On the flip side, the agency may be looking for a silver lining in the data.  Since the drug worked well for some populations (ie those with mild disease), they may be looking more closely to confirm this result.  Possibly the agency will indeed approve the drug for limited use, as I speculated in my post.  Some additional speculation can be found here from Brian Orelli at the Motley Fool.

I thought it also worth highlighting a few interesting thoughts about the value of clinical evidence and how for various reasons, initial findings of benefit can change over time.  I was particularly inspired by Jonah Lehrer’s comments in the latest edition of the New Yorker.  This is not to say that I am a skeptic of scientific thinking; in fact the opposite is true, generally.  Rather it is just to point out the complexity and difficulty inherent in trying to prove whether a therapy actually works.  So in the interest of time I am just linking to a few interesting articles below.

• Jonah Lehrer on the difficulty of proving anything, especially with the “decline effect” phenomenon: http://www.newyorker.com/reporting/2010/12/13/101213fa_fact_lehrer
• Lehrer’s blog post adding color to the article and an excerpt: http://www.wired.com/wiredscience/2010/12/the-mysterious-decline-effect/
• Interestingly Lehrer doesn’t comment much on research about the trend of rising in placebo effect in some drug trials and whether this is a thread of the larger phenomenon Lehrer describes, or something different altogether.  Here is Steve Silberman’s article on the topic: http://www.wired.com/medtech/drugs/magazine/17-09/ff_placebo_effect?currentPage=all
• Ioannidis on why most research published is false: http://www.plosmedicine.org/article/info%3Adoi%2F10.1371%2Fjournal.pmed.0020124

For unknown reasons, in lupus the immune system attacks the body, leading to a chronic inflammatory disease.  The illness can vary greatly for each patient and between patients.  Flares can appear unexpectedly and with an unpredictable severity, several times a year.  Symptoms often are “general” (fever, weight loss and fatigue) and organ-specific, including joint pain, skin lesions, kidney injury, and heart injury among others.  In severe cases, a patient may die from kidney failure, severe infection, stroke or other causes.

A lupus diagnosis is no longer a death sentence.  Sixty years ago, the 5-years survival rate was 40%, whereas since the 1980s it has been over 90%.  This may have occurred as physicians learned to identify and treat the disease sooner or use medications “off label” to help patients.  While the only FDA-approved medications for lupus are aspirin, prednisone and the anti-malarial drug hydroxychloroquine, some patients also find benefit from immune suppression drugs like cyclophosphamide and other glucocorticoids related to prednisone.  Yet taking these medicines brings many side effects, often severe.  They can include increased risk of severe infection, body weight increase and redistribution, rashes, eye cataracts, and increased risk of major cardiovascular illness including heart attack and stroke.

Enter the hope for belimumab.  Its makers, Human Genome Sciences and GlaxoSmithKline, conducted two Phase 3 trials that each enrolled 800 to 900 patients, all of whom received standard of care medicines like prednisone.  In addition, the patients either received placebo or belimumab.  Trial researchers tracked patients’ symptoms to determine whether they responded to the drug.

Did the drug reduce lupus symptoms?  In one trial with patients who were from Asia and Latin America, 58% of patients receiving the target dose of belimumab responded compared to 44% in the placebo arm.  This difference reached statistical significance, meaning it was highly unlikely for these groups to have differed solely by chance.  But in the second trial, with patients in the US, Canada and Europe, the improvement was smaller at 12 months (43% response compared to 34% with placebo) and disappeared by 18 months.  In addition, patients of African-American or African heritage worsened with belimumab.  In the first trial, 46% of these patients responded compared to 64% on placebo.  In the second trial, it was 33% for belimumab versus 39% for placebo.

Was the drug safe?  Of 2,133 patients treated, death was 1.8 times more likely for patients on belimumab (0.9% of patients) than on placebo (0.4% of patients), and this was a statistically significant result.  71% of patients treated with the drug got an infection (of any severity) compared to 67% of those receiving placebo.  Serious infections were slightly more frequent with belimumab than placebo (6% of all patients in the Phase 2 and 3 trials, versus 5% for placebo).

Considering these findings, will the FDA approve the drug?  On the one hand, some industry analysts have written, approval would encourage other lupus drug makers.  Yet lowering the bar would only benefit short-term industry interests at the expense of patients.  Difficult problems require innovation and perseverance.  Perhaps suggesting it would not lower its standards, in a briefing to the FDA’s recent advisory panel, the agency raised several major criticisms of the belimumab results.

Yet surprisingly, the panel voted 13 to 2 in support of belimumab.  Meeting minutes have not yet been released.  Reuters quoted one panel member as saying, “The efficacy is mild, but I think there is a need for drug even with mild efficacy.”   The FDA is not required to follow the panel’s recommendation, though several journalists have indicated it usually does.  One additional variable is that the drug may cost $20,000 to $30,000 per year.  While this is expensive, it is not nearly as expensive as some drugs for cancer or other rare diseases (see list here), though it would rack up costs as patients took it chronically.

One compromise is the FDA could approve the drug with restrictions.  For instance, the drug might be permitted only for patients the drug helped, such as those of Caucasian heritage or with skin or musculoskeletal forms of the illness.  Or it may be restricted to 12 months or less of continuous therapy.  In a few weeks we will learn of the decision.  Hopefully it will be made in the interest of patients first.

Further reading:

1. Mayo Clinic overview of lupus
2. Mayo Clinic overview on of glucocorticoid drugs
3. UpToDate patient information on lupus
4. Article on lupus epidemiology
5. Articles on FDA panel backing Benlysta:
1. FierceBiotech 11/16/2010
2. LA Times 11/17/2010
3. New York Times 11/17/2010
6. FDA Arthritis Advisory Commitee 2010 meeting materials
7. FDA and the clinical trial process for drugs seeking approval
8. The clinical trials discussed above are BLISS-52 (HGS press release) and BLISS-76 (press release).

For unknown reasons, in lupus the immune system attacks the body, leading to a chronic inflammatory disease.  The illness can vary greatly for each patient and between patients.  Flares can appear unexpectedly and with an unpredictable severity, several times a year.  Symptoms often are “general” (fever, weight loss and fatigue) and organ-specific, including joint pain, skin lesions, kidney injury, and heart injury among others.  In severe cases, a patient may die from kidney failure, severe infection, stroke or other causes.

A lupus diagnosis is no longer a death sentence.  Sixty years ago, the 5-years survival rate was 40%, whereas since the 1980s it has been over 90%.  This may have occurred as physicians learned to identify and treat the disease sooner or use medications “off label” to help patients.  While the only FDA-approved medications for lupus are aspirin, prednisone and the anti-malarial drug hydroxychloroquine, some patients also find benefit from immune suppression drugs like cyclophosphamide and other glucocorticoids related to prednisone.  Yet taking these medicines brings many side effects, often severe.  They can include increased risk of severe infection, body weight increase and redistribution, rashes, eye cataracts, and increased risk of major cardiovascular illness including heart attack and stroke.

Enter the hope for belimumab.  Its makers, Human Genome Sciences and GlaxoSmithKline, conducted two Phase 3 trials that each enrolled 800 to 900 patients, all of whom received standard of care medicines like prednisone.  In addition, the patients either received placebo or belimumab.  Trial researchers tracked patients’ symptoms to determine whether they responded to the drug.

Did the drug reduce lupus symptoms?  In one trial with patients who were from Asia and Latin America, 58% of patients receiving the target dose of belimumab responded compared to 44% in the placebo arm.  This difference reached statistical significance, meaning it was highly unlikely for these groups to have differed solely by chance.  But in the second trial, with patients in the US, Canada and Europe, the improvement was smaller at 12 months (43% response compared to 34% with placebo) and disappeared by 18 months.  In addition, patients of African-American or African heritage worsened with belimumab.  In the first trial, 46% of these patients responded compared to 64% on placebo.  In the second trial, it was 33% for belimumab versus 39% for placebo.

Was the drug safe?  Of 2,133 patients treated, death was 1.8 times more likely for patients on belimumab (0.9% of patients) than on placebo (0.4% of patients), and this was a statistically significant result.  71% of patients treated with the drug got an infection (of any severity) compared to 67% of those receiving placebo.  Serious infections were slightly more frequent with belimumab than placebo (6% of all patients in the Phase 2 and 3 trials, versus 5% for placebo).

Considering these findings, will the FDA approve the drug?  On the one hand, some industry analysts have written, approval would encourage other lupus drug makers.  Yet lowering the bar would only benefit short-term industry interests at the expense of patients.  Difficult problems require innovation and perseverance.  Perhaps suggesting it would not lower its standards, in a briefing to the FDA’s recent advisory panel, the agency raised several major criticisms of the belimumab results.

Yet surprisingly, the panel voted 13 to 2 in support of belimumab.  Meeting minutes have not yet been released.  Reuters quoted one panel member as saying, “The efficacy is mild, but I think there is a need for drug even with mild efficacy.”   The FDA is not required to follow the panel’s recommendation, though several journalists have indicated it usually does.  One additional variable is that the drug may cost $20,000 to 30,000 per year.  While this is expensive, it is not nearly as expensive as some drugs for cancer or other rare diseases (see list here), though it would rack up costs as patients took it chronically.

One compromise is the FDA could approve the drug with restrictions.  For instance, the drug might be permitted only for patients the drug helped, such as those of Caucasian heritage or with skin or musculoskeletal forms of the illness.  Or it may be restricted to 12 months or less of continuous therapy.  In a few weeks we will learn of the decision.  Hopefully it will be made in the interest of patients first.

Further reading:

1. Mayo Clinic overview of lupus, medically known as systemic lupus erythematosus (SLE): http://www.mayoclinic.com/health/lupus/DS00115
2. Mayo Clinic overview on the pros and cons of glucocorticoid drugs: http://www.mayoclinic.com/health/steroids/HQ01431
3. UpToDate Patient information on lupus: http://www.uptodate.com/patients/content/topic.do?topicKey=~44azQ566S.O5Xa&selectedTitle=3~150&source=search_result
4. Article about epidemiology of lupus (for instance, how common the disease is by country): http://lup.sagepub.com/content/15/5/308
5. Wikipedia article about lupus and with prevalence estimates: http://en.wikipedia.org/wiki/Lupus#Epidemiology
6. Articles on FDA panel backing Benlysta:
   1. FierceBiotech: http://www.fiercebiotech.com/story/breaking-news-hgs-wins-crucial-panel-backing-benlysta/2010-11-16
   2. LA Times: http://articles.latimes.com/2010/nov/17/news/la-heb-lupus-drug-20101117
   3. New York Times: http://www.nytimes.com/2010/11/17/health/17drug.html
7. FDA Arthritis Advisory Commitee 2010 meeting materials: http://www.fda.gov/AdvisoryCommittees/CommitteesMeetingMaterials/Drugs/ArthritisDrugsAdvisoryCommittee/ucm203434.htm
8. Overview of FDA and the clinical trial process for drugs seeking approval in the USA:  http://www.fda.gov/drugs/resourcesforyou/consumers/ucm143534.htm
9. How can a drug have a perceived benefit compared to placebo, but not actually have one due to randomness?  Read an overview of statistical significance here:  http://en.wikipedia.org/wiki/Statistical_significance
10. Interesting summary of an investigation into the high percentage of patients enrolled outside the US for drugs garnering approval in the US: http://www.nytimes.com/2010/06/22/health/research/22trial.html
11. Note the clinical trials discussed are BLISS-52 (HGS press release) and BLISS-76 (press release) as the “first” and “second” trials, respectively.

Idea 1:  Identifying malaria vaccine leads via a screen for “immunologically relevant” antigens.

File: GCE_novel antigen screen – v7

Abbreviations: MHC, major histocompatibility class. DC, dendritic cell.

Section I. A novel liver-stage malaria vaccine

The problem

Why is there no 90%+ clinically effective p. falciparum malaria vaccine after decades of research1,2,21? There are 3 explanations: 1) the vaccine must be practical to manufacture, store and administer; 2) the vaccine needs to target the parasite during a prolonged stage of infection to give T cells time to respond; 3) the immune system needs priming in order to sensitively detect and eliminate otherwise difficult-to-detect malaria-infected cells.

To date, the only vaccine approach showing 90%+ efficacy in a human study was derived from whole parasites3,4. Unfortunately this raises multiple practical problems. The vaccine requires large doses of parasites to be reared within mosquitoes and surgically isolated by skilled technicians. The vaccine is administered monthly for up to 10 months. Because it involves preserving whole cell living organisms, the vaccine must be stored at -20°C or colder3,4. Instead of a whole parasite approach, it would be more practical to use established peptide antigen-based approaches that are used for most common vaccines. These are easy to administer once, manufacture at scale, and store in the field at 4°C. We decided that a vaccine should start from the premise of being peptide-based until we are confident that possible antigens have been exhausted. Fortunately there is plenty to explore: less than 5% of malaria proteins have been studied in any way; far fewer have been investigated as vaccine antigens.

Malaria is an intracellular infection and therefore needs to be cleared by the cytotoxic T cell aspect of immunity. But after the body is exposed to an infective antigen, it takes 2-3 days to select and proliferate enough T cells to mount an effective response6-10. At the same time, p. falciparum malaria migrates through different tissues and organs during its complex life cycle within the human host. The most prolonged phase of this life cycle is the liver stage, lasting 7-10 days. This is also the stage where malaria begins to proliferate after entering the host from a mosquito bite. Each sporozoite that survives from the mosquito bite results in ~105 merozoite offspring. Most of p. falciparum malaria symptoms are caused by the extremely high burden of parasites that clog micro vessels. Thus the liver stage is a most desirable stage for attacking malaria.

The third challenge results from the fact that the cytotoxic T cell response needs lymph node priming in order to effectively identify and purge infected liver cells. If there is no such priming, as the case for an unvaccinated patient, malaria can evade immune detection in the liver. However, when dendritic cells in lymph nodes are exposed to malaria antigens, they can have a potent effect on purging infected liver cells. While these approaches have met with some success in animal models, none have yet successfully translated into effective human vaccines. A new liver stage vaccine would need to specifically focus on its ability to prime skin-based dendritic cells after the mosquito bite. This in turn would provide the immune system enough time to create a cytotoxic T cell response targeted towards malaria after it had moved on to the liver.

The solution

We propose an antigen screen that takes the above three challenges into account. The approach aims to identify p. falciparum peptides that can be presented as antigens by dendritic cells after infection, and that are also detectable as presented by infected liver cells. Finding such peptides would allow creation of a vaccine that can prime a T cell response in the skin lymph nodes, then permit the activated T cells to recognize and purge infected liver cells. The approach is a novel application, based on existing biochemical and cell biological methods.

During the first phase of the screen, human DCs will be incubated with p. falciparum sporozoites in vitro. This step will mimic the interaction of sporozoites with skin lymph node DCs that occurs within hours of the mosquito bite6-7. These antigen presenting cells digest invading parasites and present peptide fragments on their surface via MHC proteins. In the lymph node, many T cells then evaluate these antigens and proliferate if there is a strong match for the antigens. Thus we first aim to characterize the most common antigens that DCs present via MHC. Established methods exist to isolate these peptides biochemically and then sequence them with mass spectrometry11,12.

In the second phase of the screen, cultured human hepatocytes will be incubated with malaria sporozoites to mimic liver infection. A proportion of these cells will digest the malaria parasites and present malaria peptides on their cell surface via their MHC proteins. Similar to the DC protocol, these peptides will be isolated and sequenced. In the final phase of the screen, peptide sequences will be compared between DC-presented peptides and hepatocyte-presented peptides. Any peptide sequences that are similar in the two groups will be become leads for further characterization as potential vaccine antigens.

The approach is unlike all other attempts to identify vaccine antigens. Several labs have used functional genetic screens to identify proteins to target with vaccines14-17. However, the limitation of these approaches is that they identify proteins important to p. falciparum’s function, but not specifically proteins that are recognized and attacked by the immune system after the parasites have infected hepatocytes. In other words, these approaches risk creating a vaccine towards an antigen that is invisible to the immune system. Other vaccine approaches have targeted malaria surface proteins. This is a logical place to start, but does not ensure that infected hepatocytes will be targeted by T cells for destruction. Instead, our approach will screen for the most immunologically relevant antigens and therefore aims to maximize the potency of a potential vaccine.

Potential issues with the solution

It is possible our approach may not identify any common antigens between lymph node DC and hepatocyte antigen presentation. We think this unlikely because in vivo only about 3 hours transpire between mosquito bite and liver infection5,8. In this brief window, protein expression is unlikely to change dramatically. Since hepatocytes express MHC1 and sometimes MHC2 receptors7, we would expect the pathogen protein presentation to be similar for MHC1-bound antigens in DCs and hepatocytes. Furthermore, several antigens are known to be processed by DCs and hepatocytes6,7.

Our approach may also benefit from focusing on different antigen-presenting cells. We think this unlikely based on evidence to date that indicates that DCs in skin lymph nodes and hepatocytes are the main APCs for pre-erythrocytic stages of malaria7. Cells that do not have a strong role include liver-based DCs and Kupffer cells7. However, an antigen-presenting role is still unknown for liver cells such as stellate cells or liver sinusoidal endothelial cells7. In the event our initial experiments do not identify any antigens, we would modify the protocol to study these other possible antigen-presenting cells.

A common concern with malaria vaccine design is antigenic variability of malaria strains. Our approach would address this in three ways. First the screen relies on antigens presented by the host, so a vaccine targeting these antigens will destroy infected host cells and not directly kill the parasites. Thus there will not be a direct selection pressure stimulating the parasites to mutate. Second, if multiple candidate antigens are identified from the screen, next steps would include creating a multivalent vaccine from these antigens. This could further reduce the probability of parasites developing mutation resistance to the vaccine. Last, we can filter out any identified antigen candidates with genes that encode known variability regions such as the var region.

Section II. Experimental design and methods

Aim 1 ($40K; 5 months): Characterize peptides presented by hepatocytes. In our screen, hepatocytes are more likely to present difficulties in identifying strong signals of MHC-presented malaria peptides because these cells are not “professional” antigen-presenting cells (as DCs are known to be). So we will focus first on hepatocytes. An IRB protocol would be drafted and approval obtained to collect otherwise discarded surplus cells from liver biopsies and bone marrow biopsies. Primary human hepatocytes would be isolated from liver biopsies and cultured per established methods. Cryopreserved p. falciparum sporozoites (MR4, Manassas, Virginia) would be thawed and then incubated with monolayers of hepatocytes for 16-24 h20. Hepatocytes would be harvested at 3, 6, and 9 days to capture the duration of the liver stage. Peptides bound to hepatocyte MHC proteins would be isolated using the direct biochemical method11,12. Positive controls would include measuring expression of known sporozoite antigens such as CSP and TRAP in the MHC protein-isolated fractions. Negative controls would include measuring expression of intracellular proteins bound to MHC proteins. Peptides would be sequenced with mass spectrometry. Measurable outcome: sequences of hepatocyte MHC-bound malaria peptides.

Aim 2 ($40K; 5 months): Characterize peptides presented by dendritic cells. This section would be approached similarly to the hepatocyte Aim 1 with the following exceptions. Bone marrow cells would be cultured from discarded bone marrow biopsies. Bone marrow progenitors would be cultured into DCs per Plebanski et al (2005). Cryopreserved p. falciparum sporozoites (MR4, Manassas, Virginia) would be thawed and then incubated with monolayers of DCs for 16-24 h20. Positive controls would include similar sporozoite antigens to Aim 1, while negative controls would include intracellular DC proteins that would not be expressed by MHC proteins. Measurable outcome: sequences of dendritic cell MHC-bound malaria peptides.

Aim 3 ($20K; 2 months): Analyze overlapping peptide sequences to identify candidate antigens. With peptides identified , we would next begin analyzing these peptides to identify overlaps. Pep-Miner software would be used to conduct the overlap analysis12. Overlapping peptide fragments would be compared to protein databases to identify the relevant malaria proteins. Measurable outcomes: identification of shared peptides. List of likely antigenic proteins for further characterization.

Translational value and next steps: Peptides identified from the screen would be evaluated for their ability to stimulate a T cell immune response in animal models, and thus become potential future vaccine candidates.

References

1. Moran M et al. The Malaria Product Pipeline: Planning for the Future (2007)
2. Richie TL et al, Nature 415:694 (2002)
3. Hoffman et al., US Patents US2005/0208078, US2005/0220822
4. Hoffman SL et al., J Inf Diseases, 185:1155 (2002)
5. Yamauchi LM et al., Cellular Microbiology, 9:1215 (2007)
6. Prudencio M et al., Nature Reviews Microbiology, 4:849 (2006)
7. Frevert U et al. Cellular Microbiology 10:1956 (2008)
8. Amino R et al., Nature Medicine 12:220 (2006)
9. Frevert U et al., PLoS Biology, 3:e192 (2005)
10. Amino R et al., Cell & Host Microbe, 3:88 (2008)
11. Cox AL et al., Science 264:716 (1994)
12. Shoshan SH et al., Pharmacogenomics 5:845 (2004)
13. Aregawi M et al. The World Malaria Report, 2008 (WHO).
14. Florens L et al. Nature 419: 520 (2002)
15. Sam-Yellowe TY et al. Journal of Proteome Research 3: 995 (2004)
16. LaCount DJ et al. Nature 438: 103 (2005)
17. Wuchty S et al. Journal of Proteome Research 6: 1461 (2007)
18. Mueller A-K et al., Nature, 433:164 (2005)
19. van Dijk MR et al., PNAS, 102:12194 (2005)
20. Plebanski M et al., Immunol Cell Biol, 83:307 (2005)
21. Wykes M et al., International Journal for Parasitology, 27:705 (2007)
22. Graves P et al, Vaccines for preventing malaria (pre-erythrocytic) (Cochrane Collection, 2008)

Idea 2: Identifying malarious scents for a future surveillance device.

File: GCE_Scent detector – v4 doc

Section I. What is your idea? Malarious humans release odors that attract mosquitoes. We propose identifying these molecules, as a first step towards designing an environmental malaria detector.

As malaria control efforts gain momentum and continue to be successful in some areas, there is a growing need for devices that can help detect outbreaks of malaria, thus helping governments strategically prioritize their finite resources for malaria control.

A recent study demonstrated that malarious patients harboring the transmissive form of the parasite release odors that can attract mosquitoes two times more often than other malarious patients or uninfected patients (1). A device that could “sniff” these molecules would permit surveillance of a village for malaria outbreaks. This would be particularly useful in areas with insufficient coverage of community health workers (CHWs).

Here we propose to identify the odor molecules, using an established clinical study method and gas chromatography (GC). This proof-of-principle work will be a springboard for designing inexpensive, point-of-use electronic “sniffer” devices to detect malaria outbreaks in resource-poor areas. The impact of such a surveillance device would be enormous. Several countries have reduced malaria cases significantly, including Rwanda, Eritrea, and Uganda. If this progress were to be reversed by a resurgence, several million additional children would get malaria each year (2).

The first non-invasive malaria detector, and the first detector for strategic surveillance.

Our approach would be innovative for several reasons. First, no research group is currently working on identifying these molecules. Koella and colleagues have moved on to other topics (3). Second, there is no precedent for a non-invasive malaria detector, which this project would ultimately aim to accomplish. The only other options for positive identification of malaria are blood smears and rapid diagnostic tests (4,5). Finally, no technology exists that would help a country maintain the necessary vigilance after malaria prevalence has declined.

In concept the putative detector would resemble a fire alarm — highly sensitive, always sampling the environment, and only emitting a warning signal when it detects a positive. In practice, the “alarm” may not be a sound, but rather an electronic signal to an epidemiology center. The detector would require minimal maintenance and staff attention. Thus a developing country could take an evidence-based approach in deciding where to allocate limited resources to control a new outbreak of malaria.

Possible issues with our approach

Mosquitoes are attracted to bite malarious humans by both intrinsic factors (e.g., carbon dioxide, sweat, warmth, skin moisture, and body odor) (6-8) and malaria-specific factors (1). One concern is that our proposed studies could mistakenly identify intrinsic factors. However, Koella et al demonstrated that mosquitoes are attracted two-fold more to gametocytemic patients than controls. This attraction returned to baseline intrinsic attraction after treatment with anti-malarial drugs (1). Thus we plan to replicate these methods, and survey gases from patients before and after treatment with antimalarial drugs, and focus only on molecules with higher concentrations before versus after antimalarial treatment.

The technology would only identify patients with gametocytemia. These represent approximately 52% to 66% of patients, based on a 19-month longitudinal study of children in Kenya (9). One concern could be that a future technology would not be detecting all parasitemic patients (gametocytemic or not). However, our detector would not be used at the point of care, the way a rapid diagnostic test would. Instead, it would be designed to identify patients harboring the transmissible form of the parasite. Thus only identifying the gametocytemic patients is desirable, because only these patients represent transmission, and would be epidemiologically significant harbingers of an outbreak. Of course, any symptomatic patient would be treated by local health workers, regardless of the results of our scent detector.

Another concern is with detection sensitivity. GC can detect samples at a parts per billion concentration. While it may sound difficult to isolate these molecules, precedent exists even in commercial devices, for detecting and characterizing odors at the ppb range in air. More broadly, there is a precedent for commercially available detectors for gaseous molecules. These rely on a range of technologies, from relatively inexpensive detectors based on chemical reactions with substrates (e.g., Breathalyzer tests), to more expensive MS-based units (e.g., GE’s EntryScan for airport security). Our focus for this grant is identifying molecules for detection. After doing so, we would then further optimize a device to affordably detect these molecules. Other options could include electronic micro-sniffers currently in development (10).

Section II. How will you test it?

The experimental plan will use established methods: patient “olfactometer” to collect gaseous samples, GC-MS, and leverage previous clinical protocols and relationships with the Kenyan government.

Aim 1 ($20K). Establish collaboration and clinical protocols. Establish collaboration with Koella and Mukabana to extend findings from Kenya study, using olfactometers to capture gas eluants from asymptomatic patients with confirmed parasitemia or gametocytemia (1,6,11). Draft clinical research and ethics protocols, for review by collaborators and approval from Kenyan National Ethic Review Committee. Design study to recruit 10 groups of 3 children each, to identify asymptomatic patients who fall into one of 3 groups: 1) asymptomatic, 2) parasitemic but not gametocytemic, and 3) gametocytemic. Methods would follow those of LaCroix et al (2005), including immediately taking any symptomatic patients to the local health clinic for treatment. Parasitemia would be verified via thick blood films, prepared by and analyzed by our group.

Methods from LaCroix et al would be modified as follows: at sundown, when patients are monitored while sleeping in tents connected to the olfactometer, effluent gas from each tent would be collected hourly into vacuum tubes, for later analysis by GC-MS. In the beginning of the study, pilot GC-MS experiments will be conducted to verify that some molecules can be detected. Positive controls be tested by exposing a small vial of nail polish remover (acetone) for 10 seconds at sundown, and verifying identification via GC-MS. Asymptomatic patients would be analyzed before any treatment, and then 2 weeks following treatment with sulfadoxine-pyrimethamine, also per methods of LaCroix et al (2005).

In the event collaboration with the Kenyan government fails, we will leverage relationships in Ghana or Rwanda, as backup locations.

*Measurable outcomes: Completed clinical research protocol. Collaboration action plan for study activities and endpoints. Approved clinical research protocol. Kenyan site identified and relationships established with health workers and regional authorities.

*Expected time: 4-6 months.

Aim 2 ($80K). Conduct clinical study and identify odor molecules. The study would be conducted as described above. The focus of Aim 2 would be on conducting the clinical trial, analyzing samples with GC-MS, and identifying molecules based on MS profiles.

In the event that GC-MS samples are not stable during the transport each morning from research site to urban research lab, we will explore alternatives, such as transporting the asymptomatic malarious patients to an urban hospital, housing them in vacant beds, and conducting the GC-MS in real time by the patient’s bedsides. In the event the GC samples are too impure, at this stage we would experiment with column modifications, to selectively look at different aspects of the gaseous mixture. For instance, we could use a cationic filter to remove all anionic materials, then analyze the remaining cationic and neutral compounds. In the event the odors are not concentrated enough for GC detection, we would generate respiratory and other body odor concentrates (12).

*Measurable outcomes: Completed study with balance of patients in each study group. Identified molecules based on GC-MS.

*Expected time: 6-9 months.

Translational value and next steps: Identified scent compounds. Next steps would be engineering an inexpensive, sensitive detector device and conducting field tests.

Abbreviations: GC, gas chromatography. GC-MS, gas chromatography and mass spectrometry. CHW, community health worker.

References

1. Lacroix R et al., PLoS Biology, 3:e298 (2005)
2. Aregawi M et al., The World Malaria Report, 2008 (WHO)
3. Jacob Koella, personal communication, 10/14/08.
4. Moody A, Clinical Microbiology Reviews, 15:66 (2002)
5. www.rapid-diagnostics.org
6. Mukabana WR et al., Malaria Journal, 3:1 (2004)
7. Mboera LEG et al., Medical and Veterinary Entymology, 14:257 (2000)
8. Takken W et al., Annu. Rev. Entomol. 44:131 (1999)
9. Jones TR et al., Am. J. Trop. Med. Hyg., 56: 133 (1997)
10. Raman B et al., Anal. Chem, ePub 10/15/08
11. Mukabana WR et al., Malaria Journal, 1:17 (2002)
12. Ghaninia M et al., J Exp Biology 211:3020 (2008)

In my goal to master perfect pitch by the end of 2010, I think I am about 60% there.  Daily I mentally play through both hands of Bach’s First Piano Invention, which focuses mostly on the “white” keys since it is in C major.  Several times a week, I also work slowly through Beethoven’s Moonlight Sonata, which focuses more on the “black” keys of C Sharp minor.  I have finished memorizing the piece.  Now, as I’ve described before, my challenge is to play the starting note of each of the movement’s ~65 measures, then mentally play the measure to its end, then compare my mental end note to how it sounds on the piano.

It will probably take me until September to get to the point where I can mentally play the entire piece.  I am focusing mostly on the right hand arpeggios since the left hand mostly plays simple bass whole tones throughout the piece.

Because of our baby’s arrival, I wanted to see if I could keep working on mental music even when doing other daily activities.  The intent here is not to make myself crazed with multitasked activities, but just to find some way to keep my enjoyment of music going even when other priorities are the focus of my life.  I estimate I would spend 30-45 minutes each day doing mental practice or composing.  Once I have mastered perfect pitch, my ultimate goal is to use my mental music time to compose like Mozart did.

Recently I tried mentally playing through the Bach piece while walking to work and found it quite exhilarating.  I especially enjoyed walking at a relaxed pace, letting the CLAP of my feet on the pavement mark the beat.  One has to be careful not to walk too quickly, as this leads to inaccuracy and an uncomfortable sense of rushing.  Instead, walking at a slow pace encourages a relaxing indulgence in the mental sounds, without making me worried that I might invite injury from cars at intersections or obstacles like poles and recycling cans while walking, absorbed in music.

Otherwise I have not been doing much musically.  I pick up a sax about once a week, which is fine for now given my other priorities, and try to keep my embouchure in shape by doing isometric exercises (for instance, see last paragraph of this article).  Band rehearsals have been on hiatus, and I find myself wondering about finding some kirtan musicians to play with.  But that may be a pipe dream for now, I just find myself drawn lately to some really simple music like that of Krishna Das or various shakuhachi players.

I have continued working on absolute pitch exercises with Bach’s First Invention, mentally playing it from start to finish.  Also slowly memorizing Beethoven’s Op. 27 Piano Sonata #14 (first movement), “Moonlight Sonata”, so that I can start doing the same series of exercises on this tune.

One assumption I have been making is that learning perfect pitch is an associative memory process.  This means that the more ways I can associate “hearing” a note in my mind with other thoughts or mental activities, the more robust my memory of pitch will become. In practice, while I’m mentally playing through the Bach invention, trying to “hear” the sounds in my mind, I visualize the notes on sheet music, visualize my hands playing them on the piano, or sometimes imagine how the notes feel on a saxophone (not taking into account the transposition between piano and sax).

So far this has been working!  I test myself on various notes around the keyboard 4-5 times a day.  When I try to conjure a note in my mind, I am most often able to “hear” it correctly by conjuring the “feeling” I have of how that note sounds when I mentally play the Bach invention.

Here are some of the learnings and challenges I’ve faced since starting the project to learn perfect pitch in January:

1. I had to train my brain to not flatten upward leaps in pitch, or sharpen downward leaps in pitch.  This usually required identifying the interval in the piece that was causing my pitch to get off, then practicing only the interval until my mind stopped this tendency.
2. Each key change in the Bach first invention used to throw off my sense of pitch.  For instance, there’s a shift from C major to G major early on, and for a while that would disorient my sense of where C was.  With practice, that’s disappeared.
3. I’ve had more difficulty mentally “hearing” pitches that fall above or below my singing range.  Since I’m probably a tenor singing voice, anything above that took me a while to truly hear.
4. Another challenge has been, how to practice these exercises regularly in hectic daily life?  I usually practice them on a bus to work.  This almost always is fine, using the iPhone app Virtuoso Piano Pro to provide a reference pitch.  It is only challenging if the bus is playing background music or someone has headphones with really loud music on.  My mental hearing sense is improving, but it’s not strong enough yet to stay mentally in C major if other environmental music is simultaneously in another key.
5. The mind gets tired after these exercises!  It takes me 2-3 minutes to play mentally through one hand of the Bach invention.  After doing this 4-5 times I usually take a short break.
6. The more tired I am, the more distracted I become, and the more mistakes I make.  This seems obvious, but tracking my mental music performance everyday, I can see it plainly.  It’s a reminder to treat my body well and not do too much.

Here are a few milestones I’ve noted so far:

1. More often now I notice myself humming a tune, then playing the notes I’m humming on a piano and realizing I’m hearing the tune in the key it was recorded!
2. I can hear much more mentally and practice mentally for longer durations than before.
3. I can hear and enjoy the richness of deceptively simple pieces of music, like the Bach inventions, and enjoy in wonder!  Almost any type of music has been magnified in enjoyment by this “mental ear” practice I have been doing.  There is so much more to hear in what I already listen to!
4. My ears are more sensitive, so I find myself more turned off by styles of music that are really loud or not really embracing the possible subtleties that the nuances of sound provide.

Quick update on perfect pitch exercises.  After ~8 weeks I have developed “mental play” ability with Bach’s 2-part Invention No.1 so that I can hear the pitches accurately from start to finish, when I play only the starting note.  (C3 for the right hand; C2 for left).  The process of daily morning practice — starting with 1-measure increments and progressing to the full 22-measure duration of the tune — has been challenging and fun.  For the next few weeks I will be starting to memorize and then mentally play Beethoven’s Moonlight Sonata (No. 14 in C# minor).  I plan to do a longer post soon with more tips and learnings if anyone else is curious about cultivating absolute pitch.

1. Simplify to open up space for creativity
2. Listen to your passions

#1 basically means I realized that trying too hard can be an obstacle.  Every time I tried to find a better day job, free time for music disappeared.  Negative feelings about self and career began to trickle in…  But when I simplified and just tried to appreciate each day, this opened up more time for musical creation and let me realize (x) life is pretty darn good, (y) happiness is mostly what you make of it, (z) you can’t sustainably pursue an endeavor to help others if you can’t first help yourself.  On #2 this seems obvious, but it’s amazing how often my mind tries to convince me to do things that aren’t aligned with my passions.  For instance, and it’s related to #1, I decided last year to read fewer books (about 1 every 2-3 weeks instead of 1 per week) and free up more time for just listening to and enjoying music.

As a result I was fortunate to find a band I enjoy playing danceable tunes of high musical and lyrical quality.  Getback.  I also decided to become a father.  What’s up 2009!

My musical goals for 2010 are:

1. Cultivate absolute (“perfect”) pitch
2. Compose at least 1 LP’s worth of original music for recording
3. Perform public gigs with a band I dig and music I enjoy!

In thinking forward to 2010, I was about to craft #1 as “re-learn the piano” when I discovered, in reading Chang’s The Fundamentals of Piano Practice (author link & Amazon), that it might be possible to transform my “relative pitch” ability into “absolute” or perfect pitch.  Relative pitch means that, given a starting note, you can quickly if not immediately figure out other notes you are hearing based on that starting note.  Absolute pitch means that when you hear a note, you instantly know what pitch it is on a piano (e.g., “D flat”).

Chang’s approach is to memorize a few classical pieces and then practice reproducing them in your mind.  You do this until you are able to hear only the starting note of the piece, and then mentally play the rest of the tune so that you finish on the correct last pitch.  This hones your “relative pitch”.  Then you do a series of tests, repeated as often as you can manage daily, to cultivate absolute pitch.  In other words, you train your brain to recognize what each note on the piano actually sounds like, so that you can hear a note and immediately recognize its pitch on the piano.  I’m doing this to improve mental composing skills, ability to “ear learn” new music and music concentration.

I started in January learning both hands of Bach’s First 2-part invention in C major, one measure at a time.  I would play the first note of each measure, then mentally play through it and then hold the last note in my mind.  Next I would play that note on the piano and see how it compared to the note I was mentally “singing”.  I noticed I made mistakes most often when the melody was taking large leaps, or moving into flats and sharps that I thought I could hear in my mind.  Over time I worked out the mistakes I was making, and then would gradually increase the number of measures I would mentally play.  Now I am working on the 22-bar piece in two chunks of about 10 measures each.  So hopefully in another 2-3 weeks I’ll be able to mentally play the entire piece with accurate relative pitch.  The process has been really enjoyable, as I practice hearing more and more from a deceptively simple Bach tune.  I feel I can already learn music by ear faster than I could before, for instance when learning music played by bandmates or when playing along with recordings.  I also feel my general enjoyment of music has increased and find myself wanting to listen to music whenever I can!

Next, according to the method, is to work similarly on a piece that emphasizes the “black keys”, i.e. a key like C# or Eb minor.  So next I’ll be working on memorizing and then mentally practicing Beethoven’s Piano Sonata #14 in C# minor (Moonlight Sonata).  I’ll try to keep updates of how this is going on the blog.  Please ask questions (especially any music students reading) and I’ll explain more.

Last, an update on the band — we’ve decided to generate more original material so are not actively seeking gigs.  But some updates on the performance front will follow once we have finished this incubation stage!

(photo credits: Kana McKee)