DETECTION AND GENETIC ANALYSIS OF IMPORTED MALARIA IN EGYPT

Malaria is a disease that affects about 3.4 million persons1 of which 2.57 million and 2.5 million are at risk for P.falciparum and P.vivax respectively while P.ovale and P.malariae have a small percentage.2
WHO recorded in the world Malaria Report 2012 that1.2 million people died due to malaria infection 3. In Africa, malaria is considered the second cause of death among transmitting diseases after AIDS 4.
WHO considers malaria as a treatable, controllable, and preventable disease 5. So, a lot of malaria control interventions were applied including indoor residual
spray (IRS)6. Artemisinin combined treatment (ACT) was used as the 1st-line of treatment 7.
Examination of blood smears (stained by Giemsa) microscopically is considered the standard method
for malaria diagnosis 8 where parasitic stages can be detected by microscopists. Detection of malarial antigens by RDTs as well as PCR are considered other new important methods for the diagnosis of malaria 9.
Screening protocol for malaria species has 2 steps: First, a rapid real-time PCR to detect malaria parasites by targeting their gene including ssRNA genes. The second is conventional PCR including nested-PCR (nt-PCR) assays (for +ve samples) 10

Screening of malaria is necessary to detect hot areas of malaria infection where control measures are not effective11. So, the present work aimed to screen imported malaria in Egypt.
PATIENTS AND METHODS
This study is a cross-sectional one. It was performed to detect the prevailing malaria species in seven hundred and fifteen blood samples. The blood samples were collected from different medical laboratories in Cairo from travelers to endemic African areas either Egyptians or foreigners coming to Egypt within the previous eight weeks, in the period from August 2017 to August 2018. Malaria diagnosis was determined according to clinical manifestations, travel history, and the positive results of microscopic examination as well as RDTs [Abon Biopharm (Hangzhou) Co., Ltd., China]. Thin and thick blood films were prepared from all blood samples then RDT was performed to all blood samples, the rest of blood samples were collected in dry clean sterile tubes which contain EDTA, then stored at 2–8C for up to 3 days or at −20°C to store it for a longer duration for molecular studies (PCR and nPCR).
Parasitological Examination: Thin and thick blood films were prepared and stained by 10% Giemsa-stain 12, then examined microscopically by experts.
Immunological method: RDT interpretation: C band development indicates test validity. In case of presence of C band only indicate –ve result. The presence of pf band (with C band) indicates +ve result for P. falciparum (Fig.1).
Figure (1): P. falciparum + ve RDT
The presence of a pan band (with C band) indicates +ve results for P.vivax, P.ovale, or P. malariae. (Fig. 2).
Figure (2): Species – ve RDT
Molecular Studies: All microscopically positive blood samples for Plasmodium were subjected for nested-PCR (nPCR) analysis. The molecular assay was done in 3 main steps :
i.Extraction of genomic DNA from blood: We use (the Monarch PCR & DNA Cleanup Kit) to extract Genomic DNA from all dried blood samples.
ii. Amplification of extracted genomic DNA using nPCR: Extracted genomic DNA from each blood sample was used for PCR amplification, using Monarch PCR & DNA Cleanup Kit according to the manufacturer’s instructions.
Using the primers designed based on the sequence of SSU rRNA of Plasmodium 13. The sense and antisense primers of plasmodium species which were (rPLU5) 5`-CCT GTT GTT GCC TTA AAC TTC-3` and(rPLU6) 5`-TTA AAA TTG TTG CAG TTA AAA CG-3`, respectively were used in the first PCR. PCR product size 1100 bp was recovered in positive samples. PCR products from Plasmodium genomic DNA were amplified using the rPLU1 and rPLU5 primers for detection of Plasmodium species, while further amplified in nested (second) PCR 4 reactions used the sense and antisense primers of (i) P. falciparum [the sense primer (rFAL1) 5`-TTA AAC TGG TTT GGG AAA ACC AAA TAT ATT-3` and antisense primer (rFAL2) 5`-ACA CAA TGA ACT CAA TCA TGA CTA CCC GTC-3`] with PCR product size 205 bp, (ii) P.vivex [(PV18SF ) 5`-GAA TTT TCT CTT CGG AGT TTA TTC-3` and (PV18SR) 5`-GTA GAA AAG GGA AAG GGA AAC TGT TA-3`]with PCR product size 419 bp, (iii) P. malariae [(PM18SF) 5`-GAG ACA TTC ATA TAT ATG AGT GTT TCT-3` and (PM18SR)5`-GGG AAA AGA ACG TTT TTA TTA AAA AAA AC-3`] with PCR product size 423 bp, (iv) P. ovale [(PO18SF) 5`-GAA AAT TCC TTT TGG AAA TTT CTT AG-3` and (PO18SR) 5`-GGG AAA AGG ACA CTA TAA TGT ATC-3`] with PCR product size 410 bp 14.
Three microliters of the DNA template were used for PCR amplification in the reaction mixture of the master mix and 50 pmol of each primer. PCR cycling condition for 1st PCR was as follows: denaturation at 94˚C for 4 min, followed by 35 cycles of 94˚C for 30 sec, 55˚C for 1 min, 72˚C for 1 min, and then 72˚C for 4 min.
For 2ry PCR, the cycling conditions were as follows: Heating of mixture at 93 ºC for three minutes then 30 cycles of denaturation at 93 ºC for 30 s, annealing at 58 ºC for 45 s, and extension at72 ºC for 45s. The expected amplification product size was 205 bp
(P.falciparum) 419 bp(P.vivax) 423 bp (P.malariae) and 410bp (P.ovale).
iii. Detection of PCR amplification products using gel electrophoresis and ultra-violet light (U.V) trans-illumination:
DNA amplification was confirmed by running PCR products on 2% agarose gel electrophoresis then stained by ethidium bromide and visualized by UV transilluminator.
Cycle sequencing and data analysis. The Plasmodium species that were detected within DNA isolates were assessed by DNA sequencing using the positive amplified PCR products and multiple alignments of obtained nucleotide sequences. Positive PCR products were prepared as follows to be sent for sequencing.
Purification of the amplified nPCR products. This procedure is done to remove unwanted impurities and contaminants as enzymes, dyes, salts, and incorporated nucleotides. Cycle sequencing. Frederick Sanger enzymatic dideoxy DNA sequencing technique is a DNA sequencing method that depends on the selection of chain-terminating dideoxynucleotide incorporation with DNA polymerase during DNA replication in vitro15. Direct

bidirectional DNA sequencing of each purified positive nPCR product was performed.
Data Statistical analysis: Data were fed to the computer and analyzed by IBM SPSS software package version 20.0 (Armonk, NY: IBM Corp).
Quantitative data descriptions were performed using mean value. Qualitative data descriptions were performed using numbers and percentages.
P-value >0.05 was considered insignificant.
Ethical considerations: The study was approved by the ethical committee of Al-Azhar University, Egypt.
RESULTS
Among the examined persons, 250 (33.3%) of them were females and 500 (66.6%) were males. The average age was 34.6 years, ranging from 1 and 69 years. The average period between returning from the malaria-endemic area to the onset of symptoms was 14.5 days. Fever was reported in 350 cases during sampling and 400 cases had no fever during sampling. Plasmodium species were identified microscopically in 27 (3.6%) out of 750 individuals, 13 of them were diagnosed as P. falciparum, 7 were P. vivax, one was P. ovale and 6 were mixed infection with P. falciparum and P. vivax (Table1). Microscopy No. % P. falciparum 13 48.2
P. vivax
7
25.9 P. falciparum & P. vivax 6 22.2
P. ovale
1
3.7 Total 27 100
Table (1): Distribution of positive cases by microscopy (n = 27)
RDT was performed for the examined cases and positive results were obtained among 28 cases, twenty-seven of them had parasitemia and antigenemia while one had antigenemia only with a history of past infection.
Nested PCR for species identification was performed for 21 cases positive by first step PCR, 12 cases were P. falciparum, 5 cases were P. vivax and 4 were mixed P. falciparum and P. vivax infections (Table 2). No. % nPCR
P.falciparum
12
57.1 P.vivax 5 23.8
P. falciparum & P.vivax
4
19.1 Total 21 100
Table (2): Distribution of positive malaria cases according to nPCR (n = 21)
The result of nPCR was identical to the microscopic result (Table 3). Microscopy Nested PCR P. falciparum (n = 12) P. vivax (n = 5) P. falciparum + P. vivax (n = 4)
No.
%
No.
%
No.
% P.falciparum 12 100.0 0 0.0 0 0.0
P. vivax
0
0.0
5
100.0
0
0.0 P. falciparum + P. vivax 0 0.0 0 0.0 4 100.0
Table (3): Relation between microscopy and nPCR (n = 21)
The LAST results of the sequencing of nPCR products revealed 2 species P. falciparum (12 cases) and P. vivax (5 cases). While 4 nPCR products were positive for mixed infection of P. vivax and P. falciparum.
Past history of malaria among positive cases (n = 27) was present in 3 cases. The major species of imported malaria were P. falciparum and P. vivax that coming from 11 African countries (Table 4).