Long-Term Storage of Cryptosporidium parvum for In Vitro Culture.

The long-term storage of Cryptosporidium life-cycle stages is a prerequisite for in vitro culture of the parasite. Cryptosporidium parvum oocysts, sporozoites, and intracellular forms inside infected host cells were stored for 6-12 mo in liquid nitrogen utilizing different cryoprotectants (dimethyl sulfoxide [DMSO], glycerol and fetal calf serum [FCS]), then cultured in vitro. Performance in vitro was quantified by estimating the total Cryptosporidium copy number with quantitative polymerase chain reaction (qPCR) in 3- and 7-day-old cultures. Although few parasites were recovered either from stored oocysts or from infected host cells, sporozoites stored in liquid nitrogen recovered from freezing successfully. More copies of parasite DNA were obtained from culturing those sporozoites than sporozoites excysted from oocysts kept at 4 C for the same period. The best performance was observed for sporozoites stored in Roswell Park Memorial Institute (RPMI) medium with 10% FCS and 5% DMSO, which generated 240% and 330% greater number of parasite DNA copies (on days 3 and 7 post-infection, respectively) compared to controls. Storage of sporozoites in liquid nitrogen is more effective than oocyst storage at 4 C and represents a more consistent approach for storage of viable infective Cryptosporidium aliquots for in vitro culture.

An easy 'one tube' method to estimate viability of Cryptosporidium oocysts using real-time qPCR.

Viability estimation of the highly resistant oocysts of Cryptosporidium remains a key issue for the monitoring and control of this pathogen. We present here a simple 'one tube' quantitative PCR (qPCR) protocol for viability estimation using a DNA extraction protocol which preferentially solubilizes excysted sporozoites rather than oocysts. Parasite DNA released from excysted sporozoites was quantified by real-time qPCR using a ribosomal DNA marker. The qPCR signal was directly proportional to the number of oocysts excysted, and a power-law relationship was noted between oocyst age and the proportion excysting. Unexcysted oocysts released negligible amounts of DNA making the method suitable for estimating viability of as few as 10 oocysts.

In vitro evaluation of traditionally used Surinamese medicinal plants for their potential anti-leishmanial efficacy.

ETHNOPHARMACOLOGICAL RELEVANCE: Plant-based preparations are extensively used in Surinamese folk medicine for treating leishmaniasis, but often without a scientific rationale. AIM OF THE STUDY: To evaluate 25 Surinamese medicinal plants for their potential efficacy against leishmaniasis. MATERIALS AND METHODS: Concentrated plant extracts were evaluated for their effect on the viability of L. (V.) guyanensis AMC, L. (L.) major NADIM5, and L. (L.) donovani GEDII promastigotes, as well as intracellular amastigotes of L. (L.) donovani BHU814 in infected THP-1 cells. Selectivity was assessed by cytotoxicity against THP-1 cells. RESULTS: The only plant extract that showed potentially meaningful anti-leishmanial activity was that from Solanum lycocarpum that displayed mean IC50 values of about 51, 61, and <16 µg/mL against L. (V) guyanensis, L. (L) major, and L. (L) donovani promastigotes, respectively; about 374 µg/mL against L. (L) donovani amastigotes; and >500 µg/mL against THP-1 cells. The Bryophyllum pinnatum, Inga alba, and Quassia amara extracts displayed moderate to high IC50 values against promastigotes (about 51 to >500 µg/mL) and/or amastigotes (about 224 to >500 µg/mL) but were relatively toxic to THP-1 cells (IC50 values <16 to about 42 µg/mL). The remaining plant extracts exhibited in many cases IC50 values close to, around, or above 500µg/mL against promastigotes, amastigotes, and THP-1 cells. CONCLUSIONS: The S. lycocarpum preparation may be useful against leishmaniasis and may have a good safety index, warranting further investigations into its active constituents and mechanism(s) of action.

Monitoring the response of patients with cutaneous leishmaniasis to treatment with pentamidine isethionate by quantitative real-time PCR, and identification of Leishmania parasites not responding to therapy.

BACKGROUND: Leishmania (Viannia) guyanensis is believed to be the principal cause of cutaneous leishmaniasis (CL) in Suriname. This disease is treated with pentamidine isethionate (PI), but treatment failure has increasingly been reported. AIM: To evaluate PI for its clinical efficacy, to compare parasite load, and to assess the possibility of treatment failure due to other infecting Leishmania species. METHODS: Parasite load of patients with CL was determined in skin biopsies using real-time quantitative PCR before treatment and 6 and 12 weeks after treatment. Clinical responses were evaluated at week 12 and compared with parasite load. In parallel, molecular species differentiation was performed. RESULTS: L. (V.) guyanensis was the main infecting species in 129 of 143 patients (about 90%). PI treatment led to a significant decrease (P < 0.001) in parasite counts, and cured about 75% of these patients. Treatment failure was attributable to infections with Leishmania (Viannia) braziliensis, Leishmania (Leishmania) amazonensis and L. (V.) guyanensis (1/92, 1/92 and 22/92 evaluable cases, respectively). There was substantial agreement beyond chance between the parasite load at week 6 and the clinical outcome at week 12, as indicated by the κ value of 0.61. CONCLUSIONS: L. (V.) guyanensis is the main infecting species of CL in Suriname, followed by L. (V.) braziliensis and L. (L.) amazonensis. Furthermore, patient response to PI can be better anticipated based on the parasite load 6 weeks after the treatment rather than on parasite load before treatment.

Diagnosis of canine leishmaniasis in the endemic area of Belo Horizonte, Minas Gerais, Brazil by parasite, antibody and DNA detection assays.

Canine leishmaniasis caused by Leishmania chagasi (L. infantum) is found throughout the South American continent, including Brazil, and dogs are considered to be the main reservoir host for this parasite. To support the implementation of a diagnostic protocol for surveillance of the disease in the region of Belo Horizonte (Minas Gerais, Brazil) we have compared the sensitivity and specificity of two serological tests, indirect immunofluorescent antibody test (IFAT) and direct agglutination test (DAT), with the combination of direct microscopy-culture-PCR as the gold standard, using samples obtained from 103 dogs in the city of Belo Horizonte, Minas Gerais. The currently used standard serodiagnostic test, IFAT, had a sensitivity of 100% and its specificity was 74% compared to the gold standard of the study. The sensitivity and specificity of the DAT were 100% and 91%, respectively. On the basis of this study it is recommended to change from the IFAT to DAT for the serodiagnosis of canine leishmaniasis because of the superior specificity of the test combined with its user-friendliness.

Field evaluation of a fast anti-Leishmania antibody detection assay in Ethiopia.

A fast agglutination screening test (FAST) for the detection of Leishmania antibodies in human serum samples was evaluated under harsh field conditions in northern Ethiopia. Test performance was compared with a standard serological test, namely the direct agglutination test (DAT), and with parasitology. In total, 103 suspected cases were recruited for the study. Based on parasitological examination, 49 patients were confirmed of having visceral leishmaniasis (VL) and the other 54 suspected cases were parasitologically negative. Field evaluation of FAST was possible in blood samples of 89 patients. FAST had 4 false negative results and 13 false positive results. DAT had 2 false negative results and 20 false positive results. A good degree of agreement (86.9%) was observed between FAST and DAT (kappa value 0.73). In this field-based evalauation, the sensitivity and specificity of FAST were found to be 91.1% (95% CI 77.9-97.1) and 70.5% (95% CI 54.6-82.8), respectively, compared with 95.3% (95% CI 82.9-99.2) and 62.3% (95% CI 47.9-74.9) for DAT. FAST had a high predictive value of a negative test, demonstrating that FAST could be utilised to exclude rapidly non-VL patients from a large population of suspects with fever and splenomegaly in endemic areas.

Plasmodium falciparum: evaluation of a quantitative nucleic acid sequence-based amplification assay to predict the outcome of sulfadoxine-pyrimethamine treatment of uncomplicated malaria.

A quantitative nucleic acid sequence-based amplification (QT-NASBA) assay was employed to predict retrospectively the outcome of sulfadoxine-pyrimethamine (SP) treatment of uncomplicated malaria in children aged <6 years in an endemic region. Blood samples were collected at initial diagnosis and during follow-up. Mutation-specific nested PCR methods to analyse DHFR (Arg-59) and DHPS (Glu-540) mutations that are associated with SP drug resistance were applied. Parasite genotyping was performed to distinguish between re-infection and recrudescence. Eighty-six patients were recruited of which 66 were available for follow-up. Nine children were classified as early treatment failure, 13 cases were classified as late clinical failure, 32 as late parasitological failure, and only 12 children had an adequate clinical and parasitological response. DHFR and DHPS mutations conferring SP resistance were abundant in the Plasmodium population. Blood samples obtained 7 days after treatment were used to predict retrospectively the outcome of SP treatment. QT-NASBA was able to give a correct prediction of treatment outcome in 85.7% of the cases. Positive predictive value (PPV) of QT-NASBA case was 95% (95% confidence interval = 88.3-100) and negative predictive value (NPV) was 63% (95% CI = 39.5-86.5). In contrast, microscopy correctly predicted outcome in only 37.5% of the cases. PPV of microscopy was 100% (95% CI = 73.9-100) and the NPV was 25.5% (95% CI = 13.0-38.0). The analysis of a day 7 blood sample with QT-NASBA allows for the prediction of late clinical or parasitological treatment failure in the majority of the cases analysed in the present study.

Usefulness of quantitative nucleic Acid sequence-based amplification for diagnosis of malaria in an academic hospital setting.

The aim of the present study was to evaluate the usefulness of quantitative nucleic acid sequence-based amplification (QT-NASBA) to detect Plasmodium spp. in diagnostic specimens of patients suspected of having malaria in a clinical setting in a non-endemic country. During the 4-month recruitment period, 113 patients were enrolled in the study, of which 93 were diagnosed as non-malaria and 20 as malaria cases on the basis of clinical and microscopic criteria. All microscopically positive cases had QT-NASBA counts of >0.1 parasites/ micro l and there was a significant positive correlation between the parasite counts obtained with both diagnostic methods. Of the 93 microscopically negative cases, six had a positive QT-NASBA result. Three of these cases had a recent history of malaria for which specific treatment was taken. In the other three cases there was no history of malaria and QT-NASBA results in these cases were near the cut-off level (>0.1 parasites/ micro l) of the test. The results demonstrate that QT-NASBA is a useful technology for the diagnosis of malaria in a reference laboratory, and it is very helpful in cases of low parasitemia.

Development of a fast agglutination screening test (FAST) for the detection of anti-Leishmania antibodies in dogs.

A fast agglutination screening test (FAST) for the detection of anti-Leishmania antibodies in serum samples from dogs with visceral leishmaniosis was developed. The test is based on the direct agglutination test (DAT), but combines a higher parasite concentration with a smaller test volume. In contrast to the DAT, the FAST makes use of only one serum dilution and the results can be read within 3 h as opposed to 18-20 h for the DAT. The FAST was evaluated using serum samples of confirmed cases of the disease and healthy controls collected in the most important endemic regions of canine visceral leishmaniosis, import cases of canine leishmaniosis in a non-endemic country, from non-endemic healthy controls and from dogs with other diseases. The performance of the FAST was compared with standard DAT. In the present study, the FAST had a sensitivity of 93.6% and a specificity of 89.0%. The DAT had a sensitivity of 88.6% and a specificity of 96.7%. Furthermore, using a large panel of serum samples of previously examined DAT positive or negative dogs it was shown that degree of agreement between the two tests was high (95.7%; kappa value = 0.91). The FAST offers the advantages of the DAT based on freeze-dried antigen with respect to stability of the antigen, sensitivity and specificity. Moreover, the FAST allows the rapid screening of a large number of samples, which makes the test very useful for epidemiological screening of large populations of dogs.

Sero-epidemiological assessment and diagnosis of visceral leishmaniasis in an endemic locality using Fast Agglutination Screening Test (FAST).

The Fast Agglutination Screening Test (FAST) was employed on sera obtained from an endemic area of visceral leishmaniasis in southwestern Ethiopia, in February 2000. The study involved (i) active case detection among 1575 residents of two villages; and (ii) passive case detection in an outpatient clinic. Sera of 1587 individuals, including 143 sera of previously treated VL patients, were tested. Based on the size of agglutination mat, the FAST results were read qualitatively as non-reactive (-), weakly reactive (1+), moderately reactive (2+) and highly reactive (3+). All FAST reactive sera were re-tested with the Direct Agglutination Test (DAT). After clinical screening of 1625 individuals, 61 individuals with signs and symptoms of early or late VL were found; 26 sera were FAST positive. Twenty-two of these suspected VL cases were subjected to parasitological examination using lymph node aspirates. Eighteen (81.8%) were confirmed either by demonstration of amastigotes in smears or promastigotes in NNN cultures. FAST reactive anti-leishmanial antibodies were detected in 4.5% of untreated and 70.6% of previously treated patients. Forty-five sera of 1390 previously untreated asymptomatic individuals (3.2%) were found to be FAST positive. This report demonstrates that FAST is a rapid and cost-effective screening test for the diagnosis and sero-epidemiological surveillance of visceral leishmaniasis.

Development and application of 'simple' diagnostic tools for visceral leishmaniasis.

The diagnosis of visceral leishmaniasis is difficult. Due to the limitations of direct methods to detect parasites, indirect immunological methods are widely employed. The simple affordable and sensitive/specific direct agglutination test (DAT) is perhaps the most important diagnostic tool under field conditions. A significant improvement of this test is the use of a freeze-dried antigen, which is heat-stable and has a long shelf-live even under harsh conditions. The performance of this antigen in DAT has been evaluated using samples collected in East Africa. The results of these studies are presented. The detection of Leishmania infection in HIV-co-infected patients is difficult. The combination of DAT-PCR may be useful for the detection of parasite infection in these patients. Finally, we present data to show that the DAT based on the freeze-dried antigen can also be used for the detection of anti-Leishmania antibodies in dogs.

Detection and quantification of Plasmodium falciparum in blood samples using quantitative nucleic acid sequence-based amplification.

A quantitative nucleic acid sequence-based amplification (QT-NASBA) assay for the detection of Plasmodium parasites has been developed. Primers and probes were selected on the basis of the sequence of the small-subunit rRNA gene. Quantification was achieved by coamplification of the RNA in the sample with one modified in vitro RNA as a competitor in a single-tube NASBA reaction. Parasite densities ranging from 10 to 10(8) Plasmodium falciparum parasites per ml could be demonstrated and quantified in whole blood. This is approximately 1,000 times more sensitive than conventional microscopy analysis of thick blood smears. Comparison of the parasite densities obtained by microscopy and QT-NASBA with 120 blood samples from Kenyan patients with clinical malaria revealed that for 112 of 120 (93%) of the samples results were within a 1-log difference. QT-NASBA may be especially useful for the detection of low parasite levels in patients with early-stage malaria and for the monitoring of the efficacy of drug treatment.

Detection of Onchocerca volvulus DNA in pools of wild-caught Simulium ochraceum by use of the polymerase chain reaction.

The presence of Onchocerca volvulus DNA in experimentally infected flies can now be detected by use of the PCR, so that, for example, one infected Simulium damnosum can be detected in a pool of 100 uninfected flies or one S. ochraceum can be detected in pools of 20-40. As this PCR technique is specific for O. volvulus, the results are not confounded by the presence of other, unimportant, Onchocerca species, and the technique could replace time-consuming, manual dissection of flies. In 1996 and 1997, pools of 16-21 Simulium ochraceum were tested by the PCR technique. These flies had been collected biting man, between 1992 and 1994, from two hyperendemic coffee estates (fincas) in Guatemala, and stored in commercial (95%) ethanol. Collections at finca Buena Vista (869 flies in 52 pools) were made 1-2 weeks and 46 weeks after 45% of eligible subjects had been treated with ivermectin for the first time. At finca El Brote, collections (360 flies in 18 pools) were made 13 weeks before and 7 weeks after 97% of eligible subjects had received their first treatment. DNA was easily recovered from simuliids that had been stored in ethanol for up to 4 years. Of the nine pools of flies with visible blood collected at Buena Vista, each of 20 flies, eight tested positive for O. volvulus DNA. In flies without blood, 13 of 22 pools collected at Buena Vista just after treatment tested positive, whereas there were 14 positives in 22 pools taken 46 weeks later (P > 0.05). At El Brote, nine of 10 pre-treatment pools were positive, compared with three of eight taken 7 weeks post-treatment (P = 0.04), indicating that the treatments in this finca had reduced infection in the vector, and possibly transmission, by about 60%. A sub-sample of Buena Vista flies was divided into 19 sets of three separate sub-pools containing heads, thoraces and abdomens. Three pools of heads alone were positive, and had corresponding pools of positive abdomens. Three positive pools of thoraces had negative corresponding pools of heads and abdomens. These results show that PCR can be used to determine the prevalence of O. volvulus DNA in wild-caught S. ochraceum. As the infection rates observed were higher than expected from dissections reported by other workers, PCR-determined rates may not be directly comparable with traditional parameters based on the dissection of flies to reveal O. volvulus larvae.

Use of PCR for diagnosis of post-kala-azar dermal leishmaniasis.

Microscopy and PCR were compared for use in the diagnosis of post-kala-azar dermal leishmaniasis (PKDL) in 63 patients. Aspirates of lymph nodes (samples from 52 patients), skin (23 samples), and bone marrow (18 samples) were used. For 11 patients lymph node aspiration could be repeated 6 months after they recovered from PKDL. During active PKDL, PCR was positive for 42 of 52 (80.8%) lymph node aspirates and 19 of 23 (82.7%) skin aspirates, whereas microscopy was positive for only 9 of 52 (17.3%) lymph node aspirates and 7 of 23 (30.4%) skin aspirates. PCR was always positive when parasites were seen by microscopy. When the results obtained with lymph node and skin aspirates from the same patient (n = 16) were compared, there was complete agreement. Bone marrow samples were negative by microscopy and PCR for 16 patients and positive by both methods for 1 patient; for one sample only the PCR was positive. PCR confirmed the co-occurrence of visceral leishmaniasis and PKDL in one patient and confirmed the suspicion of this co-occurrence in the other patient. After recovery, no parasites were found by microscopy, but 2 of 11 (18.2%) samples were still positive by PCR. Thirty negative controls were all found to be PCR negative, and 15 positive controls were all PCR positive. Cross-reactions with Mycobacterium leprae could be ruled out. In conclusion, PCR with inguinal lymph node or skin aspirates is suitable for confirming the clinical diagnosis of PKDL. In some patients, lymph node aspirates are probably preferred because aspiration of material from the skin may leave scars.

Evaluation of PCR for diagnosis of visceral leishmaniasis.

An evaluation of Leishmania PCR was performed with bone marrow, lymph node, and blood samples from 492 patients, 60 positive controls, and 90 negative controls. Results were compared with microscopy results for Giemsa-stained smears. PCR and microscopy of lymph node and bone marrow aspirates from patients with microscopically confirmed visceral leishmaniasis (VL) were equally sensitive. However, in patients clinically suspected of having VL and in whom parasites could not be demonstrated by microscopy, PCR was positive for 12 of 23 (52.2%) lymph node aspirates and 8 of 12 (66.7%) bone marrow aspirates, thus confirming the clinical diagnosis of VL. With PCR on filter paper, Leishmania DNA was detected in the blood of 33 of 47 (70%) patients with confirmed VL and in 2 of 11 (19%) patients suspected of having VL. Positive PCR results were more frequently found for blood samples on filter paper than for samples stored in EDTA. In conclusion, PCR is a more sensitive method than microscopy for the detection of Leishmania in lymph node and bone marrow aspirates, being especially useful for the confirmation of cases of suspected VL. Blood from a finger prick may be used for the initial PCR screening of people suspected of having VL. If the PCR of blood is negative, one should perform PCR with lymph node and/or bone marrow material, because PCR with these materials is more often positive.

Polymerase chain reaction for detecting Onchocerca volvulus in pools of blackflies.

The detection of Onchocerca volvulus infected simuliids or blackflies is routinely done by dissection and microscopic examination of individual flies, but this method is tedious and time consuming. Here we describe a method of detecting single O. volvulus infected blackflies in pools of uninfected blackflies. Using a PCR with Onchocerca specific primers it is possible to reproducibly detect one heavily infected blackfly in a pool of 80 flies, or to detect one blackfly inoculated with one microfilaria in a pool of 20 flies. With the method described large numbers of blackflies can be rapidly screened for the presence of O. volvulus infected flies.

[Imported visceral leishmaniasis (kala azar) in a German infant]. / Importierte viszerale Leishmaniose (Kala Azar) bei einem deutschen Kleinkind.

We here report the case of a 1 4/12 year old girl with visceral leishmaniasis. Returning from a trip to Mallorca she presented with pancytopenia, splenomegaly and fever and was admitted to hospital with suspected malignancy. Diagnosis was established microscopically from bone marrow smear and confirmed by PCR-assisted amplification of leishmania-specific DNA from peripheral blood. Treatment was conducted with stibogluconate for 25 days. Defervescence and improvement of clinical symptoms was seen after 4 days of treatment. Infection due to Leishmania donovani can be acquired throughout the entire mediterranean area and should therefore be included in the differential diagnosis of suspected malignancies in patients with a history of travel to mediterranean countries. PCR proved to be a sensitive tool for establishing the diagnosis of visceral leishmaniasis.

Sudanese mucosal leishmaniasis: epidemiology, clinical features, diagnosis, immune responses and treatment.

The epidemiology, clinical features, pathology, immune responses, diagnosis and treatment of 14 patients with mucosal leishmaniasis in the Sudan are described. The condition occurred mainly in adult males, particularly in certain closely related tribes from the western Sudan. It affected the mucosa of the upper respiratory tract and/or the oral mucosa and sometimes followed treated kala azar. The parasites were sometimes confined to the mucosa, sometimes spread to the lymph nodes, and rarely infected the bone marrow and spleen. One of the 2 patients with both visceral and mucosal leishmaniasis differed from classical kala azar cases; his infection was longer lasting, he was leishmanin positive, and his peripheral mononuclear cells proliferated in response to leishmanial antigens. Mucosal leishmaniasis following treated kala azar is a similar phenomenon to post-kala azar dermal leishmaniasis and post-kala azar uveitis. Post-kala azar mucosal leishmaniasis can therefore be added to the other post-kala azar leishmanial infections. Using the polymerase chain reaction, Southern blot analysis with specific probes, and isoenzyme characterization, the causative parasite was identified as Leishmania donovani in 4 patients and as L. major in one. Unlike American mucocutaneous leishmaniasis, mucosal leishmaniasis in the Sudan was not preceded or accompanied by cutaneous lesions and the response to pentavalent antimony or ketoconazole was good.

Visceral leishmaniasis: use of the polymerase chain reaction in an epidemiological study in Baringo District, Kenya.

The polymerase chain reaction was applied to capillary blood spots dried on filter paper from 20 parasitologically proved cases of visceral leishmaniasis (VL), 21 subclinical cases, and 11 healthy controls in a longitudinal study of anthroponotic VL in Baringo District, Kenya. Leishmania deoxyribonucleic acid (DNA) was detected 10.5 months before diagnosis and up to 3 years after diagnosis and apparently successful treatment. Subclinical cases can have detectable circulating parasite DNA in their blood. These findings may indicate that subclinical cases can be a reservoir and formerly treated VL patients can remain a reservoir for a long time. Xenodiagnosis should be performed on subclinical cases and former VL patients to establish their role in transmission of VL in Kenya.