1. Introduction
Mortality in severe malaria ranged from none in well-equipped centers to as high as 45% in resource-poor facilities [1] [2] [3] [4] [5]. The main manifestations of severe malaria among semi-immune, residents of malaria-endemic countries are cerebral malaria and severe anaemia. Renal failure is not common [2] [4] [5]. Where renal failure occurs, damage to the kidneys might persist beyond the acute illness, as has been described in P. malariae infection and this could progress to nephrotic syndrome [6] [7] [8].
In a study of renal involvement in cerebral and uncomplicated malaria among Gambian children, Weber found Twenty-five percent of children with cerebral malaria, and 4% of children with mild malaria had an elevated serum creatinine above 62 μmol/l. Increased urinary protein excretion was frequent: 53% of children with cerebral malaria had a glomeruli-tubular pattern of protein excretion, and 46% had a tubular pattern. Median albuminuria was 68 mg/l in children with cerebral malaria, 18 mg/l in children with mild malaria, and 9 mg/l in febrile children with other diseases (P < 0.0001) [2]. Shieban also indicated that acute renal failure was a recognized complication of Plasmodium falciparum malaria in non-immune individuals, affecting up to 30% as compared to 1% - 5% in natives in endemic areas, and representing a major contributor to mortality from malaria [3]. A similar study done by Sowunmi in Ibadan reported an incidence of 45% for acute renal dysfunction in children with acute Plasmodium falciparum malaria. Mild proteinuria occurred in 40% during the acute illness which was not related to creatinine clearance or peripheral parasite density [4]. Proteinuria was absent after recovery. Other features included oliguria in addition to systemic effects of malaria [3].
During the past decade, proteinuria has taken on new importance, and recent clinical and experimental data have suggested that proteinuria is an independent risk factor for the progression of renal diseases [9]-[15]. Not only is proteinuria associated with glomerular injury and loss of perm-selective properties, but experimental data have demonstrated that protein-tubular cell interactions have inflammatory and fibrogenic consequences that can contribute to interstitial damage and fibrosis [2] [4] [9]. Albuminuria was found in children with cerebral malaria, and in children with mild malaria, as well as children with febrile illnesses from other diseases [2] [3] [4] [5]. There was no significant association between the proteinuria and height of fever or the degree of parasitaemia, and there was no significant association between death and signs of renal impairment. Renal proteinuria is common in children with malaria in The Gambia, with prerenal, glomerular, and tubulo-interstitial factors contributing. It is more pronounced in children with cerebral malaria than in those with mild malaria. However, renal dysfunction is relatively mild and does not indicate a worse prognosis [2].
2. Materials and Methods
2.1. Sample Size
The minimum sample size was determined using the formula
[16]
Where;
n = desired sample size
Z = the standard normal deviation usually set at 1.96% (or simply at 2.0) which corresponds to 95% confidence interval.
P = the proportion in the target population estimated to have a particular characteristic (renal impairment in severe malaria).
A reasonable estimated mean was 5.9% [2]. The quoted estimate was that of renal involvement in severe malaria as reported by several authors in the earlier studies [17] [18] [19].
d = tolerable margin of error usually set at 0.05
Thus, the minimum sample size:
The minimum sample size was 73
Allowing for an attrition rate of 10%, a total of 82 children with severe malaria and 82 children with uncomplicated malaria were recruited into the study.
2.2. Subject Recruitment
Children between the ages of 6 months and 11 years presenting in the EPU with features of severe malaria were recruited consecutively as they presented. Those with features of uncomplicated malaria presenting in the General Outpatient Department (GOPD) as well as EPU between the ages of 6 months to 11 years were recruited as controls. Recruitment of subjects was carried out by the researcher, with the assistance of the resident doctors assigned to EPU, the Children’s Ward and the General out Patient Department (GOPD). The children that meet the recruitment criteria were recruited serially into the study.
2.3. Ethical Considerations
Ethical clearance was obtained from the Ethics and Research Committee (ERC) of the Hospital. Informed consent was obtained from the mother, or both parents or caregivers before subject recruitment after clear explanation of the study in the language they best understood.
2.4. Study Population
A total of 164 children that meet the recruitment criteria were recruited serially into the study, out of which 82 (50%) had severe malaria and another 82, without severe malaria, served as controls. The male-to-female ratio was 1:1 in the subjects and 1.4:1 in the controls. The median age was 36.0 months in the subjects and 36.0 months in the controls, both groups were comparable (p > 0.05). Children between the ages 1 to 5 years constituted 62.2% of the entire population studied.
2.5. Urinalysis
About 15 - 20 mls of freshly voided urine was collected from each child into a sterile universal bottle. Urinalysis was performed immediately by the investigator using dipstick method. Ten milliliters of urine were tested, using Multistix 10SG (Bayer Diagnostics, with sensitivity of 98.5%) [17]. The reading of the strip is carried out by placing it at the side of the strip container against each of the parameters being tested and observing the colour change. The reading is done 30 to 40 seconds as directed by the manufacturer (i.e. Multistix(R) 10SG Bayer Diagnostics, UK.) [16] [17] [18] [19].
2.6. Physical Characterization of Urine
Urine collected in the Universal bottle was subjected to direct observation for colour and clarity.
2.7. Dipstick Reagent Strip
Procedure:
Part (10 mls) of the collected uncentrifuged urine was properly mixed before testing.
All reagent pads of the strip were immersed in the urine specimen and the strip was removed immediately.
The edge of the strip was run against the rim of the container to remove excess urine.
The test strip was held horizontally and the colour changes on the test areas were compared closely with colour chart on the container.
The colour changes were read at the times specified by the manufacturer.
All instructions as regarding the storage and handling of the reagent strip were observed as stipulated by the maker (i.e. Multistix(R) 10SG Bayer Diagnostics, UK.).
2.8. Blood Sample and Laboratory Procedure
After proper cleansing with cotton wool swab soaked in 70% ethanol, 5 mls of blood was drawn from the most visible peripheral vein on the dorsum of the hand or the forearm of each child. Sampling was done using a sterile 23G needle attached to a 5 mls syringe. The researcher collected the specimen with assistance from the registrars working in EPU. Of this, random blood sugar was determined by glucometer using a drop of whole blood; the test strips used were the ACCU-CHEK (manufactured by Roche Sensitivity is 97.5%) [17] following standard operational procedure as prescribed by the manufacturer, two milliliters was dispensed into a specimen bottle containing dipotassium salt of ethylene diamine tetra-acetic acid (EDTA) for complete blood count and malaria parasite identification and quantification [18]. The World Health Organisation (WHO) procedure was adopted for the detection and identification of malaria parasites. Thin and thick blood smears were made. The thin film was fixed in methanol. The thick film was stained in 2% Giemsa stain for 30 minutes while thin film was stained in Leishman stain. The slides were rinsed in phosphate buffer (PH 7.2) for three seconds, dried and examined with ×100 objective for malaria parasites. Parasite count was done via a count of parasites in a field containing 200 white blood cell (WBC) in the thick film, and the number of asexual forms per microlitre calculated from the WBC count using the formula [17] [18] [19] [20] [21].
where
PC = Parasite count
TLC = Total leucocytes count
An observation of a parasitaemia rate of 5% and above for the red blood cells (RBC) was regarded as severe. The counting was done under supervision of the Chief Laboratory Technologist in the Haematology Laboratory of UITH.
The packed cell volume (PCV) was expressed as the volume of erythrocytes per litre of whole blood so that it indicated the relative proportions of plasma and red cells. A heparinized capillary tube was used to take blood from the EDTA specimen bottle and centrifuged using the micro-haematocrit centrifuge at 1000 revolutions per second for five minutes. The capillary tube was transferred to the haematocrit reader and the column of the Red Blood Cells (RBCs) was measured and this corresponded to the PCV [3].
3. Results
The results obtained are summarized in Table 1.
4. Discussion
4.1. Urinalysis in the Study Population at Admission
Proteinuria occurred in 62 (75.6%) of subjects and in 26 (31.7%) controls (p = 0.01). However, no massive proteinuria was observed in the study population. Twenty-two (26.8%) and 13 (15.9%) subjects had haematuria and bilirubinuria respectively (p < 0.05). The urine pH was normal in 90.3% of the subjects and 100% of the controls. Eight (9.8%) of the subjects had alkaline pH. The specific gravity was also normal in 86.6% of the subjects, while eleven (13.4%) of the subjects had high specific gravity. Specific gravity was normal in 95.1%, high in 3.7% and low in 1.2% of the control group (p < 0.05) (Table 1). A similar study by Webber among Gambian children with malaria, the findings are considerably similar. Renal involvement is common in children with malaria in The Gambia, with prerenal, glomerular, and tubulo-interstitial factors contributing. It is more pronounced in children with cerebral malaria than in those with mild malaria. However, renal dysfunction is relatively mild and does not indicate a worse prognosis as was the case in this study where the proteinuria resolved four weeks into recovery.
4.2. Urinalysis in the Study Population at Admission and at Fourth Week
There were significant differences in the urinalysis findings at fourth week and those obtained at admission. Sixty-nine (87.3%) of the subjects four weeks later had no protein in their urine (p = 0.0001). The urine pH and specific gravity were normal in all subjects at four weeks (Table 2). The findings in this study is
Table 1. Urinalysis in the study population at admission.
Urinalysis |
Subject n = 82 (%) |
Controls n = 82 (%) |
χ2 |
P |
Proteinuria |
|
|
|
|
nil |
20 (24.4) |
56 (68.3) |
34.11 |
0.001 |
1+ |
50 (61.0) |
26 (31.7) |
15.16 |
0.001 |
2+ |
12 (14.6) |
0 (0) |
24.00 |
0.001 |
Urinary pH |
|
|
|
|
Normal pH 4.5-7.5 |
74 (90.3) |
82 (100) |
0.82 |
0.365 |
Acidic pH < 4.5 |
0 (0.0) |
0 (0) |
|
|
Alkaline pH > 7.5 |
8 (9.8) |
0 (0) |
16.00 |
0.001 |
Specific gravity (SG) |
|
|
|
|
Normal (1015~1030) |
71 (86.6) |
78 (95.1) |
0.66 |
0.417 |
High (>1030) |
11 (13.4) |
3 (3.7) |
9.14 |
0.003 |
Low (<1010) |
0 (0.0) |
1 (1.2) |
|
|
Haematuria |
13 (15.9) |
2 (2.4) |
16.13 |
0.001 |
Bilirubinuria |
22 (26.8%) |
0 (0.0) |
|
|
Table 2. Urinalysis in the study population at admission and four weeks into recovery.
Urinalysis |
Subject at admission n =82 (%) |
Subject at fourth week n =79 (%) |
χ2 |
P |
Proteinuria |
|
|
|
|
nil |
20 (24.4) |
69 (87.3) |
53.96 |
0.001 |
1+ |
50 (61.0) |
9 (11.4) |
56.98 |
0.001 |
2+ |
12 (14.6) |
1 (1.3) |
18.62 |
0.001 |
Urinary Ph |
|
|
|
|
Normal pH |
74 (90.3) |
79 (100.0) |
0.33 |
0.567 |
Acidic pH |
0 (0.0) |
0 (0.0) |
|
|
Alkaline pH |
8 (9.8) |
0 (0.0) |
|
|
Specific gravity (SG) |
|
|
|
|
Normal (1015~1030) |
71 (86.6) |
79 (100.0) |
|
|
High (>1030) |
11 (13.4) |
0 (0.0) |
|
|
Low (<1010) |
0 (0.0) |
0 (0.0) |
|
|
Haematuria |
13 (15.9) |
0 (0.0) |
|
|
Bilirubinuria |
22 (26.8) |
0 (0.0) |
|
|
similar to the findings of Webber among Gambian children and Sowunmi in Ibadan Nigeria. Finding of proteinuria at presentation indicates acute renal involvement in malaria, which demonstrated significant resolution four weeks into recovery. Urinalysis, which is simple, easy procedure to carry out and less expensive compared to Glomerular Filtration Rate (GFR) estimation is therefore advice in children with severe malaria in Nigeria and other resource-limited tropical countries. This will serve as a major tool in preventing acute and sometimes progressive kidney damage from severe malaria.
5. Conclusion
The study of 164 children with malaria, 82 children have severe malaria serves as subjects, while another 82 with uncomplicated malaria as control shows that the urine pH and specific gravity have returned to normal in all subjects after four weeks of recovery, Sixty-nine (87.3%) of the subjects four weeks later had no protein in their urine (p = 0.0001). The urine pH and specific gravity were normal in all subjects at four weeks although 12.7% of the subject still having proteinuria. However, a similar study reveals that malaria has no significant effect on the chemical composition of urine with bilirubin positively correlated with the parasite density. Dipstick urinalysis can be used together with light microscopy in resource-limited malaria-endemic areas to accurately diagnose falciparum malaria infections. Another study demonstrated that malaria accounts for a significant morbidity and mortality rates around the world, especially in communities with limited access to healthcare. Some clinical signs in urine, like haematuria, coluria and proteinuria, help for early diagnosis of severe malaria cases.
6. Recommendations
1) More studies would need to be carried out particularly in other geopolitical zones of the country to determine the prevalence of ARF secondary to malaria.
2) Routine urinalysis would be of relevance as it helps in early identification of children with severe malaria.
Severe malaria predisposes to Acute Renal Failure (ARF) and measures to reduce its incidence would be an invaluable tool in preventive nephrology in Nigeria and perhaps other parts of the Tropics.
Conflicts of Interest
The authors declare no conflicts of interest.