The Impact of Sequestration on Artemisinin-Induced Parasite Clearance in Plasmodium falciparum Malaria in Africa.

BACKGROUND: Artemisinin-resistant Plasmodium falciparum is spreading in Southeast Asia and Africa. In vivo susceptibility to artemisinin is studied by looking at the rate of decline of peripheral parasitemia (parasite clearance half-life). However, parasites that are adhered/sequestered to the endothelium and undetectable in the peripheral blood are not considered in the estimation of parasite clearance. Here, we evaluated the influence of sequestration on in vivo artemisinin efficacy in Uganda, where artemisinin resistance is spreading. METHODS: We analyzed 133 patients with P. falciparum malaria included in an in vivo study on artemisinin efficacy in northern Uganda in 2018 and 2019. The parasite clearance half-life was estimated from peripheral parasitemia after artemisinin monotherapy. P. falciparum histidine-rich protein 2 (PfHRP2) was measured in pretreatment plasma. The number of sequestered parasites was estimated from PfHRP2 concentration and peripheral parasitemia. RESULTS: The estimated number of sequestered parasites per plasma volume ranged from 0 to 2 564 000/µL. Inflammation, thrombocytopenia, and dyslipidemia were significantly associated with sequestration independent of peripheral parasitemia. The median parasite clearance half-lives were 1.65 hours in patients infected with Pfkelch13 wild-type parasites (n = 104) and 3.95 hours in those with A675V artemisinin-resistant mutant (n = 18). In the multivariable model for the wild-type population, 1 000 000/µL of sequestered parasites were estimated to delay parasite clearance by 16.8% (95% confidence interval, 5.1%-28.5%), although it was not clear in the A675V population. CONCLUSIONS: In patients with P. falciparum malaria without artemisinin-resistant mutations, intensive sequestration delays parasite clearance after treatment, which may contribute to reduced artemisinin efficacy.

Evidence of Artemisinin-Resistant Malaria in Africa.

BACKGROUND: In the six Southeast Asian countries that make up the Greater Mekong Subregion, Plasmodium falciparum has developed resistance to derivatives of artemisinin, the main component of first-line treatments for malaria. Clinical resistance to artemisinin monotherapy in other global regions, including Africa, would be problematic. METHODS: In this longitudinal study conducted in Northern Uganda, we treated patients who had P. falciparum infection with intravenous artesunate (a water-soluble artemisinin derivative) and estimated the parasite clearance half-life. We evaluated ex vivo susceptibility of the parasite using a ring-stage survival assay and genotyped resistance-related genes. RESULTS: From 2017 through 2019, a total of 14 of 240 patients who received intravenous artesunate had evidence of in vivo artemisinin resistance (parasite clearance half-life, >5 hours). Of these 14 patients, 13 were infected with P. falciparum parasites with mutations in the A675V or C469Y allele in the kelch13 gene. Such mutations were associated with prolonged parasite clearance half-lives (geometric mean, 3.95 hours for A675V and 3.30 hours for C469Y, vs. 1.78 hours for wild-type allele; P<0.001 and P = 0.05, respectively). The ring-stage survival assay showed a higher frequency of parasite survival among organisms with the A675V allele than among those with the wild-type allele. The prevalence of parasites with kelch13 mutations increased significantly, from 3.9% in 2015 to 19.8% in 2019, due primarily to the increased frequency of the A675V and C469Y alleles (P<0.001 and P = 0.004, respectively). Single-nucleotide polymorphisms flanking the A675V mutation in Uganda were substantially different from those in Southeast Asia. CONCLUSIONS: The independent emergence and local spread of clinically artemisinin-resistant P. falciparum has been identified in Africa. The two kelch13 mutations may be markers for detection of these resistant parasites. (Funded by the Japan Society for the Promotion of Science and others.).

Ex vivo susceptibility of Plasmodium falciparum to antimalarial drugs in Northern Uganda.

In Uganda, artemether-lumefantrine was introduced as an artemisinin-based combination therapy (ACT) for malaria in 2006. We have previously reported a moderate decrease in ex vivo efficacy of lumefantrine in Northern Uganda, where we also detected ex vivo artemisinin-resistant Plasmodium falciparum. Therefore, it is necessary to search for candidate partner alternatives for ACT. Here, we investigated ex vivo susceptibility to four ACT partner drugs as well as quinine and chloroquine, in 321 cases between 2013 and 2018. Drug-resistant mutations in pfcrt and pfmdr1 were also determined. Ex vivo susceptibility to amodiaquine, quinine, and chloroquine was well preserved, whereas resistance to mefloquine was found in 45.8%. There were few cases of multi-drug resistance. Reduced sensitivity to mefloquine and lumefantrine was significantly associated with the pfcrt K76 wild-type allele, in contrast to the association between chloroquine resistance and the K76T allele. Pfmdr1 duplication was not detected in any of the cases. Amodiaquine, a widely used partner drug for ACT in African countries, may be the first promising alternative in case lumefantrine resistance emerges. Therapeutic use of mefloquine may not be recommended in this area. This study also emphasizes the need for sustained monitoring of antimalarial susceptibility in Northern Uganda to develop proper treatment strategies.

Recovery and stable persistence of chloroquine sensitivity in Plasmodium falciparum parasites after its discontinued use in Northern Uganda.

BACKGROUND: Usage of chloroquine was discontinued from the treatment of Plasmodium falciparum infection in almost all endemic regions because of global spread of resistant parasites. Since the first report in Malawi, numerous epidemiological studies have demonstrated that the discontinuance led to re-emergence of chloroquine-susceptible P. falciparum, suggesting a possible role in future malaria control. However, most studies were cross-sectional, with few studies looking at the persistence of chloroquine recovery in long term. This study fills the gap by providing, for a period of at least 6 years, proof of persistent re-emergence/stable recovery of susceptible parasite populations using both molecular and phenotypic methods. METHODS: Ex vivo drug-susceptibility assays to chloroquine (n = 319) and lumefantrine (n = 335) were performed from 2013 to 2018 in Gulu, Northern Uganda, where chloroquine had been removed from the official malaria treatment regimen since 2006. Genotyping of pfcrt and pfmdr1 was also performed. RESULTS: Chloroquine resistance (≥ 100 nM) was observed in only 3 (1.3%) samples. Average IC50 values for chloroquine were persistently low throughout the study period (17.4-24.9 nM). Parasites harbouring pfcrt K76 alleles showed significantly lower IC50s to chloroquine than the parasites harbouring K76T alleles (21.4 nM vs. 43.1 nM, p-value = 3.9 × 10-8). Prevalence of K76 alleles gradually increased from 71% in 2013 to 100% in 2018. CONCLUSION: This study found evidence of stable persistence of chloroquine susceptibility with the fixation of pfcrt K76 in Northern Uganda after discontinuation of chloroquine in the region. Accumulation of similar evidence in other endemic areas in Uganda could open channels for possible future re-use of chloroquine as an option for malaria treatment or prevention.

Artemisinin-Resistant Plasmodium falciparum with High Survival Rates, Uganda, 2014-2016.

Because ≈90% of malaria cases occur in Africa, emergence of artemisinin-resistant Plasmodium falciparum in Africa poses a serious public health threat. To assess emergence of artemisinin-resistant parasites in Uganda during 2014-2016, we used the recently developed ex vivo ring-stage survival assay, which estimates ring-stage-specific P. falciparum susceptibility to artemisinin. We conducted 4 cross-sectional surveys to assess artemisinin sensitivity in Gulu, Uganda. Among 194 isolates, survival rates (ratio of viable drug-exposed parasites to drug-nonexposed controls) were high (>10%) for 4 isolates. Similar rates have been closely associated with delayed parasite clearance after drug treatment and are considered to be a proxy for the artemisinin-resistant phenotype. Of these, the PfKelch13 mutation was observed in only 1 isolate, A675V. Population genetics analysis suggested that these possibly artemisinin-resistant isolates originated in Africa. Large-scale surveillance of possibly artemisinin-resistant parasites in Africa would provide useful information about treatment outcomes and help regional malaria control.

Impact of two follow-up schemes on morbidity management and disability prevention (MMDP) programme for filarial lymphedema in Matara, Sri Lanka.

Alleviating morbidity due to lymphatic filariasis (LF)-especially in elderly patients who are rather ignorant-is presently the biggest challenge for the national filariasis campaign. We introduced two follow-up schemes and compared each other to address three key programmatic issues (1) locating patients, (2) educating patients, family members on practice of lymphoedema self-care (3) well sustained daily self-care. Hundred and seven lymphoedema patients were introduced to the new Community Home Based Care (CHBC) programme as a part of MMDP programme at their homes. Twenty seven of 107 patients were selected by purposive sampling and followed-up under two schemes, 14 in Daily follow-up (DFU) scheme and 13 in Monthly follow-up (MFU) scheme. Impact was assessed using a KAP score, number of entry lesions (EL) and number of ADL episodes, limb volume, its appearance, changes in the quality of life and gained benefits. Visiting patients in their homes to introduce lymphoedema care programme was a success. KAP scores of the more important activities on lymphoedema care were significantly higher in DFU scheme. Number of patients (51.9%; 14/27) who had EL/s at baseline reduced significantly to 18.5% (5/27) at one year follow-up. The mean numbers of ADL episodes/year reduced significantly in both schemes. Six photographs of 27 showed obvious improvement in lymphoedema and its grade. Mean volume of lymphoedema reduced significantly in both schemes at one year no significant difference between schemes. Benefit score at one year revealed that the patients in DFU scheme received significantly higher amount of benefits compared to MFU scheme. In conclusion daily instruction has significantly motivated the patient and his/her family bringing a new hope.

Erratum to: Wuchereria bancrofti infection at four primary schools and surrounding communities with no previous blood surveys in northern Uganda: the prevalence after mass drug administrations and a report on suspected non-filarial endemic elephantiasis.

[This corrects the article DOI: 10.1186/s41182-017-0060-y.].

Wuchereria bancrofti infection at four primary schools and surrounding communities with no previous blood surveys in northern Uganda: the prevalence after mass drug administrations and a report on suspected non-filarial endemic elephantiasis.

BACKGROUND: A prevalence study of Wuchereria bancrofti infection was carried out in 2014 at 4 study sites in northern Uganda using antigen and microfilaria tests. Each study site consists of a primary school and surrounding communities. These sites are inside the filariasis endemic area and have been covered by mass drug administration under the national elimination programme. However, no prevalence study had been conducted there before the present study. Without information on past and present endemicity levels, our study was meant to be an independent third-party investigation to know the latest filariasis situation. RESULTS: A total of 982 people including 570 schoolchildren (7-19 years) and 412 community people (7-25 years) were examined, all of them for filarial antigen and 695 for microfilariae. The study revealed that all subjects were negative by both methods. CONCLUSIONS: It was considered that annual mass drug administrations together with anti-malarial activities such as indoor residual spraying had contributed to the reduction of the filarial infection. However, based on the past data obtained near our study sites, we cannot exclude the possibility that filarial prevalence rates in our study sites were very low or even zero originally. During the study, we encountered several patients with lower leg edema and pachydermic (elephant skin-like), mossy skin lesion of the foot. Judging from clinical features and bare-footed life-style of people in the area, non-filarial elephantiasis, possibly podoconiosis, was suspected. This elephantiasis has been reported in areas where filariasis is not endemic.

A possible origin population of pathogenic intestinal nematodes, Strongyloides stercoralis, unveiled by molecular phylogeny.

Humans and dogs are the two major hosts of Strongyloides stercoralis, an intestinal parasitic nematode. To better understand the phylogenetic relationships among S. stercoralis isolates infecting humans and dogs and to assess the zoonotic potential of this parasite, we analyzed mitochondrial Cox1, nuclear 18S rDNA, 28S rDNA, and a major sperm protein domain-containing protein genes. Overall, our analyses indicated the presence of two distinct lineages of S. stercoralis (referred to as type A and type B). While type A parasites were isolated both from humans and dogs in different countries, type B parasites were found exclusively in dogs, indicating that the type B has not adapted to infect humans. These epidemiological data, together with the close phylogenetic relationship of S. stercoralis with S. procyonis, a Strongyloides parasite of raccoons, possibly indicates that S. stercoralis originally evolved as a canid parasite, and later spread into humans. The inability to infect humans might be an ancestral character of this species and the type B might be surmised to be an origin population from which human-infecting strains are derived.

Absence of in vivo selection for K13 mutations after artemether-lumefantrine treatment in Uganda.

BACKGROUND: Individual drug treatment may select resistant parasites in the human body, a process termed in vivo selection. Some single nucleotide polymorphisms in Plasmodium falciparum chloroquine-resistance transporter (pfcrt) and multidrug resistance gene 1 (pfmdr1) genes have been reportedly selected after artemether-lumefantrine treatment. However, there is a paucity of data regarding in vivo selection of P. falciparum Kelch propeller domain (pfkelch13) polymorphisms, responsible for artemisinin-resistance in Asia, and six putative background mutations for artemisinin resistance; D193Y in ferredoxin, T484I in multiple resistance protein 2, V127M in apicoplast ribosomal protein S10, I356T in pfcrt, V1157L in protein phosphatase and C1484F in phosphoinositide-binding protein. METHODS: Artemether-lumefantrine efficacy study with a follow-up period of 28 days was conducted in northern Uganda in 2014. The above-mentioned genotypes were comparatively analysed before drug administration and on days; 3, 7, and 28 days after treatment. RESULTS: In 61 individuals with successful follow-up, artemether-lumefantrine treatment regimen was very effective with PCR adjusted efficacy of 95.2%. Among 146 isolates obtained before treatment, wild-type alleles were observed in 98.6% of isolates in pfkelch13 and in all isolates in the six putative background genes except I356T in pfcrt, which had 2.4% of isolates as mixed infections. In vivo selection study revealed that all isolates detected in the follow-up period harboured wild type alleles in pfkelch13 and the six background genes. CONCLUSION: Mutations in pfkelch13 and the six background genes may not play an important role in the in vivo selection after artemether-lumefantrine treatment in Uganda. Different mechanisms might rather be associated with the existence of parasites after treatment.

Rapid assessment procedures to detect hidden endemic foci in areas not subjected to mass drug administration in Sri Lanka.

For the declaration of elimination of lymphatic filariasis, reliable epidemiological data in all parts of a country are required. In Sri Lanka, due to social disturbance, there are 3 provinces whose endemicity has been declared unknown. Further, a recent report revealed an endemic pocket, which is on the border with the district that was not covered by the national elimination program. These facts indicate the necessity of more extensive studies to discover hidden endemic foci. To facilitate such studies, we evaluated 2 methods of Rapid Assessment Procedure (RAP) in Hambantota district, where the filariasis endemicity was low: (1) indirect questioning by mailing a questionnaire to each local leader (IndQ), asking about the presence of clinical cases, and (2) focus group discussion (FGD) by villagers. The information given by people was validated with clinical examination by doctors (CE) and IgG4 ELISA using urine samples. In the results: there was a strong positive correlation between CE and ELISA rates. The hydrocele rates obtained by FGD or IndQ were associated significantly with CE rates. The rates by FGD or Cluster-IndQ ('modified' IndQ) were also associated significantly with ELISA rates. The IndQ was most cost-effective. Based on these findings, we have concluded that screening by IndQ and confirmation by the ELISA would be an effective and practical way in Sri Lanka to locate endemic foci in hitherto unsurveyed districts.

Enzyme-linked immunosorbent assay for the diagnosis of Wuchereria bancrofti infection using urine samples and its application in Bangladesh.

In Sri Lanka, urine ELISA showed high sensitivity and specificity in detecting filaria-specific IgG4. It also produced much higher positive rates than antigen tests in prevalence studies with young children. In this study, we have confirmed the usefulness of urine ELISA in the field of Bangladesh. The ELISA detected 89 of 105 (85%) ICT antigen test positive subjects in endemic areas. With both ICT and microfilaria positives, the sensitivity was 97% (30/31). All of 104 ICT negative people in a non-endemic area were ELISA negative (100% specificity). In a prevalence study with 319 young children (5-10 years) from a low endemic area after five rounds of MDA, seven (2.2%) were detected by the present urine test, but only one (0.3%) by ICT (P=0.075). The satisfactorily high sensitivity, 100% specificity and effective case detection among young ages along with scope for analyzing the titers will indicate urine ELISA to be an effective tool in the post-MDA surveys to confirm elimination or to detect resurgence in Bangladesh.

Visual detection of filaria-specific IgG4 in urine using red-colored high density latex beads.

The use of urine for the immunodiagnosis of lymphatic filariasis has a definite advantage: the sample collection is not invasive and thus well accepted by people. Urine-based ELISA to detect filaria-specific IgG4 has been used successfully. However, ELISA requires equipment such as a microplate reader, which is often not available in most endemic areas. We have developed a new visual immunodiagnosis that detects urinary IgG4 using red-colored latex beads (bead test). The sensitivity was 87.2% when ICT antigen test positive people were regarded as the standard (136/156), and the specificity was 97.2% with the non-endemic people in Japan and Bangladesh, and the urine ELISA negatives in Sri Lanka (1264/1300). In a prevalence study, the bead test could detect filarial infection more effectively than ICT test among young children in Sri Lanka, indicating the usefulness of the visual test in epidemiological studies.

ELISA with recombinant rKRP42 antigen using urine samples: a tool for predicting clinical visceral leishmaniasis cases and its outbreak.

We reported a highly sensitive and specific enzyme-linked immunosorbent assay (ELISA) that detects immunoglobulin G (IgG) in urine using rKRP42 antigen for the diagnosis of visceral leishmaniasis (VL). The ELISA was applied to study chronological change in antibody titers in five study areas in Rajshahi district, Bangladesh. A total of 585 subjects without a past VL history were examined at least three times in the 30-month follow-up period; of these subjects, 137 (23.4%) subjects became ELISA-positive at least one time during the study. Among the positive cases, 40 (29.2%) subjects developed clinical VL, and 31 (77.5%) of these subjects showed IgG titers of ≥ 1,000 U more than one time in the study period. Considering only the first ELISA results, 22 subjects with IgG titers of ≥ 1,000 U could be found, and 21 (95.5%) of these subjects turned out to be clinical cases. The high urinary IgG titers (≥ 1,000 U) will help predict possible clinical VL cases and thus, identify an outbreak in its earlier stage.

The Global Programme to Eliminate Lymphatic Filariasis: History and achievements with special reference to annual single-dose treatment with diethylcarbamazine in Samoa and Fiji.

Diethylcarbamazine (DEC), first introduced in 1947, was shown to have strong efficacy and safety for treatment of human lymphatic filariasis, which is caused mostly by a species Wuchereria bancrofti. Many studies to optimize the dosage and treatment schedule of DEC followed, and, based on the results, control programs with various regimens were implemented in different endemic areas/countries. By the mid 1970s, with endorsement by the WHO Expert Committee on Filariasis (3rd report, 1974), the standard DEC regimen for W. bancrofti infection in mass treatment had been established in principle: a total dose of 72 mg/kg of body weight given in 12 divided doses, once weekly or monthly, at 6 mg/kg each. Not long after the committee report, the efficacy of annual single-dose treatment at 6 mg/kg, which is only one twelfth of the WHO-recommended dose in a year, was reported effective in French Polynesia (study period: 1973-78), and later in Samoa (study period: 1979-81). These results were published between 1978 and 1985 in the Bulletin of WHO but received little attention. In the mid 1980s, the efficacy of ivermectin, the first-choice drug for onchocerciasis, against lymphatic filariae came to light. Since the effect at a single dose was remarkable, and often better than DEC, it was predicted that the newly introduced drug would replace DEC. Treatment experiments with ivermectin increased quickly in number. Meanwhile, annual single-dose mass drug administration (MDA) with DEC at 6 mg/kg was under scrutiny in Samoa and Fiji. In the early 1990s, the Samoan study, which covered the entire population of 160,000 with 3 annual MDAs, reported a significant reduction in microfilaria (mf) prevalence and mean mf density, while in Fiji, the efficacy of 5 rounds of annual MDA (total dose, 30 mg/kg) was shown to be as effective as 28 multi-dose MDA spread over 2 years (6 weekly plus 22 monthly treatments at 5 mg/kg; total dose, 140 mg/kg). Several additional studies carried out in Samoa in relation to the annual single-dose MDAs revealed that low density mf carriers, who have a very low mf count of 1-20/ml of venous blood, could not play a significant role in filariasis transmission.From around 1990, studies on spaced low-dose DEC treatments and various types of combination chemotherapy with DEC and ivermectin increased. Albendazole, a well-known anti-intestinal helminths agent, was later added to the combination. The main findings of these studies with W. bancrofti are: (i) a single dose of DEC at 6 mg/kg reduced mean mf density by ca. 90% 1 year after treatment; (ii) the same dose could damage/kill adult worms; (iii) a single dose of ivermectin at ca. 400 µg/kg was more effective than DEC in reducing mf density during the first year and was similarly or less effective in the second year; (iv) ivermectin probably could not kill adult worms; (v) a single combined dose of albendazole (400 mg) and DEC (6 mg/kg) was effective to reduce mf density by 85 to nearly 100% 12-24 months after treatment; and (vi) ivermectin or albendazole included in the combination chemotherapy produced "beyond-filariasis" benefits: clearance/reduction of intestinal helminths, and, additionally, in the case of ivermectin, skin-dwelling ectoparasites.The Global Programme to Eliminate Lymphatic Filariasis (GPELF) started its worldwide activities in 2000, with the target of elimination by 2020. The basic strategy is to conduct annual single-dose MDAs for 4-6 years. In 2000-2007, a minimum of 570 million individuals were treated in 48 of 83 endemic countries. The drugs used are DEC 6 mg/kg plus albendazole 400 mg in most countries, or ivermectin 200-400 µg/kg plus albendazole 400 mg particularly in onchocerciasis endemic countries in Africa. (MDAs with DEC alone had been used in India.)The GPELF achieved impressive results in terms of parasitological cure/improvement, clinical benefits, social and economic impacts, etc. However, the most impressive result of all was the programme's success in mobilizing hundreds of millions of local people, who not only took drugs but many of them actively supported MDAs as drug distributors and volunteers. Beyond filariasis, the role people can play in supplementing rural health services is now a topic of discussion and a source of hope for a new sustainable system.

Development of loop-mediated isothermal amplification method for detecting Wuchereria bancrofti DNA in human blood and vector mosquitoes.

We have developed loop-mediated isothermal amplification (LAMP) method to detect Wuchereria bancrofti DNA. The sensitivity and specificity of LAMP method were equivalent to those of PCR method which detects SspI repeat sequence in W. bancrofti genomic DNA: both methods detected one thousandth of W. bancrofti DNA from one microfilaria (Mf), and did not cross-react with DNAs of Brugia malayi, B. pahangi, Dirofilaria immitis, human and Culex quinquefasciatus. We also examined the sensitivity of LAMP using the mimic samples of patient's blood or blood-fed mosquitoes containing one W. bancrofti Mf per sample. The LAMP method was able to detect W. bancrofti DNA in 1000 µl of blood or in a pool of 60 mosquitoes, indicating its usefulness in detecting/monitoring W. bancrofti infection in humans and vector mosquitoes in endemic areas.

Effects of 5 rounds of mass drug administration with diethylcarbamazine and albendazole on filaria-specific IgG4 titers in urine: 6-year follow-up study in Sri Lanka.

ELISA for filaria-specific IgG4 in urine (urine ELISA) was applied to children in 7 schools in Sri Lanka, before and after 5 rounds of annual mass drug administration (MDA). The pre-treatment IgG4 prevalence in 2002 was 3.20%, which decreased to 0.91% in 2003 after the first MDA (P<0.001), and finally to 0.36% in 2007 after the 5th MDA. Among 5-10 year-old children, the prevalence decreased from 3.37% in 2002 to 0.51% in 2003 (P=0.009). A pattern of IgG4 titer distribution according to age and its yearly change could also provide useful information in drug efficacy analysis. In 2008, new samples from eleven 2006/07 urine ELISA-positive students and their family members (total n=56) were examined by ICT antigen test, microfilaria test, and urine ELISA. No infection was confirmed among them. Urine ELISA will be useful in monitoring elimination/resurgence in a post-MDA low endemic situation.

Sensitive, specific, and rapid detection of Leishmania donovani DNA by loop-mediated isothermal amplification.

We have applied a loop-mediated isothermal amplification (LAMP) technique to detect Leishmania donovani DNA. The LAMP technique detected 1 fg of L. donovani DNA, which was 10-fold more sensitive than a conventional polymerase chain reaction (PCR). All nested PCR-positive blood samples from visceral leishmaniasis patients were positive with the LAMP technique, and DNA samples from L. infantum, L. major, L. mexicana, L. tropica, L. braziliensis, Plasmodium falciparum, and healthy humans were negative with the LAMP technique. The advantages of the LAMP method are its shorter reaction time, a lack of requirement of sophisticated equipment, and visual judgment of positivity based on the turbidity of reaction mixture. Our LAMP technique can be a better alternative to a conventional PCR, especially under field conditions.

Enzyme-linked immunosorbent assay to detect urinary antibody against recombinant rKRP42 antigen made from Leishmania donovani for the diagnosis of visceral leishmaniasis.

We recently reported the production of the recombinant kinesin-related protein of Leishmania donovani with a molecular weight of 42 kd (rKRP42) and the value of the antigen in serum-based ELISA for the diagnosis of visceral leishmaniasis (VL). In this study, the rKRP42 antigen was validated with ELISA using urine samples (rKRP42 urine ELISA). The urine-based ELISA showed 94% sensitivity (108 positives among 115 VL samples) and 99.6% specificity (239 negatives among 240 non-VL samples). The sensitivity and specificity are almost similar to our previous results by ELISA with acetone-treated L. donovani promastigote antigen and direct agglutination test, both methods being done by use of urine samples. A comparison of the rKRP42 urine ELISA with the commercially available urinary antigen detection kit (KAtex) using 108 VL samples showed much higher sensitivity of the ELISA (96.3%) than KAtex (55.6%). The use of the rKRP42 antigen with urine samples will facilitate epidemiologic studies.