Can a non-invasive urine-based test become the next-generation diagnostic tool for malaria?

This mini review summarises the non-invasive urine-based diagnostic approaches that have been used to diagnose malaria. Amongst all urine-based diagnosis methods, commercially available Rapid Diagnostic kit/strip is most likely to be suitable for malaria detection in a cost-effective, time-consuming and user-friendly manner. With further improvement in sensitivity, specificity and accuracy, this technique may become a useful next-generation gold standard malaria diagnostic tool in resource-limited regions and in areas where invasive blood tests are restricted.

Infections and the kidney: a tale from the tropics.

Tropical infections are caused by a variety of bacteria, viruses and parasitic organisms across varying geographical regions and are more often reported in adults than in children. Most of the infections are acute, presenting as a febrile illness with involvement of multiple organ systems, including the kidney. The gamut of renal manifestations extends from asymptomatic urinary abnormalities to acute kidney injury and-albeit rarely-chronic kidney disease. Tropical infections can involve the glomerular, tubulointerstitial and vascular compartments of the kidney. Leptospirosis, malaria, dengue, rickettsial fever and schistosomiasis are the most prevalent tropical infections which affect the kidneys of children living in the tropics. In this review we discuss renal involvement in these most prevalent tropical infections.

El análisis de orina como herramienta diagnóstica en casos de malaria grave / Urinalysis as a diagnostic tool in severe malaria

Resumen La malaria produce complicaciones y muerte especialmente en poblaciones con acceso limitado a la atención en salud. La malaria grave puede reconocerse tempranamente mediante la detección en la orina de hallazgos como la hematuria, la coluria y la proteinuria. Se hizo una revisión narrativa basada en estudios sobre malaria grave y el empleo del análisis de orina mediante la consulta de 91 publicaciones. Mediante el análisis de la orina, se pueden detectar alteraciones metabólicas y lesiones en distintos órganos. En estudios recientes en Colombia se ha confirmado su utilidad como apoyo en el diagnóstico de la disfunción renal, la disfunción hepática y la anemia asociada con hemólisis, las cuales son complicaciones frecuentes en la malaria. El examen constituye una herramienta de fácil aplicación en la consulta ambulatoria y en pacientes hospitalizados para reconocer tempranamente casos complicados, y permite la detección oportuna de diferentes lesiones en el paciente con malaria, contribuyendo así a la reducción de la morbilidad grave y la mortalidad.

Abstract Malaria accounts for a significant morbidity and mortality rate around the world, especially in communities with limited access to healthcare. Some clinical signs in urine, like haematuria, coluria and proteinuria, help for the early diagnosis of severe malaria cases. A narrative review was conducted by analyzing 91 publications on studies about severe malaria cases and the use of urinalysis. A urinalysis can detect metabolic disturbances and organ injury. Its diagnostic utility for frequent complications caused by malaria, such as hepatic injury, kidney dysfunction and hemolysis, has been confirmed by recent Colombian studies. This test is an easy-to-use tool in outpatient clinics and with hospitalized patients to promptly recognize complicated cases, allowing the timely identification of different lesions in patients with malaria, thus contributing to the reduction of severe morbidity and mortality.

[Urinalysis as a diagnostic tool in severe malaria].

Malaria accounts for a significant morbidity and mortality rate around the world, especially in communities with limited access to healthcare. Some clinical signs in urine, like haematuria, coluria and proteinuria, help for the early diagnosis of severe malaria cases.A narrative review was conducted by analyzing 91 publications on studies about severe malaria cases and the use of urinalysis.A urinalysis can detect metabolic disturbances and organ injury. Its diagnostic utility for frequent complications caused by malaria, such as hepatic injury, kidney dysfunction and hemolysis, has been confirmed by recent Colombian studies.This test is an easy-to-use tool in outpatient clinics and with hospitalized patients to promptly recognize complicated cases, allowing the timely identification of different lesions in patients with malaria, thus contributing to the reduction of severe morbidity and mortality.

Minimising invasiveness in diagnostics: developing a rapid urine-based monoclonal antibody dipstick test for malaria.

OBJECTIVE: To generate monoclonal antibodies (MAbs) for developing a rapid malaria diagnostic urine-based assay (RUBDA), using Plasmodium-infected human urinary antigens. METHODS: Plasmodium-infected human urinary (PAgHU) and cultured parasite (CPfAg) antigens were used to generate mouse MAbs. The reactivity and accuracy of the MAbs produced were then evaluated using microplate ELISA, SDS-PAGE, Western blotting assay, microscopy and immunochromatographic tests. RESULTS: Ninety-six MAb clones were generated, of which 68.8% reacted to both PAgHU and CPfAg, 31.3% reacted to PAgHU only, and none reacted to CPfAg only. One promising MAb (UCP4W7) reacted in WBA, to both PAgHU and CPfAg, but not to Plasmodium-negative human urine and blood, Schistosoma haematobium and S. mansoni antigens nor measles and poliomyelitis vaccines. CONCLUSION: MAb UCP4W7 seems promising for diagnosing Plasmodium infection. Urine is a reliable biomarker source for developing non-invasive malaria diagnostic tests. SDS-PAGE and MAb-based WBA appear explorable in assays for detecting different levels of Plasmodium parasitaemia.

Detection of Plasmodium knowlesi DNA in the urine and faeces of a Japanese macaque (Macaca fuscata) over the course of an experimentally induced infection.

BACKGROUND: Diagnostic techniques based on PCR for the detection of Plasmodium DNA can be highly sensitive and specific. The vast majority of these techniques rely, however, on the invasive sampling of blood from infected hosts. There is, currently, considerable interest in the possibility of using body fluids other than blood as sources of parasite DNA for PCR diagnosis. METHODS: Urine and faeces were obtained from a Plasmodium knowlesi infected-Japanese macaque (Macaca fuscata) over the course of an experimentally induced infection. P. knowlesi DNA (PkDNA) extracted from urine and faeces were monitored by nested PCR targeting the P. knowlesi specific cytochrome b (cytb) gene. RESULTS: Urinary PkDNA was detected on day 2, but was not amplified using DNA templates extracted from the samples on day 4, day 5 and day 6. Subsequently, urinary PkDNA was detected from day 7 until day 11, and from day 20 until day 30. PkDNA in faeces was detected from day 7 until day 11, and from day 20 until day 37. Moreover, real-time quantitative PCR showed a remarkable increase in the amount of urinary PkDNA following anti-malarial treatment. This might have been due to the release of a large amount of PkDNA from the degraded parasites as a result of the anti-malarial treatment, leading to excretion of PkDNA in the urine. CONCLUSIONS: The cytb-PCR system using urine and faecal samples is of potential use in molecular epidemiological surveys of malaria. In particular, monkey faecal samples could be useful for the detection of zoonotic primate malaria in its natural hosts.

Aetiology of acute febrile episodes in children attending Korogwe District Hospital in north-eastern Tanzania.

INTRODUCTION: Although the burden of malaria in many parts of Tanzania has declined, the proportion of children with fever has not changed. This situation underscores the need to explore the possible causes of febrile episodes in patients presenting with symptoms at the Korogwe District Hospital (KDH). METHODS: A hospital based cross-sectional study was conducted at KDH, north-eastern Tanzania. Patients aged 2 to 59 months presenting at the outpatient department with an acute medical condition and fever (measured axillary temperature ≥37.5°C) were enrolled. Blood samples were examined for malaria parasites, human immunodeficiency virus (HIV) and bacterial infections. A urine culture was performed in selected cases to test for bacterial infection and a chest radiograph was requested if pneumonia was suspected. Diagnosis was based on both clinical and laboratory investigations. RESULTS: A total of 867 patients with a median age of 15.1 months (Interquartile range 8.6-29.9) were enrolled from January 2013 to October 2013. Respiratory tract infections were the leading clinical diagnosis with 406/867 (46.8%) of patients diagnosed with upper respiratory tract infection and 130/867 (15.0%) with pneumonia. Gastroenteritis was diagnosed in 184/867 (21.2%) of patients. Malaria infection was confirmed in 72/867 (8.3%) of patients. Bacterial infection in blood and urine accounted for 26/808 (3.2%) infections in the former, and 66/373 (17.7%) infections in the latter. HIV infection was confirmed in 10/824 (1.2%) of patients. Respiratory tract infections and gastroenteritis were frequent in patients under 36 months of age (87.3% and 91.3% respectively). Co-infections were seen in 221/867 (25.5%) of patients. The cause of fever was not identified in 65/867 (7.5%) of these patients. CONCLUSIONS: The different proportions of infections found among febrile children reflect the causes of fever in the study area. These findings indicate the need to optimise patient management by developing malaria and non-malaria febrile illnesses management protocols.

Improved performance with saliva and urine as alternative DNA sources for malaria diagnosis by mitochondrial DNA-based PCR assays.

Saliva and urine from malaria-infected individuals contain trace amounts of Plasmodium DNA, and therefore, could be used as alternative specimens for diagnosis. A nested PCR targeting the mitochondrial cytochrome b gene (Cytb-PCR) of four human malaria species and Plasmodium knowlesi was developed and tested with 693 blood samples from febrile patients living in diverse malaria-endemic areas of Thailand, and compared with microscopy and nested PCR targeting small-subunit rRNA (18S-PCR). Cytb-PCR was 16% and 39.8% more sensitive than 18S-PCR and microscopy, respectively, in detecting all of these malarial species in blood samples. Importantly, 34% and 17% of Plasmodium falciparum and Plasmodium vivax mono-infections, respectively, detected by microscopy were, in fact, mixed P. falciparum and P. non-falciparum infections. Analysis of matched blood, saliva and urine from 157 individuals showed that microscopy and Cytb-PCR of saliva yielded no significant difference in detecting P. falciparum and P. vivax. However, Cytb-PCR of saliva was more sensitive than microscopy for diagnosis of mixed-species infections. A combination of Cytb-PCR of saliva and of urine significantly outperformed microscopy (p 0.0098 for P. falciparum, p 0.006 for P. vivax, and p 0.0002 for mixed infections). Furthermore, Plasmodium malariae and P. knowlesi could also be identified in saliva and urine with this method. Therefore, the Cytb-PCR developed herein offers a high potential for the use of both saliva and urine for malaria diagnosis, with a sensitivity comparable with or superior to that of microscopy.

Abnormal findings on dipstick urinalysis of out-patients with malaria in Abakaliki, Nigeria.

BACKGROUND & OBJECTIVES: Malaria, one of the major health challenges of the tropics affecting about 500 million people, particularly the children and pregnant women have been associated with changes in urine compositions. The present study was undertaken to document the urinary abnormalities in malaria patients based on malaria species and the level of malaria parasitaemia. METHODS: Febrile patients (n = 365) with positive Giemsa - stained blood films for malaria recruited from Outpatient Department of Ebonyi State University Teaching Hospital, Abakaliki participated in the study. Patients were classified into two categories (+ and ++) based on parasite density. Apparently healthy individuals (n = 81), without malaria parasite on both thick and thin films of comparable age and gender acted as control group. Urine sample (10 ml) was collected from each participant and analysed using standard laboratory methods and techniques. RESULTS: Seventy - four (20.3%) of the patients had Plasmodium falciparum malaria. Although all the urine parameters were higher in the malarial patients in comparison to the control, only bilirubinuria and urobilinogenuria were statistically significant (p <0.05). Also, bilirubinuria, urobilinogenuria, haematuria and proteinuria were significantly (p < 0.05) higher in P. falciparum infection than in infections with other malaria species, but only in P. falciparum infection, bilirubinuria and urobilinogenuria were significantly (p < 0.05) higher at higher parasitaemia. CONCLUSION: Even though positive blood film for malaria parasite remains the gold standard for the diagnosis of malaria, urinary abnormalities, such as bilirubinuria, urobilinogenuria, proteinuria and haematuria may aid in identifying patients with severe malaria parasitaemia, especially the falciparum malaria.

Alterations in urine, serum and brain metabolomic profiles exhibit sexual dimorphism during malaria disease progression.

BACKGROUND: Metabolic changes in the host in response to Plasmodium infection play a crucial role in the pathogenesis of malaria. Alterations in metabolism of male and female mice infected with Plasmodium berghei ANKA are reported here. METHODS: 1H NMR spectra of urine, sera and brain extracts of these mice were analysed over disease progression using Principle Component Analysis and Orthogonal Partial Least Square Discriminant Analysis. RESULTS: Analyses of overall changes in urinary profiles during disease progression demonstrate that females show a significant early post-infection shift in metabolism as compared to males. In contrast, serum profiles of female mice remain unaltered in the early infection stages; whereas that of the male mice changed. Brain metabolite profiles do not show global changes in the early stages of infection in either sex. By the late stages urine, serum and brain profiles of both sexes are severely affected. Analyses of individual metabolites show significant increase in lactate, alanine and lysine, kynurenic acid and quinolinic acid in sera of both males and females at this stage. Early changes in female urine are marked by an increase of ureidopropionate, lowering of carnitine and transient enhancement of asparagine and dimethylglycine. Several metabolites when analysed individually in sera and brain reveal significant changes in their levels in the early phase of infection mainly in female mice. Asparagine and dimethylglycine levels decrease and quinolinic acid increases early in sera of infected females. In brain extracts of females, an early rise in levels is also observed for lactate, alanine and glycerol, kynurenic acid, ureidopropionate and 2-hydroxy-2-methylbutyrate. CONCLUSIONS: These results suggest that P. berghei infection leads to impairment of glycolysis, lipid metabolism, metabolism of tryptophan and degradation of uracil. Characterization of early changes along these pathways may be crucial for prognosis and better disease management. Additionally, the distinct sexual dimorphism exhibited in these responses has a bearing on the understanding of the pathophysiology of malaria.

Comparative detection of Plasmodium vivax and Plasmodium falciparum DNA in saliva and urine samples from symptomatic malaria patients in a low endemic area.

BACKGROUND: Definite diagnosis of malaria relies on microscopy detection of blood stages of parasites in peripheral blood and requires blood sample collection. The nested PCR method has shown to be more sensitive and superior to microscopy in detecting co-infections of Plasmodium species in circulation while Plasmodium falciparum DNA can be identified in urine and saliva specimens of patients, albeit at a lower sensitivity. METHODS: Matched blood, saliva and urine samples were collected from 100 microscopy-positive and 20 microscopy-negative febrile patients who attended a malaria clinic in Tak Province, northwestern Thailand for nested PCR analysis targeting the small subunit ribosomal RNA gene of human malaria. Both P. falciparum and Plasmodium vivax have been known to circulate at a comparable rate in the study area. RESULTS: Comparing with microscopy results, nested PCR of saliva samples had a sensitivity of 74.1% for P. falciparum detection and 84% for P. vivax detection while 44.4% and 34.0% of the corresponding values were observed for urine samples. Both nested PCR results of saliva and urine samples had a specificity of 100% for identification of P. falciparum and P. vivax when compared with nested PCR results from blood. Co-infections of both species were found in four, 26 and 8 patients by microscopy and nested PCR of blood and saliva samples, respectively. Although the positive rates of nested PCR of saliva samples for P. falciparum increased with parasite density, no tendency occurred in results from nested PCR of saliva samples for P. vivax as well as those of urine samples. CONCLUSIONS: Saliva and urine samples could be alternative noninvasive sources of DNA for molecular detection of both P. falciparum and P. vivax. Further improvement of the detection method will offer an opportunity to use these samples for diagnosis of malaria.

Quantitative detection of Plasmodium falciparum DNA in saliva, blood, and urine.

BACKGROUND: Current methods for detecting malaria parasites are invasive and associated with poor compliance when repeated sampling is required. New methods to detect and quantify parasites in a less-invasive manner would greatly enhance the potential for longitudinal surveillance in clinical trials. METHODS: Saliva, urine, and blood samples from 386 Gambian outpatients with suspected malaria infections were analyzed by nested polymerase chain reaction (nPCR) to detect infection and to evaluate diagnostic accuracy in comparison to expert microscopy. The amount of parasite DNA in malaria-positive samples was estimated using real-time quantitative PCR (qPCR). RESULTS: Blood parasite density as estimated by qPCR correlated well with parasite counts established by microscopy (p = 0.94; P < .001). qPCR results for saliva had a significant correlation with microscopy counts (p = 0.58; P < .001), whereas qPCR results for urine had a positive but poor correlation with microscopy counts (p = 0.20; P = .117). The mean amounts of parasite DNA quantified in blood were greater than the mean amounts quantified in saliva and urine samples obtained concurrently from the same individual, by approximately 600-fold and approximately 2500-fold, respectively. When nPCR results were compared with microscopy results, nPCR of saliva had a sensitivity of 73% and a specificity of 97%; its sensitivity increased to 82% in samples with a parasite density of > or = 1000 parasites/microL. nPCR of urine had a sensitivity of 32% and a specificity of 98%. CONCLUSION: Saliva sampling is a promising less-invasive approach for detecting malaria infection.

Global metabolic responses of NMRI mice to an experimental Plasmodium berghei infection.

We present a metabolism-driven top-down systems biology approach to characterize metabolic changes in the mouse resulting from an infection with Plasmodium berghei, using high-resolution (1)H NMR spectroscopy and multivariate data analysis techniques. Twelve female NMRI mice were infected intravenously with approximately 20 million P. berghei-parasitized erythrocytes. Urine and plasma samples were collected 4-6 h before infection, and at days 1, 2, 3, and 4 postinfection. Multivariate analysis of spectral data showed differentiation between samples collected before and after infection, with growing metabolic distinction as the time postinfection progressed. Our analysis of plasma from P. berghei-infected mice showed marked increases in lactate and pyruvate levels, and decreased glucose, creatine, and glycerophosphoryl choline compared with preinfection, indicating glycolytic upregulation, and increased energy demand due to P. berghei infection. The dominant changes in the urinary metabolite profiles included increased levels of pipecolic acid, phenylacetylglycine, and dimethylamine, and decreased concentrations of taurine and trimethylamine- N-oxide, which may, among other factors, indicate a disturbance of the gut microbial community caused by the parasite. Although several of the observed metabolic changes are also associated with other parasitic infections, the combination of metabolic changes and, in particular, the occurrence of pipecolic acid in mouse urine postinfection are unique to a P. berghei infection. Hence, metabolic profiling may provide a sensitive diagnostic tool of Plasmodium infection and the control of malaria more generally.

The antimalarial drug artemisinin alkylates heme in infected mice.

Heme alkylation by the antimalarial drug artemisinin is reported in vivo, within infected mice that have been treated at pharmacologically relevant doses. Adducts resulting from the alkylation of heme by the drug were characterized in the spleen of treated mice, and their glucuroconjugated derivatives were present in the urine. Because these heme-artemisinin adducts were not observed in noninfected mice, this report confirms that the alkylating activity of this antimalarial drug is related to the presence of the parasite in infected animals. The identification of heme-artemisinin adducts in mice should be considered as the signature of the alkylation capacity of artemisinin in vivo.

Leptin and leptin receptors during malaria infection in mice.

Leptin, which is involved in a range of physiological processes, could be an important factor in the pathogenesis of malaria. We found that levels of leptin in serum and urine in Plasmodium berghei-infected mice increased progressively after infection, reaching a maximum value on day 6 post-infection. Serum values were approximately five-fold higher in infected mice than in non-infected controls. A similar relation was found for values of leptin in urine. Soluble leptin receptor levels also increased significantly in serum, more or less in line with the leptin increase. Our work represents the first report of visibly augmented leptin and soluble leptin receptor secretion in malarial infection.

High-performance liquid chromatography assay for simultaneous determination of dextromethorphan and its main metabolites in urine and in microsomal preparations.

An HPLC method has been developed and validated for the determination of dextromethorphan, dextrorphan, 3-methoxymorphinan and 3-hydroxymorphinan in urine samples. Deconjugated compounds were extracted on silica cartridges using dichloromethane/hexane (95:05, v/v) as an eluent. Chromatographic separation was accomplished on a Phenyl analytical column serially connected with a Nitrile analytical column. The mobile phase consisted of a mixture of an aqueous solution, containing 1.5% acetic acid and 0.1% triethylamine, and acetonitrile (75:25, v/v). Compounds were monitored using a fluorescence detector. Calibration curves were linear over the range investigated (0.2-8.0 microM) with correlation coefficients >0.999. The method was reproducible and precise. Coefficients of variation and deviations from nominal values were both below 10%. For all the analytes, recoveries exceeded 77% and the limits of detection were 0.01 microM. The validated assay proved to be suitable for the determination of DEM metabolic indexes reported to reflect the enzymatic activity of the cytochrome P450s, CYP2D6 and CYP3A, both in vivo, when applied to urine samples from patients, and in vitro, when applied to samples from the incubation of liver microsomes with dextromethorphan.

Detection of antigens and antibodies in the urine of humans with Plasmodium falciparum malaria.

Humans infected with Plasmodium falciparum frequently have elevated levels of proteins in their urine, but it is unclear if any of these proteins are parasite antigens or antimalarial antibodies. To resolve this question, urine samples from malaria patients and controls living in Thailand and Ghana were evaluated. Urine samples from 85% of the patients had elevated protein levels and contained proteins with Mrs ranging from less than 29,000 to greater than 224,000 as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Antisera were produced against urine from infected and control subjects. Antisera raised against infected, but not control, urine were positive by indirect immunofluorescence on P. falciparum parasites and immunoprecipitated approximately 12 unique bands from extracts of parasites metabolically labeled with 35S-methionine. These data suggest that a variety of P. falciparum antigens are released into urine during acute infection. It is also likely that anti-P. falciparum antibodies are present in the urine of malaria patients because samples from these patients, but not controls, were positive in indirect immunofluorescence assays and immunoprecipitated at least 19 P. falciparum antigens from extracts of metabolically labeled parasites. The detection of malarial antigens and antibodies in urine may lead to a new approach for the diagnosis of malaria.