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Abstract
Background Hepatitis B virus (HBV) transmission is known to occur through direct contact with infected blood. There has been some suspicion that the virus can also be detected in aerosol form. However, this has never been directly shown. The purpose of this study was to sample and analyse surgical smoke from laparoscopic surgeries on patients with hepatitis B to determine whether HBV is present.
Methods A total of 11 patients who underwent laparoscopic or robotic abdominal surgeries between October 2014 and February 2015 at Korea University Anam Hospital were included in this study. A high efficiency collector was used to obtain surgical smoke in the form of hydrosol. The smoke was analysed by using nested PCR.
Results Robotic or laparoscopic colorectal resections were performed in 5 cases, laparoscopic gastrectomies in 3 cases and laparoscopic hepatic wedge resections in another 3 cases. Preoperatively, all of the patients had positive hepatitis B surface antigen (HBsAg). 2 patients had detectable HBsAb, and 2 were positive for hepatitis B e antigen. 3 patients were taking antihepatitis B viral medications at the time of the study. The viral load measured in the patients’ blood was undetectable to 1.7×108 IU/mL. HBV was detected in surgical smoke in 10 of the 11 cases.
Conclusions HBV is detectable in surgical smoke. This study provides preliminary data in the investigation of airborne HBV infection.
- Surgical smoke
- Hepatitis B virus
- HBV
- Laparoscopic surgery
- Transmission
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What this paper adds
Hepatitis B virus (HBV) can survive for ∼7 days in dried blood and even at acidic or high temperature condition.
Previous studies have discussed the possibility that HBV may be detected in medical settings; however, HBV has not been detected in the air.
In this study, HBV was isolated from surgical smoke using a high efficiency collector and being analysed by nested PCR.
The results recommend that steps should be taken to reduce surgical smoke and protect medical staff in the operating theatre when operating on patients with HBV.
Introduction
Electrosurgical devices, including electrocautery and vessel-sealing tools, are now widely used intraoperatively for haemostasis. These devices enable surgeons to perform minimally invasive surgery. However, the surgical smoke that arises from electrosurgical devices may expose the surgical team to potentially harmful chemicals, viruses and viable cells.1–5 Therefore, acquiring an infectious disease from surgical smoke represents a potential health hazard.2 ,6–8 Given this possibility, several institutions have implemented procedures to improve the safety of their operating rooms.9–11
Some viruses that were initially believed to be solely blood-borne also have other infectious routes. For example, HIV has been identified in laser smoke.12 Laser plumes containing human papilloma virus (HPV) may even produce warts on individuals who are exposed to the smoke.13 Viral particles have also been found in HPV-positive patients with laryngeal papillomatosis.14 Furthermore, the CO2 plume from a bovine papilloma virus-induced lesion was able to induce disease when it was reinoculated onto the skin of calves.15 Several groups have previously attempted to detect hepatitis B virus (HBV) from the surgical field. A study found that HBV was transmitted non-parenterally via an infected haemodialysis cannula.16 Furthermore, the filter-rinse technique has been used to isolate HBV from dried blood on stainless steel surfaces.17–19 However, HBV has never been detected in an aerosol form.
We hypothesised that HBV can be detected in surgical smoke and detected during laparoscopic surgery. The aim of this study, therefore, was to isolate and analyse HBV from the surgical smoke emitted during laparoscopic procedures.
Methods
Patients' eligibility for inclusion in study
The patients included in this study underwent either laparoscopic or robotic surgery. Both types of surgery expose major vessels and employ electrosurgical devices for haemostasis. In order to obtain a sufficient sample for analysis, we chose procedures that were at least 1 hour long in duration. These procedures were robotic or laparoscopic colorectal/gastric/hepatic resections. On routine preoperative evaluation, all of the patients were positive for hepatitis B surface antigen (HBsAg).
This study included 11 patients who underwent laparoscopic or robotic surgery between October 2014 and February 2015. It was approved by the Institutional Review Board (IRB, 2014.3, AN14019-001) of Korea University Anam Hospital. The study protocol was explained to the patients before they provided signed consent for their participation.
Sample collection
The BioSampler (SKC Inc, Eighty Four), also called the Swirling Aerosol Collector (SAC), was used in this study. It has a higher detection rate isolating smaller particles with less reaerosolisation compared with that of the All Glass Impinger-30.20 We used the SAC in connection with a vacuum pump operating at the specified flow rate. Phosphate buffered saline (PBS) was used during gas collection. PBS is preferred over physiological saline, nutrient broth and peptone water because it maintains pH and ion concentration. These factors allow for easier evaporation during liquid sample collection. After being used, BioSampler was sterilised with ethylene oxide.
Airborne particles and microorganisms generated at the surgical site were collected using the BioSampler. The BioSampler has inlet, sonic and outlet nozzles. The inlet nozzle was connected to the 5 mm laparoscopic port used to evacuate surgical smoke during the procedure (figure 1). One of the trocars in an antigravity position was used (figure 2A). They were ∼5 m between the operative site and the collector (figure 2B). A modified extension tube was used to minimise contamination by intra-abdominal blood or ascites. The collector also has three tangential nozzles, which act as sonic orifices. The outlet was connected to a vacuum pump at a flow rate of ∼12.5 L/min and a pressure drop of 0.5 atm (or 15 mm Hg). Surgical smoke was collected for 30 min after a major vessel was exposed (V (L)=Qs (L/min)×t (min), 375 L=12.5 L/mL× 30 min). The surgical particles were collected in 15 mL of sterilised PBS (figure 2C). After the sample was collected, the bottle was sealed aseptically and transferred to the laboratory for analysis.
Schematic of sampling technique used to collect surgical smoke during laparoscopic procedures.
(A) The trocar connected to the collector was located at an antigravity position. (B) A 5 m modified extension tube was used to minimise contamination. (C) Surgical smoke is collected in 15 mL of sterilised phosphate buffered saline in the SKC BioSampler.
Analysis
HBV DNA extraction
The Exgene SV mini kit (GeneAll, Seoul, Korea) was used to extract the genomic DNA and viral DNA according to the manufacturer’s protocol. The extracted DNA was either directly used for PCR or stored at −80°C until use.
Polymerase chain reaction
HBV gene amplification was carried out using 25 μL of Solg 2× Taq PCR Smart mix 2, 0.5× Band Doctor, dye (Solgent, Seoul, Korea), 0.4 μm of each primer, 5 μL of the template DNA and DNase/RNase free distilled water to make a total volume of 50 μL. The PCR reaction was performed in a Veriti 96-Well Thermal Cycler (Applied Biosystems, Foster City, California, USA). The following conditions were used: denaturation at 95°C for 5 min; 40 cycles of denaturation at 95°C for 30 s; annealing at 60°C for 40 s with a 0.1°C decrease after each cycle; elongation at 72°C for 1 min and elongation at 72°C for 5 min. The initial PCR cycle yielded minimal HBV DNA amplification. Therefore, nested PCR was performed, during which 5 μL of the initial PCR products were subjected to a second round of PCR.
PCR primers for HBV detection
The primers used for DNA amplification and HBV genome sequencing are the same as those previously described in Korea and Japan (table 1).21–23
Primers used in the analysis of nested PCR
DNA sequencing
The PCR products were separated by electrophoresis on 2% Tris-borate-EDTA agarose gel. The appropriate products were excised and purified using Expin Gel SV (GeneAll, Seoul, Korea). The purified DNA fragments were sequenced using the Big Dye terminator V.3.1 sequencing kit (ABI) and were analysed using the ABI 3730×1 DNA sequencer (ABI). Sequence analysis software was then used to read the sequences (GeneStudio Professional, http://genestudio.com/). The sequenced data were analysed using Basic Local Alignment Search Tool (BLAST) provided by the National Center Biotechnology Information (NCBI, http://www.ncbi.nlm.nih.gov/).
Terms
Some published papers use the term ‘aerosol’ with regard to viral particles in the air. ‘Aerosol’ refers to particles that are smaller than 1 μm in size and can enter the respiratory tract as small droplets. It is unclear if HBV isolated from smoke has infectivity. In this report, we used ‘aerosolised gas’ for the sample we collected from the surgical fields.
Results
Patients
There were eight male and three female patients between the ages of 38 and 74 included in this study. All of the patients were Korean with the exception of one woman from Uzbekistan. Body mass index ranged from 22.3 to 28.4. On the basis of the American Society of Anesthesiologists (ASA) classification system for describing patients' preoperative health status, nine patients had mild systemic disease (category II) and the other two had severe systemic disease (category III).24 The preoperative liver function tests and α fetoprotein values were within the normal ranges for all patients.
On routine preoperative evaluations, all of the patients were positive for HBsAg. Two patients had detectable HBsAb, and two were positive for hepatitis B e antigen (HBeAg). Three patients (cases 2, 10 and 11) were taking anti-HBV medications including entecavir (1 mg/day) for 2 years, entecavir (1 mg/day) for 7 years and lamivudine (100 mg/day), sdefovir (10 mg/day) or tenofovir (300 mg/day) for 15 years, respectively. The viral HBV DNA load in the blood ranged between undetectable and 1.7×108 IU/mL (table 2).
Patient characteristics and perioperative data
Five patients underwent colorectal resections, three had gastrectomies and three had hepatic wedge resections. The duration of the surgeries averaged 220 min (range 115–325). There were no blood transfusions performed intraoperatively. A median of 375 L of gas was collected in all but two cases. The samples were collected aseptically and delivered to the laboratory within 10 min. Three samples were temporarily stored at 4°C after collection, and transferred to the laboratory within 30–60 min (table 2).
PCR results
HBV was detected in 10 of the 11 samples of surgical smoke. For case 2, the forward response was positive for HBV; however, the backward response was negative (table 3).
PCR results of blood and surgical smoke analysis
Discussion
To the best of our knowledge, this is the first report of HBV isolated from laparoscopic surgical smoke. Our methods allowed for efficient and sensitive sample collection. Despite one negative result, HBV was successfully detected in surgical smoke using a high efficiency collector and nested PCR. Repeat analysis of the single negative case reproduced the same results: the forward response (214 sequences) was positive for HBV, while the backward response was negative. These results may reflect a failure of DNA extraction or an effect of the antiviral medications. However, the other responses were thought to be unrelated to the DNA quantitation or antiviral medication use.
HBV prevalence and transmission
According to the WHO, there are ∼240 million hepatitis B carriers worldwide. Each year, ∼780 000 people die of diseases related to HBV infection including liver cirrhosis, hepatic failure and hepatocellular carcinoma.25 Eastern Asia, sub-Saharan Africa and the Amazon are highly endemic areas of HBV.26 Although all Koreans should be vaccinated against HBV, however, immunity achieved from HBV vaccination can begin to wane after 15 years. Therefore, the National Health and Nutrition Examination Survey reported that 3.2% of the total population in Korea still were positive for HBeAg in 2011.27 HBV causes infection through vertical or horizontal transmission. In highly prevalent areas, the most common route of infection is from mother to child (perinatal transmission). Other means of infection include exposure to infected blood through intravenous drug use, or medical procedures involving mucosal surfaces.28–30 HBV can also be transmitted percutaneously through the saliva, semen, vaginal/menstrual secretions, sweat, breast milk, tears and urine.31–33
The theory of HBV detection
HBV can survive for ∼7 days in dried blood.34 Viral particles in surgical smoke may be aerosolised, such that they can be inhaled.35 Beginning in the 1970s, several studies have discussed the possibility that HBV may be detected in medical settings. It is theoretically possible for HBV to penetrate through mucosal surfaces into the circulatory system. Petersen36 was the first to investigate airborne HBV transmission. The group found that HBV transmission occurred through the direct filter-rinse technique from the dialysis machine in 7 out of 48 cases (14.6%). However, HBV was not detected in the air. This latter finding may have simply been a result of insufficient collection or analysis. Collecting and analysing gaseous particles require highly efficient sampling and analytical methods as were used in this study. It may be advantageous to collect samples during laparoscopic surgery because all of the surgical smoke that was emitted could be collected from a single port.
Detection and infectivity of other viruses in surgical smoke
In 1991, HIV DNA has been isolated in culture media using a CO2 laser.12 This experiment also demonstrated p24, which is a protein expressed in AIDS. The p24 was viable for 14 days after isolation. Regardless, the airborne infectivity of HIV is unknown.
A study reported the infectivity of HPV through the air in 1995.13 It was a population-based control study performed at Mayo Clinic between 1988 and 1992. A member of the surgical team developed a nasopharyngeal wart after exposure to HPV from a patient's genital wart. Though HPV was not isolated in the study, the authors concluded that HPV types which caused warts might infect the upper airway mucosa.
Prevention in the operating theatre
Hospital operating room staff are potentially exposed to hazardous chemicals, viable cells, bacteria and viruses in surgical smoke.2 Several professional practice, standard setting and government organisations have recommended methods to control surgical smoke generated from electrosurgical devices. These organisations include the Association of periOperative Registered Nurses (AORN), Occupational Safety and Health Administration (OSHA), American National Standards Institute (ANSI) and the National Institute for Occupational Safety and Health (NIOSH).6–8 Electrosurgical procedures that are open to the operating room environment may be more harmful than robotic or laparoscopic procedures with regard to uncontrolled surgical smoke emissions. Regardless, healthcare personnel must be trained to recognise and understand the risk when treating patients with HBV. To effectively control surgical smoke during laparoscopic procedures, a combination of proper operating room air exchanges and local exhaust ventilation (ie, laparoscopic smoke filtration devices which remove surgical smoke from the peritoneal cavity) should be used to protect healthcare workers and patients from exposure.
Limitations
This study has several limitations. Nested PCR is prone to false-positive results because there may be contamination of the first round's products. In order to minimise this error, we equipped the dedicated instruments and supplies at each stage of the procedure, and organised the workflow in the PCR area. There was no positive control to minimise the contamination by plasmid DNA-containing target nucleotide sequences.37 The protocol also continued through this set-up, regardless of the prior responses.
HBV is a robust virus that retains its immunogenicity and antigenicity on exposure to acid (pH 2.4 for at least 6 hours) and heat (98°C for 1 min; 60°C for 10 hours).38 Electrosurgical devices achieve temperatures as high as 200°C. However, the true temperature may be lower in non-target areas or when the device is only briefly activated.
It is also possible that blood and other bodily fluids were collected with the gaseous samples. The study setting was designed to prevent liquid products from being collected. The trocar connected to the collector was located at an antigravity position.
Future directions
Humans and chimpanzees were initially thought to be the only hosts of HBV. However, some studies have suggested that the virus even infects small animals such as mice.39–41 Therefore, small animal models may be useful to determine the infectivity of aerosolised HBV.
Conclusions
HBV was isolated from the surgical smoke emitted during laparoscopic abdominal surgery. This study provides preliminary data in the ongoing investigation of possible airborne transmission of HBV.
Acknowledgments
The authors thank Professor Kyong-Whan Moon from the College of Health Science, Korea University, for supplying the collector, and Kwang Mi Moon, Korea Bank for Pathogenic Viruses (KBPV) for her technical assistance.
References
Footnotes
Contributors HDK, S-HK and K-JS provided substantial contributions to the conception and design; or the acquisition, analysis or interpretation of the data. HDK, S-HK, YSS and K-JS were involved in drafting of the article or critical revision for important intellectual content. HDK and S-HK were involved in final approval of the version to be published.
Competing interests None declared.
Patient consent Obtained.
Ethics approval This study was approved by the Institutional Review Board (IRB, 2014.3., AN14019-001) of Korea University Anam Hospital.
Provenance and peer review Not commissioned; externally peer reviewed.