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Multidrug-Resistant Human Mycobacterium tuberculosis Strain Resisted Inactivation by 10% Formalin

Research Article Open Access
Oke AJ1*, Adeyemo AD2 and Oke AA3
1Department of Medical Microbiology, Bowen University Teaching Hospital, Ogbomoso, Nigeria
2Department of Medical Microbiology, College of Medicine, University College Hospital, Ibadan, Nigeria
3Department of Medical Microbiology, College of Health Sciences, Igbinedon University, Okada, Nigeria
*Corresponding authors: Dr. Oke AJ
Faculty of Basic Clinical Sciences
Bowen University Teaching Hospital
Ogbomoso, Nigeria
Tel: +2348034231902
E-mail: adefolaames@yahoo.com
 
Received September 20, 2012; Published September 27, 2012
 
Citation: Oke AJ, Adeyemo AD, Oke AA (2012) Multidrug-Resistant Human Mycobacterium tuberculosis Strain Resisted Inactivation by 10% Formalin. 1:344. doi:10.4172/scientificreports.344
 
Copyright: © 2012 Oke AJ, et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
 
Abstract
 
Multidrug-resistant strain of Mycobacterium tuberculosis (MDR-Tb) and Multidrug -sensitive Mycobacterium tuberculosis (MDS-Tb) isolated from humans were injected subcutaneously into the guinea pig. One set of infected lungs of animals was fixed in 10% formalin pH 7.2, while the other set was treated with 75% ethanol, 2 hours prior to fixation in 10% formalin.
 
After six months of fixation, MDS-Tb strains were completely inactivated by both methods of fixation. MDR-Tb strains resisted inactivation by 10% formalin, but were inactivated by the treatment with 75% ethanol 2 hours prior to 10% formalin fixation.
 
Keywords
 
Formalin; Ethanol; Inactivation; Mycobacterium tuberculosis
 
Introduction
 
Formalin is widely used to fix histological preparations, and as preservatives, in embalming solutions. It is an age-long practice in medical laboratories [1-3]. It is generally accepted that the risk of contracting tuberculosis is relatively high among Medical Laboratory workers and Pathologists. It is an assumption that once tissue is fixed in formalin, the risk for transmission and subsequent infection of Mycobacteria is greatly reduced, if not altogether eliminated [4-6]. Cadavers remain a practical teaching tool for Anatomists and Medical educators teaching gross anatomy. Infectious pathogens in cadavers that present particular risks include Mycobacterium tuberculosis, Hepatitis B and C, the AIDS virus HIV, and Prions that cause transmissible spongiform encephalopathies [7].
 
Recent studies have however, suggested that formalin does not effectively inactivate Mycobacterium tuberculosis in formalin-fixed tissue [4-13]. In this study we tried to test the inactivation ability of (a) 10% formalin fixation and (b) treatment of tissue with 75% ethanol, 2 hours prior to 10% formalin fixation. Multiple Drug Resistant (MDR) and Multiple Drugs Susceptible (MDS) strains of Mycobacterium tuberculosis were employed.
 
Methods
 
Official consent was obtained from the Head of Department, Chest Clinic, Baptist Medical Centre Ogbomoso. All subjects included in this study were volunteers.
 
Sputa were collected from subjects at the Chest clinic of Baptist Medical Centre Ogbomoso. Samples were stained by Ziehl-Neelsen’s (ZN) technique for Acid Fast Bacilli (AFB). Positive AFB samples were cultured on Lowenstein-Jensen (L-J) medium before the commencement of multiple drug therapy [14,15]. The different drugs used at the clinic include; rifampicillin, isoniazid (INH), pyrazinamide and ethambutol.
 
Primary growth on L-J was kept. Another round of sputa was collected from the same sets of subjects after the second and fifth months of directly observed multidrug therapy (DOT). The samples were stained. The positive AFB smears at the second and fifth month of DOT were considered to be MDR-Tb [16,17]. The negative AFB smears at the second and fifth months of DOT were considered MDSTb, and the primary growth from such samples were used as MDS-Tb.
 
MDR-Tb sputa samples were cultured on L-J, and incubated. One set of animals was injected subcutaneously with the primary MDSTb growth, while another set was injected with the MDR-Tb growth [14]. Control animals were injected with sterile normal physiological saline. Animals were kept, fed on pellets and sterile water. They were watched for symptoms of disease, after which they were scarified and lungs excised.
 
Parts of the lungs were processed for culture on L-J media to confirm infection in the animals. Parts of infected lung tissue were fixed in 75% ethanol for 2 hours and then in 10% formalin. Other parts of the lungs were fixed in 10% formalin pH 7.2. The fixed tissues were kept for 6 months. Lung tissues were processed for culture on L-J media, and incubated aerobically at 36°C.
 
Results
 
Fifty AFB-positive subjects were encountered in this study. Four (8%) were carrying MDR-Tb.
 
The animals injected with mycobacteria cultures developed local swellings and weight-loss after one month, and mycobacteria were isolated from their lungs when cultured on L-J medium. There was no growth from the lung tissue of the control animals. There was no growth from the lungs of MDS-Tb infected animals in both fixation methods (Figures 1 and 2). In the MDR-Tb infected lungs, there was no growth in the 75% ethanol, 10% formalin fixation, but there was growth in the 10% formalin fixation (Figures 3 and 4).
 
Figure 1: MDS-Tb fixed in 10% formalin – No growth.
 
Figure 2: MDS-Tb fixed in 75% ethanol/10% formalin – No growth.
 
Figure 3: MDR-Tb fixed in 75% ethanol/10% formalin – No growth.
 
Figure 4: MDR-Tb fixed in 10% formalin-showing growth of Mycobacterium.
 
This shows that both methods of fixation were able to inactivate MDS-Tb in lung tissue. MDR-Tb was not inactivated by 10% formalin fixation, but was inactivated by 75% ethanol/formalin fixation.
 
 
Discussion
 
The results of our study show that treatment of lung tissue with alcohol before fixation in 10% formalin was able to inactivate Mycobacterium tuberculosis in tissue including MDR-strain that resisted 10% formalin.
 
Environmental persistence of M. tuberculosis is subject of speculation. However, the reality that infected post mortem tissue can be a danger to Pathologists and embalmers has worrisome implications. A few experimental studies have demonstrated the ability of Mycobacterium tuberculosis to withstand exposure to embalming fluid and formalin. Gerston et al. succeeded in isolating live tubercle bacilli from 3 of 25 formalin fixed tissues. In a following report using a larger sample of 138 formalin-fixed lungs, they were able to obtain growth of Mycobacteria in three cases [5]. Treatment of tuberculosis is far from easy and frequently results in failure or even death [16-19]. The world is recording a high rate of death due to multiple-drug resistant tuberculosis. The results of workers suggested that there is a risk of contracting tuberculosis from tissue that has been fixed in formalin if aerosolization or accidental inoculation should occur [5]. In view of these, more effective fixative method should be employed to prevent occupational hazards to Medical Laboratory Scientists, Pathologists, Anatomists and Medical educators.
 
It has been reported that alcohols, especially 70% ethanol, have rapid mycobactericidal activity [1], and a tuberculocidal fixation may be prepared with 10% formalin in 50% ethanol [10]. Treatment of tissues with 95% ethanol prior to fixation in 10% formalin has been suggested [20]. Multiple toxic effects of alcohols on structure and metabolism are suspected, primarily due to protein denaturation and coagulation. Alcohol had been reported to disrupt the amino acid-amino acid bond to form an amino acid-alcohol hydrogen bond [1].
 
The result of this investigations are in agreement with the suggestions of other workers for the use of alcohol in addition to the conventional 10% formalin fixation of tissues [10,20,21]. This is pertinent, more so now that more people die due to MDR-TB infections.
 
Acknowledgement
 
We acknowledge Mr. Festus Obaseki, TB Lab, UCH for his great assistance in culturing process; Head of Chest Clinic, Baptist Medical Centre Ogbomoso for giving consent; Mrs. Adigun C, for her assistance in sample collections; Mr. Tayo Adebayo who assisted in procuring the animals.
 
 
References