MOLECULAR IDENTIFICATION OF PLASMODIUM FALCIPARUM ISOLATES IN OWERRI MUNICIPALITY USING NESTED POLYMERASE CHAIN REACTION (nPCR)

1. Department of Microbiology, Federal University of Technology, Owerri, Imo State, Nigeria. 2. Department of Environmental Health Science, Federal University of Technology, Owerri, Imo State, Nigeria. ...................................................................................................................... Manuscript Info Abstract ......................... ........................................................................ Manuscript History Received: 25 June 2020 Final Accepted: 30 July 2020 Published: August 2020

As Public health seek ways to annihilate malaria, there has been special attention to abrogate low-density parasite reservoirs in carriers. This research was designed to detect Plasmodium falciparum using nested polymerase chain reaction (nPCR) as a diagnostic tool to test for malaria prevalence in Owerri municipality, and the result compared to that of microscopy. A total of 300 blood samples were collected from febrile patients who sought treatment from selected hospitals in Owerri municipal. Microscopy was carried out on Giemsastained blood smears of the samples. Extraction of Plasmodium falciparum DNA from the blood samples was carried out, and the DNA products were analyzed using nPCR with genus and specie specific primers in a two-step amplification of the Pfmdr gene. The PCR products were analyzed in ethidium bromide stained 2% agarose gel. The result from microscopy showed that 130(43.3%) samples were P. falciparum positive and 170(56.7%) samples were negative, while that of nPCR, showed 202(67.3%) samples were positive and 98(32.7%) samples were negative. Out of the 170 samples that were microscopically examined as negative, 72(42.35%) were positive with nPCR. This indicates that there is high level of false negative result by microscopy, which may either lead to negation for a deserved treatment or undeserved treatment. Accurate diagnosis of Plasmodium falciparum is of importance in the treatment of malaria. Microscopic examination of blood smears remains the "gold standard" for malaria detection and speciation. In spite of the fact that there is significant drop in the number of positive cases reported from microscopy, antimalarial drugs prescriptions are on the rise as patients exhibit symptoms of malaria. This makes it tough to confirm accuracy, sensitivity and specificity of light microscopy in diagnosis of malaria in epidemic areas. Polymerase chain reaction offers an alternative to microscopy having shown to have superior sensitivity and specificity. Diagnosis of malaria parasites by nPCR will serve as a useful complement to microscopy, albeit it's high cost and time consumption. In addition, negative patients suspected to have malaria should be subjected to PCR diagnosis to better drug use.

…………………………………………………………………………………………………….... Introduction:-
Malaria is a serious problem in Africa where one in every five (20%) childhood deaths is due to the effects of the disease [121]. The World Health Organization (WHO) reckon that Africa bears almost 90% of the global burden of malaria. Nigeria due to her population of over 180 million people bears the largest share of the burden, with over 300, 000 malaria related deaths annually, especially in children under 5years [162]. It is reported that every 30 seconds, a child dies from malaria. About 3.3billion people are at risk of malaria [121]. Many children who survive an episode of severe malaria may suffer from learning impairments or brain damage [165].

Age distribution
The age-group 81-90 years had the highest malaria prevalence (100%) in both PCR and microscopy, while the age groups 41-50 and 51-60 years in microscopy and PCR respectively had the least 16.67% and 40% as shown in Table  4.2 and figure 4.3 below. The results were significant (P=0.02, p<0.05).

Educational Status
The level of education of each of the 300 patients involved in the study, were noted.
Considering the educational status of the patients in relation to positive malaria results (Table 4.4), showed no significant difference (P=0.085, p<0.05) between the groups.

Plasmodium falciparum Identification
Out of the 300 samples that were examined for Plasmodium falciparum, 130 Table 4.8 shows the results for the awareness of the occurrence of different species of Plasmodium spp. More respondents (100%) were aware of the existence of Falciparum species of Plasmodium infection than the other species. The differences in their views was not statistically significant at (p=0.128, p<0.05), meaning that the outcome did not occur by experimental chances.

Discussion:-
Malaria is a serious disease caused by a parasite belonging to the Plasmodium genus and is transmitted by female Anopheles mosquito vector [134]. In Nigeria, the disease is endemic in most areas (case study: Owerri Municipal). In this study, blood samples of patients were collected at three hospitals within the study area. The patients who visited the hospitals reside within the area, hence the samples used in the study were representative of Owerri municipality.
An important aim of the study was to employ molecular method to detect Plasmodium falciparum from patients, which is the most common in Africa and is the specie that causes the most severe disease [89]. Nested polymerase chain reaction (nPCR) was used as a diagnostic tool to detect DNA of Plasmodium falciparum from blood samples of patients. The blood samples had previously been examined by microscopy, which is one of the main traditional methods. The results from nPCR were evaluated and compared against those of microscopy.
A total of 300 blood samples were collected from patients. The samples were tested for falciparum malaria using nPCR and microscopy. Result showed that of the 300 blood samples tested, 202(67.3%) samples were positive and 98(32.7%) samples were negative using the nPCR based technique (Table 4.5). While, 130(43.3%) samples were positive and 170(56.7%) samples were negative using microscopy (Table 4.5). The overall malaria prevalence detected by nPCR at 67.3% in the study area was found to be higher than that detected by microscopy at 43.3%. The nPCR method was however able to detect the parasite DNA in some of the samples that had been categorized as malaria-negative by microscopy, this could be a typical case of low parasitemia and PCR was able to detect the 831 presence of parasite DNA at levels undetectable by microscopy, as reported by Snounou et al., 1993 [133]. This finding suggests that some samples were misdiagnosed negative by microscopy, which could be said to be falsenegative. The prevalence detected by nPCR (67.3%) was a bit lower than that of Ukpai  In Table 4.2, comparing the results from microscopy and nPCR among different age groups. It was observed that microscopy showed lesser sensitivity in the groups compared to nPCR, except in the age group 81-90 which had the highest prevalence of 100% of P. falciparum infections (table 4.2) compared to the other age groups. Prevalence of malaria in other age groups were also high which was in agreement with Uneke et al., 2005 [150], who recorded higher prevalence among the older age groups in a similar study in Jos, Nigeria. This may be due to the fact that at that age, their immunity to parasitic infections weakens and environmental conditions predisposes them to malaria. The prevalence of parasitic infections among the different age groups in the study was significant (P< 0.05) indicating that the occurrences of this infection on these age groups were not the same (table 4.4). Table 4.3 showed the prevalence of malaria in male and female as detected by nPCR as 70.9% and 63.8% respectively, and also, 45% and 41.6% respectively as detected by microscopy. nPCR showed greater sensitivity compared to microscopy. The prevalence was statistically significant (p=0.001, p<0.05). Tukey and Fisher's Pairwise Comparison showed that there was significant difference between the number of samples tested, nPCR results and microscopy at p<0.05. The study showed that P. falciparum infections were more common in the male than in the female subjects (Table 4.3). The sex prevalence supports Ukpai and Ajoku (2001) [151], who reported 78.0% and 72.0% prevalence for male and female respectively. It also agrees with the results obtained in Ogun State [124] and those by Ajero et al. 2015 [3], which reported prevalence of 27.5% and 24.3% for male and female respectively. The result conforms to the recorded higher prevalence of Plasmodium infection in males than in females in the hospital. However, studies have shown that females have better immunity to parasitic diseases and this was attributed to genetic and hormonal factors [66]. Portilo and Sullivan 1997 [111] suggested that genetic factors could play a role by endowing females with immuno-regulatory potentials to cope better with some disease infections. This may equally be attributed to the fact that males expose themselves to the bites of mosquitoes and other vectors more than females, especially when the weather is hot and during farm work. Exception is found during pregnancy and reproductive ages, when females are more vulnerable to malaria attacks due to immune suppression [9].
Prevalence of malaria was not statistically significant (P=0.621, P<0.05) among different occupational groups. It showed that people in different occupational groups were equally exposed to malaria attack. Subjecting the data further to Tukey and Fisher's Pairwise Comparison, revealed that prevalence among different occupations was not significant between nPCR and microscopy at P<0.05. Though in nPCR, prevalence was highest in unemployed, this could be as a result of inadequate protection greater exposure to mosquito bites due to the nature of their contact with the unclean environment exposing themselves to the risk of mosquitoes (Table 4.1 and figure 4.2). Poor people from low socio-economic status, with inadequate housing facilities and financial constraints are unable to engage in malaria preventive and control measures. They are also unable to purchase effective anti-malaria drugs [2].  (Table 4.7). The differences in their views were not statistically significant at (p=0.128, p<0.05), meaning that the outcome did not occur by experimental chance (Table 4.8). According to Markell and Voge, 1992, falciparum malaria is almost entirely confined to the tropics and subtropics. The Predominance of P. falciparum in the prevalence and intensity of malaria in blood donors in Nnewi, Anambra State, Nigeria was also reported by Umeanaeto et al. 2006 [149].
Statistically, there was no significant difference between the means of the compared characters of the departments from which the samples were collected at (p=0.641, p<0.05). It indicated that the results obtained do not depend on the department where the samples were taken from, but could have depended on the exposure of the patients to the parasite (Table 4.6).
The results of this study indicated that the examination of thick and thin blood smears by microscopy were insufficient for the diagnosis of malaria in this region (Table 4.5). This study supported the idea that sensitivity decreases with microscopical tests as parasitaemia falls below 100 parasites/mL and false negatives are observed [133].

Conclusion:-
This study emphasized that nPCR is an excellent method for obtaining accurate epidemiological data in malaria endemic Owerri municipal (Table 4.5). The diagnosis of P. falciparum by nPCR might prevent misdiagnosis, incorrect treatment, false positives, false negatives, the emergence and spread of drug resistance, and the transmission of parasites from a malaria-endemic region to other parts.