A Palette of Purples and Pinks: Gram Stain Cases

Transcription

1 A Palette of Purples and Pinks: Gram Stain Cases Steven D. Dallas, PhD, D(ABMM), MT(ASCP)SM Departments of Clinical Laboratory Sciences and Pathology University of Texas Health Science Center San Antonio University Hospital, San Antonio 1

2 Objectives Describe the real-time clinical utility of the Gram stain on specimens from various body sites Correlate gram stain results to expected culture growth Explain simple improvements that assure excellent Gram stains Address recent CAP accreditation checklist questions regarding microscopy QA Discuss specific cases where Gram stains made an immediate impact (either positive or negative) on patient care Fine print disclosures: I have not taken money, pizza, pens, or note pads from any vendors in the last three years. 2

3 The internet is a confusing place! 3

4 Three points The gram stain is still the best, fastest and cheapest multiplex microbiology test Body site and patient history give the biggest hints as to what we might see The gram stain, culture, and susceptibility are three acts in a play, not three separate movies! For the following cases (patient specimens) we will initially have the same info at our disposal that you might have if you were sitting at the scope reading a STAT gram stain. 4

5 Specimen Doug Bug Culture, 24 hours You are here Drug Gram stain, 30 minutes Rapid flu test, 20 minutes Real time PCR for some Organisms, one hour Detect, identify, eliminate. A progression of actionable information leading to treatment and cure! Antibiotics susceptibility 8 to 24 more hours 5

6 Gram stain clinical impact If we are doing them slow, like once per shift, does it matter? If the doctor is going to administer empiric antibiotics, does it matter? No organisms seen can actually be helpful If you report something, the doctor will act on it, especially if sterile body site May diagnose infections and guide appropriate therapy realtime GNR versus GPC versus yeast versus fungi, and some parasites! 6

7 Case 1 Sputum, 56 yr old male in ER, suspected CAP Gram stain: >25 PMNs/LPF <10 Sq EPIs/LPF Moderate gram pos rods Culture: Heavy growth H. influenzae Moderate growth mixed usual respiratory flora 7

8 Case 1 Results go out the door, no questions asked. CLS who worked up culture did not review or question the lack of correlation between gram and culture, why? Doctor did not complain either. Does this happen in your lab? 8

9 CAP requirement MIC

10 MIC.21530: gram to culture correlation Sputum GRAM: Less than 10 epithelial cells/lpf Greater than 25 PMNs/LPF Many small gram negative coccobacilli consistent with Haemophilus CULTURE: Haemophilus influenzae, heavy growth 10

11 Reasons for no correlation: Wrong slide? clerical or technical error Staining technique Less sensitive than culture Nonviable, fastidious, or anaerobic organisms Gram stain looks like Haemophilus and grew it Wound culture was no growth Despite GPC seen in Gram stain. Under-decolorized M. catarrhalis reported as Staph 11

12 MIC 21530: culture reading tech: Must review gram stain results while reading the culture (prelim or final.) Cerner, Soft can catch gram to culture mismatch The culture reading tech has the responsibility to investigate the discrepancy Start with looking at already stained slide, misinterpretation, bad technique, clerical error? Then stain duplicate if needed Then re-stain the specimen if needed. 12

13 Say what? Policy works better if you are nice! All I know is they write us up every time we disagree on a gram stain and enumeration of WBCs on said stain. ridiculous. I'd like to strangle some of those chicks over there, but am too busy!! quote from an anonymous friend who works in an unidentified lab in an undisclosed location on second shift. 13

14 Policy works better if you make pretty Gram stains 14

15 Blood culture, heat fixation 15

16 This is what happens to human cells when you heat fix a slide! RBCs are lysed, WBCs are distorted Lubbock, TX 16

17 Air dried/methanol fixed, from a duplicate slide made at the same time 17

18 Another example heat fixed 18

19 Same blood culture stained at same time, methanol fixed 19

20 Gram stain review policy Organism not seen in Gram but heavy growth on culture: lack of sensitivity. In a hurry, wrong slide, poor stain, too thin? Organism seen in Gram, but no growth in culture: lack of specificity. Anaerobe, dead, fastidious, wrong slide? Duplicate slide and slide retention policy: keep stained and unstained slide for 7 days, at the microscope 20

21 Case 2 29 yr old male, HIV status unknown Outpatient spinal tap Specimen from 24-hour walk-in emergency referred to hospital micro lab because reference lab could perform STAT test. 21

22 Case 2: other CSF results CSF tube #4 CSF color = colorless CSF appearance = clear CSF RBC = 1 CSF neutrophils = 0 CSF lymphocytes = 99 CSF monocytes = 1 22

23 Case 2: most likely pathogens? a) N. meningitidis, S. pneumoniae b) HSV, Enterovirus or other virus c) Cryptococcus d) Haemophilus e) b and c 23

24 Case 2 Lymphocyte Only one of these structures was seen. 24

25 Case 2 This image is consistent with: a) Some sort of contaminant b) An epithelial cell c) Intracellular Streptococcus pneumoniae d) Intracellular diptheroids 25

26 Case 2 The culture grew: What next? 26

27 CAP checklist: morphologic observation MIC Teach clinical correlation skills to new employees, train them to review results from other departments, to get a co-read, to ask: Does this make sense? 27

28 Case 3 Necrotic lung tissue 50 something male (HIPAA has no rules against the word something after patient age). Some of the tissue also went to histology and mycology labs 28

29 Case 3 29

30 Case 3 30

31 Case 3 31

32 Case 3 32

33 Case 3 The organism grew white cobweb-like growth in 48 hours on BA, CHOC and fungal media. This is most likely: a)fusarium b)pneumocystis c) Prototheca d)coccidioides e)aspergillus 33

34 Case 4 62 year old male PMH: Mental retardation Psychosis, not otherwise specified Gastro-esophageal reflux disease (GERD) Hypercholesterolemia Cataracts Benign prostatic hypertrophy (BPH) Hip fracture with surgical repair (2009) Lived in group home, constant supervision Recent history of constipation 34

35 3 hours antemortem: Unresponsive after vomiting Several vomiting episodes earlier EMS arrival on scene: Vomitus in airways Intubated and resuscitated aggressively CPR, epinephrine/vasopressin, cold fluids, ice packs Arrival at ED in full cardiopulmonary arrest Case 4 Glascow Coma Scale 3 Vomitus and blood noted in airways Nasogastric tube placed with suction of bloody fluid Chest x-ray: diffuse bilateral pulmonary infiltrates (diffuse pulmonary edema) 35

36 Case 4: lab results Hematology: Hgb 5.2 g/dl Hct 16.4% MCV 73.0 fl Plt 115k/mL Coag: INR 2.6 PTT 244 sec. Chemistry: Troponin I 0.03 Na 165 Cl 114 K 3.9 CO2 32 Total bilirubin

37 Case 4: more clinical history Multiple RBC units transfused Continued episodes of cardiac arrest DNR status per decedent s family Pronounced dead shortly thereafter Clinical diagnosis of acute severe upper GI bleed Decedent transferred to UH for organ procurement Autopsy consent provided by brother 37

38 Case 4: lymph node tissue gram stain 38

39 Case 4: organism also seen in: Duodenem Stomach lining Stomach contents Lungs Adrenal gland Liver Gram stains were all we had to go on at this point! 39

40 Case 4 The usual suspects? a) Clostridium b) Bacillus c) Corynebacterium d) Actinomyces e) Listeria f) Erysipelothrix 40

41 Case 4 Large, plump Gram-positive rods, agents of food poisoning: Bacillus cereus emesis several hours after eating reheated rice (preformed toxin) Clostridium perfringens diarrhea 8-24 hours after ingestion, toxin-mediated 41

42 Case 4: additional results Paraffin block of lymph node sent out for PCR: Corynebacterium ureicelerivorans DNA detected But not morphologically consistent with our organism (contaminant?) Paraffin blocks of lung sent to Dr. Olsen at Methodist Hospital (Houston) No B. cereus or B. anthracis identified 42

43 Case 4: for some reason.. The only thing available that was not in formalin/paraffin was stomach contents Aerobic culture: heavy growth of mixed morphotypes, gram pos and gram neg Anaerobic culture: heavy growth of mixed morphotypes, gram pos and gram neg 43

44 Case 4: So Dr. Andy Hansen asked: Suspecting either Bacillus or Clostridium, is there anything else we can do? Any ideas? 44

45 Case 4: happy hour for bacteria! Both are spore-forming Select for spores: incubate gastric contents in absolute ethanol one to one ratio for one hour and plate it Incubate selective and enriched plates aerobically and anaerobically Anaerobic growth at 24 hours! 45

46 Case 4: anaerobic blood plate Double zone of beta-hemolysis 46

47 Case 4 This organism often produces double-zone beta hemolysis: a) Bacillus anthracis b) Bacillus cereus c) Clostridium perfringens d) Clostridium botulinum 47

48 Case 4: final anatomic diagnosis C. perfringens food poisoning Vomiting Mucosal necrosis, upper GI Aspiration-induced lung injury Bacteria Congestion and edema Clinical history: vomitus in airways, infiltrates on CXR Clinical history of acute upper-gi bleed 48

49 Case 4: discussion Ultimate cause of death: aspiration-induced acute lung injury due to C. perfringens food poisoning Bacteria in lungs, blood secondary to resuscitation? Presence of bacteria in lymph node sinuses suggests earlier invasive infection Did aspiration occur prior to or after unconsciousness? Conscious aspiration is rare Unconsciousness due to electrolyte imbalance or hypoxia? C. perfringens usually presents with diarrhea. Why did this patient vomit? Constipation causing decreased intestinal transit time 49

50 Case 4: Clostridium perfringens Gas gangrene, sepsis Food poisoning, generally type A strains: Spores resistant to cooking, some survive Meats, gravy, spores germinate in leftovers 8-24 hours: sporulation in small intestine: diarrhea, abdominal pain Mild, self-limited Several reported cases of fatal necrotizing colitis in elderly (constipation) 50