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In visceral and mucocutaneous leishmaniasis, humoral immune response can reflect disease severity and parasite burden. Cutaneous leishmaniasis (CL) in Sri Lanka is caused by a usually visceralizing parasite, Leishmania donovani. We assessed the parasite burden (relative quantity-RQ) in 190 CL patients using quantitative real-time PCR (qPCR-with primers designed for this study) and smear microscopy, then correlated it with clinical parameters and IgG response. RQ of parasite DNA was determined with human-specific glyceraldehyde 3-phosphate dehydrogenase (GAPDH) as the internal control. The qPCR sensitivity was tested with serially diluted DNA from cultured L. donovani parasites. Smears were assigned a score based on number of parasites per high power field. Data from previous studies were used for comparison and correlation; nested Internal Transcribed Spacer 1 (ITS1) PCR as reference standard (RS) and IgG antibody titers to the Leishmania rKRp42 antigen as the immune response. The qPCR amplified and quantified 86.8% of the samples while demonstrating a fair and significant agreement with ITS1-PCR and microscopy. Parasite burden by qPCR and microscopy were highly correlated (r = 0.76; p = 0.01) but showed no correlation with the IgG response (r = 0.056; p = 0.48). Corresponding mean RQs of IgG titers grouped by percentiles, showed no significant difference (p = 0.93). Mean RQ was higher in early lesions (p = 0.04), decreased with lesion size (p = 0.12) and slightly higher among papules, nodules and wet ulcers (p = 0.72). Our study established qPCR's efficacy in quantifying parasite burden in Sri Lankan CL lesions but no significant correlation was observed between the parasite burden and host IgG response to the Leishmania rKRP42 antigen., Competing Interests: Declaration of competing interest The authors declare that they have no conflicts of interest., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Cryptosporidium spp. are enteric protozoan parasites that infect a wide range of hosts including humans, and domestic and wild animals. The aim of this study was to molecularly characterize the Cryptosporidium spp. found in calf faeces in Japan. A total of 80 pre-weaned beef and dairy calves' diarrhoeic faecal specimens were collected from nine different prefectures in Japan. A nested polymerase chain reaction targeting the small subunit 18S rRNA and GP60 genes were used to detect the Cryptosporidium genotypes and subtypes. 83.8% (67 out of 80) of the specimens were positive for Cryptosporidium spp.; Cryptosporidium was found in both beef and dairy calves. Cryptosporidium parvum was the predominant species, detected in 77.5% (31/40) of beef calves and 80% (32/40) of dairy calves. Cryptosporidium bovis was also detected, 5.0% (2/40) of dairy calves, and C. ryanae was also found 2.5% (1/40) of dairy calves. One mixed-species infection, 2.5% (1/40) was detected in a beef calf having C. parvum, and C. ryanae. We detected the most common subtype of C. parvum (i.e., IIaA15G2R1), as well as other subtypes (i.e., IIaA14G3R1, IIaA14G2R1, and IIaA13G1R1) that have not previously been detected in calves in Japan. Our results demonstrate the widespread diversity of Cryptosporidium infection in calves in Japan., Competing Interests: Declaration of Competing Interest None., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Lymphatic filariasis (LF) has been declared eliminated in Sri Lanka in September 2016. To maintain elimination status, a surveillance system to detect hidden endemic foci or LF resurgence is of highest priority. In this paper, we have reported an investigation of LF transmission in Trincomalee district where a surveillance program was not carried out due to 30 years of civil unrest. Proposed surveillance system included, measurement of anti-filarial IgG4 in urine of schoolchildren in areas where LF transmission could exist and assessment of circulating filarial antigen (CFA) and microfilaria (mf) in all urine antibody positive schoolchildren, their family members and 10-15 neighbours of each urine antibody positive household. Spatial distribution of the anti-filarial antibody titers in urine in a high antibody suspected area was analyzed using GPS logger data. Among 2301 school children from 11 schools studied, 41 (1.8%) urine antibody positives were found. The antibody positive rates of the schools ranged between 0 and 4.0%. Nine of the 630 (1.4%) examined became positive for CFA but were negative for mf. Although there were no mf positives, positive CFA and antibody results indicated the existence of Wuchereria bancrofti in Trincomalee. Highest antibody titres in an area correlated with the prevalences of urine antibodies and CFA. Spatial analysis showed LF transmission foci. Therefore, a combination of the non-invasive methods, urine ELISA and GPS mapping, will be a new effective surveillance system to identify hidden LF transmission foci., (Copyright © 2018. Published by Elsevier B.V.)

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., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Human African trypanosomiasis (HAT) is a disease caused by Kinetoplastid infection. Serological tests are useful for epidemiological surveillance. The aim of this study was to develop a multiplex serological assay for HAT to assess the diagnostic value of selected HAT antigens for sero-epidemiological surveillance. We cloned loci encoding eight antigens from Trypanosoma brucei gambiense, expressed the genes in bacterial systems, and purified the resulting proteins. Antigens were subjected to Luminex multiplex assays using sera from HAT and VL patients to assess the antigens' immunodiagnostic potential. Among T. b. gambiense antigens, the 64-kDa and 65-kDa invariant surface glycoproteins (ISGs) and flagellar calcium binding protein (FCaBP) had high sensitivity for sera from T. b. gambiense patients, yielding AUC values of 0.871, 0.737 and 0.858 respectively in receiver operating characteristics (ROC) analysis. The ISG64, ISG65, and FCaBP antigens were partially cross-reactive to sera from Trypanosoma brucei rhodesiense patients. The GM6 antigen was cross-reactive to sera from T. b. rhodesiense patients as well as to sera from VL patients. Furthermore, heterogeneous antibody responses to each individual HAT antigen were observed. Testing for multiple HAT antigens in the same panel allowed specific and sensitive detection. Our results demonstrate the utility of applying multiplex assays for development and evaluation of HAT antigens for use in sero-epidemiological surveillance., (Copyright © 2015 The Authors. Published by Elsevier Ireland Ltd.. All rights reserved.)

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., (© 2013 Elsevier Ireland Ltd. All rights reserved.)

Alveolar echinococcosis (AE) is one of the most lethal zoonotic parasitic infections. The diagnosis is based on the combination of the abdominal imaging including CT, MRI and PET, and serology. To develop a new diagnostic tool for AE with urine as samples, mouse-Echinococcus multilocularis (Em) model and then human cases were studied. The antibody levels of urine and serum samples from the infected mice and AE cases were well correlated with each other. The sensitivity and specificity of the method with urine were 91% and 98%, respectively, when IgG4 to crude Em was examined. Comparing with serum samples, the collection of urine is easier and safer and the urine diagnostic tool makes surveys of this silent disease easier., (© 2013.)

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., (© 2013.)

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., (Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.)

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., (Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.)

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., (Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.)

To eliminate lymphatic filariasis by means of mass drug administration, it is essential to have reliable data on the disease distribution and prevalence in targeted areas. In Matara district, Sri Lanka, self-administered questionnaires were mailed to 2105 local leaders questioning the presence and the numbers of elephantiasis and hydrocele cases. The information provided by them revealed that elephantiasis was clearly aggregated in the southern part of the district along the coast, while hydrocele was distributed rather evenly in the whole district, including Deniyaya region where no endemic filariasis had been known. To confirm active transmission of filariasis in Deniyaya, Wuchereria bancrofti antigen and filaria-specific urinary IgG4 antibody were measured with 2436 subjects. The positive rates for antigen and antibody were 0.6% and 4.3%, respectively. The titer analysis of IgG4 according to age revealed that the youngest IgG4 positive was 3 years old, and that in 10 years old or less, there were 16 positives out of 607 children examined (2.6%). It was concluded that filarial transmission at a low level was going on in the region.

A total of 14 Sri Lankan pregnant women, who were anti-Brugia pahangi urinary IgG4 positive, and their 14 newborn babies were followed up for the urinary antibody for 2 years by enzyme-linked immunosorbent assay. Eight babies showed positive IgG4 reaction, at least once within 4 months after birth. Urinary antibody titers of mothers and their babies measured around the perinatal period showed a significant positive correlation, suggesting that baby's IgG4 was transferred from the mother through the placenta. The IgG4 decreased gradually and became negative in all positive babies by day 339.3 after birth. The present result provides a basis to judge if a positive urine ELISA test among babies is due to a new filarial infection.