hiv - related kidney diseases have been reported in children since 1989 by strauss and his colleagues .
chronic kidney disease is now a major cause of morbidity among hiv - infected children since comprehensive hiv care with highly active antiretroviral therapy ( haart ) and prompt treatment of opportunistic infections enable patients to live long enough to develop kidney damage .
early stages of hiv - related chronic kidney disease are usually asymptomatic , often manifesting with mild reduction in glomerular filtration rate ( gfr ) .
it is therefore essential to make early diagnosis and institute treatment that will prevent further decline to end stage renal disease ( esrd ) requiring renal replacement therapy .
this is more pertinent in resource - poor african settings where facilities for renal replacement therapy are limited and very expensive [ 2 , 3 ] .
serum creatinine which is commonly used in the estimation of gfr is inaccurate due to the effects of diet , gender , race , tubular secretion , and increased extrarenal elimination found often in advanced hiv disease [ 4 , 5 ] .
also , muscle wasting is prevalent in hiv - infected african children , further diminishing the validity of creatinine as a measure of kidney function in this cohort [ 6 , 7 ] .
conversely , cystatin c , a 120-amino acid , 13-kda protein belonging to the cystatin super - family of competitive inhibitors of lysosomal cysteine proteases , has a constant production rate by all nucleated cells , is freely filtered at the glomeruli , and has no systemic reabsorption or renal tubular secretion [ 810 ] .
in addition , its serum level is not significantly influenced by age , gender , or muscle mass .
likewise , cystatin c - based formulae would mirror gold - standard measures of gfr and reflect impaired kidney function earlier than creatinine - based equations [ 7 , 11 , 12 ] .   in view of the foregoing , this study evaluated the kidney function of hiv - infected children using a serum cystatin c - based method so that early stages of ckd could be accurately and promptly detected to forestall esrd .
  the study was carried out from may to october 2011 at the paediatric outpatient ( pop ) hiv clinics of the only three public hospitals ( university of benin teaching hospital , central hospital , and stella obasanjo children hospital ) providing medical care including antiretroviral therapy ( art ) for children with hiv infection in benin city , southern nigeria .
the clinics are supported by the us president 's emergency plan for aids relief ( pepfar ) and non - governmental organizations .
it was a cross - sectional study , approved by the ethical committee of the university of benin teaching hospital .
written informed consent was obtained from parents / guardians and assent from children ten years old and above .
the hiv status of the participants was determined according to the who standardized serial testing algorithm using determine hiv-1/2 ( abbott , tokyo , japan ) , followed by uni - gold recombigen hiv ( trinity biotech , wicklow , ireland ) for a positive test .
diagnosis was based on documented positive hiv dna pcr tests on two separate samples , for those who were diagnosed before the age of 18 months .
all consecutive art - nave hiv - infected children , aged 18 months to 17 years at the pop clinics , were recruited .
altogether , 227 hiv - infected children attended the pop 's clinics during the study period ; 22 were excluded : nine required immediate admission for acute illnesses ; seven samples were discarded due to haemolysis ; six had no cd4 + cell count / percent results because of insufficient or spilled samples .
thus , 205 hiv - infected children were enrolled .   also , 205 age- and sex - matched apparently healthy hiv - negative children who were on follow up at general out - patient clinics of the 3 public hospitals were enrolled as controls .
age matching was done to the nearest half year for those < 5 years and to the nearest one year for those 5 years .
the matching was done prior to analysis of the samples for serum cystatin c to avoid bias .
patients who had sickle cell disease , cardiac disease , previously diagnosed kidney disease , and those on systemic steroid were excluded .
participants were recruited consecutively from the 3 public hospitals in a ratio of 5  :  2  :  1 , respectively , based on their varied patient loads . relevant
children with hiv infection were staged according to the revised who paediatric clinical staging criteria . in this study , subjects were further described as having  advanced  or  not advanced  hiv disease .
advanced hiv disease was defined as who clinical stages 3 or 4 , cd4 percent < 25% ( in children < 5 years ) , or cd4 + t cell count < 350  cells / mm ( in children  5 years ) . five milliliters ( 5  ml ) of blood was obtained from every study participant .
two milliliters out of the 5  ml of blood was allowed to clot , while the resulting serum was frozen at 8c after centrifugation until analysis for serum cystatin c. the remaining 3  ml of blood was analysed in the pepfar / ubth laboratory for cd4 + cell count or percent in the hiv - infected children , detection of hiv antibodies in the controls , and haemoglobin electrophoresis in both groups .
all samples were transported from the sites of collection to the pepfar / ubth laboratory in an ice - packed polythene container with cock - screwed lid within an hour of collection .
specimen containers were coded to ensure confidentiality . in batches of 82 samples , cystatin c was measured quantitatively by a sandwich enzyme - linked immunosorbent assay ( elisa ) technique , using kits manufactured by wkea med supplies corps , changchun china with a sensitivity of 0.01  g / l , specificity 100% , assay range : 30  g / l800 
the end product was measured spectrophotometrically at 450  nm using a micro - well diareader elx800uv ( dialab gmbh vienna / austria ) .
the actual concentration of serum cystatin c in the sample was obtained by multiplying the measured cystatin c concentration by a dilution factor of 5 .
serum cystatin c level greater than 1.00  mg / l was classified as elevated as in previous studies [ 7 , 16 ] .
the gfr ( ml / min/1.73  m ) was estimated by the cystatin c - based equation derived by filler and lepage : log  ( gfr ) = 1.962 + [ 1.123  log  ( 1/cystatin c ) ] , where 1/cystatin c is the reciprocal of the concentration of serum cystatin c in mg / l .  
the data were analysed using the software package for social science ( spss ) version 17.0 ( windows inc ; chicago , il , usa ) .
anthropometric z - scores were calculated using the who anthroplus software developed using the who child growth standards and the who reference 2009 .
then , the z - scores of the hiv - positive group were compared to those of the controls using the mann - whitney u - test .    continuous data such as age , cystatin c , and egfr were summarized as mean ( sd ) , while categorized data like sex , socioeconomic class , and hiv stages were represented as proportions .
fisher 's exact test or pearson 's chi - square was used to compare categorized data while student t test was used to assess for any significant difference between two means .
pearson 's correlation test was done for the association between egfr and cd4 + cell count .
a total of 205 children infected with hiv ( 98 males and 107 females ) and 205 uninfected ones were studied .
the mean  sd age of the children infected with hiv was 5.9  3.5 years while that of the control group was 5.4  3.5 years .
the youngest study participant was 1.5 years while the oldest was 17 years . among the 205 hiv - infected children 11 ( 5.4% ) were in upper socioeconomic class ( sec ) , 83 ( 40.5% ) in middle class , and 111 ( 54.1% ) in lower class , with a significantly larger proportion in the lower sec compared to the controls , (  = 10.77 , df = 2 , p = 0.005 ) .
the hiv - infected children were leaner ( bmi z - score 0.83 versus 0.40 ; p = 0.002 ) , shorter ( haz score 0.65 versus 0.02 ; p = 0.000 ) and lighter ( waz score 1.015 versus 0.02 ; p = 0.000 ) compared with the control ( table 1 ) . based on who paediatric clinical staging system , 129 children ( 62.9% ) had stage 1 or 2 disease ( not advanced ) while 76 ( 37.1% ) had stage 3 or 4 disease ( advanced ) .
using who immunological staging , 41 ( 38.7% ) of the hiv - infected children younger than 5 years had cd4 + cell percent < 25% while 23 ( 23.2% ) of children 5 years had a cd4 + cell count less than 350  cells / mm , both classified as advanced disease .
mg / l in the hiv - infected group compared with 0.72  0.20  mg
similarly , 48 ( 23.4% ) hiv infected children compared with 11 ( 5.4% ) in the control group had serum cystatin c > 1  mg / l ( p - value = 0.000 ) . in all age groups ,
the mean serum cystatin levels were significantly higher in the hiv - infected children than in the controls ( p < 0.05 , table 2 ) .
the correlation of age , weight , height , and bmi with serum cystatin c was not significant in both groups of children .
also , there was no significant correlation of cd4% with cystatin c in children less than 5 year old ( r = 0.184 ; p = 0.059 ) . however , cd4 + cell count correlated significantly with cystatin c in the hiv - infected children older than 5 years ( r = 0.281 ; p = 0.005 ) .  
the mean estimated glomerular filtration rate ( egfr ) of hiv infected children was 102.7  31.0  ml / min/1.73  m which was significantly lower than the value of 126.9  28.5  ml / min/1.73  m in the control group ( p = 0.014 ) . also , 44 ( 21.5% ) children with hiv infection had reduced egfr < 90  ml / min/173  m compared to 11 ( 5.4% ) in the control group . altogether , significantly
higher proportions of the hiv - infected children than the controls had egfr < 90  ml / min/173  m ( p = 0.000 ; table 3 ) .
the prevalence of chronic kidney disease ( ckd ) among hiv - infected children in this study was 10.7% .
children infected with hiv were 12.2 times more likely to have ckd compared to the controls ( fisher 's exact test = 17.703 ; or = 12.202 ; 95% c.i .
children with hiv infection who had ckd were similar in mean age and body mass indices to those without ckd .
those with ckd had a male - female rate of 1.2  :  1 , and 13 ( 59.0% ) of them were in advanced immunological stage of hiv disease . also , they had a significantly lower mean cd4 count and higher serum cystatin c compared to others .
cd4 count < 200 was predictive of ckd in hiv - infected children ( or = 5.926 ; c.i .
there was a statistically significant association between estimated glomerular filtration rates ( egfrs ) and immunological stages of hiv - infected children  5 year old ( p = 0.028 ) .
however , there was no such association in those < 5 years ( p = 0.112 ; table 5 ) .
also , there was no significant association between egfr and clinical stages of hiv infection at all ages : ( < 5 years old : df = 3 ;  = 2.801 , p = 0.462 ; 5 years : df = 3 ,   = 4.897 , p = 0.153 ) .
there was a significant positive correlation of kidney function ( assessed using cystatin c - based egfr ) with cd4 count in children who were 5 years and older in this study ( r = 0.23 ; p = 0.022 ; figure 1 ) .
mg / l ) is higher than the mean value ( 0.77  0.29  mg / l ) obtained by esezobor et al .   in hiv - infected children in lagos
. the difference could be partly due to the larger proportion of children with elevated serum cystatin c ( > 1  mg / l ) in this study than in the lagos study ( 23.4% versus 16.7% ) .
however , the high level of serum cystatin c of hiv - infected children found in this study is similar to the mean value ( 1.03  0.02  mg /
l ) found by jones et al .   among 250 hiv - positive adults in the nutrition for healthy
mg / l and 0.92 + 0.22  mg / l ) obtained by jaroszewicz et al .   in poland and odden et al .  
furthermore , the high mean serum cystatin c in this work is consistent with prior reports of increased burden of hiv - related kidney diseases in blacks .
compared to the controls , the hiv - infected children had significantly higher serum cystatin c levels in all age groups in this study .
this is consistent with the findings of esezobor et al .   and reports from other regions of the world [ 19 , 20 ] .
this trend suggests that the kidneys , while serving as reservoirs for hiv , suffer subclinical damage causing persistently elevated serum cystatin c in hiv - infected persons .
thus , nearly a quarter of the children with hiv infection in this survey had elevated serum cystatin c ( > 1  mg / l ) which is prognostic of poor renal and cardiovascular outcomes as well as death in this cohort .
it is important to note that the serum cystatin c levels of the controls in all age groups consistently remain within the reference ranges published by finney et al .   for healthy children . since cystatin
c has no significant extraglomerular excretion routes , the stable level obtained in these controls reflects the constancy of gfr in healthy children after infancy .
this study found no correlation between cystatin c and body mass indices of the participants , confirming previous reports in an african study   and researches from other regions [ 11 , 24 ] .
this implies that cystatin c remained an accurate marker of kidney function in the general population and even among hiv - infected african children who are often malnourished . in this study , only cd4 count significantly correlated with serum cystatin c. this is contrary to the findings of jaroszewicz et al .   in polish adults who found no correlation between serum cystatin c levels and cd4 counts .
this may be due to the dual effects of art on the serum cystatin c levels and cd4 counts of their study participants .
the mean cystatin c - based egfr of the hiv positive group was 102.7  31.0  ml / min/1.73  m , significantly lower than that of the age and sex matched controls consistent with previous reports [ 16 , 25 ] . also , the fact that 21.5% of the hiv - infected group had cystatin c - based egfr below normal range ( < 90  ml / min/1.73  m ) further attests to the detrimental effects of hiv on kidney function .
hence , the hiv medicine association of the infectious disease society of america ( idsa ) recommended regular monitoring of gfr in hiv - infected children in order to promptly detect renal impairment and institute measures that may halt or slow its progression .
also , an accurate measure of gfr with serum cystatin c will enable recommended drug dosage adjustments to avoid systemic toxicities of drugs excreted via the kidneys .
therefore , as shown in this work , accurate measure of gfr in hiv infection before therapy and during follow - up is pertinent to a good renal outcome .
the high prevalence of ckd ( 10.7% ) among hiv - infected children in this work is consistent with the report of the previous nigerian study of hiv - infected children using a cystatin c - based method .
recently in hiv - infected children in one of the study centres ( ubth ) using their creatinine - based egfrs .
however , the prevalence in the present study is lower than the 15.2% found by jones et al .   among 250 hiv - infected adults using cystatin
was apparently due to longer duration of hiv disease , use of nephrotoxic drugs , and comorbidities like liver disease , diabetes mellitus and viral hepatitis in some of their study participants .
hiv infected children are more likely to develop ckd as observed in this study ( or = 12.202 ; c.i .
= 2.83052.608 ; p < 0.05 ) , consistent with previous reports of increased burden of ckd among hiv - infected children highlighting an increasing need for renal replacement therapy which the health system is currently ill - prepared for in this sub - region [ 2 , 3 ] .
there was male preponderance among children with ckd in the study , consistent with prior reports among africans [ 28 , 29 ] .
the peak age group ( 59 years ) for ckd in this study coincided with that reported by anochie et al .   among children with hiv - related ckd in port harcourt .
this may be due to the fact that a majority of perinatally infected children followed a downhill course with complications involving the kidneys and other organs by school age , or perhaps due to similar virulence / nephropathic effect of hiv serotypes prevalent in this region .
moreover , consistent with prior reports [ 16 , 21 ] , children with hiv infection in this study were significantly leaner than the controls , and more likely to be in the lower social economic class , portraying poverty - malnutrition - disease cycle .  
other significant predictors of ckd found in this study include nadir cd4 count < 200  cells / mm , consistent with the findings of emem et al .   in a multicentre study in southern nigerian hiv - infected persons
and other researchers [ 18 , 21 ] in the usa found that low cd4 count and/or elevated serum cystatin c were predictive of kidney disease and sometimes death in hiv infected persons .
furthermore , jaroszewicz et al .   in poland identified high viral loads and prolonged use of art as risk factors for ckd .
however , the viral loads of the children recruited in this study were not determined .
the egfr of the hiv - infected children positively correlated with their cd4 count in this study , contrary to the report of the previous nigerian study which found no relationship between the kidney function and immunological / clinical stages of hiv - infected children , possibly underpowered by a small sample size .
other investigators [ 7 , 29 , 31 ] confirmed worsening of kidney function in advanced immunological stages of hiv disease .
surprisingly , the correlation of kidney function with immune status was not significant in children less than 5 years old , possibly due to the shorter duration of their hiv infection .
in this study , kidney function showed no significant association with clinical stages of hiv disease in all age groups , despite its correlation with the immunological stages of the children older than 5 years .
this is an instance of clinical - immunological discordance in hiv infection consistent with the report of  wools - kaloustian et al .  
that renal impairment was significantly associated with severe immunosuppression , but independent of several parameters for clinical staging of hiv infection .
this may be due to the fact that common comorbidities like tuberculosis , found in over 10% of the seropositive group in this study , classify patients into advanced clinical stages , irrespective of their renal or immunological status .
conversely , renal impairment may be the first manifestation of hiv disease in some patients .
winston et al .   found in a large series that about 50% of patients later confirmed to have severe immunosuppression and hivan were otherwise clinically asymptomatic at presentation .
thus , they were inadvertently classified into early clinical stages of hiv disease until further evaluation confirmed advanced immunological stages and renal disease .
the strengths of our study are the recruitment of only art - nave patients and the analysis of serum cystatin c in a single laboratory , avoiding systematic errors
.   the limitations of this study include the lack of assessment of the cause of ckd and the lack of data on proteinuria , an important marker of kidney damage .
also , we used a single measurement of gfrs without repeating 3 or more months later to exclude transient reduction in gfr .
also , like other formulae for the estimation of gfr , the filler 's formula has not been validated in hiv - infected children .   in conclusion , there was a high prevalence of ckd among the hiv - infected children and nadir cd4 count < 200  cells / mm was a potent risk factor for ckd in this study .
children aged  5 years with advanced immunological stages of hiv disease had significantly worse kidney function than those in earlier stages .
the high burden of ckd and elevated serum cystatin c indicative of early renal dysfunction found in this study should be taken into cognizance while providing basic health care / art programmes in this medically under - served region of the world .
 
 background . human immunodeficiency virus ( hiv ) is now a confirmed risk factor for kidney disease with an increased burden in persons of african descent . method . 
 we measured the serum cystatin c levels of 205 art - naive , hiv - infected children by an elisa technique and compared them with the levels of apparently healthy children . result . 
 the mean  sd serum cystatin c level of children with hiv infection was 1.01  0.44  mg / l , significantly higher than the mean value in the control group , that is , 0.72  0.20  mg / l ( p = 0.000 ) . 
 the mean  sd cystatin c - based estimated gfr of children with hiv infection was 102.7  31.0  ml / min/1.73  m2 , significantly lower than 126.9  28.5  ml / min/1.73  m2 in the control group , ( p = 0.014 ) 
 . a significantly higher proportion of hiv - infected children compared to controls had egfr < 90  ml / min/1.73  m2 ( 21.5% versus 5.4% ; p = 0.00 ) . 
 the prevalence of chronic kidney disease ( ckd ) among the hiv - infected children was 10.7% . 
 the cystatin c - based egfr of the hiv - infected children 5 years old correlated positively with their cd4 count ( r = 0.23 ;  p = 0.022 ) . 
 conclusion . 
 there is a high prevalence of ckd among hiv - infected children , requiring regular monitoring of their kidney function using a cystatin c - based method . 
1. Introduction
2. Methods
3. Results
4. Discussion