Archive for May, 2014

The tuberculin skin test: how safe is safe? -the tuberculins contain unknown forms capable of reverting to cell-wall-deficient mycobacteria

May 27, 2014

Alexander P. Lysenko, Vladimir V.Vlasenko, Lawrence Broxmeyer, Artem P. Lemish, Tatiana P. Novik, Andrei N. Pritychenko

Clinical and Experimental Medical Sciences, Vol. 2, 2014, no. 2, 55 – 73.


® U.S. Library of Congress


Tuberculin is made from proteins derived from tubercle bacilli that have been “killed” by heating. Yet in both Zwadyk’s 1994 study and Bemer-Melchior’s 1999 investigations ‘heat-killed’ tuberculosis and its related mycobacteria, whether in tuberculin, vaccination or otherwise, have dormant, practically indestructible cell-wall-deficient forms which can revert back to virulent TB bacilli “killed” ― by neither heat nor sterilization. The ability and actual preference of mycobacteria such as Mycobacterium tuberculosis and Mycobacterium bovis to form filterable, multi-shaped cell-wall-deficient (CWD) forms and spores in order to survive unfavorable conditions has in fact been known for some time. But the possibility of PPD tuberculins for human use containing such potentially virulent CWD forms, even after autoclaving, sterilizing and ultrafiltration, has not. Autoclaved ultra-filtrates of the various mycobacteria used to produce tuberculin skin tests, consisting of M. Tuberculosis, M. bovis, and M. avium were investigated. All samples were mixed with growth stimulant, incubated, and placed on a special nutrient medium with a 1% agar base. Within 2-10 days after incubation colonies of a variety of non-acid-fast forms were noted, yet all of these proved, through PCR real time with FAM probe to still have antigens in common with their classic tubercular parent form, from which they originated. Moreover, in true cell-wall-deficient fashion, the isolates, upon guinea pig inoculation, did not immediately produce visible lesions, but nevertheless persisted. However, tissue homogenates of the infected animals, once placed on a growth-enhancing medium showed cell-wall-deficient mycobacterial forms interspersed with classical acid-fast rods. And a repeated passage of such tissue homogenates back into non-infected guinea pigs, not only induced small mycobacterial granulomas in their livers, but a distinct increase in acid-fast rods. Moreover, similar cell-wall-deficient mycobacterial forms with acid-fast rods occurred when embryonated chicken eggs were inoculated with PPD tuberculins as well.
The autoclaved and supposedly “sterilized” purified protein derivative [PPD] used in tuberculin skin tests contain cell-wall-deficient forms capable of eventually reverting back to virulent acid-fast tuberculosis, both typical and atypical.

KEYWORDS: Tuberculin; Mycobacterium bovis; thermo stability; Cell-Wall-Deficient mycobacteria


1.WHO Report Global Tuberculosis Control 2010 World Health Organization; WHO Library Cataloguing-in- Publication Data 2010; 218pp p.16
2.Livingston V, Allen RM. Presence of consistently recurring invasive mycobacterial forms in tumor cells.Microscop Soc Bull 1948; 2: 5–18.
3.Livingston, Virginia Wuerthele-Caspe. Cancer: a new breakthrough, Los Angeles: Nash Publishing; 1972. 269pp.
4.Cantwell, A The Cancer Microbe. Los Angeles: Aries Rising Press; 1990. 283pp.
5.Guliang H, Tefu L.Mycobacterium tuberculosis L-forms. Microbial Ecology in Health and Disease 1999; 10: 129-133.
6.Song L-Y, Yan W-S, Zhao T Detection of Mycobacterium tuberculosis in lung cancer tissue by indirect in situ nested PCR; Journal of First Military Medical University; 2002:11.
7.Nalbandian A, Yan BS, Pichugin A, Bronson T, Kramnik I. Lung carcinogenesis induced by chronic tuberculosis infection: the experimental model and genetic control. Oncogen
2009 28(17): 1928-1938.
 8.Anestad G, Hoel T, Scheel O, Vainio K. Atherosclerosis and tuberculosis: are they
both chronic infectious diseases? Scand J Infect Dis 2001;33 (10):797.
9.Livingston VW, Alexander-Jackson E. Mycobacterial forms in myocardial vascular
disease. J. Amer Med Wom Assoc. 1965; 20: 449-452.
10.Broxmeyer L. Is mad cow disease caused by a bacteria? Med Hypotheses. 2004; 63 (4):731-739.
11.Cantwell AR. Variably acid-fast cell wall-deficient bacteria as a possible cause of dermatologic disease.In, Domingue GJ (Ed.) Cell Wall Deficient Bacteria.Reading: Addison-Wesley Publishing Co; 1982. pp. 321-360.
12.Cantwell AR Jr, Kelso DW. Variably acid-fast bacteria in a fatal case of Hodgkin’s
disease. Arch Dermatol 1984; 120 (3):401-402.
13.Centkowski P, Sawczuk-Chabin J, Prochorec M, Warzocha K. Hodgkin’s lymphoma and tuberculosis coexistence in cervical lymph nodes.Leuk Lymphoma; 2005: 46 (3):471-475.
14.Mattman LH Cell Wall Deficient Forms – Stealth Pathogens. CRC Press Boca Ra-
ton 3rd ed., 2001 416pp.
15.Marcova N, Slavchev G, Michailova L, Unique biological properties of Mycobacterium tuberculosis L-form variants: impact for survival under stress. Int Microbiol; 2012: 15 (2): 61-68.
16.Ghosh J, Larsson P, Singh B, Petterson BMF, Islam NM, Sarkar SN, Dasgupta S, Kirsbom LA Sporulation in mycobacteria. Proc Natl Acad Sci USA 2009, 106 (26):10781-10786.
17.Singh B, Ghosh J, Islam NM, Dasgupta S., Kirsebom LA. Growth, cell division and
sporulation in mycobacteria. Antonie van Leewenhoek 2010; 98(2):165-177.
18.Vlasenko VV Tuberculosis in focus of problem contemporarily (in Russian) Vinnica: Nauka, 1998. 350 pp.
19.Lysenko AP, Lemish AP, Krasnikova EL Investigation of the thermal resistance of Mycobacteria tuberculosis. Probl. Tuberk Bolezn. Legk. 2007;(2):42-46.
20.Сsillag A. The mycoccocus form of mycobacteria. J. Gen. Microbiol 1964; 34:.341-
21.Chandrasekhar S, Ratnam S Studies on cell-wall-deficient non-acid fast variants of Mycobacterium tuberculosis. Tuber Lung Dis 1992; 73 (5):273-279.
22.Beran V, Havelkova M, Kaustova L, Dvorska J, Pavlik I Cell-Wall-Deficient forms of
mycobacteria: a review. Veterinarni Medicina 2006; 51, (7): 365-389.
23.Slavchev G, Michailova L, Marcova N Stress-induced L-forms Mycobacterium bovis: a challenge to survivability. New Microbiologica 2013; 36:157-166.
24.Mukamolova G, Turapov OA, Young DI, Kaprelyants AS., Kell DB, Young M A family of autocrine growth factors in Mycobacterium tuberculosis Molecular Microbiology 2002; 46 (3): 623-35.
25.Lysenko AP, Archipov IN, Lemisch АP, Novik TP, Bogdanovich SV Features of antigenic composition of changed forms Mycobacteria tuberculosis. Probl. Tuberk Bolezn. Legk. 2010; 4:41-45.
26.Ma XL et al. Experimental studies on pathogenicity of Mycobacterium tuberculosis
L-form. Chinese Journal of Microekology 1995; 01.
27.Markova N, Slavchev G, Michailova L, Jordanova M Survial of Escherichia coli under lethal heat stress by L-form conversion. Int. J. of Biological Sciences 2010; 6 (4): 303-315.
28. Robinson DH. Pleomorphic mammalian tumor-derived bacteria self-organize as mul- ticellular mammalian eucaryotic like organisms: morphogenetic properties in vitro, possible origin, and possible roles in mammalian «tumor ecologies».Medical Hypothesis
2005; 64 (1):177-185.
29.Seibert FB, Feldmann FM, Davis RL, Richmond IS Morphological, biological, and immunological studies on isolates from tumors and leukemic bloods Ann. N.Y. Acad.
Sci 1970: 174: 690-728.
30.Foddai A., Elliot CT, Grant IR. Rapid assessment of the viability of M.avium subsp. paratuberculosis cells after heart treatment, using an optimized phage amplification
assay. Appl Environ Microbiol 2010; 76:1777-82.
31.Thom M, Morgan JH, Hope JC, Villareal-Ramos B., Martin M., Howard CJ The effect of repeated tuberculin skin testing of cattle on immune responses and disease following experimental infection with Mycobacterium bovis. Vet. Immunol Immunopathol 2004; 102:399-412.
32.Zwadyk P, Down JA, Myers N, et al. Rendering of mycobacteria safe for molecular
diagnostic studies and development of a lysis method for strand displacement ampli-fication and PCR. J Clin Micobiol 1994;32:2140–6.
33.Bemer-Melchior P, Drugeon HB. Inactivation of Mycobacterium tuberculosis for DNA typing analysis. J Clin Microbiol 1999;37:2350–1
34.Pence CD, Ohls, HG. Tuberculosis of the Eye; with Specific Reference to Treatment. Illinois Medical Journal. Chicago April, 1916. 29:4: 307-39
Received: April 29, 2014