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Recognizing Laboratory Cross-ContaminationCross-Contamination in Culture-Positive TB Cases: Two False-Positive Cultures of Mycobacterium tuberculosis—Oklahoma, 2011 FREE TO VIEW

Matthew G. Johnson, MD; Phillip H. Lindsey, MD; Charles F. Harvey, DO; Kristy K. Bradley, DVM, MPH
Author and Funding Information

From the Acute Disease Service (Drs Johnson, Lindsey, and Harvey), Oklahoma State Department of Health, Oklahoma City, OK; Epidemic Intelligence Service (Dr Johnson), Centers for Disease Control and Prevention, Atlanta, GA; and Office of the State Epidemiologist (Dr Bradley), Oklahoma State Department of Health, Oklahoma City, OK.

Correspondence to: Matthew G. Johnson, MD, 1000 NE 10th St, Oklahoma City, OK 73117; e-mail: mgjohnson33@gmail.com


Reproduction of this article is prohibited without written permission from the American College of Chest Physicians. See online for more details.


Chest. 2013;144(1):319-322. doi:10.1378/chest.12-2294
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Mycobacterium tuberculosis (MTB) isolation from clinical specimens is the standard for TB diagnosis. Positive MTB cultures are rarely questioned, but false-positive culture rates range from 2% to 4%. In December 2011, two smear-negative, culture-positive TB cases were reported to the Oklahoma State Department of Health (OSDH) in people without TB signs or symptoms. OSDH TB control officers interviewed physicians and laboratory personnel, reviewed patient charts, traced epidemiologic links, and performed microbiologic studies to determine if these were true TB cases. Both specimens were found to have been processed on the same day, at the same laboratory, under the same hood, and by the same technician sequentially after a strongly smear-positive TB specimen. No epidemiologic links were identified among the three patients. Spoligotyping and 24-locus mycobacterial interspersed repetitive unit typing of the three specimens were identical. Only liquid media grew MTB in the two questionable specimens. A laboratory splash incident was suspected, whereby all three liquid media sample lids were open during inoculation rather than being opened one at a time, causing cross-contamination. Also, the two specimens were incubated for 2-3 weeks longer than standard protocol before MTB growth was observed. Patient 1 was not treated for TB because her physician doubted the culture result. Patient 2, an organ transplant recipient, began four-drug anti-TB therapy, and an investigation was initiated for transplant-associated TB. Adherence to strict laboratory techniques and recognizing the possibility of false-positive MTB cultures, especially when inconsistent with clinical data, are essential in preventing erroneous TB diagnoses.

The isolation of Mycobacterium tuberculosis (MTB) from a clinical specimen is the standard for the diagnosis of TB. Positive MTB cultures can initiate a complicated and expensive series of events, including long-term antibiotic therapy, possible social stigmatization, and potentially extensive contact investigations. Positive MTB cultures are rarely questioned; however, false-positive culture rates are reported to be approximately 2% to 4%.15 The most common mechanisms for false-positive cultures are clerical errors, contamination of clinical equipment (eg, bronchoscopes), and laboratory cross-contamination.35 Of these, laboratory cross-contamination is the most frequently described mechanism in medical literature. Certain factors during the processing of clinical specimens have been implicated in laboratory cross-contamination, including faulty exhaust systems, tainted decontamination solutions, contaminated equipment, and malfunctioning heating elements.5,6 Intricate mycobacterial laboratory procedures (particularly batch processing), radiometric growth-detection methods, and the hardiness of MTB organisms can result in conditions conducive to laboratory cross-contamination.3,5,6

During December 2011, a smear-negative, culture-positive TB case (patient 1) was reported to the Oklahoma State Department of Health (OSDH). The patient had no history or symptoms consistent with TB. Another unexpected smear-negative, culture-positive TB case (patient 2) was detected the following day. Initial inquiries based upon clinical suspicion revealed that specimens from patients 1 and 2 were both processed at the same commercial reference laboratory, on the same day, under the same hood, and by the same laboratory technician sequentially after a smear-positive specimen with many acid-fast bacilli (AFB) (from patient 3). Laboratory cross-contamination was suspected, and a public health investigation was initiated.

Two physicians from the OSDH TB control program discussed the cases with the attending infectious disease physicians. All patient clinical and laboratory records were reviewed. An epidemiologic investigation involving interviews with the patient and/or family members was performed to determine if any epidemiologic links existed among the patients. In this context, an epidemiologic link was defined as any person who could have shared the same airspace with patients 1, 2, or 3.

The commercial laboratory is accredited through the Clinical Laboratory Improvement Amendments, as well as by the College of American Pathologists. Mycobacterial laboratory personnel were interviewed, and specimen processing techniques were observed. The parallel culturing technique for MTB involved decontamination with sodium hydroxide, concentration by centrifugation, rehydration in a phosphate buffer solution, inoculation of both solid and liquid broth media to maximize sensitivity, and incubation for 6 weeks.7,8 The culture media included Middlebrook 7H10 plates, Lowenstein-Jensen slants, and BacT/ALERT MP broth (bioMérieux Inc). These techniques are typically performed through batch processing, with multiple specimens processed at the same time. Spoligotyping and 24-locus mycobacterial interspersed repetitive unit tests were performed for genotyping of isolates.9

Patient 1 is a US-born woman aged 36 years with a longstanding staphylococcal abdominal wall abscess related to ventral hernia repair with porcine mesh. She complained of abdominal pain and fever. On October 11, 2011, she underwent surgical exploration of the abscess. Specimens were sent to a regional reference laboratory to be tested for a variety of pathogens, including MTB. On December 14, 2011, a culture was reported positive for MTB in liquid media. The infectious disease physician consulting on the case stated that the results were completely unexpected and inconsistent with the patient’s history and clinical findings. He reported that the patient had no known contact with TB, no risk factors for TB, and no clinical evidence of TB. Specimen AFB smear and QuantiFERON-TB Gold (QFT-G; Cellestis Ltd) were both negative. Because of the marked incongruity of the findings, OSDH TB physicians suspected cross-contamination.

Patient 2 is a US-born man aged 66 years with a history of right lung transplantation in 2008. He presented to the hospital in respiratory distress in September 2011 after a recent bout of bacterial pneumonia. He was intubated for respiratory failure and had a prolonged hospital course. Bronchoscopy was performed on October 12, 2011; a BAL specimen was tested for a variety of pathogens. BAL culture was positive for MTB on December 6, 2011. His only risk factor for TB was immunosuppressive therapy (prednisone and mycophenolate). The United Network for Organ Sharing began an investigation for possible transplant-associated TB.

Patient 3 is a US-born woman aged 61 years with longstanding fever, night sweats, and cough. She presented to the hospital on October 10, 2011; a right upper-lobe cavitary lesion was noted on chest radiograph. Her sputum was smear-positive with many AFB. Bronchoscopy was performed on October 11, 2011; a BAL culture was positive for MTB on November 3, 2011. She had multiple positive smears and cultures for MTB, and active pulmonary TB was diagnosed.

No epidemiologic links were identified among the three patients. All three isolates were sent for genotyping in December 2011. Spoligotyping and 24-locus mycobacterial interspersed repetitive unit patterns were indistinguishable, consistent with what was suspected based on clinical and epidemiologic evidence. Therefore, the culture results for patients 1 and 2 were concluded to be falsely positive because of laboratory cross-contamination from patient 3. Patient 1 was never treated for TB because her physician doubted the culture results. Patient 2 was treated for TB during December 7-20, 2011. His anti-TB therapy and the transplant-associated TB investigation were discontinued after his culture was determined to be a false-positive result.

Two deviations from laboratory protocol likely accounted for the false-positive MTB cultures. First, interviews with the laboratory supervisor suggested that a splash incident occurred during the liquid media inoculation of the three specimens by a newly trained laboratory technician. Because only the liquid media grew MTB in the two false-positive specimens, the supervisor speculated that the three liquid media sample lids were opened at the same time during inoculation rather than one at a time, and that a splash event from patient 3’s specimen (with many AFB) had introduced MTB into the liquid media samples from patients 1 and 2. Solid media did not grow MTB, and aerosolization was thought unlikely.

Second, the two false-positive TB cultures were incubated for 2-3 weeks longer than standard protocol before MTB growth was observed. The specimens from patients 1 and 2 were reported to OSDH 64 and 55 days after specimen collection, respectively. The laboratory supervisor stated that they only had one technician in the AFB laboratory, and sometimes the reading of culture results was pushed back by > 1 week, depending on workload.

This investigation highlights the need to recognize the possibility of false-positive MTB cultures. Results always need to be interpreted in the context of the patient’s clinical presentation.10 Indicators of potential false-positive MTB cases include the following: clinical course inconsistent with TB, discordant results between solid and liquid media, or a single positive MTB culture with all specimens AFB smear-negative.3,4,8 Identical genotyping results can also help define false-positive MTB cases. The delayed culture growth observed for patients 1 and 2 suggests that a low number of organisms were inoculated into the culture, consistent with the presumed splash incident. However, delayed culture growth is not always caused by cross-contamination; it can also indicate paucibacillary TB, a scant specimen, or over-decontamination of the specimen.

Coordination among health-care providers, laboratory personnel, and TB control programs is essential to preventing incorrect TB diagnoses. Health-care providers and TB control programs need to be alert for culture results that are inconsistent with clinical data.3,10 Health-care providers should also ensure that testing for MTB is ordered judiciously and is guided by clinical suspicion. For patients 1 and 2, MTB cultures were ordered as part of a general battery of tests, although TB was not suspected in either patient. Regular analysis of regional surveillance data by TB control programs might help identify possible laboratory cross-contamination by detecting clusters of positive MTB cultures from the same laboratory.3,10 Standardized mycobacterial laboratory procedures must be followed carefully, and a protocol should be in place to monitor, detect, and report suspected false-positive MTB cultures.3,4,11

This investigation illustrates the two options available when managing patients with unexpected MTB culture results. Patient 1 was not treated for TB because the attending physician and OSDH TB physicians questioned the validity of the MTB culture result. However, patient 2 was treated for TB for 14 days before treatment was discontinued. Approximately 67% of patients with false-positive MTB cultures receive treatment of active TB, which always carries the risk for toxicity.5 In addition, the incorrect diagnosis of TB could delay appropriate therapy for the true underlying pathology.

In Oklahoma, OSDH TB physicians speak with the health-care provider for all suspect TB cases. When a culture result is unexpected or inconsistent with the clinical presentation, cross-contamination is presumed, and an investigation is initiated. The OSDH laboratory database is reviewed for other positive MTB specimens that were collected or processed at the same laboratory on or about the same date in question. If there are any other positive MTB cultures identified, an epidemiologic investigation is performed to determine if any epidemiologic links exist between the two people. In addition, upon receipt of a laboratory report indicating many AFB in a specimen, OSDH TB physicians query the laboratory database to look for other positive MTB cultures that were collected or processed at the same laboratory on or about the same day. These cases are investigated if there is only a single positive MTB culture, different solid and liquid media culture results, or an essentially normal chest radiograph with a positive MTB respiratory specimen. Although these steps do not prevent laboratory cross-contamination from happening, they help to identify it early and minimize patient harm. Statewide education to hospitals, clinicians, and laboratory staff about prevention steps and early recognition of potential false-positive MTB cultures is an important measure to reduce the occurrence of laboratory cross-contamination.9

Serious clinical, social, legal, and economic repercussions exist when TB is misdiagnosed. Local and national TB surveillance numbers are also adversely affected by false-positive MTB cultures.11 Thus, possible false-positive MTB cultures should be investigated and classified quickly.4,6 Despite appropriate accreditation through College of American Pathologists and Clinical Laboratory Improvement Amendments proficiency testing, a failure in laboratory methodology occurred. Additional ongoing quality-assurance processes, such as implementing the Association of Public Health Laboratories’ quality-assurance guidelines, might have prevented these errors.5,12 Maintaining suspicion for false-positive MTB cultures, clear communication among health-care providers, laboratory personnel, and TB control programs, and judicious laboratory testing will help to prevent erroneous TB diagnoses. The axiom that any MTB organism isolated from a clinical specimen indicates TB is generally true; however, health-care providers should be aware that false-positive MTB cultures occur and should consider this possibility for patients with incongruous culture results.7,13

Financial/nonfinancial disclosures: The authors have reported to CHEST that no potential conflicts of interest exist with any companies/organizations whose products or services may be discussed in this article.

Other contributions: We thank Julie Magri, MD, MPH, from the Centers for Disease Control and Prevention for reviewing this manuscript. Also, we thank to Amy Hill, RN, from the Oklahoma State Department of Health Tuberculosis Program for her tireless work, and Matthew England, BS, from the Oklahoma State Department of Health Public Health Laboratory for his assistance in reviewing laboratory protocols. Special thanks to Renee Funk, DVM, MPH, and Maryam Haddad, MSN, MPH, from the Centers for Disease Control and Prevention for their support of this investigation. The findings and conclusions in this report are those of the authors and do not necessarily represent the official position of the Centers for Disease Control and Prevention. The authors obtained patient permission to publish this information.

AFB

acid-fast bacilli

MTB

Mycobacterium tuberculosis

OSDH

Oklahoma State Department of Health

de Boer AS, Blommerde B, de Haas PEW, et al. False-positiveMycobacterium tuberculosiscultures in 44 laboratories in The Netherlands (1993 to 2000): incidence, risk factors, and consequences. J Clin Microbiol. 2002;40(11):4004-4009. [CrossRef] [PubMed]
 
Jasmer RM, Roemer M, Hamilton J, et al. A prospective, multicenter study of laboratory cross-contamination ofMycobacterium tuberculosiscultures. Emerg Infect Dis. 2002;8(11):1260-1263. [CrossRef] [PubMed]
 
Centers for Disease Control and Prevention (CDC). Multiple misdiagnoses of tuberculosis resulting from laboratory error—Wisconsin, 1996. MMWR Morb Mortal Wkly Rep. 1997;46(34):797-801. [PubMed]
 
Lai CC, Tan CK, Lin SH, et al. Molecular evidence of false-positive cultures forMycobacterium tuberculosisin a Taiwanese hospital with a high incidence of TB. Chest. 2010;137(5):1065-1070. [CrossRef] [PubMed]
 
Burman WJ, Reves RR. Review of false-positive cultures forMycobacterium tuberculosisand recommendations for avoiding unnecessary treatment. Clin Infect Dis. 2000;31(6):1390-1395. [CrossRef] [PubMed]
 
Ruddy M, McHugh TD, Dale JW, et al. Estimation of the rate of unrecognized cross-contamination withMycobacterium tuberculosisin London microbiology laboratories. J Clin Microbiol. 2002;40(11):4100-4104. [CrossRef] [PubMed]
 
Chang CL, Kim HH, Son HC, et al. False-positive growth ofMycobacterium tuberculosisattributable to laboratory contamination confirmed by restriction fragment length polymorphism analysis. Int J Tuberc Lung Dis. 2001;5(9):861-867. [PubMed]
 
Poynten M, Andresen DN, Gottlieb T. Laboratory cross-contamination ofMycobacterium tuberculosis: an investigation and analysis of causes and consequences. Intern Med J. 2002;32(11):512-519. [CrossRef] [PubMed]
 
National TB Controllers Association/CDC Advisory Group on Tuberculosis Genotyping.Guide to the Application of Genotyping to Tuberculosis Prevention and Control. Navin T, Rosenblum L, Wallace C, Etkind S, eds. Atlanta, GA: US Department of Health and Human Services, CDC; 2004.http://www.cdc.gov/tb/programs/genotyping/images/TBGenotypingGuide_June2004.pdf. Accessed November 8, 2012.
 
Fitzpatrick L, Braden C, Cronin W, et al. Investigation of Laboratory cross-contamination ofMycobacterium tuberculosiscultures. Clin Infect Dis. 2004;38(6):e52-e54. [CrossRef] [PubMed]
 
Braden CR, Templeton GL, Stead WW, Bates JH, Cave MD, Valway SE. Retrospective detection of laboratory cross-contamination ofMycobacterium tuberculosiscultures with use of DNA fingerprint analysis. Clin Infect Dis. 1997;24(1):35-40. [CrossRef] [PubMed]
 
Association of Public Health Laboratories.Mycobacterium tuberculosis: assessing your laboratory. 2009. Association of Public Health Laboratories.website.http://www.aphl.org/aphlprograms/infectious/tuberculosis/Documents/Mycobacteria_TuberculosisAssessingYourLaboratory.pdf. Accessed December 3, 2012.
 
Burman WJ, Stone BL, Reves RR, et al. The incidence of false-positive cultures forMycobacterium tuberculosisAm J Respir Crit Care Med. 1997;155(1):321-326. [CrossRef] [PubMed]
 

Figures

Tables

References

de Boer AS, Blommerde B, de Haas PEW, et al. False-positiveMycobacterium tuberculosiscultures in 44 laboratories in The Netherlands (1993 to 2000): incidence, risk factors, and consequences. J Clin Microbiol. 2002;40(11):4004-4009. [CrossRef] [PubMed]
 
Jasmer RM, Roemer M, Hamilton J, et al. A prospective, multicenter study of laboratory cross-contamination ofMycobacterium tuberculosiscultures. Emerg Infect Dis. 2002;8(11):1260-1263. [CrossRef] [PubMed]
 
Centers for Disease Control and Prevention (CDC). Multiple misdiagnoses of tuberculosis resulting from laboratory error—Wisconsin, 1996. MMWR Morb Mortal Wkly Rep. 1997;46(34):797-801. [PubMed]
 
Lai CC, Tan CK, Lin SH, et al. Molecular evidence of false-positive cultures forMycobacterium tuberculosisin a Taiwanese hospital with a high incidence of TB. Chest. 2010;137(5):1065-1070. [CrossRef] [PubMed]
 
Burman WJ, Reves RR. Review of false-positive cultures forMycobacterium tuberculosisand recommendations for avoiding unnecessary treatment. Clin Infect Dis. 2000;31(6):1390-1395. [CrossRef] [PubMed]
 
Ruddy M, McHugh TD, Dale JW, et al. Estimation of the rate of unrecognized cross-contamination withMycobacterium tuberculosisin London microbiology laboratories. J Clin Microbiol. 2002;40(11):4100-4104. [CrossRef] [PubMed]
 
Chang CL, Kim HH, Son HC, et al. False-positive growth ofMycobacterium tuberculosisattributable to laboratory contamination confirmed by restriction fragment length polymorphism analysis. Int J Tuberc Lung Dis. 2001;5(9):861-867. [PubMed]
 
Poynten M, Andresen DN, Gottlieb T. Laboratory cross-contamination ofMycobacterium tuberculosis: an investigation and analysis of causes and consequences. Intern Med J. 2002;32(11):512-519. [CrossRef] [PubMed]
 
National TB Controllers Association/CDC Advisory Group on Tuberculosis Genotyping.Guide to the Application of Genotyping to Tuberculosis Prevention and Control. Navin T, Rosenblum L, Wallace C, Etkind S, eds. Atlanta, GA: US Department of Health and Human Services, CDC; 2004.http://www.cdc.gov/tb/programs/genotyping/images/TBGenotypingGuide_June2004.pdf. Accessed November 8, 2012.
 
Fitzpatrick L, Braden C, Cronin W, et al. Investigation of Laboratory cross-contamination ofMycobacterium tuberculosiscultures. Clin Infect Dis. 2004;38(6):e52-e54. [CrossRef] [PubMed]
 
Braden CR, Templeton GL, Stead WW, Bates JH, Cave MD, Valway SE. Retrospective detection of laboratory cross-contamination ofMycobacterium tuberculosiscultures with use of DNA fingerprint analysis. Clin Infect Dis. 1997;24(1):35-40. [CrossRef] [PubMed]
 
Association of Public Health Laboratories.Mycobacterium tuberculosis: assessing your laboratory. 2009. Association of Public Health Laboratories.website.http://www.aphl.org/aphlprograms/infectious/tuberculosis/Documents/Mycobacteria_TuberculosisAssessingYourLaboratory.pdf. Accessed December 3, 2012.
 
Burman WJ, Stone BL, Reves RR, et al. The incidence of false-positive cultures forMycobacterium tuberculosisAm J Respir Crit Care Med. 1997;155(1):321-326. [CrossRef] [PubMed]
 
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