الفهرس 
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Abstract
Malaria is one of the most prevalent parasitic infections in the world and certainly the most detrimental. It causes greater morbidity and mortality rates than any other disease worldwide. In 2019, 229 million malaria cases and 405,000 deaths were recorded worldwide. The African continent had the highest estimates of cases, followed by the Southeast Asian and the Eastern Mediterranean areas. Kuwait is a malaria-free state in the Arabian Gulf’s northwestern corner, but great economic development in Kuwait during the last decades has resulted in a massive influx of immigrant workers coming from malaria-endemic countries of tropical zones.
The present study aimed to study the prevalence and demographic risk factors of malaria among suspected malaria cases in Kuwait and to determine the species and density of malaria parasitaemia among the infected participants
Blood samples were collected from 100 immigrant workers attending the Infectious Diseases Hospital Laboratory (IDHL), Kuwait after taking informed consents and obtaining ethical approval. Pre-designed, structured questionnaires were used to collect socio-demographic characteristics and clinical data of participants. The sample collection extended from February2020 to February 2021. One part of the blood sample was used for preparation of Giemsa stained thin and thick blood films. The second portion was used for the immuno-chromatographic assay. The third portion was used to perform the Complete Blood Count (CBC) test. The fourth portion was kept at -80oC for DNA extraction and testing by a multiplex real-time PCR to identify the infecting Plasmodium species. The molecular investigation was performed on all microscopy and ICT- positive samples. Randomly selected 16 negative samples were also run in multiplex real-time PCR. Discrepant test results were resolved by pan-Plasmodium 18S rRNA conventional PCR and sequencing of the amplicons. The real-time PCR was performed at the Department of Microbiology, Faculty of Medicine, Kuwait University. Sequencing was performed at OMICSRU-Research Core Facility, Kuwait
The age of participants ranged from 20 to 73 years with a mean of 36.9±11 years. Males represented 80% of the study sample. All participants were from Asian and African countries (60% and 40% respectively). The greatest numbers were from India (34%). 66% were from rural areas. As for the educational level, 44% had school-level education, 26% were illiterate, 17% had university-level education and 13% read and write only. Regarding occupation, 46% were workers, 36% were shepherds,16% were housemaids and two were students.
Fever and headache were the most frequent symptoms, being present in 79% and 42% of participants respectively, 22% complained of nausea and vomiting and 6% had diarrhea. Most participants (80%) were returning to Kuwait after visiting a malaria-endemic area. Only eight persons mentioned that they had received malaria chemoprophylaxis before visiting the malaria-endemic area.
Microscopic examination revealed that 27% of subjects were positive for Plasmodium spp. parasite density was low in (44.4%), medium in (40.7%) and high in (14.8%). The parasites were identified as P. falciparum in 13 samples, mixed P. falciparum and P. vivax in 11 samples and mixed P. falciparum and P. malariae in three samples. ICT revealed that 17% of participants had positive results, 47.1% were P. falciparum, 52.9 were P. vivax.
Statistical analysis showed a Kappa index of 0.71 indicating good agreement between microscopy and ICT in Plasmodium detection. ICT gave false negative results in 10 samples. The parasite density was significantly higher in ICT positive samples (median =2707parasite/ µl) compared to parasite negative samples (median = 95 parasites/µl).
Positive PCR results were found in all microscopy positive samples. P. falciparum was identified in 14 samples, P. vivax in 11 samples, P. ovale in one sample and mixed P. falciparum and P. malariae in one sample.
Statistical analysis showed a strong negative correlation between parasite density in thick blood films and Ct values in the RealStar PCR. On the other hand, the RealStar PCR Ct values were significantly lower in ICT positive samples (median Ct=26) compared to ICT negative samples (median Ct = 31) (p=0.13). There was a very good agreement in species identification between RealStar PCR and ICT (Kappa index =1).
Samples with discordant species identification in microscopy and RealStar PCR(n=13) were subjected to PCR amplification using a pan-Plasmodium primer set that amplifies a fragment of the18S rRNA gene. All samples showed bands close to 300 bp in gel electrophoresis. Sanger sequencing of the amplified products was successful in 11 samples and BLAST analysis revealed the same species as the RealStar PCR.
The molecularly confirmed P. falciparum and P. vivax infections revealed that the mean age of P. falciparum cases was significantly lower than that of P. vivax cases (28.5±7.1 versus 36.6±9.58 years). A significant gender difference was observed; all P. vivax cases were males whereas P. falciparum was identified in eight males and seven females. All cases belonged to African or Asian countries with significant differences between P. falciparum and P. vivax cases. The difference was not statistically significant for residence and education level.
Fever and headache were the most frequent symptoms in both types of malaria. A positive past history of malaria was reported by 40% of P. falciparum and 27% of P. vivax patients. About 70% of P. vivax (8/11) and 40% (6/15) of P. falciparum patients were returning to Kuwait after visiting a malaria-endemic area. The intake of malaria chemoprophylaxis before visiting malaria-endemic areas was reported by one P. falciparum and two P. vivax infected patients only. None of these variables showed significant difference between both types of malaria.
The patients with falciparum malaria had significantly lower levels of haemoglobin as well as lower counts of total WBCs and neutrophils and significantly higher lymphocytes, basophils and platelet counts compared to patients with vivax malaria.