Mouse monoclonal antibodies against human urinary protein Sm-UP(2)IP of Schistosoma mansoni.

There are innumerable clinical and pathological problems associated with schistosomiasis that have necessitated various control programs. Successful control would naturally depend on effective rapid diagnosis in the field. However, the overlapping distribution of urinary and intestinal schistosomiasis in hyperendemic areas calls for differential diagnosis. This study was aimed at producing anti-Schistosoma mansoni monoclonal antibodies (MAbs) for possible utilization in assays to detect antigens in the urine of infected persons. In order to raise antibodies to less immunogenic urinary parasite antigens, BALB/c mice were immunized with Schistosoma mansoni soluble worm antigens (Sm-SWA) while urinary proteins (Sm-UP(2)IP), isolated from infected human urine samples, was used as a final booster before cell fusion. Hybridoma cells were obtained by the fusion of mouse myeloma and spleen cells from the immunized mice, which were screened by microplate ELISA and then studied further to obtain anti-S. mansoni specific MAbs. The MAbs analyzed presented IgM isotypes. The reactivity of anti-S. mansoni MAbs with Sm-UP(2)IP, 13/43 (30.2%), MAbs showed stronger reactivity. It was observed that one of the MAbs cross-reacted with antigen associated with S. haematobium urinary antigen (Sh-UP(2)IP). Nine (9/13, 69.2%) MAbs recognized glycoprotein antigenic epitopes of Sm-UP(2)IP and Sm-SWA. On the other hand, 4/13 (30.8%) MAbs recognized carbohydrate antigenic epitopes. Band size of 8.9 kDa associated with Sm-UP(2)IP was detected by the 13 MAbs. With Sm-SWA, all the MAbs detected band sizes of 177.8 and 158.5 kDa. In addition, three MAbs recognized a 38.9 kDa band. The generation of anti-S. mansoni species-specific MAbs offers opportunities to develop a specific MAb-based diagnostic tool for use in the field to detect Schistosoma mansoni infection in Ghana.

A survey of female genital schistosomiasis of the lower reproductive tract in the volta basin of Ghana.

OBJECTIVE: To determine the prevalence of female genital schistosomiasis in riparian communities in the Volta basin of Ghana, DESIGN: The study was a cross-sectional study conducted among women 15-49 years in the Volta Basin. Urinary schistosomiasis prevalence was determined using microscopy. A structured questionnaire was also administered to collect information on the demography, obstetric history and reproductive health experiences. Cervical punch biopsy was collected from women who consented to be screened for FGS. Descriptive statistics was used to determine frequency of occurrence, chi squared and logistic regression to identify associated variables RESULTS: Urinary schistosomiasis prevalence among the women was 24.8% while 10.6% of them diagnosed with FGS. More FGS diagnosed women (57.7%, p value =0.04%) were observed to report copious discharge, vaginal itch (80.8%, p=0.042) and lower abdominal pain (66.7%, p= 0.041) compared to FGS negative women. The predominant abnormal observation of the lower genital tract made was erythematous cervix (18.8%). CONCLUSION: The study confirms the reproductive health symptoms associated with FGS and recommends awareness creation on FGS among women in endemic communities to facilitate early treatment.

Sero-epidemiology of toxoplasmosis amongst pregnant women in the greater accra region of ghana.

OBJECTIVES: To investigate Toxoplasma infection among pregnant women in relation to exposure to infection risk, age and pregnancy-related risk factors. DESIGN AND METHODS: This cross-sectional study involved 294 pregnant women attending ante-natal clinic in Accra who consented to participate. Personal and Toxoplasma infection risk related data were obtained by questionnaire interviews. Venous blood was safely drawn from each participant and spun to obtain sera. Each of the 159 randomly selected serum samples was tested for specific anti-Toxoplasma (anti-T. gondii) antibodies IgG, IgA and IgM using a commercial ELISA kit (Calbiotech Inc., CA). ELISA results were correlated with exposure to possible infection risk factors as well as age and pregnancy-related risk factors. RESULTS: The 159 women aged 15-40 years in their first, second and third trimesters, numbered 29, 70 and 60, respectively. An overall anti-T. gondii antibodies IgG, IgA and IgM seroprevalence of 92.5% (147/159) was recorded, with 4.1% (6/147) of them having anti-IgG only. The remaining 88.7% (141/159) had anti-Toxoplasma antibodies IgG, IgA and IgM in various combinations and consisted of 17.7% (25/141) in their first, 44.0% (62/141) in their second, and 38.3% (54/141) in their third, trimesters. Twelve women (7.6%) were seronegative for all 3 antibodies CONCLUSIONS: Seroprevalence was high among the women and exposure to contact with cats' faeces was found to be the major T. gondii infection risk factor. Age and pregnancy-related risk factors did not have association with T. gondii infection within the limitations of this study.

Infant schistosomiasis in Ghana: a survey in an irrigation community.

We used a rapid, visually read, field applicable monoclonal antibody (MoAb)-dipstick assay for specific diagnosis of urinary schistosomiasis together with microscopy to determine the prevalence of infant schistosomiasis in a community in the Awutu-Efutu Senya District in the Central Region of Ghana. The study group consisted of 97 infants (51 males and 46 females) aged 2 months to 5 years. A total of 75 of 97 (77.3%) subjects submitted stool samples; none had Schistosoma mansoni. Three individuals (3.1%) had hookworms but there were no other intestinal helminths. The urinary schistosomiasis prevalence by MoAb-dipstick (30%) was higher (P < 0.05) than that estimated by microscopy (11.2%). However, three of nine (33.3%) microscopically confirmed cases tested MoAb-dipstick positive after pre-treatment of the urine specimen with heat. The youngest infant to be found infected with S. haematobium microscopically was 4 months old. Fifteen of 71 S. haematobium egg negative individuals tested dipstick positive, giving a dipstick specificity of 78.9% as compared with microscopy as gold standard test. The relative sensitivity of the dipstick was 100%.

Determinants of health-seeking behaviour for schistosomiasis-related symptoms in the context of integrating schistosomiasis control within the regular health services in Ghana.

Morbidity control of schistosomiasis through integration within existing health care delivery systems is considered a potentially sustainable and cost-effective approach. We conducted a questionnaire-based field study in a Ghanaian village endemic for both urinary and intestinal schistosomiasis to determine whether infected individuals self-reported to health centres or clinics and to identify factors that influenced their decision to seek health care. A total of 317 subjects were interviewed about having signs and symptoms suggestive of schistosomiasis: blood in urine, painful urination, blood in stool/bloody diarrhoea, abdominal pain, diarrhoea, swollen abdomen and fatigue within 1 month of the day of the interview. Fever (for malaria) was included as a disease of high debility for comparison. Around 70% with blood in urine or painful urination did not seek health care, whilst diarrhoea, blood in stool, abdominal pain and fever usually led to action (mainly self-medication, with allopathic drugs being used four to five times more often than herbal treatment). On average 20% of schistosomiasis-related signs and symptoms were reported to health facilities either as the first option or second and third alternative by some of those that self-medicated. A few of those who visited a clinic or health centre as first option still self-medicated afterwards. Children under 10 years and adults were more likely to seek health care than teenagers. Also, females were more likely to visit a health facility than males of the same age groups. Socio-economic status and duration of symptoms did not appear to affect health-seeking behaviour. 'Do not have the money' (43%) and 'Not serious enough' (41%) were the commonest reasons for not visiting a clinic, reported more frequently by lower and higher socio-economic classes, respectively, for both urinary or intestinal schistosomiasis. The regular health service shows some potential in passive control of schistosomiasis as some, but far too few, people visit a health facility as first or second option.

Cryptosporidium oocysts in Ghanaian AIDS patients with diarrhoea.

BACKGROUND: Although Cryptosporidiumspp. infections in acquired immunodeficiency syndrome patients (AIDS) with chronic diarrhoea have been reported in several African countries, there is no information regarding cryptosporidial diarrhoea in Ghanaian AIDS patients. OBJECTIVE: To investigate the occurrence of C. parvum and other gastrointestinal parasitic agents in Ghanaian AIDS patients with chronic diarrhoea. DESIGN: Prospective study of HIV/AIDS patients with diarrhoea over a nine month period. SETTING: Korle-Bu Teaching Hospital and Korle-Bu Polyclinic Accra, Ghana. RESULTS: Analysis of stool specimens from clinically diagnosed HIV/AIDS (n = 21; mean CD4 count was 288 cells per microliter, 95% confidence interval of 237 to 340 cells per microliter) and HIV-seronegative (n = 27) patients revealed C. parvum in six (28.6%) of HIV/AIDS and 10 (37.0%) of the HIV-seronegative patients, respectively. Three other HIV/AIDS cases had other infections involving Strongyloides stercoralis 4.8% (1/21) and Salmonella spp. 9.5% (2/21). There was no concomitant association between C. parvum and any other parasites found. Also, no enterobacteria was found in the HIV-seronegative patients. CONCLUSION: This study demonstrates the prevalence of Cryptosporidium sp. in both HIV/ AIDS and HIV-seronegative individuals in Ghana. However, there was no statistical association between cryptosporidiosis and HIV/AIDS (p > 0.05).

The antibody responses to adult-worm antigens of Schistosoma haematobium, among infected and resistant individuals from an endemic community in southern Ghana.

Antibody responses to antigens from adult Schistosoma haematobium were investigated in an endemic community in Ghana, using microplate-ELISA. The results of a survey of egg output in urine and of a questionnaire-based investigation of water-contact activities were used to select 'endemic normal' (EN) and patently infected (PI) individuals as subjects. The plasma levels of antibodies reacting with the adult-worm antigens were determined and compared and the correlations between these levels and the age, water-contact index and egg output of each subject were evaluated. Compared with the EN subjects, the PI generally had higher levels of anti-worm IgG and IgE but lower levels of anti-worm IgA. When the data for the EN and PI groups were combined, the levels of anti-worm IgG and IgE were found to be positively correlated with egg output and with each other. Whichever the antibody class considered, levels of anti-worm antibodies were never negatively correlated with egg output. These results indicate that anti-worm IgE and IgG could be used as markers to reflect current infection intensity, and that anti-worm antibodies may not act as protective antibodies in the natural course of urinary schistosomiasis.

The seroprevalence of toxoplasmosis in pigs in Ghana.

A serological survey of toxoplasmosis in pigs in Ghana was carried out between October 1997 and April 1998 in the three ecological zones of Ghana: the Coastal Savannah, the Forest Belt and the Guinea Savannah. Antibody against Toxoplasma gondii was measured in pig serum using a microplate-ELISA which had a sensitivity and specificity of 90.2 and 92.3%, respectively when compared with IFAT. A national seroprevalence of 39% was obtained in pigs, with the ecological distribution being 43.9, 30.5 and 42.5% for the Coastal Savannah, the Forest Belt and the Guinea Savannah, respectively. The age of the animal, the breed, the environmental conditions and the management practices appeared to be the major determinants of prevalence of antibodies against T. gondii. The prevalence of anti-T. gondii antibodies was found to increase with age (P<0.05). Pigs from the two Savannah zones had a significantly higher (P<0.05) antibody prevalence than those sampled from the Forest belt. Antibody prevalence (46.8%) in crossbreed pigs was significantly higher (P<0.05) than that of the exotic Large White breed (38.8%).

The prevalence of anti-Toxoplasma gondii antibodies in Ghanaian sheep and goats.

The enzyme-linked immunosorbent assay (ELISA) was used to detect anti-Toxoplasma gondii antibodies in 1258 small ruminants (732 sheep and 526 goats) sampled from 28 different locations in the three ecological zones of Ghana. The animals sampled had an overall seroprevalence of 30.5% (384 of the total). Sheep had a higher overall prevalence (33.2%) compared to the goats (26.8%). Animals sampled from the Coastal Savannah and the Forest zones had prevalences of 39.4% and 39.1%, respectively, which were significantly higher (P<0.01) than the prevalence recorded for the drier Guinea Savannah zone (20%). Prevalence of antibodies in female animals (35.8%) was significantly higher (P<0.01) than that for males (21.1%). Significant differences were also observed between breeds and age groups. The ELISA was found to be both highly sensitive (92%) and specific (91%) when compared to the IFAT, which was used as a reference test.

Detection of Trypanosoma brucei, T. congolense and T. vivax infections in cattle, sheep and goats using latex agglutination.

A monoclonal antibody-based latex agglutination test for detection of circulating trypanosome antigens in animal serum was evaluated for the ability to detect natural T. brucei, T. congolense and T. vivax infections in cattle, sheep and goats in Ghana. The test detected antigens in 180/422 (42.7%) of cattle, 27/131 (20.6%) of sheep and 14/79 (17.7%) of the goats. By comparison, the microplate-based antigen-ELISA gave similar results (P > 0.01), detecting trypanosome antigens in 41.7% of the cattle, 19.8% of the sheep and 17.7% of the goats. Trypanosomes were demonstrated in the blood of 30 (7.2%) cattle, 7 (5.3%) sheep and 3 (3.8%) goats using the buffy coat technique (BCT). Of these, 26 cattle (86.7%), 6 sheep (85.7%) and all 3 goats (100%) were antigenaemic. The most prevalent single infection in all 3 animal species involved T. vivax, and the most common mixed infection involved all 3 trypanosome species in cattle and sheep. There was no mixed infection in goats. Compared with the antigen-ELISA, the sensitivity of the latex agglutination test was 98.3% in cattle and 100% in both sheep and goats, whilst the specificity was 97.2% in cattle, 99% in sheep and 100% in goats. False positivity with the latex agglutination test was 3.9% in cattle and 3.7% in sheep. There were no false-positive reactions with the test in goats. The latex agglutination assay promises to be ideal for testing small numbers of animals under field conditions.

A monoclonal antibody-based dipstick assay for diagnosis of urinary schistosomiasis.

A Schistosoma haematobium species-specific mouse immunoglobulin (Ig) G1 monoclonal antibody (mab) Sh2/15.F that bound a 29 kDa peptide was utilized to develop a membrane-based dipstick enzyme-linked immunosorbent assay for specific diagnosis of urinary schistosomiasis. Strips of polyvinylidene difluoride membrane were wetted with methanol and stored in distilled water. The strips were used to capture urinary antigens which were then revealed by incubation in a mixture of specific mab and peroxidase-conjugated goat anti-mouse IgG. The assay correctly identified 26/30 (87%) of egg-negative control individuals and 53/54 (98%) of parasitologically confirmed cases including all of 6 individuals treated with praziquantel (40 mg/kg) but not cured. Also, the assay detected S. haematobium antigens in the urine of 3 individuals from whom 2 specimens had to be examined microscopically to confirm infection, thus suggesting that the mab detection method may have greater sensitivity than microscopy.

Limited field evaluation of a rapid monoclonal antibody-based dipstick assay for urinary schistosomiasis.

A rapid, visually read monoclonal antibody (MoAb)-based dipstick assay for specific diagnosis of urinary schistosomiasis was field tested with microscopy and the use of hematuria and proteinuria in a schistosomiasis hematobia endemic area in Southern Ghana. The study group consisted of 229 individuals (114 males and 115 females) aged 1 to 86 years; 145/229 (63.3%) of the subjects submitted stool samples from which no S. mansoni eggs were detected. However, infections with Necator americanus (hookworms) 33.1%, Ascaris lumbricoides 2.8%, Trichuris trichiura (whipworm) 2.8%, and Strongyloides stercoralis 0.7% were detected but did not appear to influence the results of the MoAb-dipstick assay. Urinary schistosomiasis prevalence was estimated as 47.6% by microscopy, 48% by MoAb-dipstick, 39.7% by microhematuria, and 23.6% by proteinuria. The MoAb-dipstick correctly identified 108/109 (99.1%) of microscopically confirmed cases and 118/120 (98.3%) of egg-negative individuals, thereby giving a sensitivity of 99.1% and a specificity of 98.3%. On the other hand, microhematuria and proteinuria were, respectively, 76.1% and 40.4% sensitive, and 94.2% and 92.5% specific when compared to microscopy. Microhematuria and proteinuria had significantly lower sensitivity (P < 0.001) than either microscopy or dipstick.

Extraction of Schistosoma haematobium antigens from infected human urine and generation of potential diagnostic monoclonal antibodies to urinary antigens.

Proteins in Schistosoma haematobium infected human urine were concentrated by precipitation with saturated ammonium sulphate 50% (v/v) and various fractions obtained at different stages of precipitation tested for presence of schistosome antigens (ShAgs) by dot-ELISA. The protein fraction (UP2S) obtained following two-times precipitation was found to contain high concentrations of ShAg. Fraction UP2S was dialysed against phosphate-buffered saline (pH 7.4) and further purified by Sephadex G-200 column chromatography. Two protein peaks were eluted of which the first peak UP2S(pkI) was found to contain high concentrations of ShAgs as determined by microplate-ELISA. The second peak UP2S(pkII) consisted of human urine proteins. Further analysis of UP2S(pkI) revealed that ShAgs were mainly in the form of immune complexes with human IgG, IgM, IgA, IgE and complement C3. The ShAgs in both UP2S and UP2S(pkI) were found to be active as they induced immune responses in mice which produced antibodies reactive with S. haematobium worm as well as soluble egg antigens (SEA). Pure ShAgs were obtained from UP2S following dissociation of immune complexes with a carbonate buffer (pH 11.42) and further purification on Sephadex G-200. Immunizations with UP2S led to the generation of MoAbs which could bind both SEA and UP2S.

Characterization of monoclonal antibodies reactive with Schistosoma haematobium-soluble egg and infected human urinary antigens.

Six murine monoclonal antibodies (MAbs) [Sh1/71.7 (IgM), Sh2/15.F (IgG1), Sh3/15.28 (IgG1), Sh3/38.2 (IgM), Sh4/14.3 (IgG1), and Sh5/32.30 (IgM)] produced against S. haematobium were extensively characterized. All the MAbs stained the surface membranes of miracidia as determined by the indirect fluorescent antibody test, yet three of them (Sh1/71.7, Sh3/15.28, and Sh3/38.2) also stained internal cytoplasmic antigens. Proteinase-K digestion and periodate oxidation studies showed that these three MAbs bound glycoprotein antigenic determinants, while the rest detected protein epitopes. Only Sh2/15.F, Sh3/15.28, and Sh4/14.3 could bind antigens with the western immunoblot assay. Sh2/15.F bound a 29-kDa antigen and Sh3/15.28 bound three antigen bands (53, 57, and 66 kDa) all in the soluble egg extract of an Egyptian strain of S. haematobium (SEAEgy), while Sh4/14.3 reacted with a 29-kDa antigen present in SEAEgy, as well as in the adult worm antigen extracts of Ghanaian strain(s) of S. haematobium and S. japonicum. Sh4/14.3 also bound a 78-kDa antigen in the S. japonicum worm. Cross-reactivity studies with S. haematobium, S. mansoni, S. japonicum, and Necator americanus revealed that Sh2/15.F and Sh3/15.28 were S. haematobium species-specific. Each of the remaining MAbs detected the three major human schistosomes without cross-reacting with N. americanus egg antigens. Three MAbs, Sh2/15.F, Sh4/14.3 and Sh5/32.30, could detect S. haematobium antigens in infected human urine.

Field evaluation of a dot-ELISA for the detection and differentiation of trypanosome species in infected tsetse flies (Glossina spp.).

A rapid, visually read, dot-ELISA developed for the detection and differentiation of trypanosome species in tsetse flies (Glossina spp.), was field tested alongside the standard fly dissection method on a range in south eastern Kenya. Of 104 G. pallidipes dissected, 2 were found to be infected with trypanosomes in their midguts. By the dissection method the infecting trypanosome species could not be identified, as both flies had no salivary gland infections. However, using the dot-ELISA, the 2 flies were shown to be infected with Trypanosoma congolense in their midguts. The midguts of an additional 6 (5.8%) of the 104 G. pallidipes tested positive for T. congolense int he dot-ELISA, even though no trypanosomes were seen on dissection. The infection rate for this fly species as determined using the dot-ELISA, therefore, was 7.7% for T. congolense in midgut infections compared to 1.9% identified by fly dissection. The salivary glands and mouthparts of the 6 additional tsetse flies identified by dot-ELISA were all negative as determined by the 2 techniques. None of 390 G. longipennis flies dissected and examined for trypanosomes in the midgut, salivary glands and mouthparts was shown, by this method, to be infected. Using the dot-ELISA, however, 17 (4.4%) of the flies tested positve for T. congolense in the midgut, whilst the salivary glands and mouthparts of the same flies were negative. Thus, the dot-ELISA appears to be more sensitive than the fly dissection method under field conditions. Moreover, the dot-ELISA can be performed in the field without electricity. It is simple to perform, and was not affected by high ambient temperatures (22-32 degrees C), or by contamination of reactants with dust.

Differentiation between culture-derived insect stages of T. brucei, T. vivax, T. congolense and T. simiae using a monoclonal antibody-based dot-ELISA.

A sensitive and specific nitrocellulose (NC) membrane-based dot-ELISA, utilizing a panel of monoclonal antibodies (mAbs), was developed for differentiation between in vitro-derived procyclic forms of Trypanosoma brucei, T. congolense and T. simiae, and epimastigotes of T. vivax. Trypanosomes in suspension were applied onto NC membrane in dots and probed with unlabelled trypanosome species-specific mAbs. Bound mAb was revealed by enzyme labelled anti-mouse IgG and precipitable chromogenic substrate. The assay detected the aforementioned trypanosome species in both single and artificially mixed preparations. Ten T. brucei, 4 T. vivax, 7 T. congolense and 3 T. simiae procyclic stocks and clones from different geographical areas were tested and identified using the specific mAbs in the dot-ELISA which had a specificity of 100%. Some of the T. brucei, T. congolense and Nannomonas-specific mAbs could detect as few as 10 trypanosomes/dot, whilst 1 T. vivax mAb was able to detect a minimum of 100 trypanosomes/dot in monospecies preparations. A concentration of 1 x 10(4) trypanosomes/microliters/dot was eventually determined as ideal for testing in the dot-ELISA. Antigen dots stored at 4 degrees C under desiccated conditions did not show any loss in activity for up to 90 days. However, when stored under similar conditions at room temperature (17-26 degrees C), the T. congolense-specific antigen remained unaffected up to 60 days, and then showed decreased activity when tested on day 90.

Production and characterization of a monoclonal antibody specific for Trypanosoma simiae.

Monoclonal antibodies (MoAb) were produced against invariant antigens of vector forms of Trypanosoma simiae. X63/AG8.653, NSI/1AG401 and Sp20Ag14 myeloma cells were fused with splenic lymphocytes from BALB/c mice that had been immunized with various preparations of T. simiae procyclics. A T. simiae-specific MoAb [KNS7/14.X (IgG1)] was detected in the hybridoma culture supernatants, which were screened for antibody activity by micro-plate and dot ELISA. Immunofluorescence studies showed that KNS7/14.X stained cytoplasmic antigens in T. simiae procyclics. Proteinase-K digestion and periodate oxidation studies revealed that KNS7/14.X binds to a carbohydrate antigenic determinant in glycoprotein or glycolipid. Cross-reactivity studies using vector forms of T. brucei, T. vivax, T. congolense, T. simiae and T. grayi showed that KNS7/14.X only reacted with T. simiae. Attempts to generate other T. simiae-specific MoAb, using 107-, 75- or 41.7-43.6-kDa peptides selected by western blotting analysis, did not yield positive results.

Hydrogen peroxide destaining: a new method for removing non-specific stains in nitrocellulose membrane-based dot-ELISA for the detection of trypanosomes in tsetse flies (Glossina spp.).

Gut samples prepared from laboratory-reared tsetse flies and applied in dots onto nitrocellulose (NC) membrane were found to stain the membrane with differing coloration and intensity. The stains were, predominantly, either reddish to brown or blackish-brown to black and occasionally greenish to almost colourless, depending on the stage of digestion of the bloodmeal in the fly. NC membrane strips applied with tsetse gut samples from T. brucei infected and uninfected control flies were tested with the standard antigen detection dot enzyme-linked immunoassay (dot-ELISA), using a T. brucei specific monoclonal antibody (MoAb) and horseradish peroxidase goat anti-mouse conjugate. The stains in both infected and uninfected sample dots persisted through the assay. Furthermore, the staining intensity of some assayed uninfected sample dots were enhanced as a result of non-specific reactivity, making it difficult to distinguish between the infected and uninfected flies. This necessitated the development of a simple technique by which the non-specific stains and reactions could be removed. Sample 'dotted' NC membrane strips were destained by incubation with 5% hydrogen peroxide (H2O2) diluted in 5% skimmed milk in Tris buffer, pH 8.0. After washing, the destained strips were tested in the dot-ELISA. This method gave satisfactory reproducible results, since the most intense stains could be removed, and it had no effect on trypanosome antigens detected by a panel of four T. brucei species-specific, three T. vivax species-specific, four T. congolense species-specific and four Nannomonas subgenus-specific MoAbs. Using the destaining process in a modified dot-ELISA, 86 out of 95 (90.5%) of Glossina morsitans centralis flies experimentally infected with T. brucei, were identified. The destaining method was also used successfully to decolorize NC membrane bound tsetse faecal material.

Detection and differentiation between trypanosome species in experimentally infected tsetse flies (Glossina spp.) using dot-ELISA.

A modified NC membrane-based dot-ELISA was used to detect and differentiate between Trypanosoma brucei, T. congolense and T. simiae procyclics in the midguts of experimentally infected tsetse flies. The modification of the assay consisted of (a) the lysis of T. congolense or T. simiae in NC membrane applied sample dots using Triton X-114, and (b) treatment of sample applied NC membrane strips with hydrogen peroxide to remove non-specific stains. Also, T. brucei was detected in the salivary glands, and T. congolense and T. vivax were detected in the mouthparts, however, in dot-ELISA without modification. In all the assays, T. brucei and T. congolense parasites were detected directly using MoAbs specific to each of them, whereas T. simiae parasites were detected by exclusion using a T. congolense specific and Nannomonas subgenus-specific MoAbs. The sensitivity of the assay for detecting midgut infections was 90.5%, 84.6% and 94.4% in detecting T. brucei, T. congolense and T. simiae, respectively. Sample dots stored at room temperature (19-26 degrees C) under desiccated conditions did not show any loss in activity in 90 days. However, after 7 days of storage, a ring-pattern reaction appeared on some sample dots that were tested with T. brucei specific MoAb, irrespective of whether T. brucei antigens were present or not. These ring reactions, however, did not interfere with correct interpretation of the assay results. The specificity of the assay for detection of T. brucei in the salivary glands was 100% and the sensitivity was 90%. Also, T. vivax and T. congolense organisms were each detected in the mouthparts of infected tsetse flies, with 100% specificity. The sensitivity was, however, lower, 43.8% for T. vivax and 55.6% for T. congolense.