Factors involved in immunity to Nematospiroides dubius infections in mice

Thesis (M.Sc.) -- University of Adelaide, Dept. of Microbiology and Immunology, 198

An improved method for undertaking limiting dilution assays for in vitro cloning of Plasmodium falciparum parasites

Abstract. Background: Obtaining single parasite clones is required for many techniques in malaria research. Cloning by limiting dilution using microscopy-based assessment for parasite growth is an arduous and labor-intensive process. An alternative method for the detection of parasite growth in limiting dilution assays is using a commercial ELISA histidine-rich protein II (HRP2) detection kit. Methods. Detection of parasite growth was undertaken using HRP2 ELISA and compared to thick film microscopy. An HRP2 protein standard was used to determine the detection threshold of the HRP2 ELISA assay, and a HRP2 release model was used to extrapolate the amount of parasite growth required for a positive result. Results: The HRP2 ELISA was more sensitive than microscopy for detecting parasite growth. The minimum level of HRP2 protein detection of the ELISA was 0.11ng/ml. Modeling of HRP2 release determined that 2,116 parasites are required to complete a full erythrocytic cycle to produce sufficient HRP2 to be detected by the ELISA. Under standard culture conditions this number of parasites is likely to be reached between 8 to 14 days of culture. Conclusions: This method provides an accurate and simple way for the detection of parasite growth in limiting dilution assays, reducing time and resources required in traditional methods. Furthermore the method uses spent culture media instead of the parasite-infected red blood cells, enabling culture to continue

Accuracy of a Commercial IgM ELISA for the Diagnosis of Human Leptospirosis in Thailand

The Leptospira immunoglobulin M enzyme-linked immunosorbent assay (IgM ELISA) has been recommended for the rapid diagnosis of leptospirosis in endemic areas. We conducted a retrospective case-control study of 218 patients (109 leptospirosis cases confirmed by Leptospira culture and/or microscopic agglutination test and 109 control patients with acute febrile illness) to evaluate the diagnostic accuracy of a commercial IgM ELISA (Panbio) in northeast Thailand. Paired serum samples taken on admission and at least 10 days after the onset of symptoms were tested. Using the cutoff value recommended by the manufacturer (11 Panbio units), sensitivity and specificity of IgM ELISA on paired sera were 90.8% and 55.1%. A receiver operating characteristic curve was used to determine the optimal cutoff value. This was 20 Panbio units, which gave a sensitivity and specificity of 76.1% and 82.6%, respectively, on paired sera. We conclude that using either cutoff value, the accuracy of IgM ELISA is limited in our setting

FACTORS ASSOCIATED WITH ACUTE RENAL FAILURE IN ADULTS WITH SEVERE FALCIPARUM MALARIA

Abstract. We conducted a retrospective study of patients with severe falciparum malaria to determine factors associated with malarial acute renal failure (MARF). We reviewed 262 medical records of adults hospitalized with severe falciparum malaria in Thailand from 2004 to 2008. The incidence of MARF in our study population was 44% (115/262); 75% (86/115) of these had MARF on admission and 25% (29/115) developed MARF during hospitalization. The majority of MARF patients presented in a hypercatabolic state (62%, 68/109) and were non-oliguric (48%, 55/115) or oliguric (44%, 51/115). Forty-six percent of MARF patients (53/115) required renal replacement therapy for a median duration of 4.5 days. Patients with MARF had significantly higher complication rates (p<0.001), longer duration of hospitalization (p<0.001) and a higher case fatality rate (p=0.001). Using stepwise multiple logistic regression analysis by backward selection method, factors associated with MARF were advanced age [odds ratios (OR); 95% confidence intervals (CI) 1.037 (1

Estimation of the Total Parasite Biomass in Acute Falciparum Malaria from Plasma PfHRP2

BACKGROUND: In falciparum malaria sequestration of erythrocytes containing mature forms of Plasmodium falciparum in the microvasculature of vital organs is central to pathology, but quantitation of this hidden sequestered parasite load in vivo has not previously been possible. The peripheral blood parasite count measures only the circulating, relatively non-pathogenic parasite numbers. P. falciparum releases a specific histidine-rich protein (PfHRP2) into plasma. Quantitative measurement of plasma PfHRP2 concentrations may reflect the total parasite biomass in falciparum malaria. METHODS AND FINDINGS: We measured plasma concentrations of PfHRP2, using a quantitative antigen-capture enzyme-linked immunosorbent assay, in 337 adult patients with falciparum malaria of varying severity hospitalised on the Thai–Burmese border. Based on in vitro production rates, we constructed a model to link this measure to the total parasite burden in the patient. The estimated geometric mean parasite burden was 7 × 10(11) (95% confidence interval [CI] 5.8 × 10(11) to 8.5 × 10(11)) parasites per body, and was over six times higher in severe malaria (geometric mean 1.7 × 10(12), 95% CI 1.3 × 10(12) to 2.3 × 10(12)) than in patients hospitalised without signs of severity (geometric mean 2.8 × 10(11), 95% CI 2.3 × 10(11) to 3.5 × 10(11); p < 0.001). Parasite burden was highest in patients who died (geometric mean 3.4 × 10(12), 95% CI 1.9 × 10(12) to 6.3 × 10(12); p = 0.03). The calculated number of sequestered parasites increased with disease severity and was higher in patients with late developmental stages of P. falciparum present on peripheral blood smears. Comparing model and laboratory estimates of the time of sequestration suggested that admission to hospital with uncomplicated malaria often follows schizogony—but in severe malaria is unrelated to stage of parasite development. CONCLUSION: Plasma PfHRP2 concentrations may be used to estimate the total body parasite biomass in acute falciparum malaria. Severe malaria results from extensive sequestration of parasitised erythrocytes

Paracheck-Pf® accuracy and recently treated Plasmodium falciparum infections: is there a risk of over-diagnosis?

BACKGROUND: An assessment of the accuracy of Paracheck Pf, a malaria rapid diagnostic test (RDT) detecting histidine rich protein 2 was undertaken amongst children aged 6-59 months in eastern Democratic Republic of Congo. METHODS: This RDT assessment occurred in conjunction with an ACT efficacy trial. Febrile children were simultaneously screened with both RDT and high quality microscopy and those meeting inclusion criteria were followed for 35 days. RESULTS: 358 febrile children were screened with 180 children recruited for five weeks follow-up. On screening, the RDT accurately diagnosed all 235 true malaria cases, indicating 100% RDT sensitivity. Of the 123 negative slides, the RDT gave 59 false-positive results, indicating 52.0% (64/123) RDT specificity. During follow-up after treatment with an artemisinin-based combination therapy, 98.2% (110/112), 94.6% (106/112), 92.0% (103/112) and 73.5% (50/68) of effectively treated children were still false-positive by RDT at days 14, 21, 28 and 35, respectively. CONCLUSION: Results show that though the use of Paracheck-Pf is as sensitive as microscopy in detecting true malaria cases, a low specificity did present a high frequency of false-positive RDT results. What's more, a duration of RDT false-positivity was found that significantly surpassed the 'fortnight' after effective treatment reported by its manufacturer. Though further research is needed in assessing RDT accuracy, study results showing the presence of frequent false positivity should be taken into consideration to avoid clinicians inappropriately focusing on malaria, not identifying the true cause of illness, and providing unnecessary treatment

PfHRP2 and PfLDH antigen detection for monitoring the efficacy of artemisinin-based combination therapy (ACT) in the treatment of uncomplicated falciparum malaria

<p>Abstract</p> <p>Background</p> <p>An assessment of the accuracy of two malaria rapid diagnostic tests (RDT) for the detection of <it>Plasmodium falciparum </it>histidine-rich protein 2 (<it>Pf</it>HRP2) or <it>Pf </it>lactate dehydrogenase (<it>Pf</it>LDH) was undertaken in children aged between six and 59 months included in an anti-malarial efficacy study in Benin.</p> <p>Methods</p> <p>In Allada (Benin), 205 children aged 6-59 months with falciparum malaria received either artesunate-amodiaquine (ASAQ), artemether-lumefantrine (AL), or sulphadoxine-pyrimethamine (SP). Children included in the study were simultaneously followed by both RDT and high-quality microscopy for up to 42 days.</p> <p>Results</p> <p>At the time of inclusion, <it>Pf</it>HRP2-based tests were positive in 203 children (99%) and <it>Pf</it>LDH-based tests were positive in 204 (99.5%). During follow-up, independent of the treatment received, only 17.3% (28/162) of children effectively cured were negative with the <it>Pf</it>HRP2 RDT at day 3, with a gradual increase in specificity until day 42. The specificity of antigen detection with the <it>Pf</it>LDH test was 87% (141/162) on day 3, and between 92% and 100% on days 7 to 42. A statistical difference was observed between the persistence of <it>Pf</it>HRP2 and <it>Pf</it>LDH antigenaemia during follow-up in children treated with artemisinin-based combination therapy (ACT) but not with SP.</p> <p>Conclusion</p> <p>Although both RDTs are as sensitive as microscopy in detecting true malaria cases, the <it>Pf</it>HRP2 RDT had very low specificity during follow-up until day 28. On the other hand, the <it>Pf</it>LDH test could be used to detect failures and, therefore, to assess anti-malarial efficacy.</p

Immunoassays Based on Penicillium marneffei Mp1p Derived from Pichia pastoris Expression System for Diagnosis of Penicilliosis

BACKGROUND: Penicillium marneffei is a dimorphic fungus endemic in Southeast Asia. It can cause fatal penicilliosis in humans, particularly in HIV-infected people. Diagnosis of this infection is difficult because its clinical manifestations are not distinctive. Specialized laboratory tests are necessary to establish a definitive diagnosis for successful management. We have demonstrated previously that a cell wall mannoprotein Mp1p, abundant in P. marneffei, is a potential biomarker for diagnosis of P. marneffei infections. In the present study, we describe immunoassays based on Mp1p derived from the yeast Pichia pastoris expression system. METHODOLOGY/PRINCIPAL FINDINGS: We generated monoclonal antibodies (MAbs) and rabbit polyclonal antibodies (PAbs) against Mp1p expressed in P. pastoris. Subsequently, we developed two Mp1p antigen capture ELISAs which employed MAbs for both the capture and detecting antibodies (MAb-MAb pair) or PAbs and MAbs as the capture and detecting antibodies (PAbs-MAb pair) respectively. The two Mp1p antigen ELISAs detected Mp1p specifically in cultures of P. marneffei yeast phase at 37-40 degrees C and had no cross-reaction with other tested pathogenic fungi. The sensitivities and specificities of the two antigen assays were found to be 55% (11/20) and 99.6% (538/540) for MAb-MAb Mp1p ELISA, and 75% (15/20) and 99.4% (537/540) for PAbs-MAb Mp1p ELISA performed using 20 sera with culture-confirmed penicilliosis, and 540 control sera from 15 other mycosis patients and 525 healthy donors. Meanwhile, we also developed an anti-Mp1p IgG antibody ELISA with an evaluated sensitivity of 30% (6/20) and a specificity of 98.5% (532/540) using the same sera. Furthermore, combining the results of Mp1p antigen and antibody detection improved the sensitivity of diagnosis to 100% (20/20). CONCLUSIONS/SIGNIFICANCE: Simultaneous detection of antigen and antibody using the immunoassays based on Mp1p derived from P. pastoris greatly improves detection sensitivity. The procedures should be useful for the routine diagnosis of penicilliosis.published_or_final_versio