Prediction of cervical cancer precursor lesions by quantitative methylation specific PCR: A retrospective study.

OBJECTIVE: This study was undertaken to evaluate the performance of FAM19A4 and hsa-mir-124-2 hypermethylation as a triage tool for women who are at risk of developing cervical cancer or high-grade cervical cancer precursor lesions by taking into consideration the cytology report, histology diagnosis, and human papillomavirus (HPV) status. METHODS: A total of 330 cervical ThinPrep samples were retrospectively collected and used for DNA isolation. HPV DNA was detected by real-time polymerase chain reaction (PCR), and HPV genotypes were identified by Sanger-based sequencing. DNA extracts were bisulphite-treated, and hypermethylation of FAM19A4 and hsa-mir-124-2 genes was detected by a quantitative methylation-specific PCR (qMSP) test using the QIAsure Methylation assay. RESULTS: Hypermethylated genes were detected in 27 (9.6%) cervical samples, mostly found in women diagnosed with high-grade squamous intraepithelial legions (77.8%) or cervical intraepithelial neoplasia grade 3 (CIN3) (72.7%). The sensitivity and the specificity of the qMSP test for predicting CIN3 lesions among women with high-risk HPV was 75% and 91%, respectively. DISCUSSION/CONCLUSION: There was a significant correlation between high-grade cervical cancer precursor lesions and detection of hypermethylated genes in samples positive for high-risk HPV. Our results suggest that the QIAsure Methylation test can be used as a triage tool to identify women at risk for cervical cancer progression.

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.

Predictors of intensive care unit admission and mortality in SARS-CoV-2 infection: A cross sectional study at a tertiary care hospital.

Background: The transmissibility and associated morbidity and mortality of severe acute respiratory syndrome-related coronavirus (SARS-Cov-2), have overwhelmed worldwide healthcare systems, resulting in an urgent need to understand this virus and its associated effects. The aim of our study was to identify patient symptoms, clinical characteristics, laboratory, and radiology findings that are associated with serious morbidity and mortality in COVID-19 patients. Methods: A cross sectional study was conducted in Jaber Al Ahmad Hospital, the designated COVID-19 center in Kuwait between August 1st, 2020 and January 31st, 2021. The main outcomes measured in this study were to identify variables associated with intensive care unit (ICU) admission, as proxy for serious morbidity, and in hospital mortality. Results: Two hundred and seventy-six patients were included in the study. Thirty-six (13%) patients were admitted to intensive care unit (ICU) and 33 (12%) patients expired. On multivariate analysis we found having elevated fibrinogen [OR 1.39, 95% CI 1.08-1.64, P = 0.04], low estimated glomerular filtration rate (eGFR) [OR 0.89, 95% CI 0.81-0.95, P = 0.02], and having bilateral patchy lung shadowing [OR 6.68, 95% CI 1.85-15.28, P < 0.01] to be significantly associated with increase odds of ICU admission. Elevated CRP [OR 1.25, 95% CI 1.10-1.98, P < 0.01], low eGFR [OR 0.95, 95% CI 0.90-0.99, P = 0.05] and having ischemic heart disease [OR 7.03, 95% CI 1.60-46.42, P = 0.04] were independently associated with increased odds of mortality. Conclusion: Certain inflammatory and coagulopathy markers, and having certain lung radiological features, in addition to having medical comorbidities, specifically, ischemic heart disease and renal impairment are key predictors for serious morbidity and mortality in patients infected with COVID-19. These should be incorporated into medical institutes risk assessment tools used by physicians and policy makers to instigate, prioritize, and reprioritize care in patients with COVID-19 and instigate preventative strategy to reduce the impact of future outbreak.

Immune Cells Profiles In The Peripheral Blood Of Patients With Moderate To Severe COVID-19 And Healthy Subjects With and Without Vaccination With The Pfizer-BioNTech mRNA Vaccine.

Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), the causative agent of Coronavirus disease 2019 (COVID-19), has caused a global crisis. Patients with COVID-19 present with a range of clinical manifestations, from no symptoms to severe illness. However, little is known about the profiles of immune cells required to protect against SARS-CoV-2. This study was performed to determine the immune cells profiles in the peripheral blood of COVID-19 patients with moderate to severe disease (n=52), and compare the findings with those from healthy subjects vaccinated with Pfizer BioNTech mRNA vaccine (VS) (n=62), and non-vaccinated healthy subjects (HS) (n=30) from Kuwait. Absolute counts and percentages of total lymphocytes and lymphocyte subsets (CD3+ T cells, CD4+ T cells, CD8+ T cells, CD19+ B cells, and CD16+CD56+ NK cells) in the peripheral blood of the three groups were analyzed using flow cytometry. The results showed that the absolute counts of total lymphocytes, CD3+, CD4+, and CD8+ T cells, CD19+ B cells, and CD56+ NK cells, were significantly lower in COVID-19 patients than normal healthy controls and vaccinated subjects. The percentages of CD3+ and CD4+ T lymphocytes were also significantly lower in the COVID-19 patients. However, the percentage of CD16+CD56+ NK cells was significantly higher in the peripheral blood of COVID-19 patients, compared to the HS and VS groups with no detectable differences in the percentages of CD8+ T cells and CD19+ B cells between the three groups. Analysis of the monocyte subsets has showed a significantly higher percentage of CD14+HLA-DR+ monocytes in COVID-19 patients compared to HS whereas the inflammatory CD14+CD16+ HLA-DR+ monocytes, and the non-classical CD16+HLA-DR+ monocytes showed significantly lower frequency in the blood of the patients than that of HS. These findings demonstrate perturbations of both innate and adaptive immune cell subsets that reflect dysregulated host responses in COVID-19 patients with moderate to severe disease.

CODEHOP-Mediated PCR Improves HIV-1 Genotyping and Detection of Variants by MinION Sequencing.

HIV-1 is genetically heterogeneous, having different subtypes and circulating recombinant forms (CRFs). HIV-1 genotyping is used to determine drug resistance profiles and is based on the use of a mixture of consensus and degenerate primers targeting the pol gene. However, the use of this type of primers is associated with either PCR bias or PCR failure. Consensus-degenerate hybrid oligonucleotide primers (CODEHOPs) can detect and identify unknown and distantly related gene sequences by PCR. CODEHOPs designed using different HIV-1 subtypes and CRFs were evaluated for HIV-1 genotyping by Sanger and MinION sequencing. A total of 321 plasma samples were used for the validation of CODEHOP-mediated HIV-1 genotyping. CODEHOP-mediated PCR showed 100% sensitivity and specificity, with limits of detection and genotyping below 200 copies/ml. The head-to-head evaluation of CODEHOP-mediated PCR and standard PCR showed 97 to 98% and 82 to 84% PCR success rates, respectively. There was 100% agreement between the CODEHOP and the reference method in the drug resistance profiles determined by Sanger-based sequencing. Using MinION sequencing, the CODEHOP-mediated PCR scheme resulted in better depth of genome coverage and detection of more drug resistance variants in the protease and reverse transcriptase genes than the standard amplification scheme. The overall prevalences of drug resistance mutations were 17.1% in treatment-experienced patients and 1.2% in treatment-naive patients. They were mainly associated with resistance to reverse transcriptase inhibitors and were linked to virological failure and the patient's treatment history. Findings from this study suggest that the performance of HIV-1 genotyping is improved by using CODEHOP-mediated PCR. IMPORTANCE HIV-1 drug resistance is the main cause of treatment failure. Regular surveillance of resistance-associated mutations in HIV-1 genomes is essential for the optimal management of HIV-1 infections. Due to HIV-1's genetic diversity, different HIV-1 genotypes are circulating worldwide. Standard primers used in the amplification of HIV-1 RNA have not been designed to cover all HIV-1 genotypes and are the main cause of amplification and drug resistance test failure. In this study, new sets of PCR primers targeting the protease, reverse transcriptase, and integrase genes were designed using the CODEHOP approach. They were compared to primers recommended in part by WHO for drug resistance testing using in-house PCR. Unsuccessful HIV-1 RNA amplification was less likely to occur with CODEHOP primers, leading to fewer test failures and lower cost. Furthermore, CODEHOP primers were more effective than standard primers for the detection of minority resistant variants by MinION sequencing.

Presence of HPV, EBV and HMTV Viruses Among Egyptian Breast Cancer Women: Molecular Detection and Clinical Relevance.

BACKGROUND: Oncogenic viruses, their possible association with breast cancer (BC) and effect on its clinical course are interesting issue. The present study evaluates the presence of human papillomavirus (HPV), EpsteinBarr virus (EBV), and human mammary tumor virus (HMTV) in BC and their relation with clinico-pathological characteristics. PATIENTS AND METHODS: This study was conducted on 80 Egyptian women with BC and 30 control women without known oncological disease. Forty formalin-fixed paraffin-embedded (FFPE) tissues, forty fresh tissue samples, and white blood cells (WBCs) of BC patients and WBCs of controls were subjected to a qualitative polymerase chain reaction (PCR). Quantitative real-time PCR was used to measure viral loads in fresh tissues of BC. The result was correlated with clinico-pathological characteristics of BC. RESULTS: HPV was detected in 33 (41.25%), EBV in 30 (37.5%) and HMTV in 33 (41.25%) BC patients. None of the control women was positive for HPV or EBV while HMTV was detected in 7 (23.3%). Among 40 BC WBCs specimens, HPV/HMTV were found together in 25%, followed by EBV/HMTV in 2.5% and EBV/HPV in 2.5%. However, the three viruses (HPV/EBV/HMTV) were found together in only 5%. In the 40 fresh BC tissues, the three viruses were found together in 12 (30%), EBV/HMTV in 7 (17.5%), HPV/HMTV in 4 (10%), and HPV/EBV in 4 (10%). EBV, HMTV, or multiple viral infections were associated with younger age of BC women. HPV, EBV, and HMTV median loads in fresh tissues were 4.8×103 copies/µL, 6.3×103 copies/µL, and 97 copies/µL, respectively. CONCLUSION: WBCs could be a more suitable specimen instead of fresh tissue for HMTV detection in BC patients to avoid invasive procedures. The presence of HPV, EBV, and HMTV together in Egyptian women with BC was significantly associated with younger age.

Virologic failure after 48 weeks of raltegravir-based regimen in low HIV-1 incidence setting.

BACKGROUND: With the advent of next generation integrase strand transfer inhibitors, the rates of virologic failure in treated subjects are expected to decrease. In this study, we analyzed the mutation patterns leading to virologic failure before and after starting integrase strand transfer inhibitor-based regimen as first-line or salvage therapy. METHODS: Between 2016 and 2019, blood samples were received from 258 patients with HIV-1 infection. Plasma HIV-1 RNA concentrations, and pol gene sequences were determined at baseline, and 16-48 weeks of treatment with integrase strand transfer inhibitor-based regimen. Only patients who did not achieve viral suppression at 48 weeks of integrase strand transfer inhibitor-based treatment were eligible for the current study. RESULTS: Virologic failure was observed in seven patients on raltegravir-based regimen. All patients with virologic failure but one were infected with CRF01_AE virus subtype. Raltegravir based-regimen was offered as first-line therapy for four patients, and as salvage therapy for three patients. M184V mutation associated with high level resistance to lamivudine and emtricitabine was detected in six out of seven patients. Primary mutations (Y143C, N155H, T66I, G118R, E138K) conferring high level resistance to raltegravir were detected in only three patients. Pre-existing polymorphic integrase mutation (T97A) was detected in two patients. Furthermore, two patients reported low adherence to treatment. CONCLUSIONS: Emergence of primary mutations in the integrase gene can account for virologic failure in less than half of patients on raltegravir-based regimen. Low adherence to treatment, pre-existing accessory mutations, and resistance to reverse transcriptase inhibitors may have some role in virologic outcome.

Prevalence of HPV Genotypes in Adult Male Patients with Cutaneous Warts: A Cross-Sectional Study.

AIM: This study was aimed at determining the distribution of type-specific human papillomavirus (HPV) in men with cutaneous warts and correlating this with the clinical and morphological presentation of warts. METHODS: Cutaneous wart samples were obtained from 167 adult men presenting to a dermatology clinic. The tissues were fixed and screened for HPV DNA using real-time PCR. The HPV genotype was determined by PCR-based sequencing. RESULTS: Nine different HPV genotypes were detected, comprising 6 from the α genus (HPV2, 6, 27b, 57b, 57c, and 94), 2 from the γ genus (HPV4 and 65), and HPV1a from the mu genus. Single HPV infection was encountered in 93.4% of the patients, whereas multiple infections were encountered in only 6.6%. The prevalence of HPV27b was highest among four body sites, followed by HPV57c, 1a, and 2. HPV1a was the most common genotype encountered in multiple infections, followed by HPV27b. Patient age, the number of warts, the duration of the presence of warts, and contact with people who have warts were not predictors of wart location. However, a high number of patients with palmar or common body warts had wart sizes of <1 cm. CONCLUSIONS: This study shows that genus α HPV types are detected in about 82% of patients with cutaneous warts.

Association of HPV genotypes with external anogenital warts: a cross sectional study.

BACKGROUND: This study was undertaken to determine the distribution of type-specific human papillomavirus (HPV) in external anogenital warts, and the correlation with clinical presentation of warts and demographic data of patients. METHODS: Genital warts specimens were obtained from 129 men and 27 women attending a dermatology clinic, who had been advised surgical excision. The tissues were fixed and screened for HPV DNA by using real-time PCR. HPV genotype was determined by PCR-based sequencing. RESULTS: Sixteen different HPV genotypes were detected, comprising 4 oncogenic HPV genotypes (16, 18, 33, 38), 2 low-risk HPV types (LR) (6, 81), HPV 9, and other types associated with common warts (1a, 2, 4, 7, 27b, 27, 57b, 57c, 65). Oncogenic HPV types were found in 34.62% of patients, LR HPV types in 14.4%, HPV 9 in 0.64%, and common warts type in 50.6%. The prevalence of HPV infection with a single type was 88.4, 9.0% for two types, and 2.6% for three types. Multiple logistic regression model showed that age, gender, nationality, number of warts, size of each wart, and positive history of wart in sexual partner, were not predictors of HPV type. However, patients with anogenital warts of one to six months duration were three times more likely to have oncogenic HPV infection compared to those with less than one month. CONCLUSIONS: This study shows that oncogenic HPV types are detected in around 35% of patients with genital warts, and are prevalent in warts of one to six months duration.

Effect of Human Coronavirus OC43 Structural and Accessory Proteins on the Transcriptional Activation of Antiviral Response Elements.

OBJECTIVES: The molecular mechanisms underlying the pathogenesis of human coronavirus OC43 (HCoV-OC43) infection are poorly understood. In this study, we investigated the ability of HCoV-OC43 to antagonize the transcriptional activation of antiviral response elements. METHODS: HCoV-OC43 structural (membrane M and nucleocapsid N) and accessory proteins (ns2a and ns5a) were expressed individually in human embryonic kidney 293 (HEK-293) cells. The transcriptional activation of antiviral response elements was assessed by measuring the levels of firefly luciferase expressed under the control of interferon (IFN)-stimulated response element (ISRE), IFN-ß promoter, or nuclear factor kappa B response element (NF-κB-RE). The antiviral gene expression profile in HEK-293 cells was determined by PCR array. RESULTS: The transcriptional activity of ISRE, IFN-ß promoter, and NF-κB-RE was significantly reduced in the presence of HCoV-OC43 ns2a, ns5a, M, or N protein, following the challenge of cells with Sendai virus, IFN-α or tumor necrosis factor-α. The expression of antiviral genes involved in the type I IFN and NF-κB signaling pathways was also downregulated in the presence of HCoV-OC43 structural or accessory proteins. CONCLUSION: Both structural and accessory HCoV-OC43 proteins are able to inhibit antiviral response elements in HEK-293 cells, and to block the activation of different antiviral signaling pathways.

Varicella infection in the Middle East: Prevalence, complications, and vaccination.

Varicella (chickenpox) is the primary infection of varicella-zoster virus (VZV), it is a mild self-limiting infection, but it is also highly contagious and can cause severe complications among high-risk group of individuals. It is usually a childhood infection providing lifelong immunity, but adults without varicella history are also susceptible to infection. High-risk group of individuals is more likely to develop serious complications. Varicella vaccine was introduced to protect this group of individuals and to prevent epidemic spread of VZV infection in a community. Thus, it was added to the recommended vaccination schedules for children in most developed countries. This review aimed to outline varicella status, seroprevalence, complications, and vaccination in the Middle East region. Based on our findings, children were the most affected age group, but there are also adult cases due to high number of expatriates, especially in Gulf Cooperation Council countries. Central nervous system involvements and skin diseases followed by varicella pneumonia were the most varicella-associated complications. Varicella vaccine was introduced in most Middle East countries, either mandatory by the Ministries of Health or optional in the private clinics. Few numbers of studies have reported an obvious reduction in varicella prevalence, hospitalizations, and deaths in the Middle East following varicella vaccination. A basic database about varicella infection before the initiation and implementation of a vaccination policy is essential to determine the target group of individuals. As far as our knowledge, this is the first review about varicella infection in the Middle East.

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.

PCR array profiling of antiviral genes in human embryonic kidney cells expressing human coronavirus OC43 structural and accessory proteins.

Human coronavirus OC43 (HCoV-OC43) is a respiratory virus that usually causes a common cold. However, it has the potential to cause severe infection in young children and immunocompromised adults. Both SARS-CoV and MERS-CoV were shown to express proteins with the potential to evade early innate immune responses. However, the ability of HCoV-OC43 to antagonise the intracellular antiviral defences has not yet been investigated. The potential role of the HCoV-OC43 structural (M and N) and accessory proteins (ns2a and ns5a) in the alteration of antiviral gene expression was investigated in this study. HCoV-OC43M, N, ns2a and ns5a proteins were expressed in human embryonic kidney 293 (HEK-293) cells before challenge with Sendai virus. The Human Antiviral Response PCR array was used to profile the antiviral gene expression in HEK-293 cells. Over 30 genes were downregulated in the presence of one of the HCoV-OC43 proteins, e.g. genes representing mitogen-activated protein kinases, toll-like receptors, interferons, interleukins, and signaling transduction proteins. Our findings suggest that similarly to SARS-CoV and MERS-CoV, HCoV-OC43 has the ability to downregulate the transcription of genes critical for the activation of different antiviral signaling pathways. Further studies are needed to confirm the role of HCoV-OC43 structural and accessory proteins in antagonising antiviral gene expression.

Drug Resistance-Associated Mutations in Antiretroviral Treatment-Experienced Patients in Kuwait.

OBJECTIVES: To investigate the prevalence of nonpolymorphic resistance-associated mutations (RAM) in HIV-1 patients on first-line antiretroviral therapy in Kuwait. SUBJECTS AND METHODS: Total RNA was isolated from plasma samples of 42 patients who received a first-line nonnucleoside reverse transcriptase inhibitor (NNRTI)-based regimen. HIV-1 protease and reverse transcriptase genetic regions were then amplified by nested reverse transcription-polymerase chain reaction and directly sequenced. The HIV-1 subtype was identified using the Bayesian phylogenetic method, and RAM were identified using the Stanford University genotypic resistance interpretation algorithm. RESULTS: The HIV-1 viral load at sampling ranged from < 20 to 8.25 × 104 copies/ml. CRF01_AE, C, and B were the most predominant HIV-1 subtypes. Nonpolymorphic mutations associated with resistance to antiretroviral drugs were detected in 11 (26.2%) of the 42 patients; 5 (11.9%) patients had mutations associated with a high-level resistance to nucleoside reverse transcriptase inhibitors (NRTI), 4 (9.5%) patients had mutations associated with resistance to NNRTI, 1 (2.4%) patient had mutations associated with resistance to both NRTI and NNRTI, and 1 (2.4%) patient had mutations potentially associated with low-level resistance to both protease inhibitors and NNRTI. All patients with RAM had a detectable plasma HIV-1 RNA level. CONCLUSION: Our results indicate the development of RAM during an NNRTI-based regimen and highlight the importance of considering other regimens to avoid treatment failure.

Adenovirus types associated with severe respiratory diseases: A retrospective 4-year study in Kuwait.

Human adenovirus (HAdV) infection can result in a severe respiratory disease. The aim of this study was to identify HAdV types detected in patients hospitalized for severe respiratory illness. The study population consisted of 743 patients with severe respiratory disease admitted to four major hospitals in Kuwait between January 2013 and December 2016. Respiratory specimens were retrospectively screened for 20 respiratory viruses by real-time PCR. The HAdV hexon gene was amplified and directly sequenced, and HAdV types were identified by performing Bayesian phylogenetic analysis. HAdV DNA was detected in 27 (3.6%) patients, with peaks in November and March. Most patients were infants and young children suffering from pneumonia or acute bronchiolitis. The detected HAdV types were C1, C2, C5, B3, and B7. Clusters of HAdV C1, C2, and C5 were observed with high posterior probability. All patients infected with HAdV C5 and 50% of patients infected with HAdV C2 or B7 were admitted to the intensive care unit (ICU). Co-infection with other viruses was detected in 44.4% of patients. The most common co-infecting virus was rhinovirus (HRV). HAdV/HRV co-infection was detected in two children who presumably developed disseminated HAdV infection and died. This is the first report describing the circulation of HAdV types associated with severe outcomes in Kuwait. These findings highlight the need for a national surveillance system to monitor changes in predominant HAdV types and increased numbers of severe respiratory infections.

Resistance-Associated Mutations and Polymorphisms among Integrase Inhibitor-Naïve HIV-1 Patients in Kuwait.

OBJECTIVES: Resistance-associated mutations (RAMs) in the integrase of different HIV-1 subtypes were investigated in a cohort of patients never exposed to integrase strand transfer inhibitors (INSTIs). METHODS: The viral RNA was extracted from plasma samples of 53 INSTI-naïve patients, and the integrase genetic region was sequenced and analyzed for subtype assignment and drug resistance. RESULTS: The median viral load at sampling was 5.28 × 104 RNA copies/mL. Bayesian phylogenetic analysis showed 85% of the HIV-1 isolates were non-B subtypes, with a predominance of subtypes C (22.6%) and CRF01_AE (26.4%). A total of 52 and 110 mutations were found in the integrase region of HIV-1 B and non-B subtypes, respectively. Nonpolymorphic INSTI-RAMs were not detected in this study. However, the accessory mutation E157Q was found in 1 patient with CRF02_AG, and the polymorphic mutations L74M/I that may contribute to a reduced susceptibility to INSTIs in the presence of major mutations were observed in 6 (13.3%) patients with non-B subtypes and 1 (12.5%) patient with the B subtype. Polymorphic mutations at positions known to harbor primary and accessory RAMs were also detected in this study. CONCLUSION: Our results highlight the importance of monitoring the emergence of INSTI-RAMS before and after the initiation of INSTI-based therapy.

Human Washington University Polyomavirus in Patients with Respiratory Tract Infection in Kuwait.

OBJECTIVE: To determine Washington University (WU) polyomavirus strains circulating among hospitalized patients with respiratory tract infections (RTI) in Kuwait. MATERIALS AND METHODS: Samples from 459 hospitalized children and adult RTI patients were screened for respiratory viruses by polymerase chain reaction from April 2013 to April 2016. The VP2 gene from WU virus (WUV)-positive samples was sequenced and subjected to phylogenetic analysis. RESULTS: Of the 459 hospitalized RTI patients, 18 (3.9%) patients were positive for WUV infection. WUV infection was common among children aged ≤11 years (9 patients, 50%). Among the 18 WUV-infected hospitalized patients, viral co-infection was detected in 9 patients (50%). The most common viruses associated with mixed infection were respiratory syncytial virus and human rhinovirus (2 patients, 11.1% each). Of the 18 WUV-infected patients, 4 were sequenced and subjected to phylogenetic analysis. The circulating strains belong to type Ia and IIIb. CONCLUSION: This study enabled us to detect WUV among hospitalized RTI patients. Co-infection with other respiratory viruses was notable. Two circulating WUV genotypes (Ia and IIIb) were identified among hospitalized RTI patients in Kuwait.

Molecular characterization of three enteroviral strains isolated in Kuwait from young children with serious conditions.

INTRODUCTION: Human enteroviruses are single stranded RNA viruses associated with many serious diseases such as encephalitis and myocarditis. They consist of up to 100 immunologically and genetically distinct types. Three enteroviral isolates, 2104, 3936 and 3988, were previously isolated from patients with neurological disorders or sepsis-like illness. In this study, the molecular characterization of the three isolates was investigated. METHODOLOGY: A full genome sequencing was performed by Sanger method, followed by phylogenetic and bootscanning analyses. A detailed analysis of genetic differences between the clinical and prototype isolates were investigated by mapping polymorphisms at nucleotide and amino acid levels, and by comparing RNA secondary structure in the noncoding regions. RESULTS: Based on the phylogenetic analysis of the VP1 gene and complete genome, 2104 was typed as coxsackievirus B1, 3936 as coxsackievirus B5, and 3988 as echovirus 7. Similarity and bootscan plots provided support for intra- and intertypic recombination crossover points occurring mainly along the nonstructural coding regions of the isolates. A sequence divergence of 12 to 14% was detected in the 5'-noncoding region between the clinical isolates and their corresponding prototype strains. Synonymous and nonsynonymous substitutions could be also mapped to different coding regions of the isolates, including those coding for the Puff, Knob and the hydrophobic pocket of the capsid. Examination of relative frequencies of synonymous and nonsynonymous substitutions in different coding regions of enteroviral isolates showed no evidence for selective pressure. CONCLUSION: The results provided a better understanding of the genetic variations, evolution and adaptation of enteroviruses in Kuwait.

Evaluation of the xTAG Gastrointestinal Pathogen Panel Assay for the Detection of Enteric Pathogens in Kuwait.

OBJECTIVE: To evaluate the utility of the Luminex xTAG gastrointestinal pathogen panel (GPP) assay in the detection of enteric pathogens from diarrheal stool samples in Kuwait. MATERIALS AND METHODS: The Luminex xTAG GPP assay was used according to the manufacturer's instructions to evaluate single diarrheal stool samples from 109 hospitalized patients at Mubarak Al-Kabeer Hospital, Kuwait, from March 2014 to June 2015. The assay procedure involved nucleic acid extraction from stool samples, amplification of the target by reverse transcriptase polymerase chain reaction, hybridization of the amplified target by probe, detection of the target by the Luminex instrument and computerized data analysis. Conventional microbiological assays were used as the gold standard for comparison. RESULTS: From the 109 diarrheal stool samples, 20 (18.4%) pathogens were detected by the xTAG GPP assay compared to 10 (9.2%) pathogens using conventional assays. Both methods detected 3 Salmonella spp., 3 Clostridium difficile, 2 rotavirus and 2 norovirus. In addition, the xTAG GPP assay detected 1 Shigella sp., 6 Campylobacter spp., 1 Cryptosporidium sp. and 2 Giardia lamblia which were missed by conventional assays. CONCLUSIONS: In this study, xTAG GPP detected twice as many pathogens as the conventional assays. We recommend the introduction of this assay in routine diagnostic laboratories for a rapid and better diagnosis and treatment of diarrheal disease.