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.

Biochemical and molecular characterization of Leishmania parasites isolated from an endemic focus in eastern Sudan.

Twelve Leishmania isolates from visceral leishmaniasis patients in eastern Sudan were characterized using isoenzyme analysis, Southern blotting and polymerase chain reaction (PCR) 'fingerprinting'. Isoenzyme analysis revealed the presence of 3 zymodemes: MON-18, MON-30 and MON-82, corresponding to Leishmania donovani sensu stricto, L. infantum and L. archibaldi (still of uncertain taxonomic status), respectively. Southern blotting and PCR 'fingerprinting' revealed identical patterns for all 3 zymodemes, which were indistinguishable from those of L. donovani s.s.

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.

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.

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.

Development and application of the polymerase chain reaction for the detection and identification of Leishmania parasites in clinical material.

Detection, diagnosis and identification of Leishmaniasis may be difficult owing to low numbers of parasites present in clinical samples. The PCR has improved the sensitivity and specificity of diagnosis of several infectious diseases. A leishmania specific PCR assay was developed based on the SSUrRNA genes which amplifies DNA of all Leishmania species. Point mutations occurring within the rRNA genes allow differentiation of the Leishmania complexes using primers constructed with the 3/ ends complementary to the specific point mutations present in the SSU rRNA genes of the Leishmania species. Biopsy material, blood, lesion impressions and blood spots on filter paper can be used in the assay. In a longitudinal study on the incidence rates of VL, subclinical cases and PKDL in an endemic region of Sudan, filter paper blood spots from proven and suspected VL patients, PKDL and control samples from an endemic region in Sudan are being taken. The blood spots were analyzed in the DAT and by PCR and results compared with clinical and parasitological data. The first results indicate that the PCR on blood spots is a simple and sensitive means of detecting active VL; in PKDL patients parasites are detectable in the skin.

Identification of Leishmania from mucosal leishmaniasis by recombinant DNA probes.

Three cases of mucosal leishmaniasis are described. Parasites isolated from mucosal lesions were identified by Southern blot analysis of their genomic deoxyribonucleic acids (DNAs) using recombinant DNA probe pDK20. Parasites from 2 patients were identified as Leishmania donovani s.l. One of the patients had pure mucosal lesions, while in the second patient there was dissemination of the parasite to other organs. The spectrum of the disease caused by L. donovani is discussed. The parasite from the third patient was identified as L. major.

Characterization of serovars of the genus Leptospira by DNA hybridization with hardjobovis and icterohaemorrhagiae recombinant probes with special attention to serogroup sejroe.

Recombinant DNA probes derived from genomic libraries of serovars hardjobovis and icterohaemorrhagiae were applied for the characterization of leptospires. Differences in hybridization signals in combination with the banding pattern appear to provide good characteristics for strain typing. The banding patterns were easy to distinguish, since the recombinant DNA probes hybridized with a limited number of fragments. They were also indicative of genomic relationships between serovars. The probes suggested the existence of four subgroups with extensive genomic homology within the serogroup Sejroe. A number of serovars outside the serogroup Sejroe showed genomic homology with these subgroups. Amplification with the polymerase chain reaction showed a correlation with the genomic homologies demonstrated by Southern analysis. Knowledge about genomic relationships between leptospiral strains, as revealed by Southern analysis, may lead to a more rational approach for primer selection for polymerase chain reaction or cloning of particular genes.