggest that V82I confers minimal or no resistance to the available protease inhibitors (PIs),(49-51) although it occasionally emerges during PI therapy.(52) Mutations at positions 20, 36, and 93 are accessory resistance mutations that occur more commonly in some non-B subtypes.(44)
A few studies have tested the in vitro susceptibility of non-subtype B HIV-1 isolates to antiretroviral drugs. Although group O isolates often demonstrate intrinsic resistance to the NNRTIs,(53,54) most studies have shown that non-B group M isolates are as susceptible as subtype B isolates to each of the three HIV drug classes.(55,56-58) Although limited data are available, there is no evidence yet for novel drug resistance mutations in non-B HIV-1 isolates.
Intersubtype genetic variability may complicate HIV-1 genotyping because primers used for reverse transcription, PCR, and sequencing may have a lower rate of annealing to non-B compared with subtype B sequences. The extent to which this occurs has not yet been reported. Preliminary studies confirm that many of the current sequencing methods are not optimized for amplifying and sequencing non-B isolates,(59) although one study using the ViroSeq kit on isolates showed a high sensitivity at detecting and sequencing subtype A, C, and D viruses from Uganda.(60)
| Phenotypic Resistance Testing|
Until the late 1990s, the most commonly used phenotypic assays were plaque reduction and PBMC assays. Plaque reduction assays were used primarily for testing laboratory isolates. Because they required the co-cultivation of virus with HeLa-CD4+ cells (which lack the CCR5 receptor), plaque reduction assays could only test the minority of clinical isolates that were syncytium inducing (CXCR4 tropic). In contrast, PBMC-based assays were suitable for testing most clinical isolates. PBMC assays, however, were laborious because they required a preliminary 10- to 14-day period to create a virus stock from a clinical isolate by cocultivating patient PBMCs with mitogen-stimulated PBMCs from HIV-seronegative individuals. Moreover, testing was expensive because virus growth in PBMCs cannot be monitored visually but requires the specific measurement of a virus product (eg, p24 antigen).
In the late 1990s, two companies developed standardized assays amenable to high-throughput performance: Tibotec-Virco (Antivirogram; Mechelen, Belgium) and ViroLogic (PhenoSense; South San Francisco, California).(61,62) Both assays use PCR to amplify the entire protease, much of RT and some of gag from HIV-1 RNA extracted from patient plasma. The amplified material is incorporated into a pol-deleted recombinant virus construct to create a recombinant HIV-1 isolate. A standardized virus inoculum is then used to infect a cell line, and virus replication is measured in the presence and absence of a range of concentrations of different antiretroviral drugs.
The first few steps of sample preparation (RNA extraction, reverse transcription, and PCR amplification) are the same for both of the phenotypic assays. After amplification, the ViroLogic PhenoSense assay, uses direct ligation, and the Tibotec-Virco Antivirogram assay uses homologous recombination in cell culture to combine the patient's pol gene with the pol-deleted HIV-1 vector. The ViroLogic assay tests the recombinant construct during a single cycle of replication using a luciferase reporter gene assay designed to be highly sensitive to HIV-1 replication. The Tibotec-Virco assay cultures the virus for several replication cycles using cell killing as the measure of virus replication.
Both companies report the concentration of drug (µM) required to inhibit HIV-1 replication by 50% (IC50), as well as how this concentration compares with the concentration of drug required to inhibit a wild-type HIV-1 clone. Both assays have undergone rigorous internal testing and have been shown to produce little variation (less than twofold) when the same sample is tested repeatedly.(63) However, there is only one comparative study of isolates with and without known drug- resistance mutations. A preliminary study in which both assays were run on 34 isolates from untreated persons and 16 isolates from treated persons demonstrated 92% concordance.(64) However, most of these isolates were wild type, so further comparisons of isolates with known genotypes would be useful.
Recombinant virus susceptibility assays have several advantages over older nonrecombinant assays. Recombinant virus assays can be done using plasma, whereas nonrecombinant assays require the isolation of PBMCs. Recombinant virus assays can be performed under highly uniform conditions because the backbone of the virus construct, which remains constant, can be tailored for replication in the cells used for susceptibility testing. Finally, recombinant virus assays use PCR to amplify the protease and RT genes, dramatically decreasing the need for virus culture.
The use of recombinant viruses for susceptibility testing, however, has a theoretical limitation for testing susceptibility to PIs that has not been adequately addressed in the published literature. PI resistance is modulated by mutations at gag-pol cleavage sites,(65-72) and although four of the nine cleavage sites in the recombinant virus come from the patient virus sample, five come from the laboratory virus construct (Figure
3). The cleavage sites with the most compensatory changes (p7/p1 and p1/p6) are included in both recombinant virus assays. However, if a patient's virus sample contains a critical compensatory mutation at one of the five cleavage sites absent from the recombinant virus, then the recombinant virus may fail to replicate or may produce inaccurate drug susceptibility results.
| Drug Resistance Interpretation|
| General Principles|
HIV-1 drug resistance is rarely an all-or-none phenomenon. Clinicians treating infected patients usually need the answers to the following two questions: 1) Does the result suggest that the patient will respond to a drug in a manner comparable to a patient with a wild-type isolate?, and 2) Does the result suggest that the patient will obtain any antiviral benefit from the drug? The second question distinguishes antiviral susceptibility testing from antibacterial susceptibility testing. In the case of bacteria, it is usually possible to avoid using any drug with reduced susceptibility against a pathogen. This is usually not possible in the case of HIV, however, because of the extent of cross-resistance within each class of HIV drugs. To answer both these questions it is necessary to grade the extent of inferred resistance relative to wild type (eg, low-level, intermediate, high-level).
The clinical significance of both genotypic and phenotypic data have been difficult to establish for the following reasons: 1) all antiretroviral drugs are used in combinations, many of which are synergistic (reduced susceptibility to a drug may not interfere with the drug's beneficial effect on the antiretroviral activity of another drug used in the same regimen); 2) a drug may have some benefit even in the setting of resistance, because many drug-resistant variants are less fit than drug-susceptible variants; and 3) the serum levels of some drugs, particularly the PIs, can be highly variable. Low levels of PI resistance may be overcome in some cases if higher serum PI levels can be obtained. Therefore, most genotypic and phenotypic interpretations depend on a combination of in vitro as well as in vivo data.
| Genotypic Test Results|
Genotypic results bear little resemblance to those of a typical antimicrobial susceptibility assay. Rather than receiving a result such as "susceptible" or "resistant" for each of the available HIV drugs, the ordering clinician receives a list of mutations present in the virus isolate. The fact that genotypic interpretation is independent of the process of genotyping makes genotypic interpretation an ideal application for a computerized expert system. Laboratories doing HIV-1 genotyping can provide physicians with the option of receiving a file with the sequence data (string of nucleotides or list of amino acid differences from consensus). Such data can then be analyzed by interpretation systems other than those used by the sequencing laboratory.(73) Table
1 describes several of the most commonly used systems for HIV-1 genotypic interpretation. During the next 1-2 years, these algorithms will evolve and most likely converge through an ongoing process of interalgorithm comparison and validation using clinical data sets. This is because there is probably more concordance among clinical virologists than is currently reflected in published algorithms.
The VirtualPhenotype (Tibotec-Virco) is a pattern-matching algorithm that uses a large genotypic-phenotypic correlative database to infer phenotypic properties based on sequence data.(74) The analysis includes a tabulation of the number of matches in the database for each drug, and the distribution of phenotypes (fold increase in IC50) for the matching samples. The mean IC50 of the matching samples is interpreted using drug-specific cutoff values, providing a quantitative prediction of drug resistance. Although the VirtualPhenotype has been described in several abstracts, a complete description of the workings of this approach has not been published.
Specifically, it is not known which mutations are used to match a new sequence to those sequences that are already in the database. In addition, the database and pattern-matching algorithm are proprietary.
| HIV RT and Protease Sequence Database|
The HIV RT and Protease Sequence Database at Stanford University is an online database (http://hivdb.stanford.edu) that links sequence data to the HIV drug treatments of the patients from whom the sequenced isolates were obtained and to drug susceptibility results. The database also contains two sequence analysis programs. The first, HIV-SEQ, accepts user-submitted RT and protease sequences, compares them with a reference sequence, and uses the differences as query parameters for interrogating the sequence database.(75) This allows users to detect unusual sequence results immediately so that the person doing the sequencing can check the primary sequence output while it is still on the desktop. In addition, unexpected associations between sequences or isolates can be discovered by immediately retrieving data on isolates sharing one or more mutations with the sequence.
The second program, "Drug Resistance Interpretation," is an expert system that accepts user-submitted protease and RT sequences and returns inferred levels of resistance to the 16 FDA-approved anti-HIV drugs. Each drug resistance mutation is assigned a drug penalty score; the total score for a drug is derived by adding the penalty scores of each mutation associated with resistance to that drug. Using the total drug score, the program reports one of the following levels of inferred drug resistance: susceptible, potential low-level resistance, low-level resistance, intermediate resistance, and high-level resistance. Genotypic interpretations do not necessarily correlate with the inferred level of phenotypic resistance because the genotypic interpretation also uses correlations between genotype and clinical outcome in deciding how a drug's susceptibility should be graded. A listing of all mutation/drug score pairs can be found with the program's release notes.
| Phenotypic Interpretation |
To interpret a drug susceptibility result, it is important to know the reproducibility of the assay for a given drug ("technical cutoff" or "reproducibility cutoff"), the range of IC50's (or IC90's) required to inhibit wild-type viruses ("biological cutoff"), and the clinical significance associated with different levels of reduced drug susceptibility ("clinical cutoff"). There should ideally be at least two clinical cutoffs: the IC50 at which there is some reduction in drug activity and the IC50 at which a drug no longer has specific antiviral activity.
Both Tibotec-Virco and ViroLogic have published data on the reproducibility of their assay and the range in susceptibility results obtained in testing wild-type isolates from untreated persons.(76,77,78) However, the results are not readily accessible and may have been calculated differently for each assay (Table
2). Clinical cutoffs are still being developed, although important correlations between the level of susceptibility to a given drug and clinical outcome have recently been reported in the following settings: intensification with abacavir(79) or tenofovir,(80) switching from zidovudine to stavudine,(81) and salvage therapy with lopinavir and efavirenz in patients who developed virologic failure on an earlier PI-containing regimen.(82) Both assays have also recently lowered their clinical cut-points to detect low-level resistance to didanosine, zalcitibine, stavudine, and tenofovir.
The interpretation of phenotype results would also be aided by the publication of drug-susceptibility data obtained with both assays on isolates with common patterns of drug-resistance mutations. Although both the Antivirogram (Tibotec-Virco) and PhenoSense (ViroLogic) assays are recombinant virus assays, there are differences that might be expected to influence test performance. PhenoSense should theoretically be more reproducible because it measures drug activity over a single cycle of HIV replication using a very sensitive marker (Tat production measured by a luciferase reporter gene). In contrast, the Antivirogram compares the extent of cell killing in the presence and absence of a drug. Cell killing requires several rounds of replication (usually 4-7 days) and is likely to be a much less reproducible marker of HIV replication. However, it is difficult to determine for both assays the extent to which the technical and biological cutoffs overlap with the clinical cutoffs for these difficult-to-test drugs.
Three main factors explain apparently discordant results between genotypic and phenotypic susceptibility tests: 1) phenotypic tests are more likely to detect resistance caused by uncommon mutations that have not been included in genotypic interpretation algorithms; 2) genotypic tests are more likely to detect resistance caused by mutations present as part of a mixture, transitional mutations, and mutations that are suppressed by another mutation in the virus; and 3) genotypic tests are more likely to detect nucleoside reverse transcriptase inhibitor (NRTI) resistance caused by nucleotide excision mutations (NEMs). Examples of each of these scenarios are shown in Table
| Limitations of Drug Resistance Testing |
Several factors limit the usefulness of both genotypic and phenotypic testing: 1) There is a complex relationship between drug resistance and clinical failure, often making it difficult to discern the contribution of drug resistance to virologic failure; 2) the HIV-1 population within an individual consists of innumerable variants and minor variants that often go undetected; 3) because of extensive cross-resistance within each drug class, the results of resistance testing often leave clinicians with few options for treatment.
| Complex Relationship Between Drug Resistance and Disease Progression |
Drug resistance is not the only cause of treatment failure. Nonadherence, the use of insufficiently potent treatment regimens, and pharmacokinetic factors that decrease the levels of one or more drugs in a treatment regimen also contribute to treatment failure. In addition, the natural history of HIV-1 infection is highly variable and dependent on a complex set of host-virus interactions.(83) In the absence of therapy some patients progress to advanced immune deficiency within 3 years after infection, whereas other patients remain healthy for more than 15 years. It is likely that the same host-virus interactions that so greatly influence disease progression in the absence of drug therapy also influence the risk of virologic failure in patients receiving antiretroviral therapy.
Two recent observations underscore the complexity of the relationship between drug resistance and disease progression. The first is that patients developing virologic failure on their first treatment regimen are usually found to have HIV-1 isolates with resistance to only one of the drugs in the regimen.(84,85,86,87,88,89) The drugs to which resistance most commonly develops in this situation are lamivudine and the NNRTIs; resistance to PIs and NRTIs other than lamivudine is less common in patients with initial virologic failure. The observation that virus becomes detectable and replication ensues despite the fact that the replicating virus remains sensitive to at least two drugs in the treatment regimen suggests that factors in addition to drug resistance are contributing to virologic failure. Possibly the remaining drugs in the regimen are not potent enough to fully suppress virus even though they remain active. Alternatively, one of the presumably active drugs in the regimen may be present at insufficient levels because of nonadherence or pharmacokinetic factors.
The second observation is that virologic failure in patients receiving HAART is not always followed by immunologic and clinical deterioration.(90-93) This may be because the immunologic benefits of virus suppression persist beyond the period of virus suppression or because drug-resistant viruses may be less virulent, particularly when they first emerge and are associated with fewer compensatory mutations.(94,95) However, the duration of immunologic benefit in the face of ongoing virus replication is not known. In patients experiencing their first virologic failure, treatment is usually changed because there is an ongoing risk of accumulating additional drug resistance mutations that would interfere with the success of salvage therapy.(96)
Several lines of evidence suggest that drug-resistant viruses are less fit than drug-susceptible viruses. First, in vitro experiments have consistently shown that isolates containing protease and/or RT drug resistance mutations replicate less well in cell culture, and that purified enzymes with these mutations usually have less activity than wild-type enzymes (reviewed in (97)). There are conflicting data, however, on whether multidrug-resistant variants are less cytopathic in specific types of cells (eg, thymus).(98-100) Second, drug resistance mutations are often replaced in vivo by wild-type variants within weeks to months after removal of selective drug pressure.(95,101-104) The rate at which this occurs depends on the extent to which archived wild-type viruses exist within an individual patient. If there are no archived wild-type viruses, a significant interlocking of primary and compensatory mutations may limit reversion to wild type.(105)
One clinical trial in patients with detectable viremia and multidrug-resistant virus showed that in those patients randomized to discontinue antiretroviral therapy, plasma HIV-1 RNA increased by 0.84 log10 and CD4 cell counts decreased by 128 cells/µL as the dominant virus population reverted to wild type.(91) This study suggests that the decreases in fitness of drug-resistant viruses seen in vitro are clinically significant, and that continuing drug therapy in the face of resistance may have utility in patients with few other therapeutic options. However, the possibility cannot be excluded that many of the isolates in this study retained some degree of susceptibility to one or more drugs in treatment regimens that were used.
| Quasispecies Nature of HIV-1 |
A recognized limitation of HIV-1 drug susceptibility testing by either genotypic or phenotypic methods is the unreliability of these tests in detecting minority HIV-1 variants in the virus population of the patient tested. This limitation is particularly troublesome in patients with complicated treatment histories or those who have discontinued one or more antiretroviral drugs.(103,104,106) To maximize the likelihood of identifying drug-resistance mutations present within the virus population of a patient, it is important to obtain plasma samples for resistance testing before stopping or changing antiretroviral drugs and to consider a patient's treatment history when interpreting the results of resistance testing.
In some patients, the treatment history can be used to infer the presence of archived drug-resistance mutations. For example, if a patient previously received lamivudine as part of an incompletely suppressive treatment regimen, it is likely that M184V exists within the virus population of that patient, even if it is not detected at the time of genotyping. The same principle would apply to patients who received NNRTIs as part of an incompletely suppressive treatment regimen; however, in this situation, it would not be possible to know specifically which NNRTI mutations are likely to be archived. In contrast, patients receiving lamivudine and NNRTIs as part of completely suppressive treatment regimens are not expected to harbor variants resistant to these drugs.
If a patient once harbored drug-resistant variants, these variants may persist at low levels in latently infected cells even if a subsequent treatment regimen brings about complete virus suppression.(106,107-111) In patients in whom previous resistance tests have documented extensive drug resistance, the clinical usefulness of repeated resistance testing is likely to be minimal, because many resistant variants selected by previous treatment regimens will go undetected in future tests, yet are likely to emerge during attempts at salvage therapy.
| Cross-Resistance |
Most mutations arising during drug therapy contribute resistance to multiple drugs within the same drug class. Because there are only three drug classes available, and combinations of drugs from at least two classes are usually required to achieve durable HIV-1 suppression, cross-resistance is a significant problem. Genotypic assays frequently do not identify enough fully active non-cross-resistant drugs to completely block HIV-1 replication, and many patients changing regimens because of virologic failure will have to use a regimen containing drugs that are partially compromised at the start of therapy.
Although cross-resistance is not a direct limitation of genotypic or phenotypic testing, it limits the usefulness of resistance testing, particularly in heavily treated patients. Nonetheless, by providing prognostic data and helping to avoid unnecessary drugs, resistance testing can have a role even in these patients. When fully active drugs are not available, salvage therapy in heavily treated patients may include drug combinations that exploit antagonistic mutational interactions or generate high in vivo drug levels.
Robert W. Shafer is supported in part by NIH grant AI46148-01 and has received unrestricted educational grants from various pharmaceutical and diagnostic companies for maintenance of the HIV RT and Protease Sequence Database. A list of the companies who have supported the database can be found at http://hivdb.stanford.edu/pages/acknowledgements.html.
Matthew J. Gonzales assisted with manuscript preparation, including creation of many of the tables and figures, and is supported in part by a supplement to NIH grant AI46148-01.
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|| ||Kemp SD, Salin M, Stammers D, Wynhoven B, Larder BA, Harrigan PR. A mutation in HIV-1 reverse transcriptase at codon 318 (Y-F) confers high-level non-nucleoside reverse transcriptase inhibitor resistance in clinical samples. Antivir Ther 2001;6(Supplement 1):36.|
|| ||Ruiz L, Telles F, Smith M, Maillard A, Domingo C, Heiken H, Holtzer C. Inter-laboratory and inter-lot reproducibility and quality control of HIV-1 genotyping with a commercial kit [abstract 55]. Antivir Ther 2000;5(2):176-7.|
|| ||Dileanis J, Marlowe N, Hoo B, Team. PBHD, Kunstman K, Wolinsky S, Eshleman S. RT-PCR analysis of clinical specimens with the PE Biosystems Version 2 HIV-1 Genotyping System [abstract 47]. Antivir Ther 2000;5 (Suppl 3):171.|
|| || Vahey M, Nau ME, Barrick S, Cooley JD, Sawyer R, Sleeker AA, Vickerman P, Bloor S, Larder B, Michael NL, Wegner SA. Performance of the Affymetrix GeneChip HIV PRT 440 platform for antiretroviral drug resistance genotyping of human immunodeficiency virus type 1 clades and viral isolates with length polymorphisms. J Clin Microbiol 1999;37(8):2533-7.|
|| || Van Laethem K, Van Vaerenbergh K, Schmit JC, Sprecher S, Hermans P, De Vroey V, Schuurman R, Harrer T, Witvrouw M, Van Wijngaerden E, Stuyver L, Van Ranst M, Desmyter J, De Clercq E, Vandamme AM. Phenotypic assays and sequencing are less sensitive than point mutation assays for detection of resistance in mixed HIV-1 genotypic populations. J Acquir Immune Defic Syndr 1999;22(2):107-18.|
|| || Servais J, Lambert C, Fontaine E, Plesseria JM, Robert I, Arendt V, Staub T, Schneider F, Hemmer R, Burtonboy G, Schmit JC. Comparison of DNA Sequencing and a Line Probe Assay for Detection of Human Immunodeficiency Virus Type 1 Drug Resistance Mutations in Patients Failing Highly Active Antiretroviral Therapy. J Clin Microbiol 2001;39(2):454-9.|
|| ||De Smet K, Celis L, Schmit J, Colebunders R, Miller V, Soriano V, Quinn T, A. M, Stuyver L. Identification of key mutations in the HIV-1 protease using the INNO-LiPA HIV protease assay [abstract 50]. Antivir Ther 2000;5:172-3.|
|| || Demeter LM, D'Aquila R, Weislow O, Lorenzo E, Erice A, Fitzgibbon J, Shafer R, Richman D, Howard TM, Zhao Y, Fisher E, Huang D, Mayers D, Sylvester S, Arens M, Sannerud K, Rasheed S, Johnson V, Kuritzkes D, Reichelderfer P, Japour A. Interlaboratory concordance of DNA sequence analysis to detect reverse transcriptase mutations in HIV-1 proviral DNA. ACTG Sequencing Working Group. AIDS Clinical Trials Group. J.Virol.Methods 1998;75:93-104.|
|| || Kusumi K, Conway B, Cunningham S, Berson A, Evans C, Iversen AK, Colvin D, Gallo MV, Coutre S, Shpaer EG, et al. Human immunodeficiency virus type 1 envelope gene structure and diversity in vivo and after cocultivation in vitro. J Virol 1992;66(2):875-85.|
|| ||Kuiken CL, Foley B, Hahn BH, Marx P, McCutchan FE, Mellors J, Mullins JI, Wolanski S, Korber B. Human retroviruses and AIDS: a compilation and analysis of nucleic and amino acid sequences. Los Alamos, NM: Los Alamos National Laboratory;1999.|
|| ||Robertson DL, Anderson JL, Bradac JA, Carr JK, Foley B, Funkhouser RK, Gao F, Hahn BH, Kalish ML, Kuiken CL, Learn GH, Jr, Leitner T, McCutchan FE, Osmanov S, Peeters M, Pieniazek D, Salminen MO, Sharp PM, Wolinsky S, Korber B. HIV-1 nomenclature proposal: A reference guide to HIV-1 classification. In: Kuiken, CL;Foley, B;Hahn, BH et al., editors, translator and editor Human retroviruses and AIDS: a compilation and analysis of nucleic and amino acid sequences. Los Alamos, NM: Los Alamos National Laboratory;2000;p. 492-505. |
|| || Pieniazek D, Rayfield M, Hu DJ, Nkengasong J, Wiktor SZ, Downing R, Biryahwaho B, Mastro T, Tanuri A, Soriano V, Lal R, Dondero T. Protease sequences from HIV-1 group M subtypes A-H reveal distinct amino acid mutation patterns associated with protease resistance in protease inhibitor-naive individuals worldwide. HIV Variant Working Group. AIDS 2000;14(11):1489-95.|
|| || Vergne L, Peeters M, Mpoudi-Ngole E, Bourgeois A, Liegeois F, Toure-Kane C, Mboup S, Mulanga-Kabeya C, Saman E, Jourdan J, Reynes J, Delaporte E. Genetic diversity of protease and reverse transcriptase sequences in non-subtype-B human immunodeficiency virus type 1 strains: evidence of many minor drug resistance mutations in treatment-naive patients. J Clin Microbiol 2000;38(11):3919-25.|
|| ||Brown AJ, Precious HM, Whitcomb J, Simon V, Daar ES, D'Aquila R, Keiser P, Connick E, Hellmann N, Petropoulos C, Markowitz M, Richman D, Little SJ. Reduced susceptibility of HIV-1 to protease inhibitors from patients with primary HIV infection by three distinct routes [abstract 424]. In 8th Conference on Retroviruses and Opportunistic Infections. Chicago, Il.;2001.|
|| || Maguire M, Shortino D, Klein A, Harris W, Manohitharajah V, Tisdale M, Elston R, Yeo J, Randall S, Xu F, Parker H, May J, Snowden W. Emergence of resistance to protease inhibitor amprenavir in human immunodeficiency virus type 1-infected patients: selection of four alternative viral protease genotypes and influence of viral susceptibility to coadministered reverse transcriptase nucleoside inhibitors. Antimicrob Agents Chemother 2002;46(3):731-8.|
|| || Fontaine E, Riva C, Peeters M, Schmit JC, Delaporte E, Van Laethem K, Van Vaerenbergh K, Snoeck J, Van Wijngaerden E, De Clercq E, Van Ranst M, Vandamme AM. Evaluation of two commercial kits for the detection of genotypic drug resistance on a panel of HIV type 1 subtypes A through J. J Acquir Immune Defic Syndr 2001;28(3):254-8.|
|| || Hertogs K, de Bethune MP, Miller V, Ivens T, Schel P, Van Cauwenberge A, Van Den Eynde C, Van Gerwen V, Azijn H, Van Houtte M, Peeters F, Staszewski S, Conant M, Bloor S, Kemp S, Larder B, Pauwels R. A rapid method for simultaneous detection of phenotypic resistance to inhibitors of protease and reverse transcriptase in recombinant human immunodeficiency virus type 1 isolates from patients treated with antiretroviral drugs. Antimicrob.Agents Chemother. 1998;42:269-76.|
|| || Petropoulos CJ, Parkin NT, Limoli KL, Lie YS, Wrin T, Huang W, Tian H, Smith D, Winslow GA, Capon DJ, Whitcomb JM. A novel phenotypic drug susceptibility assay for human immunodeficiency virus type 1. Antimicrob Agents Chemother 2000;44(4):920-8.|
|| || Kaufmann GR, Suzuki K, Cunningham P, Mukaide M, Kondo M, Imai M, Zaunders J, Cooper DA. Impact of HIV type 1 protease, reverse transcriptase, cleavage site, and p6 mutations on the virological response to quadruple therapy with saquinavir, ritonavir, and two nucleoside analogs. AIDS Res Hum Retroviruses 2001;17(6):487-97.|
|| ||Kempf D, King M, Brun S, Sylte J, Marsh T, Stewart K, Sun E. Association of the nucleocapsid-p1 (NC-p1) Gag cleavage site alanine to valine mutation with mutational patterns in HIV protease producing protease inhibitor resistance. Antivir Ther 2001;6(Supplement 1):46.|
|| ||Maguire M, MacManus S, Griffin P, Guinea C, Harris W, Richard N, Wolfram J, Tisdale M, Snowden W, Klein J-P. Interaction of HIV-1 protease and gag gene mutations in response to amprenavir-selective pressure exerted in amprenavir-treated subjects - contribution of gag p6 changes L449F and P453L. Antivir Ther 2001;6(Supplement 1):48.|
|| ||Shafer RW, Gonzales MJ, Brun-Vezinet F. Online comparison of HIV-1 drug resistance algorithms identifies rates and causes of discordant interpretations. Antivir Ther 2001;6(Supplement 1):101.|
|| ||Verbiest W, Peeters M, Hertogs K, Schel P, Bloor S, Rinehart A, Graham N, Cohen C, Larder BA. VirtualPhenotype is concordant with actual phenotype: a retrospective analysis of data from VIRA3001 [abstract 81]. Antivir Ther 2000;5 (Suppl 3):62.|
|| ||Hellmann N, Johnson P, Petropoulos C. Validation of the performance characteristics of a novel, rapid phenotypic drug susceptibility assay, PhenoSense HIV [abastract 51]. Antivir Ther 1999;4(Suppl. 1):34-5.|
|| ||Vingerhoets J, Bloor S, Michiels L, Scholliers A, Dehertogh P, Van Den Eynde C, De Vroey V, Van Cauwenberge A, de Koning I, Alcorn T, Larder B, Hertogs K. The accuracy and reproducibility of high througput genotypic and phenotypic HIV-1 resistance testing under EN45001 and CLIA accreditation labels [abstract 77]. Antivir Ther 1999;4 (Suppl. 1):53.|
|| ||Lanier RE, Danehower S, Daluge SM, Cutrell A, Tisdale M, Pearce G, Spreen B, Lafon S, Kemp S, Bloor S, Larder BA. Genotypic and phenotypic correlates of response to abacavir (ABC, 1592). In 2nd International Workshop on HIV Drug Resistance, Treatment, and Eradication, June 24-27. Lake Maggiore, Italy;1998. Abstract 52 p.|
|| ||Shulman NS, Winters MA, Shafer RW, Zolopa AR, Liou SH, Hughes M, Whitcomb J, Petropoulos C, Hellmann N, Katzenstein D. Subtle changes in susceptibility to stavudine predict virological response to stavudine monotherapy after zidovudine treatment. Antivir Ther 2001;6(Supplement 1):91.|
|| ||Kempf D, Brun S, Rode R, Isaacson J, King M, Xu Y, Real K, Hsu A, Granneman R, Lie Y, Hellmann N, Bernstein B, Sun E. Identification of clinically relevant phenotypic and genotypic breakpoints for ABT-378/r in multiple PI-experienced, NNRTI-naive patients [abstract 89]. Antivir Ther 2000;5(Supplement 3):70-1.|
|| || Descamps D, Flandre P, Calvez V, Peytavin G, Meiffredy V, Collin G, Delaugerre C, Robert-Delmas S, Bazin B, Aboulker JP, Pialoux G, Raffi F, Brun-Vezinet F. Mechanisms of virologic failure in previously untreated HIV-infected patients from a trial of induction-maintenance therapy. Trilege (Agence Nationale de Recherches sur le SIDA 072) Study Team. JAMA 2000;283(2):205-11.|
|| || Gallego O, de Mendoza C, Perez-Elias MJ, Guardiola JM, Pedreira J, Dalmau D, Gonzalez J, Moreno A, Arribas JR, Rubio A, Garcia-Arata I, Leal M, Domingo P, Soriano V. Drug resistance in patients experiencing early virological failure under a triple combination including indinavir. AIDS 2001;15(13):1701-6.|
|| || Murphy RL, Gulick RM, DeGruttola V, D'Aquila RT, Eron JJ, Sommadossi JP, Currier JS, Smeaton L, Frank I, Caliendo AM, Gerber JG, Tung R, Kuritzkes DR. Treatment with amprenavir alone or amprenavir with zidovudine and lamivudine in adults with human immunodeficiency virus infection. AIDS Clinical Trials Group 347 Study Team. J Infect Dis 1999;179(4):808-16.|
|| || Mouroux M, Yvon-Groussin A, Peytavin G, Delaugerre C, Legrand M, Bossi P, Do B, Trylesinski A, Diquet B, Dohin E, Delfraissy JF, Katlama C, Calvez V. Early virological failure in naive human immunodeficiency virus patients receiving saquinavir (soft gel capsule)-stavudine-zalcitabine (MIKADO trial) is not associated with mutations conferring viral resistance. J Clin Microbiol 2000;38(7):2726-30.|
|| || Deeks SG, Wrin T, Liegler T, Hoh R, Hayden M, Barbour JD, Hellmann NS, Petropoulos CJ, McCune JM, Hellerstein MK, Grant RM. Virologic and immunologic consequences of discontinuing combination antiretroviral-drug therapy in HIV-infected patients with detectable viremia. N Engl J Med 2001;344(7):472-80.|
|| || Finzi D, Blankson J, Siliciano JD, Margolick JB, Chadwick K, Pierson T, Smith K, Lisziewicz J, Lori F, Flexner C, Quinn TC, Chaisson RE, Rosenberg E, Walker B, Gange S, Gallant J, Siliciano RF. Latent infection of CD4+ T cells provides a mechanism for lifelong persistence of HIV-1, even in patients on effective combination therapy. Nat Med 1999;5(5):512-7.|
|| || Martinez-Picado J, DePasquale MP, Kartsonis N, Hanna GJ, Wong J, Finzi D, Rosenberg E, Gunthard HF, Sutton L, Savara A, Petropoulos CJ, Hellmann N, Walker BD, Richman DD, Siliciano R, D'Aquila RT. Antiretroviral resistance during successful therapy of HIV type 1 infection. Proc Natl Acad Sci U S A 2000;97(20):10948-53.|
|| || Imamichi H, Crandall KA, Natarajan V, Jiang MK, Dewar RL, Berg S, Gaddam A, Bosche M, Metcalf JA, Davey Jr RT, Lane HC. Human immunodeficiency virus type 1 quasi species that rebound after discontinuation of highly active antiretroviral therapy are similar to the viral quasi species present before initiation of therapy. J Infect Dis 2001;183(1):36-50.|


The Bible tells us that the poor will always be with us, and as long as they are around there will always be politicians willing to punish them for their poverty. It might be the Wisconsin State Assembly, which has just approved legislation banning food stamp recipients from eating junk food and also ‘luxury foods’ such as ‘crab, lobster, shrimp, or any other shellfish.’ Or Hackney Council, which has just launched a ‘Public Space Protection Order’ (PSPO), which will enable police to impose £100 on-the-spot fines for homeless people sleeping in doorways – a fine that can rise to £1,000 in court.
Hackney Council’s punitive response to homelessness is not original. In America 31 cities have introduced restrictions preventing individuals and organizations from sharing food to the homeless. Others have made it illegal for homeless people to possess personal items in public, or banned the homeless from city centers. In 2013 the Budapest city council made it illegal for homeless people to occupy parks, underpasses, playgrounds and other public spaces, and threatened violators with fines and imprisonment.
State-sanctioned attacks on the homelessness are one component of a broad swathe of punitive and persecutory measures introduced by national and regional governments and municipalities in some of the richest countries in the world, that include cuts in benefits, means tests, sanctions, increasingly draconian conditions for state assistance, fines for begging, and forced unpaid work placements.
These persecutory tendencies are both new and old. In the Middle Ages vagrants or the ‘wandering poor’ were regarded as dangerous outsiders, and likely to be excluded from the parish or city states or arrested if they didn’t have a begging license or permission to be where they were. In sixteenth century Spain, vagrancy was a criminal offence in many cities. During the Great Depression, homeless ‘bums’ who rode the railroads in the United States in search of work were hounded and beaten by police.
Even when the poor received charity or relief they are often regarded with suspicion and contempt. In 1834 the Poor Law Commission recommended that no able-bodied person was to receive money or any other help from the Poor Law authorities except in a workhouse, that workhouses were to be built in every parish, where ‘conditions were to be made very harsh to discourage people from wanting to receive help.’
You don’t have to be a sociologist or historian to find explanations for such behavior: societies that accept an unequal distribution of wealth as the natural order of things do not like to be reminded of the negative consequences of such acceptance. Hungry people, beggars, and vagrants are persistent evidence of social failure and often gross injustice. Rather than analyse or seek to change the social and economic structures that create poverty, it is far more convenient to blame the poor themselves for their condition. For this reason the poor are regarded again and again in different historical epochs as feckless, idle, and unwilling to improve their situation.
At best they are regarded as a burden that the rest of society must carry – if only out of self-interest. At worst the poor are depicted as parasites, criminals, as a potentially destabilising force or as an aesthetically unpleasing and deviant intrusion into our daily lives. Such hostility can often be more visceral in societies characterized by brazen injustice and inequality.
In Renaissance Spain, much of the population lived close to starvation, while a small elite of aristocrats who paid no taxes engaged in conspicuous and ostentatious consumption. In the early 21st century we have witnessed a massive transfer of wealth to the rich and the super-rich in the UK and the United States and many other countries, that has accelerated during the financial crisis.
In these circumstances, the punitive and vindictive response to 21st century poverty is not surprising. It will always make the wealthy feel better about their own wealth if they can blame the homeless and the jobless for their predicament, and politicians whose political futures are dependent on the wealthy and the powerful are only too willing to engage in the same stigmatisation. Unfortunately too many people who are not rich prefer to kick down – particularly when they are bombarded on almost daily basis with fake rhetoric about ‘hard-working families’ that presents the ‘taxpayer’ as a victim of the parasitic ‘welfare scrounger’.
After all, poverty is discomforting and disturbing and even upsetting, particularly when you see it with your own eyes, and who wants to be made to feel uncomfortable when you’re just going about your business? The homeless are the most visible expression of a phenomenon that would otherwise remain hidden and unseen, and which many people would prefer remained unseen. This is the context in which Hackney – a Labour council – has included homelessness amongst its attempts to eliminate ‘persistent antisocial behavior’.
Some might think that punishing homeless people for being homeless itself constitutes antisocial behavior, and that such a response is yet another turn of the screw and another manifestation of institutionalized callousness and inhumanity. But maybe I’m wrong. After all, it’s extremely unlikely people sleeping in the street are going to be able to pay these fines, so they may end up going to jail for non-payment.
So maybe Hackney Council is not really trying to criminalise homelessness – it’s merely trying to house the homeless by a more circuitous route that won’t cause the same political offense that might occur if ‘the taxpayer’ provided them with social housing.
Maybe, but I really doubt it.


Teaching a Nation’s Leaders
Considered by many to have been the foremost educator in the South, Moses Waddell was of Irish parentage and was born in Rowan (now Iredell) county, North Carolina, on July 29th, 1770. He received his academic education at a school which was opened in the neighborhood under the name of Clio’s Nursery. For four years, beginning at the age of fourteen, he was engaged as a teacher (1784-1788) at various places in North Carolina and Georgia.
Leaving his employment as a teacher, he enrolled as a student at the Hampden-Sydney College, graduating there in 1791. The next year he was licensed to preach by the Hanover Presbytery, of Virginia, on May 12, 1792.
About 1793, Waddell opened his first school in Columbia county, Georgia, then another in 1801, in Vienna, Abbeville District, South Carolina. He remained in that work until 1804, when he removed to Willington, six miles south of Vienna, and it was at Willington where he founded the famous Willington Academy. It was common for Presbyterian pastors to maintain an academy, in part for the extra income, and in part because they could thus guide the moral, religious and intellectual education of the children of their parish.
All of these schools were designed as preparatory schools, utilizing a classical education model. As the fame of the Willington Academy grew, it came to be called the “Eton in the woods”. To give one example of the school’s rigor, students were required to memorize, translate and recite some 250 lines of Greek or Latin every night. A Willington graduate, South Carolina governor George McDuffie, held the record, having once recited over 2200 lines of the poet Horace.
In 1818, Waddell was elected President of what was then Franklin College, later to become the University of Georgia. However, he did not actually step into the duties of this office until May, 1819. While serving as an educator, he also labored as a pastor, founding the Presbyterian Church in Athens, Georgia in 1820. During his tenure at the University, the school prospered greatly, and he continued here as President until 1829. Resigning his post, he returned to Willington. For forty-five years he had labored as a teacher. His labors as a pastor continued another six or seven years more, and the Rev. Dr. Moses Waddell’s life drew to a close on July 21, 1840.
Dr. James McLeod provides the following account of Dr. Waddell as a teacher:
“The boys called him ‘Old Moses,’ and while he believed in corporal punishment, he never spanked in a passion, and it finally evolved that he did this only upon a verdict of a peer jury of students. He never spanked for a deficient lesson but chiefly for defects in morals or actions that had to be punished.
“He was a cheerful man even playful in his disposition. He maintained a personal interest in each boy. He had a wry sense of humor. When boys on second floor dumped water on him as he went in a door, he said nothing, but later raised an umbrella as he went in the door to the delight of the boys.
“His strength seems to have been to analyze the boys accurately, then demanded accordingly. He was not a man who used sentiment to escape facing the laziness of adolescence. He demanded. They groaned, they gave, they griped, they worshiped him later. There was a chestnut tree outside the Doctor’s study window that the boys remembered watching as they waited to see the Doctor if they had done anything wrong. Others would climb it to see if anyone was punished by him.
“Dr. Smith, the president of Princeton College, was quoted as saying that he received no students from any school in the United States who stood better examinations than those of Dr. Waddel.”
As a pastor, Alfred Nevin notes that “he was pious, zealous, and well versed in theology generally. His style of preaching was plain, simple, earnest. He addressed himself much more to the understanding than to the imagination or passions. As a teacher he stands almost unrivaled.”
Words to Live By:
In The Great Doctor Waddell, by Dr. James McLeod, the author provides a compilation of the students educated under Waddell. The list includes two Vice-Presidents, three Secretaries of State, three Secretaries of War, one Assistant Secretary of War, one US Attorney-general, Ministers to France, Spain and Russia, one US Supreme Court Justice, eleven governors, seven US Senators, thirty two members of the US House of Representatives, twenty two judges, eight college presidents, seventeen editors of newspapers or authors, five members of the Confederate Congress, two bishops, three Brigadier-generals, and one authentic Christian martyr.
In light of which, this might be a good time to review again the words of Dr. R. B. Kuiper, posted here this past July 15th:
“God has seen fit to reveal Himself to man in two books—the Bible, the book of special revelation, and nature and history, the book of general revelation. Now it is the duty of the organized Church to teach men the content of the former of these books, while it is the special task of the school to open the latter. To be sure, the two may not be separated. Truth can hardly be dealt with so mechanically. After all, truth is one because God is one. Truth is organic. And only he who has learned to understand the Bible can really know history and nature. Yet the distinction is a valid one. The Church can hardly be expected to teach the intricacies of mathematics, physics, astronomy, or the history of the Balkans. Nor does any one demand of the school that it preach the gospel. But Church and school together must declare the whole of God’s revealed truth.”


Many math lovers and geeks alike celebrate “Pi Day” on March 14. In fact, when using the American style for dates where the month precedes the day, today is 3/14. The most committed among us will even go so far as to keep an eye on their watch or set an alarm to go off at the Pi Minute, celebrated at 1:59 p.m., or even Pi Second at 1:59:26 p.m.
This tradition started in the late 80s and is now celebrated all over the world, particularly in North America, where parties and free pies are available on many campuses.
This year Pi Day is an ever bigger deal, because it’s no longer just a fun celebration of mathematics observed by a few incorrigible geeks. The US Congress approved the H.RES.224, sponsored by Rep. Bart Gordon and 15 cosponsors, titled “Supporting the designation of Pi Day, and for other purposes.”. Thanks to this, March 14, 2009 is now officially National Pi Day. More importantly the resolution includes the following statement:
Whereas Pi can be approximated as 3.14, and thus March 14, 2009, is an appropriate day for `National Pi Day’: Now, therefore, be it
Resolved, That the House of Representatives–
(1) supports the designation of a `Pi Day’ and its celebration around the world;
(2) recognizes the continuing importance of National Science Foundation’s math and science education programs; and
(3) encourages schools and educators to observe the day with appropriate activities that teach students about Pi and engage them about the study of mathematics.
Math-Blog applauds the sponsors of this resolution, which passed with 391 Yeas and 10 Nays. For once, both parties supported the initiative, and there is no doubt that the sponsors of this resolution will receive a great deal of thank you notes for acknowledging, albeit just symbolically, the importance of mathematics and science in our society. Sadly, 10 representatives felt the need to oppose this acknowledgment, and for those who are curious (without getting too political here) all 10 of them happen to be Republican.
There is now an official Pi Day website with cool merchandise, and a Facebook group you can join. To help you enjoy this day perhaps consider picking up a good book on the history of this fascinating transcendental number. While categorically rejecting any numerological implication regarding Pi Day, it’s a good occasion to celebrate mathematics and talk about it with those who otherwise usually wouldn’t be interested. And that could be the most important aspect to come out of the formal recognition of Pi Day.
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U.S. Study Finds Climate Change Impacts Significant
Climate change is fueling forest fires, creating water scarcity, harming animal habitats, and causing other significant changes throughout the United States that will only worsen as global temperatures increase, concludes a new federal government assessment of current and future climate change impacts.
The analysis, prepared by the U.S. Climate Change Science Program (CCSP), an inter-agency structure that coordinates climate research efforts across the federal government, reinforces the need for Congress to take action on climate change, according to officials at the World Wildlife Fund (WWF).
Richard Moss, Ph.D., WWF’s vice president and managing director for climate change, who led the CCSP coordination office during the Clinton and Bush administrations, said the impacts detailed in the report should serve as a wake-up call for policymakers.
“The findings of this report highlight the urgency of the climate change problem, the need to slow its growth by reducing greenhouse gas emissions, and the importance of preparing for its impacts, which ultimately will worsen with each additional political cycle of inaction,” Moss said. “The U.S. Senate has an opportunity to demonstrate leadership … when it votes on the Lieberman-Warner bill. As this assessment shows, squandering that opportunity is not an option we can afford.”
The report, “The Effects of Climate Change on Agriculture, Land Resources, Water Resources, and Biodiversity,” details significant impacts of climate change already being observed in every region of the country:
"The number and frequency of forest fires and insect outbreaks are increasing in the interior West, the Southwest, and Alaska. Precipitation, streamflow, and stream temperatures are increasing in most of the continental United States. The western United States is experiencing reduced snowpack and earlier peaks in spring runoff. The growth of many crops and weeds is being stimulated. Migration of plant and animal species is changing the composition and structure of arid, polar, aquatic, coastal, and other ecosystems."
The report also details the impacts of climate change on wildlife, noting that of 1,598 animal species examined in more than 800 studies, nearly 60 percent were found to have been affected by climate change.
“Climate change is one of the greatest long-term threats to wildlife and threatens to unravel decades of conservation efforts,” said Thomas Dillon, WWF’s senior vice president for field programs.
CCSP is expected to issue a second, broader report “Scientific Assessment of the Effects of Global Change on the United States," which will detail additional impacts on human health, transportation, energy supplies and use, and communities.
To read the report, visit www.climatescience.gov/Library/sap/sap4-3/default.php.


Damodar is a small river tributary to the Hugli River. Due to its devastating floods the river is called 'sorrow of Bengal'. The project has been planned on the pattern of Tennessee Valley Authority (TVA) of the United States and is managed through Damodar Valley Corporation (DVC).
The project involves compact, unified multipurpose river basin development programmes, viz., controlling floods, promoting irrigation, improving water supply and drainage,
Generating electric power, facilitating navigation, encouraging afforestation, soil conservation and promoting the growth of agriculture, industry, and tourism in the area.
The project has to be completed in two phases. The first phase programme comprised: (a) four dams at Tilaiya, Konar, Maithon and Panchet Hill with hydel power stations attached to each of them
(except at Konar) with a total capacity of 104 mw, (b) three thermal power stations at Bokaro, Durgapur and Chandrapura with a total capacity of 9,57,000 km, (c) a grid covering 1,280 km of transmission lines, and (d) a barrage at Durgapur with about 2,500 km of irrigation-cum navigation channels. The second phase included construction of four dams at Balphari, Bokaro, Aiyar and Bermo with hydroelectric stations and corresponding extensions to transmission system. Some subsidiary activities like a forestation, soil conservation, and malaria eradication and fisheries development were also to be taken up.
The work under the first phase has been completed. The work under the second phase has been started. The work is continuing on the construction of a thermal station at Mazia (Bankura district) with installed capacity of 3 x 210 mw and a gas turbine station at Maithon with installed capacity of 3 x 30 mw. The progress of the project up-till now has been as follows:
Tilaiya Dam-the construction of this dam has been completed in 1953. It is an all-concrete dam across the Barakar River, about 208 km upstream from its confluence with the Damodar. It is 33 m high and 366 m long with gross storage capacity of 394.7 m cu. m. It will provide irrigation to about 7,500 hectares of agricultural land. The underground power station with installed capacity of 60,000 km will provide cheap power to the mica mines in Kodarma a Hazaribagh.
Konar Dam-this dam was completed in 1955. The dam has been built across the Konar River in Hazaribag district, about 24 km east of the confluence of the Konar with the Damodar. It is an earthen dam with concrete spillway 49 km high and 3,546 m long. It has gross storage capacity of 337 m cu m. which will irrigate about 16,200 hectares of agricultural land. It will also provide cooling water to Bokaro thermal power station. The hydel station located near the dam generates about 40,000 km of electricity.
Maithon Dam-this dam was completed in 1958. It is an earthen dam built across the Barakar River at some distance upstream from the confluence of the Barakar with the Damodar River. It is 56 m high and 4,357 m long with storage capacity of 1,357 m cu m. It provides irrigation to about 50,000 hectares of agricultural land. Besides, the underground power station near the dam generates 60,000 kw of electricity.
Panchet Hill Dam-it has been completed in 1959. It has been built across the Damodar River about 20 km south of the Maithon dam. It is 45 m high and 2,545 m long with gross storage capacity of 1,497 m cu m. It irrigates about 2.75 lakh hectares of agricultural land. The power station near the dam has installed capacity of 40,000 kw of electricity.
This barrage was completed in 1955. It is a concrete dam 12 m high and 672 m long. It receives water from the storage dams and distributes the same through two canals (2,495 km long) which irrigate about 4.75 lakh hectares of area in Barddhaman, Hugli and Haora districts. The right bank main canal is 89 km long and the left bank main canal 137 km long. The latter is constructed mainly for navigation to the Hugli River.
Bokaro Thermal Station-it has installed capacity of 140 mw. An additional unit of 75 mw has since been added to this station. The power station is supplying electricity to the adjoining areas of Bihar and West Bengal.
Durgapur Thermal Power Station-it has one unit of 140 mw and two units of 75 mw. The power station is located near Oyaria railway station. The electricity is used in the adjoining parts of West Bengal and Bihar.
Chandrapura Thermal Power Station-the power station lies 306 km away from Kolkata. It has three units of 140 mw each and two units of 120 mw each.
The Damodar Valley Project has total installed capacity of generating 1,181 mw of electricity and providing irrigation facilities to 5.15 lakh hectares of agricultural area. When fully commissioned it will help in allround development of24,235 square km of adjoining area covering industrial belt of Bihar and West Bengal and rural areas of Patna, Gaya, Shahabad, Singhbhum (Bihar) and Medinipur (West Bengal), districts. The D.V.C. accounts for nearly 10 per cent of the country's total power output. The major industries located in Jamshedpur, Durgapur, Burnpur and Kulti and the coal mines of Jharia and Raniganj operate with D.V.C. power. A portion of electricity is also utilised in operating trains on the Eastern Railway.
Other benefits of the project include : (a) erosion-resisting structures like Adivasi, Deochanda, Bachhi and Gauria Karma dams built in the catchment area to hold back rain water and prevent gully erosion, (b) scientific regeneration of denuded forests, (c) large-scale plantation of timber, mulberry and other fast growing trees, (d) promotion of culture, (e) development of aquacukure in the reservoirs of the D.V.C. (cukurable area being 20,235 ha; production potential being 800 tones), (0 improvement in the mining of coal, iron ore, bauxite, limestone, mica, etc. (g) development of paper metallurgical, engineering, aluminium, cement and other industries, and (h) promotion of tourism, horticulture and dairying.


Olaudah Equiano was the first political activist within Britain’s African community during the 18th century. According to his famous autobiography, he was kidnapped at the age of 11 and enslaved.
The ill-treatment he received and the suffering of Africans inspired Equiano to join the campaign to abolish the so called ‘transatlantic slave trade’.
Latest News & Events
The Equiano Society
The Equiano Society was founded by Arthur Torrington and Samuel B. King in London in November 1996. Its main objective is too publicise and celebrate the life and work of Olaudah Equiano.
Equiano an exhibition of an extraordinary life
Gas Hall, Birmingham Museum & Art Gallery
The Equiano Project
In December 2006, Birmingham Museums and Art Gallery (BM&AG) and The Equiano Society received a significant grant from the Heritage Lottery Fund. With support from Birmingham City Council and Renaissance in the Regions (Museums, Libraries and Archives Council), the partnership delivered a major project that celebrated the life of Olaudah Equiano.


In the melting of commercial non-ferrous metals and alloys (aluminum, magnesium, copper, zinc, and lead), various auxiliary molten metal processing steps are necessary other than melting and alloying.
Generally, the historic practices of fluxing, metal refining, deoxidation, degassing, and grain refining have been used, and they apply to virtually all nonferrous metal systems. In addition, molten metal pumping and filtration are two somewhat newer but now commonly practiced technologies in nonferrous molten metal processing. This article will describe the molten metal processing methodologies currently used in conventional nonferrous molten metal operations. These process methodologies pertain not only to foundry melting and casting but also to smelting, refining, and in certain cases mill product operations.
In the melting of commercial non-ferrous metals and alloys (aluminum, magnesium,
copper, zinc, and lead), various auxiliary molten metal processing steps are necessary
other than melting and alloying.
Generally, the historic practices of fluxing, metal refining, deoxidation, degassing,
and grain refining have been used, and they apply to virtually all nonferrous metal
systems. In addition, molten metal pumping and filtration are two somewhat newer but
now commonly practiced technologies in nonferrous molten metal processing. This article
will describe the molten metal processing methodologies currently used in conventional
nonferrous molten metal operations. These process methodologies pertain not only to
foundry melting and casting but also to smelting, refining, and in certain cases mill
The term fluxing is used in this article to represent all additives to, and treatments
of, molten metal in which chemical compounds or mixtures of such compounds are
employed. These compounds are usually inorganic. In some cases, metallic salts are
used in powder, granulated, or solid tablet form and may often melt to form a liquid
when used. They can be added manually or can be automatically injected, and they can
perform single or, in combination, various functions, including degassing, cleaning,
alloying, oxidation, deoxidation, or refining.
The term fluxing also includes the treatment of nonferrous melts by inert or reactive
gases to remove solid or gaseous impurities. Fluxes are commonly used to some extent
with virtually all nonferrous molten metal operations in both the foundry and in the
production of mill products.
Fluxing of Aluminum Alloys
In aluminum melting, and especially in the remelting of foundry returns or other
scrap, oxide formation and nonmetallic impurities are common. Impurities appear in the
form of liquid and solid inclusions that persist through melt solidification into the
casting. Inclusions can originate from dirty tools, sand and other molding debris,
sludge, metalworking lubricant residues, and the oxidation of alloying elements and/or
the base metal.
The term fluxing, in the broadest sense, applies to a treatment technique to the melt
containing such impurities and inclusions as those mentioned above. Fluxing of the melt
facilitates the agglomeration and separation of such undesirable constituents from the
Some materials that are being used as fluxes for aluminum are listed below:
- Aluminum chloride, AlCl3
- Aluminum fluoride, AlF3
- Borax, Na2B4O7
- Calcium chloride, CaCl2
- Calcium fluoride, CaF2
- Carnalite, MgCl2KCl
- Zinc chloride, ZnCl2
- Cryolite, 3NaFAlF3
- Lithium chloride, LiCl
- Magnesium chloride, MgCl2
- Potassium chloride, KCl
Four principal types of fluxes are used for aluminum alloys. They are: cover fluxes,
cleaning fluxes, drossing fluxes, and refining fluxes. Wall-cleaning fluxes are also
employed, but these are usually sprayed onto furnace walls rather than added to the
Cover fluxes are designed to be used primarily with smaller (pot,
crucible) furnaces to provide a physical barrier to oxidation of the melt or to serve
as a cleanser for alloys, scrap foundry returns, or fresh ingot being charged.
Cleaning fluxes are usually higher in chloride salt compound content
and usually contain fluorides to facilitate wetting of the oxide inclusions for easier
separation from the melt.
Drossing fluxes are designed to promote separation of the aluminum oxide
(Al2O3) dross layer that forms on the surface of the melt
from the molten metal. Drosses and liquid or solid metal are usually intermingled
in the dross layer. The drossing fluxes are designed to react with Al2O3 in the slag
or dross layer and to recover metal. The fluorides wet and dissolve thin oxide
films according to the general reaction.
6Na2SiF6 + 2Al2O3 → 4Na3AlF6 + 3SiO2 + 3SiF4
With sufficient mechanical agitation through rabbling with a rake, these films will
be broken long enough to release entrapped metal. Drossing fluxes are used to great
advantage in the aluminum industry to reduce the rich metallic content of the dross.
Untreated dross may contain 60 to 85% free metal, which, if allowed to burn or thermite,
will convert to unrecoverable Al2O3.
Wall-cleaning fluxes contain compounds that help soften the oxide
buildup that occurs on furnace walls. These fluxes can often be applied with a typical
refractory gunning device.
Fluxing of Magnesium Alloys
Magnesium and its alloys are exceptionally susceptible to oxidation, melt loss, and
fires because of the extreme reactivity between magnesium and oxygen. Protection is
therefore always required when melting this alloy family. Historically, this has
involved the so-called flux process, which uses salt fluxes as a cover, or more recently
the fluxless process, which uses inert gas.
Molten magnesium oxidizes readily to form a magnesium oxide (MgO) film. This film is
easily disturbed, and discontinuous MgO liquid film inclusions readily wet and coat
solid charge materials and can also entrain liquid metal. Fluxes are therefore used
to protect the melt from oxidation, to agglomerate nonmetallic inclusions originating
with the charge, and to break up and collect the oxide inclusions and skins that may
form during melting. These fluxes are usually low-melting mixtures of halide salts
capable of wetting both solid and liquid metal surfaces.
A typical flux composition includes approximately 49% MgCl2, 27% KCl, 20%
BaCl2, and 4% CaF2. The magnesium and potassium chloride salts
provide the low-melting eutectic; the fluoride, the surface wettability and chemical
reactivity with magnesium oxide; and the heavy barium chloride salt constituent, the
density component to effect mixing and sludging capability for separation. Other
useful cover fluxes include a simple mixture of sulfurous compounds with fluoborate
salts or boric acid.
All flux materials should be kept clean and dry and should be stored in their original
containers. All tools used with fluxes should be clean, dry, and preheated to drive off
any surface moisture and to minimize thermal shock when placed into the melt.


Mice are social creatures -- in fact, living alone can stress out a mouse. And while they typically rely on the company of other mice, mice can also be quite social when it comes to people. Naturally, a mouse may be a little hesitant to be handled by a creature such as yourself. After all, you're quite large compared to him. But if you focus on developing a relationship gradually and with positivity and patience, soon your little rodent will love spending time cupped in the palm of your hand.
Spend time with your mouse while he's inside his cage. Make yourself somewhat conspicuous by talking to him regularly, especially when doing positive things like feeding him or giving him fresh water. He will associate you with good things, and learn that you aren't a threat.
Hand-feed him treats. Put a treat in the palm of your hand and gently lower your hand into the cage. Wait for him to approach your hand and take the treat -- doing this every time you give him a treat teaches him that the approach of your hand is a good thing, and that it is safe to interact with it.
Scoop him up in the palm of your hand once he is used to being around it. If he resists, gently grab him by the base of his tail and drop him into your palm -- make that hand off quickly, though, because while being held in such a manner isn't painful, he won't necessarily thank you for it.
Hold him in your hands for a few minutes, speaking gently to him before returning him to his cage. Do this everyday for gradually longer periods of time. This builds his confidence and doesn't overwhelm him.
- Give your mouse at least one other mouse to live and play with. Mice are social creatures, and one from the pet store is undoubtedly used to living with plenty of company. If you remove him from that environment and isolate him, he'll be stressed and unhappy. If you want multiple male mice to live together, they should be adopted at the same time -- unfamiliar adult males are prone to fighting. Female mice also do well living in groups, so try not to mix sexes. Otherwise, you'll shortly have a lot more mice than you planned on.
- Do not dangle your mouse by his tail, and especially do not pick him up by the tail from anywhere but the base. Excess dangling is stressful, and picking him up from the middle or tip of his tail is painful and dangerous.
Little mouse image by Multiart from Fotolia.com
Tom Ryan is a freelance writer, editor and English tutor. He graduated from the University of Pittsburgh with a degree in English writing, and has also worked as an arts and entertainment reporter with "The Pitt News" and a public relations and advertising copywriter with the Carnegie Library of Pittsburgh.


There are hundreds of thousands of bacteria and viruses, mostly found on common surfaces such as door handles or touch pads. However, many viruses are also passed through airborne or saliva while talking. If you are high risk or have a serious health condition, it is imperative that you are careful and take the necessary precautions when you leave your house.
Visit Protected Businesses
Due to Covid 19, many businesses have set up sneeze guards. When you go out, make sure to visit businesses that have sneeze guards set up. These guards provide protection from the spread of viruses and bacteria. In any business where you have to interact with others, a sneeze guard will give you peace of mind and virus protection.
Wear a Mask
Transmission of diseases such as Covid 19, influenza, and the common cold can all be significantly reduced if people wear masks. Surgical masks, such as N95 masks are the most protective, however, scientists say that any mask that covers your mouth and nose will work. As stated by an epidemiologist, wearing a mask is the most important thing you can do to protect your health and the health of those around you.
Use Hand Sanitizer
If you are out and about running errands, it can be hard to find a sink so you can wash your hands. For that reason, you need to keep hand sanitizer with you. By sanitizing your hands, you can help prevent the spread of bacteria and viruses to yourself or others. Make sure you sanitize both before and after you touch something or someone. By sanitizing before you touch, you are cleaning your hands of anything that may have been on them. This helps protect the person you are interacting with. Then, sanitize your hands again after the interaction as this will protect you from any bacteria or viruses on the item you were handling.
Keep Your Distance
Finally, an easy way to stay healthy if you have to leave the house is to maintain your distance from others. Many diseases and viruses are passed from one person to another though the air or through small saliva particles as we breathe and talk. By keeping your distance, you drastically reduce your risk of catching anything.
While it can be difficult to leave the house if you have a lowered immune system, there are simple things you can do to stay healthy. Just be aware of your surroundings and respond accordingly.


[Haskell-cafe] Proof in Haskell
patrick.browne at dit.ie
Tue Dec 21 12:53:36 CET 2010
In a previous posting I asked was there a way to achieve a proof of
mirror (mirror x) = x
in Haskell itself. The code for the tree/mirror is below:
module BTree where
data Tree a = Tip | Node (Tree a) a (Tree a)
mirror :: Tree a -> Tree a
mirror (Node x y z) = Node (mirror z) y (mirror x)
mirror Tip = Tip
The reply from Eugene Kirpichov was:
>> It is not possible at the value level, because Haskell does not
>> support dependent types and thus cannot express the type of the
>> "forall a . forall x:Tree a, mirror (mirror x) = x",
>> and therefore a proof term also cannot be constructed.
Could anyone explain what *dependent types* mean in this context?
What is the exact meaning of forall a and forall x?
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More information about the Haskell-Cafe


Can an innovative approach to teaching school students also transform how we train entrepreneurs and smallholder farmers around the world?
A number of years ago when my daughter was in elementary school and working at the kitchen table on her homework, some of her assigned homework was to watch math videos to learn the concepts that she and her classmates were studying.
The following day, she completed in-class exercises to practice using the math concepts – and if she needed help, the teacher was there to provide support.
This is exactly the opposite of the way most traditional classrooms operate, where the teacher teaches new content in class and then assigns homework to be completed outside of class.
The new approach my daughter’s school used for teaching math flipped around how students learn and practice new skills. Learn new concepts at home; practice skills in class with teacher support. Popularized by Khan Academy, this is known as the flipped classroom model.
The flipped classroom model allows students to learn content individually – watching and listening to lessons as many times as they need – and then apply their learning in a group setting, in class and with the teacher there to support them.
The flipped classroom model has a number of benefits: its flexibility is well-suited for busy students; the ability to repeat lessons (literally rewinding and reviewing material) helps with learning difficult new concepts; and it encourages better and more interaction with teachers.
Because of these strengths, this methodology is also a model for reimagining adult training and education in the post-COVID digital era.
Reimagining TechnoServe’s Training Paradigm
Prior to digitization – which was brought to many TechnoServe programs via the pandemic – many of TechnoServe’s training programs followed the traditional classroom model: beneficiaries were taught new content in a group setting – either in a classroom workshop or a group farm demonstration plot – and then returned to their businesses and farms to implement the new practices individually, often with advisory support.
As a result of the COVID-19 pandemic, the TechnoServe entrepreneurship practice moved to a remote and blended learning model and has effectively used the flipped classroom concept to digitize its entrepreneurship and micro-retail programs. Entrepreneurs study modules individually, and then apply learning to their businesses with support from their business advisor and their peer group.
Let’s look at an example of how a rural learner might benefit from the flipped classroom.
Meet Grocery Store Owner Roselia Alonso Muhun
Roselia runs a grocery store with her husband, Walter Anibal, in Patzicía, a rural town in Guatemala. There are just over 21,000 people in Patzicía, which is miles from the nearest city. “We live at the foot of the mountains, and that is what characterizes the place,” she says.
Roselia has been running her grocery store for several years, and she’s extremely dedicated. “I start at 6:00 a.m. and the first thing I do is thank God for the new day, then we start the business.”
“We alternate days between my husband and myself to take care of the business,” she continues. “We usually close at 10:00 p.m.”
But more time at the store wasn’t helping her business grow. She needed to be more efficient.
“I didn’t keep track of my expenses and costs. I had a business that worked but I didn’t have a technical basis for my investments,” Roselia admits.
A TechnoServe program was about to help her turn all that around – with the flipped classroom model at the center.
Roselia joined Impulsa Tu Empresa (ITE), a business accelerator program that aims to help small and growing businesses fast-track their growth, and started working with a business advisor, Miguel Angel.
But she couldn’t do the training in a classroom while running her grocery store, which always took priority.
Flipped Training Makes a Difference
Now Roselia uses business training videos through a learning platform created by the program in order to get up to speed. She watched videos about strategic decision making and analysis, among others. “I find that the distance learning styles with videos is good. Having videos allows me to see things several times.”
And the ITE program brings a gender lens to the training videos. “I watch [the videos] to see myself. I particularly liked the story about Linea Rosa, a taxi driver, because she was a working woman like me.”
Then, when she meets with Miguel Angel, her TechnoServe advisor, he can help answer specific questions and help her work through ideas, instead of having to teach her the material.
“I solve my doubts there,” she says of her meetings with Miguel. “I bring him ideas and…between the two of us, we improve them.”
Roselia is using the training to reduce her work debt, expand her store, and improve her customer service. All thanks to the flipped classroom model.


"What day of the week do you go running?"
I'm not sure of DL randomises the phrase pairings, but i dont feel that "几去" makes too much sense to be paired together, and should have instead been seperate options. It confused me, but I could be wrong.
Somebody explain, "几去" for me. Google translate says ，"Go a few". Or is 几 used the same as 几点 (what time?). 几去 means, "What go?"
"几" has to be grouped with the two characters in front of it, i.e. "星期", and not with the one after. So, it has to be read "星期几", which means "What day...?"/"Which day of the week...?"
The construction was explained in the lesson Time 1:


A doll that was originally designed for use in car […]
A doll that was originally designed for use in car crash tests is about to be used as a research tool by forensic scientists who are studying brain damage. The Department of Forensic Medicine at the University of Oslo is studying “shaken baby syndrome” and the relationship between shaking and brain damage. Scientists at SINTEF ICT have installed accelerometers inside the head of the doll in order to measure the forces caused by shaking and impacts with solid surfaces.
The measurements will be stored on a computer for subsequent analysis at SINTEF. In Norway around 15 babies are severely shaken by their parents every year, and about one third of them are left with serious long-term injuries. Court cases often have to be unresolved due to lack of sufficient evidence. The forensic medical scientists hope that this situation will soon change.


Clear Pond Fall and Winter Formula
FALL AND WINTER FORMULA
Prepare Your Pond For Winter
FALL AND WINTER FORMULA – Prepare Your Pond For Winter
Our Fall and Winter Formula is a blend of cold water aerobic and facultative anaerobes that are capable of working in both an oxygen and non-oxygen environment, and also in darkness under ice. These microbes have been isolated and trained to produce large amounts of digestive enzymes when put into pond water.
• Break down sludge thru the winter months to prepare for a healthier and cleaner Spring.
• Improve Oxygen content through the winter months.
• Effective in water temperatures under 55 degrees F.


Paramecium is a protozoa that is more developed than Amoeba. It reproduces both sexually and asexually. Asexual reproduction is by binary fission and sexual reproduction is by conjugation.
(a) Binary Fission in Paramecium
Sexual Reproduction in Paramecium
Sexual reproduction by conjugation takes in Paramecium in the following steps:
- Two individuals called conjugant come together and lie side by side.
- The meganucleus of each conjugants disintegrate.
- The micronucleus of each conjugants divides into two parts twice, forming four micronuclei. Three of the four micro – nuclei in each conjugant disintegrate. The remaining micronucleus again divides into two.
- One micronucleus from each conjugal migrates into the other conjugant. In other words, the conjugants exchange nuclear material.
- The migratory nucleus of one conjugant and the stationary nucleus of the other conjugant fuse in each individual to form a fusion nucleus in each ex-conjugal divides into four new individuals, each with a new
- The fusion nucleus in each ex-conjugant divides into four individuals each, with a new meganucleus and micronucleus.
Paramecium in conjugation: Stages:
EVALUATION (POST THE QUESTION TITLE AND YOUR ANSWER IN THE QUESTION BOX BELOW FOR EVALUATION AND DISCUSSION)
- Explain sexual reproduction in paramecium
- Describe asexual reproduction in paramecium with the aid of a suitable diagram
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Good handwriting is a gift and only a few people possess it. The Sumedha Educational Academy, an English medium school at Bethamcharla in Kurnool district, recently conducted an orientation session for its students in handwriting by engaging the Hyderabad-based expert in handwriting, Mr. Y. MallikarjunRao. Parents and the teachers of the school also attend the session.
Mr. MallikarjunRao, an MBA, has made grapho-therapy as his fulltime career. After his education, Mr. Rao become a teacher in a school and had an opportunity to examine the handwriting of hundreds of students. Mr.Rao, who was conscious about handwriting since his childhood, often pestered his students to mend writing. One of his colleagues suggested him to teach handwriting itself for which very few people were qualified. Soon he enrolled the institute abroad that offered courses in handwriting and its analysis. So far, he tutored 30,000 people. According to Mr. Rao, letter connections, zone technique, spacing, slant, baseline and position of some individual letters influenced the handwriting. There are 15 letters in English that spoiled the handwriting. Five letters required special attention while connecting to other letters. Cursive handwriting helps write fast.
Zone technique was another important factor that helped beautify handwriting. English alphabet has seven upper zone letters and 13 middle zone letters which need to be positioned properly. For example, b, d, l and k are upper zone letters, while g, p, q, and y are lower zone letters. Spacing between letters and words also contribute to good handwriting.