2010 NPHL Challenge Set Summary Report

Transcription

1 2010 NPHL Challenge Set Summary Report General remarks 24 laboratories signed up for the 2010 NPHL Challenge Set Exercise (NCSE), 20 laboratories (83%) returned results. NCSE shipped on Tuesday July 6, 2010 via NPHL courier or FedEx. Only one laboratory reported delay in delivery of NCSE in excess of 1 week. NCSE cultures should have been incorporated in your routine daily laboratory workflow and instructions shared with bench testing personnel. If your facility deemed it necessary (recognized trigger points) to follow the LRN special pathogens recognize, rule out and refer (ROAR) procedures, all manipulation of the organism should have been performed under a certified Class II Biological Safety Cabinet Trigger points to indicate the need for the ROAR sentinel guidelines may include: o Source sterile body site (blood, CSF or other body fluid) o Patient history or diagnosis o Direct Gram Stain Morphology (important to review specimen gram stain prior to o working with culture plates) sterile site with Gram negative diplococci or coccobacilli o Poor growth or failure to grow after 48 hours o Better growth on Chocolate agar than on Sheep blood agar o Gram negative rod growth on Sheep blood agar but not on MacConkey agar o Any culture with mould If Special Pathogen agents were ruled out, organism could be returned to normal bench protocol and worked up according to your laboratory guidelines. To test communications between LRN Sentinel laboratories and your LRN Reference Lab (NPHL), exercise participants were requested to contact NPHL if, after performing the established LRN sentinel rule out and refer guidelines, they are unable to rule out an agent of bioterrorism. Only 4 notifications were received. 13 laboratories utilized online method (SurveyMonkey) to submit results, 7 laboratories faxed results. General Laboratory Question Summary 1-2. Does your microbiology laboratory have a Class II biological safety cabinet? What type? Yes No All laboratories (20 of 20) indicated that they did have a Class II biological safety cabinet (BSC). Some laboratories were not sure if they had category A or B. The main difference between A and B is that Class IIA exhausts some HEPA-filtered air into the laboratory work area. Class II B exhausts all HEPAfiltered air to the outside through dedicated ductwork. This information is found on a sticker on front of BSC. 3. Are the ASM Sentinel Laboratory Guidelines for Suspected Agents of Bioterrorism and/or LRN Sentinel Laboratory protocols provided by your state public health laboratory part of into the standard operating procedure of your laboratory? Yes No 18 of 20 laboratories indicated that they did have the protocols as part of their standard operating procedure. We recommend that the protocols are readily available as a reference in your laboratory. The most recent protocols can be downloaded from the NPHL website at The protocols instruct laboratorians how they can recognize, rule-out and refer (ROAR) special pathogens. The NPHL periodically offers laboratory training workshops based on these protocols. For further information about these workshops please contact Karen Stiles at or kstiles@unmc.edu.

2 4. Have you incorporated procedures in your laboratory for recognizing, referring or destroying, and properly documenting select agents, per the federal requirements of the APHIS/CDC Select Agent Program? Yes No The law (42 CFR Part 73) does require that laboratories, after confirmation of a select agent or toxin, transfer the specimen and isolate to a facility eligible to receive them or destroys the material on-site by some means sufficient to cause inactivation of the agent. As a part of this process, the laboratory is required to prepare a record of the identification and transfer or destruction on a form called, Guidance Document for Reporting the Identification of a Select Agent or Toxin in a Clinical or Diagnostic Laboratory (Animal and Plant Health Inspection Service [APHIS]/CDC Form 4). NOTE: NO TRANSFER IS TO OCCUR UNTIL FORM 2 HAS BEEN PROCESSED AND AN AUTHORIZATION NUMBER HAS BEEN ISSUED BY THE CDC. 17 of 20 laboratories indicated that they have incorporated procedures in their laboratory for recognizing, referring, destroying, and properly documenting Select Agents per federal regulations. We recommend that the protocols be readily available as a reference in your laboratory. For information on properly documenting the isolation, see boratory.pdf 5. Does your laboratory have policies in place for documentation of potential exposures to bioterrorism agents or agents that can cause laboratory acquired infections? Yes No Again, it is federal law ALL laboratory personnel handling a specimen ultimately determined to contain a viable agent or toxin must file the CDC form of 20 laboratories indicated that they have incorporated policies that document potential exposures that can cause laboratory acquired infections (LIA). Contact your infection control staff if you do not have a policy in place. 6. What does your laboratory use for transporting packages of agents of bioterrorism and emerging infectious diseases? Private courier Commercial courier NPHL courier Other All laboratories use either one or multiple couriers. Only 9 laboratories documented they have an NPHL courier. If your facility is not aware if they have a means to transport to NPHL, please contact us. 7. Does your laboratory have designated personnel trained in the LRN rule out procedures? Yes No 18 of 20 laboratories indicated that they do have designated personnel trained in the LRN recognize, rule out and refer (ROAR) procedures. We recommend that if you are trained but would like review, or if you have not been trained, to consider attending NPHL wet workshops. Onsite training or online courses are also available for continuing education credits. For more information, contact Karen Stiles at kstiles@unmc.edu. 8. Does your laboratory have designated personnel trained for the packaging and shipping of Division 6.2 Hazardous Materials? Yes No 16 of 20 laboratories indicated that they do have designated personnel trained for packaging and shipping of Division 6.2 Hazardous Materials. If your facility has not been trained, we highly recommend attending one of the workshops available in Omaha on Sept 21 st or in North Platte on Sept 23 rd. You can register at Online courses are also available, but are not recommended if they have not yet been certified.

3 Organism Results NCSE-01 A 25 year old female developed onset microbial keratitis in left eye 2 weeks after LASIK surgery. Specimen: Test: Eye Aerobic culture with gram stain No susceptibility testing required Reference: J Refract Surg Mar:22(3): Bacillus megaterium delayed onset lamellar keratitis after LASIK, Ramo- Esteban, JC; Servat JJ; Tauber S; Bia F. Trigger Points Rapid growth with large colonies on SAB/Choc only, nonhemolytic. Other trigger points to consider if this was a sterile site, or characteristic Gram positive bacilli seen in a direct Gram stain. Organism 1 Isolate was Bacillus megaterium (#25) Satisfactory answers: Bacillus species, not anthracis NOS (#20) Bacillus species, unable to rule out anthracis (#21) Bacillus megaterium (#25) Bacillus species, non-hemolytic, non-motile, NOS (#28) Gram-positive bacillus, suspicious for Bacillus anthracis, unable to further identify (#77) Unsatisfactory answers: Non - BT Bacillus species, non-hemolytic, motile, NOS (#29) This isolate was non-motile. Gram-positive rod (#147) This answer may be satisfactory is the next question was answered as continue with identification if needed. Organism 1 s Satisfactory #of responses #20 Bacillus species, not anthracis NOS 6 #21 Bacillus species, unable to rule out anthracis 9 #77 Gram-positive bacillus, suspicious for Bacillus anthracis, unable to further ID 3 #22 Bacillus species, NOS 0 #25 Bacillus megaterium 0 #28 Bacillus species, non-hemolytic, non-motile, NOS (#28) 0 Unsatisfactory #of responses #147 Gram-positive bacillus NOS 2 # 29 Bacillus species, non-hemolytic, motile, NOS 0 #22 Bacillus species, NOS 0 We at the NPHL would prefer that labs not give this as an answer because of the motility media that is provided to laboratories free of charge (media available upon training).

4 We would like to acknowledge the laboratorians at Fremont, York, Alliance, Columbus, Bellevue, Methodist, and Norfolk hospitals for participating in capturing images with their STATPack. B anthracis shows growth on Sheep Blood Agar: 2-5 mm, tenacious, non-hemolytic colonies after hr. Flat, slightly convex, irregularly round colonies with irregular wavy, border and ground glass appearance. Left side: NCSE-1 Bacillus megaterium Right side: Bacillus anthracis ** Perform In Bio Safety Cabinet ** Gram Stain: Large box car shaped Gram positive rods often in long chains, with oval, central-to-subterminal spores which do not cause significant swelling of cells India Ink:

5 Reference:

6 2. What would be the next step that your laboratory would take in regards to the identification stated above? Continue with identification 5.0% 1 Contact the NPHL and refer the isolate for confirmation 65.0% 13 No further action 30.0% 6 Call the Centers for Disease Control and Prevention Comments: Unknown cannot rule out by biochemical s alone. No further action required if confident that morphology does not fit Bacillus anthracis. If questionable or not confident to rule out by morphology, please confer with NPHL. 3. Organism grows on: (Check all the apply) SBA 95.0% 19 Chocolate 95.0% 19 MAC - Fermenter MAC - Nonfermenter Comment: All in agreement that it grows well on SBA and Chocolate.

7 4. Hemolysis: 5.0% 1 Alphahemolytic Betahemolytic Nonhemolytic 90.0% 18 Not applicable 5.0% 1 Comment: Hemolysis should be applicable in this scenario. B. megaterium can be variable on the hemolysis, especially if held >72hrs. 5. Gram Stain: Positive 95.0% 19 Negative 5.0% 1 Comment: Unknown should be Gram positive but obviously de-colorizes easily with age. Repeat stain especially if large cells and spores are seen. 6. Morphology: Bacilli 100.0% 20 Cocci Coccobacilli Diplococci Spores present

8 7. Chaining: Yes 60.0% 12 No 40.0% 8 Comment: Subjective depending on how manipulated organism was handled while making stain, especially from plate. Broth or thio shows more characteristic morphology. ** Perform In Bio Safety Cabinet ** 8. Did you use any of the following to identify the organism? If yes, please indicate reaction: Positive Negative Not Performed Betalactamase 0.0% (0) 0.0% (0) 100.0% (18) 18 Catalase 80.0% (16) 5.0% (1) 15.0% (3) % (1) 0.0% (0) 94.4% (17) 18 Indole 0.0% (0) 5.6% (1) 94.4% (17) 18 Motility 26.3% (5) 57.9% (11) 15.8% (3) 19 Oxidase 0.0% (0) 11.1% (2) 88.9% (16) 18 Nitrate 0.0% (0) 5.6% (1) 94.4% (17) 18 Satellite 0.0% (0) 0.0% (0) 100.0% (18) 18 Urease 5.6% (1) 0.0% (0) 94.4% (17) 18 Addtional tests 6.3% (1) 0.0% (0) 93.8% (15) 16 Other 3 Comment: Key reactions in red. ** Perform In Bio Safety Cabinet ** Motility can be difficult to read if commercial media used. Encourage those trained in the ROAR protocol, to consider using our motility media which can be ordered via the NPHL website. If your laboratory does not have the capability to test these key reaction, contact Karen Stiles at kstiles@unmc.edu.

9 9. Colistin disk: No zone Zone Not performed 100.0% India Ink: Encapsulated 5.0% 1 Nonencapsulated 5.0% 1 Not performed 90.0% 18 Comment: This optional test is used to improve visualization of encapsulated B. anthracis in clinical samples such as blood, blood culture bottles, or CSF. This is not an ASM recommended procedure; performance of this test requires staff that is trained/experienced with this procedure. 11. Polymyxin B disk: No zone Zone Not performed 100.0% 20

10 12. TSI or KIA: Butt red (no change) Butt yellow Slant red (no change) Slant yellow Not performed 100.0% Did you attempt to ID this organism on a commercial system? Yes No (Skip questions 14-16) 100.0% 20 In Summary, Bacillus megaterium can mimic biochemically a Bacillus anthracis, however with training, can be recognized by colony morphology and ruled out. If questionable, please consider consulting with NPHL.

11 Organism Results NCSE-02 A 5 year old girl in ICU presented with pneumonia based on clinical findings as diffuse rhonchi, rales and wheezing. Child classified as being in mild to moderate respiratory distress. Predisposing conditions include Chronic Granulomatous Disease. Specimen: Test: Blood Aerobic culture with gram stain No susceptibility testing required Reference: European Journal of Clinical Microbiology & Infectious Diseases, Vol 20, no 2, , Olegella ureolytica in Bood Culture: Contaminate or Infection?, Lechner, A;Bruckner, DA. Trigger points Sterile site, slow growth >48hrs, direct gram stain Organism 2 - Isolate was Oligella ureolytica (#105) Satisfactory answers: Gram-negative coccobacillus, suspicious for Brucella sp, unable to further identify (#75) Unable to rule out Brucella sp Oligella ureolytica (#105) Oligella species NOS (#104) Unsatisfactory responses: Brucella sp, NOS (#38) (also answered next question refer to NPHL ) The protocol as written, does not utilize a sufficient number of procedures to a make a determination of Brucella species. It is important to not overcall or project an answer from the limited number of tests. The majority of commercial identification systems do not contain Brucella sp in their data banks. Gram negative rods, NOS (#148) or would refer According to the LRN protocol, Gram negative rods would be more in line with Yersinia but can be ruled out by the oxidase and growth characteristics on MacConkey agar. This answer may be satisfactory is the next question was answered as continue with identification if needed. Gram negative rods, suspicious of Burkholderia Burkholderia is oxidase positive. However, it is more rod like in gram stain and good growth on MacConkeys in 48hr. Organism 2 s Satisfactory #of responses #75 GNCB suspicious for Brucella 12 #105 Oligella ureolytica 3 #104 Oligella sp NOS 0 Unsatisfactory #of responses # 38 Brucella sp NOS 2 #148 Gram negative rod NOS 1 Gram Negative Rod, would refer 1 Gram Negative Rod, Suspicious of Burkholderia 1

12 We would like to acknowledge the laboratorians at Fremont, York, Alliance, Columbus, Bellevue, Methodist, and Norfolk hospitals for participating in capturing images with their STATPack. Brucella sp Slow growth >48hr non-hemolytic, moist, convex, smooth with smooth, shiny surface. Left side: NCSE-2 Oligella ureolyticus Right side: Brucella sp Left side: NCSE-2 Oligella ureolyticus Right side: Burkholderia sp Left side: NCSE-2 Brucella sp Right side: Burkholderia sp

13 ASM SENTINEL LABORATORY GUIDELINES for Brucella sp Oligella is indistinguishable from Brucella sp. if you strictly follow the LRN rule out and refer protocol (similar morphology & growth rates, both positive for oxidase, catalase and rapid production of urease). In order to feel confident that Brucella is ruled out and to continue testing by an automated system, a laboratory would have to do additional testing beyond the LRN protocol (such as PDA or Nitrite).

14 ASM SENTINEL LABORATORY GUIDELINES for Brucella sp Comments: Brucellosis is a zoonotic infection, with four species being recognized as causing infection in humans: Brucella abortus (cattle), Brucella melitensis (goats, sheep, and camels), Brucella suis (pigs), and Brucella canis (dogs). The disease has been known by several terms, including Malta fever, undulant fever, Rock of Gibraltar fever, and Bang s disease. There are between 50 and 100 cases of Brucella infection in humans each year in the United States. Infections are seen in essentially two patient populations. The first is individuals who work with animals which have not been vaccinated against brucellosis. This patient population includes farmers, veterinarians, and slaughterhouse workers. B.abortus (cattle) and B. suis (pigs) are the agents most likely to cause infections in this group of individuals. They become infected either by direct contact with or aerosolization from infected animal tissues. Brucellosis is also seen in individuals who ingest unpasteurized dairy products contaminated with Brucella. This is most likely to occur in individuals who travel to or migrate from rural areas of Latin American and the Middle East, where disease is endemic in dairy animals, particularly goats and camels. B. melitensis is the most common agent seen in this patient population.

15 2. What would be the next step that your laboratory would take in regards to the identification stated above? Continue with identification 5.0% 1 Contact the NPHL and refer the isolate for confirmation 80.0% 16 No further action 15.0% 3 Call the Centers for Disease Control and Prevention Comments: If organism was identified as suspicious for Brucella sp, correct answer should be Refer to NPHL. If organism was identified as Oligella species/ Oligella ureolytica, correct answer should not refer for confirmation or further identification (provided additional testing was done in BSC to rule out Brucella sp before testing on automated system). 3. Organism grows on: (Check all the apply) SBA 95.0% 19 Chocolate 100.0% 20 MAC - Fermenter MAC - Nonfermenter 20.0% 4 Comments: Growth on MacConkeys possible, but usually seen after 48hrs.

16 4. Hemolysis: Alphahemolytic Betahemolytic Nonhemolytic 90.0% 18 Not applicable 10.0% 2 Comments: Both are acceptable answers as hemolysis is not a predictable characteristic of Gram negative organisms. 5. Gram Stain: Positive Negative 100.0% Morphology: Bacilli 50.0% 10 Cocci Coccobacilli 55.0% 11 Diplococci Spores present Comments: Oligella is described as a small Gram negative coccobacilli. Some rods may be possible, but smaller than Enterics.

17 7. Chaining: Yes 30.0% 6 No 70.0% 14 Comments: Not a predictable characteristic. 8. Did you use any of the following to identify the organism? If yes, please indicate reaction: Positive Negative Not Performed Betalactamase 5.9% (1) 5.9% (1) 88.2% (15) 17 Catalase 66.7% (12) 5.6% (1) 27.8% (5) % (1) 5.9% (1) 88.2% (15) 17 Indole 0.0% (0) 44.4% (8) 55.6% (10) 18 Motility 5.9% (1) 17.6% (3) 76.5% (13) 17 Oxidase 90.0% (18) 0.0% (0) 10.0% (2) 20 Nitrate 5.9% (1) 17.6% (3) 76.5% (13) 17 Satellite 0.0% (0) 29.4% (5) 70.6% (12) 17 Urease 85.0% (17) 0.0% (0) 15.0% (3) 20 Addtional tests 0.0% (0) 9.1% (1) 90.9% (10) 11 Other 3 Comments: ** Perform In Bio Safety Cabinet ** Key reactions in red. Catalase reaction should be positive. If negative, consider doing tube catalase. If your laboratory does not have the capability to test these key reaction, contact Karen Stiles at kstiles@unmc.edu.

18 9. Colistin disk: No zone Zone 15.0% 3 Not performed 85.0% India Ink: Encapsulated Nonencapsulated 5.0% 1 Not performed 95.0% 19 Comment: This optional test is used to improve visualization of capsules in clinical samples such as blood, blood culture bottles, or CSF. This is not an ASM recommended procedure; performance of this test requires staff that is trained/experienced with this procedure. 11. Polymyxin B disk: No zone Zone Not performed 100.0% 20

19 12. TSI or KIA: Butt red (no change) 5.0% 1 Butt yellow Slant red (no change) 5.0% 1 Slant yellow Not performed 90.0% 18 Comments: The Kliger s Iron Agar (or Triple Sugar Iron) tube is used in the following algorithms to test for glucose: OXIDASE + GLUCOSE + MOTILITY + MOTILITY = GLUCOSE = MOTILITY + MOTILITY = Pseudomonas species, Burkholderia species, Alcaligenes xylosoxidans, Agrobacterium Chryseobacterium meningosepticum (MAC V), Chryseobacterium species (MAC V + ) Alcaligenes species, Oligella ureolytica Myroides odoratum, Moraxella species. 13. Did you attempt to ID this organism on a commercial system? Yes 20.0% 4 No (Skip questions 14-16) 80.0% 16

20 14. Which system? API 25.0% 1 Microsc an Vitek Vitek % 1 Phoenix Other (Remel RapID NF plus) 50.0% If an automated machine was used to ID the organism please provide the Bionumber, Profile, or Biotype number (Located on Microscan or Vitek report). 16. If an automated machine was used to ID the organism please provide the percentage of probability of the ID. >98% Summary: Other organisms that can be confused with Brucella sp species because they are urease positive are Oligella ureolytica (usually found only in the urine), Psychrobacter phenylpyruvicus, Psychrobacter immobilis, and Bordetella bronchiseptica (motile). Oligella ureolytica can mimic biochemically a Brucella sp, however additional biochemicals are required to rule out possible Brucella sp prior to testing on an automated system [motility positive, PDA positive, nitrite positive or ruled out by RapidID (not recommended by LRN)]. It is recommended to consult with NPHL. NOTE: Brucella sp has been responsible for many laboratory-acquired infections. If Brucella is suspected or the Gram stain shows a small, gram-negative coccobacillus, avoid aerosols and perform subcultures in a biosafety cabinet. Plates should be taped shut, and all further testing should be performed only in the biosafety cabinet, using Biosafety Level 3 practices.

21 Conclusion: In all circumstances, if your laboratory has isolated an organism that is suspicious for a Special Pathogen (Francisella tularensis, Bacillus anthracis, Yersinia pestis, Brucella species, or Burkholderia mallei/pseudomallei) DO NOT send it to your routine reference laboratory. The reference laboratory may not be able to do any further identification, and they will need to send it on to a public health laboratory such as the Nebraska Public Health Laboratory (NPHL) for confirmation, increasing the delay in identification. Second, by sending these isolates to your reference laboratory you are possibly exposing more individuals to these infectious agents. If you identify an isolate that cannot be ruled out as one of these agents please page the Nebraska Public Health Laboratory at or and we will assist you. We urge your laboratory to modify your standard operating procedures to avoid testing slow growing fastidious gram-negative coccobacilli on any automated identification systems. The risk to your staff from the potential aerosolization of these infectious agents is too great. The key learning points from this exercise are: o Commercial systems are typically not able to correctly identify gram-negative coccobacilli and laboratory standard operating procedures should be written to address this issue. o Placement of suspect organisms on automated instrumentation may result in further exposure of the technologist.