In Ontario, numbers for August show there were 319 cases of C.difficile, a deadly form of infectious diarrhea, found among the 228 hospital sites in Ontario. The provincial C.diff rate was 0.39 per 1,000 patient days.
Information from patientsafetyontario.net.
Monday, October 6, 2008
Disinfectants Can Make Bacteria Resistant To Treatment
Chemicals used in the environment to kill bacteria could be making them stronger, according to a paper published in the October issue of the journal Microbiology. Low levels of these chemicals, called biocides, can make the potentially lethal bacterium Staphylococcus aureus remove toxic chemicals from the cell even more efficiently, potentially making it resistant to being killed by some antibiotics.
Biocides are used in disinfectants and antiseptics to kill microbes. They are commonly used in cleaning hospitals and home environments, sterilizing medical equipment and decontaminating skin before surgery. At the correct strength, biocides kill bacteria and other microbes. However, if lower levels are used the bacteria can survive and become resistant to treatment.
"Bacteria like Staphylococcus aureus make proteins that pump many different toxic chemicals out of the cell to interfere with their antibacterial effects," said Dr Glenn Kaatz from the Department of Veterans Affairs Medical Center in Detroit, USA. "These efflux pumps can remove antibiotics from the cell and have been shown to make bacteria resistant to those drugs. We wanted to find out if exposure to biocides could also make bacteria resistant to being killed by the action of efflux pumps."
The researchers exposed S. aureus taken from the blood of patients to low concentrations of several biocides and dyes, which are also used frequently in hospitals. They looked at the effect of exposure on the bacteria and found that mutants that make more efflux pumps than normal were produced.
"We found that exposure to low concentrations of a variety of biocides and dyes resulted in the appearance of resistant mutants," said Dr Kaatz. "The number of efflux pumps in the bacteria increased. Because the efflux pumps can also rid the cell of some antibiotics, pathogenic bacteria with more pumps are a threat to patients as they could be more resistant to treatment."
If bacteria that live in protected environments are exposed to biocides repeatedly, for example during cleaning, they can build up resistance to disinfectants and antibiotics. Such bacteria have been shown to contribute to hospital-acquired infections.
"Scientists are trying to develop inhibitors of efflux pumps. Effective inhibitors would reduce the likelihood of additional resistance mechanisms emerging in bacteria," said Dr Kaatz. "Unfortunately, inhibitors evaluated to date do not work on a wide range of pathogens so they are not ideal to prevent resistance."
"Careful use of antibiotics and the use of biocides that are not known to be recognised by efflux pumps may reduce the frequency at which resistant strains are found," said Dr Kaatz. "Alternatively, the combination of a pump inhibitor with an antimicrobial agent or biocide will reduce the emergence of such strains and their clinical impact."
--------------------------------------------------------------------------------
Adapted from materials provided by Society for General Microbiology, via EurekAlert!, a service of AAAS.
Source - Science Daily
Biocides are used in disinfectants and antiseptics to kill microbes. They are commonly used in cleaning hospitals and home environments, sterilizing medical equipment and decontaminating skin before surgery. At the correct strength, biocides kill bacteria and other microbes. However, if lower levels are used the bacteria can survive and become resistant to treatment.
"Bacteria like Staphylococcus aureus make proteins that pump many different toxic chemicals out of the cell to interfere with their antibacterial effects," said Dr Glenn Kaatz from the Department of Veterans Affairs Medical Center in Detroit, USA. "These efflux pumps can remove antibiotics from the cell and have been shown to make bacteria resistant to those drugs. We wanted to find out if exposure to biocides could also make bacteria resistant to being killed by the action of efflux pumps."
The researchers exposed S. aureus taken from the blood of patients to low concentrations of several biocides and dyes, which are also used frequently in hospitals. They looked at the effect of exposure on the bacteria and found that mutants that make more efflux pumps than normal were produced.
"We found that exposure to low concentrations of a variety of biocides and dyes resulted in the appearance of resistant mutants," said Dr Kaatz. "The number of efflux pumps in the bacteria increased. Because the efflux pumps can also rid the cell of some antibiotics, pathogenic bacteria with more pumps are a threat to patients as they could be more resistant to treatment."
If bacteria that live in protected environments are exposed to biocides repeatedly, for example during cleaning, they can build up resistance to disinfectants and antibiotics. Such bacteria have been shown to contribute to hospital-acquired infections.
"Scientists are trying to develop inhibitors of efflux pumps. Effective inhibitors would reduce the likelihood of additional resistance mechanisms emerging in bacteria," said Dr Kaatz. "Unfortunately, inhibitors evaluated to date do not work on a wide range of pathogens so they are not ideal to prevent resistance."
"Careful use of antibiotics and the use of biocides that are not known to be recognised by efflux pumps may reduce the frequency at which resistant strains are found," said Dr Kaatz. "Alternatively, the combination of a pump inhibitor with an antimicrobial agent or biocide will reduce the emergence of such strains and their clinical impact."
--------------------------------------------------------------------------------
Adapted from materials provided by Society for General Microbiology, via EurekAlert!, a service of AAAS.
Source - Science Daily
HIV/AIDS Emerged as Early as 1880s
October 1, 2008
The AIDS pandemic in humans originated at least three decades earlier than previously thought, and it may have been triggered by rapid urbanization in west-central Africa during the early 20th century, according to an international team of researchers.
A better understanding of the conditions that helped fuel the pandemic could be key in controlling the disease and preventing future outbreaks of other emerging viruses.
"Rapid urbanization was the turning point that allowed the pandemic to start," said Michael Worobey, an evolutionary biologist at the University of Arizona, Tucson, and lead author of the study.
"We as human beings made some changes that took a virus that could not exist on its own and turned it into a successful epidemic," he added.
Birth of AIDS
Until now it was thought that HIV-1 Group M, the strain of HIV that causes the most infections worldwide, originated in 1930 in Cameroon.
Epidemic levels of AIDS and HIV-1 infections started appearing in Leopoldville, Belgian Congo (now Kinshasa, Democratic Republic of the Congo), around 1960.
Findings from the new study, however, suggest that the virus most likely started circulating among humans in sub-Saharan Africa sometime between 1884 and 1924.
Worobey and his colleagues made the discovery while analyzing tissue samples collected between 1958 and 1960 from Kinshasa. One of them, acquired in 1960, contained bits of HIV-1 RNA, the virus's genetic material.
The researchers compared the 1960 virus with the oldest known HIV-1 strain, which was obtained in 1959 and evolved independently of the 1960 variant. They found that the 1960 version was significantly different.
Next the researchers constructed an evolutionary family tree of the HIV-1 virus, made up of both the 1959 and 1960 strains along with more than a hundred modern viral sequences.
Using a mathematical model, Worobey and his colleagues discovered that the 1960 strain must have been evolving for at least 40 years to account for the number of differences from the 1959 strain.
The model also traced the most recent common ancestor of both strains to 1908.
The team's findings appear today in the journal Nature.
Lurking Danger
"This confirms that this was a virus that was lurking around for many decades before it exploded into the human population to become a noticeable pandemic, as opposed to something that started in the '70s or '80s," said Anthony Fauci, director of the National Institute of Allergy and Infectious Diseases (NIAID), in Maryland.
NIAID funded the study along with the National Institutes of Health.
"It solidifies our understanding of the timetable of how this virus emerged from the chimpanzees to establish itself as a human infection," Fauci added.
"[HIV-1] flew below the radar level for decades until social conditions such as the end of colonization, migration of people to cities, increase in prostitution, and promiscuous sexual activity made it much easier for the disease to explode into a pandemic," Fauci explained.
More Mutations
Robert Garry is a microbiologist at New Orleans's Tulane University who was not involved with the study.
In the late 1980s, Garry was the first scientist to examine tissues samples taken from the U.S.'s first confirmed AIDS patient, who died in 1969.
"This study is very important, and what they are finding here is when the human virus started circulating in people," he said. "We still don't know when exactly the virus jumped from chimpanzees to humans, but it may be pushed back even further with this study.
"There will be other emerging viruses in the future, and what we learn about the conditions that help viruses spread, be it social changes or changes within the virus itself, will make us better prepared for other epidemics," he said.
Garry also argues that, though the rapid growth of cities in west-central Africa may have sparked the spread of infections, the virus itself underwent some sort of genetic change to facilitate transmission.
"We have to figure out what that change was," he cautioned.
Source:Amitabh Avasthi - National Geographic News
The AIDS pandemic in humans originated at least three decades earlier than previously thought, and it may have been triggered by rapid urbanization in west-central Africa during the early 20th century, according to an international team of researchers.
A better understanding of the conditions that helped fuel the pandemic could be key in controlling the disease and preventing future outbreaks of other emerging viruses.
"Rapid urbanization was the turning point that allowed the pandemic to start," said Michael Worobey, an evolutionary biologist at the University of Arizona, Tucson, and lead author of the study.
"We as human beings made some changes that took a virus that could not exist on its own and turned it into a successful epidemic," he added.
Birth of AIDS
Until now it was thought that HIV-1 Group M, the strain of HIV that causes the most infections worldwide, originated in 1930 in Cameroon.
Epidemic levels of AIDS and HIV-1 infections started appearing in Leopoldville, Belgian Congo (now Kinshasa, Democratic Republic of the Congo), around 1960.
Findings from the new study, however, suggest that the virus most likely started circulating among humans in sub-Saharan Africa sometime between 1884 and 1924.
Worobey and his colleagues made the discovery while analyzing tissue samples collected between 1958 and 1960 from Kinshasa. One of them, acquired in 1960, contained bits of HIV-1 RNA, the virus's genetic material.
The researchers compared the 1960 virus with the oldest known HIV-1 strain, which was obtained in 1959 and evolved independently of the 1960 variant. They found that the 1960 version was significantly different.
Next the researchers constructed an evolutionary family tree of the HIV-1 virus, made up of both the 1959 and 1960 strains along with more than a hundred modern viral sequences.
Using a mathematical model, Worobey and his colleagues discovered that the 1960 strain must have been evolving for at least 40 years to account for the number of differences from the 1959 strain.
The model also traced the most recent common ancestor of both strains to 1908.
The team's findings appear today in the journal Nature.
Lurking Danger
"This confirms that this was a virus that was lurking around for many decades before it exploded into the human population to become a noticeable pandemic, as opposed to something that started in the '70s or '80s," said Anthony Fauci, director of the National Institute of Allergy and Infectious Diseases (NIAID), in Maryland.
NIAID funded the study along with the National Institutes of Health.
"It solidifies our understanding of the timetable of how this virus emerged from the chimpanzees to establish itself as a human infection," Fauci added.
"[HIV-1] flew below the radar level for decades until social conditions such as the end of colonization, migration of people to cities, increase in prostitution, and promiscuous sexual activity made it much easier for the disease to explode into a pandemic," Fauci explained.
More Mutations
Robert Garry is a microbiologist at New Orleans's Tulane University who was not involved with the study.
In the late 1980s, Garry was the first scientist to examine tissues samples taken from the U.S.'s first confirmed AIDS patient, who died in 1969.
"This study is very important, and what they are finding here is when the human virus started circulating in people," he said. "We still don't know when exactly the virus jumped from chimpanzees to humans, but it may be pushed back even further with this study.
"There will be other emerging viruses in the future, and what we learn about the conditions that help viruses spread, be it social changes or changes within the virus itself, will make us better prepared for other epidemics," he said.
Garry also argues that, though the rapid growth of cities in west-central Africa may have sparked the spread of infections, the virus itself underwent some sort of genetic change to facilitate transmission.
"We have to figure out what that change was," he cautioned.
Source:Amitabh Avasthi - National Geographic News
Saturday, September 27, 2008
Australia in biosecurity 'hotspot'
Australia is "encased by a ring of fire" with modelling by international experts showing Asia to be the most likely source of the next global infectious disease outbreak.
The panel of biosecurity and infectious disease experts also warn that a "fortress Australia" approach is not enough to stop bio-threats entering the country.
Instead Australia must also help develop the region's capacity to deal with and prevent infectious disease outbreaks, they say.
The presentation came ahead of today's launch, by the Australian Biosecurity Cooperative Research Centre, of the Biosecurity Risk Intelligence Scanning Committee.
Committee chair Professor John Edwards, Dean of the School of Veterinary and Biomedical Sciences at Murdoch University, says the group aims to predict emerging threats and inform research priorities.
"Australia has some of the best biosecurity systems in the world," Edwards says.
"But biosecurity is something you can never do well enough."
Edwards says greater attention needs to be given to animal viruses as up to 70% have zoonotic potential - can be passed to humans.
Priority assessments
He says the new committee will as a priority assess the risk of bat-borne viruses such as the Hendra-like Nipah virus, the dengue-like Chikungunya virus and Bluetongue virus. The later is spread by a biting midge and causes serious disease in livestock, particularly sheep.
Dr Peter Daszak, executive director of the Consortium for Conservation Medicine, says modelling of the likely areas where new viruses will emerge shows the region to the north of Australia to be most at risk.
"Australia is surrounded by the hotspot for emerging diseases and they are areas with incredibly low surveillance effort," he says.
"To understand the risk of new viruses we have to know what viruses wildlife carry, and of course we don't.
"We need to understand what makes them emerge and deal with that before they emerge."
'Hottest of hotspots'
Daszak says more than half of all emerging disease come from wildlife, yet estimates suggest 99.8% of viral diversity is unknown.
While it is hard to predict what the next pandemic will be, Daszak says "we can get a handle on where the next one will come from".
"The countries that border Australia are becoming the hottest of the hot spots," he says, adding we are "encased" by a biosecurity "ring of fire".
"The networks of trade and travel mean we are extremely connected to areas where diseases are emerging and therefore [Australia is] at high risk," Daszak says.
His view is supported by World Health Organization deputy regional advisor for communicable disease surveillance and response Dr Julie Hall.
Hall says the Asia-Pacific region has all the "drivers that enhance or create an environment" where new disease can emerge.
This includes high population density, significant poultry populations, and natural disasters and climate change causing large migrations of population.
'One new disease a year'
Hall says 70% of new emerging diseases are expected to come from animals and globally during the past 30 years on average one new disease a year has emerged.
She says the emerging threats in the region at present come from vector-borne disease, spread by mosquitoes.
This includes Dengue fever and Chikungunya virus.
However, Hall warns the most serious threat to the nation's biosecurity is "influenza fatigue" where interest wanes in the field because the threatened pandemic does not arrive.
This would mean a cut in funding to safeguards and the erosion of systems that prevent the pandemics from occurring.
Source: ABC
The panel of biosecurity and infectious disease experts also warn that a "fortress Australia" approach is not enough to stop bio-threats entering the country.
Instead Australia must also help develop the region's capacity to deal with and prevent infectious disease outbreaks, they say.
The presentation came ahead of today's launch, by the Australian Biosecurity Cooperative Research Centre, of the Biosecurity Risk Intelligence Scanning Committee.
Committee chair Professor John Edwards, Dean of the School of Veterinary and Biomedical Sciences at Murdoch University, says the group aims to predict emerging threats and inform research priorities.
"Australia has some of the best biosecurity systems in the world," Edwards says.
"But biosecurity is something you can never do well enough."
Edwards says greater attention needs to be given to animal viruses as up to 70% have zoonotic potential - can be passed to humans.
Priority assessments
He says the new committee will as a priority assess the risk of bat-borne viruses such as the Hendra-like Nipah virus, the dengue-like Chikungunya virus and Bluetongue virus. The later is spread by a biting midge and causes serious disease in livestock, particularly sheep.
Dr Peter Daszak, executive director of the Consortium for Conservation Medicine, says modelling of the likely areas where new viruses will emerge shows the region to the north of Australia to be most at risk.
"Australia is surrounded by the hotspot for emerging diseases and they are areas with incredibly low surveillance effort," he says.
"To understand the risk of new viruses we have to know what viruses wildlife carry, and of course we don't.
"We need to understand what makes them emerge and deal with that before they emerge."
'Hottest of hotspots'
Daszak says more than half of all emerging disease come from wildlife, yet estimates suggest 99.8% of viral diversity is unknown.
While it is hard to predict what the next pandemic will be, Daszak says "we can get a handle on where the next one will come from".
"The countries that border Australia are becoming the hottest of the hot spots," he says, adding we are "encased" by a biosecurity "ring of fire".
"The networks of trade and travel mean we are extremely connected to areas where diseases are emerging and therefore [Australia is] at high risk," Daszak says.
His view is supported by World Health Organization deputy regional advisor for communicable disease surveillance and response Dr Julie Hall.
Hall says the Asia-Pacific region has all the "drivers that enhance or create an environment" where new disease can emerge.
This includes high population density, significant poultry populations, and natural disasters and climate change causing large migrations of population.
'One new disease a year'
Hall says 70% of new emerging diseases are expected to come from animals and globally during the past 30 years on average one new disease a year has emerged.
She says the emerging threats in the region at present come from vector-borne disease, spread by mosquitoes.
This includes Dengue fever and Chikungunya virus.
However, Hall warns the most serious threat to the nation's biosecurity is "influenza fatigue" where interest wanes in the field because the threatened pandemic does not arrive.
This would mean a cut in funding to safeguards and the erosion of systems that prevent the pandemics from occurring.
Source: ABC
World Faces Global Pandemic Of Antibiotic Resistance, Experts Warn
Sep. 18, 2008) — Vital components of modern medicine such as major surgery, organ transplantation, and cancer chemotherapy will be threatened if antibiotic resistance is not tackled urgently, warn experts on bmj.com.
A concerted global response is needed to address rising rates of bacterial resistance caused by the use and abuse of antibiotics or "we will return to the pre-antibiotic era", write Professor Otto Cars and colleagues in an editorial.
All antibiotic use "uses up" some of the effectiveness of that antibiotic, diminishing the ability to use it in the future, write the authors, and antibiotics can no longer be considered as a renewable source.
They point out that existing antibiotics are losing their effect at an alarming pace, while the development of new antibiotics is declining. More than a dozen new classes of antibiotics were developed between 1930 and 1970, but only two new classes have been developed since then.
According to the European Centre for Disease Prevention and Control, the most important disease threat in Europe is from micro-organisms that have become resistant to antibiotics. As far back as 2000, the World Health Organisation was calling for a massive effort to address the problem of antimicrobial resistance to prevent the "health catastrophe of tomorrow".
So why has so little been done to address the problem of resistance, ask the authors?
Antibiotics are over prescribed, still illegally sold over the counter in some EU countries, and self medication with leftover medicines is commonplace.
There are alarming reports about serious consequences of antibiotic resistance from all around the world. However, there is still a dearth of data on the magnitude and burden of antibiotic resistance, or its economic impact on individuals, health care, and society. This, they suggest, may explain why there has been little response to this public health threat from politicians, public health workers, and consumers.
In addition, there are significant scientific challenges but few incentives to developing new antibiotics, state the authors.
The authors believe that priority must be given to the most urgently needed antibiotics and incentives given for developing antibacterials with new mechanisms of action. In addition, "the use of new antibiotics must be safeguarded by regulations and practices that ensure rational use, to avoid repeating the mistakes we have made by overusing the old ones", they say.
They point out that reducing consumer demand could be the strongest force to driving change—individuals must be educated to understand that their choice to use an antibiotic will affect the possibility of effectively treating bacterial infections in other people.
But, they claim, the ultimate responsibility for coordination and resources rests with national governments, WHO and other international stakeholders.
Not only is there an urgent need for up-to-date information on the level of antibiotic resistance, but also for evidence of effective interventions for the prevention and control of antibiotic resistance at national and local levels, while more focus is needed on infectious diseases, they conclude.
--------------------------------------------------------------------------------
Adapted from materials provided by BMJ-British Medical Journal, via EurekAlert!, a service of AAAS.
Source: ScienceDaily
A concerted global response is needed to address rising rates of bacterial resistance caused by the use and abuse of antibiotics or "we will return to the pre-antibiotic era", write Professor Otto Cars and colleagues in an editorial.
All antibiotic use "uses up" some of the effectiveness of that antibiotic, diminishing the ability to use it in the future, write the authors, and antibiotics can no longer be considered as a renewable source.
They point out that existing antibiotics are losing their effect at an alarming pace, while the development of new antibiotics is declining. More than a dozen new classes of antibiotics were developed between 1930 and 1970, but only two new classes have been developed since then.
According to the European Centre for Disease Prevention and Control, the most important disease threat in Europe is from micro-organisms that have become resistant to antibiotics. As far back as 2000, the World Health Organisation was calling for a massive effort to address the problem of antimicrobial resistance to prevent the "health catastrophe of tomorrow".
So why has so little been done to address the problem of resistance, ask the authors?
Antibiotics are over prescribed, still illegally sold over the counter in some EU countries, and self medication with leftover medicines is commonplace.
There are alarming reports about serious consequences of antibiotic resistance from all around the world. However, there is still a dearth of data on the magnitude and burden of antibiotic resistance, or its economic impact on individuals, health care, and society. This, they suggest, may explain why there has been little response to this public health threat from politicians, public health workers, and consumers.
In addition, there are significant scientific challenges but few incentives to developing new antibiotics, state the authors.
The authors believe that priority must be given to the most urgently needed antibiotics and incentives given for developing antibacterials with new mechanisms of action. In addition, "the use of new antibiotics must be safeguarded by regulations and practices that ensure rational use, to avoid repeating the mistakes we have made by overusing the old ones", they say.
They point out that reducing consumer demand could be the strongest force to driving change—individuals must be educated to understand that their choice to use an antibiotic will affect the possibility of effectively treating bacterial infections in other people.
But, they claim, the ultimate responsibility for coordination and resources rests with national governments, WHO and other international stakeholders.
Not only is there an urgent need for up-to-date information on the level of antibiotic resistance, but also for evidence of effective interventions for the prevention and control of antibiotic resistance at national and local levels, while more focus is needed on infectious diseases, they conclude.
--------------------------------------------------------------------------------
Adapted from materials provided by BMJ-British Medical Journal, via EurekAlert!, a service of AAAS.
Source: ScienceDaily
QualSec's eNose Detects Emerging Global Health Threats
NanoNose Detector Turns Epidemic Intelligence into a Predictive Science
CHAPEL HILL, N.C.--(BUSINESS WIRE)--QualSec (OTCBB: QLSCB):
In 1918, a strain of influenza, popularly known as the Spanish flu, ravaged the globe, killing an estimated 2.5% of the world's population. Now, concerns about new pandemics have led the global public health community to seek early warning systems to identify disease "hot spots" and enable rapid responses to prevent epidemics. The technological breakthrough that epidemiologists are looking for exists with QualSec’s NanoNose.
As environmental changes worldwide bring people in greater contact with animals, zoonoses - diseases that can transfer between humans and animals – are increasingly emerging as threats, including diseases like avian influenza, hantavirus, Ebola and West Nile viruses. QualSec’s NanoNose uses patented breakthroughs in nanotechnology for rapid data collection, allowing health officials better disease surveillance and vulnerability mapping.
This electronic nose technology developed by QualSec will be able to rapidly identify "hot spots" that could become epidemic nodes. Rapid detection is crucial for effective epidemic intelligence.
QualSec’s NanoNose is highly portable and rugged, and can be easily deployed to remote areas and used by operators with minimal training to detect epidemics before they spread. Though the sensor is designed to process data individually, it can be linked into a wireless network and transmit findings of disease to a central response center, where global health organizations can react to disease outbreaks.
For further details on the company and its products please visit the company's website at www.qualsensors.com.
QualSec believes that this press release contains forward-looking statements as that term is defined by the Private Securities Litigation Reform Act of 1995. Such forward-looking statements are subject to risks and uncertainties. Such statements are based on management's current expectations and are subject to facts that could cause results to differ materially from the forward-looking statements. For further information you are encouraged to view QualSec's filings with the Securities and Exchange Commission, including its annual report on Form 10K for the period ending December 31, 2007. The Company assumes no obligation to update the information contained in this press release.
CHAPEL HILL, N.C.--(BUSINESS WIRE)--QualSec (OTCBB: QLSCB):
In 1918, a strain of influenza, popularly known as the Spanish flu, ravaged the globe, killing an estimated 2.5% of the world's population. Now, concerns about new pandemics have led the global public health community to seek early warning systems to identify disease "hot spots" and enable rapid responses to prevent epidemics. The technological breakthrough that epidemiologists are looking for exists with QualSec’s NanoNose.
As environmental changes worldwide bring people in greater contact with animals, zoonoses - diseases that can transfer between humans and animals – are increasingly emerging as threats, including diseases like avian influenza, hantavirus, Ebola and West Nile viruses. QualSec’s NanoNose uses patented breakthroughs in nanotechnology for rapid data collection, allowing health officials better disease surveillance and vulnerability mapping.
This electronic nose technology developed by QualSec will be able to rapidly identify "hot spots" that could become epidemic nodes. Rapid detection is crucial for effective epidemic intelligence.
QualSec’s NanoNose is highly portable and rugged, and can be easily deployed to remote areas and used by operators with minimal training to detect epidemics before they spread. Though the sensor is designed to process data individually, it can be linked into a wireless network and transmit findings of disease to a central response center, where global health organizations can react to disease outbreaks.
For further details on the company and its products please visit the company's website at www.qualsensors.com.
QualSec believes that this press release contains forward-looking statements as that term is defined by the Private Securities Litigation Reform Act of 1995. Such forward-looking statements are subject to risks and uncertainties. Such statements are based on management's current expectations and are subject to facts that could cause results to differ materially from the forward-looking statements. For further information you are encouraged to view QualSec's filings with the Securities and Exchange Commission, including its annual report on Form 10K for the period ending December 31, 2007. The Company assumes no obligation to update the information contained in this press release.
Tuesday, August 26, 2008
Changing Pattern of Clostridium Difficile Associated Diarrhoea in a Tertiary Care Hospital: A 5 Year Retrospective Study
Changing Pattern of Clostridium Difficile Associated Diarrhoea in a Tertiary Care Hospital: A 5 Year Retrospective Study
Posted on: Tuesday, 26 August 2008, 03:00 CDT
By Chaudhry, Rama Joshy, Lovely; Kumar, Lalit; Dhawan, Benu
Background & objectives: Frequent use of broad spectrum antibiotics in hospitalized patients has increased the incidence of Clostridium difficile diarrhoea in recent years. In our tertiary care hospital in north India, C. difficile was responsible for 15 per cent of cases of nosocomial diarrhoea in 1999. A retrospective study was carried out to determine the frequency of C. difficile associated diarrhoea (CdAD) in our hospital, and to assess the effect of awareness among the hospital personnel and control measures taken to present C. difficile infection following the previous report. Methods: A retrospective chart review of all suspected cases of CdAD diagnosed at the hospital from January 2001 to December 2005 was done. Clinical specimens comprised 524 stool samples. All the samples were analyzed for C. difficile using culture and ELISA for toxin A and B. Attempts were made to type isolates using antibiogram, SDS-PAGE, gas liquid chromatography (GLC), PCR for toxin A and B gene fragments and restriction fragment length polymorphism (RFLP).
Results: A total of 37 (7.1%) specimens were positive for C. difficile toxin (11.2% in 2001, 9.4% in 2002, 8.6% in 2003, 5% in 2004 and 4% in 2005). The highest number of C. difficile toxin positive cases were from stool samples of patients hospitalized in the haematology/oncology ward (67.5% of all positive cases) followed by gastrointestinal surgery, neurology and nephrology wards. Of the C. difficile toxin positive samples, 15 (41%) were also positive for C. difficile culture. The isolates were grouped in to one, 3 and 5 groups using antibiogram, SDS-PAGE and PCR RFLP respectively. We observed an increase in the number of stool specimens tested for C. difficile infection but a decrease in C. difficile positives.
Interpretation & conclusions: A decrease in the number of C. difficile positive cases were noted during the 5 year study period though number of samples tested was increased. This may be due to stringent surveillance and an improved antibiotic policy followed in the hospital.
Diarrhoea is one of the most frequent side effects of antibiotic treatment. The symptoms may vary from slight abdominal discomfort to severe diarrhoea to colitis1. The aetiology of antibiotic associated diarrhoea (AAD) varies. The disruption of normal enteric flora caused by antibiotics may lead to overgrowth of pathogens and functional disturbances of the intestinal carbohydrate and bile acid metabolism, resulting in osmotic diarrhoea1. Allergic, toxic and pharmacological effects of antibiotics may also affect the intestinal mucosa and motility2. Cytotoxin producing Clostridium difficile has been reported to be the causative agent of approximately 20 per cent of AAD and of nearly all cases of pseudomembraneous colitis, the most severe manifestation of AAD1. Because of the frequent use of broad spectrum antibiotics, the incidence of C. difficile diarrhoea has risen dramatically in recent years3,4. Established guidelines should be followed to minimize exposure to the pathogen which include judicious use of antibiotics, rapid detection of C. difficile by immunoassays for toxin A and B, isolation of patients who had C. difficile associated diarrhoea (CdAD), proper disinfection of objects and education of staff members5. In our hospital which is a tertiary care hospital in north India, C. difficile was responsible for 15 per cent of cases of nosocomial diarrhoea in 1999(6). Standard control measures were implemented in our hospital to minimize the spread of this nosocomial pathogen after this report. This retrospective analysis was carried out in continuation of our earlier study6 to determine the effect of awareness and control measures taken to contain C. difficile infection in our hospital during the subsequent years.
Material & Methods
The study comprised retrospective analysis of faecal specimens from 524 patients suspected on clinical grounds to have CdAD2. The patients were hospitalized in All India Institute of Medical Sciences, New Delhi, India, over a period of 5 yr (January 1, 2001 - December 31, 2005). These included 80 patients in 2001, 96 in 2002, 92 in 2003, 106 in 2004 and 150 patients in 2005 respectively. Of these, 53 per cent were males and 82.4 per cent were in all age group > 12-60 yr (Table I).
Clinical information about the cause of diarrhoea underlying disease and antimicrobial therapy was obtained by reviewing the patient charts. A patient was considered to have CdAD if AAD was present and a stool specimen was positive in a toxin dependent C. difficile assay.
Sample collection and isolation of C. difficile: All the stool specimens were processed immediately for culture of C. difficile and stool aliquots were stored at -20[degrees]C for <72 h till they were tested for C. difficile toxin A and B. Spore selection was performed using 95 per cent ethanol and culture for C. difficile was done on cycloserine cefoxitin fructose agar (CCFA) and brain heart infusion agar (BHIA) as described elsewhere6. Concurrently, a loopful of stool specimen was inoculated into Robertsons cooked meat broth and incubated at 37[degrees]C for 48 h.
The plates were incubated anaerobically at 37[degrees]C in an anaerobic jar for 48 h. After incubation, the plates were examined and colonies which resembled C. difficile were Gram stained and identified by biochemical reactions using standard methods7.
When culture plate were negative for C. difficile, subcultures were made from cooked meat broth onto CCFA and BHIA and incubated anaerobically at 37[degrees]C up to 5 days before being discarded as negative.
Enzyme immunoassay for toxin A and B: Detection of enterotoxin and cytotoxin (toxin A and toxin B) of C. difficile was performed on the stool specimens by a double sandwich enzyme-linked immunosorbent assay technique using a commercial kit (Premier toxins A & B; Meridian Diagnostics, Inc., Cincinnati, Ohio, USA). The assay was performed according to the manufacturer's instructions.
Characterization of C. difficile isolates: All the C. difficile isolates were characterized phenotypically using antibiogram, SDS- PAGE6, gas liquid chromatography (GLC)7, and genotypically using PCR for toxin A gene and RFLP8,9.
Antibiogram typing - Antibiogram patterns were determined by disc diffusion method10. The antibiotics tested were chloramphenicol (30 [mu]g), penicillin G (10 units), clindamycin (2 [mu]g), vancomycin (5 [mu]g); metronidazole (5 [mu]g), tetracycline (30 [mu]g) and erythromycin (10 [mu]g). The results were expressed as susceptible or resistant.
Analysis of volatile fatty acids by gas liquid chromatography (GLC): All isolates were inoculated to cooked meat broth and incubated anaerobically for 48 h or more for GLC analysis to detect volatile fatty acids produced as metabolic end products. 1 ml of RCM broth was acidified with 0.2 ml of 50 per cent sulphuric acid and extracted with 1 ml of diethyl ether. The mixture was shaken vigorously and centrifuged at 176 g for 3 min; 1.5 [mu]l of the extracted ether layer was injected to the injection port of preconditioned GLC column with a 10 [mu]l Hamilton syringe. Chromatography was performed on a Nucon Series 5700, fitted with a flame ionization detector (FID)7. Operating conditions were as follows: carrier gas (oxygen free nitrogen): 60 ml/min oven temperature: detector 240[degrees]C, column 175[degrees]C, injector 240[degrees]C, attenuation 4X, sensitivity of the detector was set at 1000X. Fatty acids were identified by comparing the retention times of peaks in the test samples with those of known standard solutions which were examined each day7.
PCR assay for toxin gene fragments: The presence of toxin A gene in all isolates of C. difficile was determined by specific PCR using published primers8. PCR to detect the toxin B gene was performed in the C. difficile isolates using primers that had been developed and validated by Gumerlock et at9 to yield a 399-bp fragment for toxin B gene. PCR was performed in a 25 [mu]l reaction volume. Each reaction tube contained 1 X buffer (10 mm Tris HCl, pH 8.3, 50 mm KCl, 2.5 mm MgCl^sub 2^, 0.001% gelatin), each deoxynuclotide at a concentration of 100 mm (MBI, Fermentas, USA), each primer at a concentration of 20 pmol, 1.25 U Taq polymerase (MBI, Fermantas, USA) and 10 [mu]l of DNA. PCR was performed for 2 min at 95[degrees]C followed by 30 cycles of 1 min of denaturation at 95[degrees]C, 1 min of annealing at 52[degrees]C and 1 min of extension at 72[degrees]C. After the 30th cycle, extension was continued for an additional 10 min. 10 [mu]l of the amplified product was analyzed in 1 per cent agarose gel stained with ethidium bromide.
PCR-restriction fragment length polymorphism (RFLP) analysis: The amplified toxin A gene fragment was then digested with Alu I(10 units) restriction enzyme, under conditions recommended by the supplier (MBI-Fermentas). These digests were then subjected to electrophoresis on 2.5 per cent agarose gel at 60 V, along side a PCR size marker (100 bps, Sigma, USA).
Comparisons of patterns were performed visually. Strains with patterns differing alteast by one band were assigned to different types.
Results
A total of 524 stool specimens were analyzed for C. difficile from suspected cases of CdAD. The maximum number of C. difficile suspected cases were from oncology ward (378 cases, 72%), followed by other wards such as gastrointestinal surgery, neurology, nephrology and other medical wards. A total of 95 per cent of the analyzed group were on multiple antibiotics which included, 65 per cent on cephalosporins, 35 per cent on quinolones, 43 per cent on aminoglycosides, 12 per cent on macrolides, 69 per cent in vancomycin and metronidazole. Of the analyzed group, 37 (7.1%) patients were positive for C. difficile infection by the toxin dependent assay. Of these, 9 samples (11.2%) were positive in 2001, 9 (9.4%) in 2002, 8 (8.6%) in 2003, 5 (5%) in 2004 and 6 patients (4%) in 2005 (Fig. 1). Fifteen (41%) of the 37 toxin positive stool samples were also positive for C. difficile by culture. Eight of the 37 toxin positive cases expired, the cause of death was not directly related to C. difficile diarrhoea, although this might have been a contributory factor. Other pathogenic clostridia isolated from the patient group included C. perfringens (2.5%).
The highest number of C. difficile toxin positive cases were from stool samples of patients hospitalized in the haematology/oncology ward (25 samples, 67.5% of all positive cases), followed by gastrointestinal surgery, neurology and nephrology wards. Recovery rates of C. difficile in patient populations surveyed and summarized in Table II.
Of the 37 positive cases, 19 (51%) were males; 32 patients (86%) experienced diarrhoea during antibiotic treatment or within 15 days after the start of antibiotic treatment. The median time of occurrence of symptoms was 7 days (ranges 0-16 days) after start of antibiotic treatment and 8 days after admittance to hospital. All the patients were on multiple drugs and 50 per cent of the positive cases were on 3rd generation cephalosporins. None of the positive cases was on clindamycin. C. difficile positive cases were treated with metronidazole or vancomycin.
Antibiogram grouped all 15 isolates together as all were sensitive to erythromycin, chloramphenicol, penicillin, tetracyclin, clindamycin, vancomycin and metronidazole.
The identical fatty acid producers were grouped into 2 groups based on the production of isocaproic acid. Except one, all the isolates were producing isocaproic acid.
Based on the protein profiles observed on SDSPAGE, the isolates were placed into 3 groups; 12 isolates in group A, 2 in group B and 1 in group C.
PCR and RFLP analysis: All the ...lates were positive for toxin A (1.2 kb fragment) ... (399 bp) gene by PCR. Five different restriction profiles were obtained using Alu I endonucleases. The isolates were classified into five RFLP groups. The most frequent RFLP type was group I (6 isolates) group II, III, IV and V had 5, 2, 1 and 1 isolates respectively.
Discussion
C. difficile is considered as the most frequent aetiological agent of nosocomial diarrhoea occurring in hospitalized patients, spreading easily to the environment, the hands of health care workers and subsequently to other patients, particularly in large hospitals12. A trend of increasing prevalence of C. difficile has been reported in Europe and USA during the past 10 years13.
In our hospital C. difficile was found to be responsible for 15 per cent of the cases of nosocomial diarrhoea in our earlier study6. In this study, C. difficile was isolated mainly from patients in the haematology/ oncology wards. This points to the high risk areas for nosocomial spread of C. difficile isolates14. However, the percentage of infection showed a gradual decrease during the recent years.
Standard laboratory methods for diagnosing these infections include stool culture and identification of bacterial isolate, faecal toxin detection and C. difficile antigen detection. The culture lacks specificity due to the possible faecal carriage of non- toxigenic isolates, therefore many laboratories rely on toxin detection rather than culure for diagnosis of C. difficile infection15. A European survey of diagnostic methods for C. difficile, showed that culture of the organism is performed only in few countries. Mostly C. difficile toxin EIAs were used for diagnosis of CdAD16. In this study we used ELISA for toxin A, B and culture for diagnosing C. difficile infection. However, to the previous study6 we used C. difficile toxin A dependent ELISA for the analysis.
There was a gradual decrease in the percentage of ... infection during 2001 and 2005. The fact ... the 22 culture negative cases were on ... or vancomycin at the time of sample ... might be responsible for the decrease in isolation of organism as compared with the ELISA.
Older age, female gender and a prolonged hospital stay have been identified as risk factors in hospitalized CdAD patients17. In the current study, there was no gender prevalence among the positive cases and the median age of positive cases were 39 yr. However, highest percentage of culture positives was seen among patients >60 yr of age. Prolonged courses of antibiotic treatment have been related to an increased risk of AAD18,19. The median time for occurrence of symptoms was 7 days after the start of treatment in the present study, which was in accordance with other studies1,20. This suggests that disturbance of the normal colonic flora, eventually resulting in diarrhoea, takes place within about one week of antibiotic treatment. Prolonged duration of hospital stay has also been reported to be associated with AAD and CdAD19,21. In the present study, the median time of hospital stay was 8 days.
AAD was found to be frequently associated with cephalosporins, clindamycin and broad spectrum penicillins and quinolones22-25. In this study, about 50 per cent of our CdAD cases were on cephalosporins. However, since all the patients were on multiple antibiotics, the association with a particular group was not identified.
Discontinuation of antibiotic therapy withdraws the offending agents but is often not appropriate if the indication for such therapy was correct. An alternative is to change to antibiotics that do not belong to the high risk groups for induction of CdAD, such as quinolones, sulphonamides, parenteral aminoglycosides, cotrimoxazole, etc26. Metronidazole is suggested as the first line drug for the treatment of C. difficile infection2, and therefore the policy of the use of metronidazole in the treatment of suspected CdAD in our hospital is justified.
No nosocomial outbreak of C. difficile was reported during the study period. In this study we found antibiogram was least discriminatory of the typing strategies evaluated. The detection of short chain fatty acids by GLC is commonly utilized in bacteriological laboratories to identify anaerobes27. As all the isolates were positive for toxin A gene, we looked forward to analyze the variability of toxin A gene among C. difficile isolates by RFLP analysis. As the sequence analysis of the amplified 1.2 kbp toxin A gene fragment does not show any restriction sites for the previously reported restriction enzymes like Hinc II, Hind III, Ace I, EcoR I28, we decided to examine the amplified gene structure using restriction enzyme AIu I (5' AG[arrow down]CT 3', 3'TC[arrow up]GA 5'), which showed multiple restriction sites (8 sites) in the amplicon.
Although PCR-RFLP types 1 and II clustered some patient isolates, there was no epidemiological association between them. The locations where these patients were housed were different, and were admitted at different time periods. Better discriminatory methods such as pulsed field gel electrophoresis (PFGE) or ribotyping may be used to analyze the epidemiology of the pathogen.
In our recent prospective study, all the C. perfringens isolates were analyzed for the presence of enterotoxin by reverse passive later agglutination (RPLA), ELISA and by PCR assay for the presence of enterotoxin gene29. Of these, two were positive by PCR, RPLA and ELISA for C. perfringens enterotoxin. None of these samples had a co- infection with C. difficile.
Prevention of C. difficile infection is challenging. A change in antibiotic policy and implementation of standard infection control measures reduce the incidence of C. difficile symptomatic infections30,31. Combined approach, involving effective control measures, the use of rapid and sensitive techniques for laboratory diagnosis as well as prudent use of antibiotics, is necessary to reduce morbidity and mortality due to C. difficile associated infections in hospitalized patients.
In conclusion, we observed a decrease in the number of C. difficile toxin positive cases during the 5 yr of the study though there was an increase in the number of stool specimens tested per year for C. difficile. This possibly could be a result of stringent surveillance and antibiotic policy followed in our hospital especially in high risk areas such as haematology/oncology wards. Secondly, the use of clindamycin has been minimized in the hospital. Thirdly, antibiotics effective against C. difficile such as metronidazole have been included as the first line drugs in suspected CdAD cases. Isolation of the patients having C. difficile infection and regular awareness programmes conducted in the hospital might also have contributed.
Acknowledgment
Authors thank Ms. Sonam, Ms. Poornima and Shri Madho Prasad for technical assistance and acknowledge the Indian Council of Medical Research (ICMR), New Delhi, for financial suppport.
References
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2. Hogenauer C, Hammer HF, Krejs GJ, Reisinger EC. Mechanisms and management of antibiotic-associated diarrhea. Clin Infect Dis 1998; 27 : 702-10.
3. Dallal RM, Harbrecht BG, Boujoukas AJ, Sirio CA, Farkas LM, Lee KK, et al. Fulminant Clostridium difficile: an underappreciated and increasing cause of death and complications. Ann Surg 2002; 235 : 363-72. 4. Kyne L, Hamel MB, Polavaram R, Kelly CP. Health care costs and mortality associated with nosocomial diarrhea due to Clostridium difficile. Clin Infect Dis 2002; 34 : 346-53.
5. Schroeder MS. Clostridium difficile-associated diarrhea. Am Fam Physician 2005; 71 : 921-8.
6. Dhawan B, Chaudhry R, Sharma N Incidence of Clostridium difficile infection: a prospective study in an Indian hospital. J Hosp Infect 1999; 43 : 275-80.
7. Sutter VL, Citron DM, Edelstein MAC, Finegold SM. Wadsworth Anaerobic Manual. Belmont, California: Star Publishers, 1985. p. 71- 5.
8. Kato N, Ou CY, Kato H, Bartley SL, Brown VK, Dowell VR Jr, et al. Identification of toxigenic Clostridium difficile by the polymerase chain reaction. J Clin Microbiol 1991; 29 : 33-7.
9. Gumerlock PH, Tang YJ, Weiss JB, Silva J Jr. Specific detection of toxigenic strains of Clostridium difficile in stool specimens. J Clin Microbiol 1993; 3 : 507-11.
10. Lambe DW Jr., Laslie WW. A standard disc diffusion method for antibiotic susceptability testing for anaerobes; four years antibiotic data. In: Lambe DW Jr, Gena RJ, Carson KJM, editors. Anaerobic bacteria-selected topics. New York: Plenum Press, 1980.
11. Laemmli UK. Cleavage of structural proteins during the assembly of the head of bacteriophage. Nature 1970; 227 : 680-5.
12. Wroblewska MM, Swoboda-Kopec E, Rokosz A, Nurzynska G, Bednarska A, Luczak M. Detection of Clostridium difficile and its toxin A (TcdA) in stool specimens from hospitalised patients. Pol J Microbiol 2005; 54 : 111-5.
13. Wongwanich S, Pongpech P, Dhiraputra C, Huttayananont S, Sawanpanyalert P. Characteristics of Clostridium difficile strains isolated from asymptomatic individuals and from diarrheal patients. Clin Microbiol Infect 2001; 7: 438-41.
14. Blot E, Escande MC, Besson D, Barbut F, Granpeix C, Asselain B, et al. Outbreak of C. difficile related diarrhea in an adult oncology unit: risk factors and microbiological characteristics. J Hosp Infect 1993; 53: 187-92.
15. Wilcox MH, Fawley WN, Settle CD, Davidson A. Recurrence of symptoms in Clostridium difficile infection-relapse or reinfection? J Hosp Infect 1998; 38 : 93-100.
16. Barbut F, Delmee M, Brazier JS, Petit JC, Poxton IR, Rupnik M, et al. ESCMID Study Group on Clostridium difficile (ESGCD). A European survey of diagnostic methods and testing protocols for Clostridium difficile. Clin Microbiol Infect 2003; 9 : 989-96.
17. Al-Eidan FA, McElnay JC, Scott MG, Kearney MP. Clostridium difficile-associated diarrhoea in hospitalised patients. J Clin Pharm Ther 2000; 25 : 101-9.
18. Spencer R. The role of antimicrobial agents in the aetiology of Clostridium difficile-associated disease. J Antimicrob Chemother 1998; 41 : 21-7.
19. Bignardi GE. Risk factors for Clostridium difficile infection. J Hosp Infect 1998; 40 : 1-15.
20. Thamilikitkul V, Danpakdi K, Chokloikaew S. Incidence of diarrhea and Clostridium difficile toxin in stools from hospitalized patients receiving Clindamycin, beta lactams, and nonantibiotic medications. J Clin Gastroenterol 1996; 22 : 161-3.
21. Clabots CR, Johnson S, Olson MM, Peterson LR, Gerding DN. Acquisition of Clostridium difficile by hospitalized patients: evidence for colonized new admissions as a source of infection. J Infect Dis 1992; 166 : 561-7.
22. Impallomeni M, Galletly NP, Wort SU, Starr JM, Rogers TR. Increased risk of diarrhea caused by Clostridium difficile in elderly patients receiving cefotaxime. BMJ 1995; 311 : 1345-6.
23. Pear SM, Williamson TH, Bettin KM, Gerding DN, Galgiani JN. Decrease in nosocomial Clostridium difficile-associated diarrhea by restricting clindamycin use. Ann Intern Med 1994; 720 : 272-7.
24. Pepin J, Saheb N, Coulombe MA, Alary ME, Carrivean MP, Authier S, et al. Emergence of fluoroquinolones as the predominant risk factor for Clostridium difficile-associated diarrhea: a cohort study during an epidemic in Quebec. Clin Infect Dis 2005; 41 : 1254- 60.
25. Muto CA, Pokrywka M, Shutt K, Mendelsohn AS, Nouri K, Posey K, et al. A large outbreak of Clostridium difficile-associated disease with an unexpected proportion of deaths and colectomies at a teaching hospital following increased fluoroquinolone use. Infect Control Hosp Epidemiol 2005; 26 : 273-80
26. Bartlett JG. Antibiotic associated diarrhea. Clin Infect Dis 1992; 15 : 573-81.
27. Pepersack F, Labbe M, Nonhoff C, Schoutens E. Use of gas- liquid chromatography as a screening test for toxigenic Clostridium difficile in diarrhoeal stools. J Clin Pathol 1983; 36 : 1233-6.
28. McFarland LV, Surawicz CM, Rubin M, Fekety R, Elmer GW, Greenberg RN. Recurrent Clostridium difficile disease: Epidemiology and clinical characteristics. Infect Control Hosp Epidemiol 1999; 20 : 43-50.
29. Joshy L, Chaudhry R, Dhawan B, Kumar L, Das BK. Incidence and characterization of Clostridium perfringens isolated from antibiotic- associated diarrhoeal patients: a prospective study in an Indian hospital. J Hosp Infect 2006; 63 : 323-9.
30. Khan R, Cheesbrough J. Impact of changes in antibiotic policy on Clostridium difficile-associated diarrhoea (CDAD) over a five- year period in a district general hospital. J Hosp infect 2003; 54 : 104-8.
31. Riley TV. Nosocomial diarrhoea due to Clostridium difficile. Curr Opin Infect Dis 2004; 17 : 323-7.
Rama Chaudhry, Lovely Joshy, Lalit Kumar* & Benu Dhawan
Departments of Microbiology, * Medical Oncology, Institute- Rotary Cancer Hospital
All India Institute of Medical Sciences, New Delhi, India
Received December 7, 2006
Reprint requests: Dr Rama Chaudhry, Professor, Department of Microbiology, All India Institute of Medical Sciences
New Delhi 110029, India
e-mail: drramach@rediffmail.com, ramach003@yahoo.com
Copyright Indian Council of Medical Research Apr 2008
(c) 2008 Indian Journal of Medical Research. Provided by ProQuest LLC. All rights Reserved.
Source: Indian Journal of Medical Research
Posted on: Tuesday, 26 August 2008, 03:00 CDT
By Chaudhry, Rama Joshy, Lovely; Kumar, Lalit; Dhawan, Benu
Background & objectives: Frequent use of broad spectrum antibiotics in hospitalized patients has increased the incidence of Clostridium difficile diarrhoea in recent years. In our tertiary care hospital in north India, C. difficile was responsible for 15 per cent of cases of nosocomial diarrhoea in 1999. A retrospective study was carried out to determine the frequency of C. difficile associated diarrhoea (CdAD) in our hospital, and to assess the effect of awareness among the hospital personnel and control measures taken to present C. difficile infection following the previous report. Methods: A retrospective chart review of all suspected cases of CdAD diagnosed at the hospital from January 2001 to December 2005 was done. Clinical specimens comprised 524 stool samples. All the samples were analyzed for C. difficile using culture and ELISA for toxin A and B. Attempts were made to type isolates using antibiogram, SDS-PAGE, gas liquid chromatography (GLC), PCR for toxin A and B gene fragments and restriction fragment length polymorphism (RFLP).
Results: A total of 37 (7.1%) specimens were positive for C. difficile toxin (11.2% in 2001, 9.4% in 2002, 8.6% in 2003, 5% in 2004 and 4% in 2005). The highest number of C. difficile toxin positive cases were from stool samples of patients hospitalized in the haematology/oncology ward (67.5% of all positive cases) followed by gastrointestinal surgery, neurology and nephrology wards. Of the C. difficile toxin positive samples, 15 (41%) were also positive for C. difficile culture. The isolates were grouped in to one, 3 and 5 groups using antibiogram, SDS-PAGE and PCR RFLP respectively. We observed an increase in the number of stool specimens tested for C. difficile infection but a decrease in C. difficile positives.
Interpretation & conclusions: A decrease in the number of C. difficile positive cases were noted during the 5 year study period though number of samples tested was increased. This may be due to stringent surveillance and an improved antibiotic policy followed in the hospital.
Diarrhoea is one of the most frequent side effects of antibiotic treatment. The symptoms may vary from slight abdominal discomfort to severe diarrhoea to colitis1. The aetiology of antibiotic associated diarrhoea (AAD) varies. The disruption of normal enteric flora caused by antibiotics may lead to overgrowth of pathogens and functional disturbances of the intestinal carbohydrate and bile acid metabolism, resulting in osmotic diarrhoea1. Allergic, toxic and pharmacological effects of antibiotics may also affect the intestinal mucosa and motility2. Cytotoxin producing Clostridium difficile has been reported to be the causative agent of approximately 20 per cent of AAD and of nearly all cases of pseudomembraneous colitis, the most severe manifestation of AAD1. Because of the frequent use of broad spectrum antibiotics, the incidence of C. difficile diarrhoea has risen dramatically in recent years3,4. Established guidelines should be followed to minimize exposure to the pathogen which include judicious use of antibiotics, rapid detection of C. difficile by immunoassays for toxin A and B, isolation of patients who had C. difficile associated diarrhoea (CdAD), proper disinfection of objects and education of staff members5. In our hospital which is a tertiary care hospital in north India, C. difficile was responsible for 15 per cent of cases of nosocomial diarrhoea in 1999(6). Standard control measures were implemented in our hospital to minimize the spread of this nosocomial pathogen after this report. This retrospective analysis was carried out in continuation of our earlier study6 to determine the effect of awareness and control measures taken to contain C. difficile infection in our hospital during the subsequent years.
Material & Methods
The study comprised retrospective analysis of faecal specimens from 524 patients suspected on clinical grounds to have CdAD2. The patients were hospitalized in All India Institute of Medical Sciences, New Delhi, India, over a period of 5 yr (January 1, 2001 - December 31, 2005). These included 80 patients in 2001, 96 in 2002, 92 in 2003, 106 in 2004 and 150 patients in 2005 respectively. Of these, 53 per cent were males and 82.4 per cent were in all age group > 12-60 yr (Table I).
Clinical information about the cause of diarrhoea underlying disease and antimicrobial therapy was obtained by reviewing the patient charts. A patient was considered to have CdAD if AAD was present and a stool specimen was positive in a toxin dependent C. difficile assay.
Sample collection and isolation of C. difficile: All the stool specimens were processed immediately for culture of C. difficile and stool aliquots were stored at -20[degrees]C for <72 h till they were tested for C. difficile toxin A and B. Spore selection was performed using 95 per cent ethanol and culture for C. difficile was done on cycloserine cefoxitin fructose agar (CCFA) and brain heart infusion agar (BHIA) as described elsewhere6. Concurrently, a loopful of stool specimen was inoculated into Robertsons cooked meat broth and incubated at 37[degrees]C for 48 h.
The plates were incubated anaerobically at 37[degrees]C in an anaerobic jar for 48 h. After incubation, the plates were examined and colonies which resembled C. difficile were Gram stained and identified by biochemical reactions using standard methods7.
When culture plate were negative for C. difficile, subcultures were made from cooked meat broth onto CCFA and BHIA and incubated anaerobically at 37[degrees]C up to 5 days before being discarded as negative.
Enzyme immunoassay for toxin A and B: Detection of enterotoxin and cytotoxin (toxin A and toxin B) of C. difficile was performed on the stool specimens by a double sandwich enzyme-linked immunosorbent assay technique using a commercial kit (Premier toxins A & B; Meridian Diagnostics, Inc., Cincinnati, Ohio, USA). The assay was performed according to the manufacturer's instructions.
Characterization of C. difficile isolates: All the C. difficile isolates were characterized phenotypically using antibiogram, SDS- PAGE6, gas liquid chromatography (GLC)7, and genotypically using PCR for toxin A gene and RFLP8,9.
Antibiogram typing - Antibiogram patterns were determined by disc diffusion method10. The antibiotics tested were chloramphenicol (30 [mu]g), penicillin G (10 units), clindamycin (2 [mu]g), vancomycin (5 [mu]g); metronidazole (5 [mu]g), tetracycline (30 [mu]g) and erythromycin (10 [mu]g). The results were expressed as susceptible or resistant.
Analysis of volatile fatty acids by gas liquid chromatography (GLC): All isolates were inoculated to cooked meat broth and incubated anaerobically for 48 h or more for GLC analysis to detect volatile fatty acids produced as metabolic end products. 1 ml of RCM broth was acidified with 0.2 ml of 50 per cent sulphuric acid and extracted with 1 ml of diethyl ether. The mixture was shaken vigorously and centrifuged at 176 g for 3 min; 1.5 [mu]l of the extracted ether layer was injected to the injection port of preconditioned GLC column with a 10 [mu]l Hamilton syringe. Chromatography was performed on a Nucon Series 5700, fitted with a flame ionization detector (FID)7. Operating conditions were as follows: carrier gas (oxygen free nitrogen): 60 ml/min oven temperature: detector 240[degrees]C, column 175[degrees]C, injector 240[degrees]C, attenuation 4X, sensitivity of the detector was set at 1000X. Fatty acids were identified by comparing the retention times of peaks in the test samples with those of known standard solutions which were examined each day7.
PCR assay for toxin gene fragments: The presence of toxin A gene in all isolates of C. difficile was determined by specific PCR using published primers8. PCR to detect the toxin B gene was performed in the C. difficile isolates using primers that had been developed and validated by Gumerlock et at9 to yield a 399-bp fragment for toxin B gene. PCR was performed in a 25 [mu]l reaction volume. Each reaction tube contained 1 X buffer (10 mm Tris HCl, pH 8.3, 50 mm KCl, 2.5 mm MgCl^sub 2^, 0.001% gelatin), each deoxynuclotide at a concentration of 100 mm (MBI, Fermentas, USA), each primer at a concentration of 20 pmol, 1.25 U Taq polymerase (MBI, Fermantas, USA) and 10 [mu]l of DNA. PCR was performed for 2 min at 95[degrees]C followed by 30 cycles of 1 min of denaturation at 95[degrees]C, 1 min of annealing at 52[degrees]C and 1 min of extension at 72[degrees]C. After the 30th cycle, extension was continued for an additional 10 min. 10 [mu]l of the amplified product was analyzed in 1 per cent agarose gel stained with ethidium bromide.
PCR-restriction fragment length polymorphism (RFLP) analysis: The amplified toxin A gene fragment was then digested with Alu I(10 units) restriction enzyme, under conditions recommended by the supplier (MBI-Fermentas). These digests were then subjected to electrophoresis on 2.5 per cent agarose gel at 60 V, along side a PCR size marker (100 bps, Sigma, USA).
Comparisons of patterns were performed visually. Strains with patterns differing alteast by one band were assigned to different types.
Results
A total of 524 stool specimens were analyzed for C. difficile from suspected cases of CdAD. The maximum number of C. difficile suspected cases were from oncology ward (378 cases, 72%), followed by other wards such as gastrointestinal surgery, neurology, nephrology and other medical wards. A total of 95 per cent of the analyzed group were on multiple antibiotics which included, 65 per cent on cephalosporins, 35 per cent on quinolones, 43 per cent on aminoglycosides, 12 per cent on macrolides, 69 per cent in vancomycin and metronidazole. Of the analyzed group, 37 (7.1%) patients were positive for C. difficile infection by the toxin dependent assay. Of these, 9 samples (11.2%) were positive in 2001, 9 (9.4%) in 2002, 8 (8.6%) in 2003, 5 (5%) in 2004 and 6 patients (4%) in 2005 (Fig. 1). Fifteen (41%) of the 37 toxin positive stool samples were also positive for C. difficile by culture. Eight of the 37 toxin positive cases expired, the cause of death was not directly related to C. difficile diarrhoea, although this might have been a contributory factor. Other pathogenic clostridia isolated from the patient group included C. perfringens (2.5%).
The highest number of C. difficile toxin positive cases were from stool samples of patients hospitalized in the haematology/oncology ward (25 samples, 67.5% of all positive cases), followed by gastrointestinal surgery, neurology and nephrology wards. Recovery rates of C. difficile in patient populations surveyed and summarized in Table II.
Of the 37 positive cases, 19 (51%) were males; 32 patients (86%) experienced diarrhoea during antibiotic treatment or within 15 days after the start of antibiotic treatment. The median time of occurrence of symptoms was 7 days (ranges 0-16 days) after start of antibiotic treatment and 8 days after admittance to hospital. All the patients were on multiple drugs and 50 per cent of the positive cases were on 3rd generation cephalosporins. None of the positive cases was on clindamycin. C. difficile positive cases were treated with metronidazole or vancomycin.
Antibiogram grouped all 15 isolates together as all were sensitive to erythromycin, chloramphenicol, penicillin, tetracyclin, clindamycin, vancomycin and metronidazole.
The identical fatty acid producers were grouped into 2 groups based on the production of isocaproic acid. Except one, all the isolates were producing isocaproic acid.
Based on the protein profiles observed on SDSPAGE, the isolates were placed into 3 groups; 12 isolates in group A, 2 in group B and 1 in group C.
PCR and RFLP analysis: All the ...lates were positive for toxin A (1.2 kb fragment) ... (399 bp) gene by PCR. Five different restriction profiles were obtained using Alu I endonucleases. The isolates were classified into five RFLP groups. The most frequent RFLP type was group I (6 isolates) group II, III, IV and V had 5, 2, 1 and 1 isolates respectively.
Discussion
C. difficile is considered as the most frequent aetiological agent of nosocomial diarrhoea occurring in hospitalized patients, spreading easily to the environment, the hands of health care workers and subsequently to other patients, particularly in large hospitals12. A trend of increasing prevalence of C. difficile has been reported in Europe and USA during the past 10 years13.
In our hospital C. difficile was found to be responsible for 15 per cent of the cases of nosocomial diarrhoea in our earlier study6. In this study, C. difficile was isolated mainly from patients in the haematology/ oncology wards. This points to the high risk areas for nosocomial spread of C. difficile isolates14. However, the percentage of infection showed a gradual decrease during the recent years.
Standard laboratory methods for diagnosing these infections include stool culture and identification of bacterial isolate, faecal toxin detection and C. difficile antigen detection. The culture lacks specificity due to the possible faecal carriage of non- toxigenic isolates, therefore many laboratories rely on toxin detection rather than culure for diagnosis of C. difficile infection15. A European survey of diagnostic methods for C. difficile, showed that culture of the organism is performed only in few countries. Mostly C. difficile toxin EIAs were used for diagnosis of CdAD16. In this study we used ELISA for toxin A, B and culture for diagnosing C. difficile infection. However, to the previous study6 we used C. difficile toxin A dependent ELISA for the analysis.
There was a gradual decrease in the percentage of ... infection during 2001 and 2005. The fact ... the 22 culture negative cases were on ... or vancomycin at the time of sample ... might be responsible for the decrease in isolation of organism as compared with the ELISA.
Older age, female gender and a prolonged hospital stay have been identified as risk factors in hospitalized CdAD patients17. In the current study, there was no gender prevalence among the positive cases and the median age of positive cases were 39 yr. However, highest percentage of culture positives was seen among patients >60 yr of age. Prolonged courses of antibiotic treatment have been related to an increased risk of AAD18,19. The median time for occurrence of symptoms was 7 days after the start of treatment in the present study, which was in accordance with other studies1,20. This suggests that disturbance of the normal colonic flora, eventually resulting in diarrhoea, takes place within about one week of antibiotic treatment. Prolonged duration of hospital stay has also been reported to be associated with AAD and CdAD19,21. In the present study, the median time of hospital stay was 8 days.
AAD was found to be frequently associated with cephalosporins, clindamycin and broad spectrum penicillins and quinolones22-25. In this study, about 50 per cent of our CdAD cases were on cephalosporins. However, since all the patients were on multiple antibiotics, the association with a particular group was not identified.
Discontinuation of antibiotic therapy withdraws the offending agents but is often not appropriate if the indication for such therapy was correct. An alternative is to change to antibiotics that do not belong to the high risk groups for induction of CdAD, such as quinolones, sulphonamides, parenteral aminoglycosides, cotrimoxazole, etc26. Metronidazole is suggested as the first line drug for the treatment of C. difficile infection2, and therefore the policy of the use of metronidazole in the treatment of suspected CdAD in our hospital is justified.
No nosocomial outbreak of C. difficile was reported during the study period. In this study we found antibiogram was least discriminatory of the typing strategies evaluated. The detection of short chain fatty acids by GLC is commonly utilized in bacteriological laboratories to identify anaerobes27. As all the isolates were positive for toxin A gene, we looked forward to analyze the variability of toxin A gene among C. difficile isolates by RFLP analysis. As the sequence analysis of the amplified 1.2 kbp toxin A gene fragment does not show any restriction sites for the previously reported restriction enzymes like Hinc II, Hind III, Ace I, EcoR I28, we decided to examine the amplified gene structure using restriction enzyme AIu I (5' AG[arrow down]CT 3', 3'TC[arrow up]GA 5'), which showed multiple restriction sites (8 sites) in the amplicon.
Although PCR-RFLP types 1 and II clustered some patient isolates, there was no epidemiological association between them. The locations where these patients were housed were different, and were admitted at different time periods. Better discriminatory methods such as pulsed field gel electrophoresis (PFGE) or ribotyping may be used to analyze the epidemiology of the pathogen.
In our recent prospective study, all the C. perfringens isolates were analyzed for the presence of enterotoxin by reverse passive later agglutination (RPLA), ELISA and by PCR assay for the presence of enterotoxin gene29. Of these, two were positive by PCR, RPLA and ELISA for C. perfringens enterotoxin. None of these samples had a co- infection with C. difficile.
Prevention of C. difficile infection is challenging. A change in antibiotic policy and implementation of standard infection control measures reduce the incidence of C. difficile symptomatic infections30,31. Combined approach, involving effective control measures, the use of rapid and sensitive techniques for laboratory diagnosis as well as prudent use of antibiotics, is necessary to reduce morbidity and mortality due to C. difficile associated infections in hospitalized patients.
In conclusion, we observed a decrease in the number of C. difficile toxin positive cases during the 5 yr of the study though there was an increase in the number of stool specimens tested per year for C. difficile. This possibly could be a result of stringent surveillance and antibiotic policy followed in our hospital especially in high risk areas such as haematology/oncology wards. Secondly, the use of clindamycin has been minimized in the hospital. Thirdly, antibiotics effective against C. difficile such as metronidazole have been included as the first line drugs in suspected CdAD cases. Isolation of the patients having C. difficile infection and regular awareness programmes conducted in the hospital might also have contributed.
Acknowledgment
Authors thank Ms. Sonam, Ms. Poornima and Shri Madho Prasad for technical assistance and acknowledge the Indian Council of Medical Research (ICMR), New Delhi, for financial suppport.
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Rama Chaudhry, Lovely Joshy, Lalit Kumar* & Benu Dhawan
Departments of Microbiology, * Medical Oncology, Institute- Rotary Cancer Hospital
All India Institute of Medical Sciences, New Delhi, India
Received December 7, 2006
Reprint requests: Dr Rama Chaudhry, Professor, Department of Microbiology, All India Institute of Medical Sciences
New Delhi 110029, India
e-mail: drramach@rediffmail.com, ramach003@yahoo.com
Copyright Indian Council of Medical Research Apr 2008
(c) 2008 Indian Journal of Medical Research. Provided by ProQuest LLC. All rights Reserved.
Source: Indian Journal of Medical Research
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