R Diaz, F Crespo, S Herrera, J Sevy-Court, A Marrero, D van Soolingen
cross-contamination, dna fingerprinting, is6110 rflp, m. tuberculosis
R Diaz, F Crespo, S Herrera, J Sevy-Court, A Marrero, D van Soolingen. Laboratory Cross-Contamination Of Mycobacterium Tuberculosis During Preparation Of Smears. The Internet Journal of Third World Medicine. 2004 Volume 2 Number 2.
A possible cross-contamination of Mycobacterium tuberculosis at the Epidemiology and Hygiene Center of Havana (EHCH) occurred in 1999. Nine M. tuberculosis isolates from two acid-fast bacilli (AFB) smear-positive sputa and seven AFB smear-negative specimens were analyzed by IS6110 DNA fingerprinting. All isolates had identical fingerprints indicating a laboratory cross-contamination. No breaking rules were registered on the specimen transportation, sample labeling neither during sputum processing for culturing. Additional investigations in the peripheral laboratory (ML-HC2) showed that the recent entrance of new personnel in conjunction with severe difficulties in preparing smears for microscopy might have provoked the cross-contamination incident. The use of only one metal wire inoculation loop for smearing all series of sputa, a sand flask without alcohol and a faulty burner for loop sterilization, indicating the cross-contamination most likely occurred in ML-LC2. Multiple actions taken in the ML-LC2 have allowed that none laboratory contamination event has been reported after this.
After several decades of decline, a resurgence of tuberculosis occurred in Cuba in the last decade (Marrero et al. 2000). This led to a two-three fold increase in the number of specimens being processed for the detection of
In recent years, to detect cross-contaminations and to identify possible sources, IS
In this report, we applied IS
Materials And Methods
This study concerns persons with respiratory symptoms (cough and expectoration for more than 14 days) and positive
Sputa were analyzed in the microbiology laboratory of a peripheral health center (ML-HC2) in Havana by acid-fast microscopy using the Ziehl-Neelsen method (Kent and Kubica 1985). Specimens were processed in the reference tuberculosis laboratory (RTL) at the EHCH by the Petroff´s NaOH method and the final sediment was inoculated onto Löwenstein-Jensen slants (Kent and Kubica 1985).. Identification of
The laboratory records and procedures for all false-positive cultures were reviewed to try to determine at which step the cross-contamination may have occurred.
Nine mycobacterial primary cultures all processed on May 31, 1999 by the same technician in the RTL-EHCH, were identified as
The extent of growth of isolates from smear negative specimens varied from a single colony (1) to a dozen colonies (12) per slant.
Public health officials were informed of the suspicion that
Figure 1: IS
Lane one, DNA from
Lanes 2 and 10, isolates from AFB smear-positive sputa of a patient with a clinical and radiological evidence of pulmonary tuberculosis (source case).
Lane 3-9, culture isolates of
Numbers on the left indicate the size of standard DNA fragments (in kilobase pairs).
Because the specimens had been processed at the same day, and by the same technician, but in batches with other culture-negative specimens from geographically separated patients and because at the RTL-EHCH a well-trained and experienced technician had strictly followed all laboratory procedures recommended by the National Reference Laboratory at IPK, the laboratory cross-contamination was hard to imagine at this level.
A detailed investigation in the RTL-EHCH showed there was no evidence of the incorrect application of a certain technique in this laboratory. Specimens were divided in batches and processed in a properly maintained, vertical laminar down flow biosafety cabinet. Work surfaces were disinfected regularly with phenol. Each specimen was carefully manipulated to avoid accidental transfer of bacteria from one sample to another via pipettes, caps of tubes or common reservoirs of reagents or containers to discard material. A separate sterile glass pipette for each specimen or each reagent aliquot was used in all cases. Specimens were decontaminated, digested and neutralized with ready-to-use working solutions that were discarded later on. After centrifugation, the decontaminated specimen sediments were inoculated on Löwenstein-Jensen slants with a separate pipette.
On the other hand, the transportation procedure of specimens for culturing, from ML-HC2 to RTL-EHCH, was also evaluated. The personnel who transported the containers were interviewed to detect any incident of mix of samples in the sputum containers. However, no irregularity was observed in both cases.
A different investigation done in the ML-HC2, which does not have biological safety cabinet, revealed some difficulties during the preparation of smears for microscopy and the presence of new personnel in the laboratory. There was only one nichrome wire inoculation loop to pick up the sputa for smearing, which was passed through a sand flask without alcohol in between the processing of different samples.
Additionally, it was inadequately heated for a few seconds (but not sterilized) under the yellow flame of a faulty and old Bunsen burner (no blue cone was observed in the burner flame). An AFB smear-positive sputum
In the last decade, many cross-contamination events have been adequately confirmed by the high resolution IS
In Cuba, some possible cross-contamination incidents have been found earlier (Diaz et al. 1998, 2001), but these have been documented only partly.
We here describe seven “patients” whose
Finished our investigation, some actions were taken in the ML-HC2 to implement the good laboratory practice in this setting and therefore to minimize the risk of cross-contamination. These actions included changing the Bunsen burner, adding 95% alcohol to the sand flask, supplying five new nichrome wire inoculation loops, training of laboratory technician and professional personnel, re-evaluating of general conditions of the laboratory. None laboratory contamination event has been reported for sputum samples from ML-HC2 in the last five years.
In Cuba, wire loops to prepare smears of sputa are commonly used. We are now recommending using disposable wooden sticks for smear microscopy in Cuba in stead of nichrome wire loops. Single-use wooden sticks are cheaper and avoid the transfer of mycobacteria from a sample to another during the smear process.
The episode mentioned here was an alert calling to the National Control Program for improving the quality assurance of smear microscopy in peripheral laboratories.
Although in this event none of the seven false-positive patients were treated with antituberculous drugs, false-positive cultures resulted in unnecessary physician consultations and diagnostic tests, including radiography and bronchoscophy, for the respective patients. It would also have affected their social, laboral and familiar environment as suspected cases of active tuberculosis.
To our knowledge, this is the first report of laboratory cross-contamination during the acid-fast microscopy procedure, at the primary level health services, adequately confirmed by IS
Because many laboratories in Third World settings still work in this way, this may disclose another pathway of laboratory cross-contaminations leading to over diagnosis of tuberculosis.
This event again shows that good laboratory practice is of the utmost importance to prevent laboratory mishaps that have serious consequences for the concerned patients.
Raul Diaz, PhD Fax: (537) 204-6051. E-mail: firstname.lastname@example.org