Luiz Fernando Job Jobim

Polymerase chain reaction for the laboratory diagnosis of aseptic meningitis and encephalitis

A protocol for testing cerebrospinal fluid specimens using a range of PCR assays for the diagnosis of central nervous system infection was developed and used to test prospectively 383 specimens. PCR assays were used for the detection of adenovirus, Borrelia burgdorferi, enteroviruses, Epstein Barr virus, cytomegalovirus, herpes simplex virus, human herpes virus type 6, JC virus, Leptospira interrogans, Listeria monocytogenes, lymphocytic choriomeningitis virus, measles virus, mumps virus, Mycobacterium sp. , Mycoplasma pneumoniae, Toxoplasma gondii and varicella zoster virus. Of the 383 specimens tested in this study, 46 (12.0%) were found to be positive. The microorganisms detected were CMV, enterovirus, Epstein Barr virus, herpes simplex virus, human herpes virus type 6, JC virus, L. monocytogenes, Mycobacterium genus, Toxoplasma gondii and varicella zoster virus. The introduction of the PCR protocol described has improved the diagnosis of a range of central nervous system infections in our laboratory. We believe however that further evaluation of these assays in immunocompromised patients is necessary to better determine the predictive value of positive PCR results in these patient groups.

Study of killer immunoglobulin-like receptor genes and human leukocyte antigens class I ligands in a Caucasian Brazilian population with Crohn's disease and ulcerative colitis

Crohns disease (CD) and ulcerative colitis (UC) are chronic inflammatory diseases of the bowel, of unknown origin. Exposure to specific environmental factors by genetically susceptible individuals, leading to an inadequate response of the immune system, is one of the potential explanations for the occurrence of these diseases. Natural killer cells are part of the innate immune system recognizing class I HLA (human leukocyte antigen) molecules on target cells through their membrane receptors. The main receptors of the natural killer cells are the killer immunoglobulinlike receptors (KIRs). Our study aimed to evaluate the association between the KIR genes in patients with inflammatory bowel diseases and healthy controls. We typed 15 KIR genes and HLA class I ligands in 248 unrelated Brazilian Caucasians, of which 111 had UC and 137 had CD, and 250 healthy controls by polymerase chain reaction using sequence-specific oligonucleotides and sequence-specific primers. We found an increase in KIR2DL2 in controls (inflammatory bowel disease [IBD]: p < 0.001; UC: p = 0.01; CD: p = not significant [NS]). The genotype 2DL2+/HLA-C lys(80)+ was also more common in controls (IBD: p = 0.005; UC: p = 0.01; CD: p = NS); as well as 2DL1+/HLA-C Asn(80)+ (IBD: p = 0.026; UC: p = NS;CD: p = NS). The imbalance between activating and inhibitory KIR and HLA ligands may explain, at least in part, the pathogenesis of these inflammatory bowel diseases.

Interleukin‐10 Gene Promoter and NFKB1 Promoter Insertion/Deletion Polymorphisms in Systemic Sclerosis

Systemic sclerosis (SSc) is a connective tissue disease characterized by fibrotic, immunological and vascular abnormalities. Nuclear factor‐kB (NFKB), as a key transcription factor involved in the regulation of immune responses, appears to be a good candidate for studies on the pathogenesis of autoimmune diseases, as well as the interleukin‐10 (IL‐10) polymorphism, which other studies have suggested an association with SSc. Our objective was to study the association of NFKB and IL‐10 gene polymorphisms with SSc. One hundred and fifty‐one SSc patients and 147 healthy bone marrow donors were enrolled in a case–control study. Blood was collected for DNA extraction; typing of IL‐10 genes was made by polymerase chain reaction with sequence‐specific primers (PCR–SSP), and NFKB gene typing was made by restriction fragment length polymorphism (RFLP). Patients underwent clinical evaluation, serology, Doppler echocardiography and chest high‐resolution computed tomography. The frequency of IL‐10 (−1082) GG genotype was found to be significantly higher in SSc patients (36.4%) as compared to healthy controls (22.4%) (P = 0.012). The frequency of heterozygous genotype GA was significantly lower (P = 0.004) in patients (38.4%) in comparison with control subjects (55.8%). A predominance of the high‐producing IL‐10 phenotype (GCC+/GCC+) was observed in SSc patients compared with healthy controls (37.7% versus 24.5%, respectively; OR: 1.87, 95% CI: 1.10–3.19, P = 0.019). No significant difference was found in the allelic and genotype distribution of the NFKB promoter polymorphism between patients and controls. No statistically significant associations were found between IL‐10 or NFKB polymorphisms clinical and laboratory features of SSc. Our results confirmed the association of the high‐producing phenotype (GCC+/GCC+) with increased risk for SSc, but found no correlation with NFKB polymorphisms.

Killer cell immunoglobulin‐like receptor (KIR) genes in systemic sclerosis

A previous study has suggested that the combination KIR2DS2+/KIR2DL2‐ was related to increased risk for systemic sclerosis (SSc), while others have failed to reproduce this finding. Our objective was to study this matter further and test the association of other KIR genes with SSc. One hundred and ten SSc patients and 115 healthy bone marrow donors were enrolled in a case–control study. Blood was collected for DNA extraction; typing of 15 KIR genes and human leucocyte antigen‐C (HLA‐C) was made by polymerase chain reaction with sequence specific primers (PCR–SSP), followed by electrophoresis on agarose gel. Patients underwent clinical evaluation, serology, Doppler echocardiography and chest high‐resolution computed tomography. The frequency of the inhibitory KIR2DL2 was significantly lower in patients [29·1% versus 65·2% in controls, P < 0·0001; odds ratio (OR) = 0·22, 95% confidence interval 0·12–0·40]. When combinations of activating and inhibitory KIR genes were analysed, the presence of KIR2DS2 in the absence of KIR2DL2 (KIR2DS2+/KIR2DL2‐) was more frequent in patients than in controls (25·5% versus 1·7%, respectively; P < 0·0001; OR = 19·29, 4·24–122·26). However, the presence of both KIR2DS2 and KIR2DL2 (KIR2DS2+/KIR2DL2+) was more frequent in controls (57·4%) than in patients (28·2%, P < 0·0001), suggesting a preponderant protective effect of KIR2DL2 over KIR2DS2. Stratification for HLA‐C1 status did not change these results. No statistically significant associations were found between KIR phenotypes and clinical and laboratory features of SSc. Our results suggest a protective role of KIR2DL2+ phenotype and confirmed the association of the combination KIR2DS2+/KIR2DL2‐ with increased risk for SSc.

Combined effects of CXCL8 and CXCR2 gene polymorphisms on susceptibility to systemic sclerosis.

UNLABELLED A previous study suggested that the CXCR2 (+1208) TT genotype was associated with increased risk of systemic sclerosis (SSc). In the present study, we investigated the influence of variation in the CXCL8 and CXCR2 genes on susceptibility to SSc and combined the variant alleles of these genes to analyze their effects on SSc. METHODS One fifty one patients with SSc and 147 healthy bone marrow donors were enrolled in a case-control study. Blood was collected for DNA extraction; typing of CXCL8 (-251) T/A and CXCR2 (+1208) T/C genes was made by polymerase chain reaction with sequence specific primers (PCR-SSP), followed by agarose gel electrophoresis. RESULTS The CXCR2-TC genotype was significantly less frequent in patients (23.8% versus 55.1% in controls; P<0.001, OR=0.26, 95%CI=0.15-0.43), whereas the CXCR2-CC genotype was significantly more frequent (44.4% versus 22.4% in controls; P<0.001, OR=2.76, 95%CI, 1.62-4.72). When CXCR2 and CXCL8 combinations were analyzed, the presence of CXCR2 T in the absence of CXCL8 A (CXCR2 T+/CXCL8 A-) was more frequent in patients than in controls (34.5% versus 3.5%; P<0.001, OR=14.50, 95%CI=5.04-41.40). However, CXCR2 TT and CXCL8 A were significantly more common in controls (100%) than in patients (58.3%) (P<0.001). Likewise, the presence of CXCR2 TC and CXCL8 A was more frequent in controls (95.1%) than in patients (75%) (P=0.004). Furthermore, the CXCR2-CC genotype in CXCL8 A was more frequent in patients (59.7% versus 0% in controls; P<0.001, adjusted OR=98.67, 95%CI=6.04-1610.8). In patients, a high frequency was observed in combination with the CXCL8 TA and AA genotypes (P<0.001; OR=28.92), whereas in controls, there was a high frequency of combination with CXCL8 T (P<0.001; OR=0.03) and TT (P<0.001; OR=0.01). CONCLUSIONS These findings suggest a protective role of CXCL8 (-251) A in the CXCR2 (+1208) TT and TC genotypes and an increased risk of CXCL8 (-251) A in association with the CXCR2 (+1208) CC genotype in SSc patients.

Association of killer cell immunoglobulin-like receptors and human leukocyte antigen–C genotypes in South Brazilian with type 1 diabetes

Type 1 diabetes mellitus (T1D) is a multifactorial and chronic autoimmune disease caused by the deficiency of insulin synthesis and or by its secretion or action defects. Genetic and environmental factors are known to be involved in its pathogenesis. The human leukocyte antigen complex (human leukocyte antigen (HLA)) constitutes the most relevant region contributing with 50% of the inherited risk for T1D. Natural killer cells (NK) are part of the innate immune system recognizing class I HLA molecules on target cells through their membrane receptors, called killer immunoglobulin-like receptors (KIR). The aim of our study is to evaluate the association between the KIR genes and HLA alleles in patients with T1D and healthy controls. Two hundred forty-eight T1D patients and 250 healthy controls were typed for HLA and KIR genes by PCR-SSP. Our results showed an increase of C2 in controls (p = 0.002). The genotype 2DL1/C2+ was also more common in controls (p = 0.001), as well as haplotype association KIR2DL2/DR3/DR4+ and the combination with only DR3+ (p < 0.001; p < 0.001). The maximum protection was seen when KIR2DL2/DR3-were absent when the combination of KIR2DL1/C2+ were present (p < 0.001) and the maximum risk was observed when KIR2DL2/DR3/DR4+ were present in the absence of KIR2DL1/C2- (p = 0.005). Our results confirmed the association of the KIR2DL2/DR3 increasing risk for T1D and suggest a protective role of KIR2DL1/C2.

Analysis of KIR gene frequencies and HLA class I genotypes in prostate cancer and control group

Prostate cancer is the second most common cancer in men, with a significant increase in incidence and mortality in men over 50 years of age. Natural killer cells (NK) are part of the innate immune system recognizing class I HLA molecules on target cells through their membrane receptors, called killer cell immunoglobulin‐like receptors (KIR). The aim of our study is to evaluate the association between the KIR genes and HLA alleles in patients with prostate cancer and healthy controls. Two hundred patients with prostate cancer and 185 healthy controls were typed for HLA class I and KIR genes by PCR‐SSP. When both groups were compared, no significant differences were found for HLA‐C group 1 and group 2, HLA‐Bw4, HLA‐A3 and A11. No difference was seen either in KIR frequency between patients with prostate cancer and controls. In conclusion, our data suggest no potential role for the KIR gene system in prostate cancer.

Simultaneous detection of Neisseria meningitidis, Haemophilus influenzae and Streptococcus sp. by polymerase chain reaction for the diagnosis of bacterial meningitis.

The simultaneous detection of Neisseria meningitidis, Haemophilus influenzae, and Streptococcus sp. was assessed by polymerase chain reaction (PCR) for the diagnosis of bacterial meningitis, as well as the applicability of PCR as a routine test. A cohort study was carried out with 182 children (2 months to 12 years of age) with suspicion of bacterial meningitis. Routine tests identified the etiologic agent in 65/84 children whose clinical status and laboratory findings suggested the presence of bacterial meningitis. Bacterial meningitis was ruled out in 98 children. In 19 children, the etiologic diagnosis was not possible using standard methods; in 14 of these patients, the etiologic agent was identified by PCR (N. meningitidis=12; H. influenzae=1; Streptococcus sp.=1). The sensitivity of PCR was 88.1%; specificity, 99.0%; positive predictive value, 98.7%; and negative predictive, 90.1%. PCR is a useful complementary diagnostic technique, especially when Gram stain, culture, or antigenic detection are negative or inconclusive.

Killer cell immunoglobulin‐like receptor gene diversity in a Caucasian population of Southern Brazil

Killer immunoglobulin‐like receptors (KIR) regulate the activity of natural killer and T cells through an interaction with specific human leucocyte antigen (HLA) class I molecules on target cells. Diversity in KIR gene content, KIR allelic and haplotype polymorphism has been observed between different ethnic groups. However, most population studies on KIR variability have focused on Europe and Asia, while Americas, Oceania and Africa remain poorly studied. The aim of this study was to analyse the variability of KIR genes in 200 healthy nonrelated individuals from the Southern Brazilian population. KIR genes and HLA‐A, ‐B and ‐Cw were genotyped using polymerase chain reaction‐sequence‐specific primers. Southern Brazilian population demonstrated several similarities to states that are closer geographically and distinct differences with Northern Brazil in the frequency of genes KIR2DS1, 2DS2, 2DS3, 2DS5, 3DL1, 3DS1, 2DL1 and 2DL2. The activating gene KIR2DS5 was the least frequent locus found in our group. Interaction of KIR/HLA was more common in the 2DS1−/2DL1+/C2+ association. This study demonstrated the diversity of KIR genes and of KIR/HLA association in a Caucasian group of Southern Brazil, establishing differences and similarities to other different populations.

Occult hepatitis B in renal transplant patients

Abstract: Background. Occult hepatitis B (HB) is characterized by the presence of HBV‐DNA in patients who do not have HB surface antigen (HBsAg) detectable in sera, and is frequently described in patients with hepatitis C virus (HCV) infection. These viral liver diseases are common and may have a negative impact on the survival of renal transplant patients, especially if they are both present. In this study we aimed to evaluate the prevalence of occult HB in renal transplant patients either with or without HCV infection.