Drug resistance-associated mutations in antiretroviral treatment-naïve and -experienced patients in Kuwait.

The identification of human immunodeficiency virus (HIV) mutations leading to drug resistance enables patient-specific adaptation of the treatment regimen and predicts the risk of transmission of drug-resistant HIV. In this study, we report for the first time the prevalence in Kuwait of non-polymorphic resistance-associated mutations (RAMs) in patients under first-line antiretroviral therapy. Viral RNA was extracted from plasma samples of 64 treatment-naïve (untreated) and 64 treatment-experienced patients. The HIV-1 load was determined by real-time RT-PCR. The protease- and reverse transcriptase-encoding regions were analyzed by subtyping, and for drug resistance. The HIV-1 load at sampling in treatment-naïve patients ranged from 1.61 x 104 to 1.91 x 106 copies/ml, whereas that in treatment-experienced patients ranged from bitors (PIs) and NNRTIs. These results necessitate efforts to be made for reducing emergence of resistance-associated mutations in treated patients, and highlight the need for continuous monitoring of drug resistance patterns in Kuwait.

Clinical Utility of Viral Load in the Management of Cytomegalovirus Infection in Solid Organ Transplant Patients in Kuwait.

BACKGROUND: Infection with human cytomegalovirus (CMV) in solid organ transplant patients remains an unresolved challenge, despite improvements in immunosuppressive therapy, post-transplantation care, viral prevention, and therapy. METHODS: We conducted quantitative real-time polymerase chain reaction (PCR) assays of CMV on plasma samples of 1,168 patients in Kuwait who received solid organ transplants from 2012 to 2014 to detect and monitor CMV DNA viral load. RESULTS: Of the 1,168 patients, 180 (15.4%) were positive for CMV DNA. Among the CMV DNA-positive patients, 119 (66.1%) remained without symptoms and 61 (33.9%) developed CMV-related symptoms. During the follow-up period, peak viral loads were significantly (P < .05) higher in symptomatic patients (mean 970 copies/mL; range, 15-625,000 copies/mL) than in asymptomatic patients (<150 copies/mL; range, 67-2,650 copies/mL). Many symptomatic patients (n = 57) were successfully treated, and their viral loads declined. However, some symptomatic patients had irregular viral-load kinetics, with prolonged periods of symptoms despite CMV treatment; we excluded the possibility of drug resistance in these patients, because there was no evidence of clinical resistance to treatment. CONCLUSIONS: Quantitative real-time PCR of CMV DNA is useful in monitoring CMV infection and the effectiveness of CMV treatment in renal transplant recipients in Kuwait.

Cytomegalovirus genotypes gB1 and gH1 are the most predominant genotypes among renal transplant recipients in Kuwait.

BACKGROUND: The human cytomegalovirus (HCMV) is a common pathogen responsible for asymptomatic and persistent infections in healthy individuals. However, cytomegalovirus infections are a major cause of morbidity and mortality in immunocompromised patients, especially in recipients of solid-organ transplants and AIDS patients. METHODS: HCMV DNA from 42 patients who received kidney transplants between 2004 and 2008 were subjected to polymerase chain reaction and restriction fragment length polymorphism to identify HCMV gB and gH genotypes. RESULTS: HCMV gB1 and gH1 genotypes were the most the predominant HCMV genotypes (P < .05, P < .05, respectively). In addition, both HCMV gB1 and gH1 genotype were significantly more often associated with the development of fever with leukopenia and severe HCMV disease than other gB or gH2 genotypes. No significant differences were observed among viral loads between the HCMV genotypes among infected individuals. CONCLUSION: This study demonstrated the prevalence and role of HCMV genotypes in infection and disease in renal transplant patients in Kuwait.

Respiratory syncytial virus and human rhinoviruses are the major causes of severe lower respiratory tract infections in Kuwait.

Respiratory infections are very common in Kuwait, yet little is known about the cause of severe lower respiratory tract infections. This study was designed to investigate the viral cause of lower respiratory tract infections using sensitive molecular methods. PCR was applied to investigate 10 respiratory viruses in respiratory samples from 1,014 patients aged between 3 days to 76 years with acute lower respiratory tract infections. Of the 1,014 patients with lower respiratory tract infections, 288 (28.4%) had a viral infection. One hundred fifty-five (53.8%) presented with bronchiolitis, 100 (43.7%) with pneumonia, and 33 (11.5%) with croup. One hundred six (36.8%) and 99 (34.4%) patients had evidence of respiratory syncytial virus and human rhinoviruses infections, respectively. Adenoviruses were detected in 44 (15.2%) patients, while influenza A virus in 21 (7.3%) patients. The majority of respiratory syncytial virus infections (84%) were among patients aged <1 year. Similarly, of the 99 patients infected by human rhinoviruses, 50 (50.5%) were also among this age group. In contrast, most of influenza A virus infections, 12 of 21 (57.1%), were among patients aged over 16 years. Parainfluenza virus-2 and human coronaviruses were not detected in any of the patients' samples. Over the 3-year period, most of the hospitalized patients were seen during the autumn and winter months from October through March. These data show that respiratory syncytial virus and human rhinoviruses may be the major causes of lower respiratory tract infections in children admitted to hospital in Kuwait.

Low levels of Th1-type cytokines and increased levels of Th2-type cytokines in kidney transplant recipients with active cytomegalovirus infection.

BACKGROUND: Cytomegalovirus (CMV) infection is a major complication after kidney transplantation. It is clear that Th1 and Th2 cell subsets are of major importance in determining the class of immunoprotective function in infectious diseases. Given the strong influence exerted by Th1- and Th2-type immunity on the outcome of infections, we felt it important to elucidate the levels of Th1- and Th2-type cytokines to CMV-related antigens in kidney recipients and to identify antigens that play an essential role in preventing the development of CMV infection and/or disease. METHODS: One hundred twenty subjects were followed for CMV infection by the antigenemia assay. We investigated peripheral blood mononuclear cells (PBMCs) responses to five CMV-related peptide antigens (pp65, gB, pp150, pp28, and pp38). Stimulation index was determined by radioactive thymidine uptake, while the production of Th1-type cytokines (interferon-gamma and tumor necrosis factor-alpha) and Th2-type cytokines (interleukins-4 and -10) were measured by enzyme-linked immunosorbent assay. RESULTS: The levels of Th1-type cytokine production after stimulating PBMCs with CMV-related antigens gB and pp150 resulted in significant decreases in the levels of interferon-gamma, while pp65, pp150, and pp38 produced significant decreases in the level of tumor necrosis factor-alpha between the two groups (P < .05). For Th2-type cytokines only pp28 produced a significant increase in the level of interleukin-10 between the two groups (P < .05). Regarding the Th1:Th2 ratios, a lower Th1-bias was observed among the CMV-positive patients for PBMCs stimulated with three CMV-related antigens (pp65, pp38, and pp28). CONCLUSION: Low levels of Th1-type cytokines and increased levels of Th2-type cytokines upon stimulation with CMV-related peptide antigens were associated with reduced cell-mediated immunity to CMV, thus seeming to correlate with active CMV infections.

Is combined pretransplantation seropositivity of kidney transplant recipients for cytomegalovirus antigens (pp150 and pp28) a predictor for protection against infection?

OBJECTIVE: This study was aimed at detecting antibodies to the antigens which may contribute to protection against cytomegalovirus (CMV) infection after organ transplantation. MATERIALS AND METHODS: A total of 203 kidney transplant patients were enrolled in the study. Based on CMV antigenemia assay, 23 patients were antigen-positive and of the remaining 180 antigen-negative patients, 46 were selected as controls matched for age, gender and source of kidney. The 69 kidney recipients (KR) had CMV antibody due to previous infection and were followed up for a period of 6 months after transplantation for the development of active CMV infections by the antigenemia assay. Antibody responses to five CMV-related peptide antigens (pp65, gB, pp150, pp28 and pp38) were investigated by enzyme immunoassay and their presence was correlated with the results of the CMV antigenemia assay. RESULTS: Of the five CMV-related peptide antigens, only gB antigen showed response to the antibody in 10/23 (43.5%) antigen-positive patients and 9/46 antigen-negative patients and the difference was statistically significant (p = 0.048). On the other hand, there was no significant difference in antibody responses between the antigen-positive and antigen-negative KR to the other four CMV peptide antigens (p > 0.05). However, among the antigen-positive KR there was only 1 patient who had antibodies to both pp150 and pp28 antigen, while among the antigen-negative KR, 22 of 46 (47.8%) had the antibodies (p < 0.001). CONCLUSION: The findings suggest that the combined presence of antibodies against the pp150 and pp28 antigens may indicate a lower risk of CMV reactivation after kidney transplantation.

Genotypic characterization of rotaviruses and prevalence of serotype-specific serum antibodies in children in Kuwait.

There are no data on the serotypes of rotaviruses prevalent in Kuwait, which has a large expatriate population and hence a focal point for transmission of pathogens. The serotype information will contribute to the fund of knowledge on the world epidemiology of rotavirus serotypes and will predict the outcome of vaccination in Kuwait. Of the 75 rotavirus-positive samples from 172 children (aged <5 years) with severe diarrhoea, 69 were genotyped. The distribution of genotypes was G1 (63.8%) followed by G9 (10.2%), G2 (7.3%), G4 (7.3%) and G3 (4.4%). Among the P types, P[8] was the most common type found across all G types. By fluorescent focus neutralization test, serum antibodies to genotypes G1 (94%), G4 (68%) and G9 (46%) were found in 120 other children. These results show that G1 is the predominant serotype in Kuwait and that a vaccine that contains G1 will be most effective.

BK virus nephropathy in renal transplant recipients in Kuwait: a preliminary report.

INTRODUCTION: BK virus nephropathy (BKVN) is a significant cause of graft loss among renal transplant recipients. The treatment outcomes of BKVN have been variably reported in the literature. PATIENTS AND METHODS: We prospectively investigated BKV infection and BKVN among a population of renal transplant recipients with suspected BKV infection. The 42 subjects who all had acute allograft dysfunction, were categorized in three groups: those with clinical, laboratory, and histological findings that did not suggest acute rejection, drug toxicity, or obstruction (group 1, n = 24); those with findings that suggested probable acute cellular rejection but did not respond to antirejection treatment (group 2, n = 10); and those whose renal histology suggested BKVN (group 3, n = 8). Polymerase chain reaction analysis was done to detect BKV DNA in urine and blood samples from each subject. BKV DNA was detected in 19 (45%) urine samples with 11 of these subjects (26.1% of total) having BK viremia as well. RESULTS: No evidence of BKVN was detected histologically in seven subjects with isolated BK viruria, while the others proved to be JC virus infections. Among the 11 subjects with BK viremia, eight had BKVN based on renal histology at the time of diagnosis with BKV infection, while the other three subsequently developed histological features of BKVN. BKVN developed after 5.3 +/- 2.5 (2 to 44) months after transplantation. The serum creatinine at time of BKVN diagnosis was 158.9 +/- 58 (87 to 285) micromol/L. All subjects were initially treated with a 50% reduction in immunosuppressive drug doses. Further decreases in immunosuppression were performed in all patients with close monitoring of renal function. All subjects were followed up for a of 18.2 +/- 5 (12 to 26) months. Two grafts were lost not due to BKVN, and one patient was lost to follow-up during this period. The latest serum creatinine in eight recipients is 113 + 20 (81 to 138) micromol/L, which is better than the renal function at diagnosis. CONCLUSION: The prevalence of BKVN in suspected BKV infection was 26%. Although the study period was short (30 months), BK viremia strongly correlated with BKVN, which seemed to be successfully treated with reduction in immunosuppression.

Correlation between CMV genotypes, multiple infections with herpesviruses (HHV-6, 7) and development of CMV disease in kidney recipients in Kuwait.

The possible correlation between cytomegalovirus, human herpesvirus types 6, 7 and cytomegalovirus-related clinical symptoms was studied in kidney transplant patients in Kuwait. Cytomegalovirus infection was diagnosed using the pp65 antigenemia assay. DNA of cytomegalovirus was detected by nested polymerase chain reaction (nested-PCR). PCR was also used to amplify the genes coding for structural proteins of human herpesvirus-6 (240 bp) and human herpesvirus-7 (186 bp). Glycoprotein B genotypes of cytomegalovirus were determined by restriction fragment length polymorphism. The average number of cells positive for cytomegalovirus pp65 antigen showed a steady increase with the severity of the cytomegalovirus-related symptoms. Furthermore, cytomegalovirus pp65 antigen positivity was significantly more frequent among recipients of cadaver kidney (45.5%) than among those who received live related kidneys (22.6%). Cytomegalovirus gB genotype 1 was detected more frequently (P<0.036) in recipients with live related donor kidney (38%) than in patients of cadaver kidney (13%). The genome of human herpesvirus-6 was detected at the same rate in patients with or without cytomegalovirus-related symptoms. However, the genome of human herpesvirus-7 was detected significantly more frequently (P<0.0001) in asymptomatic patients (41.7%) than in recipients with symptomatic cytomegalovirus infection (17%). We conclude that cytomegalovirus gB genotypes are not associated with the outcome of a cytomegalovirus infection in kidney transplant patients, that human herpesvirus-6 does not play a role in cytomegalovirus pathogenesis and that the role of human herpesvirus-7 in cytomegalovirus-related morbidity in kidney recipients remains unclear.

Lymphocyte responses to human cytomegalovirus in different groups of patients in Britain and in adults from west Africa and the Middle East.

Antibody prevalence and lymphocyte proliferation responses to cytomegalovirus (CMV) and herpes simplex virus (HSV) were compared in several different groups of patients: genitourinary medical (GUM) patients, hemophiliacs, men with clinical acquired immunodeficiency syndrome (AIDS) and cases of primary CMV mononucleosis, and also in adults in the general population (control subjects) comprising separate groups native to Britain, West Africa, and the Middle East. Among the British control subjects who were positive for CMV IgG, all were also positive against CMV antigen by the lymphocyte transformation test (LTT). However, among those who were CMV IgG-positive in the various groups of patients, 20-86.9% gave positive responses to CMV antigen by the LTT; moreover, 75.7% and 55.5% of the CMV IgG-positive healthy control subjects from West Africa and the Middle East, respectively, gave positive LTT responses to CMV antigen. When the same groups of patients were tested for responsiveness to HSV antigen by the LTT, there was good agreement between a positive result by this test and by serology in all except those with primary CMV mononucleosis (42.8%). Overall, lymphocyte responses to CMV were significantly impaired in healthy, CMV antibody-positive subjects from West Africa and the Middle East compared to similar subjects from Britain.

Rapid culture-amplified immunofluorescent test for the detection of human rhinoviruses in clinical samples: evidence of a common epitope in culture.

Ohio HeLa cells in multichamber slides were inoculated with nasal samples from patients presenting with common cold symptoms and incubated at 33 degrees C with gentle shaking for 48 hours. The cultures were fixed with cold acetone, and viral antigens were detected by immunofluorescence using an antirhinovirus type 2 (HRV-2) polyclonal serum. Of 158 samples, 58 (36.7%) and 57 (36%) were positive for HRV by virus isolation (confirmed by acid lability test) and by culture-amplified immunofluorescent (CAIF) test, respectively. The correlation between the two tests was highly significant (P = 0.0001). Nasal washings or nasal/throat swabs were equally suitable for detecting virus by isolation but not by CAIF. On the other hand, nasal washings were better than nasal/throat swabs for detecting HRV by CAIF. In an ELISA system, the polyclonal anti-HRV-2 serum recognized a rhinovirus antigen expressed in situ within 48 hr postinfection by all the 11 HRV serotypes investigated. However, 60 hr postinfection, the anti-HRV-2 serum recognized only homologous and closely related HRV antigens. These results suggest that a rhinovirus "common" antigen may be expressed some 48 hr after infection of Ohio HeLa cells with rhinoviruses. The CAIF test provides a sensitive, rapid and reliable procedure to detect wild-type rhinovirus infection as well as a clear alternative to detection by isolation.

Effects of zinc gluconate and nedocromil sodium on performance deficits produced by the common cold.

Two studies are reported on the effects of drugs on the performance impairments induced by experimentally- produced colds. The first study examined the effects of zinc gluconate on choice reaction time, and showed that the zinc removed the cold-induced performance impairment. The second experiment used nedocromil sodium and, again, the drug was effective in reducing the drop in performance observed in volunteers with colds. While the precise mode of action of these compounds is unclear it is speculated that one mechanism involves changes in trigeminal stimulation, and this could be responsible for both the clinical efficacy and CNS effects of these drugs.

Detection of rhinovirus RNA in nasal epithelial cells by in situ hybridization.

This paper describes the development and evaluation of in situ hybridization (ISH) for the detection of rhinovirus in cells obtained from nasal washings of volunteers infected with human rhinovirus 14 (HRV-14). Twenty-five (66%) and 27 (71%) of 38 volunteers inoculated with HRV-14 had evidence of infection by virus isolation and ISH, respectively, on at least one of 4 days investigated after virus challenge. In contrast, only 14 of 38 (37%) volunteers had significant antibody rises as detected by the neutralization test. Of the 38 volunteers inoculated with HRV-14, only 13 (34%) had symptoms of colds. Of these, 12 (92%) and 10 (77%) were positive by virus isolation or ISH, respectively, on at least one day. Six (46%) had significant antibody rises by neutralization. Similarly, of the 38 volunteers challenged, 22 (58%) were asymptomatic and of these 10 (45.5%) and 12 (54.5%) were positive by virus isolation and ISH, respectively, on at least one day. Only 8 (36.4%) of these asymptomatic volunteers showed significant antibody rises by neutralization. There were significant associations between the detection of rhinoviruses by ISH and virus isolation on the third day (P less than 0.025) after virus challenge in the group as a whole and in the symptomatic group. These results show that generally rhinovirus detection by ISH compares well with virus isolation and both tests are clearly more sensitive than the neutralization test in detecting evidence of infection. It is concluded that ISH is an interesting new technique that may play an important role in the study of rhinovirus infection and pathogenesis.

Detection and differentiation of picornaviruses in clinical samples following genomic amplification.

A polymerase chain reaction (PCR) assay was used to detect and differentiate picornaviruses (PVs), using primers homologous to the 5' non-coding and VP2 regions of the PV genome. The PCR resulted in a 530 bp PCR product for human rhinoviruses (HRVs) and a 650 bp product for polioviruses, coxsackieviruses (CV) or echoviruses. The PCR assay could detect as little as 1 p.f.u. of virus in either cerebrospinal fluid (CSF) or stool, using ethidium bromide-stained gels. Standard strains of poliovirus, CV, echovirus and HRV were detected, with the exception of echovirus type 22. In contrast, heterologous viruses, such as herpes simplex virus, human cytomegalovirus, adenovirus, influenza virus and rotavirus, as well as human and monkey cell DNA, were not amplified. In nasal swabs taken from patients with respiratory infections, the PCR detected 27 of 28 HRV isolation-positive specimens. All specimens from which viruses other than HRVs were isolated were negative by PCR. The PCR definitively identified poliovirus and CVs from the CSF or stool of patients with aseptic meningitis, as well as CV in the pericardial fluid of a patient who had suffered a myocardial infarction. Specimens taken from patients with similar pathologies, and from which heterologous viruses were isolated, were uniformly negative by PCR.

Pathogenicity for humans of human rhinovirus type 2 mutants resistant to or dependent on chalcone Ro 09-0410.

Mutants of human rhinovirus type 2 (HRV-2) resistant to and dependent on the antirhinoviral compound chalcone Ro 09-0410 were selected in cell culture under clean laboratory conditions. A total of 42 volunteers were challenged with either the drug-resistant mutant [SR2-410(r)] (15 volunteers), the drug-dependent mutant [SR2-410(d)] (15 volunteers), or a wild-type HRV-2 which had a similar passage level in vitro as the mutants but without the drug (12 volunteers). Of volunteers challenged with the wild-type HRV-2, 33, 67, and 82% developed cold symptoms, shed virus, and showed serological evidence of infection, respectively. In contrast, only 13, 27, and 23% of volunteers challenged with the drug-resistant mutant developed colds, shed virus, and showed serological evidence of infection, respectively. None of the volunteers challenged with the drug-dependent virus became infected or had symptoms of colds. These results demonstrate that a drug-resistant rhinovirus was capable of infecting humans and producing disease, although its infectivity was reduced when compared with that of the wild type. In contrast, a drug-dependent virus had lost its ability to infect humans.

A host-cell-selected variant of influenza B virus with a single nucleotide substitution in HA affecting a potential glycosylation site was attenuated in virulence for volunteers.

An influenza B virus was passaged in man (virus A) and then in human embryo trachea (C) and into embryonated eggs (D) or directly into eggs (B). Virus A, B, and C had the same (cell-like) haemagglutinin phenotype on reaction with selected monoclonal antibodies while D had an "egg-like" phenotype. The viruses were administered at a dose of 1,000 TCD50 (for MDCK cells) by intranasal inoculation to groups of 27 or 28 volunteers. Viruses A, B, and C all produced disease in six to eight volunteers, whereas D produced no illness and only four volunteers were infected. The viruses shed by the volunteers were indistinguishable from those with which they were inoculated. The haemagglutinin genes of the viruses were sequenced and changes were detected indicating amino acid substitutions at position 196-198 in the attenuated egg-grown virus D whereby a potential glycosylation site present in the other viruses was lost.

The human cytomegalovirus immediate early enhancer-promoter is responsive to activation by the adenovirus-5 13S E1A gene.

The stimulatory effects of the 13S adenovirus E1A gene product on the human cytomegalovirus (HCMV) major immediate early (IE) enhancer were examined. Chimeric plasmids containing cloned portions of the HCMV major IE enhancer-promoter positioned upstream of the chloramphenicol acetyltransferase gene (cat) were cotransfected into HeLa cells with the plasmid p13S-wt which contained a cDNA encoding the adenovirus 13S E1A gene product. CAT expression from chimeric plasmids containing at least one copy of the HCMV 19 base pair (bp) repetitive motif was stimulated 10-fold in the presence of p13S-wt. The 19-bp motif contains a potential binding site for the cellular transcription factor ATF/CREB. Deletion analysis indicated that the ATF/CREB site was crucial for E1A-mediated stimulation. Insertion of a synthetic oligonucleotide homologous to a 19-bp motif and containing an ATF/CREB binding site into an HCMV chimera lacking ATF/CREB motifs conferred E1A responsivity on HCMV promoter-mediated CAT expression whereas insertion of a similar oligonucleotide containing a change of two bases in the sequence of the ATF/CREB site did not. Measurement of CAT-specific RNA verified the results of the CAT enzyme experiments. The ATF/CREB motif may be a target for stimulation of HCMV gene expression through either viral or cellular transcription factors.

The effect of intranasal nedocromil sodium on viral upper respiratory tract infections in human volunteers.

Two studies involving double-blind group comparative trials in human volunteers compared the effects of intranasal nedocromil sodium (2.6 mg active drug per nostril, q.i.d.) with placebo on clinical symptoms and performance impairment associated with the common cold. In the first study volunteers were challenged with rhinoviruses (RV9 and RV14), and in the second study with respiratory coronavirus. In both studies, active and placebo groups of volunteers were demographically similar. Infection rates in both groups were also similar. There were no withdrawals resulting from unusual symptoms related to either treatment. In the rhinovirus study (19, placebo; 20, nedocromil sodium) daily symptom scores and daily mean nasal secretion weights were significantly lower in the nedocromil sodium-treated group. In the coronavirus study (26, placebo; 27, nedocromil sodium) there was little difference in the severity of colds between the active and placebo-treated groups, but trends favoured nedocromil sodium. In both studies the impairment of performance in volunteers who developed a cold was significantly less in those treated with nedocromil sodium than in those treated with placebo.