Structure of virus identified that could lead to hepatitis C vaccine

Rutgers University scientists have determined the structure of a hepatitis C surface protein, a finding that could assist in the development of a vaccine to halt the spread of the the deadly disease.

Joseph Marcotrigiano, associate professor of chemistry and chemical biology, says this new research -- published online in Nature -- describes an outer region of hepatitis C that enables the virus to evade the body's natural immune system response, causing persistent, chronic infection.

Hepatitis C is constantly mutating, allowing it to infect a host cell and evade the immune responses, causing chronic infection that can be difficult to treat. By identifying the structure of virus's outer protein, Marcotrigiano, the study's lead author, says scientists will be better able to develop a vaccine that targets the immune system to vulnerable regions of the virus in order to prevent infection.

"Viruses are smart and it is a constant battle to keep them out," says Marcotrigiano who collaborated on the research with colleagues from the Center for Advanced Biotechnology and Medicine at Rutgers and Emory University School of Medicine. "That's why the development of a vaccine is so important. It's always better to prevent infection through an effective vaccine then to treat after a chronic infection has been established."

Hepatitis C virus is a major global health problem with 160 million people infected throughout the world, about four times more individuals than those with HIV. Most of those infected do not show symptoms until the virus -- the number one cause of liver transplantation -- has caused severe liver damage.

The virus is mainly spread through contact with an infected person's blood, such as sharing of needles. Prior to 1992, when donated blood began being tested, the virus was also spread through blood transfusions and organ donation.

Recently, the Food and Drug Administration approved several new drugs that could cure many patients infected with hepatitis C in as little as 12 weeks. However, at about $1000 per pill, this may not be a cost-effective solution to hepatitis C virus.

Developing a vaccine against hepatitis C would not only prevent people from acquiring the disease, Marcotrigiano says, but would also be the most cost-conscious health intervention.

Michael Houghton, a researcher at the University of Alberta in Canada, has been developing a vaccine that is currently being tested clinically. Houghton, who led a team that discovered the hepatitis C virus in 1989, says the Rutgers finding is important because knowing the structure of the virus will help in the development of a vaccine that enables the immune system to produce more infection-fighting antibodies that can neutralize the virus.

Journal Reference:

1. Abdul Ghafoor Khan, Jillian Whidby, Matthew T. Miller, Hannah Scarborough, Alexandra V. Zatorski, Alicja Cygan, Aryn A. Price, Samantha A. Yost, Caitlin D. Bohannon, Joshy Jacob, Arash Grakoui, Joseph Marcotrigiano. Structure of the core ectodomain of the hepatitis C virus envelope glycoprotein 2. Nature, 2014; DOI: 10.1038/nature13117

source: www.sciencedily.com

Malaria vaccine development paves way for protective therapy

Scientists have made a significant contribution towards the development of a vaccine to prevent malaria. Researchers have tested a preliminary form of a vaccine against the disease, which is spread by the bite of the mosquito and kills more than 600,000 people each year.

Until now, developing malaria vaccines has been challenging. A vaccine must incorporate key proteins from the malaria parasites, which will trigger production of antibodies by the immune system. These proteins have a complex, intricate structure that is hard to reproduce.

Scientists at the University of Edinburgh have now found a novel way to produce proteins that could lead to malaria vaccines being easy and cheap to manufacture. They have grown them inside a tiny single-celled aquatic creature, whose biological make-up is similar to that of the malaria parasite. The organism, and the protein, can multiply quickly in the lab.

In tests in mice, a vaccine developed using human malaria parasite proteins -- known as MSP-1-BBM -- enabled the immune system to produce antibodies in the bloodstream. These antibodies were shown to respond to the human malaria parasite, indicating that the vaccine would be likely to trigger an immune reaction if it were used in people.

Researchers now hope to develop the vaccine for further testing, with the aim of producing a therapy that will be effective in humans. Scientists say there is a pressing need for new treatments, as many forms of the disease are becoming resistant to existing drugs. Children and pregnant women in sub-Saharan Africa are particularly at risk.

The study, carried out in collaboration with the German company Cilia AG and published in the journal PLoS One, was funded by the European Union.

Dr. David Cavanagh, of the University of Edinburgh's School of Biological Sciences, who led the study, said: "There is a desperate need for an effective vaccine, which can be made easily in large quantities, to protect against this devastating disease. Our findings meet this challenge and, with more work, could lead to a vaccine to help those most at risk."

Journal Reference:

1. Graeme J. M. Cowan, Ulrike Bockau, Janna Eleni-Muus, Ingo Aldag, Kay Samuel, Alison M. Creasey, Marcus W. W. Hartmann, David R. Cavanagh. A Novel Malaria Vaccine Candidate Antigen Expressed in Tetrahymena thermophila. PLoS ONE, 2014; 9 (1): e87198 DOI: 10.1371/journal.pone.0087198

source: www.sciencedaily.com

Genetic secret of mosquito resistance to DDT, bed net insecticides discovered

Researchers from LSTM (Liverpool school of tropical medicine) have found that a single genetic mutation causes resistance to DDT and pyrethroids (an insecticide class used in mosquito nets). With the continuing rise of resistance the research, published in the journal Genome Biology, is key as scientists say that this knowledge could help improve malaria control strategies.

The researchers, led by Dr Charles Wondji, used a wide range of methods to narrow down how the resistance works, finding a single mutation in the GSTe2 gene, which makes insects break down DDT so it's no longer toxic. They have also shown that this gene makes insects resistant to pyrethroids raising the concern that GSTe2 gene could protect mosquitoes against the major insecticides used in public health.

Mosquitoes (Anopheles funestus) are vectors of malaria, and most strategies for combating the spread of the disease focus on control of mosquito populations using insecticides. The spread of resistance genes could hold back efforts to prevent the disease. The authors say that knowing how resistance works will help to develop tests, and stop these genes from spreading amongst mosquito populations.

Charles Wondji said: 'We found a population of mosquitoes fully resistant to DDT (no mortality when they were treated with DDT) but also to pyrethroids. So we wanted to elucidate the molecular basis of that resistance in the population and design a field applicable diagnostic assay for its monitoring.'

They took mosquitoes from Pahou in Benin, which were resistant to DDT and pyrethroids, and mosquitoes from a laboratory fully susceptible strain and did a genome wide comparison study. They identified the GSTe2 gene as being upregulated -- producing a lot of protein -- in Benin mosquitoes.

They found that a single mutation (L119F) changed a non-resistant version of the GSTe2 gene to a DDT resistant version. They designed a DNA-based diagnostic test for this type of resistance (metabolic resistance) and confirmed that this mutation was found in mosquitoes from other areas of the world with DDT resistance but was completely absent in regions without. X-ray crystallography of the protein coded by the gene illustrated exactly how the mutation conferred resistance, by opening up the 'active site' where DDT molecules bind to the protein, so more can be broken down. This means that the mosquito can survive by breaking down the poison into non-toxic substances..

They also introduced the gene into fruit flies (Drosophila melanogaster) and found they became resistant to DDT and pyrethroids compared to controls, confirming that just this single mutation is enough to make mosquitoes resistant to both DDT and permethrin.

Wondji says: 'For the first time, we have been able to identify a molecular marker for metabolic resistance (the type of resistance most likely to lead to control failure) in a mosquito population and to design a DNA-based diagnostic assay. Such tools will allow control programs to detect and track resistance at an early stage in the field, which is an essential requirement to successfully tackle the growing problem of insecticide resistance in vector control. This significant progress opens the door for us to do this with other forms of resistance as well and in other vector species.'

Journal Reference:

1. Jacob M Riveron, Cristina Yunta, Sulaiman S Ibrahim, Rousseau Djouaka, Helen Irving, Benjamin D Menze, Hanafy M Ismail, Janet Hemingway, Hilary Ranson, Armando Albert, Charles S Wondji. A single mutation in the GSTe2 gene allows tracking of metabolically-based insecticide resistance in a major malaria vector. Genome Biology, 2014; 15 (2): R27 DOI: 10.1186/gb-2014-15-2-r27 

source: www.sciencedaily.com

3D structure discovered of enzyme that helps defend against bacteria

An  essential weapon in the body's fight against infection has come into sharper view. Researchers at Princeton University have discovered the 3D structure of an enzyme that cuts to ribbons the genetic material of viruses and helps defend against bacteria.

The discovery of the structure of this enzyme, a first-responder in the body's "innate immune system," could enable new strategies for fighting infectious agents and possibly prostate cancer and obesity. The work was published Feb. 27 in the journal Science.

Until now, the research community has lacked a structural model of the human form of this enzyme, known as RNase L, said Alexei Korennykh, an assistant professor of molecular biology and leader of the team that made the discovery.

"Now that we have the human RNase L structure, we can begin to understand the effects of carcinogenic mutations in the RNase L gene. For example, families with hereditary prostate cancers often carry genetic mutations in the region, or locus, encoding RNase L," Korennykh said. The connection is so strong that the RNase L locus also goes by the name "hereditary prostate cancer 1." The newly found structure reveals the positions of these mutations and explains why some of these mutations could be detrimental, perhaps leading to cancer, Korennykh said. RNase L is also essential for insulin function and has been implicated in obesity.

The Princeton team's work has also led to new insights on the enzyme's function.

The enzyme is an important player in the innate immune system, a rapid and broad response to invaders that includes the production of a molecule called interferon. Interferon relays distress signals from infected cells to neighboring healthy cells, thereby activating RNase L to turn on its ability to slice through RNA, a type of genetic material that is similar to DNA. The result is new cells armed for destruction of the foreign RNA.

The 3D structure uncovered by Korennykh and his team consists of two nearly identical subunits called protomers. The researchers found that one protomer finds and attaches to the RNA, while the other protomer snips it.

The initial protomer latches onto one of the four "letters" that make up the RNA code, in particular, the "U," which stands for a component of RNA called uridine. The other protomer "counts" RNA letters starting from the U, skips exactly one letter, then cuts the RNA.

Although the enzyme can slice any RNA, even that of the body's own cells, it only does so when activated by interferon.

"We were surprised to find that the two protomers were identical but have different roles, one binding and one slicing," Korennykh said. "Enzymes usually have distinct sites that bind the substrate and catalyze reactions. In the case of RNase L, it appears that the same exact protein surface can do both binding and catalysis. One RNase L subunit randomly adopts a binding role, whereas the other identical subunit has no other choice but to do catalysis."

To discover the enzyme's structure, the researchers first created a crystal of the RNase L enzyme. The main challenge was finding the right combination of chemical treatments that would force the enzyme to crystallize without destroying it.

After much trial and error and with the help of an automated system, postdoctoral research associate Jesse Donovan and graduate student Yuchen Han succeeded in making the crystals.

Next, the crystals were bombarded with powerful X-rays, which diffract when they hit the atoms in the crystal and form patterns indicative of the crystal's structure. The diffraction patterns revealed how the atoms of RNase L are arranged in 3D space.

At the same time Sneha Rath, a graduate student in Korennykh's laboratory, worked on understanding the RNA cleavage mechanism of RNase L using synthetic RNA fragments. Rath's results matched the structural findings of Han and Donovan, and the two pieces of data ultimately revealed how RNase L cleaves its RNA targets.

Han, Donovan and Rath contributed equally to the paper and are listed as co-first authors.

Finally, senior research specialist Gena Whitney and graduate student Alisha Chitrakar conducted additional studies of RNase L in human cells, confirming the 3D structure.

Now that the human structure has been solved, researchers can explore ways to either enhance or dampen RNase L activity for medical and therapeutic uses, Korennykh said.

"This work illustrates the wonderful usefulness of doing both crystallography and careful kinetic and enzymatic studies at the same time," said Peter Walter, professor of biochemistry and biophysics at the University of California-San Francisco School of Medicine. "Crystallography gives a static picture which becomes vastly enhanced by studies of the kinetics.

Journal Reference:

1. Y. Han, J. Donovan, S. Rath, G. Whitney, A. Chitrakar, A. Korennykh. Structure of Human RNase L Reveals the Basis for Regulated RNA Decay in the IFN Response. Science, 2014; DOI: 10.1126/science.1249845

source: www.sciencedaily.com

"

Noninvasive Prenatal Testing Detects Most Chromosomal Abnormalities

Noninvasive prenatal testing detected 83.2% of chromosomal abnormalities normally picked up by invasive diagnostic testing strategies, such as chorionic villus sampling (CVS) or amniocentesis.

Image: Fetal chromosome test showing trisomy 13, indicated by red arrow (Photo courtesy of the National Human Genome Research Institute).

Noninvasive prenatal testing (NIPT) using cell free DNA provides accurate screening for the common trisomies, including trisomy 13, Patau syndrome, trisomy 18, Edwards syndrome, and trisomy 21, Down syndrome.

Specialist in maternal-fetal medicine at the University of California, San Francisco (CA, USA) carried out a study on rare chromosome abnormalities detected by current prenatal screening compared to expected performance using noninvasive prenatal testing. There were 68,990 of 1,324,607 women who tested positive for trisomy 18 or 21 when they underwent prenatal screening between March 2009 and December 2012.

Invasive diagnostic testing with CVS or amniocentesis was performed on 26,059 women who tested positive, and 2,993 were found to have abnormal results. Of those chromosomal abnormalities, 2,489 (83.2%) were abnormalities that would be detectable with NIPT, while 16.8% were less common aneuploidies that would not be detected. The scientists found that more of the abnormal results were detectable in the women over the age of 40, who are at higher risk for trisomy 13, 18, or 21. Conversely, fewer of the abnormalities in younger women would be detected by NIPT, as the risk for common trisomies is lower in this group, while the rare aneuploidies are not typically associated with maternal age.

Mary Norton, MD, one of the study’s authors, said, “While noninvasive prenatal testing with cell free DNA presents some real advantages in accuracy of screening for Down syndrome, as with everything there is a trade-off. Traditional aneuploidy screening with serum and ultrasound markers has higher false positive rates, but in these false positive cases are some fetuses with significant abnormalities that would not be found with NIPT.”

Dr. Norton added, “In prenatal genetic testing, patient preferences are really the most important driver. With this test, the patient makes a tradeoff between NIPT, which is noninvasive and detects most, but not all chromosome abnormalities, and is somewhat better in older women, and amniocentesis or CVS, which detect more chromosome abnormalities, 8% to 25% more, depending on age, but with a small risk of miscarriage due to the procedure. For an older woman, detecting 83% with the noninvasive test may be good enough, while for a 25-year-old, failing to detect 25%, which may include rare aneuploidies not usually associated with age, may be of concern.” The study was presented on February 6, 2014, at the Society for Maternal-Fetal Medicine's annual meeting, The Pregnancy Meeting, held in New Orleans (LA, USA). 

source: www.labmedica.com

New Capping System Simplifies Storage of Blood and Urine Samples

A novel system for re-capping sample tubes permits secure storage of blood and urine specimens for several days in case additional tests need to be performed.

Image: The Novitain Capcluster-50 offers a rapid and economical solution for capping 50 individual sample tubes (Photo courtesy of Micronic Europe BV).

Blood and urine samples are usually collected in vacuum tubes before being sent to a central laboratory for analysis. Following analytical testing, the vacuum tubes require storage for several days in case additional or follow-up tests need to be performed. To simplify the sample retention process Micronic Europe BV (Lelystad, The Netherlands) has introduced a unique postanalytical capping solution for blood or urine sample collection tubes.

The system comprises the Novitain Manual Capper CV750 instrument and Novitain Capcluster-50 multi-caps. The Novitain TPE Push Caps that make up the Capcluster-50 were designed to seal 13-mm diameter sample-collection tubes in a 50-tube format. The caps are manufactured from a chemically resistant medical grade hydrophobic polymer to ensure stored sample integrity.

The Novitain Manual Capper CV750 is an easy-to-use, stand-alone instrument. To seal sample tubes, a Novitain Capcluster-50 sheet is placed on the rack of sample tubes, and the rack is inserted into the Novitain Manual Capper CV750 rack entrance. A simple forward/reverse action of the black handle evenly caps all 50 tubes in less than two seconds. Following the capping step, the Novitain Capcluster-50 retaining foil can be easily removed to leave each individual tube securely capped. The tubes can be opened again using the tab attached to each cap without the need for additional tools.

 

source: www.labmedica.com

A Soda a Day Ups CVD Risk by 30%: NHANES Study

American adults consume on average about 15% of their calories from sugars added to foods during processing, with a whopping 37% of the added sugar consumed in sugar-sweetened beverages, suggests an analysis of data extending back about 25 years. Moreover, the study projects that regularly drinking as little as one 12-ounce sugary soda a day may increase the risk of cardiovascular disease by about 30%—independent of total calories, obesity, or other risk factors ^[1]. 

"Our findings indicate that most US adults consume more added sugar than is recommended for a healthy diet," write Dr Quanhe Yang (Division for Heart Disease and Stroke Prevention, Centers for Disease Control and Prevention, Atlanta, GA) and colleagues in their report published online February 3, 2014 in JAMA Internal Medicine.

 

"Our results support current recommendations to limit the intake of calories from added sugars in US diets."

Dr Kirsten Bibbins-Domingo (University of California, San Francisco), who was not involved in the study, commented to heart wire that the research suggests that clinicians need to counsel patients about the health harms from too much sugar. 

"I think that we are just really recognizing that there are significant health consequences from eating excessive amounts of sugar that go beyond not wanting to be overweight, but go to wanting to make sure that our heart and our kidneys and our brains are healthy," she said. 

Sugar and CVD Death

Epidemiologic studies have linked high consumption of added sugar with an increased risk of type 2 diabetes, obesity, and hypertension, but most studies focused only on sugar-sweetened beverages, and none looked at the relationship between added sugar intake and CVD mortality in a national sample, the authors observe 

There is disagreement about how much added sugar is acceptable in a healthy diet, they note. Added sugar should be less than 25% of total calories according to the Institute of Medicine, but less than 10% of total calories according to the World Health Organization. The American Heart Association has even stricter recommendations: less than 100 calories a day (about 5% of total daily calories) for women and 150 calories a day (about 7.5% of total daily calories) for men. 

Yang et al analyzed data from three National Health and Nutrition Examination Survey (NHANES) surveys —1988–1994, 1999–2004, and 2005–2010 —and examined CVD mortality during a mean follow-up of 14.6 years. 

During the three time periods, among US adults aged 20 and older, the adjusted mean daily calories from added sugar went from 15.7% to 16.8% to 14.9%. 

More than 70% of the added sugar came from sugar-sweetened beverages (37.1%), grain-based desserts (13.7%), fruit drinks (8.9%), dairy desserts (6.1%), and candy (5.8%). 

In 2005–2010, most adults (71.4%) consumed 10% or more of calories from added sugar, and about 10% consumed 25% or more calories from sugar. 

Adjusted hazard ratio (95% CI) of CVD mortality* for different percentages of calories from added sugar

Calories from added sugar (% of total)			p
0 to <10	10.0 to <25	> 25
1.00	1.30 (1.09– 1.55)	2.75 (1.40–5.42)	0.004

*Adjusted for age, sex, race/ethnicity, education, smoking, alcohol consumption, physical activity, antihypertensive medication, family history of CVD, Healthy Eating Index score, body-mass index, systolic BP, total serum cholesterol, and total calories 

 

Blacks were more likely than others to consume more than 25% of calories as added sugar, but there was also no significant link between sugar consumption and CVD mortality among blacks —possibly due to the small number of events in this subgroup, the researchers write. 

"Yang et al inform this debate by showing that the risk of CVD mortality becomes elevated once added sugar intake surpasses 15% of daily calories—equivalent to drinking one 20-ounce Mountain Dew soda in a 2000-calorie daily diet," Dr Laura A Schmidt (University of California, San Francisco) writes in an invited commentary ^[2]. The risk rises exponentially as sugar intake increases, peaking with a fourfold increased risk of CVD death for individuals who consume one-third or more of their daily calories in added sugar, she adds. 

"Until federal guidelines are forthcoming, physicians may want to caution patients that, to support cardiovascular health, it is safest to consume less than 15% of their daily calories as added sugar," she advises. 

No conflicts of interest were reported.

References

1. Yang Q, Zhang Z, Gregg EW, et al. Added sugar intake and cardiovascular diseases mortality among US adults. JAMA Intern Med 2014; DOI:10.1001/jamainternmed.2013.13563. Abstract
   
2. Schmidt LA. New unsweetened truths about sugar. JAMA Intern Med 2014; DOI:10.1001/jamainternmed.2013.12991. Commentary 
   
   source: www.medscape.net
   
   
    

Bacteremia Appears to Increase 30-Day Risk of MI or Stroke

Patients who had bacteremia —mainly urinary-tract infections, pneumonia, or sepsis —when admitted to hospital were much more likely to have an MI or stroke within 30 days, compared with healthy controls or patients hospitalized for other reasons, in a new study ^[1].

"Our study corroborates that acute infection may trigger cardiovascular events," Dr Michael Dalager-Pedersen (Aalborg University Hospital, Denmark) told heart wire in an email. "It is the first study to demonstrate that many different bacterial infections may affect MI and stroke risk," he added.
The research suggests that "bacteremia (a severe and acute infection) should be considered a risk factor for MI and stroke, but only for a short period of time after onset of infection," and it hints that infection with Staphylococcus aureus may confer a particularly high risk.

"Patients admitted with signs of acute infection and bacteremia/sepsis should be monitored closely for complications, and treated early . . . with fluid therapy, oxygen, and antibiotics," Dalager-Pedersen continued. "Moreover, it seems prudent to increase vaccination efforts (eg, influenza and pneumococcal vaccination), in particular in patients who already have established cardiovascular disease, since infection may trigger new cardiovascular events."

Future studies are needed to clarify whether specific cardiovascular therapies (eg, antithrombotic or anti-inflammatory drugs) may reduce the risk of cardiovascular complications in patients with bacteremia, he said.

The study was published online February 12, 2014 in Circulation.

Infection a Trigger for MI, Stroke
An estimated one million Americans have an acute MI or stroke each year, and it would be useful to understand how acute infections might trigger these events, but most previous studies lacked laboratory confirmation of infection, the researchers write.

Using population-based databases, they identified 4389 patients in Northern Denmark who had positive blood cultures when admitted to hospital from 1992 to 2010. The pathogens were Escherichia coli, Streptococcus pneumoniae, S aureus, other bacteria, and fungi. Most patients had urinary-tract infections or pneumonia, while others had central nervous system infections, endocarditis, and other infections.

The mean age of patients was 73 years. Based on age, gender, and date of admission, each patient was matched with about five patients hospitalized for other reasons and about 10 individuals in the general population.

Researchers identified all incident MI and stroke events that occurred within 0 to 30 days, 31 to 180 days, and 181 to 365 days after the day of hospitalization.

Patients with community-acquired bacteremia had a greatly increased risk of MI or stroke within 30 days. At 31 to 180 days, these patients had a modestly higher risk of MI or stroke compared with healthy controls, but not compared with other hospitalized patients. No differences in cardiovascular risk were seen after more than six months.

Relative Risk (95% CI) of Acute MI or Stroke Among Patients Admitted to Hospital (Multivariate Adjusted)

Time	Community-acquired bacteremia patients vs healthy controls	Community-acquired bacteremia patients vs hospitalized controls
0–30 days	20.86 (15.38–28.29)	2.18 (1.80–2.65)
31–180 days	1.64 (1.18–2.27)	0.95 (0.69–1.32)

"I was surprised by the magnitude of increased risk for AMI and stroke in bacteremia patients, especially because the risk was increased in all the examined subgroups," said Dalager-Pedersen.
"An important step in improving the prognosis for patients with severe acute infection is to recognize the association between acute infection and MI/stroke, in order to raise the level of suspicion for vascular complications and diagnose and treat these complications at an early stage."

Call for Improved Vaccination Rates, More Studies
"If Dalager-Pedersen and colleagues' results are accurate and generalizable it is likely that 30 000 to 40 000 new strokes or MIs occur after bacteremia each year in the US," Dr Allan J Walkey (Boston University School of Medicine, MA) writes in an accompanying editorial ^[2].

"Adding in previously described risks of cardiovascular events following pneumonia or urinary-tract infection, we estimate that 5% to 10% of acute MIs or strokes may be associated with acute infection."
The study identified that " 'being sick' in general was not the only mechanism for increased risk" of MI or stroke, Walkey observed.

Like the study authors, he calls for improved vaccination rates and more research. "Increased efforts should be made to improve suboptimal vaccination rates among patients with cardiovascular disease . . . [and] further mechanistic studies and large randomized trials would be required to assess whether antiplatelet, beta-blocker, or statin therapies might result in lower cardiovascular complications and possibly better outcomes after acute infection."

The researchers reported no conflicts of interest. Walkey received funding from the National Heart, Lung, and Blood Institute for a study on atrial fibrillation in sepsis.

References

1. Dalager-Pedersen M, Søgaard M, Schønheyder HCC, et al. Risk for myocardial infarction and stroke after community-acquired bacteremia: A 20-year population-based cohort study. Circulation 2014; DOI:10.1161/CIRCULATIONAHA.113.006699. Abstract
   
2. Walkey AJ. Preventing cardiovascular complications of acute infection: A missed opportunity? Circulation 2014; DOI:10.1161/CIRCULATIONAHA.114.008712. Editorial
   

 

source: www.medscape.net

Maternal Hair Selenium Levels as a Possible Long-term Nutritional Indicator of Recurrent Pregnancy Loss

Viju V Thomas, Robert Knight, Stephen J Haswell, Stephen W Lindow, Zephne M van der Spuy

BMC Womens Health. 2013;13(40)

 Abstract

Background Approximately 1% of all couples trying to conceive will suffer from recurrent pregnancy loss (RPL). Nutritional deficiencies have been postulated as a possible cause of RPL and in particular, selenium deficiency has been associated with reproductive failure in animal studies and more recently, in some human studies. This study was undertaken to assess the maternal hair selenium levels in women with RPL without an identified cause and to compare these results with those of women with successful reproductive histories.
Methods Twenty four patients with RPL and twenty four control subjects with at least one successful pregnancy and no pregnancy failures, who were matched for age and ethnicity, were recruited. A questionnaire was completed, which included demographic and social information and a dietary history. Hair samples were collected and analyzed for selenium content by inductively coupled plasma mass spectrometry.
Results The control subjects had a higher mean income and had completed more years of education compared with the RPL patients. There was no significant difference in the intake of selenium rich foods between the 2 groups. The patients, however, consumed significantly more fruit, cheese, potatoes and chocolate than the controls. The median (range) selenium content was 0.80 ppm (0.19–4.15) and 0.68 ppm (0.43–3.76) in patients and controls respectively (Mann Whitney U test 209.5 p = 0.74).
Conclusions While there were significant differences in the 2 groups with regard to resources, education and diet our results show that hair selenium concentrations and dietary selenium intake, were similar in the two groups. Both groups had low levels of this important element.

Background

Miscarriage is the most common complication in pregnancy with an incidence of 12–31%. ^[1] In South Africa it contributes to 30% of "pregnancy related sepsis" and is directly responsible for 3.5% of all maternal deaths. ^[2] Recurrent pregnancy loss (RPL) is defined as the loss of three or more consecutive pregnancies before 20 weeks gestation and is estimated to affect 1% of all couples trying to conceive. ^[3,4]
Malnutrition is a global problem. In developed countries it is often related to inappropriate diets or eating disorders while in developing countries, chronic ill health and food scarcity contribute to malnutrition. ^[5] Micronutrient deficiencies associated with malnutrition, such as selenium, copper, zinc and iron have been implicated in miscarriage and selenium deficiency has been identified as a possible cause for RPL in several studies. ^[6–10]
Selenium is a key component of a number of selanoproteins, of which the best known is the antioxidant glutathione peroxidase, which contributes to the delicate balance between pro-oxidants and anti-oxidants, a critical aspect for survival in aerobic organisms.
The concentration of selenium in the soil is reflected in the plants and the animals that feed on these plants. Reproductive failure as a consequence of selenium deficiency has been demonstrated in a number of animal and agricultural studies. Soil selenium levels differ between countries and local studies in herbivores have demonstrated that the selenium concentration in South African soils is low. ^[11] Humans obtain selenium through their diet. Studies from the UK have demonstrated that selenium concentrations are highest in foods like Brazil nuts (245 ug/100 g), kidney (146 ug/100 g), crab meat (84 ug/100 g) and liver (42 ug/100 g). Other sources of selenium include bread, cereals, meat, poultry and fish. ^[12]
A number of women with RPL have no identifiable cause for their pregnancy failure. An expert review article suggests that reduced antioxidant protection may contribute to early pregnancy loss. ^[13] Given the data on selenium, the findings of other studies and the suggested selenium deficiency in the Western Cape, this study was undertaken to determine whether selenium deficiency was present in women with RPL and to compare this group to women with an uncomplicated obstetric/pregnancy history. In view of the proven value of determining micronutrients and other substances in hair, the hair selenium status was measured as a reflection of long-term nutritional status.

Methods

Our aim was to assess the association between RPL and selenium deficiency in a population living in a low selenium environment.
This study was approved by the Research Ethics Committee of the Faculty of Health Sciences, University of Cape Town (Research Ethics Committee Reference no: 119/94 and 041/2006). Written informed consent was obtained from all participants. This case–control study included a total of 24 patients and 24 control subjects. RPL was defined as women with 3 consecutive first trimester miscarriages or 2 second trimester miscarriages. Patients consisted of non-pregnant women, not using hormonal contraception who suffered from RPL. All patients were recruited from the Reproductive Failure Clinic at Groote Schuur Hospital, Cape Town, and had no identifiable pathology when screened for known causes of RPL, e.g. anatomical defects, chromosomal abnormalities, endocrinopathies, immunologic and thrombotic pathology.
Control subjects were healthy, non-pregnant women with at least one live child who were not using hormonal contraception and had not been pregnant during the previous 6 months. These controls and subjects were matched for age (±2 years) and ethnicity. Controls were recruited from women attending our outpatient clinics and staff members who fulfilled the criteria. Inclusion and exclusion criteria are tabulated in .

Table 1.  Inclusion and exclusion criteria

Inclusion criteria		Exclusion criteria
CASES	CONTROLS	CASES	CONTROLS
Non pregnant women	Non pregnant women	Currently pregnant or lactating	Currently pregnant or lactating
Three consecutive 1st trimester miscarriage or two 2nd trimester losses	Uncomplicated obstetric history	Cause for RPL identified.	Any underlying medical condition that may cause RPL
No live births after miscarriages	One or more successful pregnancies	Use of hormonal contraception in preceding 6 months	Use of hormonal contraception in preceding 6 months
No diagnosis for RPL established	N/A	Not living in Cape Town for more than 2 years	Not living in Cape Town for more than 2 years
No hormonal contraception in preceding 6 months	Not on hormonal contraception

A datasheet was completed for each recruit incorporating demographic details, proximity to industrial areas, types of dwelling/roofing, cooking utensils, contraceptive use, personal habits (smoking and alcohol consumption), dietary history, hair products/treatments and reproductive history. We sought information regarding diet, hair treatments and contraception for 6 months prior to sampling. Hair was sampled from the nape of the neck, just above the hair line and marked proximally to identify the newest growth. The RPL and control subjects were compared using a chi square or t test as appropriate.

Analysis of Hair Samples

Sharp surgical scissors were used to cut hair from the nape of the neck. All samples were stored in a plastic container at room temperature and couriered to the University of Hull, Department of Chemistry, where preparation and analysis was performed and reported. The samples were washed with lipsol detergent in pure water to remove surface debris, then air dried overnight. The hair was weighed and put into Teflon digestion vessels with 0.5 ml nitric acid, sealed and left overnight. They were then placed in a microwave oven (CEM MARS Xpress) and heated at high temperature, then cooled to room temperature. Once opened the contents were diluted in 5 ml water and analysed using the Perkin Elmer Elan DRCII inductively coupled plasma mass spectrometry (ICPS) instrument. Selenium was calibrated at 0, 1, 5, 10, 20 parts per billion (ppb), which gave linear calibrations. The measured concentrations of selenium were then calculated to give the amount in the original dry hair samples by multiplying the measured concentration by the dilution factor. Any dilutions of the digests were allowed for at this point. Changing the units gave values in ppm, or micrograms per gram of original dry solid. Gaps in the tables were filled in by recalculation of the data in the Elan software sample by sample. The limit of detection was 1.016 ppb. Except for 4 values (3 RPL and 1 control), the measured concentrations of selenium were all above these values.

Results

Twenty four women were recruited to each group (patients and controls). All the patients who were invited agreed to participate, while in the control group, out of 31 potential subjects, 7 were unsuitable and 24 satisfied all criteria and agreed to participate. Subjects gave written informed consent. Cases and controls were matched for ethnicity. Each group had 17 participants of mixed race origin, 6 black African and 1 Indian subject. presents the general characteristics of the two groups. The mean ages were 32.5 years (RPL) and 33 years (control group).

Table 2.  Background patient characteristics

Characteristics	Recurrent miscarriage n = 24	Control n = 24	P
Age(y)	32.5 (5.62)	33 (5.87)	0.775
BMI (kg/m 2)	31.9 (21.8)	28.5 (6.3)	0.473
Education (y)	10.0(3.12)	14.9(4.23)	0.374
Gravidity	4.9 (1.66)	2.3 (1.22)	0.001
Household income/Rand per month	R. 6147.55 (8022.6)	R. 14761 (11351.44)	0.006
Miscarriages	3.7 (1.2)	0 (0)	0.001
Weight gain(kg)	1.9(3.43)	3.2(3.66)	0.214
Weight loss(kg)	1.0(1.79)	0.7(1.60)	0.87

Mean (SD).

The only significant differences between the two groups were obstetric outcomes, as would be expected, and household income. The mean number of miscarriages in the RPL group was 3.7 ±1.2. There was a significantly higher monthly household income among control subjects (mean income R6221 (SD 8195) v R14762 (SD 11352) p < 0.006) in RPL and controls respectively. Although not statistically significant, analysis of educational achievements showed that the controls had an average of 4.9 years more of formal education than the patients.
The results of the dietary questionnaire are presented in . Comparison of the consumption of selenium rich foods namely nuts, liver and kidney did not show any significant difference between the groups. In the RPL group, there was a statistically significant increase in weekly consumption of cheese [4.85 (sd.9.3) vs. 0.44 (sd.0.64) P = 0.02], fruit [4.88 (sd. 6.9) vs. 1.46 (sd.1.06) P = 0.025], chocolate [1.9 (sd.2.65) vs. 0.54 (sd.0.72) P = 0.016] and potato [4.58 (sd.4.9) vs. 0.98(sd.0.93) P = 0.001]. There was no difference in the consumption of alcohol or of smoking. All women were asked about the use of anti-dandruff shampoo and hair colouring agents. In the RPL group 62.5% used hair colouring agents, including one patient who had used henna products within the preceding 6 months, compared with 45.8% in the control group (P = 0.424). A review of shampoo brands used by our subjects revealed that none contained selenium, but the henna product did have selenium and this was reflected in the results of her hair analysis.

Table 3.  Results of dietary questionnaire

Food, drink and habits	Recurrent miscarriage n = 20	Control n = 23	P
Alcohol (units/day)	0.7(1.7)	0.4(1.2)	0.957
Bread (slices/day)	3.2(2.0)	3.0(2.0)	0.757
Cereals (bowls/day)	0.6(0.7)	0.6(0.5)	0.528
Ceylon tea (cups/day)	1(2.1)	1.0(1.3)	0.921
Cheese (helping/week)	4.9(9.3)	0.4(0.6)	0.026
Chocolate (bars/week)	1.9(2.7)	0.5(0.7)	0.016
Coffee (cups/day)	1.8(2.4)	2(1.5)	0.73
Cream (helpings/week)	0.1(0.3)	0.04(0.2)	0.575
Fruit (pieces/week)	4.9(6.9)	1.5(1.1)	0.025
Liver/kidney (helping/week)	0.6(1.4)	0.1(0.2)	0.129
Nuts (grams/day)	10.4(29.3)	7.9(17.4)	0.728
Peas/beans(helpings/day)	0.9(1.9)	0.7(0.5)	0.627
Potato (helping/week)	4.6(4.9)	1.0(0.9)	0.001
Rooibos tea (cups/day)	0.7(1.0)	0.4(0.9)	0.317
Smoking (cigarettes/day)	5.2(7.3)	2.6(4.8)	0.550

Selenium Levels in Hair

represents our findings of selenium levels in hair. Hair selenium levels were not normally distributed and log selenium levels were also not normally distributed therefore a non-parametric analysis was undertaken. The median (range) selenium content was 0.80 ppm (0.19–4.15) and 0.68 ppm (0.43–3.76) in patients and controls respectively (Mann Whitney U test 209.5 p = 0.74). There was no significant difference in hair selenium levels between the two groups. Figure 1 demonstrates that there is an outlier with a very high selenium concentration of 20.4 ppm. A review of this patient's questionnaire revealed that she was the only subject who had used henna hair products in the preceding six months and this result was excluded from the analysis. This patient fell into the lower income category and her dietary history did not show any difference in any food consumption. The concentrations from 3 RPL and 1 control subject fell below the limit of detection. These 4 values together with the one outlier were excluded from the statistical analysis.

Table 4.  Hair selenium concentrations

	Selenium (ppm) median	Range (ppm)
RPL n = 20	0.80	0.19–4.15
CONTROLS n = 23	0.68	0.43–3.76

Mann Whitney U test 209.5 ( p = 0.74). (Analysis excluding 5 women, 3 RPL and one control whose selenium concentrations fell below the limit of detection and one outlier in the RPL group who had used henna hair products).

Figure 1.
  Selenium concentrations on vertical axis in ppm.

Because the control group had significantly better incomes, the income and hair selenium level was correlated across the combined group of women. Hair selenium concentrations were not significantly correlated with income (Pearson correlation coefficient −0.183, p = 0.219).

Discussion

The aetiology of RPL is probably multi-factorial and may include nutritional and micronutrient deficiencies.
Undernutrition and malnutrition are recognised as having a potential impact on reproductive performance. The women who attend our Reproductive Failure Clinic often do not have an identified cause for their pregnancy loss, which makes future management and counselling difficult. Nutritional deficiencies have been identified as causes of failure of both fertility and fecundity. Animal work makes a compelling case for a role for selenium in successful reproduction and recent studies in women with RPL have suggested that selenium deficiency may contribute to RPL. ^[10,14–16] Our study was undertaken in an attempt to identify the selenium status in women with RPL of unknown cause and compare this to those in women with successful reproductive histories. If selenium is deficient in our clinic population replacement therapy may prove helpful in affected patients.
Using hair as the tissue for study offers an opportunity to identify chronic/long term selenium status. Forensic science has utilized hair for analysis of chronic drug exposure, pesticide exposure and post mortem toxicology and these methods are validated and reproducible. ^[17–21] Al- Kunani et al., in a study undertaken in Hull, UK, using blood and hair, demonstrated selenium deficiency in the RPL group in hair samples but not blood samples. ^[10]
As hair selenium levels may be affected by use of hair products, such as certain anti-dandruff shampoos and henna, ^[19,21] we reviewed all products used by subjects in the preceding 6 months. One patient was using henna hair dye, and this is reflected in the abnormally high selenium levels in her hair. The analysis was done after excluding this outlier. Further confounding factors were taken into account such as environmental exposure from roofing, type of housing, proximity to industrial areas, access to running water and cooking utensils. All subjects had been living in Cape Town for at least two years and comparison between the two groups showed no difference in environmental exposure.
Studies have demonstrated a positive correlation between serum estradiol levels and glutathione peroxidase and we therefore excluded any women who had used hormonal contraception over the preceding 6 months. ^[22,23]
The two groups differed significantly in family incomes, with the median income of the controls almost four times that of the patients (R12850 vs. R3450). There was a non-significant difference in their education levels, where the controls studied on average 4.9 years more than patients. This result reflects the different populations selected for each group, where control subjects included appropriate hospital staff members. Although not intended, the difference between the two groups may mean that controls had more access to dietary information, enabling them to eat healthier, and possibly consume food types with higher selenium content. Comparison of the selenium rich foods, however, showed no difference in their intake, possibly suggesting no advantage among the control subjects in terms of dietary information supplied to them, despite their educational and economic advantages.
Patients and controls had different dietary habits, but not with regard to selenium rich foods. Obviously in the controls, the levels may have been different during their successful pregnancies but there is no way of ascertaining this and it seems likely that their diet was fairly constant over time.
The results show a significantly higher intake in the RPL group of potato, cheese, fruit and chocolate and these foods are relatively low in selenium. In South Africa, fruit is readily affordable and potato is relatively cheap and more portions was eaten by patients per week. A fungus, Phytophthora infestans, was identified as the cause for potato blight in the Irish Famine of 1844 and although there is no evidence in the literature that there is a direct link to miscarriage however the possibility that this fungus may cause miscarriage should be considered.
The selenium levels from the study by Al Kunani et al. and this study were analysed in the same laboratory at the University of Hull, and in comparison with the UK (Al Kunani ^[10]) results, women in South Africa had comparatively higher selenium concentrations, possibly suggesting higher selenium in South African soils and foods.
In contrast to our findings, Ejezie et al. ^[24] recruited 40 women (n = 120) in each trimester and 35 control subjects and measured serum selenium concentration during pregnancy and postpartum and found significantly decreased selenium concentrations in pregnancy, when compared to non pregnant controls. The authors concluded that selenium supplementation might be indicated in pregnancy.

Conclusion

Micronutrient deficiency has been documented in a number of reproductive disorders and in a population with absolute and relative nutritional compromise. This study did not demonstrate selenium differences in the two groups studied but a nutritional component, still to be defined, in RPL is still a possibility particularly when one reviews the socioeconomic deprivation in many of our communities. In our opinion selenium supplementation in RPL cannot be recommended until more research has been performed.
Future research possibilities include exploring and comparing different tissue types to evaluate selenium and other micronutrients and employing prospective studies to evaluate selenium concentrations in RPL.

References

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2. Pattinson RC, Snyman LC: Miscarriage. In Clinical Gynaecology. 3rd edition. Edited by Kruger TF, Botha MH. Cape Town: Juta; 2007::188–197.
   
3. Katz VL, Kuller JA: Recurrent miscarriage. Am J Perinatol 1994, 11(6):386–397.
   
4. Rai R, Regan L: Recurrent miscarriage. Lancet 2006, 368(9535):601–611.
   
5. van der Spuy ZM: Nutrition and reproduction. Clin Obstet Gynaecol 1985, 12(3):579–604.
   
6. Barrington JW, Lindsay P, James D, Smith S, Roberts A: Selenium deficiency and miscarriage: a possible link? Br J Obstet Gynaecol 1996, 103(2):130–132.
   
7. Barrington JW, Taylor M, Smith S, Bowen-Simpkins P: Selenium and recurrent miscarriage. J Obstet Gynaecol 1997, 17(2):199–200.
   
8. Kocak I, Aksoy E, Ustun C: Recurrent spontaneous abortion and selenium deficiency. Int J Gynaecol Obstet 1999, 65(1):79–80.
   
9. Munoz C, Carson AF, McCoy MA, Dawson LE, Irwin D, Gordon AW, et al.: Effect of supplementation with barium selenate on the fertility, prolificacy and lambing performance of hill sheep. Vet Rec 2009, 28;164(9):265–271.
   
10. Al-Kunani AS, Knight R, Haswell SJ, Thompson JW, Lindow SW: The selenium status of women with a history of recurrent miscarriage. BJOG 2001, 108(10):1094–1097.
    
11. Van Ryssen JBJ: Geographical distribution of the selenium status of herbivores in South Africa. S Afr J Animal Sci 2001, 31:1–8.
    
12. Barclay M, MacPherson A, Dixon J: Selenium content of a range of UK foods. J Food Composit Anal 1995, 8:307–318.
    
13. Mistry HD, Broughton Pipkin F, Redman CWG, Poston L: Selenium in reproductive health. Am J Obstet Gynecol 2012, 206(1):21–30.
    
14. Zachara BA, Dobrzynski W, Trafikowska U, Szymanski W: Blood selenium and glutathione peroxidases in miscarriage. BJOG 2001, 108(3):244–247.
    
15. Kumar KS, Kumar A, Prakash S, Swamy K, Jagadeesan V, Jyothy A: Role of red cell selenium in recurrent pregnancy loss. J Obstet Gynaecol 2002, 22(2)):181–183.
    
16. Nicoll AE, Norman J, Macpherson A, Acharya U: Association of reduced selenium status in the aetiology of recurrent miscarriage. Br J Obstet Gynaecol 1999, 106(11):1188–1191.
    
17. Boumba VA, Ziavrou KS, Vougiouklakis T: Hair as a biological indicator of drug use, drug abuse or chronic exposure to environmental toxicants. Int J Toxicol 2006, 25(3):143–163.
    
18. Mantzouranis EC, Bertsias GK, Pallis EG, Tsatsakis AM: Hair analysis differentiates chronic from acute carbamazepine intoxication. Pediatr Neurol 2004, 31(1):73–75.
    
19. Musshoff F, Madea B: Analytical pitfalls in hair testing. Anal Bioanal Chem 2007, 388(7):1475–1494.
    
20. Tsatsakis AM, Tzatzarakis MN, Tutudaki M: Pesticide levels in head hair samples of Cretan population as an indicator of present and past exposure. Forensic Sci Int 2008, 176(1):67–71.
    
21. Spallholz JE, Boylan LM, Palace V, Chen J, Smith L, Rahman MM, Robertson JD: Arsenic and selenium in human hair: a comparison of five countries with and without arsenicosis. Biol Trace Elem Res 2005, 106(2):133–144.
    
22. Massafra C, Gioia D, De FC, Picciolini E, De LV, Bonifazi M, et al.: Effects of estrogens and androgens on erythrocyte antioxidant superoxide dismutase, catalase and glutathione peroxidase activities during the menstrual cycle. J Endocrinol 2000, 167(3):447–452.
    
23. Massafra C, Buonocore G, Berni S, Gioia D, Giuliani A, Vezzosi P: Antioxidant erythrocyte enzyme activities during oral contraception. Contraception 1993, 47(6):590–596.
    
24. Ejezie FE, Okaka AC, Nwagha UI: Reduced maternal selenium levels in pregnant and lactating Nigerian women: should routine selenium supplementation be advocated? Niger J Med 2012, 21(1):98–102.
    

 

Competing interests

The authors declare that they have no competing interests.
Authors' contributions

VT collected the data and drafted the manuscript. RK and SH carried out the laboratory work. ZVDS and SL participated in the design of the study. SL performed the statistical analysis. SL and ZVDS conceived of the study and participated in its design and coordination and helped to draft the manuscript. All authors read and approved the final manuscript.
Acknowledgements

The authors would like to acknowledge the following:

• Associate Professor Landon Myers, University of Cape Town, for assisting with the statistical analysis.

• Dr Sascha Edelstein, Subspecialist trainee, Department of Obstetrics and Gynaecology, University of Cape Town, who assisted in the initial recruitment of subjects.

• The patients of the Reproductive Failure Clinic at Groote Schuur Hospital and control subjects who agreed to participate in this study.

• The members of the Reproductive Medicine Unit, and in particular, Sister A. Hoffman, who maintains the patient database for the RMU and who assisted in recruitment of patients.
Condensation

This study measured hair selenium levels in women with recurrent miscarriage and compared them to those with uncomplicated reproductive histories in a tertiary unit in South Africa. While differences in dietary habit and socioeconomic status were demonstrated, selenium levels in the two groups were not significantly different.
BMC Womens Health. 2013;13(40) © 2013  BioMed Central, Ltd.

Urine Specimen Collection

How a Multidisciplinary Team Improved Patient Outcomes Using Best Practices

Vickie J. Dolan, APRN-NP, APRN-CNS, ACNP, CCNS, Nancy E. Cornish MD, FCAP, FASCP

Urol Nurs. 2013;33(5):249-256. 

Abstract and Introduction

Abstract

The Institute of Medicine challenged the health care system to ensure safe care for all patients. This article reviews one hospital's attempt to minimize contamination of urine specimens by using a multidisciplinary team to implement evidencebased best practices. The clinical nurse specialist (CNS) collaborated with microbiology and laboratory personnel to review literature, collect and analyze data, provide staff education, and facilitate change in urine collection kits and urine processing that significantly improved the quality of urine specimens in a 350-bed hospital in the Midwest.
Objectives:
1. Discuss the potentially negative impact of poorly collected urine specimen on patient care.
2. Explain the proper urine collection method for males.
3. Explain the proper urine collection method for females.
4. Describe benefits of implementing a multidisciplinary approach to urine collection.

Introduction

Specimen management in microbiology includes selection, collection, transport, storage, analysis, and reporting. When errors occur at any point in this process, the result may adversely affect the patient (i.e., misdiagnosis, extended length of hospital stay, or inappropriate therapy). Clinicians base treatment decisions on urine culture results, and patients are affected either positively or negatively by the accuracy of those results. A chain of people with varying levels of education, diverse backgrounds, and different foci on patient care impact this process.
In 2007, the director of micro biology at a 350-bed community hospital observed that the incidence of culture results that grew only coagulase-negative staphylococcus greater than 100,000 colony forming units (CFU)/ml in urine specimens was increasing. This is an unusual finding because coagulase-negative staphylococcus (CoNS) is an uncommon cause of urinary tract infection (UTI) (McCarter, Burd, Hall, & Zervos, 2009). Additionally, CoNS is a common contaminate of microbiology specimens, including urine specimens (Versalovic et al., 2011). The urine specimens were, for the most part, collected by staff nurses via straight catheterization in the emergency department (ED) and inpatient care areas. This raised a question of possible contamination of the specimens or another quality assurance issue, such as prolonged transport (defined as greater than 2 hours) or storage at incorrect temperatures (urines should be stored at a refrigerator temperature of 4 degrees centigrade) (McCarter et al., 2009).
The microbiology director alerted the medical director of pathology, the infection prevention department, and the patient multidisciplinary meeting was convened and included nursing leaders, infection prevention staff, laboratory leaders/supervisors, information technology specialists, and patient safety and process improvement (PI) leaders and staff. A committee was formed to investigate the issue, make recommendations if problems were found, and facilitate implementation of solutions.
The purpose of this article is to 1) review how one hospital addressed the investigation of a potentially flawed pre-analytic urine collection/transport process by forming a multidisciplinary performance improvement team; 2) share "best practices" found during the literature review regarding obtaining, transporting, and processing urine specimens; and 3) discuss implementation of these best practices in our institution as recommended in the literature.

Significance

Patients may be negatively impacted when results from faulty urine specimens are re ported. Inappropriate use of anti biotic therapy can occur when prescribed based on the result of a contaminated specimen. The mean cost to treat a catheter-associated urinary tract infection (CAUTI) is $759 (Anderson et al., 2007). In the intensive care unit (ICU), the mean cost of a CAUTI is $1,955 (Chen, Wang, Liu, & Chou, 2009). In addition to being costly, antibiotic overuse is a factor in development of Clostrid ium difficile(C. difficile) and/or multidrug-resistant organisms. Fluoroquinolone use has a strong association with the development of C.difficile (Cohen et al., 2010). A prospective study of 1,703 patients by Loo et al. (2005) found C.difficile to be associated with fluoroquinolone use in 82% of cases. A retrospective chart review by Khawcharoenporn, Vasoo, Ward, and Singh (2010) found the fluoroquinolone resistance rate for health care-associated UTIs to be 38% vs. 10% in the community. They also identified other evolving drug resistances: trimethoprim-sulfamethoxazole (TMPSMZ) (26% vs. 17%), amoxicillin (53% vs. 45%), and amoxicillinclavulanate (16% vs. 6%).

Performance Improvement Processes

A multidisciplinary microbiology performance improvement team was formed to address the suspected faulty urine collection process. Representatives from eight departments were members of this team; these included nursing, performance improvement, medicine, pathology, laboratory, specimen control, microbiology, and information technology (IT). Each member brought a unique perspective and best practice recommendations from review of the literature regarding urine specimen collection and processing. Discussion content focused on current practice, evidence-based practices (EBPs), and improvement opportunities. To complete the project, current practices were evaluated, the literature was reviewed, and practice changes were made. Each of those processes will be addressed in further detail in this article.

Evaluation of Current Practice

Urine Specimen Collection. The clean-catch midstream void collection process was found to have differing instructions in outpatient (ED) and inpatient settings. Patients were not routinely instructed about the importance of collecting a true clean-catch urine specimen correctly and how it may influence results.
Observation of urine collection processes via straight catheterization revealed that the initial (first void) 10 ml of urine was being collected because the catheter was pre-attached by the manufacturer to the specimen collection container, which only had a total volume of 10 ml. This method allowed bacteria from the urethra to be processed. Urine specimens from chronic indwelling catheters were frequently collected from catheter tubing without changing the catheter prior to collection, even when the catheter had been in place for longer than two weeks. This allowed for collection of biofilm build up along the catheter tubing wall instead of a true specimen of urine from the bladder.
Specimen Labeling. A patient identification label was affixed to the specimen container and dated. The hospital used a paper requisition format with no requirement for all elements to be completed and no consequence to the collector for incomplete forms. In March 2008, staff compliance with completing the requisition form correctly was 30%. Staff indicated that barriers to completing the form correctly were lack of time and lack of clear instructions on what elements were required.
Transportation. Data were collected during three separate one-month time periods. Data collected for the month of August 2009 revealed that urine specimens remained at room temperature for between 4 to 120 minutes prior to being sent to the laboratory. This was often well over the 20-minute doubling time it takes for some common bacteria to replicate at room temperature (see ).

Table 1.  Urine Specimen Transport Time

Date	Average Time from Collection to Arrive in Laboratory (at room temperature)	Average Time from in Laboratory to Being Processed (UA)	Average Time from Collection to Being Processed
January	Range: 10 to 81 minutes	Range: 13 to 28 minutes	Range: 26 to 109 minutes
June	Range: 8 to 62 minutes	Range: 11 to 37 minutes	Range: 19 to 79 minutes
August	Range: 4 to 120 minutes	Range: 13 to 29 minutes	Range: 23 to 133 minutes

PICOT Question

A PICOT question was formulated to guide the literature review search. PICOT is an abbreviation for the Population affected, Intervention being studied, Comparison group, Outcome evaluated, Time frame (Polit & Beck, 2012). The question was: "What is the best practice for urine specimen collection process in acute care hospitals to decrease incidence of contaminated urine culture results?"
Following development of the PICOT question, the clinical nurse specialist (CNS) and director of microbiology reviewed the research literature and practice guidelines related to urine specimen collection. The Institute of Medicine (IOM) (2000) recommended implementation of best practices at the bedside and use of a multidisciplinary team to help ensure patient safety.

Literature Review

The purpose of the literature review was to research best practices for obtaining and processing urine specimens. The literature review is organized under three headings: pathology, asymptomatic versus symptomatic infections, and specimen collection processes.
Pathology. Wilson (2008) describes the female and male urethra and presence of normal flora found. A normal female urethra is 3.8 cm in length. It is colonized with 2 to 11 different organisms, with the total population between 105 and 106 viable microbes. Examples of urethral bacteria include lactobacilli (protective), coagulase-negative Staphylococcus, diptheroids, anaerobes, and mycoplasma. Age and sexual maturity result in changes in bacteria present; older age groups have greater numbers and greater variety of organisms present, including gram-negative rods. Treatment with antibiotics can result in colonization with gram-negative rods, such as Escherichia coli, Klebsiella species, and Proteus mirabilis.
The normal male urethra is 20 cm in length; the first 6 cm is colonized with bacteria, which may account for fewer urinary tract infections in males. Types of bacteria vary with age and sexual activity just as in females. Sexual activity results in a greater number and diversity of bacteria ( Gardnerella vaginalis, gram-negative rods, anaerobes, Group B streptococcus, and yeast).
Asymptomatic vs. Symptomatic Infections. Diagnostic criteria for a UTI from both the clinical and epidemiologic perspective can be likened to a moving target. It is not within the scope of this article to provide an exhaustive list of all the criteria and definitions for UTIs. Mul ti ple guidelines are available, all of which vary slightly in their definitions because they were published at different times by different au thors and organizations (such as the Infection Diseases Society of America [IDSA], Centers for Disease Control and Prevention [CDC] National Healthcare Safety Network [NHSN], Ferr [2010], and the Association for Professionals in Infection Control and Epi demiology [APIC]/Society for Healthcare Epidemioloysis of America [SHEA]). The CDC NHSN hosts a Web site ( www.cdc.gov/nhsn/) that provides guidelines on health care-associated infections, including urinary tract infection. These guidelines are periodically updated as new information becomes available. The literature reviewed will be described under additional categories of asymptomatic and symptomatic urinary tract infections.
Asymptomatic Urinary Tract Infection: According to the IDSA (Nicolle et al., 2005) guidelines, asymptomatic bacteriuria is defined as follows:

• In women: two consecutive clean-catch mid-void specimens with same bacteria at greater than or equal 10 ⁵.
  
• In men: one clean-catch mid-void specimen with single bacteria at greater than or equal to 10 ⁵.
  
• A single straight catheter specimen with one bacterial species at greater than or equal 10 ².
  

The ISDA guideline also identifies when asymptomatic bacteriuria should not be treated: pre-menopausal, non-pregnant women, diabetic women, older adults, individuals who are institutionalized, persons with spinal cord injury, catheterized patients while the catheter remains in situ, and pyuria in absence of signs/symptoms of UTI. Pyuria may reflect contamination from the vagina, urethral opening, an existing indwelling Foley catheter, urinary tract stones or foreign bodies, neoplasms (tumors), appendicitis, pancreatitis, diverticulitis, interstitial cystitis or nephritis, nephrotic syndrome, and post-streptococcal glomerulonephritis (Nicolle et al., 2005).
Symptomatic urinary tract infection: Symptomatic urinary tract infection (SUTI) is defined by clinical symptoms and a positive urine culture that demonstrates colony counts of greater than or equal to 10 ³ colony-forming units per milliliter (CFU/ml) (Ferri, 2010). Presenting symptoms can vary from person to person, as can the location or cause of the infection, including cystitis, pyelonephritis, or CAUTI (see ).

Table 2.  Location and Presenting Symptoms of Symptomatic Urinary Tract Infection

Acute uncomplicated cystitis (symptomatic bladder infection in a normal genitourinary tract)	Urinary frequency, urgency Dysuria Urge incontinence Suprapubic pain Gross or microscopic hematuria
Acute pyelonephritis	Flank or abdominal pain Chills Malaise Vomiting Diarrhea
Catheter-associated urinary tract infection (CAUTI)	New onset or worsening of fever, rigors, altered mental status, malaise, or lethargy with no other identified cause Flank pain Costovertebral angle tenderness Acute hematuria Pelvic discomfort Dysuria, urgent or frequent urination, and/or suprabuic pain and tenderness if urinary catheter has been removed

Source: Ferri, 2010; Hooten et al., 2010.

CAUTI is further defined by Hooten et al. (2010) as the presence of symptoms or signs compatible with UTI with no other identified source of infection along with greater than 10 ³ CFU/mL of greater than or equal to 1 bacterial species in a midstream voided urine or single-straight catheter specimen from a patient whose urethral, suprapubic, or condom catheter has been removed within the previous 48 hours. In addition, the CDC Division of Healthcare Quality Promotion's (2009) NationalHealthcare Safety NetworkManual states that at least one of the following criteria must be present for diagnosis of a CAUTI.

• Criteria 1 – Patient had an indwelling urinary catheter in place at the time of specimen collection or removed within the 48 hours prior to specimen collection and at least one of the following signs or symptoms with no other recognized cause: fever greater than 38 degrees Celsius, suprapubic tenderness, or costovertebral angle pain or tenderness and a positive urine culture of greater than or equal to 10 ⁵ CFU/mL with no more than 2 species of microorganisms.
  
• Criteria 2 – Same as Criteria 1, with the addition of a positive urinalysis demonstrated by at least one of the following findings: positive dipstick for leukocyte esterase and/or nitrite, pyuria, urine specimen with greater than or equal to 10 white blood cells (WBCs)/mm ³ or greater than or equal to 3 WBC/high power field of unspun urine, microorganisms seen on gram stain of unspun urine.
  

Urine Specimen Collection. The urine specimen collection consists of the following components: clean-catch mid-stream, straight catheterization, chronic indwelling catheter, and specimen labeling.
Clean-catch mid-stream: The purpose of collecting mid-stream specimens is to avoid collection of bacteria from the urethra, vaginal, and perineal areas that are colonized with gastrointestinal flora. Wilson (2005) identified numerous microbes lining the urethra that are shed during urination. Viable bacteria per ml found in this initial stream range from 0 to 10 ⁵ CFU in patients without a urinary tract infection (Wilson, 2005). See Figures 1 and 2 for specific instructions on collection steps.

Figure 1.
  Male Instructions on How to Obtain a Clean Catch Urine Specimen
Source: Adapted from South Central Association for Clinical Microbiology (n.d.). Used with permission from Nebraska Methodist Hospital, Omaha, NE.

Figure 2.
  Female Instructions on How to Obtain a Clean Catch Urine Specimen
Source: Adapted from South Central Association for Clinical Microbiology (n.d.). Used with permission from Nebraska Methodist Hospital, Omaha, NE.

Straight Catheterization: The purpose of collecting a urine specimen via straight catheterization is to minimize contamination of the specimen when patients are unable to provide a reliable selfcollected specimen. This is a sterile procedure, and the skin must be prepped appropriately. Once the catheter is inserted, the initial urine (first void) stream should be discarded to eliminate the risk of false-positive cultures caused by urethra flora that may have been collected in/on the catheter during insertion (McCarter et al., 2009). Approximately 30 ml of mid-stream urine should be collected in a sterile cup (Perry & Potter, 2006).
Chronic Indwelling Catheter: Formation of biofilm is addressed by several authors (Hooton et al., 2010; Smith et al., 2008; Trautner & Darouiche, 2004). Biofilm build up occurs along the inner and outer walls of urinary catheters from urethral organisms. Chronic catheter placement results in 100% colonization of the urine by bacteria due to biofilm development.
Bacterial colonization of a urethral catheter can begin within a few days of catheter insertion. Encrustation from proteins, electrolytes, and other organic molecules build along the catheter walls. Antibiotics do not frequently clear resistant bacteria in biofilms from catheter surfaces (Trautner & Darouiche, 2004). According to Hooten et al. (2010), if a chronic indwelling catheter has been in place for more than two weeks, the catheter should be changed prior to obtaining a urine specimen for culture if a UTI is suspected.This practice has been associated with improved resolution of UTI, as well as accurate urine culture results.
Specimen Labeling. The College of American Pathologists (CAP) (2013), Centers for Medicare and Medicaid Services (CMS) (2003), and Miller (1999) specify that the following elements must accompany a urine specimen to the laboratory: patient's first and last name, hospital identification number, specific culture source site, and date and time of collection.
Specimen Transport: Escherichia coli can have a doubling time as short as 20 minutes when urine is kept at room temperature (Lewin, Krebs, Goldstein, & Kilpatrick, 2011). Within two hours, one E. coli per mL will replicate to the quantity of 105 E.coli per mL. Urine specimens should be kept cold to prevent growth, and thus, prevent falsepositive results due to low levels of bacterial contamination when present, and should be sent to the laboratory as soon as possible (CAP, 2008; McCarter et al., 2009; Miller, 1999).
Summary of the Literature Review. After the literature re view was completed, the following conclusions were used as the basis for practice changes: urine specimens should be collected via mid- or late-stream; all specimens should be labeled with the patient's first and last name, hospital identification number, specific culture site, date and time of collection. Urine specimens should be kept cold to prevent bacterial replication, and thus, prevent false-positive results due to contamination, and should be sent to the laboratory as soon as possible (Bekeris, Jones, Walsh, & Wagar, 2008; CMS, 2003). Findings were shared with the multidisciplinary microbiology PI subcommittee, and action plans were developed to implement best practices to minimize re porting of inaccurate urine results.

Practice Changes Based On Best Evidence

Clean-catch Mid-stream. Standardized steps to obtain midstream voided urine specimen in outpatient and inpatient areas were developed collaboratively by the CNS and the director of clinical microbiology. Rationale on the importance of collecting the specimen correctly was provided to patients on the new collection instructions: "Important: It is important to follow the instructions below exactly, to properly clean the bacteria from the skin and urinary opening. A dirty specimen may result in the need for a second test, or worse yet, may be the cause of a wrong diagnosis, unnecessary medicine, or the wrong medicine being given to you." Changes to the hospital's nursing service policy were implemented to reflect evidence-based recommendations. Nursing staff were educated through e-mails and fliers posted on each inpatient unit and in the ED. E-mails and updated instructions were sent to outpatient clinics.
Straight-catheterization Collection. Quick-cath kits were removed from the central supply department and nursing units, and replaced with straight catheterization kits to collect mid- to late-stream urine specimens rather than early stream specimens. The hospital's nursing service policy for urine collection via straight catheterization was revised. Practice changes were reviewed at the Nursing Practice Council.
Chronic Indwelling Urinary Catheter. The director of microbiology and the hospital patient safety department facilitated discussions with physicians. Physicians expressed concern regarding making a blanket statement to change all chronic indwelling catheters prior to collecting a urine culture without the clinician having the final say. The potential inability of nurses to reinsert an indwelling catheter was a concern. Physicians unanimously agreed to having chronic catheters changed prior to collecting a urine specimen only with a physician order first. Practice change of contacting the physician to change a chronic in dwelling catheter prior to urine collection was shared with nursing through the Nursing Practice Council, e-mails, and fliers posted on each inpatient unit and in the ED. Additional communication to nursing staff included collaboration with the information technology (IT) department. A reminder was placed on the electronic physician urine collection order and the urine requisition form.
Specimen Labeling. Patient identification labels are affixed to the specimen container and dated. Collaboration with IT facilitated the development of an electronic requisition form and automation of the form's appearance on the nurse's computer when a urine collection order is entered. This electronic requisition form has required elements to mandate compliance with the laboratory's recommendations. Once completed, it prints at the nurse's front desk for the nurse to pick up and send to the laboratory with the specimen. A clinician order triggers an electronic requisition documentation form for the nurse to complete.
To promote compliance, a rejection policy was developed. Any required elements not completed prompt laboratory personnel to reject the specimen and notify nursing that another specimen must be collected. This policy was disseminated widely to nursing staff before implementation. Magnets with "reminders" of the new policy were placed at stations where specimens were sent to the laboratory. E-mails were sent to nursing staff, and fliers explaining the new electronic requisition form were posted on each inpatient unit and in the ED. Content was also shared at the Nursing Practice Council.
Specimen Transport. Urine specimens must be sent to the laboratory within 15 minutes of collection to prevent prolonged time at room temperature. Collaboration with the Pathology and Laboratory Specimen departments helped develop a rejection policy to assist nursing compliance. If the specimen is not received in the laboratory within 15 minutes of documented specimen collection time, laboratory personnel reject the specimen. The same guidelines apply as when the specimen labeling elements are not complete. Fliers were posted on each in-patient unit and emergency department; magnets with this reminder were placed at stations where specimens are sent to the laboratory. In addition, this time requirement was added to the electronic requisition form as another reminder for nursing staff.

Cost Benefits and Outcomes

Data on incidence of urine cultures positive for coagulasenegative Staphylococcus (CoNS) were collected by the pathology department to determine effectiveness of practice changes. Laboratory findings of CoNS in urine decreased by 250 in one year, indicating a reduction of contaminated urine specimens following changes in urine specimen collection practices. The laboratory urine evaluation (urinalysis, culture and sensitivity) costs $141.00 per specimen. This reduction in evaluation of probable contaminated specimens saved $35,250 in laboratory fees in one year. Additionally, at a cost savings of up to $3,000 per patient (depending on medication used), another potential $750,000 per year was saved in antibiotic use.
Staff compliance with correct specimen labeling and completion of the requisition form was evaluated by the laboratory personnel. Prior to implementation of changes (March 2008), compliance was only 30%. After the rejection policy was completely in place, compliance increased to 100% (see ).

Table 3.  Specimen Labeling Data

Data Collection Dates	Inpatient Floor Compliance	Outpatient Compliance	Overall Compliance	Inpatient Number of Specimens Rejected
Before Practice Change	55%	36%	46%	–
After Practice Change.	100%	96%	98%	1

Staff compliance with getting the urine specimen to the laboratory department within 15 minutes was also evaluated. The goal was to have greater than 95% compliance by nursing staff. Prior to instituting the rejection policy, urine specimens were getting to the laboratory within 15 minutes 87% of the time (2010). After the rejection policy was implemented, compliance rose to 97% (see ).

Table 4.  Urine Transport Data

Data Collection Dates	Floor to Laboratory Less Than 15 Minutes (Inpatient Areas Only)	Urine Analysis to Micro Set-Up Less Than 3 Hours (7:00 a.m. to 10:00 p.m., Inpatient Areas Only)
Before Practice Change	87%	84%
After Practice Change	97%	90%

Nursing Implications

This performance improvement project supported a multidisciplinary collaboration approach to improve patient outcomes. Staff compliance with practice changes was facilitated with several interventions. Evidence-based practice provided the rationale to support practice change. IT support to hardwire practices provided automation and visual reminders for health care providers. Additionally, consequences for not following recommended practice improved compliance. Providing optimal health care to patients takes a team effort. The purpose of collecting a urine specimen for culture is to detect pathological organisms in a patient suspected of having an infection. The consequence of reporting inaccurate results is costly to the patient and the health care system. Accurate and standardized collection, transportation, and handling of urine specimens are essential to provide the best outcome for all patients.

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Urol Nurs. 2013;33(5):249-256. © 2013  Society of Urologic Nurses and Associates