Laboratory acquired melioidosis

Case Reports

Laboratory ROBERT

PETER

Acquired N.

M.D., c.M.,

GREEN,

G. TUFFNELL,

M.R.c.s.,

Toronto,

Melioidosis*

L.R.C.P.

Ontario,

F.R.c.P.~ (LOND.),

and XC.

PATH.

Canada

Described herein is a case of laboratory acquired melioidosis resulting probably from the inhalation of an infectious aerosol. The course of the disease suggested that the acute form of melioidosis was arrested by kanamycin therapy and that tetracycline, sulfonamides and ampicillin contributed to the resolution of residual lesions with complete recovery. The epidemiology of melioidosis and the identification of Pseudomonas pseudomallei are briefly reviewed. in man with Pseudomonas pseudomallei is thought to be most commonly acquired through breaks in the skin. Subsequently the infection may fail to spread, may cause insignificant illness detectable only by serologic tests or may progress to peracute, acute disseminated, chronic or local forms of the disease. Although infection by the oral route is common in animals it is probably uncomman in man. Arthropod-borne infection has not been shown to occur naturally, but experimentally the rat flea and the mosquito have transmitted the infection [1,2]. The epidemiology of melioidosis has been reviewed by Nigg [3]. Mice and hamsters can be readily infected by aerosols, variation in the dose resulting in differing incubation periods and acuteness of the disease [4]. The course in mice after this mode of infection closely resembles the types of infection and the lesions described in man. The recent recognition of melioidosis in service personnel in Viet Nam [5] has increased interest in the diagnosis and management of this disease. Accurate diagnosis in the acute cases necessitates identification of the organism and although fluorescent antibody technics may be applicable to direct identification in specimens, isolation of the organism will remain the standard procedure. The potential danger of

I

the organism to laboratory personnel is illustrated by this case report. The most effective treatment has not yet been defined, the majority of case reports emphasizing the unpredictable response to antibacterial therapy. Kanamycin appeared to be an effective drug in this case and deserves trial in other cases to determine its place in the treatment of this disease.

NFECTION

CASE REPORT A forty-eight year old woman Ph.D. bacteriologist at the University of Toronto (H.H.) was admitted to the Toronto General Hospital on August 27, 1966. Recent past history consisted of two chest colds with cough and pleural pain in the preceding twelve months. For one year prior to admission she had been working with a type collection strain of Ps. pseudomallei, and recently she had been concentrating the organism by centrifugation in order to isolate and study the deoxyribonucleic acid (DNA) content. Three days prior to admission the continuous flow centrifuge developed a leak and a concentrated suspension of organisms was sprayed into the air and over the walls and benches of her laboratory. Using her bare hands she cleaned up this mixture of media and organisms. She had an ulcerative lesion at the base of her right index finger. Three days later she had chills, fever and malaise, tenderness in the right axilla and pleural pain in the right side of her chest. These

* From the Department of Medicine, and the Division of Microbiology, Toronto General Hospital, University of Toronto, Toronto, Ontario, Canada. Manuscript received April 26, 1967. t Present address: Department of Medicine, Kingston General Hospital, Kingston, Ontario, Canada. VOL.

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and the

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Melioidosis-Green,

Kanamycin

FIG. 1. Response to antibiotics during the first eighteen days in hospital. symptoms continued throughout the day and she presented herself at the Emergency Department of Toronto General Hospital at 8 P.M. On physical examination she was found to be an intelligent, cooperative, middle-aged woman who appeared to be ill and rather anxious. The temperature was 103.2’~., pulse 100 per minute and regular, blood pressure 130/90 mm. Hg, respirations 22 per minute. Chest examination was within normal limits although she complained of pleuritic pain over the right anterior portion of her chest on deep inspiration or on coughing. No pleural friction rub was heard. A small tender node was present in the right axilla. The skin was clear except for a small encrusted nontender lesion at the base of her right index finger. A chest roentgenogram was within normal limits. Hemoglobin was 12 gm-per cent, white cell count 9,000 per cu. mm. with 86 per cent neutrophils. The clinical impression on admission was that of an insipient lobar pneumonia, but because of the recent exposure melioidosis was also considered as a possible diagnosis. The patient informed us that her

Tt@nell previous studies had shown this strain of Ps. pseudomallei to be sensitive to penicillin (sensitivity disc of 10 units). Ten separate blood specimens were taken for culture in the first twelve hours in the hospital. Following this, a continuous intravenous infusion of penicillin, 24 million units per twenty-four hours, was started. Samples of the Ps. pseudomallei strain involved were obtained from her laboratory immediately and antibiotic sensitivity studies were carried out. The organism was sensitive to kanamycin and chloramphenicol, and moderately sensitive to tetracycline, ampicillin and penicillin. The organism was sensitive to penicillin at a level of 20 units per ml., but resistant to streptomycin 100 pg. per ml. Day 2. Twenty-four hours after starting the penicillin infusion the lesion at the base of her right index finger was noted to be tender and a margin of erythema had developed around it. The scab was lifted off and a small amount of whitish exudate present was transferred directly to culture broth and to blood agar. Her temperature remained at 101”~. for most of the day (see fever chart, Fig. 1). Headache and moderate pleuritic pain continued. A chest film showed faint areas of infiltration in the upper lobes of both lungs and a small pleural effusion at the right costophrenic angle. An irregular area of consolidation could also be seen in the lower zone of the right lung (Fig. 2A) and a solitary, well defined, nodular lesion with an eccentric radiolucency suggestive of a cavity at the base of the left lung (Fig. 2B). Days 3, 4 and 5. The intravenous penicillin infusion was continued at 24 million units daily. During this time her temperature spiked to lOlo and 102’~. with recurrent pleuritic pain in the right side of her chest, headache and a moderate mental de-

2

FIG. 2. Posteroanterior B, solitary parenchymal

chest film of August 29. A, ill defined area of consolidation nodule with an eccentric small cavity. AMERICAN

at the base of the right lung.

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Melioidosis- - Green! pression. ‘L’he white cell count with 71 per cent neutrophils,

was 7,700

per cu. mm.

hemoglobin

12.3

gm.

per cent: erythrocyte sedimentation rate 103 mm. per ltour. Chest film on the fifth hospital day showed faint parenchymal infiltrations in the upper lobes of both lungs. the pleural reaction at the baqe of the right lung having cleared. Da? 0. At 4 A.M. the intravenous infusion was no longer functioning and there was interstitial swelling. The patient had a temperature of IOO’F., headache and was quite depressed. She refused permission for the House Staff to re-start her intravenous infusion. For the next thirty-six hours she was given no antibiotics. None of the previous cultures had grown any organisms, and with some clinical and roentgenologic evidence of diminution in the chest symptoms, it was elected to observe the further progress of her illness. Da? 7. At 4 P.M. a fever developed with a temperature of 104’F., chills and rigors. Blood was taken for routine cultures and viral studies, and the intramuscular administration of kanamycin, 250 mg. every eight hours, was started. Temperature of 104’~. continued throughout the night and most of the following day, with recurrent chills and rigors. The right index finger was noted to be more swollen. red and tender in the region of the previous lesion. Duvs 8, 9 and 70. The patient continued to have fever with recurrent chills and moderate pleural discomfort in the right side of her chest. She also complained of generalized weakness, muscle aches and pains, and mild headache. On the tenth hospital day she began coughing up thick, greenish sputum which was cultured on blood agar and in broth. Chest film the following day revealed faint areas of infiltration

601

Tu@wll

in the upper

lobes with small

sions. She continued

bilateral

to have recurrent

pleural

effu-

mild pleuritic

pain and temperatures of 101” to 104’~. Day 72. ‘The white cell count was 6,200 per cu. mm. with 65 pvr cent neutrophils. hemoglol)in 9.5 gm. per cent, erythrocyte sedimentation ratr 118 mm. per hour. Tetracycline, 250 mg. every six hours, was added to the regimen of kanamycin (250 mg. every eight hours). On this day it way learned that the culture originally taken from her right index fingel on the second hospital day grew Ps. pseudomallei. Her fever subsided the following day and she remained afebrile for the rest of her course in hospital. Her symptoms of pleural pain and hradachr: were minimal. Day 77. After ten days of kanamycin therapy the patient complained of a change in the quality of her hearing. Kanamycin treatment was discontinued and her hearing improved on the following day. An audiogram showed a loss of 25 to 30 d.c. in the low pitch region in her right ear and a 10 d.c. loss in the low pitch region in her left ear. Her hearing returned to normal within ten days after administration of the drug was stopped. She continued to receive tetracycline, 250 mg. every six hours. and to complain of moderate headache and vague recurrent chest pain. Day 20. A chest film taken two days previously had shown bilateral pleural effusions with multiple small areas of infiltration throughout the lung fields and elevation of the right diaphragm. ‘l‘he lesions seen previously (Fig. 2) showed significant change. The solitary well defined nodular lesion in the lower left side of the chest had resolved and this area had reverted to normal (Fig. 3B). The irregular area of

3h

‘IG. 3. Posteroanterior chest film of September 14. A, note increase in extent of consolidation at base of right lung md small pleural effusion. B, simultaneous witii this there has been compiete clearing of the nodule at the base ,f the left lung. VOL.

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Melioidosis-Green, consolidation in the lower right side of the chest (Fig. 2A) had increased in size (Fig. 3A). White cell count was 7,400 per cu. mm with 50 per cent neutrophils; hemoglobin was 9.5 gm. per cent, and the sedimentation rate 116 mm. per hour. Cultures of the greenish yellowish sputum which the patient had been coughing up were reported at this time to have grown Ps. pseudomallei. Day 24. Because of the persistence of chest signs and symptoms and in view of the serious nature of this illness, it was decided that a more vigorous course of antibiotic therapy be embarked upon. The patient was given 8 gm. of ampicillin daily and this dose was gradually diminished over the next five days to a maintenance dose of 4 gm. daily in divided doses. Tetracycline therapy was discontinued twenty-four hours after the administration of ampicillin was started. After five days on ampicillin therapy the patient was also given 6 gm. of triple sulfa (Trulfa tabs. Frosst) for three days and then maintained on 4 gm. of triple sulfa daily throughout the remainder of her hospital stay. The patient remained in the hospital receiving ampicillin and triple sulfa (4 gm. of each daily for twenty-two more days) and made a gradual but definite clinical recovery. A chest film prior to discharge revealed no evidence of active intrathoracic disease. Roentgenograms of both hands showed no evidence of bony involvement or other changes. She was discharged after forty-seven days in the hospital. Follow-Up Studies. A chest film obtained four weeks after discharge showed no change, the raised right diaphragm with pleural thickening in the right costophrenic angle remained. The hemoglobin was 10.9 gm. per cent, white cell count 4,300 per cu. mm. with 39 per cent neutrophils, sedimentation rate 26 mm. per hour. The dose of ampicillin and triple sulfa was reduced to 2 gm. of each daily. Six weeks after discharge the patient looked and felt well. The only abnormality detected was a slight dullness with reduced breath sounds over the lower right side of the chest anteriorly and laterally. Fluoroscopy showed the left diaphragm to move 3 cm. and the right diaphragm 2 cm. with adhesions on the right anteriorly and laterally. Triple sulfa therapy was discontinued and ampicillin was continued at dosage of 2 gm. daily. Fifteen weeks after discharge the patient was working full time and feeling quite well. She had no cough or shortness of breath. Chest movement was slightly reduced on the right side and there was some dullness at the base of the right lung with diminished breath sounds anterolaterally. Fluoroscopy again showed the adhesions of the right diaphragm anterolaterally, but the diaphragm appeared to be moving more freely. The white cell count was 6,400 per cu. mm. with 49 per cent neutrophils; hemoglobin 10.9 was gm. per cent and the sedimentation rate 26 mm. per hour. Ampicillin therapy was continued (2 gm. daily) for an additional nine weeks, resulting in a full six months of treatment.

Tufnell

Twenty-four weeks after discharge, antibiotic treatment was discontinued. Clinical and laboratory findings were unchanged from her assessment nine weeks earlier. BACTERIOLOGY

From a specimen of the hand lesion on August 29 and two separate specimens of sputum (September 6 and 7) organisms were isolated which resembled in all particulars studied the stock strain N.T.C.C. No. 7383. The morphologic characters of these isolates were those of gram-negative bacilli 2 to 5 p X 0.4 to 0.6 p arranged singly or in pairs and very occasional chains. Bipolar staining was common, the ends were rounded and many cells were definitely oval. Very occasional longer forms were seen but pleomorphism was not marked. The cells were actively motile by polar flagella. On plates of blood agar after twenty-four hours’ incubation at 37” C. the colonies were 1 mm. in diameter, transparent, shiny, convex, entire and gray with slight greening of the blood around confluent growth. At forty-eight hours the diameter was 2 mm., the center becoming nodular, the surface semimatt with a slight metallic sheen upon the surface of the confluent part of the growth. The fully developed colonies after five days were 6 mm. in diameter, slightly raised and circular with a regular crenated edge and a dull surface, with regularly radiate rounded ridges peripherally but serpentine centrally and of a light cafe au lait color. The odor was ammoniacal-earthy. More crowded colonies developed a convex center, finely nodular with the rounded ridges radiating regularly from the center, giving an appearance strikingly reminiscent of a daisy head. The metallic sheen of such convex centers was accentuated where growth was confluent to create an appearance suggestive of aluminum paint. Occasional colonies lacked the radiating ridges and were low convex, finely granular colonies with the metallic sheen. Beta type lysis was evident around heavy growth but not around individual colonies. On MacConkey agar (Difco) the colonies were similar but became red in forty-eight hours. There was no growth on S.S. agar (Difco). In broth culture a moderate turbidity with surface growth at twenty-four hours became a definite pellicle in forty-eight hours. The optimum temperature for growth was 37”c., the minimum growth temperature was below 18”c., the maximum above 42”~. In phenol red broth sugars (Difco) there was AMERICAN

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Melioidosis--Green, no acid production in twenty-four hours. In forty-eight hours weak acid production was evident in dextrose, glycerol, galactose, raffinose and sorbitol. In five days these reactions were stronger and acid was evident in mannitol, dulcite, trehalose and xylose. Lactose, sucrose, maltose and salicin remained negative at twelve days. Low peptone-base sugars showed acid production from dextrose, lactose, arabinose and xylose in the aerobic but not in oil-sealed tubes. Reactions were negative in the following tests: methyl red, Voges-Proskauer, hydrogen sulfide production, phenyl alanine deamination, indole production, gluconate oxidation and urease production. Gelatin and coagulated blood serum were rapidly liquefied and nitrogen produced from nitrates. Growth occurred on Simmons citrate agar and in malonate broth. Catalase and oxidase tests were positive. The phosphatase produced was heat stable at 70”~. for twenty minutes [6]. In studies on pathogenicity, the first isolate from sputum was inoculated (0.25 ml. of an overnight broth culture) intraperitoneally into a female guinea pig. The animal died in twelve hours. At autopsy there were no gross abnormalities except a slight, clear, peritoneal exudate. The organism was re-isolated from blood and peritoneum. Another animal inoculated with 0.1 ml. died in approximately thirty hours and demonstrated pleurisy, fibrinous peritonitis, dark red adrenals and inflamed uterine horns. Culture of heart blood and peritoneal fluid grew pure cultures of the organism. A third animal was scratched on the shaved abdomen with an infected needle. Within forty-eight hours there was an 0.5 cm. nodule at the site. At four days there was a 1 cm. ulcer with a rolled edge and marked surrounding induration. At nine days the animal was moribund and was sacrificed. At autopsy the local lesion measured 3 by 2 cm., was adherent to the peritoneum and contained a central caseous area 1 by 1.5 cm. The lungs, heart, kidney, liver, spleen and adrenals were congested. The spleen measured 3 cm. and showed 1 mm. white nodules on its surface. There wasmarkedconjunctivitis. Stained sections of the organs showed a coagulative necrosis of the skin, subcutaneous infiltration with mixed acute and chronic inflammatory cells and some central necrosis of the roughly nodular collections of these cells. Similar abscesses were present in the lungs and the spleen. The liver showed fatty degeneration. VOL.

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The identification of Ps. pseudomallei includes differentiation from (1) Actinobacillus mallei [7], which is nonmotile, liquefies gelatin only slowly, does not grow on Simmons citrate agar or on MacConkey agar or at 42’c.; (2) Ps. stutzeri [8], which grows with an adherent colony, grows on S.S. agar, does not liquefy gelatin, does not grow at 42”~. and is nonpathogenie to guinea pigs; and (3) Ps. aeruginosa, which usually produces pigment, is gluconatepositive, grows on S.S. agar and is nonpathogenie for guinea pigs. According to Liu [6], only Ps. pseudomallei and Ps. aeruginosa produce a phosphatase that resists 70”~. for twenty minutes. An exception was Ps. boreopolis, a nonpathogenic species that does not grow at 37’~. [S]. The characters of the organisms isolated from the patient establishes its identity as Ps. pseudomallei. A specimen of the patient’s serum taken on the fifteenth day after infection was compared in an agglutination test with a second specimen taken on the twenty-eighth day. The antigen was a suspension of the organism isolated from the first specimen of sputum, autoclaved for one hour at 121”~. Suspensions prepared in phenol saline solution or after boiling for one hour were moderately autoagglutinable whereas the autoclaved suspension was smooth. The titer of the first specimen was 1: 20, and of the second 1: 640. The antibiotic sensitivities of the first isolate were by the disc technic, sensitive to 10 pg. of chloramphenicol, 5 pg. of kanamycin, 2 pg. of gentamycin, 10 units of penicillin G (resistant to 2 units), 30 pg. of tetracycline (resistant to 5 pg.), 10 pg. of ampicillin (resistant to 2 pg.) and 250 mg. of sulfadiazine. It showed resistance to 300 units of polymyxin B and 10 pg. of streptomycin. By a tube dilution method the organism was sensitive to 20 units per ml. of penicillin G, 10 pg. per ml. of ampicillin and 30 pg. per ml. of kanamycin. It was resistant to 100 pg. per ml. of streptomycin. COMMENTS The sequence of events enable a reasonable assumption that infection occurred at the time of the accident with the centrifuge. Inhalation of the resulting aerosol causing primary pulmonary infection is more likely than either systemic spread from the infected hand or oral infection from contaminated hands. Signs of active infection of the hand abrasion appeared

Melioidosis-Green, at the time of obvious systemic manifestations of disease, suggesting simultaneous infection. Pulmonary involvement was the major feature of the subsequent course, with no evidence of septicemia or localization elsewhere. Considering the conditions in which the infection was acquired, the incubation period of three days is probably no indication of the length of the incubation period in natural infection, but was similar to that described in mice infected by aerosol with small doses of Ps. pseudomallei [4]. Prompt treatment with large doses of penicillin G appeared to modify the course of the disease slightly. The level of sensitivity of the organism in vitro and the continued progress of the disease during treatment suggested that successful treatment with penicillin alone was most unlikely. Although sulfonamide and chloramphenicol have been the drugs most frequently mentioned as producing clinical benefit, the response in most cases has been disappointing, even when the organism has been demonstrably sensitive in vitro 1.91. Kanamycin has been used in melioidosis only once to our knowledge [70] and failed to produce any detectable response in a patient with chronic pulmonary melioidosis after treatment for five days despite the sensitivity of the organism to the drug Zn vitro. Kanamycin was selected for treatment in our case because of its effectiveness in vitro and its tidal mode of action. Extensive experience with this antibiotic has demonstrated its effectiveness in a wide range of infections, especially those due to gram-negative bacilli, with a low incidence of toxic effects in persons with normal renal function given the drug for a short time [ 771. The early detection of auditory symptoms prompted cessation of the drug to prevent the further development of cochlear changes. The addition of tetracycline to the regimen after five days of kanamycin therapy was based on the probability that the kanamycin could not be given for a prolonged period. Sulfonamides and ampicillin were considered to be best kept in reserve or for continuing treatment. Chloramphenicol was withheld because the advantage of demonstrated in vitro sensitivity did not outweigh the serious side effects which may occur with this drug. The multiple areas of pulmonary infiltration resolved slowly whereas the pleural effusion never became large and resolved without the need for aspiration. It seems probable that kanamycin was the effective agent in pro-

7itfneli ducing rcnlission of the acute phase because the fall in tentperature occurred before the tetracycline could reasonabl>- be expected to be exerting a significant effect. Trrracvcline on its own was shown by Cruickshank / !.?I to be irleffective in experimental melioidosis. Because of the presence of residual pulrnonar!. lesions and the possibility of lesions in other organs front hematogenous spread it seetned obligatory to continue antibacterial therapy after the administration of kanamycin was stopped. Tetracycline therapy was continued for ten days and sulfonamides and ampicillin were chosen for continuing treatment. Although it is impossible to be certain that there would have been recrudescence of the infection without this treatment, the possibility had to be considered. Airborne transmission of Ps. pseudomallei has not been demonstrated in man under natural conditions. That infection by this route is possible is demonstrated by this case. It is reasonable to suppose that infected dust or atomized water under natural conditions could likewise cause infection. If such cases occur, it may be expected that the incubation period and the acuteness of the disease would vary with the size of the dose inhaled. ADDENDUM

When last seen (January 1968), the patient was in good health and with no demonstrable physical signs, a total of eighteen months after infection. Acknowledgment: We wish to thank Dr. K. J. R. Wightman, Professor of Medicine, Dr. C. R. Woolf and Dr. L. F. W. Loach for assistance with clinical studies and Dr. D. E. Sanders for assistance with roentgenologic studies. Special thanks to our patient for her cooperation and bacteriologic advice [ 731. REFERENCES G. and BALTAZARD, M. Transmission of Whitmore’s bacillus by rat flea Xenopsylla cheopis. Compt. rend. Acad. d. SC., 213: 541, 1941. Quoted by Beamer, P. R., Varney, P. L., Brown, W. G. and McDowell, F. Studies on Makomyces gseudomallei isolated from melioidosis originating in the western hemisphere. Am. J. Clin. Path., 24: 1231, 1954. 2. Ibid., p. 670. 3. Nrcc, C. and JOHNSTON, M. J. The complement fixation test in experimental clinical and subclinical melioidosis. J. Bact., 82: 159, 1961. 1. BLANC,

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Melioidosis-Green, 4. DANNENBERG. A. M. and SCOTT, E. M. Melioidosis, pathogenesis and immunity in mice and hamsters. I. Studies with virulent strains of Malleomyces pseudomallei. J. Exper. Med., 107: 153, 1958. 5. TIGERTT, W. D., COL., MC, Director, U. S. Army Medical Service Laboratories. Personal communication. 6. Ln., P. V. Differentiation of pathogenic pseudomonads by heat resistance of alkaline phosphatase. rim. J. Clin. Path., 45: 639, 1966. 7. WRTMORE, P. W. and GOCHENOW, W. S. Comparative studies of the genus Malleomyces and selected pseudomonas species. I. Morphological and cultural characters. J. Bat., 72: 79, 1956. 8. VON. GRAEVENITZ, A. Pseudomonas stutzeri isolated

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from clinical specimens. Arrr. ./. C.‘/l!/. Path.. 43: 357, 1965. MON.I.GOMERY, I<. Melioidosis. Report on a fatal case in a British soldier. J. Knl,. :ll-ml M. &p.r., 109: 223, 1963. MAEGRAITH, B. G. and LEITHEAD. (:. S. Melioidosis: .\ case report. Lancct, 1: 862, 1764. FINEGOLD, S. M. Toxicity of kanamyrin in adults. .4nn. Xew York Acad. SC., 132. .\rt. 2. 042, 1966. CRUICKSHANK,J. C. Failure ol‘aureomycin in experimental meliodiosis. Brit. *21. J.. 2: 410, 1949. FARKAS-HIMSLEY, H. Selection and r,lpid identification of Pseudomonas pseudornallei from other gram-negative bacteria. .Im. ./. C//n. Path., in press.