Background Anopheles gambiae  Giles is the most important vector of human malaria in sub-Saharan Africa. Knowledge of the factors that influence its daily oviposition pattern is crucial if field interventions targeting gravid females are to be successful. This laboratory study investigated the effect of oviposition substrate and time of blood feeding on daily oviposition patterns of  An. gambiae  mosquitoes.
Background
Anopheles gambiae  Giles is the most important vector of human malaria in sub-Saharan Africa. Knowledge of the factors that influence its daily oviposition pattern is crucial if field interventions targeting gravid females are to be successful. This laboratory study investigated the effect of oviposition substrate and time of blood feeding on daily oviposition patterns of  An. gambiae  mosquitoes.Anopheles gambiaeAn. gambiaeMethods Greenhouse-reared gravid and hypergravid (delayed oviposition onset)  An. gambiae sensu stricto  and wild-caught  An. gambiae sensu lato  were exposed to three types of substrates in choice and no-choice cage bioassays: water from a predominantly anopheline colonised ground pool (anopheline habitat water), swamp water mainly colonised by culicine larvae (culicine habitat water) and distilled water. The daily oviposition pattern and the number of eggs oviposited on each substrate during the entire egg-laying period were determined. The results were subjected to analysis of variance using the General Linear Model (GLM) procedure.
Methods
Greenhouse-reared gravid and hypergravid (delayed oviposition onset)  An. gambiae sensu stricto  and wild-caught  An. gambiae sensu lato  were exposed to three types of substrates in choice and no-choice cage bioassays: water from a predominantly anopheline colonised ground pool (anopheline habitat water), swamp water mainly colonised by culicine larvae (culicine habitat water) and distilled water. The daily oviposition pattern and the number of eggs oviposited on each substrate during the entire egg-laying period were determined. The results were subjected to analysis of variance using the General Linear Model (GLM) procedure.An. gambiae sensu strictoAn. gambiae sensu latoResults The main oviposition time for greenhouse-reared  An. gambiae s.s.  was between 19:00 and 20:00 hrs, approximately one hour after sunset. Wild-caught gravid  An. gambiae s.l.  displayed two distinct peak oviposition times between 19:00 and 20:00 hrs and between 22:00 and 23:00 hrs, respectively. During these times, both greenhouse-reared and wild-caught mosquitoes significantly ( P  < 0.05) preferred anopheline habitat water to the culicine one. Peak oviposition activity was not delayed when the mosquitoes were exposed to the less preferred oviposition substrate (culicine habitat water). However, culicine water influenced negatively ( P  < 0.05) not only the number of eggs oviposited by the mosquitoes during peak oviposition time but also the overall number of gravid mosquitoes that laid their eggs on it. The differences in mosquito feeding times did not affect the daily oviposition patterns displayed.
Results
The main oviposition time for greenhouse-reared  An. gambiae s.s.  was between 19:00 and 20:00 hrs, approximately one hour after sunset. Wild-caught gravid  An. gambiae s.l.  displayed two distinct peak oviposition times between 19:00 and 20:00 hrs and between 22:00 and 23:00 hrs, respectively. During these times, both greenhouse-reared and wild-caught mosquitoes significantly ( P  < 0.05) preferred anopheline habitat water to the culicine one. Peak oviposition activity was not delayed when the mosquitoes were exposed to the less preferred oviposition substrate (culicine habitat water). However, culicine water influenced negatively ( P  < 0.05) not only the number of eggs oviposited by the mosquitoes during peak oviposition time but also the overall number of gravid mosquitoes that laid their eggs on it. The differences in mosquito feeding times did not affect the daily oviposition patterns displayed.An. gambiae s.s.An. gambiae s.l.PPConclusion This study shows that the peak oviposition time of  An. gambiae s.l.  may be regulated by the light-dark cycle rather than oviposition habitat characteristics or feeding times. However, the number of eggs laid by the female mosquito during the peak oviposition time is affected by the suitability of the habitat.
Conclusion
This study shows that the peak oviposition time of  An. gambiae s.l.  may be regulated by the light-dark cycle rather than oviposition habitat characteristics or feeding times. However, the number of eggs laid by the female mosquito during the peak oviposition time is affected by the suitability of the habitat.An. gambiae s.l.

Background Although  An. gambiae s.l.  mosquitoes are nocturnal in their feeding and oviposition activities, the probable time of oviposition is determined by many factors including ambient temperature and light conditions, and the time the mosquito obtains its blood meal [ 1 , 2 ]. In addition, we hypothesised that the availability of a suitable larval habitat would also affect the mosquito's predisposition to oviposit.  Anopheles gambiae  is discriminative in its oviposition behaviour [ 3 ]. Its preferred larval habitats are fresh water pools that are generally small, transient and sunlit, devoid of vegetation and often turbid [ 4 - 6 ]. Oviposition tendency might therefore be related to location and availability of such sites. In this study, we compared the daily oviposition patterns and the number of eggs laid by  An. gambiae s.s.  and wild-caught  An. gambiae s.l.  on aqueous collections from habitats colonised by anopheline or culicine larvae respectively, and distilled water.
Background
Although  An. gambiae s.l.  mosquitoes are nocturnal in their feeding and oviposition activities, the probable time of oviposition is determined by many factors including ambient temperature and light conditions, and the time the mosquito obtains its blood meal [ 1 , 2 ]. In addition, we hypothesised that the availability of a suitable larval habitat would also affect the mosquito's predisposition to oviposit.  Anopheles gambiae  is discriminative in its oviposition behaviour [ 3 ]. Its preferred larval habitats are fresh water pools that are generally small, transient and sunlit, devoid of vegetation and often turbid [ 4 - 6 ]. Oviposition tendency might therefore be related to location and availability of such sites. In this study, we compared the daily oviposition patterns and the number of eggs laid by  An. gambiae s.s.  and wild-caught  An. gambiae s.l.  on aqueous collections from habitats colonised by anopheline or culicine larvae respectively, and distilled water.An. gambiae s.l.Anopheles gambiaeAn. gambiae s.s.An. gambiae s.l.Methods Mosquitoes Anopheles gambiae s.s.  (MBITA strain; colonised since February 2001) mosquitoes from Mbita Point, western Kenya, were reared in a greenhouse [ 7 ] in water obtained from a natural ground pool colonised by anopheline larvae. Average temperatures and relative humidities were 31°C, 52 % during the day and 24°C, 72% at night. The mosquitoes were exposed to the natural photoperiod, 00° 25' South of the equator. A data logger (HOBO™) was used to record ambient conditions. Larvae were fed on Tetramin ®  fish food. Adult mosquitoes were kept in standard mosquito rearing cages (30 × 30 × 30 cm) made of a metal wire frame with a solid metal base and covered with white nylon mosquito netting. They were offered a 6% glucose solution soaked in white paper towel wicks. Three-to-four-day-old females were offered two blood meals, one each day at 18.00 hrs, from the forearm of a human volunteer. The unfed mosquitoes were removed from the cage after each blood meal. Fully engorged females were left in the cages until they were gravid or hypergravid. Gravid mosquitoes are those that were provided with oviposition substrates on the third evening after their first blood meal. Hypergravid mosquitoes were provided with oviposition substrates one day later. Wild, indoor-resting, blood fed anopheline mosquitoes were collected during early morning hours from houses in Lwanda village of Suba district, western Kenya, by means of aspirators. They were immediately transported to the greenhouse, sorted out to obtain  An. gambiae s.l.  females and provided with 6% glucose solution. They were used in periodicity experiments on the second evening after collection, as described below. Oviposition substrates Turbid water taken from a natural ground pool colonised by anopheline larvae (anopheline habitat water), yellow-brown water from a reed swamp colonised by culicine larvae (culicine habitat water), and distilled water were used as oviposition substrates. Presence of larvae was determined by making five random dips using a 350 ml standard dipper. Oviposition substrate preference The experiments were carried out under greenhouse conditions in 25 cm cubic Plexi ® -glass cages, each fitted with a white netting top and a side sleeve opening. To determine oviposition substrate preference, individual gravid  An. gambiae s.s.  mosquitoes were exposed to 20 ml of each of the above substrates in a three-choice bioassay (n = 55). The substrates were held in black plastic oviposition cups (2 cm depth, 4 cm diameter), placed at equal distances from one another. Individual mosquitoes were released into the cages at about 17.00 hours and left overnight. The following morning, eggs oviposited on each substrate were counted under a dissection microscope. In subsequent replications, oviposition cups containing substrates were rotated such that they occupied different positions every time in the oviposition cages. Daily oviposition patterns in a no-choice bioassay Daily oviposition patterns of  An. gambiae  female mosquitoes on test oviposition substrates, which were offered individually, were determined as follows. Groups of five greenhouse-reared gravid and hypergravid  An. gambiae s.s.  females were held in separate cages into which anopheline or culicine habitat water or distilled water were introduced. Each mosquito and substrate combination treatment was replicated four times on each experimental day and the experiment repeated on three different days. At the end of the experiment, the mosquitoes that had laid in each group were identified by dissecting each under a dissection microscope and examining their ovaries for the presence of either retained eggs, coiled or uncoiled tracheolar skeins [ 8 ]. Daily oviposition patterns in a choice bioassay Groups of five gravid and hypergravid  An. gambiae s.s.  (ten cages of each) were placed in separate cages and allowed to choose from the three types of oviposition substrates. Similarly, groups of five wild-caught  An. gambiae s.l.  mosquitoes were offered a choice of the three substrates and their daily oviposition patterns monitored. The experiment was replicated twice on each experimental day and repeated on five different days with new mosquito batches. Individual species within the wild-caught  An. gambiae  mosquitoes that had laid were identified using polymerase chain reaction (PCR) [ 9 ]. Effect of the time of blood feeding on daily oviposition patterns The effect of the time of blood feeding of  An. gambiae s.s.  on its daily oviposition pattern was determined as follows. Four groups of three-to-four-day-old females were given two blood meals, one each day at 06.00 hrs, 18.00 hrs, 22.00 hrs or at 00.00 hrs, respectively. Unfed females were removed from the cages after every blood meal. Gravid mosquitoes were then provided with oviposition cups on the third day at 06.00 hrs and their daily oviposition patterns monitored. In all experiments, the oviposition cups were removed from the cages after every hourly interval, for 24 hours, starting at 18.00 hrs and replaced with freshly prepared ones. The eggs laid on each substrate were counted under a dissection microscope. To minimise disturbance that might have been due to exposure to white light, red light was used at night while replacing the oviposition cups. Data analysis Since oviposition trends for gravid and hypergravid females were similar, data for the two were pooled for analysis. The differences in the number of eggs laid on different oviposition substrates were compared statistically by analysis of variance using the General Linear Model (GLM) procedure. The effect of oviposition substrate on the number of either gravid or hypergravid mosquitoes contributing to the total egg number was similarly compared. Means were separated by the least significant difference (LSD) procedure. Data were subjected to log 10  (n+1) transformation to normalise their distribution. All the analyses were carried out using the SPSS ®  statistical package, version 11.0.
Methods
Mosquitoes Anopheles gambiae s.s.  (MBITA strain; colonised since February 2001) mosquitoes from Mbita Point, western Kenya, were reared in a greenhouse [ 7 ] in water obtained from a natural ground pool colonised by anopheline larvae. Average temperatures and relative humidities were 31°C, 52 % during the day and 24°C, 72% at night. The mosquitoes were exposed to the natural photoperiod, 00° 25' South of the equator. A data logger (HOBO™) was used to record ambient conditions. Larvae were fed on Tetramin ®  fish food. Adult mosquitoes were kept in standard mosquito rearing cages (30 × 30 × 30 cm) made of a metal wire frame with a solid metal base and covered with white nylon mosquito netting. They were offered a 6% glucose solution soaked in white paper towel wicks. Three-to-four-day-old females were offered two blood meals, one each day at 18.00 hrs, from the forearm of a human volunteer. The unfed mosquitoes were removed from the cage after each blood meal. Fully engorged females were left in the cages until they were gravid or hypergravid. Gravid mosquitoes are those that were provided with oviposition substrates on the third evening after their first blood meal. Hypergravid mosquitoes were provided with oviposition substrates one day later. Wild, indoor-resting, blood fed anopheline mosquitoes were collected during early morning hours from houses in Lwanda village of Suba district, western Kenya, by means of aspirators. They were immediately transported to the greenhouse, sorted out to obtain  An. gambiae s.l.  females and provided with 6% glucose solution. They were used in periodicity experiments on the second evening after collection, as described below.
Mosquitoes
Anopheles gambiae s.s.  (MBITA strain; colonised since February 2001) mosquitoes from Mbita Point, western Kenya, were reared in a greenhouse [ 7 ] in water obtained from a natural ground pool colonised by anopheline larvae. Average temperatures and relative humidities were 31°C, 52 % during the day and 24°C, 72% at night. The mosquitoes were exposed to the natural photoperiod, 00° 25' South of the equator. A data logger (HOBO™) was used to record ambient conditions. Larvae were fed on Tetramin ®  fish food. Adult mosquitoes were kept in standard mosquito rearing cages (30 × 30 × 30 cm) made of a metal wire frame with a solid metal base and covered with white nylon mosquito netting. They were offered a 6% glucose solution soaked in white paper towel wicks. Three-to-four-day-old females were offered two blood meals, one each day at 18.00 hrs, from the forearm of a human volunteer. The unfed mosquitoes were removed from the cage after each blood meal. Fully engorged females were left in the cages until they were gravid or hypergravid. Gravid mosquitoes are those that were provided with oviposition substrates on the third evening after their first blood meal. Hypergravid mosquitoes were provided with oviposition substrates one day later. Wild, indoor-resting, blood fed anopheline mosquitoes were collected during early morning hours from houses in Lwanda village of Suba district, western Kenya, by means of aspirators. They were immediately transported to the greenhouse, sorted out to obtain  An. gambiae s.l.  females and provided with 6% glucose solution. They were used in periodicity experiments on the second evening after collection, as described below.Anopheles gambiae s.s.An. gambiae s.l.Oviposition substrates Turbid water taken from a natural ground pool colonised by anopheline larvae (anopheline habitat water), yellow-brown water from a reed swamp colonised by culicine larvae (culicine habitat water), and distilled water were used as oviposition substrates. Presence of larvae was determined by making five random dips using a 350 ml standard dipper.
Oviposition substrates
Turbid water taken from a natural ground pool colonised by anopheline larvae (anopheline habitat water), yellow-brown water from a reed swamp colonised by culicine larvae (culicine habitat water), and distilled water were used as oviposition substrates. Presence of larvae was determined by making five random dips using a 350 ml standard dipper.Oviposition substrate preference The experiments were carried out under greenhouse conditions in 25 cm cubic Plexi ® -glass cages, each fitted with a white netting top and a side sleeve opening. To determine oviposition substrate preference, individual gravid  An. gambiae s.s.  mosquitoes were exposed to 20 ml of each of the above substrates in a three-choice bioassay (n = 55). The substrates were held in black plastic oviposition cups (2 cm depth, 4 cm diameter), placed at equal distances from one another. Individual mosquitoes were released into the cages at about 17.00 hours and left overnight. The following morning, eggs oviposited on each substrate were counted under a dissection microscope. In subsequent replications, oviposition cups containing substrates were rotated such that they occupied different positions every time in the oviposition cages.
Oviposition substrate preference
The experiments were carried out under greenhouse conditions in 25 cm cubic Plexi ® -glass cages, each fitted with a white netting top and a side sleeve opening. To determine oviposition substrate preference, individual gravid  An. gambiae s.s.  mosquitoes were exposed to 20 ml of each of the above substrates in a three-choice bioassay (n = 55). The substrates were held in black plastic oviposition cups (2 cm depth, 4 cm diameter), placed at equal distances from one another. Individual mosquitoes were released into the cages at about 17.00 hours and left overnight. The following morning, eggs oviposited on each substrate were counted under a dissection microscope. In subsequent replications, oviposition cups containing substrates were rotated such that they occupied different positions every time in the oviposition cages.An. gambiae s.s.Daily oviposition patterns in a no-choice bioassay Daily oviposition patterns of  An. gambiae  female mosquitoes on test oviposition substrates, which were offered individually, were determined as follows. Groups of five greenhouse-reared gravid and hypergravid  An. gambiae s.s.  females were held in separate cages into which anopheline or culicine habitat water or distilled water were introduced. Each mosquito and substrate combination treatment was replicated four times on each experimental day and the experiment repeated on three different days. At the end of the experiment, the mosquitoes that had laid in each group were identified by dissecting each under a dissection microscope and examining their ovaries for the presence of either retained eggs, coiled or uncoiled tracheolar skeins [ 8 ].
Daily oviposition patterns in a no-choice bioassay
Daily oviposition patterns of  An. gambiae  female mosquitoes on test oviposition substrates, which were offered individually, were determined as follows. Groups of five greenhouse-reared gravid and hypergravid  An. gambiae s.s.  females were held in separate cages into which anopheline or culicine habitat water or distilled water were introduced. Each mosquito and substrate combination treatment was replicated four times on each experimental day and the experiment repeated on three different days. At the end of the experiment, the mosquitoes that had laid in each group were identified by dissecting each under a dissection microscope and examining their ovaries for the presence of either retained eggs, coiled or uncoiled tracheolar skeins [ 8 ].An. gambiaeAn. gambiae s.s.Daily oviposition patterns in a choice bioassay Groups of five gravid and hypergravid  An. gambiae s.s.  (ten cages of each) were placed in separate cages and allowed to choose from the three types of oviposition substrates. Similarly, groups of five wild-caught  An. gambiae s.l.  mosquitoes were offered a choice of the three substrates and their daily oviposition patterns monitored. The experiment was replicated twice on each experimental day and repeated on five different days with new mosquito batches. Individual species within the wild-caught  An. gambiae  mosquitoes that had laid were identified using polymerase chain reaction (PCR) [ 9 ].
Daily oviposition patterns in a choice bioassay
Groups of five gravid and hypergravid  An. gambiae s.s.  (ten cages of each) were placed in separate cages and allowed to choose from the three types of oviposition substrates. Similarly, groups of five wild-caught  An. gambiae s.l.  mosquitoes were offered a choice of the three substrates and their daily oviposition patterns monitored. The experiment was replicated twice on each experimental day and repeated on five different days with new mosquito batches. Individual species within the wild-caught  An. gambiae  mosquitoes that had laid were identified using polymerase chain reaction (PCR) [ 9 ].An. gambiae s.s.An. gambiae s.l.An. gambiaeEffect of the time of blood feeding on daily oviposition patterns The effect of the time of blood feeding of  An. gambiae s.s.  on its daily oviposition pattern was determined as follows. Four groups of three-to-four-day-old females were given two blood meals, one each day at 06.00 hrs, 18.00 hrs, 22.00 hrs or at 00.00 hrs, respectively. Unfed females were removed from the cages after every blood meal. Gravid mosquitoes were then provided with oviposition cups on the third day at 06.00 hrs and their daily oviposition patterns monitored. In all experiments, the oviposition cups were removed from the cages after every hourly interval, for 24 hours, starting at 18.00 hrs and replaced with freshly prepared ones. The eggs laid on each substrate were counted under a dissection microscope. To minimise disturbance that might have been due to exposure to white light, red light was used at night while replacing the oviposition cups.
Effect of the time of blood feeding on daily oviposition patterns
The effect of the time of blood feeding of  An. gambiae s.s.  on its daily oviposition pattern was determined as follows. Four groups of three-to-four-day-old females were given two blood meals, one each day at 06.00 hrs, 18.00 hrs, 22.00 hrs or at 00.00 hrs, respectively. Unfed females were removed from the cages after every blood meal. Gravid mosquitoes were then provided with oviposition cups on the third day at 06.00 hrs and their daily oviposition patterns monitored.An. gambiae s.s.In all experiments, the oviposition cups were removed from the cages after every hourly interval, for 24 hours, starting at 18.00 hrs and replaced with freshly prepared ones. The eggs laid on each substrate were counted under a dissection microscope. To minimise disturbance that might have been due to exposure to white light, red light was used at night while replacing the oviposition cups.Data analysis Since oviposition trends for gravid and hypergravid females were similar, data for the two were pooled for analysis. The differences in the number of eggs laid on different oviposition substrates were compared statistically by analysis of variance using the General Linear Model (GLM) procedure. The effect of oviposition substrate on the number of either gravid or hypergravid mosquitoes contributing to the total egg number was similarly compared. Means were separated by the least significant difference (LSD) procedure. Data were subjected to log 10  (n+1) transformation to normalise their distribution. All the analyses were carried out using the SPSS ®  statistical package, version 11.0.
Data analysis
Since oviposition trends for gravid and hypergravid females were similar, data for the two were pooled for analysis. The differences in the number of eggs laid on different oviposition substrates were compared statistically by analysis of variance using the General Linear Model (GLM) procedure. The effect of oviposition substrate on the number of either gravid or hypergravid mosquitoes contributing to the total egg number was similarly compared. Means were separated by the least significant difference (LSD) procedure. Data were subjected to log 10  (n+1) transformation to normalise their distribution. All the analyses were carried out using the SPSS ®  statistical package, version 11.0.Results Oviposition substrate preference The mean number ± standard error (39.4 ± 6.1) of eggs oviposited on anopheline habitat water was significantly higher than that on the culicine (16.1 ± 4.6;  P  = 0.01) or distilled water (23.7 ± 5.3;  P  = 0.02). Daily oviposition patterns Daily oviposition patterns of  An. gambiae s.s.  on different substrates, offered in either no-choice or choice assays, are presented in Figures  1  and  2 , respectively. In both cases, the main oviposition time was between 19:00 and 20:00 hrs, approximately one hour after sunset, followed by a steady reduction in the number of eggs laid as the night progressed. In the choice bioassays, the gravid mosquitoes showed significant preference for anopheline habitat water over distilled ( P  = 0.004) or culicine habitat water ( P  = 0.001) throughout the daily cycle. In the no-choice bioassay, although the total number of eggs laid throughout the cycle on the different substrates was different, this was not statistically significant ( P  = 0.4). However, during the peak oviposition time, the eggs laid on anopheline habitat water were significantly more than those on the culicine one ( P  = 0.01) but not significantly more than those on distilled water ( P  = 0.07). Egg-laying by mosquitoes of different ovary development stages was influenced considerably by the type of oviposition substrate ( P  = 0.02). The hypergravid/ anopheline habitat water combination had the highest average number of mosquitoes (4.4 ± 0.3) laying their eggs, whereas gravid/culicine combination yielded the lowest response (2.5 ± 0.4; Table  1 ). Figure 1 Daily oviposition patterns of  Anopheles gambiae s.s.  on different oviposition substrates in a no-choice bioassay . Mean percentage (± SE) of the total eggs laid on each of three different oviposition substrates during 1-h time intervals. n = 24 cages containing five females each. Mosquitoes in each cage were exposed to one type of substrate under a natural LD cycle (sunset at 18:00). Figure 2 Daily oviposition patterns of  Anopheles gambiae s.s.  on different oviposition substrates in a choice bioassay . Mean percentage (± SE) of the total eggs laid on each of the three different oviposition substrates during 1-h time intervals. n = 20 cages containing five females each. Mosquitoes could choose from different substrates placed in the same cage under a natural LD cycle (sunset at 18:00). Table 1 The number of mosquitoes (Mean ± SE 1 ) contributing to the total eggs laid in each mosquito/ substrate combination. Mosquito/ Substrate Mean ± SE 1 Gravid/ Distilled water 3.3 ± 0.4 bc Gravid/ Anopheline habitat water 3.5 ± 0.4 ab Gravid/ Culicine habitat water 2.5 ± 0.4 c Hypergravid/ Distilled water 3.8 ± 0.4 ab Hypergravid/ Anopheline habitat water 4.4 ± 0.3 a Hypergravid/ Culicine habitat water 3.6 ± 0.4 ab 1 SE: Standard Error. n = 12 cages each containing five mosquitoes. Any two means sharing a letter in common are not significantly different at 5% level (LSD test). Unlike the greenhouse-reared  An. gambiae s.s. , the wild-caught  An. gambiae s.l. , which consisted of 23.9%  An. gambiae s.s. ,71.7%  An. arabiensis  and 4.4% unidentified gravid females (n = 46), displayed two main oviposition times early in the night, between 19:00 and 20:00 hrs and between 22:00 and 23:00 hrs, respectively (Figure  3 ). These mosquitoes also showed significant preference ( P  = 0.01) for anopheline habitat water over distilled or culicine habitat water. Figure 3 Daily oviposition patterns of wild-caught  Anopheles gambiae s.l.  on different oviposition substrates in a choice bioassay . Mean percentages (± SE) of the total eggs oviposited on each of the three different oviposition substrate during 1-h time intervals. n = 10 cages containing five females each. Mosquitoes could choose from different substrates placed in the same cage under a natural LD cycle (sunset at 18:00). An. gambiae s.s.  females that obtained their blood meals later in the night displayed a somewhat broader oviposition peak time interval, ranging from 19:00 hrs to 22:00 hrs (Figure  4 ), than those that had fed earlier on, whose peak oviposition time interval was narrower (19:00 hrs to 21:00 hrs). Figure 4 Daily oviposition patterns of  Anopheles gambiae s.s.  fed at different times . Mean number (± SE) of eggs oviposited during 1-h time intervals. n = 8 cages containing five females each. Mosquitoes were kept under a natural LD cycle (sunset at 18:00).
Results
Oviposition substrate preference The mean number ± standard error (39.4 ± 6.1) of eggs oviposited on anopheline habitat water was significantly higher than that on the culicine (16.1 ± 4.6;  P  = 0.01) or distilled water (23.7 ± 5.3;  P  = 0.02).
Oviposition substrate preference
The mean number ± standard error (39.4 ± 6.1) of eggs oviposited on anopheline habitat water was significantly higher than that on the culicine (16.1 ± 4.6;  P  = 0.01) or distilled water (23.7 ± 5.3;  P  = 0.02).PPDaily oviposition patterns Daily oviposition patterns of  An. gambiae s.s.  on different substrates, offered in either no-choice or choice assays, are presented in Figures  1  and  2 , respectively. In both cases, the main oviposition time was between 19:00 and 20:00 hrs, approximately one hour after sunset, followed by a steady reduction in the number of eggs laid as the night progressed. In the choice bioassays, the gravid mosquitoes showed significant preference for anopheline habitat water over distilled ( P  = 0.004) or culicine habitat water ( P  = 0.001) throughout the daily cycle. In the no-choice bioassay, although the total number of eggs laid throughout the cycle on the different substrates was different, this was not statistically significant ( P  = 0.4). However, during the peak oviposition time, the eggs laid on anopheline habitat water were significantly more than those on the culicine one ( P  = 0.01) but not significantly more than those on distilled water ( P  = 0.07). Egg-laying by mosquitoes of different ovary development stages was influenced considerably by the type of oviposition substrate ( P  = 0.02). The hypergravid/ anopheline habitat water combination had the highest average number of mosquitoes (4.4 ± 0.3) laying their eggs, whereas gravid/culicine combination yielded the lowest response (2.5 ± 0.4; Table  1 ). Figure 1 Daily oviposition patterns of  Anopheles gambiae s.s.  on different oviposition substrates in a no-choice bioassay . Mean percentage (± SE) of the total eggs laid on each of three different oviposition substrates during 1-h time intervals. n = 24 cages containing five females each. Mosquitoes in each cage were exposed to one type of substrate under a natural LD cycle (sunset at 18:00). Figure 2 Daily oviposition patterns of  Anopheles gambiae s.s.  on different oviposition substrates in a choice bioassay . Mean percentage (± SE) of the total eggs laid on each of the three different oviposition substrates during 1-h time intervals. n = 20 cages containing five females each. Mosquitoes could choose from different substrates placed in the same cage under a natural LD cycle (sunset at 18:00). Table 1 The number of mosquitoes (Mean ± SE 1 ) contributing to the total eggs laid in each mosquito/ substrate combination. Mosquito/ Substrate Mean ± SE 1 Gravid/ Distilled water 3.3 ± 0.4 bc Gravid/ Anopheline habitat water 3.5 ± 0.4 ab Gravid/ Culicine habitat water 2.5 ± 0.4 c Hypergravid/ Distilled water 3.8 ± 0.4 ab Hypergravid/ Anopheline habitat water 4.4 ± 0.3 a Hypergravid/ Culicine habitat water 3.6 ± 0.4 ab 1 SE: Standard Error. n = 12 cages each containing five mosquitoes. Any two means sharing a letter in common are not significantly different at 5% level (LSD test). Unlike the greenhouse-reared  An. gambiae s.s. , the wild-caught  An. gambiae s.l. , which consisted of 23.9%  An. gambiae s.s. ,71.7%  An. arabiensis  and 4.4% unidentified gravid females (n = 46), displayed two main oviposition times early in the night, between 19:00 and 20:00 hrs and between 22:00 and 23:00 hrs, respectively (Figure  3 ). These mosquitoes also showed significant preference ( P  = 0.01) for anopheline habitat water over distilled or culicine habitat water. Figure 3 Daily oviposition patterns of wild-caught  Anopheles gambiae s.l.  on different oviposition substrates in a choice bioassay . Mean percentages (± SE) of the total eggs oviposited on each of the three different oviposition substrate during 1-h time intervals. n = 10 cages containing five females each. Mosquitoes could choose from different substrates placed in the same cage under a natural LD cycle (sunset at 18:00). An. gambiae s.s.  females that obtained their blood meals later in the night displayed a somewhat broader oviposition peak time interval, ranging from 19:00 hrs to 22:00 hrs (Figure  4 ), than those that had fed earlier on, whose peak oviposition time interval was narrower (19:00 hrs to 21:00 hrs). Figure 4 Daily oviposition patterns of  Anopheles gambiae s.s.  fed at different times . Mean number (± SE) of eggs oviposited during 1-h time intervals. n = 8 cages containing five females each. Mosquitoes were kept under a natural LD cycle (sunset at 18:00).
Daily oviposition patterns
Daily oviposition patterns of  An. gambiae s.s.  on different substrates, offered in either no-choice or choice assays, are presented in Figures  1  and  2 , respectively. In both cases, the main oviposition time was between 19:00 and 20:00 hrs, approximately one hour after sunset, followed by a steady reduction in the number of eggs laid as the night progressed. In the choice bioassays, the gravid mosquitoes showed significant preference for anopheline habitat water over distilled ( P  = 0.004) or culicine habitat water ( P  = 0.001) throughout the daily cycle. In the no-choice bioassay, although the total number of eggs laid throughout the cycle on the different substrates was different, this was not statistically significant ( P  = 0.4). However, during the peak oviposition time, the eggs laid on anopheline habitat water were significantly more than those on the culicine one ( P  = 0.01) but not significantly more than those on distilled water ( P  = 0.07). Egg-laying by mosquitoes of different ovary development stages was influenced considerably by the type of oviposition substrate ( P  = 0.02). The hypergravid/ anopheline habitat water combination had the highest average number of mosquitoes (4.4 ± 0.3) laying their eggs, whereas gravid/culicine combination yielded the lowest response (2.5 ± 0.4; Table  1 ).An. gambiae s.s.PPPPPPThe number of mosquitoes (Mean ± SE 1 ) contributing to the total eggs laid in each mosquito/ substrate combination.Mosquito/ SubstrateMean ± SE 11 SE: Standard Error. n = 12 cages each containing five mosquitoes. Any two means sharing a letter in common are not significantly different at 5% level (LSD test).Unlike the greenhouse-reared  An. gambiae s.s. , the wild-caught  An. gambiae s.l. , which consisted of 23.9%  An. gambiae s.s. ,71.7%  An. arabiensis  and 4.4% unidentified gravid females (n = 46), displayed two main oviposition times early in the night, between 19:00 and 20:00 hrs and between 22:00 and 23:00 hrs, respectively (Figure  3 ). These mosquitoes also showed significant preference ( P  = 0.01) for anopheline habitat water over distilled or culicine habitat water.An. gambiae s.s.An. gambiae s.l.An. gambiae s.s.An. arabiensisPAn. gambiae s.s.  females that obtained their blood meals later in the night displayed a somewhat broader oviposition peak time interval, ranging from 19:00 hrs to 22:00 hrs (Figure  4 ), than those that had fed earlier on, whose peak oviposition time interval was narrower (19:00 hrs to 21:00 hrs).An. gambiae s.s.Discussion In the present study, the daily oviposition patterns of greenhouse-reared  An. gambiae s.s.  were well defined with oviposition peak times between 19:00 and 20:00 hrs, regardless of the type of oviposition substrate used. Haddow and Ssenkubuge [ 10 ] obtained comparable results using  An. gambiae s.s.  (KISUMU strain, western Kenya): about half of the eggs were laid during the first three hours of the night (18:00 – 21:00 hrs). On the other hand, oviposition by wild-caught mosquitoes from the coast of Kenya used by McCrae [ 1 ], comprising mostly  An. gambiae s.s. , peaked much later at night in the hour following midnight. This suggests differences in oviposition patterns between our strain and that of Haddow and Ssenkubuge representing Lake Victoria populations, on one hand, and that used by McCrae representing the Kenyan coastal population, on the other. Studies of oviposition patterns of populations from different parts of eastern Africa may help shed further light on the question. In the current study, wild-caught  An. gambiae s.l. , which were shown to contain a mixture of  An. gambiae s.s.  and  An. arabiensis  gravid females, displayed two distinct oviposition peak times in the first half of the night. The two peaks may be attributed to the two sibling species and suggests that this may also be an important factor in the diversity of oviposition patterns in the field in different geographical locations. The differences in the mosquito feeding times did not affect the timing of peak oviposition, although females that obtained their blood meals later in the night displayed a somewhat broader oviposition peak interval. Peak oviposition consistently occurred approximately one hour after sunset; therefore, a fall in light intensity might be one of the important cues that trigger oviposition in female  An. gambiae  that are physiologically ready to oviposit. On the other hand, McCrae [ 1 ] observed that the time of oviposition was a function of the time of blood feeding and not a result of an endogenous rhythm. Given the uniform oviposition peak times of mosquitoes that were fed at different times, daily oviposition among  An. gambiae s.l.  may also be endogenously regulated. Detailed experiments to demonstrate a free-running oviposition periodicity would clarify this. There was no difference in oviposition patterns displayed by gravid and hypergravid mosquitoes. Since significantly more gravid females exposed to the preferred substrate oviposited their eggs than those exposed to the less preferred one, gravid females that fail to find a suitable oviposition site on the night they are due may retain their eggs and oviposit early the next night as hypergravids. Gravid mosquitoes are generally attracted to water; however, the decision to oviposit may depend on additional olfactory signals [ 11 ] and /or contact stimuli received when the insects land on the water surface [ 12 ]. In this study and others [ 13 ], the gravid mosquitoes showed marked preference for the water taken from a site naturally inhabited by anopheline larval populations. This suggests 'memory' of similar information gathered by contact with the oviposition water at emergence or during larval period as in the case of  Culex quinquefasciatus  [ 14 ]. In this regard, gravid females might associate specific site characteristics from conspecific and heterospecific immatures, soil microbial activity [ 11 ], colour and turbidity of the oviposition substrate [ 13 ] with their suitability for sustaining progeny development.
Discussion
In the present study, the daily oviposition patterns of greenhouse-reared  An. gambiae s.s.  were well defined with oviposition peak times between 19:00 and 20:00 hrs, regardless of the type of oviposition substrate used. Haddow and Ssenkubuge [ 10 ] obtained comparable results using  An. gambiae s.s.  (KISUMU strain, western Kenya): about half of the eggs were laid during the first three hours of the night (18:00 – 21:00 hrs). On the other hand, oviposition by wild-caught mosquitoes from the coast of Kenya used by McCrae [ 1 ], comprising mostly  An. gambiae s.s. , peaked much later at night in the hour following midnight. This suggests differences in oviposition patterns between our strain and that of Haddow and Ssenkubuge representing Lake Victoria populations, on one hand, and that used by McCrae representing the Kenyan coastal population, on the other. Studies of oviposition patterns of populations from different parts of eastern Africa may help shed further light on the question.An. gambiae s.s.An. gambiae s.s.An. gambiae s.s.In the current study, wild-caught  An. gambiae s.l. , which were shown to contain a mixture of  An. gambiae s.s.  and  An. arabiensis  gravid females, displayed two distinct oviposition peak times in the first half of the night. The two peaks may be attributed to the two sibling species and suggests that this may also be an important factor in the diversity of oviposition patterns in the field in different geographical locations.An. gambiae s.l.An. gambiae s.s.An. arabiensisThe differences in the mosquito feeding times did not affect the timing of peak oviposition, although females that obtained their blood meals later in the night displayed a somewhat broader oviposition peak interval. Peak oviposition consistently occurred approximately one hour after sunset; therefore, a fall in light intensity might be one of the important cues that trigger oviposition in female  An. gambiae  that are physiologically ready to oviposit. On the other hand, McCrae [ 1 ] observed that the time of oviposition was a function of the time of blood feeding and not a result of an endogenous rhythm. Given the uniform oviposition peak times of mosquitoes that were fed at different times, daily oviposition among  An. gambiae s.l.  may also be endogenously regulated. Detailed experiments to demonstrate a free-running oviposition periodicity would clarify this. There was no difference in oviposition patterns displayed by gravid and hypergravid mosquitoes. Since significantly more gravid females exposed to the preferred substrate oviposited their eggs than those exposed to the less preferred one, gravid females that fail to find a suitable oviposition site on the night they are due may retain their eggs and oviposit early the next night as hypergravids.An. gambiaeAn. gambiae s.l.Gravid mosquitoes are generally attracted to water; however, the decision to oviposit may depend on additional olfactory signals [ 11 ] and /or contact stimuli received when the insects land on the water surface [ 12 ]. In this study and others [ 13 ], the gravid mosquitoes showed marked preference for the water taken from a site naturally inhabited by anopheline larval populations. This suggests 'memory' of similar information gathered by contact with the oviposition water at emergence or during larval period as in the case of  Culex quinquefasciatus  [ 14 ]. In this regard, gravid females might associate specific site characteristics from conspecific and heterospecific immatures, soil microbial activity [ 11 ], colour and turbidity of the oviposition substrate [ 13 ] with their suitability for sustaining progeny development.Culex quinquefasciatusConclusions This study shows that the peak oviposition time of  An. gambiae s.l.  may be regulated by the light-dark cycle rather than oviposition habitat characteristics or feeding times. However, the number of eggs laid during the peak oviposition time is affected by the suitability of the habitat. This suggests that there is a relationship between the investment made by the female mosquito with respect to the number of eggs laid in a given habitat and the potential fitness of the progeny. Females may use a series of site characteristics, including olfactory cues, to locate and oviposit at such sites. Our results on oviposition patterns differ from those reported on a coastal population, and suggest that a lot more work needs to be done to elucidate differences in this regard between different populations.
Conclusions
This study shows that the peak oviposition time of  An. gambiae s.l.  may be regulated by the light-dark cycle rather than oviposition habitat characteristics or feeding times. However, the number of eggs laid during the peak oviposition time is affected by the suitability of the habitat. This suggests that there is a relationship between the investment made by the female mosquito with respect to the number of eggs laid in a given habitat and the potential fitness of the progeny. Females may use a series of site characteristics, including olfactory cues, to locate and oviposit at such sites. Our results on oviposition patterns differ from those reported on a coastal population, and suggest that a lot more work needs to be done to elucidate differences in this regard between different populations.An. gambiae s.l.Competing interests The authors declare that they have no competing interests.
Competing interests
The authors declare that they have no competing interests.Authors' contributions LAS and KO conducted all the experimental work. AH, ALD, BGJK, JCB and JG co-ordinated and/or supervised the work. All authors actively contributed to the interpretation of the findings and development of the final manuscript and approved the final manuscript.
Authors' contributions
LAS and KO conducted all the experimental work. AH, ALD, BGJK, JCB and JG co-ordinated and/or supervised the work. All authors actively contributed to the interpretation of the findings and development of the final manuscript and approved the final manuscript.


In the present article we review the main published data on the application of Tissue Doppler Imaging (TDI) to stress echocardiography for the detection of myocardial ischemia. TDI has been applied to stress echocardiography in order to overcome the limitations of visual analysis for myocardial ischemia. The introduction of a new technology for clinical routine use should pass through the different phases of scientific assessment from feasibility studies to large multicenter studies, from efficacy to effectiveness studies. Nonetheless the pro-technology bias plays a major role in medicine and expensive and sophisticated techniques are accepted before their real usefulness and incremental value to the available ones is assessed. Apparently, TDI is not exempted by this approach : its applications are not substantiated by strong and sound results. Nonetheless, conventional stress echocardiography for myocardial ischemia detection is heavily criticized on the basis of its subjectivity. Stress echocardiography has a long lasting history and the evidence collected over 20 years positioned it as an established tool for the detection and prognostication of coronary artery disease. The quantitative assessment of myocardial ischemia remains a scientific challenge and a clinical goal but time has not come for these newer ultrasonographic techniques which should be restricted to research laboratories.

Pharmacologic stress echocardiography is an established cost-effective technique for the detection of coronary artery disease [ 1 ]. The widespread use in the clinical practice has become possible only after evidence collected through large scale multicenter studies that demonstrated its feasibility, safety, diagnostic and prognostic accuracy [ 4 - 8 ]. According to the guidelines of ACC/AHA – pharmacological stress echocardiography with either dobutamine or dipyridamole is a class I indication (of documented effectiveness and usefulness) for the diagnosis of coronary artery disease and for the prognostic stratification of patients with known coronary artery disease [ 2 , 3 ]. Its major limitation is related to a high inter-observer variability and to operator-dependent expertise that might be overcome by an appropriate training and the use of strict reading criteria [ 9 - 11 ]. Nonetheless the hunt for an objective, operator-independent technique to be applied to the conventional black and white regional wall motion analysis remains a major goal in stress echocardiography. Tissue Doppler Imaging (TDI) provides a quantitative analysis of regional myocardial function through the analysis of myocardial velocities [ 12 , 13 ]. Since velocity imaging is confounded by influence from velocities in other segments, the TDI – based modalities strain and strain rate imaging have been introduced to measure regional shortening fraction and shortening rate, respectively [ 14 ] Is the application of Tissue Doppler Imaging to stress echocardiography the technique that will solve it all? According to major journals the answer is yes: the diagnostic accuracy of stress echocardiography improves with TDI when analyzed in comparison with visual assessment of wall motion analysis for the detection of inducible ischemia. Inducible ischemia quantified in a number without the approximations of visual assessment. However the enthusiasm showed by some investigators is not substantiated by scientific results. In fact, a careful analysis of the data published so far raises more doubts than certainties.

Pharmacologic stress echocardiography is an established cost-effective technique for the detection of coronary artery disease [ 1 ]. The widespread use in the clinical practice has become possible only after evidence collected through large scale multicenter studies that demonstrated its feasibility, safety, diagnostic and prognostic accuracy [ 4 - 8 ]. According to the guidelines of ACC/AHA – pharmacological stress echocardiography with either dobutamine or dipyridamole is a class I indication (of documented effectiveness and usefulness) for the diagnosis of coronary artery disease and for the prognostic stratification of patients with known coronary artery disease [ 2 , 3 ]. Its major limitation is related to a high inter-observer variability and to operator-dependent expertise that might be overcome by an appropriate training and the use of strict reading criteria [ 9 - 11 ]. Nonetheless the hunt for an objective, operator-independent technique to be applied to the conventional black and white regional wall motion analysis remains a major goal in stress echocardiography. Tissue Doppler Imaging (TDI) provides a quantitative analysis of regional myocardial function through the analysis of myocardial velocities [ 12 , 13 ]. Since velocity imaging is confounded by influence from velocities in other segments, the TDI – based modalities strain and strain rate imaging have been introduced to measure regional shortening fraction and shortening rate, respectively [ 14 ] Is the application of Tissue Doppler Imaging to stress echocardiography the technique that will solve it all? According to major journals the answer is yes: the diagnostic accuracy of stress echocardiography improves with TDI when analyzed in comparison with visual assessment of wall motion analysis for the detection of inducible ischemia. Inducible ischemia quantified in a number without the approximations of visual assessment. However the enthusiasm showed by some investigators is not substantiated by scientific results. In fact, a careful analysis of the data published so far raises more doubts than certainties.What we talk about when we talk about TDI The TDI modalities include myocardial velocity imaging, displacement imaging, strain rate imaging and strain imaging. TDI measures velocities by the Doppler shift of reflected ultrasound. Velocities are measured in the conventional imaging planes, from apical views as longitudinal velocities and from parasternal views as radial velocities. When we employ TDI, the velocities within a myocardial segment are the net result of motion caused by contractions in that segment, motion due to tethering to other segments, and overall motion of the heart. This tethering effect is the reason why longitudinal velocities increase progressively from the apex toward the base, when measured in an apical window. Therefore ischemia in the apical region reduces myocardial velocities not only in the apex, but also in the nonischemic basal segments [ 15 ]. In practical terms, the reduction of TDI velocities in basal segments is not synonymous of reduction of function in the same segments. The opposite effect, tethering of nonischemic segments might induce increase in velocity of adjacent ischemic segments. These limitations could be overcome by the employment of strain and strain rate. Strain rate reflects how fast regional myocardial shortening or lengthening occurs measured at two locations separated by a distance. This is the reason why some authors define strain rate as a spatial velocity gradient. Strain is calculated as the time integral of strain rate and is a dimensionless quantity. The limitations of TDI have been widely outlined [ 16 - 18 ] and this is beyond the scope of the present review but they may be synthesized into two main problems: 1 – the amplitude of the estimated velocity is dependent on the angle at which the region is imaged; 2 – the overall cardiac motion, rotation, and contraction of adjacent segments will influence regional velocity estimates. Therefore, a more critical approach to this technology would have avoided the inconsistencies of scientific results when it was applied in the clinical arena.
What we talk about when we talk about TDI
The TDI modalities include myocardial velocity imaging, displacement imaging, strain rate imaging and strain imaging. TDI measures velocities by the Doppler shift of reflected ultrasound. Velocities are measured in the conventional imaging planes, from apical views as longitudinal velocities and from parasternal views as radial velocities. When we employ TDI, the velocities within a myocardial segment are the net result of motion caused by contractions in that segment, motion due to tethering to other segments, and overall motion of the heart. This tethering effect is the reason why longitudinal velocities increase progressively from the apex toward the base, when measured in an apical window. Therefore ischemia in the apical region reduces myocardial velocities not only in the apex, but also in the nonischemic basal segments [ 15 ]. In practical terms, the reduction of TDI velocities in basal segments is not synonymous of reduction of function in the same segments. The opposite effect, tethering of nonischemic segments might induce increase in velocity of adjacent ischemic segments. These limitations could be overcome by the employment of strain and strain rate. Strain rate reflects how fast regional myocardial shortening or lengthening occurs measured at two locations separated by a distance. This is the reason why some authors define strain rate as a spatial velocity gradient. Strain is calculated as the time integral of strain rate and is a dimensionless quantity. The limitations of TDI have been widely outlined [ 16 - 18 ] and this is beyond the scope of the present review but they may be synthesized into two main problems: 1 – the amplitude of the estimated velocity is dependent on the angle at which the region is imaged; 2 – the overall cardiac motion, rotation, and contraction of adjacent segments will influence regional velocity estimates. Therefore, a more critical approach to this technology would have avoided the inconsistencies of scientific results when it was applied in the clinical arena.TDI and Stress echocardiography for myocardial ischemia detection Feasibility studies have been published demonstrating the applicability of TDI to stress echocardiography [ 19 - 27 ] but only few studies addressed the issue of its diagnostic accuracy in a clinical context [ 28 - 31 ]. Cain et al [ 28 ] applied myocardial Doppler velocity to dobutamine stress echocardiography in order to assess its diagnostic accuracy when compared to conventional visual assessment. They first identified the normality ranges of myocardial velocities in patients with normal coronary arteries or with a very low pretest probability of having coronary artery disease. Then they selected 114 patient with coronary artery disease assessed at coronary angiography and evaluated the diagnostic accuracy: see Table 1 ( Additional file 1 ). Neither overall nor vascular territory accuracy was better for myocardial velocity when compared to visual wall motion scoring. The MYDISE Study [ 29 ] was the first multicenter study on the absolute value of TDI applied to dobutamine stress echocardiography. The study enrolled 289 patients separated in 3 groups: group 1 (n = 92) healthy volunteers or patients with normal coronary arteries, group 2 (n = 48) patients with known coronary artery disease and group 3 (n = 149) consecutive patients with known or suspected coronary artery disease. Exclusion criteria were: atrial fibrillation, previous myocardial infarction (Q waves on the electrocardiogram, or akinetic segments on the resting echocardiographic images), previous revascularization, unstable angina, complete bundle branch block, significant heart valve disease, contraindication to dobutamine or atropine). The diagnostic criteria were developed by comparing 92 normal subjects with 48 patients with coronary artery disease and applied in a prospective series of 149 patients referred to stress echo laboratory for the suspect of coronary artery disease. Velocity cut-off points were tested and discarded since they did nor perform well when compared to logistic regression models, using systolic velocities at peak stress in 7 myocardial segments and after adjusting for heart rate, age and gender [ 29 ]. The main concerns refer to strict stress echocardiographic issues:1.the lack of a comparison between conventional visual assessment of regional wall motion and TDI analysis. In absolute terms the diagnostic accuracy is not striking: see Table 1 ( Additional file 1 ) and Fig  1 . Even if we accept the hypothesis of a non-inferiority analysis of TDI versus dobutamine stress echocardiography we have to take into consideration some major limitations outlined by the authors: the optimal diagnostic accuracy was obtained by using peak systolic velocity after adjusting for maximal heart rate, age and gender: "ignoring these factors reduces both sensitivity and specificity" [ 29 ]. Moreover, authors applied a very complex regression model for diagnostic accuracy assessment. A recent meta-analysis on dobutamine stress echocardiography showed an overall sensitivity of 80% and a specificity of 87% [ 32 ] (see fig  2 ). 2 – The extent and severity of myocardial ischemia as defined by the number of ischemic segments and the pharmacologic load is never provided. The protocol was interrupted only in the presence of secondary criteria, but never for development of myocardial ischemia since the quantitative analysis was performed off-line. It has been demonstrated that diagnostic and prognostic accuracy of stress echocardiography increases when the response is stratified in the time and space domain, i.e. number of ischemic segments, severity of ischemia induced, the time of onset of ischemia and the pharmacologic dose. 3 – the apical segments have been excluded by the analysis since the systolic velocity is not reliable making the analysis possible only in 11 segments. Nonetheless, the apex and the apical segments are very often the site of stress echocardiographic positivity unless very proximal atherosclerotic lesions are present 4 – the time for performing analysis is never reported. We are informed that the comparison between systolic velocities at rest and at peak stress is disregarded since it is time consuming and increases the potential for observer variability without increasing diagnostic accuracy. 5 -, apparently, TDI cannot be applied to patients with wall motion abnormalities at rest. The exclusion of this group of patients makes this quantitative approach quite unfeasible for routine clinical application. Figure 2 Sensitivity and specificity of dobutamine stress echocardiography Voigt et al. [ 30 ] used a more realistic approach to the application of TDI to stress echocardiography. They first demonstrated in 44 patients with known or suspected coronary artery disease that strain rate quantitatively differentiates ischemic and nonischemic regional myocardial response to dobutamine stress echocardiography [ 30 ] and compared it with conventional visual assessment. The ratio of postsystolic shortening to maximal strain was the best quantitative parameter to identify dobutamine stress induced-ischemia. This quantitative analysis improved sensitivity from 81 (visual assessment) to 86% and specificity from 82% to 89%. The statistical significance is not provided in the manuscript. Then, in the same population of 44 [ 31 ], they compared the visual assessment of wall motion abnormalities with different parameters derived from TDI application such as peak systolic velocity, systolic displacement and strain rate imaging. They employed simultaneous perfusion scintigraphy as a gold standard of myocardial ischemia. The stress echocardiographic methodology employed was not a standard one for segmentation of the left ventricle (18 segments instead of 16 or 17), pharmacologic protocol (up to 2 mg atropine instead of 1 mg) and criteria for ischemia (worsening of wall motion only in 1 segment). Also in this case, the overall accuracy is not striking: Table 1 ( Additional file 1 ). On the basis of these results TDI reduces significantly the diagnostic accuracy of dobutamine stress echocardiography whereas strain rate imaging equals the diagnostic accuracy but it does not improve it. Interestingly enough, the sensitivities and specificities of strain rate imaging are slightly different in the two papers even though the analysis was conducted in the very same set of patients. Moreover, since coronary angiography was performed in all patients, the diagnostic accuracy should have been calculated on this real gold standard instead of perfusion scintigraphy. In a recent [ 33 ] article Marwick et al. questioned the hypothesis that false-negative results of dobutamine stress echocardiography reflect the underinterpretation of regional left ventricular function. On the opposite, the quantitative parameters such as strain rate and peak systolic strain rate were no different between false and true negative tests, suggesting that false-negative results are related to lack of ischemia in a functional sense. On the basis of this observation, quantitative markers are unlikely to increase the sensitivity of dobutamine stress echocardiography.
TDI and Stress echocardiography for myocardial ischemia detection
Feasibility studies have been published demonstrating the applicability of TDI to stress echocardiography [ 19 - 27 ] but only few studies addressed the issue of its diagnostic accuracy in a clinical context [ 28 - 31 ].Cain et al [ 28 ] applied myocardial Doppler velocity to dobutamine stress echocardiography in order to assess its diagnostic accuracy when compared to conventional visual assessment. They first identified the normality ranges of myocardial velocities in patients with normal coronary arteries or with a very low pretest probability of having coronary artery disease. Then they selected 114 patient with coronary artery disease assessed at coronary angiography and evaluated the diagnostic accuracy: see Table 1 ( Additional file 1 ). Neither overall nor vascular territory accuracy was better for myocardial velocity when compared to visual wall motion scoring. The MYDISE Study [ 29 ] was the first multicenter study on the absolute value of TDI applied to dobutamine stress echocardiography. The study enrolled 289 patients separated in 3 groups: group 1 (n = 92) healthy volunteers or patients with normal coronary arteries, group 2 (n = 48) patients with known coronary artery disease and group 3 (n = 149) consecutive patients with known or suspected coronary artery disease. Exclusion criteria were: atrial fibrillation, previous myocardial infarction (Q waves on the electrocardiogram, or akinetic segments on the resting echocardiographic images), previous revascularization, unstable angina, complete bundle branch block, significant heart valve disease, contraindication to dobutamine or atropine). The diagnostic criteria were developed by comparing 92 normal subjects with 48 patients with coronary artery disease and applied in a prospective series of 149 patients referred to stress echo laboratory for the suspect of coronary artery disease. Velocity cut-off points were tested and discarded since they did nor perform well when compared to logistic regression models, using systolic velocities at peak stress in 7 myocardial segments and after adjusting for heart rate, age and gender [ 29 ].The main concerns refer to strict stress echocardiographic issues:1.the lack of a comparison between conventional visual assessment of regional wall motion and TDI analysis. In absolute terms the diagnostic accuracy is not striking: see Table 1 ( Additional file 1 ) and Fig  1 . Even if we accept the hypothesis of a non-inferiority analysis of TDI versus dobutamine stress echocardiography we have to take into consideration some major limitations outlined by the authors: the optimal diagnostic accuracy was obtained by using peak systolic velocity after adjusting for maximal heart rate, age and gender: "ignoring these factors reduces both sensitivity and specificity" [ 29 ]. Moreover, authors applied a very complex regression model for diagnostic accuracy assessment. A recent meta-analysis on dobutamine stress echocardiography showed an overall sensitivity of 80% and a specificity of 87% [ 32 ] (see fig  2 ). 2 – The extent and severity of myocardial ischemia as defined by the number of ischemic segments and the pharmacologic load is never provided. The protocol was interrupted only in the presence of secondary criteria, but never for development of myocardial ischemia since the quantitative analysis was performed off-line. It has been demonstrated that diagnostic and prognostic accuracy of stress echocardiography increases when the response is stratified in the time and space domain, i.e. number of ischemic segments, severity of ischemia induced, the time of onset of ischemia and the pharmacologic dose. 3 – the apical segments have been excluded by the analysis since the systolic velocity is not reliable making the analysis possible only in 11 segments. Nonetheless, the apex and the apical segments are very often the site of stress echocardiographic positivity unless very proximal atherosclerotic lesions are present 4 – the time for performing analysis is never reported. We are informed that the comparison between systolic velocities at rest and at peak stress is disregarded since it is time consuming and increases the potential for observer variability without increasing diagnostic accuracy. 5 -, apparently, TDI cannot be applied to patients with wall motion abnormalities at rest. The exclusion of this group of patients makes this quantitative approach quite unfeasible for routine clinical application.Voigt et al. [ 30 ] used a more realistic approach to the application of TDI to stress echocardiography. They first demonstrated in 44 patients with known or suspected coronary artery disease that strain rate quantitatively differentiates ischemic and nonischemic regional myocardial response to dobutamine stress echocardiography [ 30 ] and compared it with conventional visual assessment. The ratio of postsystolic shortening to maximal strain was the best quantitative parameter to identify dobutamine stress induced-ischemia. This quantitative analysis improved sensitivity from 81 (visual assessment) to 86% and specificity from 82% to 89%. The statistical significance is not provided in the manuscript. Then, in the same population of 44 [ 31 ], they compared the visual assessment of wall motion abnormalities with different parameters derived from TDI application such as peak systolic velocity, systolic displacement and strain rate imaging. They employed simultaneous perfusion scintigraphy as a gold standard of myocardial ischemia. The stress echocardiographic methodology employed was not a standard one for segmentation of the left ventricle (18 segments instead of 16 or 17), pharmacologic protocol (up to 2 mg atropine instead of 1 mg) and criteria for ischemia (worsening of wall motion only in 1 segment). Also in this case, the overall accuracy is not striking: Table 1 ( Additional file 1 ). On the basis of these results TDI reduces significantly the diagnostic accuracy of dobutamine stress echocardiography whereas strain rate imaging equals the diagnostic accuracy but it does not improve it. Interestingly enough, the sensitivities and specificities of strain rate imaging are slightly different in the two papers even though the analysis was conducted in the very same set of patients. Moreover, since coronary angiography was performed in all patients, the diagnostic accuracy should have been calculated on this real gold standard instead of perfusion scintigraphy.In a recent [ 33 ] article Marwick et al. questioned the hypothesis that false-negative results of dobutamine stress echocardiography reflect the underinterpretation of regional left ventricular function. On the opposite, the quantitative parameters such as strain rate and peak systolic strain rate were no different between false and true negative tests, suggesting that false-negative results are related to lack of ischemia in a functional sense. On the basis of this observation, quantitative markers are unlikely to increase the sensitivity of dobutamine stress echocardiography.Conclusions The quantitative interpretation of stress echocardiography is not superior to expert wall motion assessment. Open issues in the quantitative analysis remain at stake: which technique to be employed among systolic velocities, strain and strain rate, the assessment of normality criteria of myocardial velocities and how to interpret their values, the management of patients with regional and global left ventricular dysfunction, the analysis of the apical segments, the complexity of the analysis in a real clinical environment, the applicability to unselected populations, its unsuitability to exercise, the most widely used stressor in the clinical practice [ 34 ]. What is presented as a breakthrough technology should have already answered to these issues and when exported into the clinical arena should have provided an incremental value to the established and more easily accessible methods. It is at this point that we are lost in clinical translation: authoritative journals provide data that cannot be transferred into the daily life of a busy stress echocardiographic laboratory, although the general message is optimistic and tend to ignore flaws and limitations of the technique [ 35 , 36 ]). The advantage/disadvantage balance of a new technology should clearly be stated. The potential advantages should always outweigh the disadvantages related to the higher costs and higher complexity of analysis. Perhaps, the shape of the quantitative technology to come has not been designed yet [ 37 , 38 ]. TDI is one of the tools in our hands but apparently this is not its time. At least not on the basis of this evidence. Figure 1 In the left panel sensitivities and specificities in the three main vascular districts and overall sensitivities and specificities without correcting results for age, gender and heart rate. In the right panel, the same data corrected for age, gender and heart rate (modified from ref.29).
Conclusions
The quantitative interpretation of stress echocardiography is not superior to expert wall motion assessment. Open issues in the quantitative analysis remain at stake: which technique to be employed among systolic velocities, strain and strain rate, the assessment of normality criteria of myocardial velocities and how to interpret their values, the management of patients with regional and global left ventricular dysfunction, the analysis of the apical segments, the complexity of the analysis in a real clinical environment, the applicability to unselected populations, its unsuitability to exercise, the most widely used stressor in the clinical practice [ 34 ]. What is presented as a breakthrough technology should have already answered to these issues and when exported into the clinical arena should have provided an incremental value to the established and more easily accessible methods. It is at this point that we are lost in clinical translation: authoritative journals provide data that cannot be transferred into the daily life of a busy stress echocardiographic laboratory, although the general message is optimistic and tend to ignore flaws and limitations of the technique [ 35 , 36 ]). The advantage/disadvantage balance of a new technology should clearly be stated. The potential advantages should always outweigh the disadvantages related to the higher costs and higher complexity of analysis. Perhaps, the shape of the quantitative technology to come has not been designed yet [ 37 , 38 ]. TDI is one of the tools in our hands but apparently this is not its time. At least not on the basis of this evidence.Supplementary Material Additional File 1 Table 1.TDI vs visual assessment of myocardial ischemia during dobutamine stress echocardiography. Click here for file
Supplementary Material
Additional File 1
Table 1.TDI vs visual assessment of myocardial ischemia during dobutamine stress echocardiography.Click here for file


Background Our objective was to study tongue lesions and their significance in psoriatic patients.
Background
Our objective was to study tongue lesions and their significance in psoriatic patients.Methods The oral mucosa was examined in 200 psoriatic patients presenting to Razi Hospital in Tehran, Iran, and 200 matched controls.
Methods
The oral mucosa was examined in 200 psoriatic patients presenting to Razi Hospital in Tehran, Iran, and 200 matched controls.Results Fissured tongue (FT) and benign migratory glossitis (BMG) were the two most frequent findings. FT was seen more frequently in psoriatic patients (n = 66, 33%) than the control group (n = 19, 9.5%) [odds ratio (OR): 4.69; 95% confidence interval (CI): 2.61–8.52] (p-value < 0.0001). BMG, too, was significantly more frequent in psoriatic patients (28 cases, 14%) than the control group (12 cases, 6%) (OR: 2.55; 95% CI: 1.20–5.50) (p-value < 0.012). In 11 patients (5.5%), FT and BMG coexisted. FT was more frequent in pustular psoriasis (7 cases, 53.8%) than erythemato-squamous types (56 cases, 30.4%). On the other hand, the frequency of BMG increased with the severity of psoriasis in plaque-type psoriasis assessed by psoriasis area and severity index (PASI) score.
Results
Fissured tongue (FT) and benign migratory glossitis (BMG) were the two most frequent findings. FT was seen more frequently in psoriatic patients (n = 66, 33%) than the control group (n = 19, 9.5%) [odds ratio (OR): 4.69; 95% confidence interval (CI): 2.61–8.52] (p-value < 0.0001). BMG, too, was significantly more frequent in psoriatic patients (28 cases, 14%) than the control group (12 cases, 6%) (OR: 2.55; 95% CI: 1.20–5.50) (p-value < 0.012). In 11 patients (5.5%), FT and BMG coexisted.FT was more frequent in pustular psoriasis (7 cases, 53.8%) than erythemato-squamous types (56 cases, 30.4%). On the other hand, the frequency of BMG increased with the severity of psoriasis in plaque-type psoriasis assessed by psoriasis area and severity index (PASI) score.Conclusions Nonspecific tongue lesions are frequently observed in psoriasis. Further studies are recommended to substantiate the clinical significance of these seemingly nonspecific findings in suspected psoriatic cases.
Conclusions
Nonspecific tongue lesions are frequently observed in psoriasis. Further studies are recommended to substantiate the clinical significance of these seemingly nonspecific findings in suspected psoriatic cases.

Background The occurrence of psoriatic lesions on oral mucous membranes was a subject of controversy [ 1 , 2 ]. Some investigators stated that they do not occur [ 3 ]; others, have claimed that they are uncommon. Still others say that they occur only in generalized pustular psoriasis (GPP) [ 4 , 5 ]. Nowadays, there is sufficient evidence that a subset of patients have oral lesions in association with skin disease [ 2 ]. Oppenheim, in 1903, was the first to substantiate oral psoriasis with biopsy [ 6 ]. Since then, various lesions have been described, including grey, yellowish, white or translucent plaques or annular forms, diffuse areas of erythema, geographic tongue and fissured tongue [ 7 - 16 ]. In all the cases reported in the literature, a positive biopsy showing a psoriasiform pattern has been the crucial component of the diagnosis [ 5 , 17 - 20 ]. Thus hyperkeratosis, parakeratosis, and an inflammatory infiltrate consisting of lymphocytes, polymorphonuclear leukocytes and histiocytes have been noted as well as Munro's microabscesses and spongiform pustules of Kogoj. In addition, many investigators believe that the presence of cutaneous lesions with a course parallel to that of oral lesions is necessary for establishing the diagnosis of oral psoriasis [ 2 , 5 , 20 ]. However, it is impossible to perform an oral biopsy in psoriatic cases in everyday clinical practice. On the other hand, some of the lesions seen more frequently in psoriatic patients are not specific histologically. In fact, similar changes are seen in otherwise healthy people (although with a lower frequency) leading to an underestimation of the value of these findings in psoriatic patients. In order to substantiate further the relationship between these oral disorders and psoriasis, we compared 200 patients with psoriasis to a matched control group.
Background
The occurrence of psoriatic lesions on oral mucous membranes was a subject of controversy [ 1 , 2 ]. Some investigators stated that they do not occur [ 3 ]; others, have claimed that they are uncommon. Still others say that they occur only in generalized pustular psoriasis (GPP) [ 4 , 5 ]. Nowadays, there is sufficient evidence that a subset of patients have oral lesions in association with skin disease [ 2 ].Oppenheim, in 1903, was the first to substantiate oral psoriasis with biopsy [ 6 ]. Since then, various lesions have been described, including grey, yellowish, white or translucent plaques or annular forms, diffuse areas of erythema, geographic tongue and fissured tongue [ 7 - 16 ].In all the cases reported in the literature, a positive biopsy showing a psoriasiform pattern has been the crucial component of the diagnosis [ 5 , 17 - 20 ]. Thus hyperkeratosis, parakeratosis, and an inflammatory infiltrate consisting of lymphocytes, polymorphonuclear leukocytes and histiocytes have been noted as well as Munro's microabscesses and spongiform pustules of Kogoj. In addition, many investigators believe that the presence of cutaneous lesions with a course parallel to that of oral lesions is necessary for establishing the diagnosis of oral psoriasis [ 2 , 5 , 20 ].However, it is impossible to perform an oral biopsy in psoriatic cases in everyday clinical practice. On the other hand, some of the lesions seen more frequently in psoriatic patients are not specific histologically. In fact, similar changes are seen in otherwise healthy people (although with a lower frequency) leading to an underestimation of the value of these findings in psoriatic patients.In order to substantiate further the relationship between these oral disorders and psoriasis, we compared 200 patients with psoriasis to a matched control group.Methods Two hundred psoriatic patients (70 women and 130 men) attending the dermatology clinics of Razi Hospital, a major referral center in Tehran, from September 2000 till February 2001, were enrolled in this study using simple nonrandom (sequential) sampling. The diagnosis was made mainly on clinical data. The control group included 200 healthy subjects among the visitors of Surgery wards in a general hospital, matched one by one for age and sex. The skin and oral mucosa were examined in the two groups and, in addition to demographic and clinical data, PASI score [ 21 ] was recorded in plaque-type psoriasis. The data were analyzed by Epi-Info (version 6) software, and frequency, mean, standard deviation, OR and p-value were calculated.
Methods
Two hundred psoriatic patients (70 women and 130 men) attending the dermatology clinics of Razi Hospital, a major referral center in Tehran, from September 2000 till February 2001, were enrolled in this study using simple nonrandom (sequential) sampling. The diagnosis was made mainly on clinical data. The control group included 200 healthy subjects among the visitors of Surgery wards in a general hospital, matched one by one for age and sex. The skin and oral mucosa were examined in the two groups and, in addition to demographic and clinical data, PASI score [ 21 ] was recorded in plaque-type psoriasis. The data were analyzed by Epi-Info (version 6) software, and frequency, mean, standard deviation, OR and p-value were calculated.Results The mean age of the patient group was 33.8+/- 18.2 years (4–79 years). The mean age of onset of disease was 26+/-17.7 years (0–74 years), 23 +/-18.8 years in women and 27.6 +/- 17.0 years in men. Age and sex were matched between patients and control subjects. Family history of psoriasis was positive in 34 patients. Different clinical types of psoriasis were as follows: Chronic plaque-type psoriasis (n = 140); generalized pustular psoriasis (n = 10); flexural psoriasis (n = 10); erythrodermic psoriasis (n = 9); localized pustular psoriasis (n = 3); guttate psoriasis (n = 9); palmoplantar psoriasis (n = 15); scalp (n = 95); nail alone (n = 3). Oral findings were detected in 87 (43.5%) and 39 (19.5%) cases in the psoriatic and control groups, respectively. They are presented in table  1 . FT was seen more frequently in psoriatic patients (66 patients, 33%) than the control group (19, 9.5%) (OR: 4.69; 95% CI: 2.61–8.52) (p-value < 0.0001). BMG, too, was significantly more frequent in psoriatic patients (28 cases, 14%) than the control group (12, 6%) (OR: 2.55; 95% CI: 1.20–5.50) (p-value < 0.012). BMG was seen in 18.2% of patients with FT, and 42.9% of patients with BMG suffered from FT. In other words, in 12 patients (6%) FT and BMG coexisted. In the control group, FT and BMG coexisted in 2 cases (1%). Table 1 The frequency of oral findings in psoriasis patients and control group Psoriasis Control Fissured tongue 66 19 Benign migratory glossitis 28 12 Diffuse oral and tongue erythema 11 3 Hairy tongue 2 4 Rhomboid glossitis 2 3 Depapillated tongue 2 3 One hundred eighty-four patients (92%) suffered from erythemato-squamous lesions and 13 cases (6.5%) from pustular lesions. The frequency of FT in the erythemato-squamous and pustular groups was 30.4% (56 cases) and 53.8% (7 cases), respectively. On the other hand, the frequency of BMG in the erythemato-squamous and pustular groups was 14.1% (26 cases) and 15.4% (2 cases), respectively. The severity of chronic plaque-type psoriasis cases assessed by PASI score was as follows: mild, 53 cases (37.9%); moderate, 60 cases (42.9%); and severe 27 cases (19.3%). The corresponding frequency of FT and BMG in the three severity groups is presented in table  2 . The frequency of BMG increased with the severity of skin lesions (p-value < 0.001). Table 2 Frequency of fissured tongue and benign migratory glossitis according to severity in plaque-type psoriasis Fissured tongue Benign migratory glossitis Mild 14 (26.4%) 3 (5.7%) Moderate 23 (38.3%) 10 (16.7%) Severe 7 (25%) 9 (32.1%)
Results
The mean age of the patient group was 33.8+/- 18.2 years (4–79 years). The mean age of onset of disease was 26+/-17.7 years (0–74 years), 23 +/-18.8 years in women and 27.6 +/- 17.0 years in men. Age and sex were matched between patients and control subjects. Family history of psoriasis was positive in 34 patients. Different clinical types of psoriasis were as follows: Chronic plaque-type psoriasis (n = 140); generalized pustular psoriasis (n = 10); flexural psoriasis (n = 10); erythrodermic psoriasis (n = 9); localized pustular psoriasis (n = 3); guttate psoriasis (n = 9); palmoplantar psoriasis (n = 15); scalp (n = 95); nail alone (n = 3).Oral findings were detected in 87 (43.5%) and 39 (19.5%) cases in the psoriatic and control groups, respectively. They are presented in table  1 . FT was seen more frequently in psoriatic patients (66 patients, 33%) than the control group (19, 9.5%) (OR: 4.69; 95% CI: 2.61–8.52) (p-value < 0.0001). BMG, too, was significantly more frequent in psoriatic patients (28 cases, 14%) than the control group (12, 6%) (OR: 2.55; 95% CI: 1.20–5.50) (p-value < 0.012). BMG was seen in 18.2% of patients with FT, and 42.9% of patients with BMG suffered from FT. In other words, in 12 patients (6%) FT and BMG coexisted. In the control group, FT and BMG coexisted in 2 cases (1%).The frequency of oral findings in psoriasis patients and control groupOne hundred eighty-four patients (92%) suffered from erythemato-squamous lesions and 13 cases (6.5%) from pustular lesions. The frequency of FT in the erythemato-squamous and pustular groups was 30.4% (56 cases) and 53.8% (7 cases), respectively. On the other hand, the frequency of BMG in the erythemato-squamous and pustular groups was 14.1% (26 cases) and 15.4% (2 cases), respectively.The severity of chronic plaque-type psoriasis cases assessed by PASI score was as follows: mild, 53 cases (37.9%); moderate, 60 cases (42.9%); and severe 27 cases (19.3%). The corresponding frequency of FT and BMG in the three severity groups is presented in table  2 . The frequency of BMG increased with the severity of skin lesions (p-value < 0.001).Frequency of fissured tongue and benign migratory glossitis according to severity in plaque-type psoriasisDiscussion In general oral lesions in psoriasis can be divided into two major categories. The first one includes authentic psoriatic lesions proved by biopsy and with a parallel clinical course with skin lesions. It's not known whether these lesions are truly rare, or they remain undetected, as mucosal biopsy is seldom done in known psoriatic cases. The second group comprises the majority of oral findings in psoriasis and includes nonspecific lesions such as FT and psoriasiform lesions such as BMG [ 22 ]. These lesions are underestimated in the literature, but deserve more attention due to their high frequency. We will discuss the main oral findings observed in our study as well as those reported in the literature. Fissured tongue, also termed lingua fissurata, lingua plicata, scrotal tongue, and grooved tongue is recognized clinically by an antero-posteriorly oriented fissure, often with branch fissures extending laterally. It's believed by most authors to be an inherited trait. The frequency of FT increases with age and has been associated with Down's syndrome and the Melkerson-Rosenthal syndrome [ 5 , 20 ]. According to our study, FT was the most common oral finding in the psoriasis group: Nearly one-third of patients suffered from FT. It was significantly more frequent in psoriasis patients than the control group (9.5%) (p-value < 0.0001). The previously reported figures of the frequency of FT in the general population vary markedly in the literature depending on the study design and the target study. Axell reported a figure of 6.5% [ 10 ] and Morris found FT in 20.3% of its target study [ 17 ]. Aboyans et al reported a frequency of 2.56% in Iran [ 23 ]. On the other hand, FT was reported in 6–16.7% of psoriatic patients in different studies [ 3 , 10 , 14 , 15 , 20 ]. BMG or geographic tongue presents clinically as one or more erythematous patches with a raised white or yellow serpiginous border. Lesions may migrate across the tongue by healing on one edge while extending on another. BMG has no known cause, but it has been associated with atopic conditions, diabetes mellitus, reactive bronchitis, anemia, stress [ 20 ], hormonal disturbances, Down's syndrome and lithium therapy [ 24 ]. Lesions identical to BMG have been described in patients with Reiter's syndrome and psoriasis. The association of both psoriasis and BMG with HLA-CW6 provides further evidence that the two disorders are related [ 25 ]. In our study, BMG was significantly more frequent in psoriatic patients (14%) than the control group (6%) (p-value < 0.012). According to the literature, the estimated frequency of BMG in the general population is from 1–5% [ 1 , 10 , 20 , 26 ] and varies from 1–10.3% in psoriatic patients [ 3 , 10 , 13 - 15 , 20 , 26 ]. Only Hietanen found a figure of 1% in psoriasis [ 10 ]. FT was more frequent in patients with pustular lesions compared with the erythemato-squamous types. Contrary to previous studies, this finding was not seen for BMG, a disease generally considered accompanied with GPP [ 27 , 28 ]. This may be due to the low frequency of GPP in our study group. On the other hand, the frequency of BMG increased with the severity of psoriasis in plaque-type disease, a finding not seen in Morris's study [ 20 ], perhaps due to different definition for the severity of the disease. According to our study, the frequency of FT didn't increase by increasing severity of psoriasis. SAM was first described by Cooke in 1955 as an idiopathic inflammatory condition of the nonlingual oral mucosa [ 10 , 15 ]. It is also denoted using different terms: Geographic stomatitis, ectopic geographic tongue, erythema circinate migrans, and migratory stomatitis. These lesions are similar in appearance to BMG, but occur on the oral mucosal surfaces as well as the dorsum of the tongue [ 1 , 29 , 30 ]. As seen in Van der Wal's study, [ 14 ] we didn't find SAM in psoriatic patients. The reported frequency of this oral finding in psoriatic patients in the literature is between 0–19% [ 15 , 20 ]. Furthermore, this lesion seems to be very rare in the general population, too: Bouquot found no patients with SAM in 231616 white American dental patients [ 20 ]. Diffuse oral and tongue erythema was another positive finding in the psoriasis group with a frequency of 5.5%. This lesion, too, was reported previously in the literature, although with a lower frequency (1%) [ 10 ]. An association between FT and BMG is well established in the literature [ 31 , 32 ]. In our study, BMG was seen in 18.2% of patients with FT (results consistent with Pindorf's) [ 14 , 16 ].
Discussion
In general oral lesions in psoriasis can be divided into two major categories. The first one includes authentic psoriatic lesions proved by biopsy and with a parallel clinical course with skin lesions. It's not known whether these lesions are truly rare, or they remain undetected, as mucosal biopsy is seldom done in known psoriatic cases. The second group comprises the majority of oral findings in psoriasis and includes nonspecific lesions such as FT and psoriasiform lesions such as BMG [ 22 ]. These lesions are underestimated in the literature, but deserve more attention due to their high frequency.We will discuss the main oral findings observed in our study as well as those reported in the literature.Fissured tongue, also termed lingua fissurata, lingua plicata, scrotal tongue, and grooved tongue is recognized clinically by an antero-posteriorly oriented fissure, often with branch fissures extending laterally. It's believed by most authors to be an inherited trait. The frequency of FT increases with age and has been associated with Down's syndrome and the Melkerson-Rosenthal syndrome [ 5 , 20 ].According to our study, FT was the most common oral finding in the psoriasis group: Nearly one-third of patients suffered from FT. It was significantly more frequent in psoriasis patients than the control group (9.5%) (p-value < 0.0001). The previously reported figures of the frequency of FT in the general population vary markedly in the literature depending on the study design and the target study. Axell reported a figure of 6.5% [ 10 ] and Morris found FT in 20.3% of its target study [ 17 ]. Aboyans et al reported a frequency of 2.56% in Iran [ 23 ]. On the other hand, FT was reported in 6–16.7% of psoriatic patients in different studies [ 3 , 10 , 14 , 15 , 20 ].BMG or geographic tongue presents clinically as one or more erythematous patches with a raised white or yellow serpiginous border. Lesions may migrate across the tongue by healing on one edge while extending on another. BMG has no known cause, but it has been associated with atopic conditions, diabetes mellitus, reactive bronchitis, anemia, stress [ 20 ], hormonal disturbances, Down's syndrome and lithium therapy [ 24 ]. Lesions identical to BMG have been described in patients with Reiter's syndrome and psoriasis. The association of both psoriasis and BMG with HLA-CW6 provides further evidence that the two disorders are related [ 25 ].In our study, BMG was significantly more frequent in psoriatic patients (14%) than the control group (6%) (p-value < 0.012). According to the literature, the estimated frequency of BMG in the general population is from 1–5% [ 1 , 10 , 20 , 26 ] and varies from 1–10.3% in psoriatic patients [ 3 , 10 , 13 - 15 , 20 , 26 ]. Only Hietanen found a figure of 1% in psoriasis [ 10 ].FT was more frequent in patients with pustular lesions compared with the erythemato-squamous types. Contrary to previous studies, this finding was not seen for BMG, a disease generally considered accompanied with GPP [ 27 , 28 ]. This may be due to the low frequency of GPP in our study group. On the other hand, the frequency of BMG increased with the severity of psoriasis in plaque-type disease, a finding not seen in Morris's study [ 20 ], perhaps due to different definition for the severity of the disease. According to our study, the frequency of FT didn't increase by increasing severity of psoriasis.SAM was first described by Cooke in 1955 as an idiopathic inflammatory condition of the nonlingual oral mucosa [ 10 , 15 ]. It is also denoted using different terms: Geographic stomatitis, ectopic geographic tongue, erythema circinate migrans, and migratory stomatitis. These lesions are similar in appearance to BMG, but occur on the oral mucosal surfaces as well as the dorsum of the tongue [ 1 , 29 , 30 ].As seen in Van der Wal's study, [ 14 ] we didn't find SAM in psoriatic patients. The reported frequency of this oral finding in psoriatic patients in the literature is between 0–19% [ 15 , 20 ]. Furthermore, this lesion seems to be very rare in the general population, too: Bouquot found no patients with SAM in 231616 white American dental patients [ 20 ].Diffuse oral and tongue erythema was another positive finding in the psoriasis group with a frequency of 5.5%. This lesion, too, was reported previously in the literature, although with a lower frequency (1%) [ 10 ].An association between FT and BMG is well established in the literature [ 31 , 32 ]. In our study, BMG was seen in 18.2% of patients with FT (results consistent with Pindorf's) [ 14 , 16 ].Conclusions Overall, although oral lesions might not be considered authentic oral psoriasis unless proven histologically and with a parallel clinical course, nonspecific tongue lesions are significantly more frequent in psoriatic cases. Further studies are recommended to evaluate the clinical significance of these seemingly nonspecific lesions in a suspected psoriatic case. Furthermore, more thorough studies are recommended regarding the relationship of oral psoriasis and disease severity in plaque-type psoriasis.
Conclusions
Overall, although oral lesions might not be considered authentic oral psoriasis unless proven histologically and with a parallel clinical course, nonspecific tongue lesions are significantly more frequent in psoriatic cases. Further studies are recommended to evaluate the clinical significance of these seemingly nonspecific lesions in a suspected psoriatic case. Furthermore, more thorough studies are recommended regarding the relationship of oral psoriasis and disease severity in plaque-type psoriasis.Competing interests The author(s) declare that they have no competing interests.
Competing interests
The author(s) declare that they have no competing interests.Authors' contributions All authors contributed equally in the study design, literature search, data analysis and manuscript preparation. All authors read and approved the final manuscript.
Authors' contributions
All authors contributed equally in the study design, literature search, data analysis and manuscript preparation. All authors read and approved the final manuscript.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: