1. Applying IS6110: Restriction Fragment Length Polymorphism Analysis to Aide in the Identification of
Laboratory Cross-Contamination
Erick Cortes, MPH 1 Henry Fraimow, MD 2 Barry Kreiswirth, PhD 3 Natalia Kurepina , PhD 3 Elena Shashkina , PhD 3
1
New Jersey Department of Health and Senior Services, 2 Cooper University Medical Center, 3 Public Health Research Institute and UMDNJ/ NJMS-Global Tuberculosis Institute
AbstrAct bAckground Methods results Case Study
Background: The New Jersey Department of Health & Senior • Since cooperating within the Universal Genotype Project in early • In late December 2009, the State TB Genotype Coordinator (GC) • LJ slants for all M.TB isolates identified at the hospital during the
Services has established a partnership with the Public Health 2007, the State TB Program has continued to work closely with was notified of a possible episode of laboratory contamination, period from October 2009 through January 2010 were submitted In late December of 2009, a New Jersey University Hospital and Trauma Center
Research Institute (PHRI) to obtain IS6110-RFLP analysis for all all Hospitals and reference labs operating throughout the State of with 5 independent samples having cultured positive for MTB, to the Public Health Research Institute for IS6110- RFLP analysis reported cultures positive for M tuberculosis on samples from five independent
New Jersey. from a New Jersey Tertiary Care University Hospital. patients. Each of the five samples were collected between 10/3/2009 and
positive M. tuberculosis (M.TB) cultures prior to submission to • These included 5 specimens collected in October 2009 and
10/31/10, all from different source sites, and all were initially reported as growth
the regional genotyping laboratory in Michigan. In December • Submission of Mycobacterium tuberculosis (MTB) isolates for • Six weeks later, in February 2010, that same hospital laboratory 9 collected in December 2009.
positive in MGITTM culture system between 11/17/2009 and 11/27/2009.
of 2009, the Director of Microbiology at a New Jersey hospital reported an additional 9 independent samples as having cultured • Of five initial specimens, only the patient with the BW562 strain
IS6110 RFLP analysis has continued to increase, resulting in On January 11, 2010 the State TB Genotype Coordinator (GC) was contacted
notified the state TB Genotyping Coordinator of potential positive for MTB. was considered “high probability ” of having TB disease. This
universal coverage for all reported and available positive cultures by the Hospital Director of Microbiology, concerned of the possibility for lab
cross contamination of specimens from five individual patients patient had clinical and radiographic findings consistent with
for the State of New Jersey. • During this three month time period, the number of patients contamination. In cooperation with the State TB Program, and the Public Health
submitted to the laboratory in the same week and culture Pulmonary tuberculosis, and his was the only specimen that was Research Institute, cultures for the isolates in question were submitted for IS6110-
with cultures positive for MTB far exceeded the laboratory
positive for M.TB. In February of 2010, the same laboratory • In addition to isolate submission, many Hospitals and Reference both smear as well as culture positive. RFLP Analysis. Central State Case reporting for the cultures had been halted,
average for the previous seven years (Table 1).
reported nine additional potentially contaminated specimens Labs have also requested summaries of specific IS6110 Restriction pending molecular beacon results.
testing culture positive for M.TB. Fragment Length Polymorphism (RFLP) results to aid in Quality • Upon the request of the Hospitals Director of Microbiology, the • The other four were identified as H37Ra, a laboratory quality
Assurance from their institutions. GC was asked to provide assistance in differentiating between control strain serving as the contaminant, classifying these
Methods: LJ slants of all cultures were sent to PHRI for M.TB true and false positive results. patients as “low” to have TB. Treatment had been started on IS6110-RFLP analysis was completed for all five cultures 9 days later. Molecular
strain genotyping. Standard IS6110-based RFLP genotyping • Continued collaboration between the State TB Program, and two patients in this group, but was stopped when cultures were analysis indicated that four of the cultures isolated produced a fingerprint identical
all operational Mycobacteriology Laboratories, responsible for • Bio-Images were compared with the PHRI image database (over confirmed as laboratory contaminants to strain H37Ra, a standard non-pathogenic laboratory quality control strain and
method was applied to individually fingerprint each M.TB
culturing specimens reported by the State of New Jersey, has 10,000 images / 28,000 M.TB isolates) for strain identification. thus a likely contaminant. The fifth culture was found to have a unique RFLP
isolate. Images were compared with the PHRI image database • Of the nine subsequent specimens sharing the BW562 RFLP, only
(over 10,000 images, 28,000 M.TB isolates) for strain resulted in identifying instances where probable laboratory • RFLP results were then compared to the clinical impression of fingerprint labeled BW562, and was considered a likely true-positive M.Tuberculosis
two were classified by the physician as “moderate probability” of
identification. RFLP results were compared to the clinical cross-contamination has occurred. a physician at the Hospital knowledgeable in TB diagnosis and culture. This patient also had 3 subsequent sputum cultures that also grew
having TB. TB treatment was initiated in one of these patients,
impression of a physician knowledgeable in TB diagnosis and treatment who either personally evaluated or reviewed the medical M.tuberculosis in November, 2009. IS6110 RFLP results were immediately disclosed
• We describe our recent experience in which the timely a 26 year old Vietnamese immigrant with a positive PPD, to the State TB Progam’s Surveillance Unit, the Hospital Microbiology Director, and
treatment. This physician categorized each patient as clinically availability of RFLP data enabled rapid determination of a records and radiology of all patients with positive MTB cultures. Quantiferon test, and pulmonary nodule.
“low”, “moderate”, or “high” probability to have TB. to the TB specialist on staff at ths hospital, who was also the consultant for the
laboratory contamination problem and limited unnecessary • Categorization of each patient was established to be clinically Tuberculosis Program in the county where the hospital was located . Immediate
Results: Of the first five specimens analyzed by IS6110-RFLP, anti-tuberculosis treatment of a large number of patients. “low,” “moderate” or “high” probability of having TB Disease. action taken by the Hospital Laboratory Staff, in cooperation with the State TB
four were identified as H37Ra, a laboratory quality control Control Program had simultaneously avoided erroneous State Case Reporting,
as well as prolonged treatment for the four false-positive cases. The Hospital
strain serving as the contaminant. The last specimen was a true
M.TB culture yielding a unique RFLP identifier of BW562. The
conclusions laboratory also amended the microbiology reports for the specimens with likely
subsequent nine specimens, analyzed separately from the first • Of the 13 false-positive cases, only 3 patients were placed on contaminants with the statement “FURTHER GENETIC TESTING STRONGLY
five, shared an RFLP identifier of BW562, matching the true treatment until IS6110 RFLP results were available, and TB disease SUGGESTS THIS ISOLATE IS A CONTAMINANT”
Table 1. Hospital Cure History
M.TB culture from the first cluster. had been ruled out. In all but one patient with a false positive
2002 2003 2004 2005 2006 2007 2008 1/9/2009 to 9/30/2009 10/1/2009 to 12/20/2009* culture, clinical findings and negative culture data were sufficient
Total # of Patients with Positive MTB Cultures 5 4 7 10 12 13 7 2 14
On February 3, 2010 the same New Jersey Hospital once again contacted the
Conclusions: Of five initial specimens, only the patient * includes those specimens subsequently
to rule out a diagnosis of TB disease. GC for assistance with an additional nine specimens that cultured positive for
Total # of Specimens Growing MTB 15 4 27 30 45 36 28 4 17
with the BW562 strain was considered “likely” to have TB. identified as likely contaminant
• Laboratory cross contamination of multiple-aliquot diluent M.Tuberculosis. Each of the 9 specimens were collected on different dates between
TB was classified as “unlikely” for all four patients sharing solutions was most likely the source of contamination in the 12/8/2009 and 12/18/2009 and from multiple source sites and all were initally
the H37Ra strain. Treatment was initiated for two of these Culture Date Site Smear Date MGIT Positive Date Probe Positive Probability of TB PPD/IGRA Meds Started Meds Stopped Date To PHRI Date of PHRI Result IS6110 Identification
10/05/09 BAL negative 11/20/09 12/03/09 low negative no NA 12/16/09 01/20/10 H37Ra
second cluster reported as positive in the MGITTM culture system between 12/31/2009 and
patients, but discontinued when RFLP results were known. 10/02/09 T10 Spine negative 11/27/09 12/03/09 moderate PPD 3 mm yes yes 12/16/09 01/20/10 H37Ra • The local availability of IS6110-RFLP Analysis provides real time 1/18/2010. Previous cooperation between the Hospital Laboratory, and the State
Of the nine subsequent specimens sharing the BW562 RFLP, 10/05/09 Peritonsilar abscess negative 11/27/09 12/03/09 low not done no NA 12/16/09 01/20/10 H37Ra actionable information earlier than results generated from the TB Program had ensured an efficient system for identifying lab contamination. In
only one was classified by the physician as “possibly” having 10/31/09 sputum rare 11/16/09 12/03/09 high positive yes no 12/16/09 01/20/10 BW562
Regional Genotyping Laboratory that directly impacted on clinical similar fashion as the previous episode, the 9 cultures were submitted to the Public
TB and treatment was initiated, but stopped when disease 10/06/09 parasacral fluid negative 11/17/10 11/19/10 low negative yes yes 12/16/09 01/20/10 H37Ra Health Research Institute for IS6110 RFLP Analysis. IS6110-RFLP results returned 12
decision making.
was eventually ruled out. Due to suspicion of contamination days later with all specimens producing a single identical fingerprint result, BW562.
12/15/09 sputum negative 01/18/10 01/28/10 moderate positive no NA 02/05/10 02/11/10 BW562 • Continued effective working relationships have expanded the This RFLP fingerprint was the identifier of the previous true positive case. Immediate
by the laboratory and the rapid availability of IS6110-RFLP to 12/18/09 BAL negative 01/18/10 01/28/10 low no data no NA 02/05/10 02/11/10 BW562
State TB Program’s Genotyping Project from a surveillance dissemination of results were issued to the State TB Program’s Surveillance Unit,
confirm this fact, therapy was never initiated for 10 of these 12/14/09 inguinal LN negative 12/31/10 01/14/10 low no data no NA 02/05/10 02/11/10 BW562
12/18/09 BAL negative 01/12/10 01/28/10 moderate positive yes no 02/05/10 02/11/10 BW562 resource, to a systematic Quality Assurance method for all the Hospital Laboratory, and to the TB physician on staff. As a result of the
13 false-positive cases. Laboratory cross contamination of
12/08/09 stool negative 01/18/10 01/28/10 low no data no NA 02/05/10 02/11/10 BW562 reported samples. identification of these contamination problems, procedures and practices in the
multiple-aliquot diluent solutions was most likely the source of 12/15/09 sputum negative 01/04/10 01/14/10 low negative no NA 02/05/10 02/11/10 BW562
contamination in the second cluster. No subsequent episodes of 12/18/09 BAL negative 01/18/10 01/28/10 low no data no NA 02/05/10 02/11/10 BW562
• Timely dissemination of IS6110-RFLP information has resulted Hospital Laboratory were reviewed and modified to eliminate potential cross-
cross contamination have occurred at this laboratory. 12/12/09 sputum negative 01/18/10 01/28/10 low no data no NA 02/05/10 02/11/10 BW562 in a more proactive coordination between reference laboratories contamination, including pre-aliquoting to avoid use of multi-dose diluent vials and
12/11/09 BAL negative 01/16/10 01/28/10 low no data no NA 02/05/10 02/11/10 BW562 across the US and the State TB Control Program. enhanced decontamination procedures.