Edwin K. Wiredu

Cerebrospinal fluid and serum biomarkers of cerebral malaria mortality in Ghanaian children

BackgroundPlasmodium falciparum can cause a diffuse encephalopathy known as cerebral malaria (CM), a major contributor to malaria associated mortality. Despite treatment, mortality due to CM can be as high as 30% while 10% of survivors of the disease may experience short- and long-term neurological complications. The pathogenesis of CM and other forms of severe malaria is multi-factorial and appear to involve cytokine and chemokine homeostasis, inflammation and vascular injury/repair. Identification of prognostic markers that can predict CM severity will enable development of better intervention.MethodsPostmortem serum and cerebrospinal fluid (CSF) samples were obtained within 2–4 hours of death in Ghanaian children dying of CM, severe malarial anemia (SMA), and non-malarial (NM) causes. Serum and CSF levels of 36 different biomarkers (IL-1β, IL-1ra, IL-2, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-12 (p70), IL-13, IL-15, IL-17, Eotaxin, FGF basic protein, CRP, G-CSF, GM-CSF, IFN-γ, TNF-α, IP-10, MCP-1 (MCAF), MIP-1α, MIP-1β, RANTES, SDF-1α, CXCL11 (I-TAC), Fas-ligand [Fas-L], soluble Fas [sFas], sTNF-R1 (p55), sTNF-R2 (p75), MMP-9, TGF-β1, PDGF bb and VEGF) were measured and the results compared between the 3 groups.ResultsAfter Bonferroni adjustment for other biomarkers, IP-10 was the only serum biomarker independently associated with CM mortality when compared to SMA and NM deaths. Eight CSF biomarkers (IL-1ra, IL-8, IP-10, PDGFbb, MIP-1β, Fas-L, sTNF-R1, and sTNF-R2) were significantly elevated in CM mortality group when compared to SMA and NM deaths. Additionally, CSF IP-10/PDGFbb median ratio was statistically significantly higher in the CM group compared to SMA and NM groups.ConclusionThe parasite-induced local cerebral dysregulation in the production of IP-10, 1L-8, MIP-1β, PDGFbb, IL-1ra, Fas-L, sTNF-R1, and sTNF-R2 may be involved in CM neuropathology, and their immunoassay may have potential utility in predicting mortality in CM.

Human papillomavirus prevalence and type distribution in invasive cervical cancer in sub‐Saharan Africa

In sub‐Saharan Africa, invasive cervical cancer (ICC) incidence and mortality are among the highest in the world. This cross‐sectional epidemiological study assessed human papillomavirus (HPV) prevalence and type distribution in women with ICC in Ghana, Nigeria, and South Africa. Cervical biopsy specimens were obtained from women aged ≥21 years with lesions clinically suggestive of ICC. Histopathological diagnosis of ICC was determined by light microscopy examination of hematoxylin and eosin stained sections of paraffin‐embedded cervical specimens; samples with a confirmed histopathological diagnosis underwent HPV DNA testing by polymerase chain reaction. HPV‐positive specimens were typed by reverse hybridization line probe assay. Between October 2007 and March 2010, cervical specimens from 659 women were collected (167 in Ghana, 192 in Nigeria and 300 in South Africa); 570 cases were histologically confirmed as ICC. The tumor type was identified in 551/570 women with ICC; squamous cell carcinoma was observed in 476/570 (83.5%) cases. The HPV‐positivity rate in ICC cases was 90.4% (515/570). In ICC cases with single HPV infection (447/515 [86.8%]), the most commonly detected HPV types were HPV16 (51.2%), HPV18 (17.2%), HPV35 (8.7%), HPV45 (7.4%), HPV33 (4.0%) and HPV52 (2.2%). The prevalence of single and multiple HPV infections seemed higher among HIV‐positive women and HPV type distribution appeared to differ according to tumor type and HIV status. In conclusion, HPV16, 18, 45 and 35 were the most common HPV types in sub‐Saharan African women with ICC and HPV infections were more common in HIV‐positive women.

High-level cerebellar expression of cytokines and adhesion molecules in fatal, paediatric, cerebral malaria

Abstract Although the roles played by systemic tumour necrosis factor (TNF) and interleukin 1β (IL-1β), and their upregulation of intercellular adhesion molecule 1 (ICAM-1), vascular cell adhesion molecule 1 (VCAM-1) and E-selectin, in the pathogenesis of human cerebral malaria (CM) are well established, the role of local cytokine release, in the brain, remains unclear. Immunohistochemistry was therefore used to compare the expression of ICAM-1, VCAM-1, E-selectin, IL-1β, TNF and transforming growth factor β (TGF-β) at light-microscope level, in cryostat sections of cerebral, cerebellar and brainstem tissues collected, post-mortem, from Ghanaian children. Among the 21 children investigated were 10 cases of CM, five of severe malarial anemia (SMA), one of purulent bacterial meningitis (PBM), two of non-central-nervous-system infection (NCNSI) and three children who had no infection (NI) when they died. Parasitised erythrocytes were detected in all of the sections from the cases of fatal malaria (CM and SMA), and sequestered leucocytes were present in most of the sections from the CM cases (but none of the sections from the SMA cases). Significantly elevated vascular expression of all three adhesion molecules investigated was detected in the brains of the 15 cases of fatal malaria and one of the cases of NCNSI (a child with Salmonella septicaemia), and in the malaria cases this showed highly significant co-localization with the areas of erythrocyte sequestration. In terms of the levels of expression of ICAM-1, VCAM-1 and E-selectin, there were, however, negligible differences between the CM and SMA cases. Although TGF-β showed intravascular and perivascular distribution in all the subjects, its expression was most intense in the PBM case and the CM group. Only in the sections from the PBM and CM cases did TNF and IL-1β show prominent brain parenchymal staining, in addition to the intravascular and perivascular staining seen in all subjects. The highest observed expression of each of the six antigens studied was in the cerebellar sections of the malaria cases. Endothelial activation in the brain therefore appears to be a feature of fatal malaria and Salmonella sepsis, and in cases of fatal malaria is closely associated with leucocyte sequestration. In the present study, IL-1β and TNF were only up-regulated in the brains of children with neurodegenerative lesions, whereas TGF-β was present in all cases.

Toxicity potentials of the nutraceutical Moringa oleifera at supra-supplementation levels.

Moringa oleifera Lam. (order -Moringales, family -Moringaceae and genus -Moringa) is a well known nutraceutical used in the treatment of hypercholesterolemia and hyperglycemia, and also, as a nutritional supplementation. Its popularity use raises the question of possible toxicity at supra-supplementation levels. The objective of the study was to ascertain possible acute toxicity with supra-supplementation using Sprague-Dawley (S-D) rats. In experiment 1, human peripheral blood mononuclear cells were given graded doses of Moringa oleifera aqueous leaf extract to induce cytotoxicity. In experiment 2, two groups of rats received low and high dose (LD and HD, respectively) levels (1,000 and 3,000 mg/kgb.wt, respectively) per o.s. alongside negative and positive control rats (0.9% saline and 10mg/mL N-ethyl-N-nitrosourea - administered i.m., respectively). Each group consisted of five rats. Rats were killed after 48 h and the femur bone marrow aspirate examined for polychromatic micronucleated erythrocytes (PCEMN)/normochromatic micronucleated erythrocytes (NCEMN) ratios after Giemsa/Leishman staining. In experiment 3, control, LD and HD groups were established. The LD and HD extracts were administered per o.s. to the respective groups and observed for 14 days. Each group consisted of five rats. Blood was sampled after 48 h and 14 days and examined biochemically and haematologically for acute toxicity. Experiment 1 showed that Moringa oleifera was cytotoxic at 20mg/mL. In experiment 2, PCEMN/NCEMN ratios were: negative control=2.087; LD=1.849; HD=1.397; positive control=1.257. Statistically, LD and HD ratios were significant (p=0.020). Experiment 3 showed that hepatonephro-toxicity was nil with no abnormal haematology results. Genotoxicity results have hitherto not been shown. Moringa oleifera is genotoxic at supra-supplementation levels of 3,000 mg/kg b.wt. However, intake is safe at levels ≤ 1,000 mg/kg b.wt.

Acute toxicity studies of aqueous leaf extract of Phyllanthus niruri

Acute toxicity studies of aqueous leaf extract of Phyllanthus niruri Phyllanthus niruri is a plant with medicinal properties. It is often used to treat mild malaria and the elimination of renal stones. However, studies on its toxicity are scarce. The study was carried out to determine if the aqueous leaf extract of P. niruri administered to female Sprague-Dawley rats would illicit evidence of toxicity. Fifteen female rats weighing 150-200 g were divided into 3 groups. Rats in Group 1 were given a single low dose (LD) of 2 000 mg/kg b.w. of the extract by oral gavage within 24 hrs. Rats in Group 2 were given a single high dose (HD) of 5 000 mg/kg b.w. of the extract by oral gavage within 24 hrs. Rats in Group 3 were not given any extract but drinking water and served as the control group (C). All the rats were observed for signs of toxidromes for 14 days. On the 15th day, all the rats were sacrificed. Body organs were harvested for macroscopic examination. Urine and blood samples were drawn and analyzed. Hematological tests performed included full blood count and hemoglobin. Biochemical examinations included bilirubin, alanine aminotransferase (ALT), aspartate aminotransferase (AST), total protein, albumin, globulin, alkaline phosphatse (ALP), γ-glutamyltranspeptidase (GGT), urea, and creatinine. The results of the three groups were not significantly different. Examination of the various body organs did not show any abnormality. Thus no toxicity was observed at the levels administered. The LD50 of the aqueous extract is > 5 000 mg/kg. b.w.

Genotoxicity, cytotoxicity and toxicological evaluation of whole plant extracts of the medicinal plant Phyllanthus niruri (Phyllanthaceae).

Phyllanthus niruri is a medicinal plant (commonly known as stone breaker) found in the tropics and other parts of the world. It is known for its capacity to block the formation of calcium oxalate crystals and kidney stone formation in urolithiasis. This plant has been used to treat hyperglycemia, hypertension, pain, and mild cases of malaria. We examined the geno-, cyto- and overall toxicity of P. niruri whole plant ethanolic extract. The extract was administered as a single dose of 30 or 300 mg/kg to laboratory rats by gavage, accompanied by negative (0.9% saline) and positive (10 mg/mL N-ethyl-N-nitrosourea) controls that were injected intramuscularly 48 h after extract administration. The ratio of polychromatic (PCE)/normochromatic erythrocytes (NCE) from femur bone marrow was scored for genotoxicity. Cytotoxicity was determined using descending concentrations (0.2-0.0125 g/mL) of the extract incubated with peripheral blood mononuclear cells. Lactate dehydrogenase release from damaged cells was determined and the CC(50) calculated. Subchronic administration of the extract at 30 or 300 mg/kg was done for 90 days to determine general toxicity. PCE:NCE (%) for the extract and negative control was 63, compared to 168 (positive control). The CC(50) was 26.3 mg/mL and hepato-renal toxicity after subchronic extract administration was nil. We conclude that ethanol extract of P. niruri is not cytotoxic or genotoxic, and is generally non-toxic on subchronic administration.

Acute toxicity studies of Croton membranaceus root extract

AIM OF THE STUDY Croton membranaceus root and leaf extracts are used in the Bahamas to aromatize tobacco, in Nigeria to improve digestion, and in Ghana, for benign prostate hyperplasia. Despite claims of success there is paucity of information on its toxicity. The aim of this study was to determine if Croton membranaceus has acute toxicity properties. MATERIALS AND METHODS Roots were air-dried in a solar dryer for one week before milling. The powder was extracted with 96% ethanol, freeze-dried and re-extracted with distilled water and freeze-dried. 15 male Sprague-Dawley rats (180-200 g) were divided equally into 2 treatment groups [low dose (LD) and high dose (HD)], plus a control group (C). LD and HD received 1500 and 3000 mg/kg b.wt. Croton membranaceus aqueous extract, respectively, one time and observed for 14 days. Haematological [Full Blood Count and haemoglobin (Hb)], biochemical [bilirubin, alanine aminotransferase (ALA), aspartate aminotransferase (AST), total protein, albumin, globulin, alkaline phosphatise (ALP), γ-glutamyltranspetidase (GGT), urea, creatinine, creatinine kinase - Muscle and Brain (CK-MB), creatinine kinase - Total (CK-R)] examinations were performed. RESULTS Control groups CK-MB (5444±534 U/L) and LD group CK-MB (4014±1016 U/L) were significantly different (p<0.05). Control and the HD group CK-MB (3955±1135 U/L) were significantly different (p<0.05). Both LD and HD CK-R levels (697±197U/L and 732±203 U/L, respectively), were lower than the control (1139±220 U/L) at 48 h and 14 days (p<0.05, p<0.05, respectively). γ-GT levels of the HD group was 4.8±0.4 U/L compared to the Control group value of 0.9±0.2 U/L (p<0.05). CONCLUSIONS Taking all factors into consideration, Croton membranaceus ingestion does not produce general acute toxicity. However, its creatinine kinase lowering ability could be explored.

Epidemiology of Helicobacter pylori infection in dyspeptic Ghanaian patients

Introduction Helicobacter pylori is a gram-negative urease-producing bacterium causally linked with gastritis, peptic ulcer disease and gastric adenocarcinoma. Infection is more frequent and acquired at an earlier age in developing countries compared to European populations. The incidence of Helicobacter pylori infection in dyspeptic Ghanaian patients was 75.4%. However, epidemiological factors associated with infection vary across populations. Methods This study used a cross-sectional design to consecutively sample dyspeptic patients at the Endoscopy Unit of the Korle-Bu Teaching Hospital, Accra between 2010 and 2012. The study questionnaire elicited their epidemiological clinical characteristics. Helicobacter pylori infection was confirmed by rapid-urease examination of antral biopsies at upper Gastro-intestinal endoscopy. Results The sample population of dyspeptic patients attending the Endoscopy Unit for upper GI endoscopy yielded 242 patients of which 47.5% were females. The age distribution of H. pylori-infection was even across most age – groups, ranging from 69.2% (61 – 70) years to 80% (21 – 30) years. Helicobacter pylori prevalence decreased across areas mapping to the three residential classes in accordance with increasing affluence with rural areas having the highest prevalence. The unemployed and patients in farming had relatively high Helicobacter pylori infection rates of 92.3% and 91.7% respectively. Conclusion Helicobacter pylori is endemic in Ghana but the persistently high prevalence across age groups despite significant community anti-microbial use suggests likely re-crudescence or re-infection from multiple sources in a developing country. Socio-cultural factors such as residential class and farming may be facilitating factors for its continued prevalence.

Immunoreactivity of some epitopes in longtime inappropriately stored paraffin-embedded tissues

Abstract One advantage associated with paraffin-embedded tissues is their availability for further studies and review. Where block filing facilities are not available, used blocks are often dumped in neglected rooms. This may affect their appropriateness for follow-up studies such as immunohistochemistry. The goal of this study was to perform immunohistochemical procedures on poorly stored paraffin-embedded blocks after a simple cleaning and re-embedding procedure. Thirty paraffin-embedded Onchocerca nodules, poorly stored for over 15 years, were used. The blocks were soaked overnight in tap water, rinsed in distilled water, air-dried, and re-embedded in fresh paraffin wax. Immunoperoxidase demonstrations of six major enzymes (LDH, SDHB, PDK2, G6PD, ME1, and A/BHD4) were performed on their sections and examined by light microscopy. Fifty-one 0.6% Female worm nodules in the amount of 51.6% had detectable SDHB, 56·6% had detectable levels of PDK2, 58·6% had A/BHD4, 61·1% had G6PD, 63·3% had detectable levels of ME1, and 64·5% had detectable levels of LDH. At a 99% confidence level, ≧30% of the nodules have all six enzymes detectable by immunohistochemistry and ≧35% have four detectable enzymes (LDH, PDK, A/BHD, and ME). Poorly stored archival formalin-fixed, paraffin-embedded Onchocerca nodules over a prolonged period still retain enough antigenicity for immunohistochemical demonstration of the enzymes LDH, SDHB, PDK2, G6PD, ME1, and A/BHD4 and perhaps other antigens.

Short time incubation at low temperature for retrieval of some antigens from formalin-fixed and paraffin-embedded tissues

Abstract Antibody manufacturers often recommend antigen retrieval for formalin-fixed paraffin-embedded tissue sections before immunohistochemical staining. The most commonly used methods involve high temperatures (90–120°C) in buffers. Low-temperature retrieval methods allow the use of tissue flotation baths but their incubation times are often long. This study investigated retrieval of some antigens at low temperature (65°C) for 10 minutes in citrate buffer (pH 6·0) using a tissue flotation bath. Sections were obtained from two tissue specimens, breast and O. volvulus nodules, which were formalin-fixed and paraffin-embedded. The antigens of lactate dehydrogenase, pyruvate dehydrogenase kinase-2, malic enzyme-1, succinate dehydrogenase-B, glucose-6-phosphate dehydrogenase, alpha/beta-hydrolase-4, estrogen receptors (ER), and progesterone receptors (PR) were retrieved at low temperature and in the cases of ER and PR, were also retrieved at high temperature (120°C), using a pressure cooker, for comparison. All the retrievals were carried out in citrate buffer, pH 6·0. The results showed that all the enzymes scored 2+ (i.e. a strong reaction) in staining intensity and the staining for ER and PR were similar for both methods (2+). In immunohistochemical staining, the most important stage is antigen retrieval. The low-temperature antigen retrieval in citrate buffer at pH 6·0 provided staining intensities comparable to those by high-temperature methods. The results indicate that low-temperature antigen retrieval is cost-effective, rapid, and reliable.