Frequency of Anti-HCV, HBsAg, HIV1-p24 Antigen, Acid Fast Bacilli and Plasmodium spp., in Herbal Home and Hospital Patients with Elevated tNFα (≥ 5.0 pg/ml) in Saki, West Nigeria

Infectious agents and chemical substances can trigger the production and the release of cytokines. To determine the pattern of infectious agents in herbal homes patients with elevated TNFα (≥ 5.0 pg/ml) in Saki – West-Nigeria. The subjects include patients with elevated TNFα (≥ 5.0 pg/ml) in 15 herbal homes(n=23 ; aged 5-69 years; males – 13 ; females – 10) and patients with elevated TNFα in 3 major hospitals (n = 32; aged 6 -67years; males – 12 ; females 20). Plasma TNFα, anti-HCV, HBsAg and HIV1-p24 antigen were determined by ELISA, Plasmodium spp., by Giemsa thick film staining and Acid Fast Bacilli by Ziehl-Nelson staining. The frequency of infectious agents obtained in the patients include 4.3 %(1) anti-HCV; 17.4% (4) HBsAg; 4.3 %(1) HIV1-p24 antigen; 17.4% (4) Plasmodium spp.; 8.7 %(2) Acid Fast Bacilli and 4.3 %(1) HBsAg + Plasmodium spp., in patients of herbal homes and 6.3 %(2) anti-HCV; 9.4% (3) HBsAg; 3.1 %(1) HIV1-p24 antigen; 15.6% (6)Plasmodium spp., 0%(0) Acid Fast Bacilli and 3.1 %(1) HBsAg + Plasmodium spp., in patients of the three major hospitals. The frequency of those with 5.0 – 6,0 pg/ml TNFα (65.2% (15)) was more than those whose TNFα was between 6.1 – 6.8 pg/ml (34.8% (8) in herbal home patients while the frequency of those with 5.0 – 6,0 pg/ml TNFα (40.6%(13)) was lower than those whose TNFα was between 6.1 – 6.8 pg/ml (59.4% (19)) in hospital patients. The pattern of the infectious agents was more in herbal home patients with 5.0 – 6,0 pg/ ml TNFα than those with 6.1 – 6, 8 pg/ml TNFα while in hospital patients it was more in those with 6.1 – 6,8 pg/ml TNFα than those with 5.0 – 6,0 pg/ml TNFα. The overall frequency of the infectious agents was more in herbal homes patients (58.5%(13)) than 37.5%(12) obtained in hospital patients. The frequency of viral immunochemical biomarkers, Acid Fast Bacilli and Plasmodium spp., was more in herbal home patients than the patients recruited from the hospital which varies with the TNFα range.


iNtRODUCtiON
H e r b a l t re at m e nt i nvo l ve s t h e administration of dietary supplement in form of tablets, capsules, powders, teas, extracts, and fresh or dried plants. People believe the treatment is safe and natural. Plants are basis for medical treatments in human history. Pharmaceutical drugs are made from many plant-derived compounds. According to World Health Organization (WHO) about 80 percent of the population in Asian and African countries use herbal medicine for some aspect of primary health care 1,2 . About 25% of modern drugs used in the United States were derived from plants 1,2 . At least 7,000 medical compounds in the modern pharmacopoeia are derived from plants. Out of the 120 active compounds currently isolated from plants and used in modern medicine currently, 80% show a positive correlation between their modern therapeutic use and the traditional use of the plants from which they are derived 1,2 .
Herbal treatment is more prevalent in patients with cancer, diabetes, asthma, viral infection, Plasmodiasis, hypertension/hypercholesterolemia and end-stage renal disease. Multiple factors such as gender, age, ethnicity, education and social class are also shown to have association with prevalence of herbal remedies use [1][2][3][4] . The most common administration of herbs in treatment in Nigeria is through drinking of raw liquid extract or aqueous extract through boiling 5 . Herbal treatment can cause adverse effects due to possible adulteration, inappropriate formulation, administration or lack of understanding of plant and drug interactions 6,7 .
The practice of herbalism entails that herbalists must learn wild-crafting, cultivation of herbs, diagnosis and treatment of ailments, preparations and dispensing of herbal medications 8 . Non-hygeinic and unethical practices by some herbalists could bring about cross infection and contaminations during product preparation 8,9 Tumor necrosis factor (TNFα ) is one of the cell signaling proteins known as cytokine involved in systemic inflammation; It is a cytokine that make up the acute phase reaction. It is produced by activated macrophages, CD4+ lymphocytes, NK cells, neutrophils, mast cells, eosinophils, endothelial cells, cardiac myocytes, adipose tissue, fibroblasts and neurons. It acts as an endogenous pyrogen, it induces fever, apoptotic cell death, cachexia, inflammation, inhibits tumorigenesis and viral replication 10,11 .
It acts on organ and system as proinflammatory cytokine to stimulate the release of corticotropin releasing hormone (CRH), suppressing appetite, induce fever, stimulate acute phase response which causes increase in C-reactive protein, stimulates phagocytosis, regulates bitter taste perception 12,13 . A local increase in concentration of TNF will brings about signs of Inflammation such as : heat, swelling, redness, pain and loss of function. The synthesis and release of TNFα are trigger by presence of infectious agents and chemicals that result in inflammation especially hepatitis 12,13 .
Inappropriate use of herbs, some phytochemicals, phytonutrients and infectious agents can induce inflammation 12,13 . This work was therefore designed to determine the Viral immunochemical frequency of anti-HCV, HBsAg and HIV1-p24 antigen including pattern of other infectious agents in herbal homes patients with elevated TNFα (≥5.0 pg/ml) in Saki -West-Nigeria

MAteRiAls AND MethODs study area
The study area is Saki-West local government area at the Northern part of Oyo state in Nigeria. It is a semi-urban area that shares border with Burkina faso, Kwara state, Saki-East and ATISBO local government areas in Nigeria.

Biological specimens
Five milliliters of venous blood was collected into lithium heparinized specimen bottle to determine viral biomarkers and plasma TNFα. Sputum was also obtained from each of the patients for the demonstration of Acid Fast Bacilli.

Methods TNF alpha ELISA
Plasma TNF alpha was determined in the subjects using Abcam's kit. Abcam's A monoclonal antibody specific TNF alpha has been coated onto the wells of the microtiter strips provided. Samples, including standards of known TNF alpha concentrations, control specimens or unknowns are pipetted into these wells. During the first incubation, the standards or samples and a biotinylated monoclonal antibody specific for TNF alpha are simultaneously incubated. After washing, the enzyme Streptavidin-HRP, that binds the biotinylated antibody is added, incubated and washed. A TMB substrate solution is added which acts on the bound enzyme to induce a colored reaction product. The intensity of this colored product is directly proportional to the concentration of TNF alpha present in the samples.

HIV-1 p24 Antigen ELISA using Zeptrometrix retrotek kit Principle
Microwells are coated with a monoclonal antibody specific for the p24 gag gene product of HIV-1. Viral antigen in the specimen is specifically captured onto the immobilized antibody during specimen incubation. The captured antigen is then reacted with a high tittered human anti-HIV-1 antibody conjugated with biotin. Following a subsequent incubation with Streptavidin-Peroxidase, color develops as the bound enzyme reacts with the substrate. Resultant optical density is proportional to the amount of HIV-1 p24 antigen present in the specimen.

Anti HCV ELISA Assay
This was determined by using Anti-Hepatitis C Virus Core Antigen antibody (ab50288) Abcam kit.

HBsAg ELISA Test
This was assayed using Diagnostic automation/ Cortez Diagnostics, INC kit by ELISA method Principle The HBsAg ELISA Test kit employs an (1) HBsAg + (1) (1) (1) (1) (32) antibody sandwich ELISA technique where monoclonal antibodies unique to HBsAg, are pre-coated on polystyrene microwell strips. The plasma sample was added together with a second After incubation and washing, to eliminate unwanted serum proteins and unbound HRP-conjugate, chromogen solutions containing tetramethyl-benzidine (TMB) and urea peroxide were added to the wells. The colorless chromogens were hydrolyzed by the bound HRP-conjugate to a blue-colored product. Sulfuric acid was added to stop the reaction and the blue color then turns yellow. This color intensity is directly proportional to the amount of antigen in the samples. If the blue colour remains colorless it indicates HBsAg negative.

Demonstration of Acid Fast Bacilli in sputum
Ziehl-Neelsen (Zn) staining technique was used to demonstrate Acid Fast Bacilli (Mycobacterium sp) as Described by Cheesbrough 14 .

Identification of Plasmodium species Giemsa staining-thick blood film technique
Plasmodium species were determined by Giemsa staining-thick blood film technique as described by Cheesbrough 14 .

Ethical Consideration
Ethical approval for this work was obtained from ethical and research committee of Baptist Medical center Saki-Nigeria before the commencement of this work. Informed consent was also obtained from each of the patients.

Methods of Data Analysis
Data were analysed for mean, percentage and frequency using Statistical Package for Social Sciences (IBM SPSS, version 18).

ResUlts
The overall frequency of the infectious agents was more in herbal homes patients (58.5%(13)) than 37.5% (12) obtained in hospital patients.
The pattern of the infectious agents was more in hospital patients with 5.0 -6.0 pg/ml TNFα than those with 6.1 -6.8 pg/ml TNFα while the pattern of the infectious agents was more in hospital patients with 6.1 -6.8 pg/ml TNFα than those with 5.0 -6.0 pg/ml TNFα (Table 1).

DisCUssiON
The overall frequency of the infectious agents was more in herbal homes patients (58.5%(13)) than 37.5%(12) obtained in hospital patients.
The The presence of infectious agents in patients with elevated TNFα (≥5.0 pg/ml) could be attribute to the fact that TNFα is a proinflammatory cytokine. The release is triggered by the presence of infectious agents to initiate process of inflammatory response and inhibit viral replication. It is also involved in acute phase reaction which can be caused by pathogenic agents.
In addition the pattern of the infectious agents was more in hospital patients with 5.0 -6,0 pg/ml TNFα than those with 6.1 -6,8 pg/ml TNFα while the pattern of the infectious agents was more in hospital patients with 6.1 -6,8 pg/ ml TNFα than those with 5.0 -6,0 pg/ml TNFα.
The explanation to these include the fact that the patients of herbal homes are regular set of individuals who have been accessing herbal treatment for sometimes while those recruited in the hospitals have never received any herbal treatment. This might be responsible for the differences in the frequency as un-hygienic and unethical practices by some herbalists could bring about cross infection and contaminations during product preparation and adminis-tration 8,9 . This could be due to the following reasons that drugs administered in hospitals apart from infectious agents can trigger inflammatory response leading to increase in plasma TNFα while most herbal preparations contain antiinflammatory and antioxidative agents 1,2 which may also account for the above results.
The findings of this work is consistent with the report of Yusa 15 that IL-4, IL-8, IL-10, IL-12 and TNF-α in addition to WBC counts and CRP levels could be a possible biomarkers of infections.

CONClUsiON
The frequency of viral immunochemical biomarkers, Acid Fast Bacilli and Plasmodium spp., was more in herbal home patients than the patients recruited from the hospital which varies with the TNFα range.