The emergence of an imported variant of dengue virus serotype 2 in the Jazan region, southwestern Saudi Arabia.

BACKGROUND: Dengue virus (DENV) infection is a global economic and public health concern, particularly in tropical and subtropical countries where it is endemic. Saudi Arabia has seen an increase in DENV infections, especially in the western and southwestern regions. This study aims to investigate the genetic variants of DENV-2 that were circulating during a serious outbreak in Jazan region in 2019. METHODS: A total of 482 serum samples collected during 2019 from Jazan region were tested with reverse transcription-polymerase chain reaction (RT-PCR) to detect and classify DENV; positive samples underwent sequencing and bioinformatics analyses. RESULTS: Out of 294 positive samples, type-specific RT-PCR identified 58.8% as DENV-2 but could not identify 41.2%. Based on sequencing and bioinformatics analyses, the samples tested PCR positive in the first round but PCR negative in the second round were found to be imported genetic variant of DENV-2. The identified DENV-2 imported variant showed similarities to DENV-2 sequences reported in Malaysia, Singapore, Korea and China. The results revealed the imported genetic variant of DENV-2 was circulating in Jazan region that was highly prevalent and it was likely a major factor in this outbreak. CONCLUSIONS: The emergence of imported DENV variants is a serious challenge for the dengue fever surveillance and control programmes in endemic areas. Therefore, further investigations and continuous surveillance of existing and new viral strains in the region are warranted.

Detection of Dengue Virus From Aedes aegypti (Diptera, Culicidae) in Field-Caught Samples From Makkah Al-Mokarramah, Kingdom of Saudi Arabia, Using RT-PCR.

Dengue fever (DF) is endemic to Makkah and Jeddah, the Kingdom of Saudi Arabia (KSA). However, until recently, the circulation of dengue virus (DENV) in Aedes mosquitoes in these areas was unknown. Serological surveillance of DENV in Ae aegypti is a powerful tool for early detection of dengue outbreaks and essential for developing effective control strategies. Therefore, this research aimed to examine a sample of adult Ae aegypti mosquitoes from Makkah, KSA, to detect DENV. In total, 1295 Ae aegypti mosquitoes were collected from the field from target areas of Makkah with a high incidence and prevalence of DF. The samples were divided into 259 coded pools (five mosquitoes in each) and preserved in 1.5 mL plastic tubes. The tubes were labeled, capped, and stored at-86°C until use. RT-PCR was used to detect DENV in the samples. All positive pools were confirmed by RT-PCR. The RT-PCR products were analyzed by gel electrophoresis (1.5% agarose in Tris-acetate EDTA buffer), stained with ethidium bromide, and visualized. DENV was isolated from six female Ae Aegypti collected from six pools (out of 259 pools). No other viruses were detected. Only five of the nine target localities had positive pools. Samples from the remaining four localities yielded negative results. Four DENV-positive mosquitoes were collected at the aquatic stages, and two were collected at the adult stage. These results show the circulation of DENV in adult mosquitoes and offspring, indicating vertical transmission of DENV. In conclusion, this study found that, in Makkah, DENV is circulating in dengue vectors with a high significance rate, suggesting the possibility of a dengue outbreak in the future; therefore, a sensitive surveillance system is vital to predict the outbreak and for early intervention and control.

Distribution and Molecular Identification of Culex pipiens and Culex tritaeniorhynchus as Potential Vectors of Rift Valley Fever Virus in Jazan, Saudi Arabia.

Entomologic investigations were conducted in the Al-Darb, Al-Reath, Al-Aridah, Abuareesh, Al-Ahad, Samttah, Sabyah, Damad and Beash areas by CO2-baited CDC miniature light traps in the Jazan region. Vectors were identified morphologically, as well as COI gene segment amplification and sequencing. The relative abundance (RA%) and pattern of occurrence (C%) were recorded. The presence of the Rift Valley fever virus (RVFV) in pooled mosquito samples was investigated by reverse transcriptase-polymerase chain reaction (RT-PCR). Culex pipiens (C. pipiens) and Culex tritaeniorhynchus (C. tritaeniorhynchus) were found with RA% values of 96% and 4%, respectively, in the region. Significant variations in vector population densities were observed in different districts. The C. pipiens was found highly abundant in all districts and RA% value (100%) was recorded in the Al-Darb, Al-Reath, Al-Aridah, Samttah and Damad areas, whereas RA% values (93.75%, 93.33%, 92.30% and 91.66%) were noted in Al-Ahad, Sabyah, Abuareesh and Beash districts, respectively. RA% values for C. tritaeniorhynchus were recorded as 8.33%, 7.70%, 6.66% and 6.25% in Beash, Abuareesh, Sabyah and Al-Ahad areas, respectively. The pattern of occurrence for C. pipiens and C. tritaeniorhynchus was recorded as 100% and 44.4% in the region. Phylogenetic analysis of C. pipiens and C. tritaeniorhynchus exhibited a close relationship with mosquitoes from Kenya and Turkey, respectively. All mosquito samples tested by RT-PCR were found negative for RVFV. In summary, the current study assessed the composition, abundance, distribution of different mosquito vectors and presence of RVFV in different areas of the Jazan region. Our data will help risk assessments of RVFV future re-emergence in the region.

Diagnosis and Causative Species of Visceral Leishmaniasis in Southwest Saudi Arabia.

Visceral leishmaniasis (VL) is the most severe clinical form of the disease and has been reported in the Jazan region of southwest Saudi Arabia. This study aimed to diagnose VL by real-time polymerase chain reaction (PCR) and the direct agglutination test (DAT) and to identify the causative Leishmania species. A total of 80 participants, including 30 suspected VL patients, 30 healthy endemic control individuals, and 20 malaria disease controls, were enrolled in this study. Blood samples were collected and tested for Leishmania DNA by real-time PCR and for antibody by the DAT. Sequencing of some amplified PCR products was used to identify the causative Leishmania species. The diagnosis of VL was successfully achieved by both real-time PCR and by DAT with 100% sensitivity. Leishmania donovani and Leishmania infantum species were detected by sequencing both by the kDNA and ITS1 target genes, followed a BLASTn search. The detection of VL antibody by the DAT followed by the confirmatory detection of Leishmania DNA in patient blood by PCR could promote the adoption of the much less invasive and more sensitive methods for the routine diagnosis of VL. Further study with high sample volume to evaluate the PCR and the DAT are needed, to generate more robust evidence. Based on the sequencing results, emerging studies on VL should focus on the causative Leishmania species, reservoirs, and vectors that are important in the study area.

Kelch 13-propeller polymorphisms in Plasmodium falciparum from Jazan region, southwest Saudi Arabia.

BACKGROUND: Artemisinin-based combination therapy (ACT) is recommended at the initial phase for treatment of Plasmodium falciparum, to reduce morbidity and mortality in all countries where malaria is endemic. Polymorphism in portions of P. falciparum gene encoding kelch (K13)-propeller domains is associated with delayed parasite clearance after ACT. Of about 124 different non-synonymous mutations, 46 have been identified in Southeast Asia (SEA), 62 in sub-Saharan Africa (SSA) and 16 in both the regions. This is the first study designed to analyse the prevalence of polymorphism in the P. falciparum k13-propeller domain in the Jazan region of southwest Saudi Arabia, where malaria is endemic. METHODS: One-hundred and forty P. falciparum samples were collected from Jazan region of southwest Saudi Arabia at three different times: 20 samples in 2011, 40 samples in 2016 and 80 samples in 2020 after the implementation of ACT. Plasmodium falciparum kelch13 (k13) gene DNA was extracted, amplified, sequenced, and analysed using a basic local alignment search tool (BLAST). RESULTS: This study obtained 51 non-synonymous (NS) mutations in three time groups, divided as follows: 6 single nucleotide polymorphisms (SNPs) '11.8%' in samples collected in 2011 only, 3 (5.9%) in 2011and 2016, 5 (9.8%) in 2011 and 2020, 5 (9.8%) in 2016 only, 8 (15.7%) in 2016 and 2020, 14 (27.5%) in 2020 and 10 (19.6%) in all the groups. The BLAST revealed that the 2011 isolates were genetically closer to African isolates (53.3%) than Asian ones (46.7%). Interestingly, this proportion changed completely in 2020, to become closer to Asian isolates (81.6%) than to African ones (18.4%). CONCLUSIONS: Despite the diversity of the identified mutations in the k13-propeller gene, these data did not report widespread artemisinin-resistant polymorphisms in the Jazan region where these samples were collected. Such a process would be expected to increase frequencies of mutations associated with the resistance of ACT.

Knockdown resistance mutations contributing to pyrethroid resistance in Aedes aegypti population, Saudi Arabia.

BACKGROUND: Dengue is endemic in Saudi Arabia especially in Jeddah, Makkah, Asir, and Jazan areas where pyrethroids are widely used to control the vector, Aedes aegypti. Resistance of Ae. aegypti to pyrethroid insecticides has been reported from most of these areas. AIMS: The present study was carried out in Jazan region in south-west Saudi Arabia to explore the resistance status of Ae. aegypti to pyrethroids and the consequent underlying mechanisms. METHODS: Three pyrethroids (permethrin, lambda-cyhalothrin, and cyfluthrin) were used to investigate the resistance status of Ae. aegypti adults following World Health Organization (WHO) standard methods: PCR and sequencing techniques were used to detect the S989P, V1016G and F1534C kdr mutations. RESULTS: Ae. aegypti populations were susceptible to cyfluthrin and having a possibility of resistance to permethrin while resistant to lambda-cyhalothrin. Three potential kdr mutations were detected for the first time in Ae. aegypti population, F1534C, V106G, and S989P. It was found that F1534C often co-exists with V1016G and this haplotype was strongly associated with permethrin and lambda-cyhalothrin resistance. On the other hand, S989P mutation was detected as RR in 18.8% with a low-frequency rate (R) of 18.8%, and in 55.5% as R with 58.3% frequency rate in permethrin and lambda-cyhalothrin- resistant female mosquitoes, respectively. CONCLUSIONS: Early detection of resistance alleles should be considered the essential tool for the successful implementation of insecticide resistance management strategies by providing early warning of insect resistance.