La lithiase de l’enfant au Maroc : composition de 432 calculs urinaires analysés par spectrophotométrie infrarouge

25 mars 2019

Auteurs : F. Meiouet, S. El Kabbaj, M. Daudon
Référence : Prog Urol, 2019, 3, 29, 173-182




 




Introduction


In a number of countries, adults are more prone than children to urinary calculi. The prevalence of this condition is inversely related to the socio-economic level of the populations [1, 2]. In industrialized countries, pediatric urolithiasis and especially bladder stones have considerably diminished in frequency during the XXth century compared to what has been observed before [2]. However, in recent years, some reports suggest an increased prevalence of urolithiasis in children [3, 4].


The childhood urolithiasis requires an accurate etiologic investigation and an appropriate therapeutic strategy. Especially, it is highly important to early identify hereditary diseases such as primary hyperoxaluria, cystinuria, hypercalciuria or primitive tubular acidosis in order to prevent an evolution towards nephrocalcinosis and renal insufficiency.


Our goal was to investigate the epidemiologic profile of pediatric stones collected from the whole of the Moroccan territory, and to discuss the main etiologic factors suggested by stone composition and morphology.


Material and methods


Among 9400 urinary stones analyzed in the Laboratoire de Recherches et d'Analyses Médicales de la Gendarmerie Royale between 1999 and 2016, 432 (4.6%) came from children aged less than 18 years. Among them, 130 (30.1%) came from girls and 302 (69.9%) from boys. Each calculus was checked for its morphological characteristics using a stereomicroscope to define the morphological type according to the previously reported classification [5, 6] and to select the representative parts of the stone (core, inner layers and surface) for specific infrared analysis. Then, a fraction of 2mg of each calculus sample selected under the stereomicroscope was pulverized and crushed into 200mg of pure and dry potassium bromide for IR spectroscopy analysis [7]. The mixture was transformed into a transparent pellet of 13mm in diameter using a press under a pressure of 10 tons/cm2 as previously described [7]. The pellet was then placed in a special holder and inserted into the infrared spectrophotometer for analysis. The instrument used was an Avatar 360 Fourier transform infrared spectrometer from Thermo-Nicolet. The infrared spectra were recorded on the wavelength range from 2.5 to 25μm (4000-400cm−1) with a resolution of 4cm−1. Air was used as the reference. The resulting spectra were identified by comparison with available reference spectra [7, 8].


Thereafter, in order to assess the total composition of the stone, each calculus was pulverized in its totality and a fraction of the powder was again used to prepare a pellet to be analyzed. Results are expressed as percentages or as mean±SD when appropriate. Statistical analysis of the data was performed using SPSS 17.0 software for windows. Analysis of variance or chi-squared test were used when appropriate: P values less than 0.05 were considered significant.


Results


The patient's age ranged from 3 months to 17 years among boys with an average age of 7.3±4.4 years and from 1 to 17 years in girls with an average age of 8.7±4.6 years. A strong male prevalence was observed for the age class less than 3 years (Figure 1). Thereafter, the male-to-female (M/F) ratio was constantly decreasing down to 1 in teenagers. The mean M/F ratio for the whole series was 2.32.


Figure 1
Figure 1. 

Change in the sex rario M/F according to the patient's age.




Location of calculi


The anatomical localization was specified for 330 stones (76% of cases). As shown in Table 1, the stones were found within the kidney in 36.4% of cases, in the ureter in 10.3% and in the bladder in 15.2%. More than 38% of the stones were spontaneously passed.


The mean age (± SD) of patients was 8.3±4.4 years for kidney stones, 9.0±4.4 years for ureter stones, 6.8±3.9 years for bladder stones (P <0.05 vs kidney stones; P =0.013 vs ureter stones) and 6.2±4.5 years for spontaneously passed stones (P <0.001 vs kidney or ureter stones). We noted a similar average age of 7.7±4.5 years for the two genders. No statistical difference was found between girls and boys regarding stone location.


Chemical composition of stones


Calcium oxalate was the main component of the stones (51.6%). It was followed by magnesium ammonium phosphate hexahydrate (MAP) or struvite (18.1%), ammonium urate (AmUr, 9.5%) and carbapatite (9.0%). A strong difference in the distribution of the main components between the two genders was observed only for two crystalline phases, namely calcium oxalate monohydrate (COM) and struvite (Table 2). The former was predominantly found in girls (66.9% vs 33.1% in boys, P <10−6). By contrast, struvite was more frequent in boys (22.2% vs 8.5% in girls, P <10−3). Carbapatite was the main component in 4.6% of cases in girls vs 10.9% in boys (P <0.05) while the frequency of AmUr stones was similar in both genders.


Calcium oxalate dihydrate (COD) was 5 times less frequent than COM. Among other components, cystine accounted for 5.3% of stones. Uric acid was relatively infrequent (3.5% of the cases). Xanthine stones accounted only for 0.5% of cases. Finally, drug-induced urolithiasis, made of ceftriaxone calcium salt was found in three cases.


Influence of age


As shown in Table 3, Table 4, stone composition changed in boys and girls in relation to the patient's age. In boys (Table 3), AmUr and calcium phosphates (CaP) were more prevalent in infants while both COM and COD were increasing with age. In girls (Table 4), COM was the unique crystalline species increasing with patient's age up to the age class 10≤15 years. In patients aged more than 10 years old, 83.4% of stones were mainly formed of COM.


Occurrence of stone components


In Table 5, are reported all the components identified among our 432 stones and their frequency. As expected, the commonest crystalline phase was COM (64.4%), followed by carbapatite (40.5%) and by AmUr (40.0%). The other components found in more than 10% of cases were proteins (34.3%), MAP (26.2%) and COD (22.5%). Significant differences were found regarding the frequency of some components between genders. For example, calcium oxalate was significantly more frequent in girls than in boys (80.8 vs 62.3%, P <0.001). By contrast, MAP was more frequent in boys than in girls (30.1 vs 16.9%, P <0.01).


Composition of the stone nucleus


The stone nucleus was available for infrared analysis in 426 cases, i.e., 98.6% of calculi (129 stones in girls, 297 stones in boys). Composition of the stone nucleus is shown in Table 6. Three crystalline species were more frequent than any other one: COM, AmUr and MAP that accounted for 29.1%, 22.5% and 21.8% respectively. Calcium oxalate nuclei accounted for 50% of all stone nuclei in girls and 29.9% in boys (P <10−4). Among calcium oxalate crystalline phases, COM nuclei were two times more frequent in girls than in boys and COD nuclei were 5 times more frequent in boys than in girls. While AmUr was the main component of only 9.5% of the stones, it was found as the nucleus in 22.5% of cases without any difference regarding the gender. By contrast, MAP and CaP nuclei were two times more frequent in boys than in girls.


Influence of age


In boys (Table 7), the proportion of calculi initiated from COM was increasing with age from 12.2% in infants to 47.1% in children aged 15 years or more. In the same time, CaP nuclei were constantly decreasing from 22.0% to 0%. By contrast, MAP nuclei remained stable, accounting for around 25% of cases irrespective with patient's age. A slight decrease was found from infants to teenagers regarding AmUr as stone nucleus.


By contrast with boys, COM was found as the starting point in 27.3% of calculi in female babies and accounted for around half of all nuclei in girls aged more than 3 years (Table 8). MAP was found at the origin of calculi in about 10% of stones, irrespective to the patient's age. Regarding AmUr, it was found as the nucleus of the stone in 11.8 to 30.6% of cases without any relation to age.


Stone morphology


Another major aspect of stone analysis is the morphology related to the main component. Based on the criteria described in the morpho-constitutional classification of stones previously reported in the nineties [6], we found that two morphological types were clinically relevant in our series of children stones: first, type Ic (Figure 2), described as a marker of primary hyperoxaluria [9]; second, type IIId (Figure 3), highly suggestive for chronic diarrhea in patients producing AmUr stones [10]. Among calcium oxalate calculi that accounted for 43.3% of the 432 stones in our series, we found 76 calculi (17.6% of cases: 16.3% in boys and 20.5% in girls) exhibiting a type Ic morphology. Among purine stones, AmUr was found as the main component of 9.5% of calculi. It was the component of nucleus in 22.5% of cases and was identified as a stone component in 40% of cases. AmUr stones exhibiting a type IIId morphology were found in 87 cases (20.1% of the stones). In 84 out of the 87 stones (96.6%), the nucleus of the stone exhibited a IIId morphology and was composed of pure AmUr or AmUr mixed with calcium oxalate (only three cases).


Figure 2
Figure 2. 

Examples of urinary calculi exhibiting type Ic morphology highly suggestive for primary hyperoxaluria. The stone surface is budding, brown-yellow pale. The inner structure is often granular, poorly organized and very light in color.




Figure 3
Figure 3. 

Examples of urinary calculi exhibiting type IIId morphology suggestive for chronic diarrhea episodes associated with unbalanced diet with low phosphate and protein intake. The stone surface is very heterogeneous, presenting with rough and porous areas. Color is whitish-beige to grey-brown. The inner structure is made of alternate and irregular concentric layers. Cream layers often are loose and contain small rounded grains of ammonium urate while grey-brown layers are more compact.





Discussion


In agreement with already published series [11, 12, 13], calcium oxalate was the most frequent main component of urinary calculi. We observed a male prevalence of urolithiasis in Morocco, as previously reported [13], with a M/F ratio of 2.32. This value is significantly higher than that reported in the US (M/F=0.77) [14] or in Tunisia (M/F=1.5) [12]. The M/F ratio was inversely correlated with the patient's age and was found below 1 in patients aged more than 15 years, as reported in other series [15, 16].


Now, calcium oxalate is the main component of stones originating from most developing countries [17, 18, 19], except some countries in the Sub-Saharan Africa [19, 20]. Moreover, stones are mainly formed in the upper urinary tract as observed in our series [21]. COM stones were particularly frequent in Algeria [11], in Tunisia [12] and in Morocco with a proportion six to nine times higher than that of COD stones. AmUr was the main component of only 9.5% of calculi in our series but is was found at the origin of 22.5% of the stones. Of note, ammonium urate was reported at the origin of about half of the stones observed in Sudan [22] and one third of paediatric stones in several other countries of sub-Saharan Africa (Cameroon, Senegal, Mali, Burkina Faso) [19, 20].


In the literature, the frequency of stones containing MAP as the main component was variable: 2.3% in China [23], 25% in Croatia [24] with often a high proportion of stones containing any proportion of struvite: 41.2% in Cameroon, Senegal and Mali [19], 35.7% in Burkina Faso [20] and 24.6% in Algeria [11]. In our series, MAP was present in 26.2% of the calculi and was the main component of 18.1% of the stones.


Etiological orientation suggested by stone morphology and composition in Moroccan children


Several studies emphasize the high relationship between stone composition and/or morphology and pathological condition [6, 9, 15, 25]. Unfortunately, clinical and biochemical data were not available for a majority of our patients. However, it is highly probable that the relations between stone composition (or morphology) and etiology in our series are the same as reported in other works.


Calcium oxalate stones


Among calcium oxalate stones, the crystalline phases have different etiology. As previously reported, COM was found to be oxalo-dependent whereas COD is calcium-dependent [6, 25]. Thus, differences in the relative proportions of stones made of one or another crystalline phase may reveal differences in risk factors involved in stone formation.


Calcium oxalate dihydrate


In this study (Table 2) and also in those carried out in Tunisia [12] and Algeria [11], COD was significantly less frequent as compared to the results reported in France [19]. Such data suggest that hypercalciuria could be a less frequent cause of stones in the Maghreb countries compared to industrialized ones where hypercalciuria is found in more than 45% of cases in children stone formers [26, 27].


Calcium oxalate monohydrate


The frequency of COM stones in Moroccan children was found very high (51.5%). The same was observed in Algeria (50.8%) [11], and in Tunisia (45.8%) [12]. COM is primarily oxalo-dependent and its origin in stones relies mainly five mechanisms:

an insufficient diuresis which is a frequent cause of excessive urinary oxalate concentration;
an excessive consumption of oxalate-rich foods (black chocolate, spinaches, beet, star fruit, pepper and other oxalate-rich vegetables);
inflammatory bowel diseases that increase the permeability of colon mucosa to oxalate ions;
an endogenous oxalate overproduction of dietary origin, from hydroxyprolin or ascorbic acid-rich diet;
an endogenous oxalate overproduction of genetic origin, especially an alanine glyoxylate aminotransferase deficiency in the liver induced by AGXT gene mutations, which is the most severe form of nephrolithiasis often resulting in multi-recurrent stone disease, nephrocalcinosis and progressive loss of kidney function.


Several works emphasized the link between stone morphology and the cause of hyperoxaluria [9, 28, 29] highlighting the interest of performing a morphological typing of the stones in addition to stone composition.


Primary hyperoxaluria is the leading cause of inherited calcium oxalate stones in the childhood in Morocco as it was suspected from morphological type Ic in 76 children (17.6%). Because the main component of stones is COM, i.e. a common component of urinary calculi in children and adults, such a composition does not help to suspect such a severe disease. By contrast, stone morphology may orient easily to the diagnosis [9, 28]. In all children who exhibited COM stones with type Ic morphology, primary hyperoxaluria was confirmed by oxalate measurement in urine, calculation of the oxalate to creatinine ratio and/or by genetic investigation. Because search for gene mutations is expensive, it was performed in about ten patients only, confirming in all cases mutations of the AGXT gene. For the remaining patients with Ic COM stones, heavy hyperoxaluria was found in all cases when urine sample was available for measurement of oxalate (n =37), with a very high oxalate to creatinine ratio [30] for the whole series: mean±SD=0.29±0.14mmol/mmol, range: 0.1 to 0.7mmol/mmol, vs 0.09±0.1mmol/mmol in children who do not suffer from primary hyperoxaluria, P <10−6, personal data). All these results are highly suggestive for primary hyperoxaluria in these patients exhibiting COM stones with type Ic morphology. No other cause for hyperoxaluria was identified in these patients based on clinical investigation and inquiry.


Due to the high degree of consanguinity, the prevalence of homozygous primary hyperoxaluria is significantly higher in North Africa than in the industrialized countries [31]. However, because of it scarcity, primary hyperoxaluria is often misdiagnosed or diagnosis is delayed, leading to an impairment of kidney function. Therefore, early diagnosis is crucial, implemented by a morpho-constitutional analysis of the first calculus identified in the child. Type Ic morphology is a very useful tool for the detection of the disease while stone composition is not suggestive. Of note, measurement of oxalate in urine, which is the most common criterion used to orient the diagnosis may be inconclusive due to pre-analytical problems. Genetic diagnosis, now more easily available, is mandatory to identify mutations and help for the clinical management of the patients.


Ammonium urate stones


AmUr accounted for 9.5% of stones in our series. It was reported with frequencies much higher in Sub-Saharan Africa: 29.4% in Cameroon, Senegal, Mali [19] and 48.6% in Sudan [21]. In contrast, AmUr is infrequent in the industrialized countries like in the US where its frequency was reported to be less than 3% [14]. It is endemic in developing countries characteristic of low income populations and generally accepted to be due to malnutrition and low phosphorus intake that is coupled to hyperuricosuria due to tubular immaturity. Infectious diarrhea of bacterial or viral origin is also a major risk factor for AmUr calculi with type IIId morphology [10]. Despite the frequency of AmUr was strongly decreasing in North Africa, this compound was yet found in the core of 22.5% of stones in our series.


Infection stones


It is widely accepted that infection stones are those containing any proportion of MAP. In our series, MAP-containing stones accounted for 26.2% of cases. They were significantly more frequent among boys than girls (30.1 vs 16.9%, P <0.01). This type of urolithiasis strongly decreased in industrialized countries in the past decades reflecting the improvement of their medical level. Infection stones were observed with high rates in several countries like Croatia (25%) [24], Armenia (17%) [10], and in Sub-Saharan Africa (Cameroon, Senegal, Mali) [19] where MAP was identified in 17.7% of cases. Of note, infection stones should not be restricted to MAP-containing calculi but also include CaP stones with a carbonation rate>15% [32].


Calcium Phosphate stones


CaP in the form of carbapatite are not frequent in the Moroccan child (4%). The other crystalline forms of CaP are still rarer. The two main metabolic risk factors at the origin of CaP calculi (with low carbonation rate) are hypercalciuria and a high urinary pH related, for example, to tubular acidification defect.


Uric acid stones


We noted a very weak frequency (2%) of uric acid stones. This result is in agreement with other pediatric series [11, 12, 13]. Two main causes for pure uric acid stones are:

transient nephron immaturity in newborns responsible for a defect in uric acid reabsorption by the proximal tubule;
a high consumption of fructose (fruits or fructose-rich beverages) in children aged more than 5 years.


Cystinuria


Cystine stones accounted for 4% of pediatric calculi, a frequency similar to that reported in Tunisia [12]. In other countries like China [23] and Croatia [24], cystine stones were reported as more frequent, 10 and 9% respectively.


Conclusion


The combination of two physical methods, namely stereomicroscopy and infrared spectroscopy, appeared to be a good procedure for stone analysis. In Moroccan children, urolithiasis is characterized by the preponderance of calcium oxalate monohydrate, a high proportion of which (17.6%) resulting from primary hyperoxaluria. The high occurrence of struvite-containing stones indicates that infection by urea-splitting bacteria is another leading cause of calculi in Moroccan children. In all cases, the morpho-constitutional analysis of calculi allows to detect in a useful way severe metabolic diseases orienting to specific etiological diagnostic and additional investigation.


Disclosure of interest


The authors declare that they have no competing interest.




Table 1 - Location of stones.
  Kidney 
Ureter 
Bladder 
Passed 
Total 
  n   n   n   n   n  
Boys  80  35.4  18  8.0  37  16.4  91  40.2  226 
Girls  40  38.5  16  15.4  13  12.5  35  33.6  104 
Total  120  36.4  34  10.3  50  15.2  126  38.1  330 





Table 2 - Main components of urinary stones.
  Girls 
Boys 
Total 
p B vs G  
  n   n   n    
Calcium oxalate (CaOx)   94   72.3   129   42.7   223   51.6   < 10 −6  
Calcium oxalate monohydrate (COM)  87  66.9  100  33.1  187  43.3  <10−6 
Calcium oxalate dihydrate (COD)  5.4  29  9.6  36  8.3  NS 
Calcium phophate (CaP)   6   4.6   36   11.9   42   9.7   <0.05 
Carbapatite (CA)  4.6  33  10.9  39  9.0  < 0.05 
Brushite (Br)  0.0  0.7  0.5 
Whitlockite (WK)  0.0  0.3  0.2 
Magnesium ammonium phosphate (MAP)  11  8.5  67  22.2  78  18.1  <10−4 
Uric acid   2   1.6   13   4.3   15   3.5   - 
Uric acid anhydrous (UA0)  0.8  3.0  10  2.3 
Uric acid dihydrate (UA2)  0.8  1.3  1.2 
Ammonium hydrogen urate (AmUr)  8  6.1  33  10.9  41  9.5  NS 
Potassium quadriurate (KUr)  0.0  0.3  0.2 
Cystine (CYS)  6  4.6  17  5.6  23  5.3  NS 
Xanthine (XAN)  1.6  0.0  0.5 
Proteins (PROT)  0.8  0.7  0.7 
Calcium ceftriaxonate (CEF)  0.0  1.0  0.7 
Calcite (CALC)  0.0  0.3  0.2   
Total  130  100.0  302  100.0  432  100.0   





Table 3 - Main components according to patient's age in boys.
  0- <
3- <
5- <10 
10- <15 
15- <18 years 
  n   n   n   n   n  
CaOx   5   11.6   19   36.5   53   46.9   41   53.3   11   64.7  
COM  4.65  15  28.8  43  38.0  32  41.6  47.1 
COD  7.0  7.7  10  8.9  11.7  17.6 
CaP   13   30.25   6   11.5   10   8.9   7   9.1   0   0  
MAP  18.6  14  26.9  25  22.1  16  20.8  23.5 
Uric acid   1   2.3   4   7.7   5   4.4   3   3.9   0   0  
AmUr  12  27.9  9.6  11  9.7  6.5 
KUr  0.9 
CYS  4.65  5.8  5.3  5.2  11.8 
XAN 
PROT  1.9  0.9 
CEF  4.65  0.9 
CALC  1.3 
Total  43  100,0  52  100,0  113  100,0  77  100  17  100 





Table 4 - Main components according to patient's age in girls.
  0- <
3- <
5- <10 
10- <15 
15- <18 years 
  n   n   n   n   n  
CaOx   3   27.3   10   58.7   36   75.0   30   83.4   15   83.4  
COM  27.3  10  58.7  32  66.7  28  77.8  14  77.8 
COD  8.3  5.6  5.6 
CaP   0   0   2   11.8   1   2.1   3   8.3   0   0  
MAP  18.2  5.9  10.4  8.3 
Uric acid   1   9.1   0   0   1   2.1   0   0   0   0  
AmUr  18.2  11.8  6.3  5.6 
CYS  18.2  11.8  11.1 
XAN  9.1  2.1 
PROT  2.1 
CEF 
Total  11  100,0  17  100,0  48  100,0  36  100  18  100 





Table 5 - Frequency of stone components (%).
  Girls 
Boys 
Total 
P B vs G 
  n   n   n    
CaOx   105  80.8  188  62.3  293  67.8  0,001  
COM  101  77.7  177  58.6  278  64.4  0,001 
COD  39  30.0  58  19.2  97  22.5  0,02 
CaP   46   35.4   133   44.0   179   41.4   NS  
CA  46  35.4  129  42.7  175  40.5  NS 
Br  0,0  1.0  0.7 
WK  4.6  15  5.0  21  4.9  NS 
MAP   22   16.9   91   30.1   113   26.2   0,01  
Uric acid  3.8  20  6.6  25  5.8  NS 
UA0  3.1  18  6.0  22  5.1  NS 
UA2  1.5  2.3  2.1  NS 
AmUr  45  34.6  128  42.4  173  40.0  NS 
NaUr  0.0  1.0  0.7 
KUr  0.0  0.3  0.2 
CYS  3.8  19  6.3  24  5.6  NS 
XAN  1.5  0.0  0.5 
PROT  47  36.2  101  33.4  148  34.3  NS 
CEF  0.0  1.0  0.7 
Others (CAL, triglycerides)  0.8  0.7  0.7 
Total  130    302    432     





Table 6 - Components of the stone nucleus.
  Girls 
Boys 
Total 
P B vs G  
  n   n   n    
CaOx   65   50.4   86   29.9   151   35.4   < 10 −4  
COM  63  48.8  61  20.5  124  29.1  <10−6 
COD  1.6  25  8.4  27  6.3  <0.01 
CaP   6   4.6   33   11.1   39   9.1   < 0.05  
CA  4.6  31  10.4  37  8.7  NS 
Br  0.3  0.2 
WK  0.3  0.2 
MAP  19  14.7  74  24.9  93  21.8  0,02 
Uric acid   3   2.3   9   3.0   12   2.8   NS  
AmUr  27  20.9  69  23.2  96  22.5  NS 
NaUr  0.3  0.2 
KUr  0.3  0.2 
CYS  6  4.6  18  6.1  24  5.6  NS 
XAN  0.8  0.2 
PROT  1.6  1.0  1.2 
CEF  1.0  0.7 
Total  129  100.0  297  100.0  426  100.0   





Table 7 - Composition of stone nucleus in boys according to age.
  0- <
3- <
5- <10 
10- <15 
15- <18 years 
  n   n   n   n   n  
CaOx   5   12.2   12   23.1   31   28.2   29   37.7   8   47.1  
COM  4.9  11  21.2  26  23.6  22  28.6  29.4 
COD  7.3  1.9  4.5  9.1  17.6 
CaP   9   22.0   8   15.4   9   8.2   4   5.2   0   0  
MAP  11  26.8  13  25.0  30  27.3  22  28.6  23.5 
Uric acid   1   2.4   2   5.8   5   4.5   3   3.9   0   0  
AmUr  11  26.8  13  23.1  25  22.7  14  18.2  17.6 
KUr    0.9 
CYS  2.4  5.8  7.3  5.2  11.8 
PROT  2.4  1.9  1.3 
CEF  4.9  0.9 
Total  41  100,0  52  100,0  110  100,0  77  100  17  100 





Table 8 - Composition of stone nucleus in girls according to age.
  0- <
3- < 5 
5- <10 
10- <15 
15- <18 years 
  n   n   n   n   n  
CaOx   3   27.3   8   47.1   24   51.1   18   50.0   10   55.6  
COM  27.3  47.1  18  38.3  15  41.7  50.0 
COD  12.8  8.3  5.6 
CaP   0   0   3   17.6   3   6.4   4   11.1   0   0  
MAP  9.1  11.8  12.8  8.3  11.1 
Uric acid   1   9.1   0   0   0   0   0   0   0   0  
AmUr  27.3  11.8  12  25.5  11  30.6  22.2 
CYS  18.2  11.8  11.1 
XAN  9.1  2.1 
PROT  2.1 
Total  11  100.0  17  100.0  47  100.0  36  100.0  18  100.0 




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