- Email: [email protected]
Laboratory observations during an outbreak of pertussis
as "Enteric Group 19" (6). Strains of this organism have been isolated from blood and spinal fluid, suggesting possible clinical significance, but this point requires further study. Most strains are lactose negative at 48 h but ONPGpositive and exhibit a - - + + IMViC pattern. The name is pronounced " t a y ' - l o h r - e y e " and is given in honor of the British bacteriologist Joan Taylor and the American clinical microbiologist Welton Taylor. References 1. Aldova, E., O. Hausner, and M. Gabrhelova. 1984. Budvicia--a new genus of Enterobacteriaceae. Data on phenotypic characters. J. Hyg. Epidemiol. Microbiol. Immunol. 28:234-237. 2. Aldova, E. et al. 1983. A hydrogen sulphide producing gram-negative rod from water. Zentralbl. Bakteriol. Mikrobiol. Hyg. [A] 254(Abstr):95108. 3. Bercovier, H. et al. 1984. Yersinia aldovae (formerly Yersinia enterocolitica-like group X2): a new species of Enterobacteriaceae isolated from aquatic ecosystems. Int. J. Syst. Bacteriol. 34:166-172. 4. Bouvet, O. M. M. et al. 1985. Budvicia aquatica gen. nov., sp. nov.: a hydrogen sulfide-producing member of
5.
6.
7.
8.
9.
10.
the Enterobacteriaceae. Int. J. Syst. Bacteriol. 35:60-64. Brenner, D. J. et al. 1986. Enterobacter asburiae sp. nov., a new species found in clinical specimens, and reassignment of Erwinia dissolvens and Erwinia nimipressuralis to the genus Enterobacter as Enterobacter dissolvens comb. nov. and Enterbacter nimipressuralis comb. nov. J. Clin. Microbiol. 23:1114-1120. Farmer, J. J. III. et al. 1985. Escherichia fergusonii and Enterobacter taylorae, two new species of Enterobacteriaceae isolated from clinical specimens. J. Clin. Microbiol. 21:77-81. Freney, J. et al. 1984. Klebsiella trevisanii colonisation and septicaemia. Lancet i:909. Gavini, F. et al. 1986. Priority of Klebsiella planticola Bagley, Seidler, and Brenner 1982 over Klebsiella trevisanii Ferragut, Izard, Gavini, Kersters, Diley, and Leclerc 1983. Int. J. Syst. Bacteriol. 36:486-488. Hickman-Brenner, F. W. et al. 1983. Providencia rustigianii: a new species in the family Enterobacteriaceae formerly known as Providencia alcalifaciens biogroup 3. J. Clin. Microbiol. 17:1057-1060. Hickman-Brenner, F. W. et al. 1985. Koserella trabulsii, a new genus and species of Enterobacteriaceae formerly
known as enteric group 45. J. Clin. Microbiol. 21:39-42. 11. Hickman-Brenner, F. W. et al. 1984. Moellerella wisconsensis, a new genus and species of Enterobacteriaceae found in human stool specimens. J. Clin. Microbiol. 19:460-463. 12. Hickman-Brenner, F. W. et al. 1985. Leminorella, a new genus of Enterobacteriaceae : identification of Leminorella grimontii sp. nov. and Leminorella richardii sp. nov. found in clinical specimens. J. Clin. Microbiol. 21:234-239. 13. Izard, D. et al. 1985. Separation of Escherichia adecarboxylata from the Erwinia herbicola-Enterobacter agglomerans complex and from the other Enterobacteriaceae by nucleic acid and protein electrophoretic techniques. Ann. Inst. Pasteur Microbiol. 136 B:151168. 14. Miller, J. M. 1983. New genera and species of Enterobacteriaceae. Clin. Microbiol. Newsl. 5:149-151. 15. Muller, H. E. 1983. Providenciafriedericiana, a new species isolated from penguins. Int. J. Syst. Bacteriol. 33:709-715. 16. Tamura, K. et al. 1986. Leclercia adecarboxylata gen. nov. comb. nov., formerly known as Escherichia adecarboxylata. Curt. Microbiol. 13:179-184.
Editorial II
Laboratory Observations During an Outbreak of Pertussis Scott A. Young, B.S., M.T. (ASCP) Gary L. Anderson, B.S., M.T. (ASCP) Paul D. Mitchell, Ph.D. Microbiology Section Marshfield Medical Center Laboratory Marshfield Clinic~St. Joseph's Hospital Marshfield, Wisconsin 54449
tibody (DFA) procedure. This outbreak afforded us an opportunity to assess the usefulness and effectiveness of several laboratory practices for diagnosing pertussis. Our experiences may assist other laboratories that provide this diagnostic service. We describe here: 1) specimen collection and transportation; 2) culture media and enrichment techniques; 3) direct specimen testing by immunofluorescence; and 4) identification procedures.
Specimens From June 1 to December 31, 1985, our laboratory reported 214 confirmed cases of pertussis including 40 symptomatic patients with Bordetella parapertussis. Patients ranged from 2 months to 92 years of age (Figure 1). During this time, 3,571 nasopharyngeal specimens were received for culture and testing by the direct fluorescent an-
176
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After the first several cases of laboratory-confirmed pertussis were documented, publicized information warned of a possible outbreak in Central Wisconsin. Our laboratory serves a large medical complex and is a referral laboratory, therefore we expected a significant number of specimens for microbiologic examinations. To pro-
©1987 Elsevier Science Publishing Co., Inc.
mote the collection, transportation, and handling of specimens in a consistent manner, a specimen collection kit was made available. It consisted of a microscope slide container with one slide on which two 12 mm areas were inscribed for the direct application of nasopharyngeal secretions and subsequent DFA testing, two prepackaged dacron swabs on a flexible shaft (Spectrum Laboratories, Inc., Los Angeles, Calif.), and a tube of transport medium. The transport medium consisted of half-strength Oxoid charcoal agar CM119 supplemented with 40 I~g/ml cephalexin and 10% hemolyzed, deftbrinated horse blood. Instructions were provided and pernasal nasopharyngeal secretions were indicated as the specimen of choice. Specimens submitted from our medical complex were processed within 2 to 4 h of collection while those from more remote facilities
Clinical Microbiology Newsletter 9:22,1987
4O oo t-
(3-
30 il
I
o 20 <5 Z 10
fl I I o,
<1
1-
5
6-
11-
10 15
Illl_
16- 21- 26- 31- 36- 41- 46- 51-
20 25 30 35 40 45 50 92 A g e G r o u p (years)
Figure 1. Age distribution of patients with laboratory confirmed pertussis. were not received for 10 to 24 h following collection.
Cultivation We used selective and nonselective media for the primary isolation of Bordetella. The base medium for both was Oxoid charcoal agar CM119. The nonselective charcoal agar (CA) contained 10% defibrinated horse blood and the selective charcoal agar (SCA) contained 10% defibrinated horse blood and 40 p,g/ml cephalexin. Both media were dispensed in 100 x 15 mm plates, 20 ml per plate. Swab-collected specimens were inoculated first onto the CA medium and then onto the SCA medium. Both media were streaked for isolation with a platinum loop. Plates were incubated at 35°C in a humid atmosphere of 5% CO2 and examined daily for 7 days. We found that the SCA was superior to the CA medium. Of the first 65 Bordetella isolates recovered on SCA, 17 (26%) were not recovered on CA. Significant overgrowth by indigenous flora was observed on 42% of the positive CA cultures. The nonselective medium demonstrated earlier visible growth of Bordetella in only two instances. At times, indigenous respiratory tract flora formed colonies that resembled Bordetella on the cephalexin-free medium. In contrast, the cephalexin-containing medium was highly selective and plates could be analyzed quickly and with confidence.
Clinical Microbiology Newsletter 9:22,1987
Subsequently we discontinued the use of CA medium for the primary plating of specimens, which significantly reduced the time and cost of processing specimens and evaluating culture results.
Enrichment Technique In 1977, Regan and Lowe demonstrated that the use of an enrichment technique greatly increased their yield of positive Bordetella cultures (6). After direct plating, swabs were replaced into the transport medium consisting of half-strength Oxoid charcoal agar, 10% defibrinated horse blood, and 40 txg/ml cephalexin in Bijoux bottles. After 48 h incubation, the swabs were again inoculated to selective and nonselective media. Depending on the type of transport medium used, 18 to 51% of their isolates were obtained by enrichment only. We found that the enrichment technique produced dramatic results when it was applied to diluted suspensions of B. pertussis stock cultures. Dilutions that produced 1 + growth on the primary plating media produced 4 + growth following enrichment. When we applied the direct plating and enrichment technique to 134 patient specimens, B. pertussis was recovered from 27 specimens. The primary plates recovered all 27 isolates whereas the enrichment plates recovered only 13. In no instances were isolates recovered following enrichment that were not ob-
© 1987 Elsevier Science Publishing Co., Inc.
rained on primary CA and/or SCA media. Overgrowth was observed on 6% of the primary inoculated SCA plates, but 54% of the SCA plates following enrichment. Due to the increased overgrowth and decreased recovery of B. pertussis following enrichment, the enrichment technique was discontinued.
Amphotericin B as a Selective Agent Overgrowth by filamentous fungi can be an annoying problem when culturing for Bordetella. This problem may be circumvented by using a selective medium containing an antifungal agent, such as amphotericin B, or by cutting out the sector of agar medium containing fungal growth. After initial primary plating, swabs can be placed back into the transport medium and held at room temperature. If, after 1 to 2 days incubation, the primary plating media appear in danger of fungal overgrowth, an SCA plate containing 12 ixg/ml amphotericin B may be inoculated with the original specimen. This medium can inhibit B. pertussis and consequently we do not recommend its use as the sole primary plating medium. In most instances we preferred to remove the fungal growth by cutting out the sector of medium with a sterile scalpel. We used a biological safety cabinet for this task. Wrapping plates with a sealing material may help prevent the spread of fungal spores to adjacent cultures and prevent dehydration of the medium.
Immunofluorescence We performed the DFA test to identify pertussis cases as quickly as possible, preferably the same day specimens were received in our laboratory. Although the DFA assay has been used with varying degrees of success, it can provide an earlier presumptive (if not definitive) diagnosis than culture thereby permitting prompt administration of appropriate antimicrobial therapy and institution of measures for control and prevention. The two-well slides containing nasopharyngeal secretions were stained with FITC-conjugated anti-B, pertussis and anti-B, parapertussis antisera (Difco
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Laboratories, Detroit, Mich.), according to the manufacturer's instructions. The correlations between culture and DFA results are presented in Table 1. The DFA test detected 42 (26%) of the 164 patients who were culture positive for B. pertussis and 8 (21%) of 38 who were culture positive for B. parapertussis. In other studies, the percentage of infected patients detected by the DFA assay has ranged from 18 to 95% (2-6). When cultures were positive and the corresponding DFA assays were negative, the stained slides, which had been retained at 4°C, were reviewed. In some instances, after prolonged review of the slides, 1 to 3 fluorescing bacilli were observed. In most cases no fluorescing bacteria could be detected. Approximately 40% of the slides reviewed were noted to have very little cellular material (leukocytes and brush-border epithelial cells) which indicated an inferior specimen for DFA testing and could explain, in part, the decreased test sensitivity. Other explanations include the possible lability of BordeteUa antigens on air-dried, transported slides and variances in specimen collection techniques and specimen application to the slides. In our hands, the DFA test was specific. Although occasional false-positive reactions were observed with other bacteria, the reactions were less intense and the cellular morphology clearly differed from that of Bordetella spp. Most difficulties in interpretation were overcome with experience. In those few instances in which the DFA test was positive and the culture negative, brightly staining, distinct coccobacilli were observed in significant numbers and we do not feel they represented false-positive reactions. Several of
these patients had been treated with antimicrobics which could explain the negative cultures. Although certain factors limit the diagnostic effectiveness of DFA tests, most can be overcome if personnel have expertise in immunofluorescent microscopy and if appropriate and adequate specimens are collected during the first 2 to 3 weeks of illness.
Identification Most Bordetella isolates were detected in culture within 2 to 4 days incubation, but approximately 25% were detected after 5 to 7 days. Plates were examined for small, shiny, greyishwhite, round, convex colonies. Suspicious colonies were Gram stained. Because Bordetella generally stain faintly, a 2-minute safranin counterstain was used. Elongated and pleomorphic cells were discernible at times due to the effects of the cephalexin present in the selective medium. When morphological forms consistent with Bordetella were seen, growth and DFA assays were performed. Suspicious colonies were subcultured to sheep blood agar, chocolate agar, and CA plates. B. pertussis does not grow on sheep blood agar and rarely grows on chocolate agar. B. parapertussis grows on all three media. The chocolate agar medium helps to distinguish B. pertussis from cephalexin-resistant strains of Haemophilus influenzae. H. influenzae is indistinguishable from B. pertussis on Gram stain and like B. pertussis, does not grow on sheep blood agar. In many instances H. influenzae produces characteristic colonies on chocolate agar, thus no further testing is required. The DFA test was highly specific and was used for the confirmatory identification of B. per-
Table 1 Comparison of Direct Fluorescent Antibody (DFA) Test with Culture for Detecting B. pertussis and B. parapertussis Tests DFA DFA DFA DFA
+, -, +, -,
Culture Culture Culture Culture
+ + -
B. pertussis
B. parapertussis
10~ 122 42 3,397
2 30 8 3,531
a Numberof patients.
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©1987ElsevierSciencePublishingCo.. Inc.
tussis and B. parapertussis isolates during this outbreak.
Discussion Although pertussis is a preventable and controllable disease, sporadic cases and outbreaks still occur. In 1985, 3,276 cases were reported in the United States; 1,393 more cases than the median annual number for 1980 through 1984 (1). As with other reportable diseases, these figures undoubtedly represent only a fraction of the actual number of cases. Several factors contribute to the increased incidence of pertussis: pertussis is a highly communicable disease; vaccines do not afford 100% protection; levels of immunization in different populations vary; decline in vaccine usage (and acceptance) has contributed to an increased susceptible population; inadequate supplies of vaccine have existed in some geographical areas; asymptomatic and atypical disease occur which are not confirmed as pertussis; diagnosis based on clinical manifestations alone is unreliable; and disease and infection occur in susceptible adults who may represent significant reservoirs, especially during outbreaks. To better understand the role of these factors in the epidemiology, prevention, and control of pertussis, a means for accurate laboratory diagnosis is paramount. The etiologic agents of pertussis, B. pertussis and B. parapertussis, can be isolated from nasopharyngeal specimens on several artificial media. Cultural methods are the primary means for providing a definitive laboratory diagnosis. The direct detection of B. pertussis and B. parapertussis in nasopharyngeal specimens by DFA assay is also possible, but the sensitivity and specificity of this method are limited. The success of culture and DFA testing depends on the appropriate collection of nasopharyngeal specimens during the early stages of the disease. The effectiveness of these methods decreases significantly when specimens are collected after the third week of illness. Based on our observations during an outbreak, the following recommenda-
ClinicalMicrobiologyNewsletter9:22.1987
4.
Perform DFA tests on appropriately collected nasopharyngeal secretions with B. pertussis and B. parapertussis-conjugated antisera to facilitate an earlier diagnosis.
5.
After inoculating primary plating media retain swabs in the original transport medium at room temperature. If cultures become overgrown with indigenous bacterial flora or fungi use swabs to inoculate additional media.
6.
Identify suspicious isolates with appropriate cultural and biochemical tests. The DFA test performed on growth from isolated colonies is an excellent procedure for confirmatory or definitive identification.
References 1. Centers for Disease Control. 1986. Morbidity and Mortality Weekly Report. 34:774. 2. Field, L. H., and C. D. Parker. 1977. Pertussis outbreak in Austin and Travis County, Texas, 1975. J. Clin. Microbiol. 6:154-160. 3. GiUigan, P. H., and M. C. Fisher. 1984. Importance of culture in laboratory diagnosis of BordeteUa pertussis infections. J. Clin. Microbiol. 20:891893. 4. Hakansson, S. et al. 1984. Diagnosis of whooping cough--a comparison of culture, immunofluorescence and ELISA. Scand. J. Infect. Dis. 16:281284. 5. Regan, J. 1980. The laboratory diagnosis of whooping cough. Clin. Microbiol. Newsl. 2:1-3. 6. Regan, J., and F. Lowe. 1977. Enrichment medium for the isolation of Bordetella. J. Clin. Microbiol. 6:303-309.
mission, the patient had a rectal temperature of 102.7°F (39.3°C), a heart rate of 152/min, and a respiratory rate of 40. His blood pressure was 100 mmHg systolic. His head circumference was 38 cm (5.5 cm below the fifth percentile), his length was 56 cm (12 cm below the fifth percentile) and his weight was 4.3 kg (3.12 kg below the fifth percentile). The weight-to-length ratio fell between the 25th and 50th percentile. His face, body, and extremities were grossly edematous. A scaly, erythematous rash covered the legs, diaper area, the trunk, and, to a lesser extent, the cheeks. The skin appeared thin and translucent; the capillaries lace-like. The hair was fine and the papillae on the tongue seemed prominent. The liver edge was felt at 4 cm below the right costal margin. The spleen tip was palpable. A neurological exam showed a slightly irritable, but all too easily consoled child, unable to sit up due to generalized weakness. His peripheral white blood cell count was 18,900/mm 3 with 62% neutrophils and 38% lymphocytes. Diffuse osteopenia was seen on bone films. Blood and urine cultures were obtained and the patient was started on intravenous cefuroxime (150 mg/kg/
day), which was discontinued 72 h later when the patient was afebrile and the cultures remained negative. Supportive care during this time consisted of careful attempts at renourishing. Thirty-six hours after antibiotics were discontinued, the infant became bradycardic and acidotic, required intubation, placement of a central and arterial line, and had recurrent episodes of cardiac failure and pulmonary edema. A blood culture obtained at this point, before institution of nafcillin (150 mg/kg/day), tobramycin (5 mg/kg/day) and ticarcillin (250 mg/kg/day) grew Achromobacter xylosoxidans. CSF cultures remained negative. The antimicrobial susceptibility of the organism was (MIC, p,g/ml): amikacin >32.0 (MBC >32.0), tobramycin 16.0, gentamicin >16.0, carbenicillin <4.0, ticarcillin 2 (MBC 4), piperacillin <2.0, chloramphenicol 32, ampicillin >32.0, cefoxitin >128.0, ceftazidime 4.0 (MBC 4.0), and cefotaxime >32.0. Repeat blood cultures subsequent to therapy remained negative. In spite of numerous interventions, the patient died 9 days later with anuria and progressive aseptic necrosis of the right ann as a consequence of an arterial line thrombus and Candida albicans sepsis.
be used in addition to the selective medium.
tions are made to maximize the laboratory diagnosis of pertussis: 1.
Collect nasopharyngeal specimens in the early stage of illness. Providing specimen collection kits facilitates the appropriate specimen collection and transportation.
2.
For swab-collected specimens use a transport medium consisting of half strength Oxoid charcoal agar supplemented with 10% hemolyzed, defibrinated horse blood and 40 p,g/ml cephalexin.
3.
Inoculate a selective primary plating medium composed of OXoid charcoal agar, 10% defibrinated horse blood, and 40 ~g/mL cephalexin. A nonselective medium without cephalexin may
Case Report
Achromobacter xylosoxidans Sepsis in a Child with Marasmus/ Kwashiorkor
Juan N. Walterspiel, M.D. Terry B. Snider, M.D. Helen M. Pollock, Ph.D. Departments of Pediatrics and Pathology University of South Alabama College of Medicine Mobile, Alabama 36617
A 9-month-old male was admitted for evaluation of edema and failure-tothrive. Nothing in the pre- and postnatal medical history contributed to his present problem. His discharge weight from the nursery was 2.4 kg. His mother stated that the infant refused to take milk and cereals starting at 2 months of age, and was fed mainly puddings and fruit juices. By the time he was 7 months old, his parents gave up offering him milk or proteincontaining nutrients. They reported the child had increasing complacency with a recent onset of pedal edema. On ad-
Clinical Microbiology Newsletter 9:22,1987
~ 1987 Elsevier Science Publishing Co., Inc.
0196-4399/87/$0.00 + 02.20
179
5.
6.
7.
8.
9.
10.
the Enterobacteriaceae. Int. J. Syst. Bacteriol. 35:60-64. Brenner, D. J. et al. 1986. Enterobacter asburiae sp. nov., a new species found in clinical specimens, and reassignment of Erwinia dissolvens and Erwinia nimipressuralis to the genus Enterobacter as Enterobacter dissolvens comb. nov. and Enterbacter nimipressuralis comb. nov. J. Clin. Microbiol. 23:1114-1120. Farmer, J. J. III. et al. 1985. Escherichia fergusonii and Enterobacter taylorae, two new species of Enterobacteriaceae isolated from clinical specimens. J. Clin. Microbiol. 21:77-81. Freney, J. et al. 1984. Klebsiella trevisanii colonisation and septicaemia. Lancet i:909. Gavini, F. et al. 1986. Priority of Klebsiella planticola Bagley, Seidler, and Brenner 1982 over Klebsiella trevisanii Ferragut, Izard, Gavini, Kersters, Diley, and Leclerc 1983. Int. J. Syst. Bacteriol. 36:486-488. Hickman-Brenner, F. W. et al. 1983. Providencia rustigianii: a new species in the family Enterobacteriaceae formerly known as Providencia alcalifaciens biogroup 3. J. Clin. Microbiol. 17:1057-1060. Hickman-Brenner, F. W. et al. 1985. Koserella trabulsii, a new genus and species of Enterobacteriaceae formerly
known as enteric group 45. J. Clin. Microbiol. 21:39-42. 11. Hickman-Brenner, F. W. et al. 1984. Moellerella wisconsensis, a new genus and species of Enterobacteriaceae found in human stool specimens. J. Clin. Microbiol. 19:460-463. 12. Hickman-Brenner, F. W. et al. 1985. Leminorella, a new genus of Enterobacteriaceae : identification of Leminorella grimontii sp. nov. and Leminorella richardii sp. nov. found in clinical specimens. J. Clin. Microbiol. 21:234-239. 13. Izard, D. et al. 1985. Separation of Escherichia adecarboxylata from the Erwinia herbicola-Enterobacter agglomerans complex and from the other Enterobacteriaceae by nucleic acid and protein electrophoretic techniques. Ann. Inst. Pasteur Microbiol. 136 B:151168. 14. Miller, J. M. 1983. New genera and species of Enterobacteriaceae. Clin. Microbiol. Newsl. 5:149-151. 15. Muller, H. E. 1983. Providenciafriedericiana, a new species isolated from penguins. Int. J. Syst. Bacteriol. 33:709-715. 16. Tamura, K. et al. 1986. Leclercia adecarboxylata gen. nov. comb. nov., formerly known as Escherichia adecarboxylata. Curt. Microbiol. 13:179-184.
Editorial II
Laboratory Observations During an Outbreak of Pertussis Scott A. Young, B.S., M.T. (ASCP) Gary L. Anderson, B.S., M.T. (ASCP) Paul D. Mitchell, Ph.D. Microbiology Section Marshfield Medical Center Laboratory Marshfield Clinic~St. Joseph's Hospital Marshfield, Wisconsin 54449
tibody (DFA) procedure. This outbreak afforded us an opportunity to assess the usefulness and effectiveness of several laboratory practices for diagnosing pertussis. Our experiences may assist other laboratories that provide this diagnostic service. We describe here: 1) specimen collection and transportation; 2) culture media and enrichment techniques; 3) direct specimen testing by immunofluorescence; and 4) identification procedures.
Specimens From June 1 to December 31, 1985, our laboratory reported 214 confirmed cases of pertussis including 40 symptomatic patients with Bordetella parapertussis. Patients ranged from 2 months to 92 years of age (Figure 1). During this time, 3,571 nasopharyngeal specimens were received for culture and testing by the direct fluorescent an-
176
0196-4399/87/$0.00 + 02.20
After the first several cases of laboratory-confirmed pertussis were documented, publicized information warned of a possible outbreak in Central Wisconsin. Our laboratory serves a large medical complex and is a referral laboratory, therefore we expected a significant number of specimens for microbiologic examinations. To pro-
©1987 Elsevier Science Publishing Co., Inc.
mote the collection, transportation, and handling of specimens in a consistent manner, a specimen collection kit was made available. It consisted of a microscope slide container with one slide on which two 12 mm areas were inscribed for the direct application of nasopharyngeal secretions and subsequent DFA testing, two prepackaged dacron swabs on a flexible shaft (Spectrum Laboratories, Inc., Los Angeles, Calif.), and a tube of transport medium. The transport medium consisted of half-strength Oxoid charcoal agar CM119 supplemented with 40 I~g/ml cephalexin and 10% hemolyzed, deftbrinated horse blood. Instructions were provided and pernasal nasopharyngeal secretions were indicated as the specimen of choice. Specimens submitted from our medical complex were processed within 2 to 4 h of collection while those from more remote facilities
Clinical Microbiology Newsletter 9:22,1987
4O oo t-
(3-
30 il
I
o 20 <5 Z 10
fl I I o,
<1
1-
5
6-
11-
10 15
Illl_
16- 21- 26- 31- 36- 41- 46- 51-
20 25 30 35 40 45 50 92 A g e G r o u p (years)
Figure 1. Age distribution of patients with laboratory confirmed pertussis. were not received for 10 to 24 h following collection.
Cultivation We used selective and nonselective media for the primary isolation of Bordetella. The base medium for both was Oxoid charcoal agar CM119. The nonselective charcoal agar (CA) contained 10% defibrinated horse blood and the selective charcoal agar (SCA) contained 10% defibrinated horse blood and 40 p,g/ml cephalexin. Both media were dispensed in 100 x 15 mm plates, 20 ml per plate. Swab-collected specimens were inoculated first onto the CA medium and then onto the SCA medium. Both media were streaked for isolation with a platinum loop. Plates were incubated at 35°C in a humid atmosphere of 5% CO2 and examined daily for 7 days. We found that the SCA was superior to the CA medium. Of the first 65 Bordetella isolates recovered on SCA, 17 (26%) were not recovered on CA. Significant overgrowth by indigenous flora was observed on 42% of the positive CA cultures. The nonselective medium demonstrated earlier visible growth of Bordetella in only two instances. At times, indigenous respiratory tract flora formed colonies that resembled Bordetella on the cephalexin-free medium. In contrast, the cephalexin-containing medium was highly selective and plates could be analyzed quickly and with confidence.
Clinical Microbiology Newsletter 9:22,1987
Subsequently we discontinued the use of CA medium for the primary plating of specimens, which significantly reduced the time and cost of processing specimens and evaluating culture results.
Enrichment Technique In 1977, Regan and Lowe demonstrated that the use of an enrichment technique greatly increased their yield of positive Bordetella cultures (6). After direct plating, swabs were replaced into the transport medium consisting of half-strength Oxoid charcoal agar, 10% defibrinated horse blood, and 40 txg/ml cephalexin in Bijoux bottles. After 48 h incubation, the swabs were again inoculated to selective and nonselective media. Depending on the type of transport medium used, 18 to 51% of their isolates were obtained by enrichment only. We found that the enrichment technique produced dramatic results when it was applied to diluted suspensions of B. pertussis stock cultures. Dilutions that produced 1 + growth on the primary plating media produced 4 + growth following enrichment. When we applied the direct plating and enrichment technique to 134 patient specimens, B. pertussis was recovered from 27 specimens. The primary plates recovered all 27 isolates whereas the enrichment plates recovered only 13. In no instances were isolates recovered following enrichment that were not ob-
© 1987 Elsevier Science Publishing Co., Inc.
rained on primary CA and/or SCA media. Overgrowth was observed on 6% of the primary inoculated SCA plates, but 54% of the SCA plates following enrichment. Due to the increased overgrowth and decreased recovery of B. pertussis following enrichment, the enrichment technique was discontinued.
Amphotericin B as a Selective Agent Overgrowth by filamentous fungi can be an annoying problem when culturing for Bordetella. This problem may be circumvented by using a selective medium containing an antifungal agent, such as amphotericin B, or by cutting out the sector of agar medium containing fungal growth. After initial primary plating, swabs can be placed back into the transport medium and held at room temperature. If, after 1 to 2 days incubation, the primary plating media appear in danger of fungal overgrowth, an SCA plate containing 12 ixg/ml amphotericin B may be inoculated with the original specimen. This medium can inhibit B. pertussis and consequently we do not recommend its use as the sole primary plating medium. In most instances we preferred to remove the fungal growth by cutting out the sector of medium with a sterile scalpel. We used a biological safety cabinet for this task. Wrapping plates with a sealing material may help prevent the spread of fungal spores to adjacent cultures and prevent dehydration of the medium.
Immunofluorescence We performed the DFA test to identify pertussis cases as quickly as possible, preferably the same day specimens were received in our laboratory. Although the DFA assay has been used with varying degrees of success, it can provide an earlier presumptive (if not definitive) diagnosis than culture thereby permitting prompt administration of appropriate antimicrobial therapy and institution of measures for control and prevention. The two-well slides containing nasopharyngeal secretions were stained with FITC-conjugated anti-B, pertussis and anti-B, parapertussis antisera (Difco
0196-4399/87/$0.00 + 02.20
177
Laboratories, Detroit, Mich.), according to the manufacturer's instructions. The correlations between culture and DFA results are presented in Table 1. The DFA test detected 42 (26%) of the 164 patients who were culture positive for B. pertussis and 8 (21%) of 38 who were culture positive for B. parapertussis. In other studies, the percentage of infected patients detected by the DFA assay has ranged from 18 to 95% (2-6). When cultures were positive and the corresponding DFA assays were negative, the stained slides, which had been retained at 4°C, were reviewed. In some instances, after prolonged review of the slides, 1 to 3 fluorescing bacilli were observed. In most cases no fluorescing bacteria could be detected. Approximately 40% of the slides reviewed were noted to have very little cellular material (leukocytes and brush-border epithelial cells) which indicated an inferior specimen for DFA testing and could explain, in part, the decreased test sensitivity. Other explanations include the possible lability of BordeteUa antigens on air-dried, transported slides and variances in specimen collection techniques and specimen application to the slides. In our hands, the DFA test was specific. Although occasional false-positive reactions were observed with other bacteria, the reactions were less intense and the cellular morphology clearly differed from that of Bordetella spp. Most difficulties in interpretation were overcome with experience. In those few instances in which the DFA test was positive and the culture negative, brightly staining, distinct coccobacilli were observed in significant numbers and we do not feel they represented false-positive reactions. Several of
these patients had been treated with antimicrobics which could explain the negative cultures. Although certain factors limit the diagnostic effectiveness of DFA tests, most can be overcome if personnel have expertise in immunofluorescent microscopy and if appropriate and adequate specimens are collected during the first 2 to 3 weeks of illness.
Identification Most Bordetella isolates were detected in culture within 2 to 4 days incubation, but approximately 25% were detected after 5 to 7 days. Plates were examined for small, shiny, greyishwhite, round, convex colonies. Suspicious colonies were Gram stained. Because Bordetella generally stain faintly, a 2-minute safranin counterstain was used. Elongated and pleomorphic cells were discernible at times due to the effects of the cephalexin present in the selective medium. When morphological forms consistent with Bordetella were seen, growth and DFA assays were performed. Suspicious colonies were subcultured to sheep blood agar, chocolate agar, and CA plates. B. pertussis does not grow on sheep blood agar and rarely grows on chocolate agar. B. parapertussis grows on all three media. The chocolate agar medium helps to distinguish B. pertussis from cephalexin-resistant strains of Haemophilus influenzae. H. influenzae is indistinguishable from B. pertussis on Gram stain and like B. pertussis, does not grow on sheep blood agar. In many instances H. influenzae produces characteristic colonies on chocolate agar, thus no further testing is required. The DFA test was highly specific and was used for the confirmatory identification of B. per-
Table 1 Comparison of Direct Fluorescent Antibody (DFA) Test with Culture for Detecting B. pertussis and B. parapertussis Tests DFA DFA DFA DFA
+, -, +, -,
Culture Culture Culture Culture
+ + -
B. pertussis
B. parapertussis
10~ 122 42 3,397
2 30 8 3,531
a Numberof patients.
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tussis and B. parapertussis isolates during this outbreak.
Discussion Although pertussis is a preventable and controllable disease, sporadic cases and outbreaks still occur. In 1985, 3,276 cases were reported in the United States; 1,393 more cases than the median annual number for 1980 through 1984 (1). As with other reportable diseases, these figures undoubtedly represent only a fraction of the actual number of cases. Several factors contribute to the increased incidence of pertussis: pertussis is a highly communicable disease; vaccines do not afford 100% protection; levels of immunization in different populations vary; decline in vaccine usage (and acceptance) has contributed to an increased susceptible population; inadequate supplies of vaccine have existed in some geographical areas; asymptomatic and atypical disease occur which are not confirmed as pertussis; diagnosis based on clinical manifestations alone is unreliable; and disease and infection occur in susceptible adults who may represent significant reservoirs, especially during outbreaks. To better understand the role of these factors in the epidemiology, prevention, and control of pertussis, a means for accurate laboratory diagnosis is paramount. The etiologic agents of pertussis, B. pertussis and B. parapertussis, can be isolated from nasopharyngeal specimens on several artificial media. Cultural methods are the primary means for providing a definitive laboratory diagnosis. The direct detection of B. pertussis and B. parapertussis in nasopharyngeal specimens by DFA assay is also possible, but the sensitivity and specificity of this method are limited. The success of culture and DFA testing depends on the appropriate collection of nasopharyngeal specimens during the early stages of the disease. The effectiveness of these methods decreases significantly when specimens are collected after the third week of illness. Based on our observations during an outbreak, the following recommenda-
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4.
Perform DFA tests on appropriately collected nasopharyngeal secretions with B. pertussis and B. parapertussis-conjugated antisera to facilitate an earlier diagnosis.
5.
After inoculating primary plating media retain swabs in the original transport medium at room temperature. If cultures become overgrown with indigenous bacterial flora or fungi use swabs to inoculate additional media.
6.
Identify suspicious isolates with appropriate cultural and biochemical tests. The DFA test performed on growth from isolated colonies is an excellent procedure for confirmatory or definitive identification.
References 1. Centers for Disease Control. 1986. Morbidity and Mortality Weekly Report. 34:774. 2. Field, L. H., and C. D. Parker. 1977. Pertussis outbreak in Austin and Travis County, Texas, 1975. J. Clin. Microbiol. 6:154-160. 3. GiUigan, P. H., and M. C. Fisher. 1984. Importance of culture in laboratory diagnosis of BordeteUa pertussis infections. J. Clin. Microbiol. 20:891893. 4. Hakansson, S. et al. 1984. Diagnosis of whooping cough--a comparison of culture, immunofluorescence and ELISA. Scand. J. Infect. Dis. 16:281284. 5. Regan, J. 1980. The laboratory diagnosis of whooping cough. Clin. Microbiol. Newsl. 2:1-3. 6. Regan, J., and F. Lowe. 1977. Enrichment medium for the isolation of Bordetella. J. Clin. Microbiol. 6:303-309.
mission, the patient had a rectal temperature of 102.7°F (39.3°C), a heart rate of 152/min, and a respiratory rate of 40. His blood pressure was 100 mmHg systolic. His head circumference was 38 cm (5.5 cm below the fifth percentile), his length was 56 cm (12 cm below the fifth percentile) and his weight was 4.3 kg (3.12 kg below the fifth percentile). The weight-to-length ratio fell between the 25th and 50th percentile. His face, body, and extremities were grossly edematous. A scaly, erythematous rash covered the legs, diaper area, the trunk, and, to a lesser extent, the cheeks. The skin appeared thin and translucent; the capillaries lace-like. The hair was fine and the papillae on the tongue seemed prominent. The liver edge was felt at 4 cm below the right costal margin. The spleen tip was palpable. A neurological exam showed a slightly irritable, but all too easily consoled child, unable to sit up due to generalized weakness. His peripheral white blood cell count was 18,900/mm 3 with 62% neutrophils and 38% lymphocytes. Diffuse osteopenia was seen on bone films. Blood and urine cultures were obtained and the patient was started on intravenous cefuroxime (150 mg/kg/
day), which was discontinued 72 h later when the patient was afebrile and the cultures remained negative. Supportive care during this time consisted of careful attempts at renourishing. Thirty-six hours after antibiotics were discontinued, the infant became bradycardic and acidotic, required intubation, placement of a central and arterial line, and had recurrent episodes of cardiac failure and pulmonary edema. A blood culture obtained at this point, before institution of nafcillin (150 mg/kg/day), tobramycin (5 mg/kg/day) and ticarcillin (250 mg/kg/day) grew Achromobacter xylosoxidans. CSF cultures remained negative. The antimicrobial susceptibility of the organism was (MIC, p,g/ml): amikacin >32.0 (MBC >32.0), tobramycin 16.0, gentamicin >16.0, carbenicillin <4.0, ticarcillin 2 (MBC 4), piperacillin <2.0, chloramphenicol 32, ampicillin >32.0, cefoxitin >128.0, ceftazidime 4.0 (MBC 4.0), and cefotaxime >32.0. Repeat blood cultures subsequent to therapy remained negative. In spite of numerous interventions, the patient died 9 days later with anuria and progressive aseptic necrosis of the right ann as a consequence of an arterial line thrombus and Candida albicans sepsis.
be used in addition to the selective medium.
tions are made to maximize the laboratory diagnosis of pertussis: 1.
Collect nasopharyngeal specimens in the early stage of illness. Providing specimen collection kits facilitates the appropriate specimen collection and transportation.
2.
For swab-collected specimens use a transport medium consisting of half strength Oxoid charcoal agar supplemented with 10% hemolyzed, defibrinated horse blood and 40 p,g/ml cephalexin.
3.
Inoculate a selective primary plating medium composed of OXoid charcoal agar, 10% defibrinated horse blood, and 40 ~g/mL cephalexin. A nonselective medium without cephalexin may
Case Report
Achromobacter xylosoxidans Sepsis in a Child with Marasmus/ Kwashiorkor
Juan N. Walterspiel, M.D. Terry B. Snider, M.D. Helen M. Pollock, Ph.D. Departments of Pediatrics and Pathology University of South Alabama College of Medicine Mobile, Alabama 36617
A 9-month-old male was admitted for evaluation of edema and failure-tothrive. Nothing in the pre- and postnatal medical history contributed to his present problem. His discharge weight from the nursery was 2.4 kg. His mother stated that the infant refused to take milk and cereals starting at 2 months of age, and was fed mainly puddings and fruit juices. By the time he was 7 months old, his parents gave up offering him milk or proteincontaining nutrients. They reported the child had increasing complacency with a recent onset of pedal edema. On ad-
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