Molecular and morphological identification of suspected Plasmodium vivax vectors in Central and Eastern Sudan

doi:10.21203/rs.2.22911/v1

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Molecular and morphological identification of suspected Plasmodium vivax vectors in Central and Eastern Sudan

https://doi.org/10.21203/rs.2.22911/v1

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published 04 Mar, 2021

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Background: In spite of the global effort to eliminate malaria, it remains the most significant vector-borne disease of humans. Plasmodium falciparum remains the dominant malaria parasite in Sub-Saharan Africa. However, P. vivax is becoming widely spread throughout Africa. The overuse of vector control methods has resulted in a remarkable change in the behaviour of mosquito that feeds on human as well as on vector composition. The aim of this study was to identify Anopheles mosquito species in vivax malaria endemic regions and to investigate their role in P. vivax malaria transmission.

Methods: Mosquito samples were collected from Central Sudan (Khartoum and Sennar States) and Eastern Sudan (New Halfa) using Pyrethrum Spray Catch (PSC) and CDC light traps. Mosquitoes were identified using appropriate morphological identification keys and Anopheles gambiae complex were confirmed to species level using molecular analysis. A subset of blood-fed anopheline mosquitoes were dissectedto study the presence of natural infection of malaria parasites. In addition, the rest of the blood-fed samples were investigated for presence of infective sporozoites of P. vivax that detecting the P. vivax circumsporozoite protein (pvcsp) gene using nested-PCR.

Results: A total of 644 adult anopheline blood-fed mosquitoes were collected from New Halfa (N = 214 mosquitoes), Khartoum (N = 132 mosquitoes), and Sennar (N = 298 mosquitoes). Morphological and molecular identification of the collected mosquitoes revealed the presence of An. arabiensis, An. funestus, and An. pharoensis. Out of 644 anophelines subjected to P. vivax sporozoite detection (N = 40, 6.2%) were positive for P. vivax.An. arabiensis 6.7% (39/583) and An. pharoensis 1.8% (1/56) were found positive for the parasite. While An. funestus was detected for the first time in New Halfa with low abundance (5/214 mosquitoes) and were found negative for the parasite.

Conclusion: This study documented the presence of An. funestus for the first time in New Halfa, Eastern Sudan. However, An. arabiensis is the most abundant vector detected, together with An. pharoensis. This findings suggests change in malaria epidemiology. Further studies are needed to investigate their contribution in malaria transmission.

Infectious Diseases

Malaria vectors

Anopheles funestus

Anopheles arabiensis

Anopheles pharoensis

Plasmodium vivax

Sudan

The estimated malaria cases in 2018 was 228 million cases occurred worldwide resulting in 405,000 malaria related death, owing the deadliest parasite (Plasmodium falciparum), the majority in Sub-Sahara Africa[1]. According to the World Health Organization (WHO) report in 2018, 89% and 91% of all malaria cases and related deaths respectively, were across the African continent [2].P. falciparum considered to be the most important due to its predominance and severity, responsible for more than 99.7% of malaria cases[2, 3] followed by P. vivax[4] a generally considered less pathogenic parasite causing a benign type of malaria. However, the “benign tertian malaria” description of vivax-malaria has been challenged by recent reports and documentation of severe P. vivax infections and even deaths due to P. vivax mono-infections [5, 6]. P. vivax stands for about half infections outside Africa[1, 7, 8], representing 75% of malaria cases in the WHO Region of the Americas, 53% in the WHO Region of South-East Asia[1] and 40% in the Eastern Mediterranean region[8]. However, its presence in Africa has not well documented and reported because of the very high endemicity of P. falciparum and for the accepted paradigm that Africans are protected versus P. vivax infection by genetic factors [9–11].

Plasmodium vivax parasite exploits the human Duffy antigen/chemokine receptor (DARC) to invade the red blood cell [12]. Duffy antigen is rarely expressed in African population [13], so infection prevalence was thought to be less in Africa due to negativity of Duffy binding protein among its population[9, 14]. However, several studies revealed that infection may persist lacking this protein [15, 16].

Previously, it has been reported that P. vivax prevalence was estimated to be 6.1% in Eastern and Central Sudan [17]. Interestingly, in recent years, the prevalence of P. vivax infections increased throughout the country with an overall prevalence of 27% [18], 40% in White Nile [19], and 38% in Gezira State [16].

The role of Anopheles mosquitoes in transmitting malaria parasites depends on several factors including their preference to feed on humans[20]and their innate susceptibility to the Plasmodium[21, 22]. The main malaria vectors in Africa belong to three major groups of vectors, the An. gambiae complex, An. funestus group, and An. nilicomplex[23, 24]. Methods of mosquito control still rely heavily on the use of LLINs and IRS [25, 26]that target indoor resting vectors. An updated study of anopheline mosquitoes and their behaviour is much needed to guide the vector control operations [27, 28].

The vast majority of studies stated that An. arabiensis is the main if not the solo malaria vector in Sudan [29–32]. However, few studies encountered the presence of other Anopheles species at malaria foci in Sudan, such as An. funestus s.s[33], An. pharoensis [34]and An. nilis.l.[35]. The present study was conducted to identify the anopheline mosquito fauna in regions where vivax malaria is endemic [36] and to investigate their role of transmission of P. vivax.Plasmodium circumsporozoite protein (CSP) is an abundant surface protein expressed on the surface of sporozoite and oocyst [37]. The gene encodes CSP has three distinctive variants (VK 210, VK 247 and P. vivax like) [38]. Plasmodium vivax circumsporozoite protein (pvcsp) is the major cell surface protein of the sporozoites. This protein plays many roles, including salivary gland invasion in mosquitoes, sporozoite maturation, and hepatocyte invasion in humans [39].

Study sites and mosquito collection

The study was conducted in two regions, Central Sudan (Khartoum State 15.7938° N, 32.8987° E, Sennar State 13.0317° N, 33.9750°) and Eastern Sudan (New Halfa 15.3288° N, 35.5986° E) (Figure 1). The collection of the adult anopheline mosquito samples was carried out using standard Pyrethrum Spray Catch (PSC), and CDC light traps.

Morphological identification and samples processing

Samples were identified to species level using standard entomological keys [23, 40]. A number (108) of the collected blood-fed Anopheles species were dissected to detect the natural infection of Plasmodia parasites according to WHO (1975)[41], while the rest samples were investigated molecularly.

DNA extraction and Pvcsp nested PCR

The total genomic DNA was extracted from the whole individual mosquito using Livak method[42]. Then samples were subjected to molecular identification [43, 44] and parasite detection using species-specific primers ofImwong et al. (2005)[45]. Cycling conditions for mosquito identification were followed as described by Scott et al. (1993)[43] with slight modification on the cycling number (36 cycles). Then PCR products were observed under UV illumination and documented using BioDocAnalyze gel image documentation system (Biometra Analytika Jena Company, Germany).

The nested-PCR was performed for P. vivax parasite detection following Imwong et al. (2005) and Lopez et al. (2012). Cycling condition for the outer PCR was as the following: 95ºC initial denaturation for 3 minutes, 37 cycles of: denaturation at 94ºC for 30 sec, annealing 58 for 1 min, 72ºC elongation for 1 min, 72ºC final elongation for 10 min. The second reaction was set as follows: 95ºC initial denaturation for 3 minutes, 15 cycles of: 94ºC for 30 sec, 63.8ºC for 1 min, 72ºC for 2 min, 22 cycles of: 94ºC for 30 sec, 64ºC for 1 min, 72ºC for 2 min and final elongation at 72ºC for 10 min. Each reaction included one negative PCR control and a positive control of P. vivax DNA. Following the PCR amplification, the PCR products were separated on a 1.5% Agarose gel. The PCR products were analyzedunder UV illumination and documented using BioDocAnalyze gel image documentation system (Biometra Analytika Jena Company, Germany).

A total of 644 adult anopheline blood-fed mosquitoes were collected from New Halfa (N = 214 mosquitoes), Khartoum (N = 132 mosquitoes), and Sennar (N = 298 mosquitoes) (Table 1). The morphological and molecular identification revealed that the species composition of the collected mosquito was predominately An. arabiensis 91% (583/644), An. pharoensis 8.7% (56/644), and An. funestus sesu stricto s.s0.8% (5/644) (Table 1).

Five hundred thirty-six (83%) and 108 (17%) specimens out of 644 blood-fed samples were tested for the P. vivax using molecular and dissecting techniques, respectively (Table 2). Forty out of 536 (7.5%) of the PCR-tested blood-fed mosquito samples were positive for P. vivax, revealed both An. arabiensis and An. pharoensis (Table 2). While all samples tested by dissecting technique to detect sporozoites under microscope were negative for P. vivax.

Different P. vivaxpvcsp allelic variants were found in two study areas. In New Halfa, five allelic variants (700bp, 300 bp, 250bp, 200bp and 100bp) were detected among the 34 positive samples (Fig. 2). While in Sennar State, two allelic variants (400 bp and 300 bp) were spotted in the 6 positive samples (Fig. 2). No parasite was detected in the samples from Khartoum State.

This is the first study to document the presence of An. funestus in Eastern Sudan, New Halfa. Updating the existed knowledge about the vector composition and relative density in malaria transmission area are essential entomological and epidemiological indicators for the diseases burden, transmission season, and monitoring the vector control methods [46-48].

Previous studies on Sudan of Abdalla et al. (2007)[17], Elmahdi et al. (2012)[31] and Mahgoub et al. (2012)[6] illustrated that P. falciparum is responsible for more than 95% of clinical malaria cases while revealing 3% were due to P. vivax, but an increase of infections due to P. vivax was recently encountered in so many parts [16, 19]. The present study was conducted to identify the species of Anopheles mosquito in endemic regions of vivax malaria and to investigate their role in P. vivax malaria transmission.

Results of this study showed that An. arabiensis was the most abundant Anopheles species, followed by An. pharoensis, and the least was An. funestus, which supports that An. arabiensis is the main malaria vector in Sudan [31]. However, in addition to Lewis(1956)[35] no further published data from Sudan revealing the role of An. pharoensis which considered to be a secondary vector, though we found one An. pharoensis sample positive for P. vivax unlike Elmahdi et al.[35] in Sennar State and Ahmed [30] in Gezira State where only An. arabiensis was found positive to carry the parasite.Unlike Himeidan et al. [49] and Lewis [50] who recorded the presence of An. pharoensis beside An. multicolor, the study revealed the presence of An. funestus, but in a small number.

We were not able to detect the sporozoites by the dissection technique, could be mainly because of the low prevalence of parasites in the mosquito populations and this finding is similar to previous studies [30, 31, 51].

In this study, no positive sample for P. vivax were detected in Khartoum State, this finding is similar to Ali [52]. This could be attributed to low infection rates (22.4%) in Khartoum State compared to (55.7%) in Kassala State, Eastern Sudan [18].

The diverse detected allelic forms of P. vivax among mosquito vectors could be attributed to different age of sporozoite [53], also the number of repeats in the protein repeat region may vary between alleles of one species and in some species (such as P.cynomolgi),the length ofeach repeated unit varies widely between alleles [54], this variation can result in size polymorphism.

vivax is becoming a serious health problem exhibiting a wide range of hosts children[6], adults [55] and even pregnant women [56]. Also has been detected on asymptomatic individuals [57, 58]. The underestimation of P. vivax malaria infections could be attributed to misdiagnosis of the disease using Rapid diagnostic tests RTDs [59], presence of hypnozoite on reservoirs which again cannot be detected using RTDs [60].

Assessment of the impact of vector control interventions on malaria transmission requires more data about entomological indicators including the identification of the vector composition, distribution, and density [61].

The findings of this study are very alarming mainly because it showed the expansion of the efficient malaria vector distribution, An. funestus, into Eastern Sudan. Also, it confirmed the role of An. pharoensis in the malaria transmission in Central Sudan. Both main finding suggested changes in malaria epidemiology in Sudan that requires further entomological, parasitological, and epidemiological studies to accurately determine the malaria vectors distribution and density countrywide, and to investigate the role of each vector species in the malaria transmission. Additionally, the potential associations between the different vector species and the different Plasmodium species they transmit need to be investigated thoroughly. Furthermore, the susceptibility of the malaria vectors existed in Sudan to the currently applied vector control tools must be investigated urgently.

Ethical approval and consent to participate

Not applicable.

Consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests

Funding

This research received partial funding from Third World Academy of Sciences (TWAS), Italy.

Authors’ contributions

MMAH and MMA: conception and design of project, OFA, AA and MMA: Sample collection and preservation. OFA: conducted the lab work. OFA, AE, GPM and MMAH: analysis and interpretation of data. OFA, AE, GPM, AA and MMAH: drafted and corrected the manuscript. All authors read and approved the final manuscript.

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Table (1). Represents anopheline mosquito collected from different study sites in Central and Eastern States, Sudan

*Anopheles species*	Study area			Total
*Anopheles species*	Khartoum	New Halfa	Sennar	Total
Anopheles arabiensis	132	209	242	583
An. pharoensis	0	0	56	56
An. funestus	0	5	0	5
Total	132	214	298	644

Table (2). Plasmodium vivax pvcsp gene positivity among anophline mosquitoes collected from Central and Eastern States, Sudan

*Anopheles* sp.	Infectivity rate N (%)
An. arabiensis	39 (6.7% )
An. pharoensis	1 (1.8%)
An. funestus	5 (0%)

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Editorial decision: Minor Revision
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Molecular and morphological identification of suspected Plasmodium vivax vectors in Central and Eastern Sudan

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