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Article Eur. J. Clin. Microbiol.InfectDis., September 1990,p. 654--658 0934-9723/90/09 0654-05 $ 3.00/0
Vol. 9, No. 9
Blood Culture Techniques for the Diagnosis of Melioidosis V. W u t h i e k a n u n l , D. Dance1, 2., W. C h a o w a g u l 3, Y. S u p u t t a m o n g k o P , Y. W a t t a n a g o o n l , N. Whitel,2
The effects of variations in laboratory technique on the speed and sensitivity of isolation of Pseudomonas pseudoraallei from blood were evaluated prospectively. Pseudomonas pseudomallei was isolated from 154 of 546 cultures from 325 patients with suspected or confirmed melioidosis. Subcultures after 12 to 24 and 36 to 48 hours of incubation were positiv e in 52.3 % and 80.8 % respectively. The yields from 20 mi (blood to broth ratio 1:4) and 50 ml (blood to broth ratio 1:10) brain heart infusion broth bottles were equivalent in patients not receiving treatment for melioidosis. During therapy, the 50 mi bottles grew Pseudomonas pseudomallei significantly faster than the 20 ml bottles (p < 0.01), and gave a higher overall yield for cultures processed in antimicrobial removal devices (p < 0.05). These devices themselves increased the speed of isolation of the organism from treated patients (p < 0.01). In most cases, all bottles collected from a patient before treatment were positive, and a single 20 ml bottle had an estimated relative sensitivity of 85.7 % (95 % confidence interval 7 7 . 1 - 9 4 . 3 %). Early subculture should be employed routinely for the laboratory diagnosis of septicaemic melioidosis. However, blood culture techniques do not need to be sophisticated. Culture of 5 ml blood in 20 ml broth is a simple and sensitive procedure suitable for regions where melioidosis is currently under-diagnosed.
Melioidosis is emerging as a major cause of morbidity and mortality in Northeastern Thailand (1-3). Diagnosis is difficult on clinical grounds alone and so depends on the isolation and identification of Pseudomonas pseudomallei, the causative organism, from clinical specimens. Since September 1986, we have conducted prospective clinical studies of melioidosis in Sappasitprasong Hospital, Ubon Ratchatani (2), where melioidosis accounts for nearly 20 % of admissions and 40 % of deaths caused by community-acquired septicaemia (3). Approximately 60 % of cases of melioidosis presenting in this hospital have positive blood cultures (2), and blood is often the first or only clinical specimen which yields Pseudomonas pseudomallei. We have therefore evaluated 1Bangkok Hospital for Tropical Diseases, Faculty of Tropical Medicine, Mahidol University, 420•6 Rajvithi Road, Bangkok, Thailand. 2Nuffield Department of Clinical Medicine,Universtiyof Oxford, John Radcliffe Hospital, Headington, Oxford, OX3 9DU, UK. 3Department of Medicine, Sappasitprasong Hospital, Ubon Ratchatani, Thailand.
prospectively the effect of variations in culture technique (early subculture, broth volume, antimicrobial removal devices) on the speed and sensitivity of isolation of Pseudomonas pseudomallei from blood. Materials and Methods
Patients. The study was conducted during the rainy season months (June-November) between June 1987 and November 1989. Inpatients in Sappasitprasong Hospital (n = 325) in whom the diagnosis of septicaemic melioidosis was considered likely, or from whom Pseudomonas pseudomaIlei had already been isolated in the hospital laboratory, were selected for study. Such patients were visited by the study team and specimens collected for bacteriological confirmation of the diagnosis in the team's laboratory. Clinical information was recorded on a standard form. Patients were admitted to a prospective trial of antibiotic chemotherapy for severe melioidosis if they were eligible and if they or their relatives gave fully informed consent ([4] and unpublished data).
Vol. 9,1990
Specimen Collection. Before collection of blood for culture using sterile disposable equipment and aseptic techniques, the skin and bottle tops were disinfected with 70 % isopropyl alcohol, which was allowed to dry. One blood culture set comprised 10 ml blood which was divided equally between one 20 ml and one 50 ml bottle of b r a i n heart infusion broth containing p-aminobenzoic acid, sodium polyanethol sulphonate and carbon dioxide under vacuum (Gibco, UK). On the first visit to each patient (day 1), three sets were collected 10 to 15 minutes apart, unless blood cultures had already been sent to the hospital laboratory that day, in which case one or two sets only were collected. One or two further sets were taken from surviving patients on day 3 or 4 of treatment, and again on days 7 and 14 if the previous culture was positive. Blood cultures were also taken on other days when indicated on clinical grounds, i.e. because of a deterioration in the patient's condition. If the patient was already receiving antibiotics active against Pseudomonas pseudomallei (chloramphenicol, doxycycline, co-trimoxazole, a third generation cephalosporin, ureidopenicillin or amoxycillin/clavulanic acid), one blood culture set was first processed in an antimicrobial removal device (ARD) containing resin (Amberlite, Marion Scientific, USA) when these were available. All bottles were transported to the laboratory within one hour of collection. Laboratory Methods. All bottles were vented to room air using BCB Vent/Sub units (Difco Laboratories, UK) immediately on receipt in the laboratory and were then incubated in air at 37 °C. Bottles were subcultured routinely via the Vent/Sub unit onto horse blood agar within 12 to 24 hours of receipt (day 1 subculture), between 36 and 48 hours of receipt (day 2 subculture), and finally on the seventh day of incubation (day 7 subculture), after which they were discarded. Bottles were also inspected daily and subcultured if macroscopically positive. Gram stained smears were made from macroscopically positive bottles. Plates were incubated in air at 37 °C and inspected at 24 and 48 hours. Pseudomonas pseudomallei was identified as previously reported (5).
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no such focus was evident. Shock was defined as a systolic blood pressure
Article Eur. J. Clin. Microbiol.InfectDis., September 1990,p. 654--658 0934-9723/90/09 0654-05 $ 3.00/0
Vol. 9, No. 9
Blood Culture Techniques for the Diagnosis of Melioidosis V. W u t h i e k a n u n l , D. Dance1, 2., W. C h a o w a g u l 3, Y. S u p u t t a m o n g k o P , Y. W a t t a n a g o o n l , N. Whitel,2
The effects of variations in laboratory technique on the speed and sensitivity of isolation of Pseudomonas pseudoraallei from blood were evaluated prospectively. Pseudomonas pseudomallei was isolated from 154 of 546 cultures from 325 patients with suspected or confirmed melioidosis. Subcultures after 12 to 24 and 36 to 48 hours of incubation were positiv e in 52.3 % and 80.8 % respectively. The yields from 20 mi (blood to broth ratio 1:4) and 50 ml (blood to broth ratio 1:10) brain heart infusion broth bottles were equivalent in patients not receiving treatment for melioidosis. During therapy, the 50 mi bottles grew Pseudomonas pseudomallei significantly faster than the 20 ml bottles (p < 0.01), and gave a higher overall yield for cultures processed in antimicrobial removal devices (p < 0.05). These devices themselves increased the speed of isolation of the organism from treated patients (p < 0.01). In most cases, all bottles collected from a patient before treatment were positive, and a single 20 ml bottle had an estimated relative sensitivity of 85.7 % (95 % confidence interval 7 7 . 1 - 9 4 . 3 %). Early subculture should be employed routinely for the laboratory diagnosis of septicaemic melioidosis. However, blood culture techniques do not need to be sophisticated. Culture of 5 ml blood in 20 ml broth is a simple and sensitive procedure suitable for regions where melioidosis is currently under-diagnosed.
Melioidosis is emerging as a major cause of morbidity and mortality in Northeastern Thailand (1-3). Diagnosis is difficult on clinical grounds alone and so depends on the isolation and identification of Pseudomonas pseudomallei, the causative organism, from clinical specimens. Since September 1986, we have conducted prospective clinical studies of melioidosis in Sappasitprasong Hospital, Ubon Ratchatani (2), where melioidosis accounts for nearly 20 % of admissions and 40 % of deaths caused by community-acquired septicaemia (3). Approximately 60 % of cases of melioidosis presenting in this hospital have positive blood cultures (2), and blood is often the first or only clinical specimen which yields Pseudomonas pseudomallei. We have therefore evaluated 1Bangkok Hospital for Tropical Diseases, Faculty of Tropical Medicine, Mahidol University, 420•6 Rajvithi Road, Bangkok, Thailand. 2Nuffield Department of Clinical Medicine,Universtiyof Oxford, John Radcliffe Hospital, Headington, Oxford, OX3 9DU, UK. 3Department of Medicine, Sappasitprasong Hospital, Ubon Ratchatani, Thailand.
prospectively the effect of variations in culture technique (early subculture, broth volume, antimicrobial removal devices) on the speed and sensitivity of isolation of Pseudomonas pseudomallei from blood. Materials and Methods
Patients. The study was conducted during the rainy season months (June-November) between June 1987 and November 1989. Inpatients in Sappasitprasong Hospital (n = 325) in whom the diagnosis of septicaemic melioidosis was considered likely, or from whom Pseudomonas pseudomaIlei had already been isolated in the hospital laboratory, were selected for study. Such patients were visited by the study team and specimens collected for bacteriological confirmation of the diagnosis in the team's laboratory. Clinical information was recorded on a standard form. Patients were admitted to a prospective trial of antibiotic chemotherapy for severe melioidosis if they were eligible and if they or their relatives gave fully informed consent ([4] and unpublished data).
Vol. 9,1990
Specimen Collection. Before collection of blood for culture using sterile disposable equipment and aseptic techniques, the skin and bottle tops were disinfected with 70 % isopropyl alcohol, which was allowed to dry. One blood culture set comprised 10 ml blood which was divided equally between one 20 ml and one 50 ml bottle of b r a i n heart infusion broth containing p-aminobenzoic acid, sodium polyanethol sulphonate and carbon dioxide under vacuum (Gibco, UK). On the first visit to each patient (day 1), three sets were collected 10 to 15 minutes apart, unless blood cultures had already been sent to the hospital laboratory that day, in which case one or two sets only were collected. One or two further sets were taken from surviving patients on day 3 or 4 of treatment, and again on days 7 and 14 if the previous culture was positive. Blood cultures were also taken on other days when indicated on clinical grounds, i.e. because of a deterioration in the patient's condition. If the patient was already receiving antibiotics active against Pseudomonas pseudomallei (chloramphenicol, doxycycline, co-trimoxazole, a third generation cephalosporin, ureidopenicillin or amoxycillin/clavulanic acid), one blood culture set was first processed in an antimicrobial removal device (ARD) containing resin (Amberlite, Marion Scientific, USA) when these were available. All bottles were transported to the laboratory within one hour of collection. Laboratory Methods. All bottles were vented to room air using BCB Vent/Sub units (Difco Laboratories, UK) immediately on receipt in the laboratory and were then incubated in air at 37 °C. Bottles were subcultured routinely via the Vent/Sub unit onto horse blood agar within 12 to 24 hours of receipt (day 1 subculture), between 36 and 48 hours of receipt (day 2 subculture), and finally on the seventh day of incubation (day 7 subculture), after which they were discarded. Bottles were also inspected daily and subcultured if macroscopically positive. Gram stained smears were made from macroscopically positive bottles. Plates were incubated in air at 37 °C and inspected at 24 and 48 hours. Pseudomonas pseudomallei was identified as previously reported (5).
655
no such focus was evident. Shock was defined as a systolic blood pressure
