A.S. Pacsa

Sequential production of cytokines by dengue virus-infected human peripheral blood leukocyte cultures

The study was undertaken to elucidate the sequence of appearance of T helper (Th)1‐ and Th2‐type cytokines in human peripheral blood leucocyte cultures infected in vitro with dengue type 2 virus. Commercial sandwich enzyme‐linked immunosorbent assay kits were used to assay the levels of tumour necrosis factor‐alpha (TNF‐α), interferon‐gamma (IFN‐γ), interleukin (IL)‐2, IL‐4, IL‐5, IL‐6, and IL‐10 in culture supernatants. Culture supernatants were also screened for the cytotoxic factor and the dengue virus titres determined. The cytokines that appeared in the culture supernatants on the first day post‐infection (p.i.) were cytotoxic factor, TNF‐α, IL‐2, and IL‐6; their levels were highest on the second day p.i. IFN‐γ appeared on the second day with a peak on the third day p.i. The levels of these cytokines declined quickly, except for human cytotoxic factor (hCF) and IL‐2. The cytokines that appeared later were IL‐10 and IL‐5 on the fourth day and IL‐4 on the sixth day p.i. Dengue virus replicated in the peripheral blood leucocyte (PBL) cultures and was present throughout the course of the study. The findings of the present study show that dengue virus induced a predominant Th1‐type cytokine response during the first 3 days of infection of PBL cultures that was replaced by a Th2‐type response later. J. Med. Virol. 59:335–340, 1999.

Diarrhoeagenic Escherichia coli are not a significant cause of diarrhoea in hospitalised children in Kuwait

BackgroundThe importance of diarrhoeagenic Escherichia coli (DEC) infections in the Arabian Gulf including Kuwait is not known. The prevalence of DEC (enterotoxigenic [ETEC], enteropathogenic [EPEC], enteroinvasive [EIEC], enterohemorrhagic [EHEC] and enteroaggregative [EAEC]) was studied in 537 children ≤ 5 years old hospitalised with acute diarrhoea and 113 matched controls from two hospitals during 2005–07 by PCR assays using E. coli colony pools.ResultsThe prevalence of DEC varied from 0.75% for EHEC to 8.4% for EPEC (mostly atypical variety) in diarrhoeal children with no significant differences compared to that in control children (P values 0.15 to 1.00). Twenty-seven EPEC isolates studied mostly belonged to non-traditional serotypes and possessed β and θ intimin subtypes. A total of 54 DEC isolates from diarrhoeal children and 4 from controls studied for antimicrobial susceptibility showed resistance for older antimicrobials, ampicillin (0 to 100%), tetracycline (33 to 100%) and trimethoprim (22.2 to 100%); 43.1% of the isolates were multidrug-resistant (resistant to 3 or more agents). Six (10.4%) DEC isolates produced extended spectrum β-lactamases and possessed genetic elements (blaCTX-M, blaTEM and ISEcp1) associated with them.ConclusionWe speculate that the lack of significant association of DEC with diarrhoea in children in Kuwait compared to countries surrounding the Arabian Gulf Region may be attributable to high environmental and food hygiene due to high disposable income in Kuwait.

Short-term IgM and IgG antibody responses to hepatitis E virus infection

53 adult patients with acute hepatitis caused by hepatitis E virus were identified by the presence of IgM antibody to hepatitis E virus, and followed for 12 months to evaluate the kinetics of anti-HEV antibodies. All but 1 female Kuwaiti patient were expatriate workers from the Indian subcontinent, temporarily working in Kuwait. Follow-up samples obtained at 1, 3, 6 and 12 months were evaluated for IgM and IgG antibodies to hepatitis E virus. IgM-class antibodies to hepatitis E virus were detectable in 12/27 (44%) patients at 1 months, in 0/26 at 3 months, in 0/8 at 6 months and 0/6 at 12 months. IgG antibodies to hepatitis E virus were detectable in 46/47 (98%) at onset, 26/27 (96%) at 1 month, in 26/29 (90%) at 3 months, 16/16 (100%) at 6 months and 8/8 (100%) at 12 months of follow-up. This study suggests that IgM antibodies to hepatitis E virus decline rapidly after an acute infection but IgG antibodies to hepatitis E virus persists for at least 1 year in many patients.

Genotypes of Hepatitis C Virus in Kuwait

Hepatitis C virus (HCV) genotypes in 144 patients in Kuwait were determined by the Murex Anti-HCV Serotyping Assay. Twenty-nine (38%) of 77 Kuwaiti patients were found to be infected with genotype 4 and 27% with genotype 1 while of the 41 Egyptian expatriate patients 37 (90%) were infected with genotype 4. Results show that although in native Kuwaiti patients the dominant genotype is 4, other genotypes, especially type 1, occur frequently.

Th1-type cytokines production is decreased in kidney transplant recipients with active cytomegalovirus infection

Cytomegalovirus (CMV) infection is a major complication after kidney transplantation. Despite antiviral therapy the infection contributes significantly to high morbidity. The present study was aimed at determining: (a) the stimulation index (S.I.) of phytohemagglutinin (PHA)‐stimulated peripheral blood mononuclear cells (PBMC) and (b) the levels of Th1‐ and Th2‐ related cytokines in kidney transplant recipients with and without active CMV infection. Thirty‐five patients with, and 44 without active CMV infections, as diagnosed by a CMV antigenemia assay, were inducted into this study. After PHA stimulation of PBMC from patients, stimulation index (S.I.) was determined by radioactive thymidine uptake while the production of Th1‐type cytokines (interleukin‐2 [IL‐2], interferon‐γ [IFN‐γ], and tumor necrosis factor‐α [TNF‐α]) and Th2‐type cytokines (IL‐4, IL‐10) were measured by enzyme‐linked immunosorbent assay. PBMC of patients with active CMV infection showed significantly lower S.I. values than patients without an ongoing CMV infection (P < .0001). Levels of Th2‐type cytokines in CMV‐infected and uninfected kidney recipients were similar; however, the levels of the Th1‐type cytokines were significantly lower in CMV‐infected patients. Low levels of Th1‐type cytokines seem to correlate well with active CMV infection in kidney recipients. J. Med. Virol. 60:223–229, 2000.

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.

The prevalence of antibody to human parvovirus B19 in pregnant women in Kuwait.

Infection with parvovirus B19 during pregnancy is known to be associated with various fetal damage, such as aplastic anemia and hydrops fetalis. Our objective was to study the seroprevalence of parvovirus B19 in the obstetric population in Kuwait and to compare this with that in the adult population in other regions. Blood samples from 1047 pregnant women were used in this prospective study. Information regarding patients age, parity, nationality and symptoms was obtained at the time of collection of the sample. Blood was tested for IgG and IgM antibodies specific for parvovirus B19 using the ELISA technique. The overall prevalence for IgG and IgM was 53.3 and 2.2%, respectively. IgG seropositivity was higher in non-Kuwaiti women, while IgM antibodies was more frequent in Kuwaiti women. A total of 17.4% of the acutely infected patients were symptomatic. Prevalence of parvovirus B19 infection in Kuwait is comparable with that in the other countries.

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.

Significance of Atypical Pathogens among Community-Acquired Pneumonia Adult Patients Admitted to Hospital in Kuwait

Objectives: The aim of this study is to determine the microbial etiology and severity of community-acquired pneumonia (CAP) in Kuwait. Subjects and Methods: The severity of consecutive adult CAP cases admitted to 3 hospitals over a 1-year period was classified according to the Pneumonia Outcome Research Team (PORT) severity index. The microbial etiology was determined using standard methods for bacteria and serological tests for atypical and viral pathogens. Results: The study population was 124 of the 135 admissions; 63 female, 61 male; mean age 41.3 ± 18 years. The severity class distribution was: class I 31%, class II 37%, class III 17%, class IV 13%, and class V 2%. Etiological agents were identified from 44 patients (35%), with one pathogen in 31 (25%), two in 9 (7%), and three or more in 4 (3%). The most common pathogens identified were: Mycoplasma pneumoniae in 14 patients (11%), Legionella pneumophila in 10 (8%), Chlamydia pneumoniae in 8 (6%), influenza B virus in 8 (6%), influenza A virus in 5 (4%), Haemophilus influenzae in 4 (3%), Streptococcus pneumoniae in 3 (2%), Staphylococcus aureus in 3 (2%), gram-negative enterobacteria in 5 (4%), Moraxellacatarrhalis in 2 (2%), and viruses in 4 (3%). The yields from laboratory tests were 48% for paired serology, 20% from adequate sputum sample, and 3% from blood culture. Conclusion: Our study shows that a large percentage of mild CAP cases are admitted to hospitals in Kuwait. Atypical pathogens have a significant role in the etiology of CAP. There is overtreatment of CAP with a combination treatment consisting mainly of third-generation chephalosporins and macrolides.

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.