Journal of Hospital Infection (2008) 68, 329e333
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Application of the forensic Luminol for blood in infection control* P.W.M. Bergervoet*, N. van Riessen, F.W. Sebens, W.C. van der Zwet Department of Medical Microbiology and Infection Control, Deventer Ziekenhuis, Deventer, The Netherlands Received 29 June 2007; accepted 18 January 2008 Available online 17 March 2008
KEYWORDS Hepatitis C virus; Haemodialysis; Luminol; Contamination
Summary Transmission of hepatitis C virus occurs frequently in haemodialysis units. A possible route of transmission is indirectly via the hospital environment although this has never been recorded. We investigated the haemodialysis unit in Deventer Hospital, Deventer, The Netherlands, with the forensic Luminol test. With this test, invisible traces of blood can be visualised based on the principle of biochemiluminescence. We demonstrated extensive contamination of the environment with traces of blood. The aim of this article is to introduce this method to infection control professionals, so it can be used to monitor cleaning and disinfection procedures, and alert healthcare workers to the possibility of contamination of the hospital environment with blood. ª 2008 The Hospital Infection Society. Published by Elsevier Ltd. All rights reserved.
Introduction In the haemodialysis unit, patient-to-patient transmission of bloodborne viruses, such as hepatitis C virus (HCV) remains a continuous threat.1e4 As blood and blood products are routinely screened for the presence of these viruses, more and more attention * This study was presented in part at the 6th congress of the International Federation of Infection Control, Istanbul, 13e16 October 2005. * Corresponding author. Address: Department of Infection Control, Deventer Ziekenhuis, P.O. Box 5001, 7400 GC Deventer, The Netherlands. Tel.: þ31 570666666; fax: þ31 570626477. E-mail address:
[email protected]
is focused on nosocomial transmission of HCV. Several risk factors have been identified for spread of HCV in haemodialysis units, such as contaminated dialysis machines, environmental contamination, inadequate infection control procedures and understaffing of the unit.1e4 Isolation of HCV-positive patients is not recommended, but routine serological screening of HCV-negative patients is common practice to detect seroconversion as soon as possible.5 In 1996, a cluster of four new HCV infections within one year occurred in the haemodialysis unit of the Deventer Hospital.6 Genotyping of the isolated strains demonstrated one single HCV genotype. It was concluded that multiple unrecognised transmissions of HCV had occurred in the unit,
0195-6701/$ - see front matter ª 2008 The Hospital Infection Society. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.jhin.2008.01.026
330 perhaps from shared medical equipment and bloodcontaminated gloves. We speculated on ways to prove this hypothesis. By coincidence, our attention was attracted to the Luminol test for detection of traces of blood by the popular television series ‘Crime Scene Investigation’. This forensic test has been used for many decades and can visualise minute traces of blood by providing a blue chemiluminescent signal from a chemical reaction catalysed by haemoglobin.7 The method is very sensitive although some other substances can give false-positive signals.8e11 The aim of this paper is to inform the infection control community about the use of the Luminol method for the detection of traces of blood in the environment. We also present the results of investigation of the haemodialysis unit in the Deventer Hospital by using Luminol.
Methods Description of the Luminol test Luminol (5-amino-2,3-dihydro-1,4-phthalazinedione) is dissolved in an alkaline mixture with hydrogen peroxide.9 In the presence of metal ions or metal complexes such as haemoglobin, the reaction produces a bright blue chemiluminescence. The Luminol method is easy to use, non-toxic, up to 20 times more sensitive for haemoglobin than other blood detection tests and can visualise traces of blood that are invisible to the naked eye.11 Traces of blood can be detected for a minimum of seven weeks.11 Luminol cannot differentiate between human and animal blood. The Luminol method is not specific for haemoglobin as other materials/substances can also catalyse the reaction. This has been extensively tested by forensic researchers.8,9 The intensity of brightness of the chemiluminescence caused by these substances is lower than for haemoglobin, except for copper metal and enamel paint. The mean intensity of sodium hypochlorite (NaOCl), which is frequently used in the hospital as a disinfectant, is 80% of that of the haemoglobin value. For dark green spray car paint and some vegetables (turnip, parsnip, horseradish) it is 20e70%. Other tested substances are not visible or are nearly visible to the naked eye (>5% of the intensity of haemoglobin value).8,9
Experiments We used the F-30000 Luminolset from BVDA (Bureau voor Dactyloscopische Artikelen, Haarlem,
P.W.M. Bergervoet et al. The Netherlands) which consists of three darkcoloured bottles containing NaOH solution (A), Luminol solution (B) and H2O2 solution (C). The working mix is composed of 1 part of A, 1 part of B, 1 part of C with 7 parts of water.7 After preparation this working mix can be used for several hours. For the chemiluminescence to be optimally visible, the environment has to be as dark as possible, so the experiments were carried out in a room without windows. On each occasion two pictures were made with the same digital camera after spraying with Luminol; the first with the light on and the second with the light off. Blood First we tested a drop of blood on a glass plate, before and after wiping off the glass plate with a premoistened microfibre cloth (Vecotec, Wecovi BV, Zwolle, Netherlands). Fluids used in haemodialysis After this we tested several materials frequently used in the haemodialysis unit which could possibly generate false-positive results with Luminol: (i) acid component of the bicarbonate dialysis fluid, (ii) disinfectant fluid, which is used for disinfection of the haemodialysis machine, and (iii) haemodialysis fluid. To be certain about the performance of the Luminol working mix, a drop of blood was tested as a positive control. Fluids used for cleaning/disinfection To estimate the frequency of false-positive results we tested two fluids commonly used in cleaning/ disinfection of the hospital environment with a known reactivity with Luminol: (i) sodium hypochlorite in household bleach and (ii) sodium hypochlorite supplied as a hospital disinfectant. Investigation of the haemodialysis unit On two evenings, when it was dark and the unit was unoccupied, we sprayed several objects in the haemodialysis unit. The objects were photographed twice with the same digital camera after spraying with Luminol; one with the light on and one immediately after the light was switched off. The following objects were investigated: haemodialysis chair, side table on which materials for connecting the patient to the dialysis machine are placed, haemodialysis machine, syringe pump, handles of cupboards, lid of the laundry container, telephone, keyboard of the computer and the floor of the haemodialysis unit.
Luminol for detection of invisible traces of blood
Results Experiments Blood A drop of blood resulted in a bright chemiluminescent reaction with Luminol. After wiping the glass plate with the microfibre cloth, the blue signal was no longer visible (Figure 1). Fluids used in haemodialysis None of the fluids used in haemodialysis generated a positive signal with the Luminol test. Fluids used for cleaning/disinfection After testing with Luminol there was a positive signal, although it was less intense and faded more rapidly than traces of blood.
Investigation of the haemodialysis unit No traces of blood could be detected by eye but with the Luminol method we detected traces of blood on many places in the unit. Some striking examples are shown in Figure 2. e Haemodialysis chair: Luminol showed that the chair had been cleaned but that blood had been moved to gaps in the covering.
Drop of blood on glass plate
Drop of blood on glass plate after spraying with luminol
Drop of blood on glass plate after spraying with luminol and after switching the light off
The same glass plate after wiping with microfiber cloth
Figure 1 Results from testing a drop of blood on a glass plate with Luminol in a room without windows. In the presence of metal ions or metal complexes such as haemoglobin, the reaction produces a bright blue chemiluminescence. This indicates blood traces.
331 e Side table: No blood was visible to the naked eye but after spraying Luminol a blue stain showed where there had been a spillage. e Haemodialysis machine and syringe pump: Luminol showed that the areas that were most frequently touched by fingertips were the most contaminated. e Handles of cupboards: The place where a sticker was attached to the cupboard gave a strong signal. e Lid of the laundry container: Places touched with hands were also contaminated with traces of blood. e Telephone and computer keyboard: The keys of these items showed the blue signal after spraying with Luminol in several places. e Floor: The floor of various haemodialysis rooms produced a blue glow. To investigate whether this was caused by the cleaning procedure we also tested the floor of the domestic service department of our hospital, which is of the same material and age and is cleaned in the same manner. This floor showed no chemiluminescence, so it was concluded that the floor of the haemodialysis rooms was extensively contaminated with minute blood traces.
Discussion Small outbreaks of hepatitis C infections have been reported from dialysis units for years.1e4 Since the cause could not be elucidated in most instances, it is usually concluded that a combination of contamination of the environment and poor infection control techniques played a role.6,12,13 Several studies support this hypothesis. In syringe needles contaminated with blood it has been shown by polymerase chain reaction (PCR) technique that human immunodeficiency virus can be detected for 30 days and HCV and HBV for at least one week and possibly up to eight months.14 Indeed, HCV-RNA and HbsAg have been isolated in the haemodialysis environment and HCV-RNA on the hands of healthcare workers of the unit.15,16 However, there is no direct evidence for the viability of those viruses in the environment, and the role of hospital surfaces as reservoir for nosocomial infection has not been fully elucidated.17 With the Luminol method we could demonstrate that contamination of our haemodialysis unit occurs in abundance, even after institution of strict preventive measures in response to our local HCV outbreak. As it is possible to obtain reliable PCR results from Luminol-treated bloodstains, the
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Control panel of haemodialysis machine
Side table
Handle of cupboard
Floor
Figure 2 Some examples of objects in the haemodialysis unit of the Deventer Hospital tested with Luminol. In the presence of metal ions or metal complexes such as haemoglobin, the reaction produces a bright blue chemiluminescence. This indicates blood traces. Left column: the object with the light on; right column: the object immediately after the light had been switched off.
Luminol for detection of invisible traces of blood Luminol method may facilitate future epidemiological studies.18,19 From a survey of 45 US haemodialysis centres, it is known that only a few healthcare workers on those units are aware of the fact that dialysis patients are at risk from bloodborne infections; strict adherence to recommendations for hand hygiene and the use of gloves is only 36%.20 In our opinion, a possible solution for improving this behaviour is to improve basic knowledge about possible transmission routes. This could be achieved by making invisible blood traces visible with the Luminol method. Confronting healthcare workers with photographs of the results was for most of them a real ‘eye-opener’, although we did not measure the effect of this intervention. The Luminol method is suitable for identifying contamination of the hospital environment in infection control audits. We intend to introduce the method for use in the haemodialysis unit, and also for other units at risk for contamination of the environment with blood, e.g. delivery rooms and operating theatres.21
Conflict of interest statement None declared. Funding sources None.
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