Does processed milk cause multiple sclerosis flare up?

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Brief History Of Nebulous Connection Between Milk & MS (Multiple Sclerosis)

Sparse data on this subject consists of

  • An US epidemiological study that compared 1949 to 1967 MS mortality rates and food consumption data, and found high correlation (0.8 to 0.9) with milk consumption (1).
  • A couple of cross-sectional epidemiological studies from 1976 (2) and 1992 (3) that compared MS prevalence and dairy product consumption. The second one looked across 29 populations in 27 countries and suggested MS progression could be influenced by factors in liquid cow’s milk but not in processed milk.

The story then appeared to lie dormant for the next several years until revived by a 2000 rat EAE (Experimental autoimmune encephalomyelitis – Wikipedia) model study (4). This study mechanistically showed Butyrophilin – Wikipedia, a milk fat globule membrane protein expressed only by the lactating mammary gland,

Similar experiments in a mouse model (5) also showed Butyrophilin could prevent MOG-induced EAE, i.e., that this milk component could protect against EAE.

Problem is though originally developed in the 1950s to supposedly mimic human MS, these rodent (mainly rat and mouse) models simply don’t mimic human MS very well (6), haven’t yielded much insight or practical therapies and yet have taken over basic MS research and remain the mainstay in the field.

Meantime, a couple of small human studies from France (n=44 MS versus 30 controls, 7) and the US (n= 35 MS versus 25 controls, 8) yielded contradictory data

  • The French study (7) found MS patients with higher circulating antibody levels cross-reactive to MOG and Butyrophilin.
  • The US study (8) found MS patients and controls had similar levels of circulating antibodies cross-reactive to MOG and Butyrophilin. However, this US study also compared anti-MOG and -Butyrophilin antibody responses in blood as well as CSF (Cerebrospinal fluid – Wikipedia) of MS patients and found they were specific for different epitopes (parts) of Butyrophilin, the one dominating in the CSF also cross-reacting to a homologous MOG peptide in 34% of MS patients, i.e., possible Molecular mimicry – Wikipedia between MOG and Butyrophilin.

In addition to these two human studies contradicting each other, these purely observational studies examined circulating antibody levels, i.e., B cell – Wikipedia, not T, cell response as the animal model studies did. Apples and oranges.

While Butyrophilin’s plausible role in MS progression lies in its high sequence similarity to MOG (9), i.e., Molecular mimicry – Wikipedia, data from these two small human studies are inconclusive and so far there’s no other data on milk proteins’ role in MS prognosis or disease course (10).

How Milk Or Any Other Factor Might Trigger Or Flare MS (Multiple Sclerosis)

Though there are many suspects, confirmed triggers for MS are still unknown. Most convincing data exist not for dietary factors such as milk but for vitamin D levels and its receptor polymorphisms, history of Epstein-Barr virus infection including Infectious mononucleosis – Wikipedia and smoking (11, 12).

No matter the trigger though, how might MS disease cascade follow? Prime suspect is molecular mimicry, i.e., molecular level similarity between such triggers and the target of autoimmune attack in MS, typically proteins such as MOG expressed on Oligodendrocyte – Wikipedia, myelin sheath cells.

  • However, even this is insufficient. Consider milk for example. Many more people drink milk than get MS. If molecular mimicry alone sufficed, they should all have MS and they don’t.
  • Clearly other factors are also involved. No matter the degree of molecular mimicry, nothing can ensue if a person’s immune system can’t ‘see’ it.
    • An immunologically necessary condition would thus be the HLA (Human leukocyte antigen – Wikipedia) haplotype. However, HLA haplotype alone wouldn’t suffice because far more people share HLA haplotypes than develop MS.
    • Breakdown in T cell tolerance is also necessary. After all, during their development in the thymus, T cells with the capacity to recognize oligodendrocyte proteins should get deleted. Obviously that process seems to fall short in MS patients.
  • Thus target antigen (and molecular mimic)-specific T cells should be present and such target(s) of immune attack should be properly processed and presented to these T cells. However, though appropriate HLA haplotype and CNS (Central nervous system – Wikipedia) protein(s)-specific T cells are the necessary building blocks for MS autoimmune pathology, their presence alone doesn’t suffice either.
  • Context is also necessary, i.e., predisposing and/or conditioning factors necessary to drive the pathological immune response necessary for MS expression. Totality of such factors, sufficient to explain MS in each and every case, still remain undefined.

Bibliography

1. Agranoff, BernardW, and David Goldberg. “Diet and the geographical distribution of multiple sclerosis.” The Lancet 304.7888 (1974): 1061-1066. https://deepblue.lib.umich.edu/b…

2. Butcher, J. “The distribution of multiple sclerosis in relation to the dairy industry and milk consumption.” The New Zealand Medical Journal 83.566 (1976): 427-430)

3. Malosse, D., et al. “Correlation between milk and dairy product consumption and multiple sclerosis prevalence: a worldwide study.” Neuroepidemiology 11.4-6 (1993): 304-312.

4. Stefferl, Andreas, et al. “Butyrophilin, a milk protein, modulates the encephalitogenic T cell response to myelin oligodendrocyte glycoprotein in experimental autoimmune encephalomyelitis.” The Journal of Immunology 165.5 (2000): 2859-2865. http://www.jimmunol.org/content/…

5. Mañá, Paula, et al. “Tolerance induction by molecular mimicry: prevention and suppression of experimental autoimmune encephalomyelitis with the milk protein butyrophilin.” International immunology 16.3 (2004): 489-499. https://www.researchgate.net/pro…

6. Tirumalai Kamala’s answer to Why can chemicals that block the alpha tumour necrosis factor make multiple sclerosis worse? Do inhibiting cytokines make inflammation worse?

7. De March, A. Kennel, et al. “Anti-myelin oligodendrocyte glycoprotein B-cell responses in multiple sclerosis.” Journal of neuroimmunology 135.1 (2003): 117-125.

8. Guggenmos, Johannes, et al. “Antibody cross-reactivity between myelin oligodendrocyte glycoprotein and the milk protein butyrophilin in multiple sclerosis.” The Journal of Immunology 172.1 (2004): 661-668. http://www.jimmunol.org/content/…

9. Vojdani, Aristo. “Molecular mimicry as a mechanism for food immune reactivities and autoimmunity.” Altern Ther Health Med 21.Suppl 1 (2015): 34-45. http://bant.org.uk/wp-content/up…

10. Von Geldern, Gloria, and Ellen M. Mowry. “The influence of nutritional factors on the prognosis of multiple sclerosis.” Nature Reviews Neurology 8.12 (2012): 678-689.

11. Belbasis, Lazaros, et al. “Environmental risk factors and multiple sclerosis: an umbrella review of systematic reviews and meta-analyses.” The Lancet Neurology 14.3 (2015): 263-273.

12. Tirumalai Kamala’s answer to Why does Colorado have the highest rate of multiple sclerosis?

https://www.quora.com/Does-processed-milk-cause-multiple-sclerosis-flare-up/answer/Tirumalai-Kamala

What experiments can we do with TILs to work out the mechanism of action of checkpoint inhibitors?

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Typical targets of checkpoint inhibitors (usually humanized monoclonal antibodies) are molecules such as Programmed cell death protein 1 – Wikipedia (PD-1), PD-L1 – Wikipedia, CTLA-4 – Wikipedia. Applying checkpoint inhibitors (Cancer immunotherapy – Wikipedia) when such molecules are expressed by cells within a given tumor is akin to releasing the brakes of tumor antigen-specific Tumor-infiltrating lymphocytes – Wikipedia (TILs) present within them, empowering them to eliminate such tumors. This is the purported mechanism of action (MOA) of checkpoint inhibitors. However, their major problem is non-specificity, as in potential for off-target responses since they could release the brakes off of all T cells that express them, not just those that are tumor antigen-specific. To be able to effectively harness TILs to destroy any given tumor,

  1. Tumor should have TILs within it in the first place.
  2. Tumor should express ‘neoantigens’, i.e., tumor-specific antigens.
  3. TILs being used in immunotherapy (1) should be specific for peptides derived from antigens specifically expressed by tumor (2).

2 and 3 present formidable challenges because unless both conditions are met, cancer immunotherapy could end up attacking the body itself, with tragic results, since even death is possible. For example, while details haven’t been divulged, higher than anticipated clinical trial deaths have led leading cancer immunotherapy company, Juno Therapeutics – Wikipedia, to recently scrap its lead CAR-T therapy (1).

Thus, checkpoint inhibitors notwithstanding, to assess whether TILs might be induced to specifically target a given tumor, the experimental process entails

  1. Assessing whether the tumor has TILs and if yes, then isolating them.
  2. Assessing whether tumor expresses tumor-specific antigens by comparing tumor and matched healthy tissue.
  3. Confirming tumor cells themselves can indeed process and present these tumor-specific antigens within MHC class I (to tumor-specific cytotoxic CD8+ T cells) or MHC class II (to tumor-specific helper CD4+ T cells) molecules, an extremely technically challenging task made even more arduous by the following caveats,
    1. Even if a given tumor expresses several tumor-specific antigens, it might not present peptides derived from them since tumors are known to have aberrant antigen processing and presentation pathways (2).
    2. Differentiating immunologically relevant from irrelevant peptides is turning out to be more complex than envisaged (3).
    3. MHC class II-binding peptides are longer and bind more promiscuously compared to those that bind class I, making binding predictions much more technically challenging.
    4. Tumor cells rarely express MHC class II.
  4. Assessing whether isolated TILs are tumor-specific, i.e., specifically bind tumor-derived pMHC (peptides bound to Major histocompatibility complex – Wikipedia (MHC)).
  5. Assessing effect of checkpoint inhibitors on such tumor-specific TILs. If tumor-specific TILs don’t express the targets of such checkpoint inhibitors and yet also don’t attack and eliminate the tumor,
    1. They could be T regs (Regulatory T cell – Wikipedia) in which case it may be difficult to impossible to engineer effective anti-tumor immunity using them.
    2. If not Tregs, may need to assess whether such TILs express novel ‘brakes’, targets for novel checkpoint inhibitors.

Plenty of obstacles stand in the way of these experimental imperatives. Steps 3 and 4 the most fiendishly complicated, no one method exists to reliably assess both in a high-throughput fashion nor are existing In silico – Wikipedia approaches fool-proof (4). There are other hurdles as well.

  • Not all tumors express abundant tumor-specific antigens (4, 5, 6, 7).
  • Some tumors can mutate ferociously, i.e., cancer Immunoediting – Wikipedia (8, 9), which reduces the likelihood of finding tumor-specific TILs.
  • Rather than being tumor antigen-specific, some TILs may be specific for antigens that both tumor and normal tissue cells express (cross-reactivity).
    • T cells function by recognizing and binding pMHC through their T-cell receptor – Wikipedia (TCR). Central tolerance – Wikipedia, the developmental process of eliminating T cells reactive to cells of the body in which they develop, is incomplete.
    • The TCR is inherently cross-reactive, capable of binding >1 pMHC, i.e., can bind peptides derived from different antigens presented by the same MHC molecule (cross-reactivity) (10).
  • Thus, possible for checkpoint inhibitors to trigger autoimmunity.

However, promise of this approach is sustained by examples of tumor-specific TILs (11) having been found in

  • Human non-small cell lung cancer (12).
  • Human melanoma (13, 14, 15, 16, 17, 18, 19).
  • Human AML (Acute Myeloid Leukemia) (20).
  • Human CLL (Chronic Lymphocytic Leukemia) (21, 22).

Bibliography

1. http://www.xconomy.com/seattle/2…

2. Leone, Patrizia, et al. “MHC class I antigen processing and presenting machinery: organization, function, and defects in tumor cells.” Journal of the National Cancer Institute 105.16 (2013): 1172-1187. https://oup.silverchair-cdn.com/…

3. Gilchuk, Pavlo, et al. “Discovering protective CD8 T cell epitopes—no single immunologic property predicts it!.” Current opinion in immunology 34 (2015): 43-51. https://www.researchgate.net/pro…

4. Gfeller, David, et al. “Current tools for predicting cancer-specific T cell immunity.” OncoImmunology 5.7 (2016): e1177691. https://www.researchgate.net/pro…

5. Alexandrov, Ludmil B., et al. “Signatures of mutational processes in human cancer.” Nature 500.7463 (2013): 415-421. https://www.researchgate.net/pro…

6. Gubin, Matthew M., et al. “Tumor neoantigens: building a framework for personalized cancer immunotherapy.” The Journal of clinical investigation 125.9 (2015): 3413-3421. Tumor neoantigens: building a framework for personalized cancer immunotherapy

7. Schumacher, Ton N., and Robert D. Schreiber. “Neoantigens in cancer immunotherapy.” Science 348.6230 (2015): 69-74. http://pmpathway.wustl.edu/files…

8. Matsushita, Hirokazu, et al. “Cancer exome analysis reveals a T-cell-dependent mechanism of cancer immunoediting.” Nature 482.7385 (2012): 400-404. https://www.researchgate.net/pro…

9. Rooney, Michael S., et al. “Molecular and genetic properties of tumors associated with local immune cytolytic activity.” Cell 160.1 (2015): 48-61. http://wulab.dfci.harvard.edu/si…

10. Tirumalai Kamala’s answer to How is it possible that a T Cell Receptor (TCR) recognises as few as 1-3 residues of the MHC-associated peptide?

11. McGranahan, Nicholas, et al. “Clonal neoantigens elicit T cell immunoreactivity and sensitivity to immune checkpoint blockade.” Science 351.6280 (2016): 1463-1469. https://www.ncbi.nlm.nih.gov/pmc…

12. Rizvi, Naiyer A., et al. “Mutational landscape determines sensitivity to PD-1 blockade in non–small cell lung cancer.” Science 348.6230 (2015): 124-128. https://www.ncbi.nlm.nih.gov/pmc…

13. Kvistborg, Pia, et al. “TIL therapy broadens the tumor-reactive CD8+ T cell compartment in melanoma patients.” Oncoimmunology 1.4 (2012): 409-418. http://www.tandfonline.com/doi/p…

14. Andersen, Rikke Sick, et al. “High frequency of T cells specific for cryptic epitopes in melanoma patients.” Oncoimmunology 2.7 (2013): e25374.; http://www.tandfonline.com/doi/p…

15. Robbins, Paul F., et al. “Mining exomic sequencing data to identify mutated antigens recognized by adoptively transferred tumor-reactive T cells.” Nature medicine 19.6 (2013): 747-752. http://ggdpathway.wustl.edu/file…

16. Lu, Yong-Chen, et al. “Efficient identification of mutated cancer antigens recognized by T cells associated with durable tumor regressions.” Clinical Cancer Research 20.13 (2014): 3401-3410. http://clincancerres.aacrjournal…

17. Frøsig, Thomas Mørch, et al. “Broadening the repertoire of melanoma-associated T-cell epitopes.” Cancer Immunology, Immunotherapy 64.5 (2015): 609-620. https://www.researchgate.net/pro…

18. Linnemann, Carsten, et al. “High-throughput epitope discovery reveals frequent recognition of neo-antigens by CD4+ T cells in human melanoma.” Nature medicine 21.1 (2015): 81-85.

19. Cohen, Cyrille J., et al. “Isolation of neoantigen-specific T cells from tumor and peripheral lymphocytes.” The Journal of clinical investigation 125.10 (2015): 3981-3991. Isolation of neoantigen-specific T cells from tumor and peripheral lymphocytes

20. Berlin, C., et al. “Mapping the HLA ligandome landscape of acute myeloid leukemia: a targeted approach toward peptide-based immunotherapy.” Leukemia 29.3 (2015): 647-659.

21. Rajasagi, Mohini, et al. “Systematic identification of personal tumor-specific neoantigens in chronic lymphocytic leukemia.” Blood 124.3 (2014): 453-462. ; http://www.bloodjournal.org/cont…

22. Kowalewski, Daniel J., et al. “HLA ligandome analysis identifies the underlying specificities of spontaneous antileukemia immune responses in chronic lymphocytic leukemia (CLL).” Proceedings of the National Academy of Sciences 112.2 (2015): E166-E175. http://www.pnas.org/content/112/…

https://www.quora.com/What-experiments-can-we-do-with-TILs-to-work-out-the-mechanism-of-action-of-checkpoint-inhibitors/answer/Tirumalai-Kamala

Is the placebo effect very powerful?

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The https://en.wikipedia.org/wiki/Pl… effect is powerful enough to be the bane of the https://en.wikipedia.org/wiki/Ra… (RCT), https://en.wikipedia.org/wiki/Bi…, and really of modern medicine itself. A dynamic attribute since it can be seemingly manipulated at will, it maybe more accurate to call it the Placebo Response, as proposed by https://en.wikipedia.org/wiki/Fa…. Their considerable scientific and economic impact is evidence of the shadow placebo responses cast on all of medicine.

Answer highlights

  • Placebo responses operate even for legitimate drugs and therapies.
  • Placebos elicit beneficial responses even when patients take them knowingly.
  • Placebo responses are becoming stronger, driving up drug development costs.
  • How placebo responses could be leveraged to make medicine more effective, cheaper and safer.
  • Examples that illustrate how just about anything to do with a Rx, colors, sizes, numbers, pills versus injections, even diligence in taking placebos, can trigger placebo response and influence its magnitude.

Longer answer for those interested in the details

Scientific Impact Of Placebo Responses

Placebo responses tend to be more pronounced in

  • Patient-reported outcomes rather than those independently-assessed (1).
  • Questionnaire- rather than clinical biomarker-based outcomes (2, 3).

No surprise since psychology is known to play an outsized role in pain and mental health conditions such as depression, which tend to lack objective diagnostic tests and are instead subjectively diagnosed on the basis of periodically revised, fuzzy questionnaires. Problem is most diseases lack biomarkers with proven therapeutic value.

However, even physiological attributes such as blood glucose levels entail placebo responses. Their concentrations in type II diabetes patients change commensurate to how their awareness of time is experimentally manipulated, lower and higher for those looking at clocks tweaked to run 2X slower or faster, respectively* (4). Blood glucose levels! Now that’s an objectively measured response. Already then, we can start to dimly discern the margins of modern biomedicine’s colossal problem. Unsubtly and arbitrarily splitting body (physiology) and mind (psychology) as a matter of course, it may not even be playing with a full deck since almost daily such new pieces of data force us to confront just how intertwined they actually are.

Placebo Responses: Outcome Of Expectation, Conditioning & Rx-Associated Communication/Ritual

Drugs and therapies are supposed to work specifically by targeting specific molecules and pathways involved in different diseases. Placebos, usually sugar pills, are supposed to mimic certain drug attributes. How? Three components working in tandem appear to explain not just how placebos work but also how placebo responses operate even for specific drugs and therapies.

Expectation is the outcome of learning, part of a general conditioning process of knowing one is getting Rx. The myriad components involved in getting Rx can be construed as Rx-associated communication/ritual (the physician, nurses, other care staff; are they warm, caring, supportive; do they explain what’s happening and what they’re doing; how much time they spend with the patient), etc.. Expectations and conditioning can mutually reinforce each other.

Paradigm shift lies in realizing placebo responses are ever-operational, even with legitimate drugs and therapies. Confused? The ‘open-hidden’ experiment design perfected by Benedetti’s team is not only clever but also groundbreaking because it reveals placebo response is an inherent part of response to medications and therapies.

In the original archetype of the ‘open-hidden’ experiment design (5), the powerful opioid painkiller https://en.wikipedia.org/wiki/Fe… was more effective when given openly by a doctor compared to being given secretly. Since then, the observation has been replicated in anxiety and Parkinson’s (6, 7). Knowingly getting a medication or therapy seems to set up expectations that make the medication or therapy more effective, i.e., by tapping Rx’s latent placebo response (8, See below from 9).

https://en.wikipedia.org/wiki/Te…, another leading placebo response researcher, has turned another conventional wisdom about placebos on their head. Giving patients placebos typically entails deception. Naturally this presents an ethical dilemma. If there are indeed bonafide benefits to placebos, how to reap them if doing so violates ethics?

Kaptchuk’s revolutionary idea is to turn this notion of secrecy on its head and to instead give placebos knowingly. Convention dictates knowingly taking placebos should blunt, even eliminate their benefit. Turns out to be not so. First for IBS (n=80, 10) then for lower back pain (n=97, 11), research on small patient groups shows knowingly taking a placebo in the context of an ongoing, supportive, caring, warm patient-physician relationship benefits patients. Rx-associated communication or ritual is a vital part of how placebo responses are triggered and sustained (12).

Economic Impact Of Placebo Responses

Decades-long evidence (13, 14, 15) suggests clinical trial placebo responses are becoming more powerful, especially for painkiller drug candidates (16) and especially in the US (17).

  • Placebo responses in anti-depressant trials were twice as strong in the 2000s compared to those in the 1980s (18). Even more compelling is the observation that this improvement occurred not for the more subjective patient self-rating but rather for the comparatively more objective observer rating (1).
  • Similar placebo response improvement when testing anti-psychotic drugs (19).
  • Placebo response magnitude change may vary by disease since it increased more weakly from 1991 to 2005 in trials for bipolar disorder (20).

Why could placebo responses be strengthening?

  • Could be simply because trials now last longer then they used to (21). Longer Rx-associated communication/ritual = Stronger Placebo responses.
  • Over the same time period, drug advertisements have undergone a sea change. The US legalized direct-to-consumer prescription drug advertising in 1997, which also exists in New Zealand. One unanticipated consequence? Social conditioning may be strengthening placebo responses.

Consequences of stronger placebo responses

  • Clinical trials are the necessary gatekeeper for new drug approvals. More powerful placebo responses increase the bar new drugs have to clear. No surprise new drug approvals have slowed down dramatically in recent years. Clearly some role there for the increasingly more powerful placebo responses.
  • Publication bias predicates that ‘real’ placebo response in RCTs remains unknowable. Studies with a significant drug to placebo difference are more likely to get published. Published placebo responses are thus likely lower than in studies that never got/get published. Ergo, actual placebo responses may be even higher than those revealed by clinical trial results that do get published. If this is the case with new drug candidates, no wonder new drug approvals have declined steeply in recent years.
  • One consequence of increasingly powerful placebo responses is a move away from the classic RCTs to newer designs such as comparative effectiveness research (CER) trials, which compare novel drugs to approved ones or to standard therapy, i.e., all participants get active Rx. Problem is such trials ‘increase the placebo response without being able to control for it‘ (9). Statistical analyses of such trials are also much challenging since they require showing the test compound is non-inferior to the standard Rx. As Enck et al point out (9, emphasis mine),

‘A non-inferiority trial tests the null hypothesis that the novel drug is inferior by the equivalence margin. If this null hypothesis is rejected, the novel compound can be regarded as clinically equivalent. This, on average, requires a fourfold larger sample size compared to classical RCTs and is one of the reasons why drug companies are increasingly concerned by the growing costs of drug development

How Placebo Responses Could Be Leveraged To Make Medicine More Effective, Cheaper & Safer

  • Pigeonholing placebos and placebo responses as undesirable or hindrance is problematic. Placebos are supposedly inert substances that should do nothing yet they do. OTOH, even genuine medicines induce placebo responses which increase their effectiveness. A way to reconcile this seeming contradiction would be by viewing placebo responses as an inevitable part of a virtuous feedback loop between psychology and physiology.
  • Expectations are outcome of learning, i.e., part of a conditioning process. Benefits of conditioning are well-known for allergies (22, 23) and nausea (24). See below from 9 how conditioning works.
  • Maybe encouraging placebo responses could make modern biomedicine more effective (9) by
    • Reducing healthcare costs since patients could interchangeably take drugs and placebos, and still benefit.
    • Minimizing harm from drug side effects since patients would be taking lower doses of drugs.
  • Now that would augur a true biomedical revolution. Conundrum is how to do this reproducibly since placebo’s evil twin, https://en.wikipedia.org/wiki/No…, lurks in the wings. Tapping the same pathways could yield harm just as easily as benefit and we simply don’t know enough about the feedback loops between psychology and physiology to manipulate them at will. For example, https://en.wikipedia.org/wiki/Na… can block analgesic (anti-pain) placebo response induced by opioid-driven but not non-opioid https://en.wikipedia.org/wiki/No… (NSAID) (25).

Placebo Responses: Research suggests placebo response magnitude depends on just about anything to do with Rx (26).

  • Rx Appearance (27). A systematic 1996 review (28) found red, yellow and orange pills worked better as stimulants while blue and green pills worked better as tranquilizers. Out of 49 nervous system drugs, stimulants were more often red or orange while sedatives were more often green, blue or purple.
  • Rx Frequency/number. A meta-analysis (29) of anti-duodenal ulcer RCTs found a difference in whether participants took 4 (51 trials) or 2 (28 trials) pills a day. After 4 weeks, 44% (805 of 1821) of those who took 4 placebo pills per day had healed duodenal ulcers compared to 36% (545 of 1504) of those who took 2, statistically a highly significant difference.
  • Rx Form. A meta-analysis (30) of 22 trials found placebo injections (32%, 279 of 862 patients) to be more effective against migraine (feeling better one hour later) compared to placebo pills (26%, 222 of 865 patients), statistically a highly significant difference.
  • Placebo-Taking Compliance Reduces Mortality Rates. BEST (Beta Blocker Evaluation of Survival Trial) participants (31) who took >75% of their prescribed placebos had significantly better outcomes (291 of 1038, 28%, died) compared to less compliant placebo-takers (55 of 136, 40%, died). A meta-analysis (32) of 21 RCT or cohort studies on conditions ranging from cancer-associated infections to heart attack to HIV found similar differences, lower mortality rates (4.3%, 581 of 13429 participants) for compliant placebo-takers compared to those less compliant (6.2%, 415 of 6204 participants).
  • Influence of Doctors (8, 9, 12, See below from 33 using data from 34).

Placebo Responses: Age, Culture, Ethnicity, Gender, etc. Also Matter

However, hastily generalizing such results could also be in error.

  • Different colors seem to matter differently in different countries, i.e., cultural differences are also at play (35).
  • An Italian study found women responded more strongly to blue sleeping pills compared to men who preferred orange ones (36).
  • Placebo injections may be more effective than pills at treating migraines in the US (33.6%, 151 of 449 patients for injections versus 22.3%, 25 of 112 patients for pills), not in Europe (25.1%, 80 of 318 patients for injections versus 27.1%, 182 of 670 patients for pills) (37).
  • Meta-analysis of 100 old RCTs of https://en.wikipedia.org/wiki/H2… on https://en.wikipedia.org/wiki/Pe… found placebo effects that ranged from 6% in Brazil, ~20% in Denmark and the Netherlands to >60% in Germany (38).
  • Again, would be wrong to conclude Germans are inherently more predisposed to strong placebo effects since the same analysis found them with among the lowest placebo effect for anti-hypertensive Rx.
  • Rotation-induced nausea is a condition with well-established ethnic differences (24).

Even Surgeries Aren’t Immune To Placebo Responses

Unlike medications, especially for subjective symptoms such as pain, surgery is usually done for tangible reasons yet even it isn’t immune to placebo responses, some attributable to bonafide placebo response power and some attesting to the futility of certain prevalent and expensive surgical procedures.

  • Back in the 1950s, ligating the internal mammary artery became a popular procedure to alleviate https://en.wikipedia.org/wiki/An…, the idea being restricting blood flow to a nearby region would improve supply going to the coronary arteries. Small placebo-controlled studies (39, 40) of this procedure found similar improvement (significantly less pain, lower nitroglycerine use) regardless whether patients got the ligation or just a skin incision.
  • Highly cited placebo-controlled studies (41, 42) on surgical Rx for osteoporosis found no difference in improvement magnitude (modest pain score improvement, physical function) between patients who got either https://en.wikipedia.org/wiki/Pe… (n=38, 68) or sham Rx (n=40, 63).
  • Highly cited placebo-controlled US-based studies (43, 44) on https://en.wikipedia.org/wiki/Os… found no difference in improvement magnitude between patients who got either arthroscopic debridement/ (https://en.wikipedia.org/wiki/Ar…), https://en.wikipedia.org/wiki/Ar… or simulated debridement (placebo, 3 small stab wounds in the knee). Even two years later, all patients had similar 6 point improvement on a 100 point scale that measured walking and bending. At the time these studies were done (1996-2002), 650,000 such arthroscopies were performed in the US each year, for an average cost of ~US $5000 each, obviously a colossal waste. An example of not the power of placebos but rather of uselessness of certain prevalent medical procedures.

* A preliminary study with few participants (n=47) that desperately needs replication. Also poor form to show data as Mean + Standard Error of the Mean (46). What that means is that actual variation within each group was much higher than what the authors chose to show, i.e., much more actual overlap between groups.

Bibliography

  1. Rief, Winfried, et al. “Meta-analysis of the placebo response in antidepressant trials.” Journal of affective disorders 118.1 (2009): 1-8. https://www.researchgate.net/pro…

2. Hróbjartsson, Asbjørn, and Peter C. Gøtzsche. “Placebo interventions for all clinical conditions.” The Cochrane Library (2010). http://nordic.cochrane.org/sites…

3. Howick, Jeremy, et al. “Are treatments more effective than placebos? A systematic review and meta-analysis.” PloS one 8.5 (2013): e62599. http://journals.plos.org/plosone…

4. Park, Chanmo, et al. “Blood sugar level follows perceived time rather than actual time in people with type 2 diabetes.” Proceedings of the National Academy of Sciences (2016): 201603444. http://www.pnas.org/content/113/…

5. Benedetti, Fabrizio, et al. “Open versus hidden medical treatments: the patient’s knowledge about a therapy affects the therapy outcome.” Prevention & Treatment 6.1 (2003): 1a.

6. Colloca, Luana, et al. “Overt versus covert treatment for pain, anxiety, and Parkinson’s disease.” The Lancet Neurology 3.11 (2004): 679-684. https://www.researchgate.net/pro…

7. Benedetti, F., E. Carlino, and A. Pollo. “Hidden administration of drugs.” Clinical Pharmacology & Therapeutics 90.5 (2011): 651-661. http://www.isdbweb.org/app/webro…

8. Schedlowski, Manfred, et al. “Neuro-bio-behavioral mechanisms of placebo and nocebo responses: implications for clinical trials and clinical practice.” Pharmacological reviews 67.3 (2015): 697-730. http://pharmrev.aspetjournals.or…

9. Enck, Paul, et al. “The placebo response in medicine: minimize, maximize or personalize?.” Nature reviews Drug discovery 12.3 (2013): 191-204.

10. Kaptchuk, Ted J., et al. “Placebos without deception: a randomized controlled trial in irritable bowel syndrome.” PloS one 5.12 (2010): e15591. http://journals.plos.org/plosone…

11. Carvalho, Cláudia, et al. “Open-label placebo treatment in chronic low back pain: a randomized controlled trial.” Pain 157.12 (2016): 2766. https://www.ncbi.nlm.nih.gov/pmc…

12. Welch, John S. “Ritual in western medicine and its role in placebo healing.” Journal of Religion and Health 42.1 (2003): 21-33.

13. The New York Times, Daniel Goleman, August 17, 1993. http://www.nytimes.com/1993/08/1…

14. Silberman, Steve. “Placebos are getting more effective. Drugmakers are desperate to know why.” Wired Magazine 17 (2009): 1-8. http://www.southdevonacupuncture…

15. Tuttle, Alexander H., et al. “Increasing placebo responses over time in US clinical trials of neuropathic pain.” Pain 156.12 (2015): 2616-2626.

16. Vase, Lene, Martina Amanzio, and Donald D. Price. “Nocebo vs. placebo: The challenges of trial design in analgesia research.” Clinical Pharmacology & Therapeutics 97.2 (2015): 143-150.

17. Quartz, Akshat Rathi, October 16, 2015. https://qz.com/525995/why-the-pl…

18. Walsh, B. Timothy, et al. “Placebo response in studies of major depression: variable, substantial, and growing.” Jama 287.14 (2002): 1840-1847. http://211.103.242.133:8080/ziyu…

19. Leucht, S., et al. “How effective are second-generation antipsychotic drugs? A meta-analysis of placebo-controlled trials.” Molecular psychiatry 14.4 (2009): 429-447. http://www.nature.com/mp/journal…

20. Sysko, Robyn, and B. Timothy Walsh. “A systematic review of placebo response in studies of bipolar mania.” The Journal of clinical psychiatry 68.8 (2007): 1213-1217.

21. Quessy, Steve N., and Michael C. Rowbotham. “Placebo response in neuropathic pain trials.” Pain 138.3 (2008): 479-483.

22. Vits, Sabine, et al. “Cognitive factors mediate placebo responses in patients with house dust mite allergy.” PLoS One 8.11 (2013): e79576. http://journals.plos.org/plosone…

23. Bartels, Danielle JP, et al. “Role of conditioning and verbal suggestion in placebo and nocebo effects on itch.” PloS one 9.3 (2014): e91727. http://journals.plos.org/plosone…

24. Quinn, Veronica F., and Ben Colagiuri. “Placebo interventions for nausea: a systematic review.” Annals of Behavioral Medicine 49.3 (2015): 449-462.

25. Amanzio, Martina, and Fabrizio Benedetti. “Neuropharmacological dissection of placebo analgesia: expectation-activated opioid systems versus conditioning-activated specific subsystems.” Journal of Neuroscience 19.1 (1999): 484-494.

26. Enck, Paul, Sibylle Klosterhalfen, and Katja Weimer. “Unsolved, Forgotten, and Ignored Features of the Placebo Response in Medicine.” Clinical Therapeutics (2016).

27. Buckalew, Louis W., and Kenneth E. Coffield. “An investigation of drug expectancy as a function of capsule color and size and preparation form.” Journal of clinical psychopharmacology 2.4 (1982): 245-248.

28. De Craen, Anton JM, et al. “Effect of colour of drugs: systematic review of perceived effect of drugs and of their effectiveness.” Bmj 313.7072 (1996): 1624-1626. http://pubmedcentralcanada.ca/pm…

29. De Craen, Anton JM, et al. “Placebo effect in the treatment of duodenal ulcer.” British journal of clinical pharmacology 48.6 (1999): 853-860. https://www.researchgate.net/pro…

30. de Craen, Anton JM, et al. “Placebo effect in the acute treatment of migraine: subcutaneous placebos are better than oral placebos.” Journal of neurology 247.3 (2000): 183-188.

31. Pressman, Alice, et al. “Adherence to placebo and mortality in the Beta Blocker Evaluation of Survival Trial (BEST).” Contemporary clinical trials 33.3 (2012): 492-498. https://www.ncbi.nlm.nih.gov/pmc…

32. Simpson, Scot H., et al. “A meta-analysis of the association between adherence to drug therapy and mortality.” Bmj 333.7557 (2006): 15. http://www.bmj.com/content/bmj/3…

33. Moerman, Daniel E. “Edible symbols: The effectiveness of placebos.” Annals of the New York Academy of Sciences 364.1 (1981): 256-268.

34. Sarles, H., R. Camatte, and J. Sahel. “A study of the variations in the response regarding duodenal ulcer when treated with placebo by different investigators.” Digestion 16.4 (1977): 289-292.

35. Bhugra, Dinesh, et al. “Colour, culture and placebo response.” International Journal of Social Psychiatry 61.6 (2015): 615-617.

36. Cattaneo, Angelo D., Paolo E. Lucchelli, and Giorgio Filippucci. “Sedative effects of placebo treatment.” European Journal of Clinical Pharmacology 3.1 (1970): 43-45.

37. de Craen, Anton JM, et al. “Placebo effect in the acute treatment of migraine: subcutaneous placebos are better than oral placebos.” Journal of neurology 247.3 (2000): 183-188.

38. MOERMAN, DE. “GENERAL MEDICAL EFFECTIVENESS AND HUMAN-BIOLOGY-PLACEBO-EFFECTS IN THE TREATMENT OF ULCER DISEASE.” AMERICAN JOURNAL OF PHYSICAL ANTHROPOLOGY. Vol. 54. No. 2. DIV JOHN WILEY & SONS INC, 605 THIRD AVE, NEW YORK, NY 10158-0012: WILEY-LISS, 1981.

39. Cobb, Leonard A., et al. “An evaluation of internal-mammary-artery ligation by a double-blind technic.” New England Journal of Medicine 260.22 (1959): 1115-1118.

40. Dimond, E. Grey, C. Frederick Kittle, and James E. Crockett. “Comparison of internal mammary artery ligation and sham operation for angina pectoris∗.” The American journal of cardiology 5.4 (1960): 483-486.

41. Buchbinder, Rachelle, et al. “A randomized trial of vertebroplasty for painful osteoporotic vertebral fractures.” New England Journal of Medicine 361.6 (2009): 557-568. http://www.nejm.org/doi/pdf/10.1…

42. Kallmes, David F., et al. “A randomized trial of vertebroplasty for osteoporotic spinal fractures.” New England Journal of Medicine 361.6 (2009): 569-579. http://www.nejm.org/doi/pdf/10.1…

43. Moseley Jr, J. Bruce, et al. “Arthroscopic treatment of osteoarthritis of the knee: a prospective, randomized, placebo-controlled trial: results of a pilot study.” The American journal of sports medicine 24.1 (1996): 28-34.

44. Moseley, J. Bruce, et al. “A controlled trial of arthroscopic surgery for osteoarthritis of the knee.” New England Journal of Medicine 347.2 (2002): 81-88. https://www.researchgate.net/pro…

https://www.quora.com/Is-the-placebo-effect-very-powerful/answer/Tirumalai-Kamala

How can I minimise pain after an intramuscular injection?

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Short answer

Pain a few days after an intramuscular (IM) injection could mean it wasn’t done properly. Question lacks the critical detail of injection site since there are four major sites of IM injection (see below from 1), and each has specific pros and cons, and different propensity for complications such as post-injection pain.

It’s the responsibility of the doctor who administered the injection to prescribe medications and/or other treatment to reduce post-injection pain.

Longer answer below for those interested in learning about IM injections in general, and sources of and approaches to minimize post-injection pain.

Brief Background On Intramuscular (IM) Injection Techniques

There are two main techniques used in IM injections to try to ensure injection is deposited in the muscle, and that injected material stays locked in it and doesn’t seep out into surrounding area along the needle track. They are the Z-track (see below from 2, 3) and air-lock techniques.

In the air-lock technique (3), a small amount of air is drawn up into the syringe along with the medication, the skin is stretched flat between two fingers and held taut, needle is plunged in at a right angle, injection includes medication followed by air, needle is withdrawn and taut skin is released. Rationale is the air locks in the medication in the muscle, hence air-lock, preventing it from seeping out into surrounding tissue along the needle track.

Possible Sources Of Pain After IM Injection

  • Aspiration is when the injector pushes the needle in but pulls back the syringe plunger before injecting the medication, rationale being to see if blood appears in the syringe meaning a blood vessel got punctured, i.e., need to try again. However, problems with aspiration are
    • No scientific support (4).
    • Can cause local tissue trauma and lingering pain (5, 6, 7, 8).
    • Is frequently done far too quickly to even be effective (4).
    • Isn’t recommended by National Immunization Technical Advisory Committees (9).
  • Inexperienced and/or unskilled injectors may end up injecting subcutaneously (SC) rather than IM, and cause undue local tissue trauma (10, 11).
  • Wrong choice of needle length can also increase chance of SC rather than IM injection. This is because ratio of subcutaneous to muscle varies by gender, age and weight, being higher in women (12, 13, 14, 15, 16, 17), older people and the obese (18, 19, 20, 21, 22). For example, a study infers a 12 to 25mm needle suffices for a thin person while a very obese person requires 76mm long needle for an IM injection (23).
  • Wiping needle with alcohol before injecting tracks it through the subcutaneous layer, which can be painful (24).

How To Minimize Pain After IM Injection

  • For something that’s a mainstay of routine medicine, shocking really that few sufficiently large, carefully controlled studies have compared different sites and techniques for their capacity to minimize pain after IM injection (25).
  • Consensus is slowly building that Ventrogluteal may cause less pain.
    • It isn’t close to large blood vessels, nerves, bone (1, 2, 10, 11, 25, 26, 27, 28, 29).
    • Being covered by relatively less subcutaneous tissue (15, 30) is another advantage since this reduces chances of accidental subcutaneous delivery (2, 27, 28).
    • However, there’s still insufficient conclusive data supporting it over Dorsogluteal (22).
  • OTOH, Dorsogluteal, though still much more commonly used compared to Ventrogluteal, has known risks such as
    • Sciatic nerve injury (26, 29).
    • Proximity to major blood vessels.
    • Increased thickness of subcutaneous tissue in this area compared to others (24), especially ventrogluteal (15, 30).
  • Injection techniques: A couple of small controlled studies (n=90 females aged between 18 and 60 years of age, 31; n=60; 32) suggest the air-lock technique can reduce tissue trauma and pain from IM injections.
  • Relaxed muscles can reduce injection site discomfort and pain (24). This means appropriately positioning the body, particularly the limbs, before IM injection. Studies (33, 34) suggest
    • Placing the hand on the hip relaxes the deltoid muscle.
    • Internal rotation of the femur relaxes the gluteal muscles.
  • No pain at initial point of needle contact on skin (24, 35). If there is pain, better to move needle 2 to 3mm at a time until reaching a painless point on the skin. Rationale is differential skin innervation, i.e., hitting upon a skin site with fewer or no pain receptors.
  • A randomized study (n=100) showed that changing needle after drawing up the medication and before injecting can minimize pain by ensuring needle tip used for injection remains sharp and free of medicine residue (36).
  • Briefly applying manual pressure to injection site before IM injection can minimize post-injection pain (6; n=48, 45 experimental and control, respectively, 37; n=74, one injection per arm, manual pressure randomly assigned, 38; n=63, 60 experimental and control, respectively, 39).

Bibliography

1. Ogston-Tuck, Sherri. “Intramuscular injection technique: an evidence-based approach.” Nursing Standard 29.4 (2014): 52-59.

2. Chadwick, Angelina, and Neil Withnell. “How to administer intramuscular injections.” Nursing Standard 30.8 (2015): 36-39.

3. Gabhann, L’am Mac. “A comparison of two depot injection techniques.” Nursing Standard 12.37 (1998): 39-41.

4. Ipp, Moshe, Jonathan Sam, and Patricia C. Parkin. “Needle aspiration and intramuscular vaccination.” Archives of pediatrics & adolescent medicine 160.4 (2006): 451-451.

5. Ipp, Moshe, et al. “Vaccine-related pain: randomised controlled trial of two injection techniques.” Archives of disease in childhood 92.12 (2007): 1105-1108.

6. Taddio, Anna, et al. “Physical interventions and injection techniques for reducing injection pain during routine childhood immunizations: systematic review of randomized controlled trials and quasi-randomized controlled trials.” Clinical Therapeutics 31 (2009): S48-S76.

7. Taddio, Anna, et al. “Reducing the pain of childhood vaccination: an evidence-based clinical practice guideline.” Canadian Medical Association Journal 182.18 (2010): E843-E855. https://www.researchgate.net/pro…

8. Davidson, Kathleen Marie, and Liam Rourke. “Teaching best-evidence: Deltoid intramuscular injection technique.” Journal of Nursing Education and Practice 3.7 (2013): 120. http://www.sciedupress.com/journ…

9. Cook, Ian F. “Best vaccination practice and medically attended injection site events following deltoid intramuscular injection.” Human vaccines & immunotherapeutics 11.5 (2015): 1184-1191. https://pdfs.semanticscholar.org…

10. Wynaden, Dianne, et al. “Establishing best practice guidelines for administration of intra muscular injections in the adult: a systematic review of the literature.” Contemporary nurse 20.2 (2005): 267-277.

11. Cocoman, A., and J. Murray. “Intramuscular injections: a review of best practice for mental health nurses.” Journal of psychiatric and mental health nursing 15.5 (2008): 424-434.

12. Haramati, Nogah, et al. “Injection granulomas. Intramuscle or intrafat?.” Archives of family medicine 3.2 (1994): 146-148.

13. Poland, Gregory A., et al. “Determination of deltoid fat pad thickness: implications for needle length in adult immunization.” Jama 277.21 (1997): 1709-1711. https://www.researchgate.net/pro…

14. Cook, I. F., M. Williamson, and D. Pond. “Definition of needle length required for intramuscular deltoid injection in elderly adults: an ultrasonographic study.” Vaccine 24.7 (2006): 937-940.

15. Chan, V. O., et al. “Intramuscular injections into the buttocks: are they truly intramuscular?.” European journal of radiology 58.3 (2006): 480-484.

16. Burbridge, Brent E. “Computed tomographic measurement of gluteal subcutaneous fat thickness in reference to failure of gluteal intramuscular injections.” Canadian Association of Radiologists Journal 58.2 (2007): 72. https://www.researchgate.net/pro…

17. Shankar, Nachiket, et al. “Influence of skin-to-muscle and muscle-to-bone thickness on depth of needle penetration in adults at the deltoid intramuscular injection site.” medical journal armed forces india 70.4 (2014): 338-343. http://medind.nic.in/maa/t14/i4/…

18. Greenway, Kathleen. “Using the ventrogluteal site for intramuscular injection.” Nursing Standard 18.25 (2004): 39-42. https://www.researchgate.net/pro…

19. Nisbet, Andrew Charles. “Intramuscular gluteal injections in the increasingly obese population: retrospective study.” Bmj 332.7542 (2006): 637-638. http://www.bmj.com/content/bmj/3…

20. Zaybak, Ayten, et al. “Does obesity prevent the needle from reaching muscle in intramuscular injections?.” Journal of advanced nursing 58.6 (2007): 552-556.

21. Sakamaki, Sakiko, et al. “The relationship between body mass index, thickness of subcutaneous fat, and the gluteus muscle as the intramuscular injection site.” Health 2013 (2013). http://file.scirp.org/pdf/Health…

22. Brown, Joe, Mark Gillespie, and Simon Chard. “The dorso–ventro debate: in search of empirical evidence.” British Journal of Nursing 24.22 (2015): 1132-1139. https://www.guidelines.ch/file/g…

23. Kaya, Nurten, et al. “The reliability of site determination methods in ventrogluteal area injection: A cross-sectional study.” International journal of nursing studies 52.1 (2015): 355-360. https://www.researchgate.net/pro…

24. Nicoll, Leslie H., and Amy Hesby. “Intramuscular injection: an integrative research review and guideline for evidence-based practice.” Applied Nursing Research 15.3 (2002): 149-162. http://lms.westernhealth.nl.ca/s…

25. Malkin, Bridget. “Are techniques used for intramuscular injection based on research evidence.” Nursing times 104.50/51 (2008): 48-51. https://www.choiceforum.org/docs…

26. Small, Sandra P. “Preventing sciatic nerve injury from intramuscular injections: literature review.” Journal of advanced nursing 47.3 (2004): 287-296.

27. Ferreira Oliveira, Laura, et al. “Ventrogluteal region, an alternative location to apply benzathine penicillin.” Revista Eletronica de Enfermagem 17.4 (2015). https://www.fen.ufg.br/revista/v…

28. Coskun, Halise, Cenk Kilic, and Cicek Senture. “The evaluation of dorsogluteal and ventrogluteal injection sites: a cadaver study.” Journal of clinical nursing (2016).

29. Mishra, P., and M. D. Stringer. “Sciatic nerve injury from intramuscular injection: a persistent and global problem.” International journal of clinical practice 64.11 (2010): 1573-1579.

30. Beecroft, P. C., and S. A. Redick. “Clarification of ventrogluteal site.” Pediatric nursing 16.4 (1989): 396-396.

31. Najafidolatabad, Shahla, Janmohamad Malekzadeh, and Zinat Mohebbinovbandegani. “Comparison of the pain severity, drug leakage and ecchymosis rates caused by the application on tramadol intramuscular injection in Z-track and Air-lock techniques.” Investigación y Educación en Enfermería 28.2 (2010): 24-33. https://www.researchgate.net/pro…

32. Yilmaz, Dilek K., et al. “The effect of air-lock technique on pain at the site of intramuscular injection.” Saudi medical journal 37.3 (2016): 304. https://www.ncbi.nlm.nih.gov/pmc…

33. Kruszwski, Ann Z., Susan Havens Lang, and Jean E. Johnson. “Effect of positioning on discomfort from intramuscular injections in the dorsogluteal site.” Nursing Research 28.2 (1979): 103-105.

34. Rettig, Frannie M., and Janet R. Southby. “Using different body positions to reduce discomfort from dorsogluteal injection.” Nursing Research 31.4 (1982): 219-221.

35. Jablecki, C. K. “Alternative technique for medication injections.” Nursing research 49.5 (2000): 244.

36. Ağaç, Emine, and Ülkü Yapucu Güneş. “Effect on pain of changing the needle prior to administering medicine intramuscularly: a randomized controlled trial.” Journal of advanced nursing 67.3 (2011): 563-568. http://www.hadassah.org.il/media…

37. Barnhill, Barbara J., et al. “Using pressure to decrease the pain of intramuscular injections.” Journal of pain and symptom management 12.1 (1996): 52-58.

38. Chung, Joanne WY, Winnie MY Ng, and Thomas KS Wong. “An experimental study on the use of manual pressure to reduce pain in intramuscular injections.” Journal of clinical nursing 11.4 (2002): 457-461.

39. Öztürk, Deniz, et al. “The effect of the application of manual pressure before the administration of intramuscular injections on students’ perceptions of postinjection pain: a semi‐experimental study.” Journal of Clinical Nursing (2016).

https://www.quora.com/How-can-I-minimise-pain-after-an-intramuscular-injection/answer/Tirumalai-Kamala

Is 16s rRNA sequencing a sound approach to studying bacterial communities?

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Suppose the task were to identify in a lake all the different types of fish as well as their relative abundance. Somewhat analogously 16S rRNA (16S ribosomal RNA – Wikipedia) and metagenomic sequencing (Metagenomics – Wikipedia) try to do likewise for microbial communities in various habitats. 16S rRNA is akin to doing it with a bunch of fishing rods, i.e., pool of PCR (Polymerase chain reaction – Wikipedia) Primer (molecular biology) – Wikipedia specific for defined bacterial 16S rRNA genes, while metagenomic sequencing is akin to a dragnet, high throughput sequencing of entire microbial genes, even genomes. Both start with DNA extracted from a sample followed by mapping to reference databases and annotation (see below from 1).

In that light, 16S rRNA is more a ‘biological fingerprint‘ (2) for bacterial species while metagenomics analyzes a given biological (human poop, skin swab, etc.) or environmental (soil, aquifer, etc.) sample more thoroughly and comprehensively and thus generates a more accurate snapshot of not just the bacteria (3) but also archaea, eukaryotes, fungi, protists and viruses (2) present in it.

Other problems with 16S rRNA analysis include

  • Greater scope for introducing bias since
    • Certain 16S rRNA sequences may be amplified more efficiently compared to others as different bacterial species carry different numbers of 16S rRNA genes (4) while copy numbers aren’t even known for many species, especially those found during human microbiome analyses since many of these species are novel, haven’t been cultured and may likely be ‘non-culturable’ using standard bacteriological techniques.
    • Chimeric amplification products can skew results (5) but the microbiome field is plagued with weak adherence to relatively easy technical fixes that could prevent such artifacts from occurring in the first place (6).
  • Can’t be used to identify to species level since many bacterial species have identical 16S rRNA genes, for example some Anthrax species (2).

Major problem with metagenomics is cost as in still rather prohibitively expensive.

16S rRNA Sequencing & Metagenomics Share Many Critical Bias-introducing Steps

However, debating the relative merits of 16S rRNA versus metagenomics risks missing the forest for the trees since both approaches share many critical bias-introducing steps (see below from 7).

  • These include study design (3), sample collection, processing, storage, DNA extraction and sequencing, all of which can introduce a variety of biases and errors over and above those inherent to the two techniques.
    • Differences in sample collection (8, 9), processing (10), transport, storage (11, 12, 13) and DNA extraction (14, 15) can change microbiome results.
    • Even different sequencing platforms can yield different results from the same samples (16).
    • Not only are reference databases incomplete (17), commonly used computational models vary in sensitivity and precision (18).
    • Both 16S rRNA and metagenomics also share another error common to any rapidly expanding scientific field, namely problematic assumptions that underlie the complex statistical approaches necessary for analysis of the colossal amount of data generated by such techniques. For example, a commonly used approach, Rarefaction (ecology) – Wikipedia, has the potential to dramatically skew false positive and false negative rates (19).
  • In other words, even as the microbiome field explodes and is therefore subject to constant technological innovation, critical quality control measures such as development of standardized protocols haven’t kept pace (20, 21). Since different published studies use different methods, results can’t be compared across studies either (17, 22).
  • Most microbiome studies consist of small numbers of subjects. Such studies have inherently poor statistical power meaning difficult to be conclusive about a particular hypothesis. After all, when diet (23) and location (24) dramatically change snapshot reads of microbiome composition of a single individual, stands to reason that each microbiome study be considered a stand-alone piece of data and yet it’s become standard to wildly extrapolate and exaggerate results from one small-scale study to an entire field or even to human physiology itself (7).
  • Currently many microbiome studies also do an extremely poor job of accounting for confounding factors when comparing microbiome differences between two groups of individuals, regardless of the method used to assess it in the first place.
    • An illustrative example of such errors, one study (25) reported that rugby players (n=40) have more diverse microbiome compared to age-matched controls (n=23, 23). Authors as well as popular press interpreted this apparent difference as proof that ‘exercise increases gut microbial diversity in humans‘ (25). Really? Exercise was the only difference between those two groups? Not diet, not more extensive contact with soil, to mention just two plausible differences between rugby players and age-matched controls (7)?
  • In other words, many microbiome studies continue making the fatal error of conflating correlation with causation.
  • Sample replicates are vital in attesting to the robustness of experimental approaches, especially so for extremely sensitive molecular biological techniques such as 16S rRNA sequencing or metagenomics. Vastly expanded sensitivity greatly increases the burden of separating signal from noise (26). A most crucial quality-control step, replication, i.e., test the same sample multiple times, thus becomes critical in helping to better discriminate signal from noise. Currently, microbiome research tends to woefully neglect sample replicates (see table below from 27).

Prosser rightfully nails the critical problem of sample numbers and replicates on its head thus (27),

‘At the moment, if I measured the height of one Englishmen, one American, one Australian and one African and then used the data to postulate ways in which height was determined by continent I would, correctly, be ridiculed. If I were to take a single faecal sample from each of the same individuals and performed massive, parallel, high throughput, 454-sequencing to generate sequence lists, relative abundances of different phylogenetic groups and pie-charts, and used these data to postulate continent associated differences in intestinal microbial communities, it is likely that the paper would be published in one of the highest profile, general science journals. There are already several precedents for this and, as a community, we should be embarrassed.’

Seven years on from the above comment about this predicament, little has changed regarding this status quo even as microbiome research has vastly expanded in scope, entering any and all areas of research on human physiology and disease.

Bibliography

1. Jonsson, Viktor. Statistical analysis and modelling of gene count data in metagenomics. Diss. Göteborgs universitet, 2017. https://gupea.ub.gu.se/bitstream…

2. Carlos, Nossa, Yi-Wei Tang, and Zhiheng Pei. “Pearls and pitfalls of genomics-based microbiome analysis.” Emerging Microbes and Infections 1 (2012): e45. https://www.researchgate.net/pro…

3. Meisel, Jacquelyn S., et al. “Skin microbiome surveys are strongly influenced by experimental design.” Journal of Investigative Dermatology 136.5 (2016): 947-956. https://www.med.upenn.edu/gricel…

4. Pei, Anna Y., et al. “Diversity of 16S rRNA genes within individual prokaryotic genomes.” Applied and environmental microbiology 76.12 (2010): 3886-3897. Diversity of 16S rRNA Genes within Individual Prokaryotic Genomes

5. Boers, Stefan A., John P. Hays, and Ruud Jansen. “Micelle PCR reduces chimera formation in 16S rRNA profiling of complex microbial DNA mixtures.” Scientific reports 5 (2015). https://www.ncbi.nlm.nih.gov/pmc…

6. Gohl, Daryl M., et al. “Systematic improvement of amplicon marker gene methods for increased accuracy in microbiome studies.” Nature Biotechnology 34.9 (2016): 942-949. http://metagenome.cs.umn.edu/pub…

7. Bik, Elisabeth M. “Focus: Microbiome: The Hoops, Hopes, and Hypes of Human Microbiome Research.” The Yale Journal of Biology and Medicine 89.3 (2016): 363. https://www.ncbi.nlm.nih.gov/pmc…

8. Dominianni, Christine, et al. “Comparison of methods for fecal microbiome biospecimen collection.” BMC microbiology 14.1 (2014): 103.

9. Sinha, Rashmi, et al. “Collecting fecal samples for microbiome analyses in epidemiology studies.” Cancer Epidemiology and Prevention Biomarkers 25.2 (2016): 407-416. http://cebp.aacrjournals.org/con…

10. Hsieh, Yu-Hsin, et al. “Impact of Different Fecal Processing Methods on Assessments of Bacterial Diversity in the Human Intestine.” Frontiers in Microbiology 7 (2016). https://www.ncbi.nlm.nih.gov/pmc…

11. Choo, Jocelyn M., Lex EX Leong, and Geraint B. Rogers. “Sample storage conditions significantly influence faecal microbiome profiles.” Scientific reports 5 (2015): 16350. Sample storage conditions significantly influence faecal microbiome profiles

12. Klymiuk, Ingeborg, et al. “16S based microbiome analysis from healthy subjects’ skin swabs stored for different storage periods reveal phylum to genus level changes.” Frontiers in Microbiology 7 (2016). https://www.ncbi.nlm.nih.gov/pmc…

13. Song, Se Jin, et al. “Preservation methods differ in fecal microbiome stability, affecting suitability for field studies.” mSystems 1.3 (2016): e00021-16. http://msystems.asm.org/content/…

14. Kennedy, Nicholas A., et al. “The impact of different DNA extraction kits and laboratories upon the assessment of human gut microbiota composition by 16S rRNA gene sequencing.” PloS one 9.2 (2014): e88982. http://journals.plos.org/plosone…

15. Wesolowska-Andersen, Agata, et al. “Choice of bacterial DNA extraction method from fecal material influences community structure as evaluated by metagenomic analysis.” Microbiome 2.1 (2014): 19. Choice of bacterial DNA extraction method from fecal material influences community structure as evaluated by metagenomic analysis

16. Hahn, Andrea, et al. “Different next generation sequencing platforms produce different microbial profiles and diversity in cystic fibrosis sputum.” Journal of Microbiological Methods 130 (2016): 95-99.

17. Amato, Katherine R. “An introduction to microbiome analysis for human biology applications.” American Journal of Human Biology (2016). http://onlinelibrary.wiley.com/d…

18. Weiss, Sophie, et al. “Correlation detection strategies in microbial data sets vary widely in sensitivity and precision.” The ISME journal (2016). https://www.researchgate.net/pro…

19. McMurdie, Paul J., and Susan Holmes. “Waste not, want not: why rarefying microbiome data is inadmissible.” PLoS Comput Biol 10.4 (2014): e1003531. http://journals.plos.org/ploscom…

20. Salter, Susannah J., et al. “Reagent and laboratory contamination can critically impact sequence-based microbiome analyses.” BMC biology 12.1 (2014): 87. Reagent and laboratory contamination can critically impact sequence-based microbiome analyses

21. Clavel, Thomas, Ilias Lagkouvardos, and Andreas Hiergeist. “Microbiome sequencing: challenges and opportunities for molecular medicine.” Expert review of molecular diagnostics 16.7 (2016): 795-805.

22. Lozupone, Catherine A., et al. “Meta-analyses of studies of the human microbiota.” Genome research 23.10 (2013): 1704-1714. https://www.researchgate.net/pro…

23. David, Lawrence A., et al. “Diet rapidly and reproducibly alters the human gut microbiome.” Nature 505.7484 (2014): 559-563. https://www.researchgate.net/pro…

24. Tirumalai Kamala’s answer to What effect can a change of location have on a person’s microbiome?

25. Clarke, Siobhan F., et al. “Exercise and associated dietary extremes impact on gut microbial diversity.” Gut (2014): gutjnl-2013. https://www.researchgate.net/pro…

26. Knight, Rob, et al. “Unlocking the potential of metagenomics through replicated experimental design.” Nature biotechnology 30.6 (2012): 513-520. http://citeseerx.ist.psu.edu/vie…

27. Prosser, James I. “Replicate or lie.” Environmental microbiology 12.7 (2010): 1806-1810. https://www.io-warnemuende.de/tl…

https://www.quora.com/Is-16s-rRNA-sequencing-a-sound-approach-to-studying-bacterial-communities/answer/Tirumalai-Kamala

What is the difference between sufficient and necessary in biology?

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Necessary and sufficient are elements essential for constructing causality in biology.

One way to contemplate necessary and sufficient in biology would be to consider whether suspected cause always precedes outcome (is it necessary) and whether suspected cause always produces outcome (is it sufficient).

Brief History Of Causality In Biomedicine

How to separate chance from causation in biology? Consider aspirin and pain. Have pain, take aspirin, pain disappears, i.e., clearly sequential, a key precept of causality (2). Though swallowing aspirin doesn’t always yield pain relief, it has done so enough times in enough people for their association to be not by chance or coincidence.

Causality entered the realm of biomedicine with the Henle-Koch postulates (Koch’s postulates – Wikipedia), commonly attributed to Friedrich Gustav Jakob Henle – Wikipedia in 1840 and Robert Koch, his student, in 1890 (3). These postulates were instrumental in illuminating the causal relationship between a particular microbe and a specific disease, and in bringing scientific rigor to medicine through the notion of causality. Though these postulates haven’t withstood the test of time unchanged, having undergone numerous revisions and becoming studded with umpteen caveats (4, 5, 6, 7), nevertheless they epitomize a revolutionary scientific breakthrough that eventually transformed medicine. As Robert Sackstein states (8),

‘Koch’s postulates infused scientific rigor into medicine, altering the cultural foundation of medical science from that of observation/association/correlation towards one grounded in causal relationships yielding mechanistic insights’

A recent salient example is that of the 2005 Physiology/Medicine Nobel Prize winner Barry Marshall – Wikipedia portrayed in the unabashedly hagiographic dramatization below (9) as a sort of ultimate truth-seeker who didn’t hesitate to infect himself to prove Helicobacter pylori – Wikipedia caused peptic ulcer.

Today this notion of causality permeates the entire biomedical research landscape, bestowing primacy to questions of mechanism of action. Even in Epidemiology – Wikipedia where the notion of causality typically remains foolhardy at the best of the times, postulates such as the Bradford Hill criteria – Wikipedia, originally formulated by Austin Bradford Hill – Wikipedia, attempt to decipher a causal association from among multiple factors and a particular biological outcome, typically disease (10).

Problems With Biological Causality Or Why Necessary And Sufficient Came To Be

In its simplest form, causality can be stated as A causes B. For example, step under a waterfall (A), get wet (B). However, such relatively simplistic, linear relationships are usually outliers and not the norm in biology.

  • Imagine a scenario where a particular bacterium must acquire a Pathogenicity island – Wikipedia through horizontal gene transfer of discrete gene segments to become virulent (necessary) to a particular animal and doing so should always yield Virulence – Wikipedia (sufficient). Sufficiency is thus a minimum requirement to recapitulate a biological feature, in this case virulence.
  • Acquisition of a pathogenicity island in this particular case could be both necessary and sufficient to bestow virulence if experiments showed inactivation of genes within it caused a measurable loss of virulence (11).

Causality becomes even trickier to decipher in epidemiology.

  • For example, a relationship between smoking (A) and lung cancer (B) is now well-established. However, to say smoking causes lung cancer isn’t strictly accurate because not every smoker develops lung cancer, which by the way can also occur in those who never smoked. The reality is smoking isn’t sufficient to induce lung cancer but can increase the probability of developing it. Thus, smoking could be somewhat necessary but not sufficient to cause lung cancer, simply because it’s more common among smokers than non-smokers.
  • Another cause and effect example from cancer is the case of HPV (Human papillomavirus infection – Wikipedia) (A) and Cervical cancer – Wikipedia (B), where the association is much stronger though still not absolute, since not all HPV infections result in cervical cancer. The WHO states (12)

‘Nearly all cases of cervical cancer can be attributable to HPV infection’ .

Such examples thus help break down biological causality into two distinct components,

  • Does HPV (A) always precede cervical cancer (B), i.e., issue of necessity? Here the answer is a qualified yes.
  • Does HPV (A) always produce cervical cancer (B), i.e., issue of sufficiency? Here the answer is no.

In a recent example, by painstakingly knocking out ~5400 genes in yeast two at a time, creating ~23 million yeast strains over a 17-year odyssey, biologists at University of Toronto identified those genes that when removed as pairs, resulted in sickness or death (13, see below from 14), i.e., both necessary and sufficient for viability. ~1000 yeast genes are already known to be essential for viability individually. This process uncovered ~3300 additional genes.

By assessing the relative contribution of each factor involved in a particular biological process and thereby helping to more accurately pinpoint the nature of their involvement, necessary and sufficient become the critical means for attempting to establish biological causality. This is because unlike the examples that made 19th century Bacteriology its Golden Age, most biological phenomena and diseases cannot be reduced to a simple, linear cause and effect between one particular agent (cause, A) and biological outcome (effect, B). Rather, most entail the interplay of multiple, overlapping, maybe even redundant, not necessarily contemporaneous factors where many individual ones may be necessary but not sufficient.

Causality’s Value In Biomedicine

The English philosopher R. G. Collingwood – Wikipedia stated (15),

‘the cause of an event in nature is the handle so to speak, by which we can manipulate it’

The hope is the greater details with which biological factors can be parsed as necessary and/or sufficient, greater the capacity to better understand where, when, how to act on, intervene in and/or manipulate what (16) to better prevent, treat or cure illnesses.

Bibliography

1. Hara, Kenta, et al. “Amino acid sufficiency and mTOR regulate p70 S6 kinase and eIF-4E BP1 through a common effector mechanism.” Journal of Biological Chemistry 273.23 (1998): 14484-14494. Amino Acid Sufficiency and mTOR Regulate p70 S6 Kinase and eIF-4E BP1 through a Common Effector Mechanism

2. Morabia, Alfredo. “Hume, Mill, Hill, and the sui generis epidemiologic approach to causal inference.” American journal of epidemiology 178.10 (2013): 1526-1532. Hume, Mill, Hill, and the Sui Generis Epidemiologic Approach to Causal Inference

3. Evans, Alfred S. “Causation and disease: A chronological journey The Thomas Parran Lecture.” American Journal of Epidemiology 108.4 (1978): 249-258.

4. Fredericks, D. N., and David A. Relman. “Sequence-based identification of microbial pathogens: a reconsideration of Koch’s postulates.” Clinical microbiology reviews 9.1 (1996): 18-33. http://cmr.asm.org/content/9/1/1…

5. Falkow, Stanley. “Molecular Koch’s postulates applied to bacterial pathogenicity—a personal recollection 15 years later.” Nature Reviews Microbiology 2.1 (2004): 67-72. http://faculty.ucmerced.edu/kjen…

6. Gradmann, Christoph. “A spirit of scientific rigour: Koch’s postulates in twentieth-century medicine.” Microbes and Infection 16.11 (2014): 885-892.

7. Byrd, Allyson L., and Julia A. Segre. “Adapting Koch’s postulates.” Science 351.6270 (2016): 224-226.

8. Sackstein, Robert. “Fulfilling Koch’s postulates in glycoscience: HCELL, GPS and translational glycobiology.” Glycobiology (2016): cww026. HCELL, GPS and Translational Glycobiology

9. Marshall, Barry. “Helicobacter connections.” ChemMedChem 1.8 (2006): 783-802. https://www.researchgate.net/pro…

10. Hill, Austin Bradford. “The environment and disease: association or causation?.” Proceedings of the Royal society of Medicine 58.5 (1965): 295. https://www.ncbi.nlm.nih.gov/pmc…

11. Falkow, Stanley. “Molecular Koch’s postulates applied to microbial pathogenicity.” Review of Infectious Diseases 10.Supplement 2 (1988): S274-S276. http://mbib.med.harvard.edu/page…

12. Human papillomavirus (HPV) and cervical cancer

13. Costanzo, Michael, et al. “A global genetic interaction network maps a wiring diagram of cellular function.” Science 353.6306 (2016): aaf1420. A global genetic interaction network maps a wiring diagram of cellular function

14. Giant Genetic Map Shows Life’s Hidden Links | Quanta Magazine

15. Collingwood, R. G. “Causation in practical natural science.” RG. Collingwood, An Essay on Metaphysics. Revised Edition. R Martin, ed (1940): 286-312.

16. Gillies, Donald Angus. “Establishing Causality in Medicine and Koch’s Postulates.” (2016). http://ijhpm.org/index.php/IJHPM…

 

Should blood banks be testing for Zika?

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Recent data suggest blood transfusions could transmit Zika so yes, blood banks in Zika-endemic areas should test for it. The link between maternal Zika infection and baby microcephaly (1) and other serious birth defects (2) is now compelling. Were Zika-infected blood transfused to pregnant women, risk could be unacceptably high for fetuses.

Though Zika fever is a Vector (epidemiology) -borne disease spread by the bite of infected Aedes mosquitoes, a steady drumbeat of data shows it can also spread by other routes.

Zika persists in semen (3, 4), vaginal tract (5, 6) and in circulation of pregnant women (7) for surprisingly long periods of time, and can be transmitted sexually (8, 9, 10, 11). Since Zika’s asymptomatic in ~80% of those infected, Zika-infected blood transfusions could be a risk factor to fetuses not just if given to pregnant women but also if given to their sexual partners.

Preliminary data suggest blood transfusion could transmit Zika

  • During the 2013-2014 French Polynesia Zika virus outbreak it was detected by Reverse transcription polymerase chain reaction (RT-PCR) in 42 of 1505 (2.8%) blood donors who were asymptomatic when they donated (12). 11 of these 14 reported having Zika fever-like syndrome about 3 to 10 days later.
  • In the US territory of Puerto Rico, a total of 68 of 12777 (~0.5%) blood donations from April 3 to June 11, 2016 were identified as presumptive Zika viremic based on Nucleic acid test (NAT) with rates rising by ~2.2 over a 9 week period during the summer (see figure below from 13).

These studies revealed Zika’s potential for spreading through blood transfusion.

More recently, a couple of case reports suggest blood transfusions could indeed transmit Zika

  • In Brazil (14), concentrated Platelet from the blood of an asymptomatic 54 year old man was transfused into a 55 year old liver cancer patient undergoing liver transplant.
    • Four days post-transplant, the transfused recipient was serum Zika virus positive by RT-PCR.
    • Suspicion fell on the platelet donor after he contacted the blood donor facility 3 days after his donation to report he’d just developed dengue-like symptoms. His stored serum sample was then tested and found positive not for Dengue but for Zika virus by RT-PCR.
    • Though source of recipient’s Zika could have been other transplant related tissues and blood products, sequence analysis of 10 partial nucleotide sequences of the Zika virus isolated from the donor compared with the complete genome sequence of that isolated from the recipient matched 99.8%, strongly suggesting Zika transmission through transfusion.
    • Recipient coming from a Zika non-epidemic area and being hospitalized in a mosquito-free area for 5 days before his Zika positive test further increased the likelihood he got Zika from his platelet transfusion.
  • In Brazil again (15), an asymptomatic person donated platelets through Apheresis on January 16, 2016. These were transfused into two different patients on January 19.
    • On January 21, the donor called the blood bank to report Zika symptoms (skin rash, eye pain, pain in both knees) starting on January 18.
    • Donor samples before and after donation were negative for related Dengue virus and Chikungunya by RT-PCR. However, donor’s plasma and urine samples were Zika positive 14 days after initial blood donation.
    • Plasma samples from both recipients were Zika positive, 6 and 23 days post-transfusion, respectively.

Given the high risk of newborn microcephaly from maternal Zika infection, far better to err on the side of caution and start screening blood donations for Zika. This may be why on August 26, 2016, the US FDA (16, emphasis mine)

issued a revised guidance recommending universal testing of donated Whole Blood and blood components for Zika virus in the U.S. and its territories‘.

Based on the available evidence the FDA concluded (17)

ZIKV meets the conditions for an RTTI [relevant transfusion-transmitted infection ] as described in 21 CFR 630.3(h)(2)

Specifically (17, emphasis mine),

FDA has determined that ZIKV meets the criteria in 21 CFR 630.3(h)(2) for an RTTI because of the sufficient incidence and prevalence of ZIKV to affect the potential donor population in the United States and because of the availability of appropriate screening tests for ZIKV‘ .

Bibliography

1. Johansson, Michael A., et al. “Zika and the Risk of Microcephaly.” New England Journal of Medicine (2016). http://www.nejm.org/doi/pdf/10.1…

2. Rasmussen, Sonja A., et al. “Zika virus and birth defects—reviewing the evidence for causality.” New England Journal of Medicine 374.20 (2016): 1981-1987. http://www.nejm.org/doi/pdf/10.1…

3. Mansuy, Jean Michel, et al. “Zika virus: high infectious viral load in semen, a new sexually transmitted pathogen.” Lancet Infect Dis 16.405 (2016): 00138-9. https://www.researchgate.net/pro…

4. Barzon, L., et al. “Infection dynamics in a traveller with persistent shedding of Zika virus RNA in semen for six months after returning from Haiti to Italy, January 2016.” Euro surveillance: bulletin Européen sur les maladies transmissibles= European communicable disease bulletin 21.32 (2016). http://www.eurosurveillance.org/…

5. Davidson, Alexander. “Suspected female-to-male sexual transmission of Zika virus—New York City, 2016.” MMWR. Morbidity and Mortality Weekly Report 65 (2016). http://www.cdc.gov/mmwr/volumes/…

6. Prisant, N. et al. Zika virus in the female genital tract. Lancet Infectious Diseases, 2016, July 11. http://www.thelancet.com/pdfs/jo…

7. Meaney-Delman, Dana, et al. “Prolonged Detection of Zika Virus RNA in Pregnant Women.” Obstetrics & Gynecology (2016). Prolonged Detection of Zika Virus RNA in Pregnant Women. : Obstetrics & Gynecology

8. Foy, Brian D., et al. “Probable non-vector-borne transmission of Zika virus, Colorado, USA.” Emerg Infect Dis 17.5 (2011): 880-2. https://www.researchgate.net/pro…

9. Coelho, Flávio Codeço, et al. “Sexual transmission causes a marked increase in the incidence of Zika in women in Rio de Janeiro, Brazil.” bioRxiv (2016): 055459. http://www.biorxiv.org/content/b…

10. D’Ortenzio, Eric, et al. “Evidence of sexual transmission of Zika virus.” New England Journal of Medicine 374.22 (2016): 2195-2198. http://www.nejm.org/doi/pdf/10.1…

11. Fréour, Thomas, et al. “Sexual transmission of Zika virus in an entirely asymptomatic couple returning from a Zika epidemic area, France, April 2016.” Eurosurveillance 21.23 (2016). http://www.e-sciencecentral.org/…

12. Musso, D., et al. “Potential for Zika virus transmission through blood transfusion demonstrated during an outbreak in French Polynesia, November 2013 to February 2014.” Euro Surveill 19.14 (2014): 20761. http://www.eurosurveillance.org/…

13. Kuehnert, Matthew J. “Screening of blood donations for Zika virus infection—Puerto Rico, April 3–June 11, 2016.” MMWR. Morbidity and Mortality Weekly Report 65 (2016). https://www.cdc.gov/mmwr/volumes…

14. Barjas‐Castro, Maria L., et al. “Probable transfusion‐transmitted Zika virus in Brazil.” Transfusion 56.7 (2016): 1684-1688. http://onlinelibrary.wiley.com/d…

15. Motta, Iara JF, et al. “Evidence for Transmission of Zika Virus by Platelet Transfusion.” New England Journal of Medicine (2016). http://www.nejm.org/doi/pdf/10.1…

16. FDA advises testing for Zika virus in all donated blood and blood components in the US

17. http://www.fda.gov/downloads/Bio…

https://www.quora.com/Should-blood-banks-be-testing-for-Zika/answer/Tirumalai-Kamala

Why is it difficult to develop a serological (blood) test to detect antibodies to the Zika virus?

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Zika virus – Wikipedia, Dengue virus – Wikipedia, West Nile fever – Wikipedia, Chikungunya – Wikipedia, Yellow fever – Wikipedia belong to the same virus family, Flavivirus – Wikipedia and are endemic (Endemism – Wikipedia) in overlapping regions of the world.

  • One consequence of these viruses being related is structural similarity of their Antigen – Wikipedia. This in turn means antibodies against one of these flaviviruses can cross-react (Cross-reactivity – Wikipedia) with another flavivirus.
  • Chances of such antibody cross-reactivity increase in regions endemic for these flaviviruses since a person suspected of being currently infected with Zika in Brazil for example may in the past have been infected with Dengue or Chikungunya or may have been vaccinated with Yellow fever vaccine – Wikipedia.

Under such circumstances, it becomes difficult to conclude whether Zika-reactive antibody detected in a current serological (blood) test truly indicates current Zika infection. In other words, are such antibodies Zika-specific or cross-reactive to antigen bits (Epitope – Wikipedia) Zika shares with other flaviviruses such as Dengue?

Thus, structural similarity with other related viruses and spread across similar geographic regions of the world, these are the two main factors that make a serological (blood) diagnosis test for Zika-specific antibodies particularly thorny. This is why Zika diagnosis gold standard currently consists of Reverse transcription polymerase chain reaction – Wikipedia (RT-PCR) for Zika virus RNA in serum or urine collected < 2 weeks after symptom onset (1).

That said, a study published in Science in July 2016 (2) isolated novel antibody candidates from Zika-infected patients. While this study found that most of the circulating antibodies elicited by Zika in these patients cross-reacted with Dengue, antibodies against Zika’s non-structural protein 1 (NS1) were largely Zika-specific.

  • These results suggest that assays to identify and measure antibodies specific for Zika NS1 might have specific diagnostic potential for Zika.
  • Of course, it remains to be seen if other studies concur with these results.
  • Also important to keep in mind that cross-reactivity with Dengue may not be the only confounder. Rather depending on the region and an individual’s life history, cross-reactivity with Chikungunya, Yellow fever, West Nile fever, etc., may also need to be ruled out in serological (blood) antibody-based diagnostic tests.

Bibliography

1. Oduyebo, Titilope. “Update: interim guidance for health care providers caring for pregnant women with possible Zika virus exposure—United States, July 2016.” MMWR. Morbidity and Mortality Weekly Report 65 (2016). https://www.cdc.gov/mmwr/volumes…

2. Stettler, Karin, et al. “Specificity, cross-reactivity and function of antibodies elicited by Zika virus infection.” Science (2016): aaf8505. http://icmr.nic.in/zika/publicat…

https://www.quora.com/Why-is-it-difficult-to-develop-a-serological-blood-test-to-detect-antibodies-to-the-Zika-virus/answer/Tirumalai-Kamala

If a child is conceived while the father has Zika, but the mother never gets infected, can the fetus be affected?

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Though bite of infected Aedes – Wikipedia mosquito is its primary mode of spread, since 2011, several case reports have

  • Suggested Zika can be sexually transmitted.
  • Documented Zika’s prolonged presence in semen of infected men.

In August 2016, the WHO (1) listed a total of 17 published reports on possible sexual transmission of Zika and 8 others on its presence in semen. Thus, as of Oct 2016, there’s plenty of scientific evidence that it’s possible for a Zika-infected man to infect a previously uninfected woman during sexual intercourse, in which case a fetus could also be affected. In other words, counting on a woman remaining uninfected following sex with a Zika-infected man is highly risky, especially for the fetus.

Reports Of Sexual Transmission From Symptomatic Males To Females

In each of these cases, the previously uninfected women developed symptoms suggestive of Zika even though they hadn’t been exposed to Zika-infected mosquitoes, i.e., highly likely they were infected through sexual transmission.

Cases reported from Argentina and France (2), Canada (3), Chile (4), France, where in one case the suspected route was oral sex (5) while the other case suggested male to female sexual transmission occurred 32 to 41 days after the man got infected with Zika (6), Germany (7), Italy (8), a case where Zika virus RNA was found in the man’s semen even 62 days after first symptoms of infection, New Zealand (9), a case where semen samples from the man tested positive for Zika virus RNA even 76 days after symptom onset and only tested negative on days 99 and 117, Peru (10), Spain (11), USA (12, 13, 14, 15).

Other Reports Of Sexual Transmission Include

  • Asymptomatic male to female, one each in France (16) and USA (17). Since ~80% of Zika-infected people remain asymptomatic (18), prolonged presence of potentially infectious Zika in semen makes its sexual transmission a very important means of spreading through the population. Data also shows a pregnant woman being asymptomatic doesn’t preclude Zika virus from affecting the fetus.
  • One case of male to male transmission (anal sex), in Texas, USA (19).
  • One case of female to male transmission in New York City, USA (20).

Documented Cases Of Zika’s Presence In Semen

Live virus: Researchers were able to isolate Zika virus from semen

  • In one case from the 2013 French Polynesia – Wikipedia Zika outbreak (21) two weeks after self-reported symptoms started.
  • In a French case where semen virus load was 100000 times that in blood (22) two weeks after self-reported symptoms started.
  • Even 62 days after first fever symptom in a case from Scotland (23).

Virus RNA: Detectable in a Netherlands patient up to 47 days after symptom onset (24), 80 (25) and 93 days (26) from two cases in France, and even 181 (27) and 188 days (28) in two cases from Italy.

These data suggest live virus is not only present in semen of Zika-infected men but can also stay there for extended periods of time. This is why the WHO recommends (1) that

  • In regions with active Zika virus transmission,

‘Pregnant women should practice safer sex or abstain from sexual activity for at least the whole duration of the pregnancy. Their partners should also be informed about this recommendation.’

  • And in regions with no active Zika virus transmission,

‘a. Men and women returning from areas where transmission of Zika virus is known to occur should adopt safer sex practices or consider abstinence for at least 6 months upon return to prevent Zika virus infection through sexual transmission.

b. Couples or women planning a pregnancy, who are returning from areas where transmission of Zika virus is known to occur, are advised to wait at least 6 months before trying to conceive to ensure that possible Zika virus infection has cleared.

c. Sexual partners of pregnant women, returning from areas where transmission of Zika virus is known to occur, should be advised to practice safer sex or abstain from sexual activity for at least the whole duration of the pregnancy.’

Bibliography

1. World Health Organization. “Prevention of sexual transmission of Zika virus: interim guidance update.” World Health Organization, Geneva, Switzerland (2016). http://apps.who.int/iris/bitstre…

2. Zika virus infection – Argentina and France

3. Statement from the Chief Public Health Officer of Canada and Ontario’s Chief Medical Officer of Health on the first positive case of sexually transmitted Zika Virus – Canada News Centre

4. Zika virus infection – Chile

5. D’Ortenzio, Eric, et al. “Evidence of sexual transmission of Zika virus.” New England Journal of Medicine 374.22 (2016): 2195-2198. http://www.nejm.org/doi/pdf/10.1…

6. Turmel, Jean Marie, et al. “Late sexual transmission of Zika virus related to persistence in the semen.” The Lancet (2016). http://ac.els-cdn.com/S014067361…

7. Frank, Christina, et al. “Sexual transmission of Zika virus in Germany, April 2016.” Eurosurveillance 21.23 (2016). http://www.eurosurveillance.org/…

8. Venturi, G., et al. “An autochthonous case of Zika due to possible sexual transmission, Florence, Italy, 2014.” Euro Surveill 21.8 (2016): 30148. http://www.eurosurveillance.org/…

9. Harrower, Jay, et al. “Sexual transmission of Zika virus and persistence in semen, New Zealand, 2016.” Emerging Infectious Diseases 22.10 (2016): 1855. http://wwwnc.cdc.gov/eid/article…

10. Zika virus infection – Peru

11. Spain records first case of sexually transmitted Zika virus

12. Foy, Brian D., et al. “Probable non–vector-borne transmission of Zika virus, Colorado, USA.” Emerging infectious diseases 17.5 (2011): 880. http://www.ncbi.nlm.nih.gov/pmc/…

13. Sex After a Field Trip Yields Scientific First. Martin Enserink, Science, April 6, 2011. Sex After a Field Trip Yields Scientific First

14. Zika Infection Transmitted by Sex Reported in Texas. The New York Times, Donald G. McNeil Jr., Sabrina Tavernise, Feb 2, 2016. Log In – New York Times

15. Hills, Susan L. “Transmission of Zika virus through sexual contact with travelers to areas of ongoing transmission—continental United States, 2016.” MMWR. Morbidity and mortality weekly report 65 (2016). http://www.cdc.gov/mmwr/volumes/…

16. Fréour, Thomas, et al. “Sexual transmission of Zika virus in an entirely asymptomatic couple returning from a Zika epidemic area, France, April 2016.” Eurosurveillance 21.23 (2016). http://www.eurosurveillance.org/…

17. Brooks, Richard B. “Likely sexual transmission of Zika virus from a man with no symptoms of infection—Maryland, 2016.” MMWR. Morbidity and Mortality Weekly Report 65 (2016). http://www.cdc.gov/mmwr/volumes/…

18. Duffy, Mark R., et al. “Zika virus outbreak on Yap Island, federated states of Micronesia.” New England Journal of Medicine 360.24 (2009): 2536-2543. http://www.nejm.org/doi/pdf/10.1…

19. Deckard, D. Trew. “Male-to-male sexual transmission of Zika virus—Texas, January 2016.” MMWR. Morbidity and mortality weekly report 65 (2016). https://www.cdc.gov/mmwr/volumes…

20. Davidson, Alexander. “Suspected female-to-male sexual transmission of Zika virus—New York City, 2016.” MMWR. Morbidity and mortality weekly report 65 (2016). http://www.cdc.gov/mmwr/volumes/…

21. Musso, Didier, et al. “Potential sexual transmission of Zika virus.” Emerg Infect Dis 21.2 (2015): 359-61. https://www.researchgate.net/pro…

22. Mansuy, Jean Michel, et al. “Zika virus: high infectious viral load in semen, a new sexually transmitted pathogen.” Lancet Infect Dis 16.405 (2016): 00138-9. https://www.researchgate.net/pro…

23. Atkinson, Barry, et al. “Detection of Zika virus in semen.” Emerg Infect Dis 22.5 (2016). Emerging Infectious Disease journal

24. Reusken, Chantal, et al. “Longitudinal follow-up of Zika virus RNA in semen of a traveller returning from Barbados to the Netherlands with Zika virus disease, March 2016.” Eurosurveillance 21.23 (2016). http://www.eurosurveillance.org/…

25. Matheron, Sophie, et al. “Long-lasting persistence of Zika virus in semen.” Clinical Infectious Diseases 63.9 (2016): 1264-1264. Long-Lasting Persistence of Zika Virus in Semen

26. Mansuy, Jean Michel, et al. “Zika virus in semen of a patient returning from a non-epidemic area.” The Lancet Infectious Diseases 16.8 (2016): 894-895. http://www.thelancet.com/pdfs/jo…

27. Barzon, Luisa, et al. “Infection dynamics in a traveller with persistent shedding of Zika virus RNA in semen for six months after returning from Haiti to Italy, January 2016.” Eurosurveillance 21.32 (2016). http://www.eurosurveillance.org/…

28. Nicastri, Emanuele, et al. “Persistent detection of Zika virus RNA in semen for six months after symptom onset in a traveller returning from Haiti to Italy, February 2016.” Eurosurveillance 21.32 (2016). http://www.eurosurveillance.org/…

 

https://www.quora.com/If-a-child-is-conceived-while-the-father-has-Zika-but-the-mother-never-gets-infected-can-the-fetus-be-affected/answer/Tirumalai-Kamala

What are the chances that there’s something else like Zika coming soon?

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Global Zika-like outbreaks are increasingly inevitable largely due to unprecedented rates of

  • Population mobility and density
  • Rapid global transport
  • Human ecosystem alteration
  • Climate change

existing cheek by jowl with

  • Vast health and wealth disparities
  • Vast global differences in public health infrastructure including sanitation and active disease surveillance capacity
  • Surfeit of neglected and/or little known diseases, many of them tropical, that lack drugs or vaccines to treat or prevent them and whose geographic reach spreads as climate change expands the range of the vectors capable of transmitting them.

Several of these factors also increase human-wildlife interactions, which in turn increase risk of Zoonosis – Wikipedia, diseases transmissible between animals and humans.

Originally discovered in 1947 in the Zika forest adjacent to Lake Victoria in Uganda, for decades Zika was but one of several RNA viruses known only to a handful of aficionados. Its obscurity began changing starting in 2007 when it caused an outbreak on the Micronesian island of Yap (1) followed by a larger 2013-2014 outbreak in French Polynesia (2) culminating of course in the global headlines of the 2015-2016 outbreak in Brazil and beyond (3).

So, clearly a case of a virus that until quite recently remained confined to a remote African forest and yet in less than a decade it’s spread across the globe, not only infecting large swaths of previously unexposed human populations but also expanding its capability in terms of disease outcomes from GBS (Guillain–Barré syndrome – Wikipedia) to fetal abnormalities (Microcephaly – Wikipedia). While less than a decade may sound shocking, not being unprecedented is even more so since Zika’s only following in the footsteps of related viruses, Chikungunya – Wikipedia and Dengue virus – Wikipedia, all zoonoses. Having similarly triumphantly marched across the globe in recent decades, their spread eerily echoes that of their carrier mosquito, Aedes – Wikipedia.

A 2008 study (4) estimated 335 emerging infectious diseases (EID) between 1940 and 2004. Averaging yields a very deceptive ~5 per year, deceptive because frequency between Pandemic – Wikipedia has steadily shrunk in recent years. Consider for example the high-profile global outbreaks of SARS (Severe acute respiratory syndrome – Wikipedia) in 2003, Bird flu (Influenza A virus subtype H5N1 – Wikipedia) in 2007, Swine flu (2009 flu pandemic – Wikipedia) in 2009, MERS (Middle East respiratory syndrome – Wikipedia) in 2012, Ebola virus disease – Wikipedia in 2014 and Zika fever – Wikipedia in 2015.

~60% of EIDs are primarily zoonotic. For e.g., Chikungunya, Dengue Ebola, HIV, Lyme, SARS, West Nile, Zika, to mention just a few.

  • The 2008 analysis (4) concluded majority arose from wildlife and that human population density was the single common predictor for all types of EIDs (zoonotic or not, drug-resistant or not, vector-borne or not).
  • EIDs seem to be a ‘hidden’ cost of human economic development (see below from 4, 5).

Population Mobility & Rapid Global Transport

Practically anywhere in the world is today a mere plane ride away even as these anywheres remain vastly different in basic public health infrastructure and active disease surveillance capacity. One sick person is all it takes for infectious diseases to spread beyond borders, i.e., vastly expanded potential for diseases to rapidly spread globally.

Consider for example how global air travel has exponentially expanded in <100 years. A paltry 1205 total global plane tickets sold in 1914 had increased to a whopping 2,595,448,927 in 2010. Yet large swaths of the world still lack adequate sanitation (see below from 6 and 7).

Human-driven Ecosystem Alterations & Climate Change Tilt The Balance In Favor Of Increased Pandemic Risk

More than ever before, massive human -driven ecosystem alterations have become the norm since the Industrial Revolution – Wikipedia with this process only further globalizing in the current era (8; see below from 9, emphasis mine).

‘With roughly half the temperate and tropical forests cut down, nearly half the icefree, desert-free terrestrial landscape converted to croplands or pasture, and more than 800,000 dams impeding the flow through more than 60% of the world’s rivers, alterations to our planet’s land use and land cover represent some of the most pervasive changes humanity has made to Earth’s natural systems

Human-driven ecological changes increase human encroachments into wildlife habitat. Such human-wildlife interactions increase zoonoses risk.

  • For example, studies suggest such processes may have kick-started the initial Ebola and HIV outbreaks (10).
  • No surprise then that ~75% of EIDs are zoonoses (4, 5, 11).
  • New disease outbreaks become more inevitable when previously unimaginable mobility and enormous human-driven ecological changes exist alongside crippling poverty consisting of acute food scarcity and no sanitation, hygiene or running water since the more malnourished are weaker and likelier to get sick, especially with new EIDs.
  • A corollary is increased hunting and consumption of wild meat, Bushmeat – Wikipedia (12).
    • For example, ‘ground zero’ for the 2013-2016 West African Ebola outbreak is suspected to be hungry children living in the remote Meliandou – Wikipedia village in southern Guinea – Wikipedia who killed and ate infected fruit bats (13, 14).
    • Something so seemingly inconsequential and yet it triggered a global public health emergency with a total of 28616 cases and 11310 fatalities from 10 countries according to West African Ebola virus epidemic – Wikipedia.
  • Mapping such outbreaks only emphasizes that infectious disease transmissions have become that much easier given how fluidly, rapidly and easily humans can traverse the globe these days, and the increasingly porous divides between previously more strongly demarcated divisions such as affluence and poverty, sanitation and filth.

Thus, since lack of hygiene, sanitation and running water are today only a plane ride away so is pandemic risk.

The rapid, global expansion of mosquito species such as Aedes aegypti – Wikipedia and Aedes albopictus – Wikipedia is but one example of how climate change effects place greater selection pressures on vast numbers of species to adapt to these rapid changes, many of which end up increasing infectious disease risk not just in humans but for all types of life forms (see some other examples below from 15).

A 2009 analysis (16) concluded climate change may influence different arthropod-transmitted Arbovirus – Wikipedia diseases differently.

  • Chikungunya: A single mutation in the Chikungunya virus facilitated its adaptation to the fast expanding mosquito species, A. albopictus, i.e., Chikungunya’s spreading by latching on to this mosquito’s coat-tails, whose spread is facilitated by climate change. Human travel simply augments spread even more.
  • Rift Valley fever – Wikipedia, Bluetongue disease – Wikipedia: According to these authors, climate change helps mosquitoes spread in newly flooded areas while human activities such as irrigation projects, movements of herded animals and animal imports to feed large numbers of humans, for example during Mecca pilgrimages, also contribute to Rift Valley virus outbreaks.

Bibliography

1. Duffy, Mark R., et al. “Zika virus outbreak on Yap Island, federated states of Micronesia.” New England Journal of Medicine 360.24 (2009): 2536-2543. http://www.nejm.org/doi/pdf/10.1…

2. Cao-Lormeau, V. M., et al. “Zika virus, French polynesia, South pacific, 2013.” Emerging infectious diseases 20.6 (2014): 1085-1086. http://wwwnc.cdc.gov/eid/article…

3. Campos, Gubio S., Antonio C. Bandeira, and Silvia I. Sardi. “Zika virus outbreak, Bahia, Brazil.” Emerging infectious diseases 21.10 (2015): 1885. https://www.ncbi.nlm.nih.gov/pmc…

4. Jones, Kate E., et al. “Global trends in emerging infectious diseases.” Nature 451.7181 (2008): 990-993.

5. World Organisation for Animal Health

6. In flight

7. Total population: access to sanitation

8. Foley, Jonathan A., et al. “Global consequences of land use.” science 309.5734 (2005): 570-574. https://www.researchgate.net/pro…

9. Myers, Samuel S., et al. “Human health impacts of ecosystem alteration.” Proceedings of the National Academy of Sciences 110.47 (2013): 18753-18760. https://www.researchgate.net/pro…

10. Hahn, Beatrice H., et al. “AIDS as a zoonosis: scientific and public health implications.” Science 287.5453 (2000): 607-614. https://www.researchgate.net/pro…

11. Taylor, Louise H., Sophia M. Latham, and E. J. Mark. “Risk factors for human disease emergence.” Philosophical Transactions of the Royal Society of London B: Biological Sciences 356.1411 (2001): 983-989. http://rstb.royalsocietypublishi…

12. Wolfe, Nathan D., et al. “Naturally acquired simian retrovirus infections in central African hunters.” The Lancet 363.9413 (2004): 932-937. http://www.jhsph.edu/research/af…

13. Vogel, Gretchen. “Bat-filled tree source of Ebola epidemic?.” Science 347.6218 (2015): 142-143. Bat-filled tree may have been ground zero for the Ebola epidemic

14. Bausch, Daniel G., and Lara Schwarz. “Outbreak of Ebola virus disease in Guinea: where ecology meets economy.” PLoS Negl Trop Dis 8.7 (2014): e3056. http://journals.plos.org/plosntd…

15. Altizer, Sonia, et al. “Climate change and infectious diseases: from evidence to a predictive framework.” science 341.6145 (2013): 514-519. http://www.colorado.edu/eeb/facu…

16. Gould, Ernest A., and Stephen Higgs. “Impact of climate change and other factors on emerging arbovirus diseases.” Transactions of the Royal Society of Tropical Medicine and Hygiene 103.2 (2009): 109-121. http://www.idpublications.com/jo…

https://www.quora.com/What-are-the-chances-that-theres-something-else-like-Zika-coming-soon/answer/Tirumalai-Kamala