Genomic characterization of circulating human respiratory syncytial viruses A and B in Kuwait using whole-genome sequencing.

The human respiratory syncytial virus (RSV) is considered one of the most common viruses that infect children globally. The virus is known to have extensive gene sequence variability within and between RSV groups A and B globally; however, there is no information on the whole-genome characterization and diversity of RSV in Kuwait. Therefore, this study aimed to sequence the entire genome of RSV strains isolated from patients with acute respiratory tract infection (ARTI) in Kuwait. Therefore, this study aimed to sequence the entire genome of RSV strains isolated from patients with ARTI in Kuwait. Between January 2020 and September 2022, 7,093 respiratory samples were collected from hospitalized infants, children, and adults and were analyzed for respiratory viruses by multiplex real-time PCR. Whole-genome sequencing using the Oxford Nanopore sequencing technology was performed on 84 RSV-positive samples. The results revealed a higher prevalence of group A (76%) than group B (24%) RSV isolates. Phylogenetic analysis showed that RSV-A strains clustered with the GA2.3.5 sub-genotype and RSV-B strains clustered with the GB5.0.5a sub-genotype; however, forming new lineages of RSV-A and RSV-B circulated in Kuwait during this period. Genetic variability was higher among the group A viruses than group B viruses, and the rate of synonymous and missense mutations was high in genes other than the G protein-coding gene. We also detected several known and unique molecular markers in different protein-coding genes. This is the first study in Kuwait to characterize the whole genomes of RSV A and B to identify the circulating genotypes, comprehend the genetic diversity and the evolution of the virus, and identify important genetic markers associated with specific genotypes.IMPORTANCEWhole-genome sequencing of respiratory syncytial virus (RSV) strains in Kuwait using MinION Nanopore technology was used to characterize and analyze the genotypes and sub-genotypes of the RSV circulating among patients with acute respiratory tract infections in Kuwait. This study also identified known and unknown gene mutations and imported genetic markers associated with specific genotypes. These results will assist in establishing a framework for RSV classification and allow for a better consideration of the mechanisms leading to the generation of diversity of RSV. In addition, these data will allow a comparison of vaccine viruses with those in Kuwait, providing useful insights into future vaccine and therapy strategies for RSV in Kuwait.

Molecular epidemiology and genetic characterization of SARS-CoV-2 in Kuwait: A descriptive study.

Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) has been fatal to human health, affecting almost the entire world. Here we reported, for the first time, characterization of the genetic variants of SARS-CoV-2 circulating in Kuwait to understand their genetic diversity and monitor the accumulation of mutations over time. This study randomly enrolled 209 COVID-19 patients whose nasopharyngeal swabs were positive for SARS-CoV-2 between February 2020 and June 2021 using RT-PCR. The whole genomes of SARS-CoV-2 from the nasopharyngeal swabs were sequenced using the Oxford Nanopore sequencing technology following the ARTIC network protocol. Whole-genome sequencing has identified different clades/sub-clades circulating in Kuwait, mimicking the virus's global spread. Clade 20A was dominant from February 2020 until January 2021, and then clade 20I (Alpha, V1) emerged and dominated. In June 2021, the number of cases infected with clades 21I, 21A, and 21 J (Delta) increased and dominated. We detected several known clade-defining missense and synonymous mutations and other missense mutations in the genes encoding important viral proteins, including ORF1a, S, ORF3a, ORF8 regions and a novel mutation in the N region. ORF1ab region harbored more mutations and deletions (n = 62, 49.2%) compared to the other 12 gene regions, and the most prevalent missense mutations were P314L (97%) in ORF1b and D614G (97%) in the S glycoprotein regions. Detecting and analyzing mutations and monitoring the evolution of SARS-CoV-2 over time is essential to help better understand the spread of various clades/strains of SARS-CoV-2 and their implications for pathogenesis. In addition, knowledge of the circulating variants and genome sequence variability of SARS-CoV-2 may potentially influence the development of vaccines and antiviral drugs to control the COVID-19 pandemic.

Strain Variation Based on Spike Glycoprotein Gene of SARS-CoV-2 in Kuwait from 2020 to 2021.

Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) is the causative agent of coronavirus disease 2019 (COVID-19), which was first identified in Wuhan, China, in December 2019. With the global transmission of the virus, many SARS-CoV-2 variants have emerged due to the alterations of the spike glycoprotein. Therefore, the S glycoprotein encoding gene has widely been used for the molecular analysis of SARS-Co-2 due to its features affecting antigenicity and immunogenicity. We analyzed the S gene sequences of 35 SARS-CoV-2 isolates in Kuwait from March 2020 to February 2021 using the Sanger method and MinION nanopore technology to confirm novel nucleotide alterations. Our results show that the Kuwaiti strains from clade 19A and B were the dominant variants early in the pandemic, while clade 20I (Alpha, V1) was the dominant variant from February 2021 onward. Besides the known mutations, 21 nucleotide deletions in the S glycoprotein in one Kuwaiti strain were detected, which might reveal a recombinant SARS-CoV-2 with the defective viral genome (DVG). This study emphasizes the importance of closely perceiving the emerging clades with these mutations during this continuous pandemic as some may influence the specificity of diagnostic tests, such as RT-PCR and even vaccine design directing these positions.

Osteolytic Lesion of the Maxilla in an Undiagnosed Multiple Myeloma Patient Identified Incidentally by Cone Beam Computed Tomography.

BACKGROUND Multiple myeloma is a hematological malignancy characterized by monoclonal plasma cell proliferation. Jaw lesions are found in nearly 35% of patients with symptomatic myeloma, and lesions occur in the mandible more often than in the maxilla. However, maxillary or mandibular lesions are rarely found as a primary manifestation of the disease. This report describes a case of a 65-year-old Palestinian woman with lytic lesions in the maxilla due to undiagnosed multiple myeloma identified incidentally on cone beam computed tomography (CBCT). CASE REPORT A 65-year-old Palestinian woman presented to the Oral Maxillofacial Surgery Clinic with an expansion of the maxilla which was initially thought of as an infection. CBCT imaging revealed diffuse osteolytic lesions involving multiple osseous structures. The patient was biopsied. Histopathological examination was suspicious for plasmacytic neoplasm. She was directly referred to the Hematology Department for further laboratory tests. These included complete blood count, liver function test, bone profile, protein electrophoresis, flow cytometry, and bone marrow biopsy, which were performed to confirm the diagnosis of multiple myeloma. The patient was treated with chemotherapy including zoledronic acid, dexamethasone, bortezomib, and cyclophosphamide. She went into remission for a year but unfortunately died 2 years later. CONCLUSIONS Primary myeloma of the maxilla is a rare presentation. The present report illustrates the role of CBCT imaging supported by a multidisciplinary approach to the diagnosis and management of myeloma. Consequently, it is recommended that dental practitioners be aware of radiographic features and possible oral manifestations to avoid any delay in medical intervention.

Dynamic profile of SARS-CoV-2 infection among hospitalized patients in Kuwait: a descriptive study.

BACKGROUND: The coronavirus induced disease 2019 (COVID-19) pandemic caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in Wuhan (China) in December 2019 is currently spreading rapidly worldwide. This study aimed to analyze the dynamic profile of SARS-CoV-2 infection among hospitalized patients that would characterize the period of viral shedding and detection among patients. METHODS: Retrospectively, 103 confirmed SARS-CoV-2 patients hospitalized at Jaber hospital in Kuwait were included. Demographic and clinical characteristics of the patients were collected. Nasopharyngeal swabs were obtained at different time intervals and analyzed by Real-Time RT-PCR for SARS-CoV-2 infection. RESULTS: Of 103 hospitalized patients with SARS-CoV-2 infection, the median age was 41 years, and 64% were male. The median period from admission to the positive SARS-CoV-2 RT-PCR test was 19 days (IQR, 13-22). The median period from admission to active negative SARS-CoV-2 RT-PCR test result was 22 days (IQR, 16-26). Older patients, patients with comorbidities, and patients with symptoms were more likely to have extended viral shedding. CONCLUSION: For the first time, this descriptive study conducted in Kuwait on SARS-CoV-2 RT-PCR test results from 103 patients positive for SARS-provided solid proof and a good understanding of the dynamic profile of SARS-CoV-2 infection among patients in Kuwait. This information will further enrich the global knowledge on the emerging SARS-CoV-2.

Human bocavirus (HBoV) in Kuwait: molecular epidemiology and clinical outcome of the virus among patients with respiratory diseases.

Introduction. Globally, human bocavirus (HBoV) has been detected in respiratory samples from patients suffering from upper and lower respiratory diseases. In Kuwait, little is known about the epidemiological and clinical characterization of the virus and genetic characterization of the virus as a respiratory pathogen is unknown.Aim. This study aims to explore the molecular epidemiology and clinical features of HBoV isolates in patients with respiratory diseases.Methodology. Retrospectively, between 2018 and 2020, 5941 respiratory samples from patients with respiratory diseases were screened for respiratory viruses using multiplex real-time PCR. Samples that were positive for HBoV were then subjected to NP1 and VP1/PV2 phylogenetic analysis.Results. HBoV was detected in 1.9 % of the patients, with a peak incidence of infection among children <1 year old. Co-infection with other respiratory viruses was observed in 56.8 % of HBoV-positive patients. Fever, cough and respiratory distress were the most common clinical features of HBoV infection. Phylogenetic analysis of the Kuwaiti HBoV isolates revealed that all the isolates were of the HBoV-1 genotype, with slight sequence variations among the isolates.Conclusion. This study illustrated the predominance of the HBoV-1 genotype in patients with respiratory diseases in Kuwait with minimal genetic variability. It also highlighted the clinical features of HBoV-1 infection, verifying its role in respiratory diseases.

Analysis of viral diversity in stool samples from infants and children with acute gastroenteritis in Kuwait using Metagenomics approach.

BACKGROUND: Current molecular target-dependent methods are used to detect only known viruses. However, metagenomics based on next-generation sequencing (NGS) technique is a target-independent assay that enables simultaneous detection and genomic characterisation of all microorganisms present in a sample. In this study, we aimed to develop a metagenomics approach using NGS to identify and characterise viruses in stool samples from infants and children with Acute Gastroenteritis (AGE) in Kuwait. METHODS: We have investigated 84 stool samples from infants and children aged one month to ten years old with signs and symptoms of gastroenteritis who attended Mubarak Al-Kabeer and Al-Amiri hospitals in Kuwait from January to December 2017. A metagenomics approach using NGS to characterise viruses in clinical samples was used. Also, the commercial Real-Time PCR assay was used to detect viruses causing gastroenteritis. RESULTS: Metagenomics analysis revealed an average of 280,768 reads in which 5% of the reads were derived from viruses. The analysis of viral sequences verified that single infection of human adenovirus was the leading cause of gastroenteritis among infants and children, which was detected in 23.2% of the patients, followed by a mixed infection of human adenovirus and other viruses, which was detected in 20.9% of patients. Also, the newly discovered viruses known to cause gastroenteritis were detected, such as astrovirus MLB2, primate bocaparvovirus-1, Aichivirus A, cardiovirus, parechovirus A, astrovirus VA4, cosavirus-F, and bufavirus-3. Our results showed 71% agreement (k = 0.445, P = 0.000) between multiplex Real-Time PCR, which is used as a routine diagnostic test and metagenomics approach in the detection of viruses causing gastroenteritis in clinical samples. CONCLUSION: Despite the difficulties in sample preparation and analysis process, we showed that metagenomics approach is a powerful and promising tool for the detection and characterisation of different viruses in clinical samples.

Assessment of immune status against measles, mumps, and rubella in young Kuwaitis: MMR vaccine efficacy.

Seroprevalence studies on measles, mumps, and rubella immunoglobulin G (IgG) antibodies after the implementation of the measles-mumps-rubella (MMR) vaccine are lacking in Kuwait. This study is an age-stratified serological study to assess the herd immunity to measles, mumps, and rubella among the young Kuwaiti population to evaluate the effectiveness of the MMR vaccine. IgG antibody titers to mumps, measles, and rubella were determined with commercial immune-assay in serum samples of 1000 Kuwaitis aged 5 to 20 years. The highest level of seropositivity was to measles (94.6%), which was significantly higher in females than in males. The highest seronegativity was for mumps (29%). The percentage of the young Kuwaiti population who were serologically positive for all the components of the MMR vaccine was 47%, and 2% of the individuals were without any protective antibodies to measles, mumps, and rubella. Females aged 5 to 10 years were best protected to rubella; however, seronegativity in 8.2% of 11- to 20-year-old females makes them vulnerable to rubella virus infection and congenital complications during pregnancy. The study provided insight into the effect of the MMR vaccine on seroprevalence of antibodies against measles, mumps, and rubella in Kuwait, which will contribute to the global knowledge base of vaccine coverage and help to inform elimination strategies. The findings strengthen the need for a third dose of MMR vaccine and catch-up campaigns for the young Kuwaiti population to increase vaccination coverage and prevent waning immunity, especially among those who received only one dose of the vaccine during childhood.

Analysis of genetic variability of respiratory syncytial virus groups A and B in Kuwait.

Respiratory syncytial virus (RSV) is the most frequently identified viral agent in infants, children, and elderly people with acute respiratory tract infections (ARTIs). This study is the only one of its kind in Kuwait, and its purpose was to investigate the genetic variability of the G protein gene in RSV strains prevalent in Kuwait. Respiratory samples were collected from patients with ARTIs in various hospitals in Kuwait and subjected to reverse transcription PCR (RT-PCR) amplifying a fragment of the G gene of RSV. A total of 305 samples were collected between January and mid-December 2016, and 77 (25.2%) were positive for RSV. Group A viruses were predominant over group B viruses; the RSV-A group was detected in 52 (67.5%) of the positive samples, while the RSV-B group was detected in 25 (32.5%) of the positive samples. Phylogenetic analysis showed that all RSV-A strains grouped into eight clusters of identical sequences of untyped strains. Twelve RSV-B strains, on the other hand, belonged to the RSV-B/BA10 genotype, while the rest were untyped. These data suggest that new and untyped strains of RSV-A group likely predominated in Kuwait and that the BA10 genotype of the RSV-B group became the dominant genotype in the 2016 season.

Metagenomic analysis of viral diversity in respiratory samples from patients with respiratory tract infections in Kuwait.

A metagenomic approach based on target independent next-generation sequencing has become a known method for the detection of both known and novel viruses in clinical samples. This study aimed to use the metagenomic sequencing approach to characterize the viral diversity in respiratory samples from patients with respiratory tract infections. We have investigated 86 respiratory samples received from various hospitals in Kuwait between 2015 and 2016 for the diagnosis of respiratory tract infections. A metagenomic approach using the next-generation sequencer to characterize viruses was used. According to the metagenomic analysis, an average of 145, 019 reads were identified, and 2% of these reads were of viral origin. Also, metagenomic analysis of the viral sequences revealed many known respiratory viruses, which were detected in 30.2% of the clinical samples. Also, sequences of non-respiratory viruses were detected in 14% of the clinical samples, while sequences of non-human viruses were detected in 55.8% of the clinical samples. The average genome coverage of the viruses was 12% with the highest genome coverage of 99.2% for respiratory syncytial virus, and the lowest was 1% for torque teno midi virus 2. Our results showed 47.7% agreement between multiplex Real-Time PCR and metagenomics sequencing in the detection of respiratory viruses in the clinical samples. Though there are some difficulties in using this method to clinical samples such as specimen quality, these observations are indicative of the promising utility of the metagenomic sequencing approach for the identification of respiratory viruses in patients with respiratory tract infections.

Phylogenetic analysis of HIV-1 subtypes and drug resistance profile among treatment-naïve people in Kuwait.

Mutations associated with resistance to antiretroviral therapy are a major cause of failure to treatment, and surveillance for the emergence of HIV resistance became a component of all antiretroviral treatment programs. As transmission of resistant viruses to newly infected persons is possible, we aimed to determine the prevalence of primary mutations associated with antiretroviral resistance among treatment-naïve patients, with respect to HIV subtype. Viral RNA was extracted from plasma samples of 43 treatment-naïve patients. Protease (PR) and reverse transcriptase (RT) regions were amplified and sequenced using the TRUGENE HIV-1 Genotyping Assay. A phylogenetic analysis was performed for HIV subtype assignment. Complete sequence information could be obtained for 35 patients. A total of ten different HIV-1 subtypes and recombinant forms were found in Kuwait with predominance of subtypes B, C, and CRF01_AE. A62V and A98G were non-polymorphic resistance-associated mutations (RAMs) detected in the RT region of two and three patients, respectively. Non-polymorphic mutations associated with resistance to protease inhibitors were not detected. Our results support continuous surveillance of RAMs in newly infected individuals to assess the effectiveness of first-line antiretroviral regimen available in Kuwait.

A relatively high number of pregnant women in Kuwait remain susceptible to rubella: a need for an alternative vaccination policy.

OBJECTIVE: To measure the prevalence of anti-rubella IgG and hepatitis B surface antigen (HBsAg) among pregnant women in Kuwait in order to assess the effectiveness of the current vaccination programs. SUBJECTS AND METHODS: This retrospective study involved 4,062 pregnant women evaluated in health centers in the Hawalli Province of Kuwait. They were screened for anti-rubella IgG and HBsAg using commercially available assays. The data were obtained from medical laboratory records. RESULTS: The mean age of the pregnant women was 29.2 ± 5.26 years (range 17-49). The rubella IgG prevalence among the pregnant women was 88.4% (n = 3,589); 276 (6.8%) of the pregnant women had no antibody to rubella, and 197 (4.8%) had rubella antibody levels ≤10 IU/ml. Therefore, 473 (11.6%) of the pregnant women were susceptible to rubella. The proportion of susceptible women increased with increasing age from 3.4 to 10.3% and from 3.4 to 6.7% among women aged <20 years and those aged ≥40 years, respectively (p = 0.016). The prevalence of HBsAg was 0.3%, and it did not vary with age. CONCLUSION: The prevalence of both anti-rubella IgG and HBsAg among pregnant women in Kuwait was relatively high. However, about 11.6% of pregnant women in Kuwait remain susceptible to rubella infection and hence congenital infection and fetal malformation.

Does Cytomegalovirus Develop Resistance following Antiviral Prophylaxis and Treatment in Renal Transplant Patients in Kuwait?

The resistance of cytomegalovirus (CMV) to ganciclovir or valganciclovir is a factor in therapeutic failure and disease progression. CMV strains resistant to ganciclovir or valganciclovir have been associated with specific mutations in the UL97 and UL54 genes. Sequencing of both CMV UL97 and UL54 genes was performed to detect the presence of CMV antiviral resistance in six patients who received ganciclovir (and/or valganciclovir) and had prolonged detectable CMV DNA in their blood during antiviral treatment. Sequencing results showed no specific mutations in either UL97 or UL54 gene of CMV and therefore the CMV strains in kidney transplant patients who received ganciclovir either prophylactically or therapeutically were from the wild type. Our results suggest that CMV management and immunosuppression protocols for kidney transplant patients followed in the Organ Transplant Centre, Kuwait, is very effective in reducing the opportunity of developing CMV antiviral resistance.

Cell-mediated immune responses to complex and single mycobacterial antigens in tuberculosis patients with diabetes.

OBJECTIVE: To evaluate cell-mediated immune (CMI) response in diabetic and non-diabetic tuberculosis (TB) patients and healthy subjects in response to complex, fractionated and single antigens of Mycobacteriumtuberculosis. MATERIAL AND METHODS: Peripheral blood mononuclear cells (PBMC) were obtained from patients suffering from pulmonary TB and type II diabetes (n = 7), pulmonary TB without diabetes (n = 10) and healthy subjects without TB and diabetes (n = 10). PBMC were assessed for CMI responses in antigen-induced proliferation assays in response to complex mycobacterial antigens (whole cells, cell walls and culture filtrate of M. tuberculosis), a battery of naturally purified or recombinant produced secreted (ESAT6, MPT59, MPT64 and MTB38) and cytosolic (MTB10, MTB70, ML10, ML28, ML36, ML65 and MB65) mycobacterial antigens and fractionated culture filtrate proteins (fractions F1-F10) of M. tuberculosis. RESULTS: The majority (>70%) of diabetic and non-diabetic TB patients and healthy subjects responded to the complex antigens of M. tuberculosis. However, among the single antigens, ESAT6 was most frequently recognized by TB patients with and without diabetes, but least recognized by healthy subjects. The secreted antigens MPT59 and MPT64 were recognized by all the groups, whereas the cytosolic antigens were recognized best by healthy subjects. When tested with fractionated secreted proteins present in the culture filtrate of M. tuberculosis, the best responses in both diabetic and non-diabetic TB patients were obtained with fractions containing low-molecular-weight proteins. CONCLUSIONS: Diabetic and non-diabetic TB patients respond frequently to secreted low-molecular-weight ESAT6 antigen of M. tuberculosis, indicating that this antigen may be useful in the diagnosis of TB in both the groups.

Detection and monitoring of cytomegalovirus infection in renal transplant patients by quantitative real-time PCR.

OBJECTIVES: To establish a sensitive and specific real-time PCR for quantitation of cytomegalovirus (CMV) DNA in clinical specimens. SUBJECTS AND METHODS: In a prospective study, CMV DNA was quantified in blood samples of 255 kidney recipients with and without CMV-related symptoms between the years 2000 and 2005 in Kuwait. In a selected group of patients, the effect of anti-CMV chemotherapy was monitored by quantitative real-time PCR (qRT-PCR). RESULTS: The established qRT-PCR assay had a sensitivity to detect 30 CMV DNA copies. CMV DNA was detected in 54/255 (24%) patients; of these, 17 (31.5%) were asymptomatic, and 37 patients (68.5%) had symptomatic CMV infection. Sequential blood specimens were collected from all CMV-positive patients and tested by CMV pp65 antigenemia and qRT-PCR assays. There was a moderate positive correlation between the two assays (Pearson's correlation = 0.52). The median CMV viral load measured by qRT-PCR was higher in symptomatic (6.5 x 10(4) copies/ml) than in asymptomatic (185copies/ml) patients (p = 0.001). The estimated cut-off value of CMV DNA for CMV symptoms/disease was > or =800 copies/ml of blood. Testing of sequential samples from patients treated with symptomatic CMV infection showed that the viral load was significantly reduced after 3 weeks of anti-CMV chemotherapy (p = 0.001). CONCLUSION: The reported qRT-PCR is a sensitive method for quantitation of CMV DNA in the blood of kidney recipients and can be useful in monitoring the efficacy of anti-CMV therapy.