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Laboratory evaluation of an outbreak of nocardiosis in immunocompromised hosts
Laboratory Evaluation of an Outbreak of Nocardiosis in lmmunocompromised
DAVID
A. STEVENS,
Hosts
Seven patients in a renal unit were proved to have nocardiosis in an interval of nine months. Six of these patients had received renal transplants. Serologic investigation suggested that two additional cases of undiagnosed pulmonary disease were also nocardial, and that there were no subclinical cases in patients or staff. Clinicalserologic correlations indicate that serologic evaluation may be a useful adjunct in diagnosis of nocardiosis, if used early and repeatedly, and to follow response to therapy. Epidemiologic investigations yielded cultures of Nocardia asteroides from air and dust inside the unit and elsewhere in the hospital. Biochemical, metabolic, physical and immunologic characterization of the isolates indicated that those from patients and those from the unit environment were identical, whereas some from outside the unit could be differentiated from these. The “epidemk strain” had type Ill antigen, which surveys indicated is not the most common type in human nocardiosis (it occurs in association with a minority of human cases). The isolates were of subgroup B, which has been associated with virulence. The characterization methods employed could be useful in studies of nocardial epidemiology. The laboratory studies indicate epidemic spread within the unit of a single organism, and current epidemiologic guidelines, which do not recommend respiratory isolation of cases of pulmonary nocardiosis, may need reconsideration particularly when there are immunocompromised hosts in the environment.
M.D.
San Jose and Stanford. California ALLAN
C. PIER.
Ph.D.,
D.V.M.
Ames, lowa BLAINE
L. BEAMAN,
Ph.D.
Davis, California PIUS
A.
MOROZUMI,
M.D.
San Jose and Stanford IAN
S. LOVETT,
ELIZABETH
M.B.,
T. HOUANG,
B.S.,
M.R.C.P.
Ph.D.
London, England
From the Division of Infectious Diseases, Departments of Medicine, Santa Clara Valley Medical Center and Stanford University, San Jose and Stanford. California; tie Institute for Medical Research, San Jose, California; the Bacterial and Mycokqical Research Laboratory, National Animal Disease Center, United States Department of Agriculture, Ames, Iowa; the Department of Medical Microbiology, University of California Medical School, Davis, California; St. Peter’s Group of Hospitals and the Institute of Urology, London, England: and the Department of Medical Microbiology, London Hospital, London, England. Reprint requests should be addressed to Dr. David A. Stevens, Department of Medicine, Santa Clara Valley Medical Center, 751 South Bascom Avenue, San Jose, CA 95 128. Manuscript accepted July 16. 1981.
928
December
1981
The American
Journal
Nocardiae are bacteria found in soil and not part of normal human flora. The incidence of human infections appears to be increasing, and human nocardiosis is particularly a problem of immunocompromised hosts [ 11.Although man-t&man transmission of infection has not been documented, the incidence of infection in some subgroups of immunocompromised hosts is particularly high [2], and clustering of a few cases in immunocompromised patients was earlier suggested to represent possible contagion [3]. A recent outbreak of N. asteroides infections, involving renal transplantation patients, in a London hospital renal unit [ 41 provided the strongest argument for contagion to date. This report concerns the application of several laboratory tools to the study of the epidemiology of that outbreak. MATERIALS AND METHODS Serologic Studles. A complement-fixation antibody (CF) test, as previously described [5], was utilized to test the serum specimens. The serum samples were not collected prospectively but were salvaged from other assays or collected from patients surviving at the time the unit was closed. The serum samples were frozen until assayed. In brief, this CF test uses an extracellular antigen (culture filtrate) prepared by pooling filtrates from four N. asteroides
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LABORATORY
strains [5]. Each strain is a prototype strain for the production of an extracellular antigen, termed I, II, ill or IV, detectable by immunization of experimental animals with culture filtrate and assay of their serums by immunodiffusion [6]. In immunodiffusion, a single, immunologically unique, band is detected with these antiserums and filtrates (no reactions of identity or partial identity with the bands of the other three strains). Virtually all wild strains of N. asteroides involved in human or animal infection produce one, and in some cases two or more, of these four antigens [6]. Previous studies have shown that CF titers of 1:4 or more are reproducible and specific for nocardial infection [5] and are considered positive results. Only serums from patients with mycobacterial infections also gave positive results in this assay, as has been reported with other serologic assays for nocardiosis [5]. Recent experience has confirmed the earlier work [5], in that additional serum samples submitted from patients with other infections do not give positive CF results (J. Z. Shainhouse, P. A. Morozumi and D. A. Stevens, unpublished results). Appropriate positive and negative controls are included in each assay [5]. The serum samples were coded prior to assay. The positive control reference serum in each assay is a rabbit antiserum prepared against the antigen pool. Monospecific rabbit antiserum against each of the four prototype antigens were also available, and this enabled establishment of a CF assay using the type Ill antigen only. The optimal reagent dilutions for this assay were determined by block titration as previously described [7]. Immunologic Typing of Isolates. With the use of the monospecific rabbit antiserums described, isolates obtained during the outbreak were typed with respect to their production of extracellular antigen(s) [6]. This was done by growing the isolates in liquid medium, and assaying the culture filtrates in immunodiffusion as previously described
STUDY OF NOCARDIA
OUTBREAK-STEVENS
ET AL.
in the unit, and the same amount elsewhere in the hospital. Media used in sampling procedures included blood agar, Sabouraud’s agar, brain-heart infusion agar and LowensteinJensen medium, with appropriate inhibitors [4]. These same media were used in culturing patient specimens. From these sources, 12 of 13 Nocardia colonies obtained inside the unit and six of seven obtained elsewhere in the hospital were available for study. One colony obtained in the affiliated hospitals was not available for study. Eight of 14 isolates from patients were available for study; the six isofates not studied were from other body sites in patients contributing at least one isolate for study. Identification and Analysis of N. asteroides Strains. Colonies suspected as possibly being Nocardia colonies were initially identified [6] and reconfirmed [9] by standard methods. All the isolates described in the present study were identified as N. asteroides. Subsequent characterization by biochemical, physical and metabolic tests was performed by previously described methods [ 6,i O-l 31. Some key tests that differentiated isolates were repeated independently in one or two different laboratories. Colonial pigment production is considered a variable phenotypic characteristic, not reliable for differentiating strains, and was not used for this purpose. Fatty acid chromatography, using whole cells grown on brain-heart infusion agar for 418hours and lysed with hydrochloric acid and methanol, was performed as previously described [ 141. Comparison was made with prototype strains of the typing system of Kurup and Schmitt [ 151, a biochemical system of aping N. asteroides. strains KN 827, KN 845, KN 850, KN 851 and KN 852. All the tests on the isolates described were performed with numerically coded samples. Some isolates were included in duplicate as an internal check on reproducibility. RESULTS
1’31. Prior to and just after Isolation of N. asteroides Strains. closure and cleaning of the unit, environmental sampling was performed throughout the hospital as detailed elsewhere [4]. In brief, air was cultured using agar plates exposed overnight (“settle plates”) or a slit air sampler filtering 3,500 liters of air for 10 minutes onto agar plates. Dust obtained from bed, stores, dialysis machine and sluice areas and ventifation grills was wetted and cultured on solid media. Swabs obtained from environmental surfaces were handled similarly. Tap water was filtered, and the filters were cultured on solid media. During the months of the outbreak, N. asteroides colonies were identified on culture plates of specimens, from three patients, obtained in affiliated hospitals; these cultures had been processed in the St. Paul’s Hospital laboratory. These isolates were deemed inconsistent with a diagnosis of nocardiosis, given these patients’ clinical courses and repeated prior and subsequent microbiologic studies. The patients had no connection with St. Paul’s Hospital. These isolates were therefore considered “plate contaminants.” In total, 60,000 liters of air in the affected unit were examined, as well as 30 dygt samples and 27 settle plates. Elsewhere in the hospital, 49,000 liters of air, 32 dust specimens and 45 settle plates were examined. As a control, 14,000 liters of air in affiliated hospitals were sampled. After closure of the unit, 7,000 liters of air were sampled monthly
The inpatient area of the Chronology of the Epidemic. Renal Unit of St. Paul’s Hospital in 1978 and 1979 was divided into two areas across a corridor from each other-the intensive care area (ICA) for transplantation patients and short-term dialysis (six inpatient beds, a dialysis room, and rooms for ancillary services), and the long-term care area with inpatient and dialysis beds for long-term dialysis patients. In July 1978, a patient with chronic renal failure was admitted to the ICA for dialysis and treatment of recurrent urinary tract infections. Her urine was collected in a nephrostomy bag, which was emptied into a sluice in the ward area. In October, cultures of her urine began to grow a “diphtheroid.” She was asymptomatic and was not treated: on repeated admissions, her urine continued to grow this organlsm. In March 1979, an ICA patient who had received a renal transplant one month earlier had an undiagnosed febrile illness with cough and pleurisy, and in April, a wound infection. N. asteroides was cultured from the pus. At that time, the urine isolate from the first patient was re-examined and identified as N. asteroides. The unit had not recently recorded a Nocardia infection, except for a patient with a nocardial
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The American Journal 01 Medicine
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LABORATORY
STUDY OF NOCARDIA
OUTBREAK-STEVENS
ET AL.
pulmonary infection and brain abscess in 1976 that were successfully treated. In May 1979. pulmonary nocardiosis proved by culture developed in three ICA transplantation patients. One of these was also shown to have hepatic nocardiosis at autopsy. In early June, the ICA was closed, and all the patients mentioned were discharged or transferred to another hospital. Three other transplantation patients were transferred to another unit in St. Paul’s. Subsequently, bacteriologically proved pulmonary nocardiosis developed in two of these, in late June and early July (one was also shown to have nocardiosis in her transplanted kidney). The third (Patient FL) had chest pain and pulmonary infiltrates in late July and died in mid-August without a diagnosis for this complication: no postmortem examination was done. After closure of the ICA, it was cleaned, sprayed with 1 percent hypochlorite, fumigated with formaldehyde and then reopened. Thus, in 1979, prior to its closure, culture-proved nocardiosis developed in six of 50 patients admitted to the ICA (five pulmonary). All six had received renal transplants in that interval, and there had only been nine transplant patients admitted during that period. In addition, the patient with urinary nocardiosis who did not undergo transplantation, whose disease is presumed to have started in 1978, completes the group of seven culture-proved cases considered to represent a single outbreak. Some epidemiologic features [4] and clinical features [ 161 of this epidemic are presented in more detail elsewhere. It is worthy of emphasis that work-up of all cases of suspected nocardiosis described in this report included cultures of sputum, other body fluids and tissue, as pertinent, for mycobacteria, other bacteria and fungi, as well. Since the reopening of the ICA, there have been no subsequent cases of nocardiosis diagnosed in it. Serologic Investigations. The serologic study included the confirmed cases just discussed, with exception of the index case; no serum was available from this patient. In addition, the confirmed case from 1976, the case of Patient FL described earlier and two other cases of special interest were also studied. One of these cases was in a patient (KS) who had received a renal transplant in December 1978, after the nocardiuria of the index case had presumably begun but before recognition of the epidemic. In March 1979, KS had an undiagnosed respiratory illness with pulmonary infiltrates, and she died in August without a postmortem examination. The other case was in Patient DC, who had received a transplant in 1976 that was rejected. He had not been a patient in the ICA since that time, being in a long-term dialysis program in the long-term care area. In mid-July 1979, fever and pleurisy developed, and pulmonary nodules were noted on x-ray. Although the diagnostic work-up was unrewarding, he began
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promptly to receive amoxicillin and erythromycin to include coverage for possible nocardiosis. His x-rays showed improvement one month later. In addition, serums were obtained after the ICA closed from 27 physicians, nurses and aides who cared for the ICA patients. Thirty-two unaffected patients also provided serum samples: one of these provided two samples a month apart. The results are shown in Table 1. Patients in three of five cases associated with the epidemic were shown to have complement-fixing antibody. Two of these seropositive immunosuppressed patients had positive findings only in the first serum sample obtained from them. The second, negative, serum sample was obtained two months after the beginning of therapy that had produced progressive improvement in one (Case 2) of these two cases. In the other, the second serum sample was obtained 50 days after the beginning of a therapeutic regimen that had produced clear clinical improvement seven days after its initiation (Case 3). The two patients who had seronegative results had serum available for testing only at 55 and 122 days after onset of their nocardial disease. Therapy had started 48 days prior to the serum collection in the first of these two patients, and her disease had cleared in that interval (normal chest x-ray six days prior to serum sample). In the second of these, therapy had started three months prior to the serum collection, with progressive improvement of her disease. One patient’s serum was anticomplementary and could not be assayed. The patient with nocardiosis in the ICA three years earlier, not associated with the outbreak, had seronegative results. Study of the three patients with suspected but not bacteriologically confirmed nocardiosis gave very interesting results. A seropositive specimen was obtained from one patient four days before the development of fever and pleuritic pain. It is highly likely his disease was incubating at the time of specimen collection, because a chest x-ray obtained six days after onset of symptoms showed pulmonary nodules. His second, negative, serum specimen was obtained one month after the start of anti-Nocardia therapy, and a chest x-ray obtained eight days after this second specimen showed clear improvement. The second patient had seropositive results four months after onset of her undiagnosed pulmonary disease, one and a half months prior to her death. She had not been treated for nocardiosis. The patient who had seronegative results was tested only once, eight days after onset (10 days before she died). Typing of the Isolates: Isolates available for study from patient specimens, and from dust and environmental sampling sites and air sampling plates obtained at the time of closure of the ICA, were examined. All four of the dust isolates obtained from bed areas came from
Volume 71
LABORATORY
TABLE
I
STUDY OF NOCARDIA
OUTBREAK-STEVENS
ET AL.
Serologic Results
Daysafter Onset Proved Cases 1. Pulmonary nocardiosis 2. Pulmonary nocardiosis 3. Pulmonary and renal nocardiosis
4. Pulmonary and cerebral nocardiosis 5. Pulmonary and hepatic nocardiosis; nocardemia 6. Pulmonary nocardiosis 7. Nocardial wound infection Suspected Cases (see text) Patient DC Patient KS Patient FL Staff 26 serums 1 serum Unaffected Patients 33 serums
CF Tiler CombinedAntigen
typeIII
0
0
I:4 0 1:8 0 0 0 0 0
1:4 0 0 0 0 0 0 0
1:8
0
+I68 +122
AC 0
AC 0
-4 +36 + approx. 120 +8
1:4 0 1:8 0
1:8 0 1:8 0
+55 +31 +66 +40 +61 -l-75 +81 + approx. 3 yr + approx. 3 yr (4 days later) +29
0
0
AC
AC
0
0
NOTE: CF = complement fixing antibody; AC = anticomplementary; Type Ill = antigen of prototype strain of serogroup Ill; 0 = <1:2.
the vicinity of one of the six beds in the ICA; this bed had been used by five of the seven patients with bacteriologically-proved outbreak-associated cases at various times (the other two patients had used one other bed in the unit at various times). Also studied were environmental isolates obtained outside the ICA, which included one obtained by air sampling in the bacteriology laboratory and one in the corridor outside the ICA, and contaminants that appeared on three Petri dishes. In addition, the only positive environmental sample in the hospital obtained after closure and disinfection of the ICA, a single colony on an air sample plate in the bacteriology laboratory five months after closure, was studied. The results of the typing studies are shown in Table II. This table highlights the tests that detected differences in the 26 strains studied. As Table II indicates, all eight patient isolates and all 12 environmental isolates from the ICA itself at the time of its closure were identical in 30 of 30 tests applied to each of these 20 strains. The pattern of 30 reactions obtained enabled characterization of “the epidemic strain.” This pattern would place the isolates in N. asteroides subgroup B of Schaal [ 13,171. Of the environmental isolates obtained outside the ICA, two contaminants that appeared on different Petri dishes (Table II) had a pattern identical to that of the epidemic strain. This is perhaps not surprising, because of the number of isolates being processed and passaged
in the laboratory at that time. Another plate contaminant was probably the epidemic strain, as it differed only by f reaction in one assay in which the epidemic strain was negative. The other environmental isolates were even more clearly not related. The isolate from the air in the bacteriology lab was not the epidemic strain. It could be distinguished by its antigenic type, inability to survive 60°C and inability to utilize ethanol as a carbon source (the sorbitol test was also 4~). The other isolate, from air in the corridor, was also clearly different; it differed in antigenic type and was dextrose-negative. This isolate also was suspected of being unrelated to the epidemic on the basis of a different antibiogram
[41. Of special interest was the only isolate obtained after the ICA was reopened. It was probably the epidemic strain, in that it had the identical pattern in 29 of 29 tests, although there was a f mannose reaction. The finding that the antigenic typing assay grouped the patient isolates and ICA environmental isolates led us to further testing to be certain antigen pattern III is not merely the most common type in association with human disease. Data derived from isolates collected from human cases in the United States suggested that this was not the case. Of 26 isolates typed, only four were of antigen pattern type Ill [ 181. We determined the pattern in the United Kingdom in the present study by obtaining isolates from eight cases
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931
Acid Production
015 o/2 o/2 or3
Q/2 f f
5J5 212 2J2 3/3
f 2J2 + +
015 o/2 o/2 o/3
f o/2 i-
o/5 OJ2 o/2 OJ3
O/8
Sorbitol
2J2 + + +
515 2/2 2/2 3J3
818
for 4 hr
Survival at 60°C
+
Of2
o/5 O/2 o/2 OJ3
O/8
-
012 +
OJ3
Of2
015 a/2
O/8
Tyrosine
Decomposition
Casein
2/2 -INT +
5J5 212 2J2 313
S/8
Source
Utilization of Ethanol as Carbon
II-IV Ill (2J2) Ill 1V III
III (5/5) 111 (2/2) Ill (2/2) III (3/3)
III (B/8)
AntitentcType
’ 26J26 isolates failed to decompose xanthine; 25J25 tested decomposed urea, produced acid from rhamnose, grew on Sabouraud’s agar or Middlebrook 7HlO agar with oleic acid-dextrose-catalase and were able to utilize 1,Zpropylene glycol, 2,3-butylene glycol or propionate as carbon sources. 25J25 isolates tested failed to decompose hypoxanthine or starch and to produce acid from sucrose, galactose, maltose, mannitol or inositol, and were unable to utilize sodium citrate, quinic acid, gtuconate, protine or valine as carbon sources. t Specimens from 7 Patients included 2 sputum isolates and 1 each of bronchial washing, throat swab, and mouth swab from 5 patients with pulmonary nocardiosis, 1 from kidney specimen of patient who also contributed a sputum isolate, 1 from nephrostomy urine, and 1 from wound pus. t NO. positiveJtotal tested. Where isolate with unique pattern is represented alone, + = positive, = negative, f = equivocal or variable and NT = not tested. 9 4 from vicinity of 1 bed; 1 from dialysis machine area. It 1 from bed area: 1 from examining room.
012 + -
-
018
Marmoss
8i8
Dextrose
of Isolates*
O/8?
Arabirtose
Biochemical, Physical and Immunologic Characterization
Patient specimens (8)t Environmental isolates, ICA, June 1979 (total, 12) Dust isolates (5)9 Floor swabs (2) Air samples (2$ Sluice area air samples (3) Environmental isolates, non-ICA, 1979 (total, 5) Bacteriology lab air Plate contaminants, lab (2) Plate contaminant, lab Corridor air Environmental isolate, non-ICA, November 1979
TABLE II
LABORATORY
STUDY OF NOCARDIA
of human nocardiosis in the United Kingdom. These eight cases were unrelated to St. Paul’s Hospital. Two of the isolates were type III, two were type IV, two were type II-IV, one was type I and one was type Ill-IV. This distribution is similar to that in human nocardiosis in the United States. These results suggest that the grouping of the epidemic-associated strains using antigen typing truly represents identity of these strains. Type III is not a common human-associated type, in either the United States or the United Kingdom. In addition to the test just detailed, 25 of the strains were characterized by chromatographic methods for their fatty acid profile. They were indistinguishable by this method. The pattern derived is shown in Table III. Of five prototype strains assayed concurrently, the isolates had a profile virtually identical to that of N. asteroides strain KN852, which would place them in the type Ill pattern of Kurup [ 151. This grouping also would place the 25 strains tested in the subgroup B of Schaal [13,17].
TABLE Ill
COMMENTS
See Beaman [ 141. t Mean f standard deviation.
OUTBREAK-STEVENS
Fatty Acid Profile of 25 Nocardia from St. Paul’s Hospital
Fatty Acid (carbon number)’ Cl4
C 15 C16:1 Cl6
Cl7 c18:l Cll3
10CHsC,B* Cl9 Go:4
Go: Go
1
c22:1 c22 c24:l
C25:i c25 C26 C26:1
Other
ET AL.
Isolates
% Total Fatly Acidst 0.4 f
l
In the present study, all the patient N. asteroides isolates and all the environmental isolates from within the affected unit were shown to be identical, by all the criteria evaluated. Conversely, all the isolates studied that were not of identical type were obtained outside the unit (some isolated outside the ICA were of the “epidemic strain” type). These data provide strong evidence for the contagiousness of nocardiosis in this setting. The method of spread, whether person-to-person or person-to-environment-to-person or a mixture of these modes, is uncertain. The presence of the same type of N. asteroides in the unit’s air and dust and the predominance of pulmonary infections in the patients suggest an airborne route of spread. Spread via contaminated articles, hands or food cannot, however, be excluded. The serologic studies suggested at least two additional cases (Patients KS and DC) could be considered as related to the outbreak, bringing the total to nine cases. Failure to isolate Nocardia from sputum in patients who are diagnosed by invasive procedures is not uncommon [ 2,181. We cannot exclude tuberculosis as a possible alternate diagnosis in these two patients (although no smear or culture evidence of this was found); however, Patient DC was given therapy appropriate for nocardiosis and responded. Since he was not a patient in the ICA at the time of the outbreak, these results suggest that cases were occurring elsewhere in the hospital. Forty patients were cared for in the long-term care area between the time of the initial positive urine culture in the ICA and the closure of the ICA, with no bacteriologically confirmed cases of nocardiosis in the long-term care area. Infection in Patient DC could, of course, have been nocardial but
t Tuberculostearic
acid.
unrelated to the outbreak, or he might have been exposed in the ICA on a transient visit or exposed to a patient or article contaminated in it. That brief exposure could be sufficient for infection is suggested by the observation that two of the patients with proved cases were exposed to the ICA for less than four weeks before its closure. Another suspected case, in Patient FL, remains undiagnosed. Her fatal illness was a very brief one, which could have frustrated attempts to make a microbiologic or serologic diagnosis of nocardiosis. This experience suggests the potential utility of the CF test in the work-up of nocardiosis. This is especially important, since even when Nocardia is isolated from clinical specimens, it may take up to 28 days to produce visible growth [ 191. The serologic assay detected only three of the five outbreak-related cases that could be assayed, compared with a previously reported sensitivity of 81 percent [5]. However, the two patients with “false-negative” results only had serum samples available approximately two and four months after onset and after successful response to therapy. Seropositive results might have been detected in these cases if earlier samples had been obtained. Seroconversion to negative in late specimens from the patients who had seropositive results early in their illness also suggests that the CF test would need to be applied to serums obtained early in the illness, and probably to specimens obtained at several intervals from a patient with a suspected case. These observations are also the first indication that the CF test could be used to follow re-
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LABORATORY STUDY OF NOCARDIA OUTBREAK-STEVENS
sponse
to therapy.
We showed
reversion
ET AL.
to negative
in association with response to therapy (e.g., Cases 2 and 3 and Patient DC), whereas antibody was present late in unresolved disease in which therapy had not been given (Patient KS). The titers in the present study are similar to those reported previously [5]. The use of the specific type antigen of the “epidemic strain” did not increase the sensitivity of the serologic assay. Finally, the serologic assay contributed some evidence against subclinical infections occurring alongside the recognized cases, and against a carrier being responsible for transmission to patients. The typing studies done with the isolates provide some data of interest to future epidemiologic studies, although there were few isolates not of the same pattern. No single test differentiated the epidemic strain type from these few isolates, although the immunologic typing method did give a clear demarcation of the non-epidemic types, including duplicates added as internal controls, without equivocal or variable results as occurs with biochemical tests. The antibiogram [4], although it did differentiate one isolate from the others (the corridor isolate, confirmed in this study), also failed to detect at least one other that could be differentiated in extended testing. The survey data from the United Kingdom and the United States suggest the utility of including immunologic typing in similar future studies. A need for more clearly defined subgroups and for an improved nocardia taxonomy was indicated earlier [4],
and the series of studies reported herein meets that need in part. The isolate obtained after cleaning and closure, although not recovered in the affected unit and not recovered in subsequent attempts, may have been the epidemic strain type. It suggests this type may still be present in some environmental haven, or may be reintroduced into the hospital. The epidemic strain was shown to belong to N. asteroides subgroup B of Schaal [ 13,171. Of note, this group is reported to be more virulent than subgroup A
[171. At the present time, authorities [20] do not recommend isolation of nocardiosis patients. In any hospital environment with immunocompromised patients, this recommendation needs to be reevaluated in light of our experience. ACKNOWLEDGMENT These studies were initiated when the senior author was supported by a Wellcome Fund grant for studies on nocardiosis at the Mycology Reference Laboratory, London School of Hygiene and Tropical Medicine, on sabbatical leave from Stanford University. We thank Dr. Donald Mackenzie, Director of the Laboratory, for the United Kingdom Nocardia isolates and Mr. Rod Fichtner, National Animal Disease Center, and Mr. Steve M. States, University of California, Davis, for technical assistance.
REFERENCES 1.
2.
3.
4.
5.
a. 7.
a.
9.
934
Beaman BL. Burnside J, Edwards B, Causey W: Nocardial infections in the United States, 1972-1974. J Infect Dis 1976; 124: 266-269. Krick JA, Stinson EB, Remington JS: Nocardia infection in heart transplant patients. Ann Intern Med 1975; 32: la26. Cox F, Hughes WT: Contagiousness and other aspects of nocardiosis in the compromised host. Pediatrics 1975; 55: 135-138. Houang ET, Lovett IS, Thompson FD. Harrison AR, Joekes AM. Goodfellow M: Nocardia asteroides infection-a transmissible disease. J Hosp Infect 1980; 1: 31-40. Shainhouse JZ, Pier AC, Stevens DA: Complement fixation antibody test for human nocardiosis. J Clin Microbial 1978; 8: 516-519. Pier AC, Fichtner RE: Serologic typing of Nocardia asteroides by immunodiffusion. Am Rev Respir Dis 1971; 103: 698-707. Pan American Health Organization. Manual of standardized serodiagnostic procedures for systemic mycoses. Part II: complement fixation tests. Dept. of Research Development and Coordination, Pan American Health Organization, 1972. Goodfellow M, Schaal KP: Identificationmethods for Nocardia, Actinomadura. and Rhodococcus. In: Skinner FW, Lovelock DW, eds. Identification methods for microbiologists, 2nd ed. London: Academic Press, 1979; 261-276. Gordon MA: Aerobic pathogenic Actinomycetaceae. In: Lennette EH, ed. Manual of clinical microbiology, 3rd ed. Washington: American Society of Microbiology, 1980;
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10. 11.
12. 13. 14.
15. 16. 17.
18.
19.
20.
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180-194. Gordon RE, Mihm JM: A comparison of four species of mycobacteria. J Gen Microbial 1959; 21: 736-748. Gordon RE, Mihm JM: Identification of Nocardia caviae (Erickson) Nov. Comb. Ann NY Acad Sci 1962; 98: 628636. Gordon RE, Mihm JM: Type species of the genus Nocardia. J Gen Microbial 1962; 27: l-10. Schaal KP: Zur mikrobiologischen Diagnostik der Nocardiose. Zentralbl Bakteriol 1972; 220: 242-246. Beaman BL: Structural and biochemical alterations of Nocardia asteroides cell walls during its growth cycle. J Bacterial 1975; 123: 1235-1253. Kurup PV, Schmitt JA: Numerical taxonomy of Nocardia. Can J Microbial 1973; 19: 1035-1048. Lovett IS, Houang ET, Burge S, et al. An outbreak of Nocardia asteroides in a renal transplant unit. Q J Med (in press). Pulverer G, Schaal KP: Pathogenicity and medical importance of aerobic and anaerobic actinomycetes. Zentralbl Bakteriol 1978; suppl6: 417-424. Pier AC, Fichtner RE: Distribution of serotypes of Nocardia asteroides from animal, human and environmental sources. J Clin Microbial 1981; 13: 548-553. Palmer DL, Harvey RL, Wheeler JK: Diagnostic and therapeutic considerations in Nocardia asteroides infections. Medicine (Baltimore) 1974; 53: 391-401. Dixon RE, Brachman PS. Bennett JV: Isolation techniques for use in hospitals. 2nd ed. Atlanta: Center for Disease Control, US Dept. of Health, Education and Welfare, 1975.
DAVID
A. STEVENS,
Hosts
Seven patients in a renal unit were proved to have nocardiosis in an interval of nine months. Six of these patients had received renal transplants. Serologic investigation suggested that two additional cases of undiagnosed pulmonary disease were also nocardial, and that there were no subclinical cases in patients or staff. Clinicalserologic correlations indicate that serologic evaluation may be a useful adjunct in diagnosis of nocardiosis, if used early and repeatedly, and to follow response to therapy. Epidemiologic investigations yielded cultures of Nocardia asteroides from air and dust inside the unit and elsewhere in the hospital. Biochemical, metabolic, physical and immunologic characterization of the isolates indicated that those from patients and those from the unit environment were identical, whereas some from outside the unit could be differentiated from these. The “epidemk strain” had type Ill antigen, which surveys indicated is not the most common type in human nocardiosis (it occurs in association with a minority of human cases). The isolates were of subgroup B, which has been associated with virulence. The characterization methods employed could be useful in studies of nocardial epidemiology. The laboratory studies indicate epidemic spread within the unit of a single organism, and current epidemiologic guidelines, which do not recommend respiratory isolation of cases of pulmonary nocardiosis, may need reconsideration particularly when there are immunocompromised hosts in the environment.
M.D.
San Jose and Stanford. California ALLAN
C. PIER.
Ph.D.,
D.V.M.
Ames, lowa BLAINE
L. BEAMAN,
Ph.D.
Davis, California PIUS
A.
MOROZUMI,
M.D.
San Jose and Stanford IAN
S. LOVETT,
ELIZABETH
M.B.,
T. HOUANG,
B.S.,
M.R.C.P.
Ph.D.
London, England
From the Division of Infectious Diseases, Departments of Medicine, Santa Clara Valley Medical Center and Stanford University, San Jose and Stanford. California; tie Institute for Medical Research, San Jose, California; the Bacterial and Mycokqical Research Laboratory, National Animal Disease Center, United States Department of Agriculture, Ames, Iowa; the Department of Medical Microbiology, University of California Medical School, Davis, California; St. Peter’s Group of Hospitals and the Institute of Urology, London, England: and the Department of Medical Microbiology, London Hospital, London, England. Reprint requests should be addressed to Dr. David A. Stevens, Department of Medicine, Santa Clara Valley Medical Center, 751 South Bascom Avenue, San Jose, CA 95 128. Manuscript accepted July 16. 1981.
928
December
1981
The American
Journal
Nocardiae are bacteria found in soil and not part of normal human flora. The incidence of human infections appears to be increasing, and human nocardiosis is particularly a problem of immunocompromised hosts [ 11.Although man-t&man transmission of infection has not been documented, the incidence of infection in some subgroups of immunocompromised hosts is particularly high [2], and clustering of a few cases in immunocompromised patients was earlier suggested to represent possible contagion [3]. A recent outbreak of N. asteroides infections, involving renal transplantation patients, in a London hospital renal unit [ 41 provided the strongest argument for contagion to date. This report concerns the application of several laboratory tools to the study of the epidemiology of that outbreak. MATERIALS AND METHODS Serologic Studles. A complement-fixation antibody (CF) test, as previously described [5], was utilized to test the serum specimens. The serum samples were not collected prospectively but were salvaged from other assays or collected from patients surviving at the time the unit was closed. The serum samples were frozen until assayed. In brief, this CF test uses an extracellular antigen (culture filtrate) prepared by pooling filtrates from four N. asteroides
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LABORATORY
strains [5]. Each strain is a prototype strain for the production of an extracellular antigen, termed I, II, ill or IV, detectable by immunization of experimental animals with culture filtrate and assay of their serums by immunodiffusion [6]. In immunodiffusion, a single, immunologically unique, band is detected with these antiserums and filtrates (no reactions of identity or partial identity with the bands of the other three strains). Virtually all wild strains of N. asteroides involved in human or animal infection produce one, and in some cases two or more, of these four antigens [6]. Previous studies have shown that CF titers of 1:4 or more are reproducible and specific for nocardial infection [5] and are considered positive results. Only serums from patients with mycobacterial infections also gave positive results in this assay, as has been reported with other serologic assays for nocardiosis [5]. Recent experience has confirmed the earlier work [5], in that additional serum samples submitted from patients with other infections do not give positive CF results (J. Z. Shainhouse, P. A. Morozumi and D. A. Stevens, unpublished results). Appropriate positive and negative controls are included in each assay [5]. The serum samples were coded prior to assay. The positive control reference serum in each assay is a rabbit antiserum prepared against the antigen pool. Monospecific rabbit antiserum against each of the four prototype antigens were also available, and this enabled establishment of a CF assay using the type Ill antigen only. The optimal reagent dilutions for this assay were determined by block titration as previously described [7]. Immunologic Typing of Isolates. With the use of the monospecific rabbit antiserums described, isolates obtained during the outbreak were typed with respect to their production of extracellular antigen(s) [6]. This was done by growing the isolates in liquid medium, and assaying the culture filtrates in immunodiffusion as previously described
STUDY OF NOCARDIA
OUTBREAK-STEVENS
ET AL.
in the unit, and the same amount elsewhere in the hospital. Media used in sampling procedures included blood agar, Sabouraud’s agar, brain-heart infusion agar and LowensteinJensen medium, with appropriate inhibitors [4]. These same media were used in culturing patient specimens. From these sources, 12 of 13 Nocardia colonies obtained inside the unit and six of seven obtained elsewhere in the hospital were available for study. One colony obtained in the affiliated hospitals was not available for study. Eight of 14 isolates from patients were available for study; the six isofates not studied were from other body sites in patients contributing at least one isolate for study. Identification and Analysis of N. asteroides Strains. Colonies suspected as possibly being Nocardia colonies were initially identified [6] and reconfirmed [9] by standard methods. All the isolates described in the present study were identified as N. asteroides. Subsequent characterization by biochemical, physical and metabolic tests was performed by previously described methods [ 6,i O-l 31. Some key tests that differentiated isolates were repeated independently in one or two different laboratories. Colonial pigment production is considered a variable phenotypic characteristic, not reliable for differentiating strains, and was not used for this purpose. Fatty acid chromatography, using whole cells grown on brain-heart infusion agar for 418hours and lysed with hydrochloric acid and methanol, was performed as previously described [ 141. Comparison was made with prototype strains of the typing system of Kurup and Schmitt [ 151, a biochemical system of aping N. asteroides. strains KN 827, KN 845, KN 850, KN 851 and KN 852. All the tests on the isolates described were performed with numerically coded samples. Some isolates were included in duplicate as an internal check on reproducibility. RESULTS
1’31. Prior to and just after Isolation of N. asteroides Strains. closure and cleaning of the unit, environmental sampling was performed throughout the hospital as detailed elsewhere [4]. In brief, air was cultured using agar plates exposed overnight (“settle plates”) or a slit air sampler filtering 3,500 liters of air for 10 minutes onto agar plates. Dust obtained from bed, stores, dialysis machine and sluice areas and ventifation grills was wetted and cultured on solid media. Swabs obtained from environmental surfaces were handled similarly. Tap water was filtered, and the filters were cultured on solid media. During the months of the outbreak, N. asteroides colonies were identified on culture plates of specimens, from three patients, obtained in affiliated hospitals; these cultures had been processed in the St. Paul’s Hospital laboratory. These isolates were deemed inconsistent with a diagnosis of nocardiosis, given these patients’ clinical courses and repeated prior and subsequent microbiologic studies. The patients had no connection with St. Paul’s Hospital. These isolates were therefore considered “plate contaminants.” In total, 60,000 liters of air in the affected unit were examined, as well as 30 dygt samples and 27 settle plates. Elsewhere in the hospital, 49,000 liters of air, 32 dust specimens and 45 settle plates were examined. As a control, 14,000 liters of air in affiliated hospitals were sampled. After closure of the unit, 7,000 liters of air were sampled monthly
The inpatient area of the Chronology of the Epidemic. Renal Unit of St. Paul’s Hospital in 1978 and 1979 was divided into two areas across a corridor from each other-the intensive care area (ICA) for transplantation patients and short-term dialysis (six inpatient beds, a dialysis room, and rooms for ancillary services), and the long-term care area with inpatient and dialysis beds for long-term dialysis patients. In July 1978, a patient with chronic renal failure was admitted to the ICA for dialysis and treatment of recurrent urinary tract infections. Her urine was collected in a nephrostomy bag, which was emptied into a sluice in the ward area. In October, cultures of her urine began to grow a “diphtheroid.” She was asymptomatic and was not treated: on repeated admissions, her urine continued to grow this organlsm. In March 1979, an ICA patient who had received a renal transplant one month earlier had an undiagnosed febrile illness with cough and pleurisy, and in April, a wound infection. N. asteroides was cultured from the pus. At that time, the urine isolate from the first patient was re-examined and identified as N. asteroides. The unit had not recently recorded a Nocardia infection, except for a patient with a nocardial
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LABORATORY
STUDY OF NOCARDIA
OUTBREAK-STEVENS
ET AL.
pulmonary infection and brain abscess in 1976 that were successfully treated. In May 1979. pulmonary nocardiosis proved by culture developed in three ICA transplantation patients. One of these was also shown to have hepatic nocardiosis at autopsy. In early June, the ICA was closed, and all the patients mentioned were discharged or transferred to another hospital. Three other transplantation patients were transferred to another unit in St. Paul’s. Subsequently, bacteriologically proved pulmonary nocardiosis developed in two of these, in late June and early July (one was also shown to have nocardiosis in her transplanted kidney). The third (Patient FL) had chest pain and pulmonary infiltrates in late July and died in mid-August without a diagnosis for this complication: no postmortem examination was done. After closure of the ICA, it was cleaned, sprayed with 1 percent hypochlorite, fumigated with formaldehyde and then reopened. Thus, in 1979, prior to its closure, culture-proved nocardiosis developed in six of 50 patients admitted to the ICA (five pulmonary). All six had received renal transplants in that interval, and there had only been nine transplant patients admitted during that period. In addition, the patient with urinary nocardiosis who did not undergo transplantation, whose disease is presumed to have started in 1978, completes the group of seven culture-proved cases considered to represent a single outbreak. Some epidemiologic features [4] and clinical features [ 161 of this epidemic are presented in more detail elsewhere. It is worthy of emphasis that work-up of all cases of suspected nocardiosis described in this report included cultures of sputum, other body fluids and tissue, as pertinent, for mycobacteria, other bacteria and fungi, as well. Since the reopening of the ICA, there have been no subsequent cases of nocardiosis diagnosed in it. Serologic Investigations. The serologic study included the confirmed cases just discussed, with exception of the index case; no serum was available from this patient. In addition, the confirmed case from 1976, the case of Patient FL described earlier and two other cases of special interest were also studied. One of these cases was in a patient (KS) who had received a renal transplant in December 1978, after the nocardiuria of the index case had presumably begun but before recognition of the epidemic. In March 1979, KS had an undiagnosed respiratory illness with pulmonary infiltrates, and she died in August without a postmortem examination. The other case was in Patient DC, who had received a transplant in 1976 that was rejected. He had not been a patient in the ICA since that time, being in a long-term dialysis program in the long-term care area. In mid-July 1979, fever and pleurisy developed, and pulmonary nodules were noted on x-ray. Although the diagnostic work-up was unrewarding, he began
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promptly to receive amoxicillin and erythromycin to include coverage for possible nocardiosis. His x-rays showed improvement one month later. In addition, serums were obtained after the ICA closed from 27 physicians, nurses and aides who cared for the ICA patients. Thirty-two unaffected patients also provided serum samples: one of these provided two samples a month apart. The results are shown in Table 1. Patients in three of five cases associated with the epidemic were shown to have complement-fixing antibody. Two of these seropositive immunosuppressed patients had positive findings only in the first serum sample obtained from them. The second, negative, serum sample was obtained two months after the beginning of therapy that had produced progressive improvement in one (Case 2) of these two cases. In the other, the second serum sample was obtained 50 days after the beginning of a therapeutic regimen that had produced clear clinical improvement seven days after its initiation (Case 3). The two patients who had seronegative results had serum available for testing only at 55 and 122 days after onset of their nocardial disease. Therapy had started 48 days prior to the serum collection in the first of these two patients, and her disease had cleared in that interval (normal chest x-ray six days prior to serum sample). In the second of these, therapy had started three months prior to the serum collection, with progressive improvement of her disease. One patient’s serum was anticomplementary and could not be assayed. The patient with nocardiosis in the ICA three years earlier, not associated with the outbreak, had seronegative results. Study of the three patients with suspected but not bacteriologically confirmed nocardiosis gave very interesting results. A seropositive specimen was obtained from one patient four days before the development of fever and pleuritic pain. It is highly likely his disease was incubating at the time of specimen collection, because a chest x-ray obtained six days after onset of symptoms showed pulmonary nodules. His second, negative, serum specimen was obtained one month after the start of anti-Nocardia therapy, and a chest x-ray obtained eight days after this second specimen showed clear improvement. The second patient had seropositive results four months after onset of her undiagnosed pulmonary disease, one and a half months prior to her death. She had not been treated for nocardiosis. The patient who had seronegative results was tested only once, eight days after onset (10 days before she died). Typing of the Isolates: Isolates available for study from patient specimens, and from dust and environmental sampling sites and air sampling plates obtained at the time of closure of the ICA, were examined. All four of the dust isolates obtained from bed areas came from
Volume 71
LABORATORY
TABLE
I
STUDY OF NOCARDIA
OUTBREAK-STEVENS
ET AL.
Serologic Results
Daysafter Onset Proved Cases 1. Pulmonary nocardiosis 2. Pulmonary nocardiosis 3. Pulmonary and renal nocardiosis
4. Pulmonary and cerebral nocardiosis 5. Pulmonary and hepatic nocardiosis; nocardemia 6. Pulmonary nocardiosis 7. Nocardial wound infection Suspected Cases (see text) Patient DC Patient KS Patient FL Staff 26 serums 1 serum Unaffected Patients 33 serums
CF Tiler CombinedAntigen
typeIII
0
0
I:4 0 1:8 0 0 0 0 0
1:4 0 0 0 0 0 0 0
1:8
0
+I68 +122
AC 0
AC 0
-4 +36 + approx. 120 +8
1:4 0 1:8 0
1:8 0 1:8 0
+55 +31 +66 +40 +61 -l-75 +81 + approx. 3 yr + approx. 3 yr (4 days later) +29
0
0
AC
AC
0
0
NOTE: CF = complement fixing antibody; AC = anticomplementary; Type Ill = antigen of prototype strain of serogroup Ill; 0 = <1:2.
the vicinity of one of the six beds in the ICA; this bed had been used by five of the seven patients with bacteriologically-proved outbreak-associated cases at various times (the other two patients had used one other bed in the unit at various times). Also studied were environmental isolates obtained outside the ICA, which included one obtained by air sampling in the bacteriology laboratory and one in the corridor outside the ICA, and contaminants that appeared on three Petri dishes. In addition, the only positive environmental sample in the hospital obtained after closure and disinfection of the ICA, a single colony on an air sample plate in the bacteriology laboratory five months after closure, was studied. The results of the typing studies are shown in Table II. This table highlights the tests that detected differences in the 26 strains studied. As Table II indicates, all eight patient isolates and all 12 environmental isolates from the ICA itself at the time of its closure were identical in 30 of 30 tests applied to each of these 20 strains. The pattern of 30 reactions obtained enabled characterization of “the epidemic strain.” This pattern would place the isolates in N. asteroides subgroup B of Schaal [ 13,171. Of the environmental isolates obtained outside the ICA, two contaminants that appeared on different Petri dishes (Table II) had a pattern identical to that of the epidemic strain. This is perhaps not surprising, because of the number of isolates being processed and passaged
in the laboratory at that time. Another plate contaminant was probably the epidemic strain, as it differed only by f reaction in one assay in which the epidemic strain was negative. The other environmental isolates were even more clearly not related. The isolate from the air in the bacteriology lab was not the epidemic strain. It could be distinguished by its antigenic type, inability to survive 60°C and inability to utilize ethanol as a carbon source (the sorbitol test was also 4~). The other isolate, from air in the corridor, was also clearly different; it differed in antigenic type and was dextrose-negative. This isolate also was suspected of being unrelated to the epidemic on the basis of a different antibiogram
[41. Of special interest was the only isolate obtained after the ICA was reopened. It was probably the epidemic strain, in that it had the identical pattern in 29 of 29 tests, although there was a f mannose reaction. The finding that the antigenic typing assay grouped the patient isolates and ICA environmental isolates led us to further testing to be certain antigen pattern III is not merely the most common type in association with human disease. Data derived from isolates collected from human cases in the United States suggested that this was not the case. Of 26 isolates typed, only four were of antigen pattern type Ill [ 181. We determined the pattern in the United Kingdom in the present study by obtaining isolates from eight cases
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931
Acid Production
015 o/2 o/2 or3
Q/2 f f
5J5 212 2J2 3/3
f 2J2 + +
015 o/2 o/2 o/3
f o/2 i-
o/5 OJ2 o/2 OJ3
O/8
Sorbitol
2J2 + + +
515 2/2 2/2 3J3
818
for 4 hr
Survival at 60°C
+
Of2
o/5 O/2 o/2 OJ3
O/8
-
012 +
OJ3
Of2
015 a/2
O/8
Tyrosine
Decomposition
Casein
2/2 -INT +
5J5 212 2J2 313
S/8
Source
Utilization of Ethanol as Carbon
II-IV Ill (2J2) Ill 1V III
III (5/5) 111 (2/2) Ill (2/2) III (3/3)
III (B/8)
AntitentcType
’ 26J26 isolates failed to decompose xanthine; 25J25 tested decomposed urea, produced acid from rhamnose, grew on Sabouraud’s agar or Middlebrook 7HlO agar with oleic acid-dextrose-catalase and were able to utilize 1,Zpropylene glycol, 2,3-butylene glycol or propionate as carbon sources. 25J25 isolates tested failed to decompose hypoxanthine or starch and to produce acid from sucrose, galactose, maltose, mannitol or inositol, and were unable to utilize sodium citrate, quinic acid, gtuconate, protine or valine as carbon sources. t Specimens from 7 Patients included 2 sputum isolates and 1 each of bronchial washing, throat swab, and mouth swab from 5 patients with pulmonary nocardiosis, 1 from kidney specimen of patient who also contributed a sputum isolate, 1 from nephrostomy urine, and 1 from wound pus. t NO. positiveJtotal tested. Where isolate with unique pattern is represented alone, + = positive, = negative, f = equivocal or variable and NT = not tested. 9 4 from vicinity of 1 bed; 1 from dialysis machine area. It 1 from bed area: 1 from examining room.
012 + -
-
018
Marmoss
8i8
Dextrose
of Isolates*
O/8?
Arabirtose
Biochemical, Physical and Immunologic Characterization
Patient specimens (8)t Environmental isolates, ICA, June 1979 (total, 12) Dust isolates (5)9 Floor swabs (2) Air samples (2$ Sluice area air samples (3) Environmental isolates, non-ICA, 1979 (total, 5) Bacteriology lab air Plate contaminants, lab (2) Plate contaminant, lab Corridor air Environmental isolate, non-ICA, November 1979
TABLE II
LABORATORY
STUDY OF NOCARDIA
of human nocardiosis in the United Kingdom. These eight cases were unrelated to St. Paul’s Hospital. Two of the isolates were type III, two were type IV, two were type II-IV, one was type I and one was type Ill-IV. This distribution is similar to that in human nocardiosis in the United States. These results suggest that the grouping of the epidemic-associated strains using antigen typing truly represents identity of these strains. Type III is not a common human-associated type, in either the United States or the United Kingdom. In addition to the test just detailed, 25 of the strains were characterized by chromatographic methods for their fatty acid profile. They were indistinguishable by this method. The pattern derived is shown in Table III. Of five prototype strains assayed concurrently, the isolates had a profile virtually identical to that of N. asteroides strain KN852, which would place them in the type Ill pattern of Kurup [ 151. This grouping also would place the 25 strains tested in the subgroup B of Schaal [13,17].
TABLE Ill
COMMENTS
See Beaman [ 141. t Mean f standard deviation.
OUTBREAK-STEVENS
Fatty Acid Profile of 25 Nocardia from St. Paul’s Hospital
Fatty Acid (carbon number)’ Cl4
C 15 C16:1 Cl6
Cl7 c18:l Cll3
10CHsC,B* Cl9 Go:4
Go: Go
1
c22:1 c22 c24:l
C25:i c25 C26 C26:1
Other
ET AL.
Isolates
% Total Fatly Acidst 0.4 f
l
In the present study, all the patient N. asteroides isolates and all the environmental isolates from within the affected unit were shown to be identical, by all the criteria evaluated. Conversely, all the isolates studied that were not of identical type were obtained outside the unit (some isolated outside the ICA were of the “epidemic strain” type). These data provide strong evidence for the contagiousness of nocardiosis in this setting. The method of spread, whether person-to-person or person-to-environment-to-person or a mixture of these modes, is uncertain. The presence of the same type of N. asteroides in the unit’s air and dust and the predominance of pulmonary infections in the patients suggest an airborne route of spread. Spread via contaminated articles, hands or food cannot, however, be excluded. The serologic studies suggested at least two additional cases (Patients KS and DC) could be considered as related to the outbreak, bringing the total to nine cases. Failure to isolate Nocardia from sputum in patients who are diagnosed by invasive procedures is not uncommon [ 2,181. We cannot exclude tuberculosis as a possible alternate diagnosis in these two patients (although no smear or culture evidence of this was found); however, Patient DC was given therapy appropriate for nocardiosis and responded. Since he was not a patient in the ICA at the time of the outbreak, these results suggest that cases were occurring elsewhere in the hospital. Forty patients were cared for in the long-term care area between the time of the initial positive urine culture in the ICA and the closure of the ICA, with no bacteriologically confirmed cases of nocardiosis in the long-term care area. Infection in Patient DC could, of course, have been nocardial but
t Tuberculostearic
acid.
unrelated to the outbreak, or he might have been exposed in the ICA on a transient visit or exposed to a patient or article contaminated in it. That brief exposure could be sufficient for infection is suggested by the observation that two of the patients with proved cases were exposed to the ICA for less than four weeks before its closure. Another suspected case, in Patient FL, remains undiagnosed. Her fatal illness was a very brief one, which could have frustrated attempts to make a microbiologic or serologic diagnosis of nocardiosis. This experience suggests the potential utility of the CF test in the work-up of nocardiosis. This is especially important, since even when Nocardia is isolated from clinical specimens, it may take up to 28 days to produce visible growth [ 191. The serologic assay detected only three of the five outbreak-related cases that could be assayed, compared with a previously reported sensitivity of 81 percent [5]. However, the two patients with “false-negative” results only had serum samples available approximately two and four months after onset and after successful response to therapy. Seropositive results might have been detected in these cases if earlier samples had been obtained. Seroconversion to negative in late specimens from the patients who had seropositive results early in their illness also suggests that the CF test would need to be applied to serums obtained early in the illness, and probably to specimens obtained at several intervals from a patient with a suspected case. These observations are also the first indication that the CF test could be used to follow re-
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sponse
to therapy.
We showed
reversion
ET AL.
to negative
in association with response to therapy (e.g., Cases 2 and 3 and Patient DC), whereas antibody was present late in unresolved disease in which therapy had not been given (Patient KS). The titers in the present study are similar to those reported previously [5]. The use of the specific type antigen of the “epidemic strain” did not increase the sensitivity of the serologic assay. Finally, the serologic assay contributed some evidence against subclinical infections occurring alongside the recognized cases, and against a carrier being responsible for transmission to patients. The typing studies done with the isolates provide some data of interest to future epidemiologic studies, although there were few isolates not of the same pattern. No single test differentiated the epidemic strain type from these few isolates, although the immunologic typing method did give a clear demarcation of the non-epidemic types, including duplicates added as internal controls, without equivocal or variable results as occurs with biochemical tests. The antibiogram [4], although it did differentiate one isolate from the others (the corridor isolate, confirmed in this study), also failed to detect at least one other that could be differentiated in extended testing. The survey data from the United Kingdom and the United States suggest the utility of including immunologic typing in similar future studies. A need for more clearly defined subgroups and for an improved nocardia taxonomy was indicated earlier [4],
and the series of studies reported herein meets that need in part. The isolate obtained after cleaning and closure, although not recovered in the affected unit and not recovered in subsequent attempts, may have been the epidemic strain type. It suggests this type may still be present in some environmental haven, or may be reintroduced into the hospital. The epidemic strain was shown to belong to N. asteroides subgroup B of Schaal [ 13,171. Of note, this group is reported to be more virulent than subgroup A
[171. At the present time, authorities [20] do not recommend isolation of nocardiosis patients. In any hospital environment with immunocompromised patients, this recommendation needs to be reevaluated in light of our experience. ACKNOWLEDGMENT These studies were initiated when the senior author was supported by a Wellcome Fund grant for studies on nocardiosis at the Mycology Reference Laboratory, London School of Hygiene and Tropical Medicine, on sabbatical leave from Stanford University. We thank Dr. Donald Mackenzie, Director of the Laboratory, for the United Kingdom Nocardia isolates and Mr. Rod Fichtner, National Animal Disease Center, and Mr. Steve M. States, University of California, Davis, for technical assistance.
REFERENCES 1.
2.
3.
4.
5.
a. 7.
a.
9.
934
Beaman BL. Burnside J, Edwards B, Causey W: Nocardial infections in the United States, 1972-1974. J Infect Dis 1976; 124: 266-269. Krick JA, Stinson EB, Remington JS: Nocardia infection in heart transplant patients. Ann Intern Med 1975; 32: la26. Cox F, Hughes WT: Contagiousness and other aspects of nocardiosis in the compromised host. Pediatrics 1975; 55: 135-138. Houang ET, Lovett IS, Thompson FD. Harrison AR, Joekes AM. Goodfellow M: Nocardia asteroides infection-a transmissible disease. J Hosp Infect 1980; 1: 31-40. Shainhouse JZ, Pier AC, Stevens DA: Complement fixation antibody test for human nocardiosis. J Clin Microbial 1978; 8: 516-519. Pier AC, Fichtner RE: Serologic typing of Nocardia asteroides by immunodiffusion. Am Rev Respir Dis 1971; 103: 698-707. Pan American Health Organization. Manual of standardized serodiagnostic procedures for systemic mycoses. Part II: complement fixation tests. Dept. of Research Development and Coordination, Pan American Health Organization, 1972. Goodfellow M, Schaal KP: Identificationmethods for Nocardia, Actinomadura. and Rhodococcus. In: Skinner FW, Lovelock DW, eds. Identification methods for microbiologists, 2nd ed. London: Academic Press, 1979; 261-276. Gordon MA: Aerobic pathogenic Actinomycetaceae. In: Lennette EH, ed. Manual of clinical microbiology, 3rd ed. Washington: American Society of Microbiology, 1980;
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10. 11.
12. 13. 14.
15. 16. 17.
18.
19.
20.
Volume 71
180-194. Gordon RE, Mihm JM: A comparison of four species of mycobacteria. J Gen Microbial 1959; 21: 736-748. Gordon RE, Mihm JM: Identification of Nocardia caviae (Erickson) Nov. Comb. Ann NY Acad Sci 1962; 98: 628636. Gordon RE, Mihm JM: Type species of the genus Nocardia. J Gen Microbial 1962; 27: l-10. Schaal KP: Zur mikrobiologischen Diagnostik der Nocardiose. Zentralbl Bakteriol 1972; 220: 242-246. Beaman BL: Structural and biochemical alterations of Nocardia asteroides cell walls during its growth cycle. J Bacterial 1975; 123: 1235-1253. Kurup PV, Schmitt JA: Numerical taxonomy of Nocardia. Can J Microbial 1973; 19: 1035-1048. Lovett IS, Houang ET, Burge S, et al. An outbreak of Nocardia asteroides in a renal transplant unit. Q J Med (in press). Pulverer G, Schaal KP: Pathogenicity and medical importance of aerobic and anaerobic actinomycetes. Zentralbl Bakteriol 1978; suppl6: 417-424. Pier AC, Fichtner RE: Distribution of serotypes of Nocardia asteroides from animal, human and environmental sources. J Clin Microbial 1981; 13: 548-553. Palmer DL, Harvey RL, Wheeler JK: Diagnostic and therapeutic considerations in Nocardia asteroides infections. Medicine (Baltimore) 1974; 53: 391-401. Dixon RE, Brachman PS. Bennett JV: Isolation techniques for use in hospitals. 2nd ed. Atlanta: Center for Disease Control, US Dept. of Health, Education and Welfare, 1975.