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Monday, November 3, 2008

Middle Ages of today’s Medicine, Overlooking Quantum-Biophysical-Semeiotic Constitutions and Related Inherited Real Risk.

Sergio Stagnaro MD

Via Erasmo Piaggio 23/8,Riva Trigoso (Genoa) Europe. Founder of Quantum Biophysical Semeiotics Who's Who in the World (and America) since 1996 to 2009.
Ph 0039-0185-42315, Cell. 3338631439,

At first sight, it could seem paradoxical, absurd, incomprehensible, my former definition of Middle Ages of today’s Medicine, but it contains an important, really distressing, disheartening truth (1-9).
In this brief paper I explain in clear manner what accounts for the reason of the justification of my definition Middle Ages of present Medicine (MAM), or Age of Darkness.

To begin with, I underscore the fundamental bias, scientists all around the world agree with, i.e., according to which, “all men are created equal”, so that Evidence Based Medicine (EBM) is the only theory at the base of every research in western countries. On the contrary, beside this theory, interesting more sponsoring drug producers than single patient, I have demonstrated firstly that individuals are different from biological view-point, and secondly that Single Patient Based Medicine theory really exists (10-13).
In following, I illustrate in a simple, clear way, easy to understand, what accounts for the reason of my term MAM, subdividing the argumentation in specific, distinct, particular paragraphs.

A) All around the world scientists are considering as truth the belief, according to which cancer can involves all individuals, though with diverse incidence. In other words, almost all physicians are overlooking both Oncological Terrain (OT) and OT-dependent Inherited Real Risk, localized in one (or more) biological system (14-24). As a consequence, today’s women are told that it is unavoidable necessary, e.g., to undergo periodically mamma echotmography and mammography, while all men are controlling periodically their PSA blood level, in order to prevent breast cancer and, respectively, prostate cancer.
The truth is that only women involved by both Oncological Terrain and breast cancer Inherited Real Risk in one (rarely in more) mamma quadrant can suffer from breast malignancy (15-23).
This is valid, of course, for carcinogenesis in every biological systems.

Overlooking above-mentioned original scientific concepts, physicians think that all individuals must be enrolled in cancer primary prevention, which will result useless and expensive, generating an avoidable Psychological Terrorism.

Please, reflect on the following message of mine posted recently in

Surely, climate change is real, as states wisely Obama. On the contrary, I believe that NHS Programs are unfortunately stable all around the world, generating the present Middle Ages of Medicine and - as a consequence - Psychological Terrorism. For instance, read”.

In addition, in every present research, aiming to study drugs usefulness in cancer primary prevention, are enrolled also individuals negative for both Oncological Terrain and OT-dependent Inherited Real Risk, e.g. in the lung. Therefore, research conclusion may be that – for instance – tobacco smoking is a tool of paramount importance in lung cancer primary prevention: a paradigmatic example of MAM.

B) All around the world scientists are considering as truth the belief that all women (and men?) can be involved by osteoporosis. Overlooking osteoporotic constitution, present war against osteoporosis is lost for ever (24-26). In spite of wise, prudent, indifferent advices of drugs companies and machines producers, all women after 40 years must be regularly controlled as far as bone calcium is concerned. In fact, nowadays to prevent osteoporosis, doctors are following precise, but clearly no-updated, WHO Guide Lines, according to which periodical MOC (Bony Computerized Mineralometria), is necessary for all women over 40 years, with and without osteoporosis constitution and ostoporotic inherited real risk; This is another outstanding example of MAM.

C) All around the world scientists are considering as truth the belief that all women and men can be involved in their life by type 2 diabetes, which is a serious today’s growing epidemics. In other words, unfortunately according to present medicine knowledge, all individuals are at different risk of diabetes. Therefore, it is nowadays advisable for everybody controlling fasting and postprandial glucose blood level, aiming to recognize in “early” symptomless patients glucose metabolism impairment. Overlooking Quantum Biophysical Semiotics, a large percentage of men involved by both diabetic “and” dyslipidemic constitutions, conditio sine qua non of diabetes, aren’t controlled, and thus not recognized as diabetics.

At this point, we must remember that so-called diabetic complications are already present when diabetes early diagnosis is made, since they occur years or decades before disorder onset (10, 11, 20, 27, 28) (S., Practical Applications, Diabetes).

From the above, briefly referred, remarks, it is clear without doubts that whatever diabetes primary prevention, performed overlooking diabetic “and” dyslipidemic constitutions, results unavoidably an expensive lack of success. This is another excellent example justifying the term MAM, i.e., Middle Ages of Medicine.

D) All around the world scientists are considering as truth the belief that all individuals with high blood levels of cholesterol (especially, LDL, No-HDL,TG, a.s.o.), homocysteine, uremia, as well as “every” hypertensive subject, tobacco smokers, obese individuals, stressed humans, and so on, are at risk of cardiovascular disease (CVD), and particularly, Coronary Artery Disease (CAD). In other, few words, in spite of wise, indifferent, neutral advices of lipid lowering drugs producers, the above mentioned pathological conditions are considered risk factors (sometimes, causes!) of CVD, including acute coronary disease.
Frankly speaking, what accounts for the reason of such as great mistake, compromising the primary prevention against an epidemics of present age, is the distressing fact that the large majority of Authors, Editors, Reviewers, University Professors, General Practitioners, National Health Service Authorities, specialized journalist, and also lay-men ignore the existence of Inherited CVD (CAD) Real Risk! (29-33)

At this point, I discuss some current differential diagnosis, since they symbolizing today’s Middle Ages of Medicine.

E) All around the world scientists are considering as truth the belief that ALL individuals with Precordialgia,  “could be” affected by CAD, so that they think urgently carry out ECG (electrocardiogram). In fact, laboratory- and image department-dependent physicians advice usually, first of all, an ECG to ALL patient with pain in the central part of their chest, to ascertain possible acute coronary disorder. To ECG follow immediately thorax X-rays, oesophagus-gastro-duodeno-endoscopy, and a long series blood examination, looking for a precise diagnosis, which is really a “bedside” diagnosis, if doctors know Quantum Biophysical Semeiotics. As a matter of fact, when intense digital pressure, applied upon a single heart trigger-point, does not bring about simultaneously gastric aspecific reflex, CAD is excluded in reliable manner (7, 32-35).
Analogously, physicians are able to exclude at te bedside whatever chest disorder, when intense digital pressure, applied upon a single thorax trigger-point, does not bring about simultaneously gastric aspecific reflex. As regards the presence of hiatal hernia, even associated with cholelithiasis  and colon diverticulosis (Saint Syndrome), doctors can recognize such as syndrome in 10 seconds (36, 37).
As a consequence, further examinations will follow exclusively when pathological physical semeiotic data are observed. To summarize, current diagnostic procedure in case of precordialgia is a paradigmatic display of MAM.

F)  All around the world scientists are considering as truth the belief that ALL individuals with joint pain could be suffering from rheumatic disorders, so that they agree with the necessity, according to Guide Lines, of laboratory research, aiming to recognize possible rheumatism.
As a consequence, patients undergo lab analyzes, joint X-rays, TAC, NMR, a.s.o., in order to make possibly the differential diagnosis and diagnosis.
Unfortunately, overlooking  both biophysical semeiotic rheumatic constitution and rheumatic inherited Real Risk  doctors cannot recognize individuals who can be involved by such as disorders, separating them from those who surely will never suffer from joint disorder, rheumatic in nature (10, 11). The above remarks, briefly referred, account for the reason of psychological terrorism and today’s Middle Ages of Medicine.

I like conclude the article, illustrating in details a common problem of today’s Medicine, brought about by diffuse utilization of Echographical examination, in every patient presenting abdominal disorders, but more frequently in normal subjects!

G)  The patient with a focal liver lesion may present difficult detection and management pro­blems, in particular when upper abdominal symptoms are completely absent. In fact, the wider application of ultrasound and more recently computed tomogra­phy and NMR, have identified increasing numbers of patients with no symptoms related to their hepatic lesions.
On the contrary, there are a lot of quantum biophysical semeiotic signs valuable and reliable in bedside finding out and diagnosing focal liver lesions, even clinically silent, as well as in moni­toring the course of the diseases (1). Due to clinical phenomenology in the right upper abdominal quadrant, related to retro-ciecal and/or sub-hepatic atypi­cal localization, appendicitis must be considered in diffe­rential diagnosis (39,40). Moreover, the usefulness of quantum biophysical semeiotics in both avoi­ding unnecessary over-investigation and in selecting patients who might benefit from high quality spe­cialist studies has to be emphasized.
At least during a long period of time, focal liver lesions may occur without upper abdominal symptoms. In other words, the majority of focal lesions of the liver are clinically silent, so that an increasing numbers of patients, with no symptoms related to their hepatic disorders, has been identified by wider application of ultrasound and computed tomography (41).
Although there is no widely accepted protocol for assessing these lesions, both sophisticated exa­minations are definitely included in various suggested algorithms (41, 42). Small haemangio­mas and some hydatid cysts, however, have atypical computed tomography and ultrasound appearances (43,  44).
On the other hand, hepatic haemangioma represents the most common benign tumour of the liver and its clinical instrumental diagnosis is often difficult. The lack of diagnostic accuracy of echoscintigraphic detection, furthermore, in asses­sing a solitary hepatic lesion is well known (38). As a matter of fact, with only the aid of echothomography, for example, is not possible the differen­tial diagnosis between hepatic abscess and solid lesion. On the contrary, ultrasound scanning permits early separation into cystic or solid lesions in almost all cases and also excludes large bile ducts obstruc­tion. It may also identify multiple hepatic lesions.
However, technical difficulties with ultra­sound scanning may arise in some patients, due, for instance, to obesity and/or overlying bowel-gas and is then necessary CT, which has an increased overall accuracy compared with ultrasound (38). All patients, observed in the past year, were routinely assessed by means of AP for evidence of hepatic tumor, first by detecting CAEMH, B, II, conditio sine qua non of tumors both benign and malignant, solid or liquid; then the "boxer's test" was carefully examined in order to ascertain the cystic syndrome, in particular starting 4 sec. after test beginning.
As regards the detection of cystic syndrome, we prefer to evaluate the upper third urethral reflex. In all positive cases AP of the liver was then carried out to find one (or more) suspected area. In the patients of the series, finger pressure on cutaneous projection area of focal suspected lesions, induced gastric aspecific reflex and cystic syndrome, thus allowing the clinical evaluation of lesion shape and size. Hepatic neoplasms, primary or secondary, must be taken into account in differential diagnosis, even when primary localization is yet unknown.
AP differential diagnosis between benign and malignant liver tumours is based also on the positivity ofRHSH "complete type" and autoimmune syndrome, both of them present exclusively in the malignancies. Moreover, "simulated defecation test", as well as "simulated micturation test", has proved useful in localizing focal lesions respectively in abdominal organs and urinary tract (unpublished work). When there are abdominal symptoms in the right upper quadrant, among other differential dia­gnoses, also appendicitis - in atypical retrocecal and/ or subhepatic localization - must be kept in mind, in order to avoid a misdiagnosis full of risk.
On the other hand, a patient with a focal lesion in the liver can be also involved by an appendicitis. From the auscultatory percussion point of view, despite its position, appendicitis is characterized by RESH "complete type" and especially by "tonic gastric contraction sign" (tgc), induced by both simulated defecation test (38, 43) and digital pressure on skin projection of diseased appendix, exactly localized by AP of the cecum. The intensity of the specific sign, furthermore, is directly related to the severity of the illness. On the contrary, the latency time before tgc enhancing is inversely correlated with the seriousness of underlying disease (e.g. from 4 to 8 sec.). As a result of these observations, AP appears to be very useful in diagnosing and differential diagnosing - of course - as well as in monitoring the evolution of appen­dicitis, apart from any atypical localization.
To return to hepatic focal lesions, it seems easy to separate by mean of AP haemangiomas from both cysts and neoplasms (only in the letter ones there is RESH and autoimmune syndrome). In fact, during the boxer's test, haemangioma size increases, whereas cyst diametre clearly decreases for 3 sec. Obviously, solid focal lesions of the liver do not vary their size during the test.
The above remarks are quite important, because haemangiomas and occasionally hydatid cysts - as a wide literature reports - may have atypical apperarances on initial investigation, and percuta­neous biopsy may result in life-threatening hemor-rage, anaphylaxis or hydatic dissemination  (24, 38).

In conclusion, although above-remarks represent  partial data of my 53-year-long clinical experience, I’ am sure they are sufficient to corroborate the term Psychological Terrorism of today’s Middle Ages of Medicine. My hope is  to can go out of both, as soon as possible,  with the precious aid of Quantum Biophysical Semeiotics.


1) Stagnaro S. The Great Beyond, July 11, 2008
2) Stagnaro Sergio. Semeiotica Biofisica Quantistica: Precisazione sulla Vaccinazione anti HVP nella Prevenzione del Cancro Cervicale. , 24 ottobre 2008,
3) Stagnaro Sergio. Role of NON-LOCAL Realm in Primary Prevention with Quantum Biophysical Semeiotics., 01 Feb, 2008-05-17
4) Stagnaro Sergio. The Lancet, January 28, 2008. Bedside Biophysical-Semeiotic Osteocalcin Test in Diagnosing and Monitoring Diabetes.
5) Stagnaro Sergio e Paolo Manzelli. Semeiotica Biofisica Endocrinologica: Meccanica Quantistica e Meccanismi d’Azione Ormonali. Dicembre 2007,,
6) Stagnaro Sergio e Paolo Manzelli. Semeiotica Biofisica: Realtà non-locale in Biologia. Dicembre 2007,,
7) Stagnaro Sergio. Role of Coronary Endoarterial Blocking Devices in Myocardial Preconditioning - c007i. Lecture, V Virtual International Congress of Cardiology.
8) Stagnaro Sergio e Paolo Manzelli. 03 Gennaio 2008, Limiti della Medicina Ufficiale. L’Esperimento di Lory.
9) Stagnaro Sergio e Paolo Manzelli. L’Esperimento di Lory. Scienza e Conoscenza, N° 23, 13 Marzo 2008.
10) Stagnaro S., Stagnaro-Neri M., Le Costituzioni Semeiotico-Biofisiche.Strumento clinico fondamentale per la prevenzione primaria e la definizione della Single Patient Based Medicine. Travel Factory, Roma, 2004.
11) Stagnaro S., Stagnaro-Neri M., Single Patient Based Medicine.La Medicina Basata sul Singolo Paziente: Nuove Indicazioni della Melatonina. Travel Factory, Roma, 2005.
12) Stagnaro Sergio. Single Patient Based Medicine: its paramount role in Future Medicine. Public Library of Science.
13) Stagnaro Sergio. Single Patient Based Medicine, Therapeutic Monitoring and proper Drugs Prescription. Nature April, 4, 2008.
14) Stagnaro S., Stagnaro-Neri M. Una patologia mitocondriale ignorata: la Istangiopatia Congenita Acidosica Enzimo-Metabolica. Gazz. Med. It. - Arch. Sci. Med. 149, 67 1990. 2) Stagnaro S. New bedside way in reducing mortality in diabetic men and women. Ann. Int. Med. 200708070-00167v1
15) Stagnaro-Neri M., Stagnaro S., Cancro della mammella: prevenzione primaria e diagnosi precoce con la percussione ascoltata. Gazz. Med. It. - Arch. Sc. Med. 152, 447 1993
16) Stagnaro-Neri M., Stagnaro S. Introduzione alla Semeiotica Biofisica. Il Terreno Oncologico. Ed. Travel Factory, Roma, 2004.
17) Stagnaro S., Stagnaro-Neri M., Oncological Terrain, conditio sine qua non of Oncogenesis, 2004:
18) Stagnaro Sergio. "Genes, Oncological Terrain, and Breast Cancer" World Journal of Surgical Oncology., 2005,
19) Stagnaro S. Reale Rischio Semeiotico-Biofisico. Ruolo diagnostico e patogenetico dei Dispositivi Endoarteriolari di Blocco neoformati patologici tipo I, sottotipo a) oncologici e b). Ed Travel Factory, Roma,, in press
20) Stagnaro S. Newborn-pathological Endoarteriolar Blocking Devices in Diabetic and Dislipidaemic Constitution and Diabetes Primary Prevention. The Lancet. March 06 2007.
21) Stagnaro Sergio. Bedside diagnosing Pancreas Cancer , even in its inherited real Risk.
Cases Journal BMC. 31 October 2008.
22) Stagnaro Sergio. Bedside Detecting Lung Cancer Inherited Real Risk. Variant Baserga’s Sign. Medical News Today’s, 23 Oct 2008.
23) Stagnaro Sergio. Bedside Diagnosing Pheochromocytoma, since its initial stage of Inherited Real Risk. Cases Journal 2008,
24) Stagnaro Sergio. Bedside diagnosis of osteoporotic constitution, real risk of inheriting ostoporosis, and finally osteoporosis. Theoretical Biology and Medical Modelling 21 June 2007.
25) Stagnaro-Neri M., Stagnaro S., Diagnosi Clinica Precoce dell’Osteoporosi con la Percussione Ascoltata. Clin.Ter. 137, 21 -27 1991 [Medline].
26) Stagnaro S. Co Q10 in the prevention and treatment of primary osteoporosis. Preliminary data. Clin Ter.;146(3):215-9 [MEDLINE]
27) Stagnaro S., Stagnaro-Neri M. Valutazione percusso-ascoltatoria del Diabete Mellito. Aspetti teorici e pratici. Epat. 32, 131, 1986.
28) Stagnaro S., West PJ., Hu FB., Manson JE., Willett WC. Diet and Risk of Type 2 Diabetes. N Engl J Med. 2002 Jan 24;346(4):297-298. [Medline]
29) Stagnaro Sergio. Role of Coronary Endoarterial Blocking Devices in Myocardial Preconditioning - c007i. Lecture, V Virtual International Congress of Cardiology.
30) Stagnaro Sergio. Biophysical-Semeiotic Inherited Coronary Real Risk, conditio sine qua non of CAD.17 August 2007.
31) Stagnaro Sergio. Reale Rischio Congenito di CAD: Nosografia e Terapia. 22 maggio 2008
32) Stagnaro Sergio. Bedside recognizing Inherited CAD Real Risk. 21 May, 2008.
33) Stagnaro Sergio. Bedside Recognizing CAD Inherited Real Risk and silent CAD with Biophysical Semeiotics. Lipid in Health and Disease. (29 May 2008)
     34) Stagnaro Sergio.   Bedside Evaluation of CAD biophysical-semeiotic inherited real risk under NIR-LED treatment. EMLA Congress, Laser Helsinki August 23-24, 2008. "Photodiagnosis and photodynamic therapy", Elsevier, Vol. 5 suppl 1 august 2008 issn 1572-1000.
     35) Stagnaro Sergio. Diagnosi clinica di cuore sano in un secondo!  7 Aprile 2008.
     36) Stagnaro Sergio. Saint’s Syndrome. Bed-side Diagnosis  by means of Biophysical-Semeiotics.
     37)  Stagnaro Sergio.  Hiatal Hernia, Oesofageal Peristalsis Modificazions And Gastro-Oesofageal Reflux Disease (Gerd): Clinical Diagnosis By Means Of Biophysical Semeiotics.
  38) Stagnaro S., Stagnaro-Neri M. Auscultatory Percussion in Detection Focal Liver Leions even Clinically Silent. Acta Med. Medit. 8, 89-94, 1992.
 39) Stagnaro S. Bed-side diagnosing acute appendicitis and gastrointestinal diseases. Gut.j.on line, 2003:
40) Stagnaro-Neri M., Stagnaro S., Appendicite. Min. Med. 87, 183, 1996 [Medline]
41) Thompson J.N., Gibson R., Czerniack A., Blumgart L.H., Focal liver lesions: a plan for management,
Brit. Med. L, 1985, 290, 1643.
42) Scheible W., A diagnostic algorhitm for liver masses, Semin. Roemtgenol., 1983, 18, 84.
43) Johnson C.M., Sheedy P.P., Stanson A.W., Stephens D.H., Hattery R.R., Adson M.A., Computed
 tomography and angiography of cavernous hemangiomas of the liver, Radiology, 1981, 138, 115.
 44) Snow J.H., Goldstein H.M., Wallace S., Comparison of scintigraphy, sonography and computed
 tomography in the evolution of hepatic neoplasm, A.T.R., 1979, 132, 915.

Sergio Stagnaro MD
Via Erasmo Piaggio 23/8
Riva Trigoso (Genoa) Europe
Founder of Quantum Biophysical Semeiotics
Who's Who in the World (and America)
since 1996 to 2009
Ph 0039-0185-42315
Cell. 3338631439

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Wednesday, May 28, 2008

In Defense of Pharmacogenetics

Author: Nils Reinton
Furst Medical Laboratory, Søren Bullsv. 25, N-1051 Oslo, Norway (

Pharmacogenetics is the analysis of specific genetic markers informing you of how efficiently you metabolize a given drug. When it comes to metabolism of drugs used for treating psychiatric disorders (ranging from mild depression to severe psychosis), three genes are commonly analyzed: Cyp2C9, Cyp2C19 and Cyp2D6. Of course, other things than genetics also influence how you respond to a drug, like compliance, diet and smoking, but individual genetic variation have profound stand-alone effects. If your genetic test shows the presence of clinically relevant genetic variants you will be grouped as a Poor Metabolizer (higher risk for unwanted side effects) or Ultrarapid Metabolizer (not responding to medication or in need of very high dosage). For a patient experiencing adverse events or no response at all when taking his medicine, pharmacogenetics can be a tremendous help in choosing more fitting medications (change dosage or use another (sub)class of drugs). Pharmacogenetics is thus, a vital part of personalized medicine.

Recently a commentary - "A Case Study of Personalized Medicine" by
Katsanis et al., was published in Science (subscription may be required), where the use of pharmacogenetics before choosing upon medication was discouraged. The reason for this was the finding that doctors failed to follow up on recommendations based on test-results. I will try to argue against such a negative approach towards pharmacogenetics and attempt to show that pharmacogenetis is in fact a valuable tool, especially for patients on psychoactive drugs who experience adverse events or lack of effect.

Claim 1: Doctors fail to follow medication recommendations based on pharmacogenetic lab-results.

Counter argument: Physicians either not understanding the test-results or ignoring lab results (due to personal conviction of some sort, be it based on clinical experience or not) reflects a general problem not restricted to pharmacogenetics. Health professionals lack of understanding is especially common for genetic tests. One could argue then, that tests that are too difficult for doctors to interpret should not be made available. But, one could easily counter argue that such an approach would be a major obstacle to medical progress. New tests will always need a time-window of learning and enlightenment. To narrow this window, the following message needs to driven home:
A patient that has to try many different drugs to achieve the desired effect without adverse reactions, is mistreated in a costly manner. Pharmacogenetics constitutes a once in a life-time test allowing a targeted approach straight towards the medication most likely to be suitable. When recommendations based on lab-results are implemented in the treatment regime, pharmacogenetic testing is cost effective and in the best interest of the patient.
Claim 2: Physicians seem to be unable to pick the right patients, and pharmacogenetic testing becomes an inefficient (as well as over hyped and expensive) general screening method, also lacking subsequent changes in medication-therapy. That the frequency of mutant alleles in the normal population does not differ from that in a given patient population, supports this notion.

Counter argument: The overall frequency in a population is irrelevant to the patient, as finding his individual response to the drug is what matters. This counter argument however, is an argument favoring screening in any given field of medicine and one may still claim that the overall cost-effectiveness is insufficient. But, in our laboratory, we do not normally get requests for pharmacogenetic testing prior to medication. Physicians using our service send samples from patients already on medication. They have come to their doctor with either adverse events or a lack of clinical effect. In our opinion these are the right patients to test. We have no reason to believe that the physicians resist changing to other medications based on our lab results when the case presented to them is as defined as this. Thus, pharmacogenetics in our hands, is targeted diagnostics rather than general screening.
In addition, we have done a small study (a panel of 12 SNP's and 2D6 copynumber variation on 595 patients) to show that the allele frequency in our patient population is either at the high end of normal variation, or above that seen in a normal population (see figure, European country codes in parentheses):

Similar results (see figure) are obtained when looking at frequency of poor and ultra rapid metabolizers (PM and UM respectively).

Consequently, it seems that physicians are able to pick the right patients. And, our results support the notion that pharmacogenetic testing constitutes targeted diagnostics in the best interest of the patient.

Reinton, N.In Defense of Pharmacogenetics.

Tuesday, May 27, 2008

The Swedish Chlamydia Mystery

Authors: Nils Reinton and Amir Moghaddam
Furst Medical Laboratory, Søren Bullsv. 25, N-1051 Oslo, Norway (

In 2006, Swedish researchers noticed a peculiar trend in the number of positive Chlamydia trachomatis cases. The number of infected patients was down by as much as 25 %. This was unexpected since there had been no public health (or preventive medical) actions to explain such a drastic decrease. Also, the numbers tested for C.trachomatis was similar to previous years. In addition, this trend had not been observed anywhere else. Something strange was happening to sexually active individuals in Sweden in particular.

The reason they found, was not due to calculation error or changes in sexual habits, but in molecular diagnostics, or more precisely, the genetic flexibility of
C.trachomatis (1). A genetic change had appeared creating a novel strain that was given the name "nvC.trachomatis.". This strain had a deletion in its "cryptic plasmid". The deletion was situated in the middle of the target sequence used by diagnostic kits from Abbott and Roche. Consequently, labs using kits from these suppliers (almost everyone in Sweden were using Roche) would misdiagnose any patient infected with the variant as negative for C.trachomatis infection. As a consequence of a diagnostics driven selection pressure, nvC.trachomatis may have reached almost 40 % of total C.trachomatis infections in Sweden (2). Numbers as high as 78 % were reported in some Swedish counties (3). Rapidly then, new tests were introduced to detect the strain and subsequent changes of laboratory routines in Sweden restored normal C.trachomatis detection specificities. Thus, the problem was fixed and everyone thought the mystery was solved.

Not so it seems. The real mystery started when neighboring countries started looking for the variant. Since there is extensive traveling and exchange of labor between the Nordic countries it would seem only natural that the variant C.trachomatis should spread rapidly to other countries as well. Curiously, that did not happen. By now, there have been studies in Norway (4), Denmark (5), England and Wales (6), Ireland (7) and the Netherlands (8). The only other countries nvC.trachomatis was detected was Norway where two out of 47 positives had the variant (one of these was a Swedish citizen) and Denmark which had only two cases out of a total of 383 positives. The conclusion from S Hoffmann and JS Jensen (reference 5) summed it up nicely:
"Sexually transmitted infections are unlikely to respect national borders, especially in an extended period of time. It was therefore an unexpected finding that only one case of the new CT variant was detected among 3,770 specimens tested during a five-month period. The samples were submitted from the whole of Denmark, although the majority came from the Copenhagen area. Considering the intense daily traffic between the Copenhagen area in Denmark and southern parts of Sweden, it is surprising that the spread occurred so late."
The spread is still limited. So far (April 2008) other labs in Norway have failed to find any cases at all, while our laboratory (in Oslo Norway, doing more than 20 000 C.trachomatis analyses a year) only rarely have cases of nvC.trachomatis infection. Consequently, by large the nvC.trachomatis strain remains Sweden-specific. A bug that is specific for only one given nationality is surely a novelty in epidemiology.

Finding the reason seems be far off at the moment. Because: is it likely that Swedes have strong sexual preferences towards other Swedes only ? Or are there biological differences that makes Swedes more prone to nv
C.trachomatis infection ? Sexual behavior and biological signature-attributes either in the infectious agent or in the host, are usually the starting points for STD epidemiology. But in this case either scenario is unlikely. So far then, the mystery remains unsolved.

The lesson learned in diagnostics however, was probably useful for future development of diagnostic tests as pointed out by Björn Hermann (reference
"What can we learn from the emergence of this new variant of chlamydia? This thrilling story provides several lessons. Firstly, how to design a diagnostic test. The new variant is a striking example of diagnostics driven evolution that must be considered when new methods are designed. Since routine diagnostics for chlamydia uses high volume testing based on nucleic acid detection, it is important that the targets used are not only conserved genetic elements but also essential for the organism."
Also, comfort can be taken in knowing that the variant C.trachomatis has lost its evolutionary advantage since due to these events, diagnostics manufacturers have changed their kits to be able to detect all known variants of C.trachomatis. And, in addition, one can hope that through this curious epidemiology event, awareness of STDs have been raised further.

Constant awareness is surely needed given the continuous rise in positive
C.trachomatis cases (regardless of variant strains). Unfortunately, a growing number of C.trachomatis-cases raises the probability of other new strains emerging through natural selection. This time it was easy to adapt to the new situation by changing the diagnostic method. The next time a variant bug appears it may not be this easy to find a fix. Treating the bug and stopping its spread (to achieve eradication) is what we should aim for. This story is just another one of the many wake-up calls given to us in the fight against disease-causing microorganisms over the recent years.

Reinton, N., Moghaddam, A.The Swedish Chlamydia Mystery.

Wednesday, March 26, 2008

Use of polyethylene glycol for drying polyacrylamide gels to avoid cracking

Authors: Amir Moghaddam and Nils Reinton

Furst Medical Laboratory, Søren Bullsv. 25, N-1051 Oslo, Norway (

Electrophoretic separation of proteins in non-denaturing and denaturing polyacrylamide gels remains a common technique in life science and discovery laboratories. When polyacrylamide gel electrophoresis is the last step in experimental investigation, the gel is dried down for storage, photography or exposure to X-ray film. There are two common methods for drying down polyacrylamide gels. The first and the older method is to place the gel on filter paper, to cover the gel with a plastic film and to dry the gel under vacuum and heat. Once the gel has dried, the plastic film can be removed. This method has been reproducibly used for decades for gels with low percentage polyacrylamide, such as less than 10%. If analysing relatively low molecular weight molecules, such as peptides, then higher percentage polyacrylamide gels is required and drying these gels becomes irreproducible and sometimes impossible. The gels would often crack. Several recommendations have been made to overcome the cracking problem, mainly by soaking gels in diluted glycerol. High percentage polyacrylamide gels, soaked in glycerol do not reproducibly dry with this method without cracking.

The second and newer method of drying polyacrylamide gels is to sandwich the gel with 2 cellophane sheets and to clamp the whole cellophane sandwich. The polyacrylamide gels needs to be soaked in 1-3% glycerol before drying and the cellophane sheets need to be soaked in 10% glycerol. As the cellophane dries, it shrinks and stops the gels cracking. The cellophane method is much more reliable for drying high percentage polyacrylamide gels than drying on filer paper, although the problem of cracking remains for 12 to 16% gels. However, there is one major draw back. The cellophane cannot be removed from the gel as it is irreversibly stuck to the gel. This becomes a problem if the gel is to be exposed to X-ray film and if the radiolabel is a or b emitter, such as 35S. For example, Metabolically labelled 35S-methionine and 35S-cysteine labelled proteins, separated on a polyacrylamide gel and dried in a cellophane sandwich cannot be exposed to X-ray film as the emitted particles do not cross the cellophane membrane. If fluorography is to be used, then the gels have to be soaked in a fluorophore and that increases the chances of cracking while drying.

We have overcome the problem of drying high percentage polyacrylamide gels without cellophane sticking by simply soaking our polyacrylamide gels in 20% polyethylene glycol (PEG)-400 (1) prior to drying. This technique is reliable and versatile and works with both gel-drying methods, drying on filter paper and drying in a cellophane sandwich. Figures 1 and 2 show the effectiveness of drying a 16.5% SDS-polyacrylamide gel by soaking in a solution containing 20% PEG-400 and 50% methanol for 15 minutes compared to soaking in glycerol.

Fig. 1:

Figure 1.

16.5% SDS-polyacrylamide gel was used for separation of proteins prior to fixing and staining with coomassie brilliant blue. The gels were not destained completely to allow easier photography.After destaining, the gels were soaked in 20% PEG-400 and 50% methanol (A) or 10% glycerol (B) for 15 minutes before placing on a wet 3 mm think filter paper (Whatman) and placed in a gel dryer and dried under vacuum at 75 oC for 2 hours.

Fig. 2:

Figure 2. 16.5% SDS-polyacrylamide gel was used for separation of proteins prior to fixing and staining with Coomassie brilliant blue. The gels were not destained completely to allow easier photography.After de-staining, the gels were soaked in 20% PEG –400 + 50% methanol for 15 minutes (A) or 3% glycerol for 60 minutes before sandwiching between two cellophane sheets. The sheets were soaked in water before hand according to the manufacturer’s instructions (Bio-rad). The gels were dried overnight.

We also tested whether PEG would interfere with fluorography. We found that we could supplement the fluorophore, AmplifyTM (Amersham), with PEG-400 to aid drying of the gel.Radiolabelled proteins on these gels would produce a stronger signal on an X-ray film compared to the same preparation radiolabelled proteins exposed to film without soaking in the fluorophore (figure 3). We could not assess the intensity of bands on an X-ray film, if the gel was exposed to fluorophore alone without PEG, as the gels would consistently crack into many pieces during drying.

Fig. 3:

Figure 3. An antibody and protein-A sepharose was used for immunoprecipitation of its target antigen from cells radiolabelled with 35S-methionine. The immunoprecipitate was boiled in Laemmli buffer and was loaded onto two lanes of a 16.5% polyacrylamide gel, half in each lane. After separation, the gels was fixed. One lane was soaked in Amplify™ supplemented with PEG-400 to 20% and the other in 20% PEG-400 alone. After drying, the plastic sheet was removed and the gels were exposed to X-ray film.

Many modern laboratories are equipped with cellophane cassettes and not heated vacuum gel dryers. For exposure of gels with b-emitting radiolabels to X-ray film, it remains necessary to remove cellophane sheet from one side of gel. In this case, one side of the gel can be covered with a plastic film, that has been cut to the same size as the gel, before putting on the cellophane. After drying the gel, the plastic film and cellophane from one side of the gel can be pealed off to expose the gel (not shown).

In summary, 20% PEG-400 is a good substitute to glycerol for drying polyacrylamide gels. It works with heated vacuum gel dryers and cellophane sheets and allows cellophane sheets to be peeled off if necessary. 20% Methoxy-PEG (poly(ethylene glycol methyl ether)-350 (1) can also be for drying gels with the same apparent effectiveness and versatility (data not shown).