Pattern of Mosquito Borne Parasitic Infection in the Night Blood Samples of Patients with Elevated TNF-α of> 5.0 pg/ml

Study Background: Plasmodium spp., (Protozoan) and Wuchereria bancrofti (Nematode) are transmitted by mosquitos to cause insect borne diseases known as malaria and Lymphatic filariasis/Elephantiasis. Apart from the social implication of these parasitic infections the infections can also elicit immune responses. Aim and Objective: This work was therefore designed to determine the pattern of mosquito borne parasitic infection in the night blood samples of patients with elevated TNF-α of > 5.0 pg/ml. Materials and Methods: Seventy (70; aged 31 – 76 years; Male35; Female-35) volunteers with plasma TNFα of 5.8 ±0.7 pg/ including age-matched control participants ( n= 50 ; TNFα of 2.2 ± 0.3 pg/ml). All participants were negative to Acid Fast Bacilli, ant-HCV, HBsAg and HIV tests were recruited for the study. Night blood samples and sputum samples were obtained from the participants. Blood sample was used for determination of TNFα, HIVp24ag-Ab, anti-HCV, HBsAg by ELISA and identification of Plasmodium and Wuchereria by Geimsha thick blood film staining while sputum samples were used for the demonstration of Acid Fast Bacilli by Ziehl Neelsen staining. Results: The results showed a frequency of Plasmodium spp., in individuals with plasma TNF-α of 5.8 ±0.7 pg/ml as 31.4%(22) as against a frequency of 18%(9) in subjects with plasma TNF-α of 2.2 ± 0.3 pg/ml.. The results also showed a frequency of 5.71%(4) and2%(1) Wuchereria bancrofti in subjects with plasma TNF-α of 5.8 ±0.7 pg/ml and TNF-α of 2.2 ± 0.3 pg/ml. respectively. The overall frequency of parasitic infection obtained in both test and control subjects include: 33.3% (40)Plasmodium spp., and 4.2%(5) Wuchereria bancrofti.The overall results from both test and control subjects also showed a gender distribution of 20%(24) and 13.3%(16) Plasmodium spp.,in female and males respectively while a distribution of 1.7%(2) and 2.5%(3) Wuchereria bancrofti in females and males respectively. Conclusion: This work revealed increase in the frequency of Plasmodium spp. and Wuchereria bancrofti infections with increase in plasma TNF-α while the overall frequency of parasitic infection obtained in both test and control subjects was found to be 33.3% (40)Plasmodium spp., and 4.2%(5) Wuchereria bancrofti with possible variations in regional and gender distributions. Mosquito borne parasitic infection of Plasmodium spp., was found to be more prevalent in patients with elevated TNF-α of> 5.0 pg/ml.

Malaria and filariasis are common diseases transmitted by mosquito in rural communities. Giemsa stained thick blood film smears is the "gold standard" ( finger prick Published  test) for the identification of Plasmodium and Microfilaria (W. bancrofti). Night blood sample is preferred for the identification of W. bancrofti [5][6] [7] .
Immune system of the mosquito has not been proven to destroy W. bancrofti or Plasmodium falciparum though the parasites especially Plasmodium falciparum alters the mosquito vector's feeding habit by increasing frequency of biting in infected mosquitoes, thus increasing the chance of transmitting the parasite [8] .The life cycle of Plasmodium spp., that causes malaria and Wuchereria bancrofti the major cause of lymphatic filariasis takes place in human and mosquito. Humans is the definitive host and mosquitos as the intermediate host for W. bancrofti while human is the intermediate hosts in which asexual reproduction takes place and female anopheline mosquito is the definitive host in which sexual reproduction occurs [9][10] [11] [12][13] [14] .
Tumor necrosis factor alpha(TNF-α) is an inflammatory cytokines (cell signaling protein) and one of the cytokines that make up the acute phase reaction. It is synthesized by activated macrophages, CD4+ lymphocytes, NK cells, neutrophils, mast cells, eosinophils, and neurons TNF-α primarily regulates the immune cells. It is an endogenous pyrogen that induces fever, apoptotic cell death, cachexia, inflammation in response to parasitic infection [15] [16][17] [18] .
This work is therefore designed to determine the pattern of mosquito borne parasitic infection in the night blood samples of patients with elevated TNF-α of > 5.0 pg/ml in a rural community.

Study Area
Atisbo was curved out of the old Ifedapo local government area which has been split to 3 local governments. It is located in Okeogun the Nothern part of Oyo State in Nigeria with its headquarters in Tede. Atisbo local Government was created by former Head of State Late Gen. Sanni Abacha in 1996. It is dominated by communities and their major occupation is farming.. It shares boundaries with Orire Local Government, Republic of Benin, Saki East Local and Itesiwaju and Iwajowa Local Governments. ATISBO is an acronym for Ago-Are, Tede, Irawo , Sabe , Baasi, Ofiki and Owo communities.

Sample Collection
Night blood samples and sputum samples were obtained from the participants. Blood sample was used for TNFα, HIV, anti-HCV, HBsAg ELISA and identification of Plasmodium and Wuchereria bancrofti. Sputum sample was used for Ziehl Neelsen staining to demonstrate Acid Fast Bacilli (AFB).

Laboratory Identification of Plasmodium spp., Wuchereria bancrofti and Acid Fast Bacilli
Laboratory of Plasmodium spp., Wuchereria bancrofti was carried out by Microscopy using Geimsha-Thick film method while Acid Fast Bacilli was demonstrated in the sputum as described by Cheesbrough [19] .

Anti-HCV ELISA Assay
This was determined in the subjects using Abcam kit.

HIV ELISA Test
HIV test was carried out using Genscreen™ ULTRA HIV Ag-Ab Biorad Kit.
The Genscreen™ ULTRA HIV Ag-Ab is an enzyme immunoassay based on the principle of the sandwich technique for the detection of HIV antigen and of the various antibodies associated with HIV-1 and/or HIV-2 virus in human serum or plasma.

HBsAg ELISA Test
This was assayed using Biorad ELISA kit.

TNF alpha ELISA
Plasma TNF alpha was determined in the subjects using Abcam's kit. Abcam's.

ETHICAL CONSIDERATIONS AND CLEARANCES
Tthis work was approved by 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 patient and control subjects.

METHOD OF STATISTICAL ANALYSIS
The results obtained were subjected to statistical analysis using IBM SPSS 18.0 to determine mean, standard deviation and frequency.
The results in both test and control subjects also showed a gender distribution of 20%(24) and 13.3%(16) Plasmodium spp.,in female and males respectively while a distribution of 1.7%(2) and 2.5%(3) Wuchereria bancrofti in females and males respectively(Table1, Figure 1,2).
The frequency of Plasmodium spp., and Wuchereria bancrofti was higher in subjects with elevated plasma TNF-α than the results obtained in those with lower (normal) plasma TNF-α . This is attributable to the bioactivities of TNF-α as a pro-inflammatory cytokine as Plasmodium spp., and Wuchereria bancrofti can elicit inflammatory responses to regulate immune cells and induce fever including cell death [12][15] [16][17] [18] .
The frequency of Plasmodium spp., reported in this study was higher than the report of WHO [20] because World Health Organization [20] in 2018 reported a prevalence of Plasmodium spp., (malaria) infection of 25% in Nigeria . this difference might be due to the fact that the WHO report was an overall prevalence in Nigeria considering all regions whereas this work was carried out in a local government area in South West-Nigeria.
Prevalence of Wuchereria bancrofti found in this study was lower than the reports of previous studies in Nigeria because Okorie et al., [21] in 2013 found the prevalence of, Lymphatic Filariasis in Nigeria and reported that the mean prevalence of circulating filarial antigen (CFA) was 14.0% (in 134 locations), and by microfilaria (Mf) was 8.2% (in 162 locations). Okorie et al., [21] concluded that Nigeria has the highest burden of lymphatic filariasis (LF)/elephantiasis caused by Wuchereria bancrofti which is transmitted by mosquitoes; Mu`awiyya et al., [22] carried out a sero-prevalence of Lymphatic Filariasis in Six Communities of Talata Mafara Local Government Area, Zamfara State, Nigeria and found an overall sero-prevalence of 37.8%. with highest prevalence of 43.3% in farmers than other occupational groups and Adekunle et al., [23] reported that 27%(291) out of 1,090 blood specimens examined were positive for infection with W. bancrofti in Ose Local Government Area, Ondo State, Nigeria. They reported a ge frequency of 27%(108 out of 394) in males and 26%(183 out of 696) in females using Immunochromatographic Test (ICT) for the detection of W. bancrofti.
Generally, variations in the prevalence of these two parasitic infections considering the results obtained from some parts of Nigeria might be as a result of differences in vegetation, level of hygiene and major occupation favoring the habitation, multiplication of the transmitting mosquitoes and the transmission of the parasites [12] .
In addition this work targeted test subjects with elevated TNF-α and generally test and control participants who are free of HIV, HCV, M .tuberculosis and HBV infections which might accout for the variation in the prevalence of the two parasitic infections compared with the previous reports [