A better understanding of the pharmacodynamics of intermittent presumptive treatment, says White, will guide more rational policymaking

Intermittent presumptive treatment (IPT) in pregnancy involves giving a curative treatment dose of an effective antimalarial drug at predefined intervals during pregnancy. IPT in pregnancy was first introduced in areas of high malaria transmission as a measure to reduce the adverse impact of  Plasmodium falciparum  malaria in pregnancy [ 1 , 2 , 3 , 4 , 5 , 6 , 7 , 8 ]. Later, based on trials showing that IPT could reduce anaemia in young children and also malaria episodes in infants, it was extended as a measure to reduce morbidity and mortality in the first year of life [ 9 , 10 , 11 , 12 ].Antimalarial chemoprophylaxis for pregnant women living in endemic areas has been recommended for many years, but in practice has been limited to the use of chloroquine and pyrimethamine [ 13 , 14 ]. Unfortunately, there are few places left in the world where these drugs can still be relied upon to prevent  P. falciparum  malaria. There are insufficient safety data on the newer antimalarials to warrant their systematic use in pregnant women. IPT with sulphadoxine-pyrimethamine (SP) has been introduced as an alternative. Antimalarial chemoprophylaxis in young children has been shown to reduce the adverse impact of  P. falciparum  malaria [ 15 , 16 , 17 ], but this intervention never obtained the same endorsement as chemoprophylaxis in pregnancy.Five randomised trials of IPT in pregnancy in East Africa have been reported [ 1 , 2 , 3 , 4 , 5 ], all with SP, all in high-transmission settings, and all done between 1992 and 1999 ( Table S1 ). The alarming recent increase in resistance to SP in Africa confounds the cost-effectiveness assessments upon which subsequent policy recommendations for IPT in pregnancy were based [ 18 , 19 ].There is no consensus on how IPT works, making planning difficult. This article argues that IPT provides mainly intermittent suppressive chemoprophylaxis (as opposed to treatment effect alone or some other magical effects which have never been specified). If this is correct dosing schedules should be individualised for each antimalarial depending on the drug's pharmacokinetic and pharmacodynamic properties. As increasing resistance to SP must seriously compromise IPT regimens based on this drug, the evaluation of available new effective antimalarials is needed urgently, in both high- and low-transmission areas.Pharmacokinetics After a treatment dose of SP (25 mg sulfadoxine/1.25 mg pyrimethamine per kilogram body weight), plasma concentrations of pyrimethamine (half-life, 3 days) and sulfadoxine (half-life, 7 days) decline log-linearly [ 20 , 21 ]. The antimalarial effect depends on synergy between the two components, but the effect from one treatment dose can last as long as 60 days with fully sensitive  P. falciparum  [ 20 , 21 ]. For slowly eliminated antimalarial drugs ( Table S2 ), the terminal elimination phase crosses the in vivo dose–response curve ( Figure 1 ). Thus, if a full treatment dose is given, concentrations at the beginning of the terminal elimination phase exceed the minimum parasiticidal concentrations (MPCs)—the lowest concentrations that give maximum effect  [22] . The exceptions to this are chloroquine (and probably piperaquine), as resistance to these drugs increases, because the elimination of chloroquine is multiexponential, and the terminal elimination phase begins at concentrations that are low by comparison with the peak concentrations after treatment ( Figure 2 ).  Figure 1 In Vivo Antimalarial Pharmacodynamics The parasite burden in an adult (vertical axis) is shown in green. After parasite burden expands to the point where it causes illness, treatment is given (red arrow), which causes a log-linear decline in parasite numbers until concentrations of the antimalarial drug (grey shading) fall below the MPC. As the antimalarial blood levels fall further, the decline in parasite burden slows until it reaches a multiplication rate of one (the antimalarial concentration at this point is the in vivo MIC). The parasite population then expands to cause a recrudescence six weeks later. The sigmoid concentration–effect relationship is shown in brown; it is depicted in the reverse direction to that normally drawn. PMR, parasite multiplication rate. Figure 2 Blood Concentration Profiles of Two Antimalarials with Different Elimination Profiles The examples shown here are mefloquine (orange) and chloroquine (pink). An increase in MIC has different effects on the shortening of post-treatment suppressive prophylaxis (hatched bars). MIC R , MIC for resistant parasites; MIC S , MIC for sensitive parasites. The pharmacokinetic properties of many drugs are altered in pregnancy; lower concentrations often result from an expanded volume of distribution. Strangely, despite the wide endorsement of SP IPT in pregnancy, there are no pharmacokinetic studies of sulphadoxine or pyrimethamine in pregnancy, so it is not known whether the current dosing is optimal. The absorption and disposition of many drugs are also altered in infancy, but there are very few data on antimalarial pharmacokinetics in the first year of life. For some drugs (e.g., amodiaquine) there is insufficient information for any age group.
Pharmacokinetics
After a treatment dose of SP (25 mg sulfadoxine/1.25 mg pyrimethamine per kilogram body weight), plasma concentrations of pyrimethamine (half-life, 3 days) and sulfadoxine (half-life, 7 days) decline log-linearly [ 20 , 21 ]. The antimalarial effect depends on synergy between the two components, but the effect from one treatment dose can last as long as 60 days with fully sensitive  P. falciparum  [ 20 , 21 ]. For slowly eliminated antimalarial drugs ( Table S2 ), the terminal elimination phase crosses the in vivo dose–response curve ( Figure 1 ). Thus, if a full treatment dose is given, concentrations at the beginning of the terminal elimination phase exceed the minimum parasiticidal concentrations (MPCs)—the lowest concentrations that give maximum effect  [22] . The exceptions to this are chloroquine (and probably piperaquine), as resistance to these drugs increases, because the elimination of chloroquine is multiexponential, and the terminal elimination phase begins at concentrations that are low by comparison with the peak concentrations after treatment ( Figure 2 ).The pharmacokinetic properties of many drugs are altered in pregnancy; lower concentrations often result from an expanded volume of distribution. Strangely, despite the wide endorsement of SP IPT in pregnancy, there are no pharmacokinetic studies of sulphadoxine or pyrimethamine in pregnancy, so it is not known whether the current dosing is optimal. The absorption and disposition of many drugs are also altered in infancy, but there are very few data on antimalarial pharmacokinetics in the first year of life. For some drugs (e.g., amodiaquine) there is insufficient information for any age group.Pharmacodynamics Is the benefit of IPT gained only through clearing parasites from the placenta (“treatment effect”), or is the prevention of new infections (“prophylactic effect”) an important component? If only the treatment effect is important, then how long does the beneficial effect of eradicating an asymptomatic low-density infection persist for? If it lasts until the next infection becomes patent (i.e., detectable), then rapidly eliminated drugs will provide protection only for a few days longer than the average incubation period (about two weeks). Establishment of a new placental infection (i.e., pathologically significant placental sequestration) may take longer because the placenta selects and accumulates parasites that bind to the proteoglycans chondroitin sulphate and hyaluronic acid  [23] . If only the treatment effect is important, then for sustained benefit we must hypothesise that the parasites that persist asymptomatically before IPT is given are a selected subpopulation that is more pathological than the parasites that cause subsequent reinfection. This seems implausible in infancy, and even in pregnancy it seems unlikely that it would take more than ten weeks in high-transmission settings to re-establish a significant placental infection. This suggests that the prophylactic effect is important for the efficacy of IPT. The duration of prophylactic effect is compromised particularly by resistance. For most antimalarials the duration of antimalarial effect is a simple function of the in vivo concentration–effect (dose–response) relationship and the pharmacokinetic properties of the antimalarial drug  [22] . But for SP this function is more complicated, as synergy between the two components needs to be considered. The duration of synergy depends on resistance levels determined by mutations in the parasites' genes encoding dihydropteroate synthase and dihydrofolate reductase, the respective targets of sulphadoxine and pyrimethamine [ 20 , 21 ]. Information on this temporal pattern of reinfection following IPT in pregnancy or infancy is lacking. Such information is essential if the choice of drug and the dosing is to be rationalised. Resistance is defined by a right shift in the concentration–effect relationship and results in reduced effects for any concentration below the MPC for resistant parasites  [24]  (see  Figure 1 ). As the concentration of a slowly eliminated antimalarial in the blood declines, it continues to suppress the growth of newly acquired infections as they emerge from the liver. Eventually, however, concentrations fall below the minimum inhibitory concentration (MIC) for the prevalent parasites (i.e., the concentration at which the net multiplication rate is one), and parasite expansion is possible ( Figure 3 ). It follows, then, that the duration of “post treatment prophylaxis” (PTP) (i.e., the length of time after an antimalarial treatment dose for which newly acquired infections are suppressed) is determined by the concentrations of the drugs used (determined by dose and pharmacokinetics) and the sensitivity of the prevalent parasites. The more resistant the parasites are, the shorter is the duration of PTP; for each doubling of MIC the duration of PTP is shortened by one half-life ( Protocol S1 ). The triple dihydrofolate reductase mutants now prevalent across much of Africa have an approximate 1,000-fold reduction in pyrimethamine susceptibility, which would translate into a reduction in PTP of one month ( Figure 4 ). As a further confounder, folic acid, which is prescribed widely in pregnancy, is a competitive antagonist of pyrimethamine.  Figure 3 Hypothetical Parasite Burden Profiles during Pregnancy with SP IPT in a High-Transmission Setting Entomological inoculation rate is about 50 infectious bites per person per year. Note that many infections self-cure (each infection is depicted as a green line). The hatched bars represent the duration of “suppressive prophylactic activity”, and the solid bars represent the period during which parasite multiplication is suppressed (i.e., levels exceed the in vivo MIC). The horizontal dotted line at 10 8  parasites represents the level at which malaria can be detected on a blood film. (A) represents a drug-sensitive area; (B) represents a moderately resistant area. Figure 4 Relationship between MIC and PTP The proportional increase in malaria parasite MIC with resistance is plotted against the shortening of the duration of PTP, expressed as multiples of the terminal half-life. This applies only to drugs for which suppressive antimalarial prophylaxis occurs in the terminal elimination phase (i.e., most drugs).
Pharmacodynamics
Is the benefit of IPT gained only through clearing parasites from the placenta (“treatment effect”), or is the prevention of new infections (“prophylactic effect”) an important component? If only the treatment effect is important, then how long does the beneficial effect of eradicating an asymptomatic low-density infection persist for? If it lasts until the next infection becomes patent (i.e., detectable), then rapidly eliminated drugs will provide protection only for a few days longer than the average incubation period (about two weeks). Establishment of a new placental infection (i.e., pathologically significant placental sequestration) may take longer because the placenta selects and accumulates parasites that bind to the proteoglycans chondroitin sulphate and hyaluronic acid  [23] . If only the treatment effect is important, then for sustained benefit we must hypothesise that the parasites that persist asymptomatically before IPT is given are a selected subpopulation that is more pathological than the parasites that cause subsequent reinfection. This seems implausible in infancy, and even in pregnancy it seems unlikely that it would take more than ten weeks in high-transmission settings to re-establish a significant placental infection. This suggests that the prophylactic effect is important for the efficacy of IPT.The duration of prophylactic effect is compromised particularly by resistance. For most antimalarials the duration of antimalarial effect is a simple function of the in vivo concentration–effect (dose–response) relationship and the pharmacokinetic properties of the antimalarial drug  [22] . But for SP this function is more complicated, as synergy between the two components needs to be considered. The duration of synergy depends on resistance levels determined by mutations in the parasites' genes encoding dihydropteroate synthase and dihydrofolate reductase, the respective targets of sulphadoxine and pyrimethamine [ 20 , 21 ]. Information on this temporal pattern of reinfection following IPT in pregnancy or infancy is lacking. Such information is essential if the choice of drug and the dosing is to be rationalised.Resistance is defined by a right shift in the concentration–effect relationship and results in reduced effects for any concentration below the MPC for resistant parasites  [24]  (see  Figure 1 ). As the concentration of a slowly eliminated antimalarial in the blood declines, it continues to suppress the growth of newly acquired infections as they emerge from the liver. Eventually, however, concentrations fall below the minimum inhibitory concentration (MIC) for the prevalent parasites (i.e., the concentration at which the net multiplication rate is one), and parasite expansion is possible ( Figure 3 ). It follows, then, that the duration of “post treatment prophylaxis” (PTP) (i.e., the length of time after an antimalarial treatment dose for which newly acquired infections are suppressed) is determined by the concentrations of the drugs used (determined by dose and pharmacokinetics) and the sensitivity of the prevalent parasites. The more resistant the parasites are, the shorter is the duration of PTP; for each doubling of MIC the duration of PTP is shortened by one half-life ( Protocol S1 ). The triple dihydrofolate reductase mutants now prevalent across much of Africa have an approximate 1,000-fold reduction in pyrimethamine susceptibility, which would translate into a reduction in PTP of one month ( Figure 4 ). As a further confounder, folic acid, which is prescribed widely in pregnancy, is a competitive antagonist of pyrimethamine.Preventing Placental Pathology In a high-transmission setting infections are acquired every few days or weeks throughout life (see  Figure 3 ). Mortality is high in childhood, but by the time of adulthood and pregnancy, infections are largely asymptomatic—although they are often still patent (which requires a total burden of greater than 100 million parasites)  [22] . Thus, immunity prevents life-threatening parasite burdens, and suppresses the pro-inflammatory response (which causes illness), but it does not prevent infection. In pregnancy this immune control is impaired in the placenta, which acts as a “privileged site” for parasite multiplication. The objective of IPT in pregnancy is to reduce or eliminate the adverse effects of malaria on maternal anaemia and birth weight, and, in addition, in a low-transmission setting, to prevent severe malaria in the mother [ 25 , 26 ]. How malaria produces intrauterine growth retardation is still unresolved, but in  P. falciparum  malaria, retardation tends to be greatest in the first pregnancy, and often occurs without maternal illness. The greater the placental parasite burden, the greater is the reduction in birth weight. “Placental malaria”—histological evidence of placental accumulation of parasitized erythrocytes or malaria pigment deposition—has often been used as an endpoint in intervention studies, although the quantitative relationship between placental malaria and reduction in birth weight remains poorly characterised.
Preventing Placental Pathology
In a high-transmission setting infections are acquired every few days or weeks throughout life (see  Figure 3 ). Mortality is high in childhood, but by the time of adulthood and pregnancy, infections are largely asymptomatic—although they are often still patent (which requires a total burden of greater than 100 million parasites)  [22] . Thus, immunity prevents life-threatening parasite burdens, and suppresses the pro-inflammatory response (which causes illness), but it does not prevent infection. In pregnancy this immune control is impaired in the placenta, which acts as a “privileged site” for parasite multiplication. The objective of IPT in pregnancy is to reduce or eliminate the adverse effects of malaria on maternal anaemia and birth weight, and, in addition, in a low-transmission setting, to prevent severe malaria in the mother [ 25 , 26 ]. How malaria produces intrauterine growth retardation is still unresolved, but in  P. falciparum  malaria, retardation tends to be greatest in the first pregnancy, and often occurs without maternal illness. The greater the placental parasite burden, the greater is the reduction in birth weight. “Placental malaria”—histological evidence of placental accumulation of parasitized erythrocytes or malaria pigment deposition—has often been used as an endpoint in intervention studies, although the quantitative relationship between placental malaria and reduction in birth weight remains poorly characterised.Preventing Malaria in Infancy There are fewer data on the efficacy of IPT in infancy than in pregnancy ( Table S3 ). The pharmacodynamics of IPT in infancy are probably similar to those in pregnancy, although there is no “privileged site” for parasite multiplication. Protection in the first months of life is mediated by a variety of factors, which include transplacentally acquired maternal antibody (IgG) and a relatively high haemoglobin F content in the infants' erythrocytes. After about six months of age, protection from these factors wanes, and the infant becomes much more vulnerable to malaria than the mother (because protective immunity has yet to be acquired). As delivery of antimalarials in the rural tropics is so difficult, for operational reasons IPT is currently being given to infants at the same time as the EPI immunisations (at 2, 3, and 9 months). This regimen leaves a six-month gap between the second and third administrations, which, even for fully SP-sensitive parasites, leaves four unprotected months. This is at a time when the infant is increasingly vulnerable to severe malaria. More information is needed on the duration of protection afforded by currently available antimalarial drugs when administered to healthy infants.
Preventing Malaria in Infancy
There are fewer data on the efficacy of IPT in infancy than in pregnancy ( Table S3 ). The pharmacodynamics of IPT in infancy are probably similar to those in pregnancy, although there is no “privileged site” for parasite multiplication. Protection in the first months of life is mediated by a variety of factors, which include transplacentally acquired maternal antibody (IgG) and a relatively high haemoglobin F content in the infants' erythrocytes. After about six months of age, protection from these factors wanes, and the infant becomes much more vulnerable to malaria than the mother (because protective immunity has yet to be acquired). As delivery of antimalarials in the rural tropics is so difficult, for operational reasons IPT is currently being given to infants at the same time as the EPI immunisations (at 2, 3, and 9 months). This regimen leaves a six-month gap between the second and third administrations, which, even for fully SP-sensitive parasites, leaves four unprotected months. This is at a time when the infant is increasingly vulnerable to severe malaria. More information is needed on the duration of protection afforded by currently available antimalarial drugs when administered to healthy infants.Should IPT be Used in Low-Transmission Settings? If IPT is just a simple, albeit imperfect, way of administering chemoprophylaxis, then there are also strong arguments for evaluating this approach in low-transmission settings. The adverse impact of malaria in pregnancy is greater in low-transmission than in high-transmission settings. The reduction in the birth weight of first-borne infants is similar, but extends to the second and subsequent pregnancies; treatment failure rates are higher than in non-pregnant adults [ 27 , 28 , 29 ]; and there is a significant risk of severe malaria with attendant very high mortality. In Asia and South America, where low-transmission areas predominate,  P. vivax  is also an important cause of low birth weight, and so useful preventative measures must also be effective against this infection  [30] . Antimalarial prophylaxis has been recommended and used in low-transmission settings, but whereas chloroquine remains generally effective against  P. vivax , there are no safe and effective available drugs for  P. falciparum  infections. Use of IPT in these areas would provide a sterner test than in a high-transmission area because there would be little or no background immunity to assist antimalarial drug efficacy.
Should IPT be Used in Low-Transmission Settings?
If IPT is just a simple, albeit imperfect, way of administering chemoprophylaxis, then there are also strong arguments for evaluating this approach in low-transmission settings. The adverse impact of malaria in pregnancy is greater in low-transmission than in high-transmission settings. The reduction in the birth weight of first-borne infants is similar, but extends to the second and subsequent pregnancies; treatment failure rates are higher than in non-pregnant adults [ 27 , 28 , 29 ]; and there is a significant risk of severe malaria with attendant very high mortality. In Asia and South America, where low-transmission areas predominate,  P. vivax  is also an important cause of low birth weight, and so useful preventative measures must also be effective against this infection  [30] .Antimalarial prophylaxis has been recommended and used in low-transmission settings, but whereas chloroquine remains generally effective against  P. vivax , there are no safe and effective available drugs for  P. falciparum  infections. Use of IPT in these areas would provide a sterner test than in a high-transmission area because there would be little or no background immunity to assist antimalarial drug efficacy.What Is the Correct Dose and the Correct Interval Between Doses? The dose used in IPT is usually the full age- or weight-adjusted treatment dose derived either empirically or from dosefinding studies (usually in non-pregnant adults). The dose and dosing interval should be determined by the tolerability, absorption, distribution, and elimination kinetics of the drug used, and the in vivo MIC. Unfortunately, for the two drugs that have been used for IPT (SP and amodiaquine) there are no pharmacokinetic data in pregnant women or infants. The in vivo MIC is an important measure but it is parasite specific and difficult to assess [ 31 , 32 , 33 , 34 , 35 ]. Ideally, the interval between doses should not be more than one week longer than the time needed for plasma concentrations to fall from peak post-dose levels to the MIC value. This timing is a conservative choice as it assumes all infections are equally harmful, and it does not take into account either the delay in selecting a placenta-binding  P. falciparum  subpopulation in pregnancy or the delay in achieving full growth rates because of continued sub-MIC suppression. Although the MIC for drugs that are succumbing to resistance obviously varies considerably, for newer drugs such as lumefantrine or piperaquine the variance is considerably less and generalisations can be made.
What Is the Correct Dose and the Correct Interval Between Doses?
The dose used in IPT is usually the full age- or weight-adjusted treatment dose derived either empirically or from dosefinding studies (usually in non-pregnant adults). The dose and dosing interval should be determined by the tolerability, absorption, distribution, and elimination kinetics of the drug used, and the in vivo MIC. Unfortunately, for the two drugs that have been used for IPT (SP and amodiaquine) there are no pharmacokinetic data in pregnant women or infants. The in vivo MIC is an important measure but it is parasite specific and difficult to assess [ 31 , 32 , 33 , 34 , 35 ]. Ideally, the interval between doses should not be more than one week longer than the time needed for plasma concentrations to fall from peak post-dose levels to the MIC value. This timing is a conservative choice as it assumes all infections are equally harmful, and it does not take into account either the delay in selecting a placenta-binding  P. falciparum  subpopulation in pregnancy or the delay in achieving full growth rates because of continued sub-MIC suppression. Although the MIC for drugs that are succumbing to resistance obviously varies considerably, for newer drugs such as lumefantrine or piperaquine the variance is considerably less and generalisations can be made.Should an Artemisinin Combination Be Used for IPT? If IPT is simply prophylaxis, then a rapidly eliminated artemisinin component provides very little direct benefit for the additional cost and risk (although the risks are thought to be very small in the second and third trimesters of pregnancy and in infancy). The addition of an artemisinin component would accelerate parasite clearance and prevent gametocyte production, but the benefits of this in an asymptomatic pregnant woman or child are uncertain. The main benefit would be in providing protection against the emergence of de novo resistance to the slowly eliminated drug, although, because parasitaemias tend to be low, the probabilities of de novo selection are much lower than in acute symptomatic infection. But there is a genuine concern that if monotherapies are made available, then they will be used and abused, and resistance may develop.
Should an Artemisinin Combination Be Used for IPT?
If IPT is simply prophylaxis, then a rapidly eliminated artemisinin component provides very little direct benefit for the additional cost and risk (although the risks are thought to be very small in the second and third trimesters of pregnancy and in infancy). The addition of an artemisinin component would accelerate parasite clearance and prevent gametocyte production, but the benefits of this in an asymptomatic pregnant woman or child are uncertain. The main benefit would be in providing protection against the emergence of de novo resistance to the slowly eliminated drug, although, because parasitaemias tend to be low, the probabilities of de novo selection are much lower than in acute symptomatic infection. But there is a genuine concern that if monotherapies are made available, then they will be used and abused, and resistance may develop.Discussion: The Policy Implications Without a better understanding of the pharmacodynamic effects of IPT, it will be difficult to make rational improvements in this promising approach to malaria prevention. The most parsimonious explanation for its effectiveness is that IPT provides antimalarial prophylaxis that, if sufficiently lengthy and effective, is beneficial both to the pregnant woman and the infant. But how lengthy and how effective? For IPT in pregnancy the only drug that has been evaluated is SP, at a time when the drug was more effective than it is today. A significant improvement in birth weight was found in only two of four randomised trials. In a large prospective observational study conducted in western Kenya, IPT was associated with an odds ratio of 0.65 (95% confidence interval, 0.45 to 0.95) for low birth weight  [7] . A dose–response relationship was found, with an adjusted mean increase in birth weight of 61 g for each increment in the number of SP doses (up to three doses). So, two doses of SP did not provide maximal benefit. But given the alarming decline in SP efficacy in Africa (resulting from rapid spread of “quintuple” dihydrofolate reductase/dihydropteroate synthase mutants that are about 1,000 times less sensitive to pyrimethamine than wild-type parasites), there are grave doubts about whether the efficacy observed in these various studies would still be observed today, even if the dosing was increased. SP cure rates in children in Malawi—where IPT in pregnancy is widely used—have been consistently less than 40% for the past five years  [36] . It has been suggested that asymptomatic pregnant women in high-transmission settings may have sufficient immunity to complement a failing drug—i.e., treatment responses would be better than in symptomatic children. However, if the duration of PTP is the main determinant of benefit, then this benefit is shortened progressively by increasing resistance (see  Figure 4 ). Alternatives to SP are needed urgently. Is IPT safe? There is no evidence to date that IPT is harmful. But the incidence of serious adverse effects when amodiaquine (agranulocytosis, 1:2,000) and SP (Stevens-Johnson syndrome,1:7,000) were used as antimalarial prophylaxis by Western travellers was so high that they are contraindicated  [37] . Both drugs were associated with severe hepatitis. Single treatments are considered safer, but how much safer is not known. There are insufficient data on the safety of amodiaquine in pregnancy  [38] . The closer IPT comes to continuous prophylaxis, presumably the higher the risks of serious adverse effects. The risk–benefit assessment is difficult to make, but with the current high levels of SP resistance, these important uncertainties also argue strongly for the evaluation of alternatives. Proguanil and quinine are regarded as safe, but both are eliminated very rapidly. Treatment doses of mefloquine are not well tolerated by healthy subjects, and there are safety concerns in pregnancy  [39] . Serious contenders all require more than a single dose and will need urgent evaluation. These include artemether-lumefantrine, although more information is needed on safety and on the pharmacokinetics in pregnancy, and the duration of PTP provided by lumefantrine needs further assessment. It may be too short. Dihydroartemisinin-piperaquine may be the most promising candidate. It is very well tolerated, and piperaquine is slowly eliminated. Indirect evidence from the pattern of new infections following clinical trials suggests protracted suppressive activity. It has not yet been evaluated in pregnancy, so more information is needed on safety and pharmacokinetics in this context. For IPT in infancy, however, there seems every reason to evaluate this drug as soon as possible.
Discussion: The Policy Implications
Without a better understanding of the pharmacodynamic effects of IPT, it will be difficult to make rational improvements in this promising approach to malaria prevention. The most parsimonious explanation for its effectiveness is that IPT provides antimalarial prophylaxis that, if sufficiently lengthy and effective, is beneficial both to the pregnant woman and the infant. But how lengthy and how effective?For IPT in pregnancy the only drug that has been evaluated is SP, at a time when the drug was more effective than it is today. A significant improvement in birth weight was found in only two of four randomised trials. In a large prospective observational study conducted in western Kenya, IPT was associated with an odds ratio of 0.65 (95% confidence interval, 0.45 to 0.95) for low birth weight  [7] . A dose–response relationship was found, with an adjusted mean increase in birth weight of 61 g for each increment in the number of SP doses (up to three doses). So, two doses of SP did not provide maximal benefit. But given the alarming decline in SP efficacy in Africa (resulting from rapid spread of “quintuple” dihydrofolate reductase/dihydropteroate synthase mutants that are about 1,000 times less sensitive to pyrimethamine than wild-type parasites), there are grave doubts about whether the efficacy observed in these various studies would still be observed today, even if the dosing was increased. SP cure rates in children in Malawi—where IPT in pregnancy is widely used—have been consistently less than 40% for the past five years  [36] . It has been suggested that asymptomatic pregnant women in high-transmission settings may have sufficient immunity to complement a failing drug—i.e., treatment responses would be better than in symptomatic children. However, if the duration of PTP is the main determinant of benefit, then this benefit is shortened progressively by increasing resistance (see  Figure 4 ). Alternatives to SP are needed urgently.Is IPT safe? There is no evidence to date that IPT is harmful. But the incidence of serious adverse effects when amodiaquine (agranulocytosis, 1:2,000) and SP (Stevens-Johnson syndrome,1:7,000) were used as antimalarial prophylaxis by Western travellers was so high that they are contraindicated  [37] . Both drugs were associated with severe hepatitis. Single treatments are considered safer, but how much safer is not known. There are insufficient data on the safety of amodiaquine in pregnancy  [38] . The closer IPT comes to continuous prophylaxis, presumably the higher the risks of serious adverse effects. The risk–benefit assessment is difficult to make, but with the current high levels of SP resistance, these important uncertainties also argue strongly for the evaluation of alternatives. Proguanil and quinine are regarded as safe, but both are eliminated very rapidly. Treatment doses of mefloquine are not well tolerated by healthy subjects, and there are safety concerns in pregnancy  [39] . Serious contenders all require more than a single dose and will need urgent evaluation. These include artemether-lumefantrine, although more information is needed on safety and on the pharmacokinetics in pregnancy, and the duration of PTP provided by lumefantrine needs further assessment. It may be too short. Dihydroartemisinin-piperaquine may be the most promising candidate. It is very well tolerated, and piperaquine is slowly eliminated. Indirect evidence from the pattern of new infections following clinical trials suggests protracted suppressive activity. It has not yet been evaluated in pregnancy, so more information is needed on safety and pharmacokinetics in this context. For IPT in infancy, however, there seems every reason to evaluate this drug as soon as possible.Supporting Information Protocol S1 Calculations Showing That for Each Doubling of MIC the Duration of PTP Is Shortened by One Half-Life (27 KB DOC). Click here for additional data file. Table S1 Randomised Trials of IPT in Pregnancy (31 KB DOC). Click here for additional data file. Table S2 Terminal Elimination Half-Lives of Currently Available Antimalarial Drugs (34 KB DOC). Click here for additional data file. Table S3 Randomised Trials of IPT in Infancy (27 KB DOC). Click here for additional data file. Summary Points
 Intermittent presumptive treatment (IPT) with sulphadoxine-pyrimethamine (SP) in pregnancy and with amodiaquine or SP in infancy has been proposed for use in areas with high levels of malaria transmission. The duration of post treatment prophylaxis is likely to be an important determinant of the benefit of IPT. Because of rapidly increasing resistance, it is very unlikely that IPT in pregnancy with SP is as effective now in east Africa as it was 5–10 years ago, when it was evaluated. More effective antimalarial drugs such as artemether-lumefantrine and particularly dihydroartemisinin-piperaquine should be evaluated for IPT in both low- and high-transmission settings. Choice of drug, dosing, and dose spacing for IPT should be based on a better understanding of pharmacokinetics and pharmacodynamics.
Supporting Information
Calculations Showing That for Each Doubling of MIC the Duration of PTP Is Shortened by One Half-Life
(27 KB DOC).Click here for additional data file.Randomised Trials of IPT in Pregnancy
(31 KB DOC).Click here for additional data file.Terminal Elimination Half-Lives of Currently Available Antimalarial Drugs
(34 KB DOC).Click here for additional data file.Randomised Trials of IPT in Infancy
(27 KB DOC).Click here for additional data file.Summary Points
 Intermittent presumptive treatment (IPT) with sulphadoxine-pyrimethamine (SP) in pregnancy and with amodiaquine or SP in infancy has been proposed for use in areas with high levels of malaria transmission. The duration of post treatment prophylaxis is likely to be an important determinant of the benefit of IPT. Because of rapidly increasing resistance, it is very unlikely that IPT in pregnancy with SP is as effective now in east Africa as it was 5–10 years ago, when it was evaluated. More effective antimalarial drugs such as artemether-lumefantrine and particularly dihydroartemisinin-piperaquine should be evaluated for IPT in both low- and high-transmission settings. Choice of drug, dosing, and dose spacing for IPT should be based on a better understanding of pharmacokinetics and pharmacodynamics.
Summary Points
Intermittent presumptive treatment (IPT) with sulphadoxine-pyrimethamine (SP) in pregnancy and with amodiaquine or SP in infancy has been proposed for use in areas with high levels of malaria transmission. The duration of post treatment prophylaxis is likely to be an important determinant of the benefit of IPT. Because of rapidly increasing resistance, it is very unlikely that IPT in pregnancy with SP is as effective now in east Africa as it was 5–10 years ago, when it was evaluated. More effective antimalarial drugs such as artemether-lumefantrine and particularly dihydroartemisinin-piperaquine should be evaluated for IPT in both low- and high-transmission settings. Choice of drug, dosing, and dose spacing for IPT should be based on a better understanding of pharmacokinetics and pharmacodynamics.Intermittent presumptive treatment (IPT) with sulphadoxine-pyrimethamine (SP) in pregnancy and with amodiaquine or SP in infancy has been proposed for use in areas with high levels of malaria transmission.The duration of post treatment prophylaxis is likely to be an important determinant of the benefit of IPT.Because of rapidly increasing resistance, it is very unlikely that IPT in pregnancy with SP is as effective now in east Africa as it was 5–10 years ago, when it was evaluated.More effective antimalarial drugs such as artemether-lumefantrine and particularly dihydroartemisinin-piperaquine should be evaluated for IPT in both low- and high-transmission settings.Choice of drug, dosing, and dose spacing for IPT should be based on a better understanding of pharmacokinetics and pharmacodynamics.


Background Post-operative complications are a significant source of morbidity and mortality for patients undergoing surgery. However, there is little research in the emerging field of perioperative medicine beyond cardiac risk stratification. We sought to determine the research priorities for perioperative medicine using a cross sectional survey of Canadian and American general internists.
Background
Post-operative complications are a significant source of morbidity and mortality for patients undergoing surgery. However, there is little research in the emerging field of perioperative medicine beyond cardiac risk stratification. We sought to determine the research priorities for perioperative medicine using a cross sectional survey of Canadian and American general internists.Methods Surveys were electronically sent to 312 general internists from the Canadian Society of Internal Medicine and 130 internists from the perioperative medicine research interest group within the US based Society of General Internal Medicine. The questionnaire contained thirty research questions and respondents were asked to rate the priority of these questions for future study.
Methods
Surveys were electronically sent to 312 general internists from the Canadian Society of Internal Medicine and 130 internists from the perioperative medicine research interest group within the US based Society of General Internal Medicine. The questionnaire contained thirty research questions and respondents were asked to rate the priority of these questions for future study.Results The research topics with the highest ratings included: the need for tight control of diabetes mellitus postoperatively and the value of starting aspirin on patients at increased risk for postoperative cardiac events. Research questions evaluating the efficacy and safety of perioperative interventions had higher ratings than questions relating to the prediction of postoperative risk. Questions relating to the yield of preoperative diagnostic tests had the lowest ratings (p < 0.001 for differences across these categories).
Results
The research topics with the highest ratings included: the need for tight control of diabetes mellitus postoperatively and the value of starting aspirin on patients at increased risk for postoperative cardiac events. Research questions evaluating the efficacy and safety of perioperative interventions had higher ratings than questions relating to the prediction of postoperative risk. Questions relating to the yield of preoperative diagnostic tests had the lowest ratings (p < 0.001 for differences across these categories).Conclusion The results of this survey suggest that practicing general internists believe that interventions studies are a priority within perioperative medicine. These findings should help prioritize research in this emerging field.
Conclusion
The results of this survey suggest that practicing general internists believe that interventions studies are a priority within perioperative medicine. These findings should help prioritize research in this emerging field.

Background Over 40 million people undergo non-cardiac surgery in the United States each year [ 1 ]. Postoperative cardiac complications affect 2–18% of patients alone, costing over 20 billion dollars annually in the United States [ 2 ]. Many will suffer other potentially avoidable perioperative complications such as pneumonia, hemorrhage or infection. Efforts to minimize these complications have resulted in the development of the field of perioperative medicine. Until now, research in this emerging field has focused primarily on cardiac risk stratification [ 3 ]. Despite the significant medical and economic burden of perioperative complications, few studies evaluate diagnostic testing, risk stratification for non-cardiac complications, or interventions to prevent cardiac or non-cardiac complications. Thus, there is a growing need to expand the research base in this field. Given the wide spectrum of comorbidities in the surgical patient and the potential for postoperative complications, numerous research questions still need to be answered. The purpose of this study is to identify top perceived research priorities in this field using a survey of general internists practicing perioperative medicine.
Background
Over 40 million people undergo non-cardiac surgery in the United States each year [ 1 ]. Postoperative cardiac complications affect 2–18% of patients alone, costing over 20 billion dollars annually in the United States [ 2 ]. Many will suffer other potentially avoidable perioperative complications such as pneumonia, hemorrhage or infection. Efforts to minimize these complications have resulted in the development of the field of perioperative medicine.Until now, research in this emerging field has focused primarily on cardiac risk stratification [ 3 ]. Despite the significant medical and economic burden of perioperative complications, few studies evaluate diagnostic testing, risk stratification for non-cardiac complications, or interventions to prevent cardiac or non-cardiac complications. Thus, there is a growing need to expand the research base in this field.Given the wide spectrum of comorbidities in the surgical patient and the potential for postoperative complications, numerous research questions still need to be answered. The purpose of this study is to identify top perceived research priorities in this field using a survey of general internists practicing perioperative medicine.Methods Participants To obtain the opinions of general internists who practice perioperative medicine, from the Canada and the United States, all general internists within the Canadian Society of Internal Medicine (n = 312) and all members of the perioperative medicine interest group of the American based Society of General Internal Medicine (n = 130), were surveyed. Physicians were excluded if they practiced in a subspecialty rather than general medicine (>90% of perioperative medicine consults are performed by general internists [ 4 ]) or did not perform preoperative consultation. Survey development and administration Research questions for the survey were generated from a Medline search of perioperative medicine studies and from a focus group of five general internists active in perioperative medicine research. The questionnaire was pre-tested by four independent general internists for clarity and to confirm face validity. Modifications were made based on this pre-testing. The questionnaire was developed in English and then translated into French for French speaking Canadian physicians by a medical translator. Subsequently, a bilingual general internist validated the French translation. The questionnaire contained 30 randomly ordered research questions conceptually divided into three themes: 1. evaluating the yield of preoperative diagnostic tests (5 questions), 2. predicting postoperative risk (6 questions) and 3. determining the efficacy of perioperative interventions (19 questions). A subset of the sample (130) was asked to specifically rank the importance of these three categories as an internal check of the reliability of the survey instrument. This subset was also asked to list any other research questions that ought to be considered for future research apart from those included in the questionnaire. Respondents were asked to rate the priority for each research question to be studied in the future on a 10 point Likert scale where 1 indicates a low priority study question and 10 is a high priority research question. For questions that have been partially answered by existing studies, respondents were specifically asked to rate the priority of "further research" in these areas. The self- administered questionnaire was electronically mailed in October 2000 (faxes were sent to those without e-mail addresses). For non-responders, second and third mail-outs were sent in November and December 2000. Statistical analyses Descriptive statistics were used to analyze the demographic data and ratings for individual questions. T-tests (two- sided) were used to compare mean response scores across different subgroups. Because of the multiple planned comparisons, the alpha was set at 0.005 to determine statistical significance. Repeated measures analysis of variance was used to analyze differences across the 3 major themes of questions using SPSS statistical software.
Methods
Participants To obtain the opinions of general internists who practice perioperative medicine, from the Canada and the United States, all general internists within the Canadian Society of Internal Medicine (n = 312) and all members of the perioperative medicine interest group of the American based Society of General Internal Medicine (n = 130), were surveyed. Physicians were excluded if they practiced in a subspecialty rather than general medicine (>90% of perioperative medicine consults are performed by general internists [ 4 ]) or did not perform preoperative consultation.
Participants
To obtain the opinions of general internists who practice perioperative medicine, from the Canada and the United States, all general internists within the Canadian Society of Internal Medicine (n = 312) and all members of the perioperative medicine interest group of the American based Society of General Internal Medicine (n = 130), were surveyed. Physicians were excluded if they practiced in a subspecialty rather than general medicine (>90% of perioperative medicine consults are performed by general internists [ 4 ]) or did not perform preoperative consultation.Survey development and administration Research questions for the survey were generated from a Medline search of perioperative medicine studies and from a focus group of five general internists active in perioperative medicine research. The questionnaire was pre-tested by four independent general internists for clarity and to confirm face validity. Modifications were made based on this pre-testing. The questionnaire was developed in English and then translated into French for French speaking Canadian physicians by a medical translator. Subsequently, a bilingual general internist validated the French translation. The questionnaire contained 30 randomly ordered research questions conceptually divided into three themes: 1. evaluating the yield of preoperative diagnostic tests (5 questions), 2. predicting postoperative risk (6 questions) and 3. determining the efficacy of perioperative interventions (19 questions). A subset of the sample (130) was asked to specifically rank the importance of these three categories as an internal check of the reliability of the survey instrument. This subset was also asked to list any other research questions that ought to be considered for future research apart from those included in the questionnaire. Respondents were asked to rate the priority for each research question to be studied in the future on a 10 point Likert scale where 1 indicates a low priority study question and 10 is a high priority research question. For questions that have been partially answered by existing studies, respondents were specifically asked to rate the priority of "further research" in these areas. The self- administered questionnaire was electronically mailed in October 2000 (faxes were sent to those without e-mail addresses). For non-responders, second and third mail-outs were sent in November and December 2000.
Survey development and administration
Research questions for the survey were generated from a Medline search of perioperative medicine studies and from a focus group of five general internists active in perioperative medicine research. The questionnaire was pre-tested by four independent general internists for clarity and to confirm face validity. Modifications were made based on this pre-testing. The questionnaire was developed in English and then translated into French for French speaking Canadian physicians by a medical translator. Subsequently, a bilingual general internist validated the French translation.The questionnaire contained 30 randomly ordered research questions conceptually divided into three themes: 1. evaluating the yield of preoperative diagnostic tests (5 questions), 2. predicting postoperative risk (6 questions) and 3. determining the efficacy of perioperative interventions (19 questions). A subset of the sample (130) was asked to specifically rank the importance of these three categories as an internal check of the reliability of the survey instrument. This subset was also asked to list any other research questions that ought to be considered for future research apart from those included in the questionnaire.Respondents were asked to rate the priority for each research question to be studied in the future on a 10 point Likert scale where 1 indicates a low priority study question and 10 is a high priority research question. For questions that have been partially answered by existing studies, respondents were specifically asked to rate the priority of "further research" in these areas.The self- administered questionnaire was electronically mailed in October 2000 (faxes were sent to those without e-mail addresses). For non-responders, second and third mail-outs were sent in November and December 2000.Statistical analyses Descriptive statistics were used to analyze the demographic data and ratings for individual questions. T-tests (two- sided) were used to compare mean response scores across different subgroups. Because of the multiple planned comparisons, the alpha was set at 0.005 to determine statistical significance. Repeated measures analysis of variance was used to analyze differences across the 3 major themes of questions using SPSS statistical software.
Statistical analyses
Descriptive statistics were used to analyze the demographic data and ratings for individual questions. T-tests (two- sided) were used to compare mean response scores across different subgroups. Because of the multiple planned comparisons, the alpha was set at 0.005 to determine statistical significance. Repeated measures analysis of variance was used to analyze differences across the 3 major themes of questions using SPSS statistical software.Results After 3 mailings, we obtained 152 completed surveys (overall response rate 34%). Thirty-three respondents were then excluded because of subspecialty status or because they did not perform perioperative consults. Respondents belonging to the Society of General Internal Medicine special interest group and Canadian Society of Internal Medicine were identical in most respects except fewer Canadian Society of Internal Medicine members had academic appointments (54% vs. 100%). Table  1  shows the demographic and professional characteristics for all respondents. Table 1 Physician characteristics (n = 119) Characteristics Age (SD) 44 (9.2) Female (%) 24 Average year of graduation 1982 Number of years in practice, mean (range) 13 (0.5–35) Practice Location (%) Rural 8 Urban <50 000 8 Urban 50–250 000 25 Urban >250 000 59 Academic appointment (%) 77 Number of preoperative consults performed per month, median (range) 10 (1–100) Research questions evaluating the efficacy of perioperative interventions had higher ratings followed by questions relating to prediction of postoperative risk. Questions evaluating the yield of preoperative diagnostic tests had the lowest ratings. The differences in ratings across these general categories were statistically significant (p < 0.001) and this pattern persisted regardless of academic status or volume of consults seen. Mean scores for individual research questions, based on responses where 1 indicates low priority for future research and 10 indicates high priority, ranged from a low of 3.6 (± 2.3 standard deviation) to a high of 7.2 (± 2.1 standard deviation). Mean scores for the ten highest rated individual questions are given in Table  2 . Only one respondent suggested additional research topics that were not included in the questionnaire. The full list of 30 research topics presented to respondents in the questionnaire is presented in the appendix [see  Additional file 1 ]. Table 2 The ten highest rated perioperative research issues: mean scores* (rank) Research issues Total 95% (CI) High** volume Low volume Academic Non-academic The value of tight control of diabetes mellitus postoperatively. 7.2 (1) 6.8–7.6 7.2 (1) 7.1 (2) 7.1 (3) 7.2 (1) Starting aspirin on patients at increased risk for postoperative cardiac complications. 7.1 (2) 6.7–7.5 7.2 (2) 7.0 (4) 7.1 (4) 7.1 (2) Safety and efficacy of continuing aspirin preoperatively for those already taking aspirin. 7.0 (3) 6.6–7.4 7.0 (5) 7.1 (1) 7.1 (5) 7.0 (3) Optimal management of perioperative anticoagulation for patients with prosthetic valves. 7.0 (4) 6.5–7.4 6.9 (6) 7.1 (3) 7.0 (6) 7.0 (4) The value of starting angiotensin converting enzyme inhibitors for those at increased risk of postoperative cardiac complications. 6.9 (5) 6.5–7.3 7.2 (3) 6.7 (9) 7.0 (7) 6.9 (7) Determining the diagnostic yield of routine postoperative cardiac surveillance. 6.8 (6) 6.4–7.2 7.1 (4) 6.6 (13) 6.5 (12) 6.9 (6) Developing interventions to minimize postoperative delirium. 6.8 (7) 6.4–7.2 6.5 (9) 7.0 (6) 6.4 (14) 6.9 (5) The value of starting beta-blockers for patients at increased risk of postoperative cardiac complications. 6.7 (8) 6.2–7.2 6.4 (11) 7.0 (5) 7.2 (1) 6.6 (10) Optimal management of perioperative anticoagulation for patients with atrial fibrillation. 6.6 (9) 6.2–7.1 6.3 (12) 6.9 (7) 6.6 (11) 6.7 (9) Developing a risk stratification index for predicting postoperative pulmonary complications 6.6 (10) 6.2–7.1 6.6 (8) 6.7 (10) 6.5 (13) 6.7 (8) * Mean scores were derived from responses based on a 10-point Likert scale. A score of 10 indicates high priority and 1 indicates low priority for future research. ** Ratings from consultants who see a high volume of preoperative consults (>10/month). Mean scores for most questions were similar among physicians independent of their academic status or whether they performed a high volume of preoperative consults (defined as greater than 10 consults per month) or not. For several questions, however, differences in ranking according to academic status and volume of consultations were found, although none achieved statistical significance (Table  2 ).
Results
After 3 mailings, we obtained 152 completed surveys (overall response rate 34%). Thirty-three respondents were then excluded because of subspecialty status or because they did not perform perioperative consults.Respondents belonging to the Society of General Internal Medicine special interest group and Canadian Society of Internal Medicine were identical in most respects except fewer Canadian Society of Internal Medicine members had academic appointments (54% vs. 100%). Table  1  shows the demographic and professional characteristics for all respondents.Physician characteristics (n = 119)Research questions evaluating the efficacy of perioperative interventions had higher ratings followed by questions relating to prediction of postoperative risk. Questions evaluating the yield of preoperative diagnostic tests had the lowest ratings. The differences in ratings across these general categories were statistically significant (p < 0.001) and this pattern persisted regardless of academic status or volume of consults seen.Mean scores for individual research questions, based on responses where 1 indicates low priority for future research and 10 indicates high priority, ranged from a low of 3.6 (± 2.3 standard deviation) to a high of 7.2 (± 2.1 standard deviation). Mean scores for the ten highest rated individual questions are given in Table  2 . Only one respondent suggested additional research topics that were not included in the questionnaire. The full list of 30 research topics presented to respondents in the questionnaire is presented in the appendix [see  Additional file 1 ].The ten highest rated perioperative research issues: mean scores* (rank)* Mean scores were derived from responses based on a 10-point Likert scale. A score of 10 indicates high priority and 1 indicates low priority for future research.** Ratings from consultants who see a high volume of preoperative consults (>10/month).Mean scores for most questions were similar among physicians independent of their academic status or whether they performed a high volume of preoperative consults (defined as greater than 10 consults per month) or not. For several questions, however, differences in ranking according to academic status and volume of consultations were found, although none achieved statistical significance (Table  2 ).Discussion There are few areas within perioperative medicine that are well studied beyond the area of predicting cardiac risk. The American Heart Association perioperative guidelines highlighted the paucity of studies on interventions to prevent postoperative cardiac events [ 3 ]. Reflecting this statement, respondents globally rated studies that determine the efficacy and safety of interventions as higher priority for future research compared to studies predicting postoperative risk or determining diagnostic yield of tests. Within the category of intervention studies, questions on medical therapy to prevent postoperative cardiac complications were among the highest rated questions. This result is congruent to the significant prevalence, morbidity and mortality associated with postoperative cardiac complications. A study by Devereaux et al also identified this area as an important target for future research after finding considerable practice variation in the management of cardiac medications [ 4 ]. Innovative interventions for cardiac protection with antiplatelet agents, angiotensin converting enzyme inhibitors or tight glycemic control were highly rated. Despite the publication of small trials of beta blocker therapy, responding internists felt that there was a need to definitively determine the efficacy of perioperative beta blockade. Respondents may be more skeptical of adopting results from the small trials of beta blocker where methodological controversies have arisen [ 5 ]. Other intervention questions that were rated highly focused on understudied areas of perioperative anticoagulation. Determining optimal perioperative anticoagulation strategies for patients with prosthetic valves or atrial fibrillation was rated highly. Although perioperative risk stratification is well studied for identifying those at risk of cardiac events, pulmonary complications occur more frequent than cardiac complications and are associated with a longer hospital stay [ 6 ]. Reflecting this significant morbidity and cost of respiratory complications, development of a prediction rule for postoperative pulmonary complications was among the highest rated topics. Since the completion of the survey, the ratings also reflect ongoing research activity within perioperative medicine. Tight glycemic control was a high rated topic and a study examining the effect of tight glycemic control in postoperative patients in a critical care setting has been published since the completion of this survey. Also since the completion of this survey, a large multi-center trial examining the efficacy of perioperative beta blockade has been launched. Another highly rated research topic was developing a prediction rule for pulmonary complications. Recently, a study was published to identify those at increased risk of postoperative pneumonia. Two of the highly rated research priority topics have been recently published in major medical journals [ 7 , 8 ]. There are several potential limitations with this study. The low response rate may be felt to limit the generalizability to other general internists. However, views of those who practice perioperative medicine and who have a particular interest in the area, the research consumers, are important in developing a research agenda. We presume that physicians who practice within an area and who have a particular interest are more likely to respond to a survey than those who do not. Thus, the physicians who responded to the questionnaire and reported practicing perioperative medicine define the group of research consumers we were targeting. This study also only examined the beliefs of general internists on research priorities since greater than 90% of the perioperative medicine consultations are conducted by general internists rather than subspecialists in internal medicine in tertiary care centers [ 4 ]. Also, lower response rates are seen in physician surveys but surveys with response rates of 10–45% are published in major medical journals [ 9 - 15 ]. Another limitation is that anesthesiologists, cardiologists or primary care physicians perform perioperative medicine consultations and their opinions were not elicited in this survey. Another potential limitation is that not all important research questions could be examined. However, only one respondent indicated additional research topics suggesting that there were no major omissions in the research topics listed. Additionally, it is difficult to interpret meaningful differences in mean Likert response scores among individual questions. Individual scores and their confidence intervals should be interpreted to give a general idea of high- priority questions, rather than a strict ranking of research pursuits.
Discussion
There are few areas within perioperative medicine that are well studied beyond the area of predicting cardiac risk. The American Heart Association perioperative guidelines highlighted the paucity of studies on interventions to prevent postoperative cardiac events [ 3 ]. Reflecting this statement, respondents globally rated studies that determine the efficacy and safety of interventions as higher priority for future research compared to studies predicting postoperative risk or determining diagnostic yield of tests. Within the category of intervention studies, questions on medical therapy to prevent postoperative cardiac complications were among the highest rated questions. This result is congruent to the significant prevalence, morbidity and mortality associated with postoperative cardiac complications. A study by Devereaux et al also identified this area as an important target for future research after finding considerable practice variation in the management of cardiac medications [ 4 ]. Innovative interventions for cardiac protection with antiplatelet agents, angiotensin converting enzyme inhibitors or tight glycemic control were highly rated. Despite the publication of small trials of beta blocker therapy, responding internists felt that there was a need to definitively determine the efficacy of perioperative beta blockade. Respondents may be more skeptical of adopting results from the small trials of beta blocker where methodological controversies have arisen [ 5 ]. Other intervention questions that were rated highly focused on understudied areas of perioperative anticoagulation. Determining optimal perioperative anticoagulation strategies for patients with prosthetic valves or atrial fibrillation was rated highly.Although perioperative risk stratification is well studied for identifying those at risk of cardiac events, pulmonary complications occur more frequent than cardiac complications and are associated with a longer hospital stay [ 6 ]. Reflecting this significant morbidity and cost of respiratory complications, development of a prediction rule for postoperative pulmonary complications was among the highest rated topics.Since the completion of the survey, the ratings also reflect ongoing research activity within perioperative medicine. Tight glycemic control was a high rated topic and a study examining the effect of tight glycemic control in postoperative patients in a critical care setting has been published since the completion of this survey. Also since the completion of this survey, a large multi-center trial examining the efficacy of perioperative beta blockade has been launched. Another highly rated research topic was developing a prediction rule for pulmonary complications. Recently, a study was published to identify those at increased risk of postoperative pneumonia. Two of the highly rated research priority topics have been recently published in major medical journals [ 7 , 8 ].There are several potential limitations with this study. The low response rate may be felt to limit the generalizability to other general internists. However, views of those who practice perioperative medicine and who have a particular interest in the area, the research consumers, are important in developing a research agenda. We presume that physicians who practice within an area and who have a particular interest are more likely to respond to a survey than those who do not. Thus, the physicians who responded to the questionnaire and reported practicing perioperative medicine define the group of research consumers we were targeting. This study also only examined the beliefs of general internists on research priorities since greater than 90% of the perioperative medicine consultations are conducted by general internists rather than subspecialists in internal medicine in tertiary care centers [ 4 ]. Also, lower response rates are seen in physician surveys but surveys with response rates of 10–45% are published in major medical journals [ 9 - 15 ]. Another limitation is that anesthesiologists, cardiologists or primary care physicians perform perioperative medicine consultations and their opinions were not elicited in this survey. Another potential limitation is that not all important research questions could be examined. However, only one respondent indicated additional research topics suggesting that there were no major omissions in the research topics listed. Additionally, it is difficult to interpret meaningful differences in mean Likert response scores among individual questions. Individual scores and their confidence intervals should be interpreted to give a general idea of high- priority questions, rather than a strict ranking of research pursuits.Conclusion Perioperative medicine research is growing in response to the significant medical and economic consequences of perioperative complications. Identifying the research priorities of those who provide perioperative medical care – the consumers of research, is important. The results suggest that intervention studies are a higher priority for future research compared to studies that predict postoperative complications or determine the yield of diagnostic tests. Researchers and funding boards may use the findings of this survey to identify perceived high priority research topics within perioperative medicine.
Conclusion
Perioperative medicine research is growing in response to the significant medical and economic consequences of perioperative complications. Identifying the research priorities of those who provide perioperative medical care – the consumers of research, is important. The results suggest that intervention studies are a higher priority for future research compared to studies that predict postoperative complications or determine the yield of diagnostic tests. Researchers and funding boards may use the findings of this survey to identify perceived high priority research topics within perioperative medicine.Competing interests None declared.
Competing interests
None declared.Authors' contribution NK, TT, AF, NC, FAM, and WG contributed to the design of the project. NK and TT contributed to data collection and data analysis. NK, TT, WG contributed to writing the manuscript. AF, NC, FAM made substantive, intellectual editing contributions.
Authors' contribution
NK, TT, AF, NC, FAM, and WG contributed to the design of the project. NK and TT contributed to data collection and data analysis. NK, TT, WG contributed to writing the manuscript. AF, NC, FAM made substantive, intellectual editing contributions.Pre-publication history The pre-publication history for this paper can be accessed here:
Pre-publication history
The pre-publication history for this paper can be accessed here:Supplementary Material Additional File 1 Perioperative medicine research questions. This appendix lists all 30 perioperative research issues from the survey, along with the mean scores rated by all respondents. Click here for file
Supplementary Material
Additional File 1
Perioperative medicine research questions. This appendix lists all 30 perioperative research issues from the survey, along with the mean scores rated by all respondents.Click here for file


Background To understand mycobacterial pathogenesis analysis of gene expression by quantification of RNA levels becomes increasingly important. However, current preparation methods yield mycobacterial RNA that is contaminated with chromosomal DNA.
Background
To understand mycobacterial pathogenesis analysis of gene expression by quantification of RNA levels becomes increasingly important. However, current preparation methods yield mycobacterial RNA that is contaminated with chromosomal DNA.Results After sonication of RNA samples from  Mycobacterium smegmatis  genomic DNA is efficiently removed by DNaseI in contrast to untreated samples.
Results
After sonication of RNA samples from  Mycobacterium smegmatis  genomic DNA is efficiently removed by DNaseI in contrast to untreated samples.Mycobacterium smegmatisConclusions This procedure eliminates one of the most prevalent error sources in quantification of RNA levels in mycobacteria.
Conclusions
This procedure eliminates one of the most prevalent error sources in quantification of RNA levels in mycobacteria.

Background Mycobacterium tuberculosis  is the leading cause of death from a single infectious disease with approximately 8.8 million new cases and two million deaths per year. To understand the pathogenesis of  M. tuberculosis , analysis of gene expression by relative or absolute quantification of RNA levels using microarrays and RT-PCR (batch- and real-time) becomes increasingly important [ 1 ]. Widely used methods to isolate bacterial RNA are acid-phenol extraction or guanidinium isothiocyanate extraction combined with cesium chloride purification or nucleic acid binding resins [ 2 ]. However, the cell wall of mycobacteria is very stable and a very effective permeability barrier, and, therefore, rather refractory to lysis by chaotropic agents and detergents, hampering RNA isolation from these microorganisms [ 3 ]. Since the average half-life of mycobacterial mRNA is in the range of a few minutes, mycobacteria have to be vigorously treated (e.g. bead-beating, freeze-thawing, nitrogen decompression) to quickly isolate RNA [ 4 ]. This causes fragmentation of chromosomal DNA that contaminates RNA preparations, which is one of the most prevalent error sources in quantification of RNA levels in mycobacteria. Several methods have been suggested to circumvent this problem [ 5 , 6 ]. Virtually all RNA isolation protocols use DNaseI, which does not completely remove large amounts of DNA. Our goal was to improve the efficiency of DNaseI digestion by solubilizing chromosomal DNA with sonication prior to DNaseI treatment.  Mycobacterium smegmatis  is especially refractory to lysis and therefore was chosen as a model organism.
Background
Mycobacterium tuberculosis  is the leading cause of death from a single infectious disease with approximately 8.8 million new cases and two million deaths per year. To understand the pathogenesis of  M. tuberculosis , analysis of gene expression by relative or absolute quantification of RNA levels using microarrays and RT-PCR (batch- and real-time) becomes increasingly important [ 1 ]. Widely used methods to isolate bacterial RNA are acid-phenol extraction or guanidinium isothiocyanate extraction combined with cesium chloride purification or nucleic acid binding resins [ 2 ]. However, the cell wall of mycobacteria is very stable and a very effective permeability barrier, and, therefore, rather refractory to lysis by chaotropic agents and detergents, hampering RNA isolation from these microorganisms [ 3 ]. Since the average half-life of mycobacterial mRNA is in the range of a few minutes, mycobacteria have to be vigorously treated (e.g. bead-beating, freeze-thawing, nitrogen decompression) to quickly isolate RNA [ 4 ]. This causes fragmentation of chromosomal DNA that contaminates RNA preparations, which is one of the most prevalent error sources in quantification of RNA levels in mycobacteria. Several methods have been suggested to circumvent this problem [ 5 , 6 ]. Virtually all RNA isolation protocols use DNaseI, which does not completely remove large amounts of DNA. Our goal was to improve the efficiency of DNaseI digestion by solubilizing chromosomal DNA with sonication prior to DNaseI treatment.  Mycobacterium smegmatis  is especially refractory to lysis and therefore was chosen as a model organism.Mycobacterium tuberculosisM. tuberculosisMycobacterium smegmatisResults and Methods M. smegmatis  SMR5 [ 7 ] was grown in 10 ml Middlebrook 7H9 liquid medium (Difco Laboratories; supplemented with 0.2% glycerol, 0.05% Tween 80) to an OD 600  of 0.8 and mixed with 5 ml killing buffer (20 mM Tris-HCl, 5 mM MgCl 2 , 20 mM NaN 3 ) [ 8 ]. The cell suspension was incubated on ice for 5 min. Cells were harvested by centrifugation (20 min at 6000 × g and 4°C). 20 mg cells (dry weight) were lysed in FastRNA Blue-Tubes (Bio-101 Inc.) using a FastPrep FP120 bead-beater apparatus (Savant, USA) for 20 sec at level 6.5. The tubes were centrifuged for 10 min at 10000 × g and 4°C. The supernatant was transferred to microcentrifuge tubes containing a nucleic acid binding resin (Nucleospin RNA II; Macherey-Nagel), and further experimental steps were done as described by the manufacturer. A total of 62 μg RNA was eluted in 60 μl of RNase-free water. The RNA was diluted to 50 ng μl -1  into several aliquots. One aliquot containing 10 μg RNA was left untreated. The second aliquot was directly treated with 10U of RNase-free DNaseI (Roche) for 1 h at 37°C, while the third aliquot was sonicated two times for 20 sec with 0.9 sec intervals at 20 % power (Sonopuls HD 2070; Bandelin electronic) prior to DNaseI treatment. Between the two sonication steps the cell suspension was chilled on ice for 5 min. DNaseI was removed by precipitation with polyethylene glycol (PEG) 6000. As a control for the RNA quality, cDNA was synthesized by Omniscript reverse transcriptase and sensiscript reverse transcriptase (OneStep RT-PCR system, QIAGEN) from total RNA (100 ng) for 35 min at 50°C followed by an inactivation step of 15 min at 95°C. The 16Sr RNA was then amplified with the primers 16S-FP (5'-TGCTACAATGGCCGGTACAAA-3') and 16S-RP (5'-GCGATTACTAGCGACGCCGACTT-3') using up to 30 cycles of 1 min at 94°C, 30 sec at 53°C, and 1 min at 72°C before a final extension step of 7 min at 72°C. As a control for DNA contamination, standard PCRs were performed, to which RNA was added after the RT inactivation step. PCR products were analysed at cycles 23, 25, 27 or 30 to check the purity of the RNA. All samples apparently contained 16S rRNA (Fig.  1 ). However, DNA contamination was detected by PCR at cycle 25 in the RNA sample that was not treated with DNaseI. Conventional DNaseI treatment delayed the appearance of a signal in the sample without the RT step until cycle 27. By contrast, no amplification product was obtained in the control sample even after 30 cycles, when the RNA sample was sonicated before DNaseI treatment (Fig.  1 ).
Results and Methods
M. smegmatis  SMR5 [ 7 ] was grown in 10 ml Middlebrook 7H9 liquid medium (Difco Laboratories; supplemented with 0.2% glycerol, 0.05% Tween 80) to an OD 600  of 0.8 and mixed with 5 ml killing buffer (20 mM Tris-HCl, 5 mM MgCl 2 , 20 mM NaN 3 ) [ 8 ]. The cell suspension was incubated on ice for 5 min. Cells were harvested by centrifugation (20 min at 6000 × g and 4°C). 20 mg cells (dry weight) were lysed in FastRNA Blue-Tubes (Bio-101 Inc.) using a FastPrep FP120 bead-beater apparatus (Savant, USA) for 20 sec at level 6.5. The tubes were centrifuged for 10 min at 10000 × g and 4°C. The supernatant was transferred to microcentrifuge tubes containing a nucleic acid binding resin (Nucleospin RNA II; Macherey-Nagel), and further experimental steps were done as described by the manufacturer. A total of 62 μg RNA was eluted in 60 μl of RNase-free water. The RNA was diluted to 50 ng μl -1  into several aliquots. One aliquot containing 10 μg RNA was left untreated. The second aliquot was directly treated with 10U of RNase-free DNaseI (Roche) for 1 h at 37°C, while the third aliquot was sonicated two times for 20 sec with 0.9 sec intervals at 20 % power (Sonopuls HD 2070; Bandelin electronic) prior to DNaseI treatment. Between the two sonication steps the cell suspension was chilled on ice for 5 min. DNaseI was removed by precipitation with polyethylene glycol (PEG) 6000. As a control for the RNA quality, cDNA was synthesized by Omniscript reverse transcriptase and sensiscript reverse transcriptase (OneStep RT-PCR system, QIAGEN) from total RNA (100 ng) for 35 min at 50°C followed by an inactivation step of 15 min at 95°C. The 16Sr RNA was then amplified with the primers 16S-FP (5'-TGCTACAATGGCCGGTACAAA-3') and 16S-RP (5'-GCGATTACTAGCGACGCCGACTT-3') using up to 30 cycles of 1 min at 94°C, 30 sec at 53°C, and 1 min at 72°C before a final extension step of 7 min at 72°C. As a control for DNA contamination, standard PCRs were performed, to which RNA was added after the RT inactivation step. PCR products were analysed at cycles 23, 25, 27 or 30 to check the purity of the RNA. All samples apparently contained 16S rRNA (Fig.  1 ). However, DNA contamination was detected by PCR at cycle 25 in the RNA sample that was not treated with DNaseI. Conventional DNaseI treatment delayed the appearance of a signal in the sample without the RT step until cycle 27. By contrast, no amplification product was obtained in the control sample even after 30 cycles, when the RNA sample was sonicated before DNaseI treatment (Fig.  1 ).M. smegmatisConclusions This result shows that sonication improved DNA degradation by DNaseI most likely by rendering the chromosomal DNA more accessible to enzymatic action. This work describes a simple and efficient procedure to improve the quality of RNA preparations from  M. smegmatis  and will be of great value for RNA preparations from other microorganisms, including  M. tuberculosis .
Conclusions
This result shows that sonication improved DNA degradation by DNaseI most likely by rendering the chromosomal DNA more accessible to enzymatic action. This work describes a simple and efficient procedure to improve the quality of RNA preparations from  M. smegmatis  and will be of great value for RNA preparations from other microorganisms, including  M. tuberculosis .M. smegmatisM. tuberculosisAuthors' contributions JS carried out the RNA preparations, the RT-PCR experiments and wrote a draft of the manuscript. JGB performed RNA preparations and RT-PCR experiments. FT participated in coordinating and supervising the study. MN conceived of the study, participated in coordinating and supervising the study, and wrote the final manuscript. All authors read and approved the final manuscript.
Authors' contributions
JS carried out the RNA preparations, the RT-PCR experiments and wrote a draft of the manuscript. JGB performed RNA preparations and RT-PCR experiments. FT participated in coordinating and supervising the study. MN conceived of the study, participated in coordinating and supervising the study, and wrote the final manuscript. All authors read and approved the final manuscript.