Nada Madi

Restoration of mycobacterial antigen‐induced proliferation and interferon‐γ responses in peripheral blood mononuclear cells of tuberculosis patients upon effective chemotherapy

Peripheral blood mononuclear cells (PBMC) were obtained from culture-proven tuberculosis (TB) patients before and after 2 and 6 months of chemotherapy with a multi-drug regimen. PBMC were tested for cellular responses in antigen-induced proliferation and interferon-gamma (IFN-gamma) assays in response to complex mycobacterial antigens (whole cell Mycobacterium bovis BCG and M. tuberculosis, cell walls and short-term culture filtrate [ST-CF] of M. tuberculosis), fractionated ST-CF antigens (fractions F1-F10) and ESAT-6. The responses in TB patients before anti-TB treatment were low (median stimulation index (SI)=1-7, median delta IFN-gamma=0-12 U ml(-1), and percent responders=13-67%) to all the antigenic preparations. Following the administration of anti-TB chemotherapy for 2 months, there were significant (P<0.05) improvements in the cellular responses (median SI=9-76, median delta IFN-gamma=3-70 U ml(-1), and percent responders=33-100%) to most of the antigenic preparations tested. However, concanavalin A-induced proliferation responses of PBMC from the same patients before and after 2 months of chemotherapy were high and comparable (median SI=101 and 114, respectively, P>0.05, 100% responders). A further increase in IFN-gamma responses (median delta IFN-gamma=14-250 U ml(-1) and percent responders=43-100%) to mycobacterial antigens was observed in patients receiving chemotherapy for 6 months. Among the ST-CF fractions, F1 and F2 containing low molecular mass proteins resulted in the highest responses, whereas ESAT-6 showed responses comparable to these fractions only in a minority of the patients. HLA-DR typing of these patients showed heterogeneity in the expression of molecules encoded by HLA-DRB genes. These results show that effective chemotherapy restores cellular responses of TB patients to a large number of M. tuberculosis antigens, which could be useful in monitoring the efficacy of anti-TB treatment.

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 × 104 copies/ml) than in asymptomatic (185copies/ml) patients (p = 0.001). The estimated cut-off value of CMV DNA for CMV symptoms/disease was ≧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.

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.

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. J. Med. Virol. 87:1521–1526, 2015.

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.

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.

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.

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.

1146Prevalence of blood borne viruses in the dialysis unit, Mubarak Al-Kabeer Hospital, Kuwait

Kabeer Hospital, Kuwait Haya Altawalah, BSc, MBBS, FRCPath; Mona Al-Houli, MB, BS, BSc; Mamoun AlQaseer, MB, BS, BSc, FRCPath; Nada Madi, PhD; Naser Husaain; Virology Unit, Ministry of Health, Mubarak Hospital, Kuwait; Virology Unit, Mubarak Al-Kabeer Hospital, Kuwait; Clinical Virology Unit, Mubarak Al-Kabeer Hospital, Kuwait, Kuwait; Virology Unit, Faculty of Medicine, Kuwait; Nephrology Consultant, Renal Unit, Mubarak Al-Kabeer Hospital, Kuwait