William K. Anyan

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 2u2003months to 5u2003years. 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 (Pu2003<u20030.05) 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 4u2003months 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%.

Interleukin-4 (IL-4) and IL-13 Suppress Excessive Neutrophil Infiltration and Hepatocyte Damage during Acute Murine Schistosomiasis Japonica

ABSTRACT Due to the importance of neutrophils and proinflammatory cytokines in schistosomal liver damage, we analyzed the mechanisms underlying neutrophil and proinflammatory responses in murine schistosomiasis japonica. We found that granulomatous inflammation around parasite eggs in the liver was greater in Schistosoma japonicum-infected IL-4−/− IL-13−/− (double-knockout [DKO]) mice than in infected wild-type (WT) mice at 6 weeks, but not at 8 weeks, postinfection, suggesting the importance of Th2 responses in these typical hepatic lesions. Infected DKO mice also showed increased neutrophil infiltration accompanying more severe pathology, as shown by the enhanced necrosis of hepatocytes. This was not likely due to a Th1/Th2 imbalance, because there was no detectable increase in gamma interferon (IFN-γ) production in these DKO mice. mRNA expression of interleukin-17A (IL-17A), proinflammatory cytokines, and the neutrophil chemoattractant CXCL2 in liver was higher in infected DKO mice than in WT mice. However, in IL-4−/− IL-13−/− IL-17A−/− (triple-knockout [TKO]) mice, the absence of IL-17A was associated with only marginal differences in schistosomal liver damage, suggesting that IL-17A is only partially responsible for neutrophil-driven hepatic damage. Furthermore, the expression of mRNAs encoding proinflammatory cytokines was not under the control of IL-17A in TKO mice. These findings indicate that IL-4 and IL-13 suppress excessive neutrophil recruitment, proinflammatory cytokine production, and hepatic damage during the acute stage of S. japonicum infection, suggesting that neutrophils and proinflammatory cytokines are mainly responsible for hepatocyte damage during acute murine schistosomiasis japonica. However, neutrophil induction and the production of proinflammatory cytokines were not due solely to IL-17A.

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.

Plasmodium falciparum isolates from southern Ghana exhibit polymorphisms in the SERCA-type PfATPase6 though sensitive to artesunate in vitro

BackgroundIn 2005, Ghana replaced chloroquine with artemisinin-based combination therapy as the first-line treatment for uncomplicated malaria. The aim of this work was to determine for the first time, polymorphisms in the putative pfATPase6 and pftctp, pfmdr1, pfcrt genes in Ghanaian isolates, particularly at a time when there is no report on artemisinin resistance in malaria parasites from Ghana. The sensitivity of parasite isolates to anti-malaria drugs were also evaluated for a possible association with polymorphisms in these genes.MethodsThe prevalence of point mutations in the above Plasmodium falciparum genes were assessed from filter-paper blood blot samples by DNA sequencing. In vitro drug sensitivity test was carried out on some of the blood samples from volunteers visiting hospitals/clinics in southern Ghana using a modified version of the standard WHO Mark III micro-test.ResultsAll successfully tested parasite isolates were sensitive to artesunate; while 19.4%, 29.0% and 51.6% were resistant to quinine, amodiaquine and chloroquine respectively. The geometric mean of IC50 value for artesunate was 0.73 nM (95% CI, 0.38-1.08), amodiaquine 30.69 nM (95% CI, 14.18-47.20) and chloroquine 58.73 nM (95% CI, 38.08-79.38). Twenty point mutations were observed in pfATPase6 gene, with no L263E and S769N. All mutations found were low in frequency, except D639G which was observed in about half of the isolates but was not associated with artesunate response (p = 0.42). The pftctp gene is highly conserved as no mutation was observed, while CVIET which is chloroquine-resistant genotype at codon 72-76 of the pfcrt gene was identified in about half of the isolates; this was consistent with chloroquine IC50 values (p = 0.001). Mutations were present in pfmdr1 gene but were not associated with artemisinin response (p = 1.00).ConclusionThe pfATPase6 gene is highly polymorphic with D639G appearing to be fixed in Ghanaian isolates. These may just be spontaneous mutations as all parasite isolates that were tested displayed satisfactory in vitro response to artesunate. However, there is no improvement in susceptibility of the parasites to chloroquine five years after its proscription.

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

Abstract 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 (UP 2 S) obtained following two-times precipitation was found to contain high concentrations of ShAg. Fraction UP 2 S 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 P 2 S(pkI) was found to contain high concentrations of ShAgs as determined by microplate-ELISA. The second peak UP 2 S(pkII) consisted of human urine proteins. Further analysis of UP 2 S(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 UP 2 S and UP 2 S(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 UP 2 S following dissociation of immune complexes with a carbonate buffer (pH 11.42) and further purification on Sephadex G-200. Immunizations with UP 2 S led to the generation of MoAbs which could bind both SEA and UP 2 S.

Basophil depletion downregulates Schistosoma mansoni egg-induced granuloma formation.

Granuloma formation around parasite eggs during schistosomal infection is considered to be controlled by Th2 cytokines. However, it is still controversial which cell populations are responsible for the host Th2 cytokine-dependent granuloma formation. Basophils have recently attracted attention because of their ability to produce large amounts of IL-4. Therefore, we investigated whether basophils play an essential role in the induction of granuloma formation induced by Schistosoma mansoni eggs. Together with our previous observation that basophil numbers increased markedly in the spleen at 7 weeks postinfection, immunohistochemical staining using anti-mMCP8 monoclonal antibody (mAb) showed basophil infiltration in the granulomatous lesions formed around parasite eggs. To examine the roles of basophils more directly, we treated mice with anti-CD200R3 mAb to deplete basophils. Depletion of basophils resulted in a reduction of basophil number with concomitant downregulation of egg granuloma formation at 7 weeks postinfection. Moreover, we observed a significant reduction in the size of egg granulomas formed in basophil-depleted mice in the pulmonary granuloma model. Taken together, these findings indicated that basophils are essential for S. mansoni egg-induced granuloma formation, and this may serve as a novel therapeutic target in ameliorating the pathology of schistosomiasis.

Administrative practices of health professionals and use of artesunate-amodiaquine by community members for treating uncomplicated malaria in southern Ghana: implications for artemisinin-based combination therapy deployment

Objectiveu2002 To investigate the use of artemisinin‐based combination and monotherapy by community members and the administrative practices of health professionals in treating malaria in Ghana.

Point of care diagnosis of multiple schistosome parasites: Species-specific DNA detection in urine by loop-mediated isothermal amplification (LAMP)

Schistosomes are easily transmitted and high chance of repeat infection, so if control strategies based on targeted mass drug administration (MDA) are to succeed it is essential to have a test that is sensitive, accurate and simple to use. It is known and regularly demonstrated that praziquantel does not always eliminate an infection so in spite of the successes of control programs a residual of the reservoir survives to re-infect snails. The issue of diagnostic sensitivity becomes more critical in the assessment of program effectiveness. While serology, such as antigen capture tests might improve sensitivity, it has been shown that the presence of species-specific DNA fragments will indicate, most effectively, the presence of active parasites. Polymerase chain reaction (PCR) can amplify and detect DNA from urine residue captured on Whatman No. 3 filter paper that is dried after filtration. Previously we have detected S. mansoni and S. haematobium parasite-specific small repeat DNA fragment from filtered urine on filter paper by PCR. In the current study, we assessed the efficacy of detection of 86 urine samples for either or both schistosome parasites by PCR and loop-mediated isothermal amplification (LAMP) that were collected from a low to moderate transmission area in Ghana. Two different DNA extraction methods, standard extraction kit and field usable LAMP-PURE kit were also evaluated by PCR and LAMP amplification. With S. haematobium LAMP amplification for both extractions showed similar sensitivity and specificity when compared with PCR amplification (100%) verified by gel electrophoresis. For S. mansoni sensitivity was highest for LAMP amplification (100%) for standard extraction than PCR and LAMP with LAMP-PURE (99% and 94%). The LAMP-PURE extraction produced false negatives, which require further investigation for this field usable extraction kit. Overall high positive and negative predictive values (90% - 100%) for both species demonstrated a highly robust approach. The LAMP approach is close to point of care use and equally sensitive and specific to detection of species-specific DNA by PCR. LAMP can be an effective means to detect low intensity infection due to its simplicity and minimal DNA extraction requirement. This will enhance the effectiveness of surveillance and MDA control programs of schistosomiasis.

In Vitro Assessment of Anthelmintic Activities of Rauwolfia vomitoria (Apocynaceae) Stem Bark and Roots against Parasitic Stages of Schistosoma mansoni and Cytotoxic Study

Schistosomiasis is a Neglected Tropical Diseases which can be prevented with mass deworming chemotherapy. The reliance on a single drug, praziquantel, is a motivation for the search of novel antischistosomal compounds. This study investigated the anthelmintic activity of the stem bark and roots of Rauwolfia vomitoria against two life stages of Schistosoma mansoni. Both plant parts were found to be active against cercariae and adult worms. Within 2u2009h of exposure all cercariae were killed at a concentration range of 62.5–1000u2009µg/mL and 250–1000u2009µg/mL of R. vomitoria stem bark and roots, respectively. The LC50 values determined for the stem bark after 1 and 2u2009h of exposure were 207.4 and 61.18u2009µg/mL, respectively. All adult worms exposed to the concentrations range of 250–1000u2009µg/mL for both plant parts died within 120u2009h of incubation. The cytotoxic effects against HepG2 and Chang liver cell assessed using MTT assay method indicated that both plant extracts which were inhibitory to the proliferation of cell lines with IC50 > 20u2009μg/mL appear to be safe. This report provides the first evidence of in vitro schistosomicidal potency of R. vomitoria with the stem bark being moderately, but relatively, more active and selective against schistosome parasites. This suggests the presence of promising medicinal constituent(s).