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1.  Infection of cells expressing CXCR4 mutants lacking N-glycosylation at the N-terminal extracellular domain is enhanced for R5X4-dualtropic human immunodeficiency virus type-1 
Background
Infection with human immunodeficiency virus type-1 (HIV-1) requires binding of the viral envelope gp120 to CD4 and to the CXCR4 coreceptor. Both, gp120 and CXCR4 are subject to N-glycosylation. Here we investigated the influence of the N-linked glycans g1 and g2 present on CXCR4 for HIV-1 infection.
Methods
The two CXCR4 N-glycosylation sites g1 (NYT) and g2 (NVS) were mutated by changing the first or third amino acids N or T/S to Q and A respectively (g1; N11Q or T13A; g2, N176Q or S178A). Human osteosarcoma cells (GHOST) expressing human CD4 and the various CXCR4 glycosylation mutants were tested for infection using NL4-3-based viruses with X4, R5 or R5X4-tropism differing only in the V3 loop region.
Results
All constructed cell lines expressing the various CXCR4 glycomutants showed similar permissiveness for the X4-monotropic virus and no change in the coreceptor specificity that allows infection of a CCR5-dependent R5-monotropic virus. Interestingly, the removal of glycan g1 significantly enhanced the permissiveness of GHOST cells for the R5X4 dualtropic virus. GHOST cells expressing the CXCR4-g1 or CXCR4-g1/2 mutants were infected at higher rates by the R5X4-dualtropic virus compared to cells expressing CXCR4-wt or CXCR4-g2 coreceptors.
Conclusion
Our present observations underscore a role for glycans present on the CXCR4 coreceptor in the entry process of HIV-1. The data will help to better understand the multifaceted mechanism of HIV infection and the selective forces which drive HIV-1 evolution from mono- to dual-tropism.
doi:10.1186/1471-2334-2-31
PMCID: PMC139973  PMID: 12489987
2.  Effect of treating periodontitis on C-reactive protein levels: a pilot study 
Background
Periodontitis is associated with elevated levels of C-reactive protein and fibrinogen and it may be a coronary heart disease risk factor. We wanted to study if treatment of periodontitis can decrease the levels of these inflammatory markers.
Methods
C-reactive protein and fibrinogen levels were measured in 35 patients (21 M, 14 F, mean age 50 years) with adult periodontitis, before and after treatment.
Results
The median baseline C-reactive protein level in the patients was 1.05 mg/l and it decreased to 0.7 mg/l (p = 0.05) after periodontal treatment. Of the 30 patients who could be included in the analyses, 24 patients had a baseline level below 2 mg/l (the 95th percentile limit in Finland); 6 patients had levels higher than this. Elevation of the baseline C-reactive protein level or the magnitude of its decrease were not associated with severe form of periodontitis. The decrease in C-reactive protein levels was at least 50 % in 4/6 of those with elevated baseline levels, as compared with 3/24 of the rest of the patients (p = 0.016). No corresponding effect was observed in fibrinogen levels.
Conclusions
Periodontitis seems to increase C-reactive protein only in some individuals, presumably the ones reacting to it with a systemic inflammatory reaction. Periodontal treatment decreases C-reactive protein levels in these individuals and it may thus decrease their risk of coronary heart disease.
doi:10.1186/1471-2334-2-30
PMCID: PMC138813  PMID: 12475397
3.  Further evidence for association of hepatitis C infection with parenteral schistosomiasis treatment in Egypt 
Background
Hepatitis C virus (HCV) infection and schistosomiasis are major public health problems in the Nile Delta of Egypt. To control schistosomiasis, mass treatment campaigns using tartar emetic injections were conducted in the 1960s through 1980s. Evidence suggests that inadequately sterilized needles used in these campaigns contributed to the transmission of HCV in the region. To corroborate this evidence, this study evaluates whether HCV infections clustered within houses in which household members had received parenteral treatment for schistosomiasis.
Methods
A serosurvey was conducted in a village in the Nile Delta and residents were questioned about prior treatment for schistosomiasis. Sera were evaluated for the presence of antibodies to HCV. The GEE2 approach was used to test for clustering of HCV infections, where correlation of HCV infections within household members who had been treated for schistosomiasis was the parameter of interest.
Results
A history of parenteral treatment for schistosomiasis was observed to cluster within households, OR for clustering: 2.44 (95% CI: 1.47–4.06). Overall, HCV seropositivity was 40% (321/796) and was observed to cluster within households that had members who had received parenteral treatment for schistosomiasis, OR for clustering: 1.76 (95% CI: 1.05–2.95). No such evidence for clustering was found in the remaining households.
Conclusion
Clustering of HCV infections and receipt of parenteral treatment for schistosomiasis within the same households provides further evidence of an association between the schistosomiasis treatment campaigns and the high HCV seroprevalence rates currently observed in the Nile delta of Egypt.
doi:10.1186/1471-2334-2-29
PMCID: PMC139974  PMID: 12464161
4.  Detection of Pneumocystis DNA in samples from patients suspected of bacterial pneumonia- a case-control study 
Background
Pneumocystis jiroveci (formerly known as P. carinii f.sp. hominis) is an opportunistic fungus that causes Pneumocystis pneumonia (PCP) in immunocompromised individuals. Pneumocystis jiroveci can be detected by polymerase chain reaction (PCR). To investigate the clinical importance of a positive Pneumocystis-PCR among HIV-uninfected patients suspected of bacterial pneumonia, a retrospective matched case-control study was conducted.
Methods
Respiratory samples from 367 patients suspected of bacterial pneumonia were analysed by PCR amplification of Pneumocystis jiroveci. To compare clinical factors associated with carriage of P. jiroveci, a case-control study was done. For each PCR-positive case, four PCR-negative controls, randomly chosen from the PCR-negative patients, were matched on sex and date of birth.
Results
Pneumocystis-DNA was detected in 16 (4.4%) of patients. The median age for PCR-positive patients was higher than PCR-negative patients (74 vs. 62 years, p = 0.011). PCR-positive cases had a higher rate of chronic or severe concomitant illness (15 (94%)) than controls (32 (50%)) (p = 0.004). Twelve (75%) of the 16 PCR positive patients had received corticosteroids, compared to 8 (13%) of the 64 PCR-negative controls (p < 0.001).
Detection of Pneumocystis-DNA was associated with a worse prognosis: seven (44%) of patients with positive PCR died within one month compared to nine (14%) of the controls (p = 0.01). None of the nine PCR-positive patients who survived had developed PCP at one year of follow-up.
Conclusions
Our data suggest that carriage of Pneumocystis jiroveci is associated with old age, concurrent disease and steroid treatment. PCR detection of P. jiroveci has low specificity for diagnosing PCP among patients without established immunodeficiency. Whether overt infection is involved in the poorer prognosis or merely reflects sub-clinical carriage is not clear. Further studies of P. jiroveci in patients receiving systemic treatment with corticosteroids are warranted.
doi:10.1186/1471-2334-2-28
PMCID: PMC139972  PMID: 12445330
5.  Anti-HIV-1 activity of anionic polymers: a comparative study of candidate microbicides 
Background
Cellulose acetate phthalate (CAP) in soluble form blocks coreceptor binding sites on the virus envelope glycoprotein gp120 and elicits gp41 six-helix bundle formation, processes involved in virus inactivation. CAP is not soluble at pH < 5.5, normal for microbicide target sites. Therefore, the interaction between insoluble micronized CAP and HIV-1 was studied. Carbomer 974P/BufferGel; carrageenan; cellulose sulfate; dextran/dextrin sulfate, poly(napthalene sulfonate) and poly(styrene-4-sulfonate) are also being considered as anti-HIV-1 microbicides, and their antiviral properties were compared with those of CAP.
Methods
Enzyme linked immunosorbent assays (ELISA) were used to (1) study HIV-1 IIIB and BaL binding to micronized CAP; (2) detect virus disintegration; and (3) measure gp41 six-helix bundle formation. Cells containing integrated HIV-1 LTR linked to the β-gal gene and expressing CD4 and coreceptors CXCR4 or CCR5 were used to measure virus infectivity.
Results
1) HIV-1 IIIB and BaL, respectively, effectively bound to micronized CAP. 2) The interaction between HIV-1 and micronized CAP led to: (a) gp41 six-helix bundle formation; (b) virus disintegration and shedding of envelope glycoproteins; and (c) rapid loss of infectivity. Polymers other than CAP, except Carbomer 974P, elicited gp41 six-helix bundle formation in HIV-1 IIIB but only poly(napthalene sulfonate), in addition to CAP, had this effect on HIV-1 BaL. These polymers differed with respect to their virucidal activities, the differences being more pronounced for HIV-1 BaL.
Conclusions
Micronized CAP is the only candidate topical microbicide with the capacity to remove rapidly by adsorption from physiological fluids HIV-1 of both the X4 and R5 biotypes and is likely to prevent virus contact with target cells. The interaction between micronized CAP and HIV-1 leads to rapid virus inactivation. Among other anionic polymers, cellulose sulfate, BufferGel and aryl sulfonates appear most effective in this respect.
doi:10.1186/1471-2334-2-27
PMCID: PMC139971  PMID: 12445331
6.  Effects of Ibuprofen on the physiology and outcome of rabbit endotoxic shock 
Background
Despite major developments in the management of septic shock, the mortality rate had progressively increased. Ibuprofen has been shown to have beneficial physiological effects when used as a treatment. However, there are conflicting results with respect to survival. This study aims to investigate the effect of ibuprofen on vital functions, various physiological parameters and survival during endotoxic shock in rabbits.
Methods
Twenty-eight New Zealand rabbits were randomly separated into four groups. The first group received only saline, the second was given 2 mg/kg intravenous endotoxin at t0, the third received 30 mg/kg ibuprofen 30 minutes after endotoxin administration, whilst the fourth group received ibuprofen 30 minutes before the endotoxin. Respiratory and heart rate, mean arterial blood pressure and rectal temperature were recorded. Complete blood counts were performed and thromboxane B2 was measured every 30 minutes for the first two hours, and then hourly over the course of the experiment. Urine samples were collected at the same time points for the measurement of prostaglandin E2.
Results
Ibuprofen was found to improve respiratory rate, heart rate, and arterial pressure. However, it did not improve the negative effects of endotoxin on body temperature, haematocrit values, white blood cell count, and thrombocyte number. Thromboxane B2 levels in group IV were significantly lower than in the other groups, and the increase started at a later timepoint. In ibuprofen-treated animals, Prostaglandin E2 levels stayed low for at least 90 minutes, but started to rise thereafter. While the average survival in Group II animals was 192.9 ± 46.9 minutes, those of groups III and IV were 339.1 ± 33.5 minutes (p < 0.05) and 383.0 ± 39.6 minutes (p = 0.01), respectively.
Conclusions
Ibuprofen appears to increase survival in endotoxic shock-induced animals. Therefore, it may be helpful for the prophylaxis and treatment of patients with, or who are likely to develop, septic shock.
doi:10.1186/1471-2334-2-26
PMCID: PMC137589  PMID: 12445332
7.  Characterisation of silent and active genes for a variable large protein of Borrelia recurrentis 
Background
We report the characterisation of the variable large protein (vlp) gene expressed by clinical isolate A1 of Borrelia recurrentis; the agent of the life-threatening disease louse-borne relapsing fever.
Methods
The major vlp protein of this isolate was characterised and a DNA probe created. Use of this together with standard molecular methods was used to determine the location of the vlp1B. recurrentis A1 gene in both this and other isolates.
Results
This isolate was found to carry silent and expressed copies of the vlp1B. recurrentis A1 gene on plasmids of 54 kbp and 24 kbp respectively, whereas a different isolate, A17, had only the silent vlp1B. recurrentis A17 on a 54 kbp plasmid. Silent and expressed vlp1 have identical mature protein coding regions but have different 5' regions, both containing different potential lipoprotein leader sequences. Only one form of vlp1 is transcribed in the A1 isolate of B. recurrentis, yet both 5' upstream sequences of this vlp1 gene possess features of bacterial promoters.
Conclusion
Taken together these results suggest that antigenic variation in B. recurrentis may result from recombination of variable large and small protein genes at the junction between lipoprotein leader sequence and mature protein coding region. However, this hypothetical model needs to be validated by further identification of expressed and silent variant protein genes in other B. recurrentis isolates.
doi:10.1186/1471-2334-2-25
PMCID: PMC130189  PMID: 12377101
8.  The cost-effectiveness of the WINGS intervention: a program to prevent HIV and sexually transmitted diseases among high-risk urban women 
Background
We evaluated the cost-effectiveness of the WINGS project, an intervention to prevent HIV and other sexually transmitted diseases among urban women at high risk for sexual acquisition of HIV.
Methods
We used standard methods of cost-effectiveness analysis. We conducted a retrospective analysis of the intervention's cost and we used a simplified model of HIV transmission to estimate the number of HIV infections averted by the intervention. We calculated cost-effectiveness ratios for the complete intervention and for the condom use skills component of the intervention.
Results
Under base case assumptions, the intervention prevented an estimated 0.2195 new cases of HIV at a cost of $215,690 per case of HIV averted. When indirect costs of HIV were excluded from the analysis, the intervention's cost-effectiveness ratios were $357,690 per case of HIV averted and $31,851 per quality-adjusted life year (QALY) saved. Under base case assumptions, the condom use skills component of the intervention prevented an estimated 0.1756 HIV infections and was cost-saving. When indirect HIV costs were excluded, the cost-effectiveness ratios for the condom use skills component of the intervention were $97,404 per case of HIV averted and $8,674 per QALY saved.
Conclusions
The WINGS intervention, particularly the two sessions of the intervention which focussed on condom use skills, could be cost-effective in preventing HIV among women.
doi:10.1186/1471-2334-2-24
PMCID: PMC134456  PMID: 12377102
9.  A short purification process for quantitative isolation of PrPSc from naturally occurring and experimental transmissible spongiform encephalopathies 
Background
Transmissible spongiform encephalopathies (TSEs) are neurodegenerative diseases affecting both humans and animals. They are associated with post-translational conversion of the normal cellular prion protein (PrPC) into a heat- and protease-resistant abnormal isoform (PrPSc). Detection of PrPSc in individuals is widely utilized for the diagnosis of prion diseases.
Methods
TSE brain tissue samples have been processed in order to quantitatively isolate PrPSc. The protocol includes an initial homogenization, digestion with proteinase K and salt precipitation.
Results
Here we show that over 97 percent of the PrPSc present can be precipitated from infected brain material using this simple salting-out procedure for proteins. No chemically harsh conditions are used during the process in order to conserve the native quality of the isolated protein.
Conclusion
The resulting PrPSc-enriched preparation should provide a suitable substrate for analyzing the structure of the prion agent and for scavenging for other molecules with which it may associate. In comparison with most methods that exist today, the one described in this study is rapid, cost-effective and does not demand expensive laboratory equipment.
doi:10.1186/1471-2334-2-23
PMCID: PMC134455  PMID: 12370086
10.  Stable hepatitis C virus RNA detection by RT-PCR during four days storage 
Background
Suboptimal specimen processing and storage conditions of samples which contain hepatitis C virus (HCV) RNA may result in a decline of HCV RNA concentration or false-negative results in the detection of HCV RNA in serum. We evaluated the stability of HCV RNA in serum and clotted blood samples stored at room temperature or at 4°C for 4 days with the aim of optimizing the standard procedures of processing and storage of samples.
Methods
Blood from five HCV RNA positive patients was collected in tubes with and without separator gel, centrifuged 1 or 6 hours after collection. Samples were then left 6, 24, 48, 72 or 96 h at room temperature (21.5 – 25.4°C) or at 4°C before determining their HCV RNA level using the COBAS AMPLICOR HCV MONITOR Test, vs 2.0 (Roche Diagnostic Systems).
Results
The logarithm of the HCV RNA level measurements remained within a 0.3 value of the means for 4 days at both temperatures (room temperature or 4°C).
Conclusions
We conclude that blood samples may be collected and aliquoted within 6 h of collection and can be stored at 4°C for 72 hours as proposed by the manufacturer without significant differences in measured HCV RNA level. Our results indicate that lapses in this scheme may still yield reliable results.
doi:10.1186/1471-2334-2-22
PMCID: PMC130053  PMID: 12366870
11.  Association of circulating Chlamydia pneumoniae DNA with cardiovascular disease: a systematic review 
Background
Chlamydia pneumoniae antigens, nucleic acids, or intact organisms have been detected in human atheroma. However, the presence of antibody does not predict subsequent cardiovascular (CV) events. We performed a systematic review to determine whether the detection of C. pneumoniae DNA in peripheral blood mononuclear cells (PBMC) was associated with CV disease.
Methods
We sought studies of C. pneumoniae DNA detection in PBMC by polymerase chain reaction (PCR) among patients with CV disease or other clinical conditions. We pooled studies in which CV patients were compared with non-diseased controls. We analyzed differences between studies by meta-regression, to determine which epidemiological and technical characteristics were associated with higher prevalence.
Results
Eighteen relevant studies were identified. In nine CV studies with control subjects, the prevalence of circulating C. pneumoniae DNA was 252 of 1763 (14.3%) CV patients and 74 of 874 (8.5%) controls, for a pooled odds ratio of 2.03 (95% CI: 1.34, 3.08, P < 0.001). Prevalence was not adjusted for CV risk factors. Current smoking status, season, and age were associated with C. pneumoniae DNA detection. High prevalence (>40%) was found in patients with cardiac, vascular, chronic respiratory, or renal disease, and in blood donors. Substantial differences between studies were identified in methods of sampling, extraction, and PCR targets.
Conclusions
C. pneumoniae DNA detection was associated with CV disease in unadjusted case-control studies. However, adjustment for potentially confounding measures such as smoking or season, and standardization of laboratory methods, are needed to confirm this association.
doi:10.1186/1471-2334-2-21
PMCID: PMC130041  PMID: 12359046
12.  Expression of human beta-defensins 1 and 2 in kidneys with chronic bacterial infection 
Background
Constitutive expression and localization of antimicrobial human β-defensin-1 (HBD-1) in human kidneys as a potential mechanism of antimicrobial defense has been previously reported. Inducible expression of human β-defensin-2 (HBD-2) has been described in various epithelial organs but not for the urogenital tract.
Methods
We investigated the gene- and protein expression of HBD-1 and HBD-2 by reverse transcriptase-polymerase chain reaction, and immunohistochemistry in 15 normal human kidney samples and 15 renal tissues with chronic bacterial infection. Additionally, cell culture experiments were performed to study HBD gene expression by real-time RT-PCR in response to inflammatory cytokines TNFα and IL-1β as well as lipopolysaccharide from Gram-negative bacteria.
Results
Constitutive HBD-1 gene- and protein expression was detected in normal renal tissue and kidneys with chronic infection. As a novel finding, inducible HBD-2 gene- and protein expression was demonstrated in tubulus epithelia with chronic infection but not in normal renal tissue. In pyelonephritic kidneys HBD-1 and HBD-2 expression showed a similar pattern of localizaton in distal tubules, loops of Henle and in collecting ducts of the kidney. Furthermore, real-time RT-PCR of kidney derived cell lines stimulated with inflammatory agents TNF-α, IL-1β and LPS revealed a strong increase in relative HBD-2 transcription level and also a slight increase in relative HBD-1 transcription level.
Conclusions
Upregulated HBD-2 expression in renal tubulus epithelium indicates a role of a wider range of human defensins for antimicrobial host defense in the urogenital tract than previously recognized.
doi:10.1186/1471-2334-2-20
PMCID: PMC128826  PMID: 12238953
13.  Impact of HIV vaccination on laboratory diagnosis: case reports 
Background
It has not been clearly demonstrated whether HIV vaccination can complicate routine HIV testing. In this report, we describe the laboratory data of two prisoners who received rgp120 vaccine in a phase III trial underway in Thailand. These data indicate that previous vaccination may complicate the interpretation of screening HIV diagnostic tests.
Case presentation
The participants were identified from a cohort study on "Health factors related to HIV-1 and other viral infections among incarcerated people" that was approved by The Ethical Committee for Research in Human Subjects, Ministry of Public Health, Thailand. HIV diagnosis was definitively established with serial specimens using multi-screening tests, Western blot and diagnostic PCR.
Anti-HIV screening tests consistently exhibited either weakly reactive or inconclusive results. The band patterns of the Western blot analysis corresponded to those found in individuals who received the rgp120 vaccination. Definite results were established using diagnostic PCR, which exhibited consistently negative results with follow-up specimens. Such problems in HIV testing are not easily resolved in the routine clinical setting in Thailand.
Conclusions
These data demonstrate that HIV-1 vaccination interferes with routine diagnostic tests. Similar cases will not be uncommon in Thailand, where 2,545 people have already participated in a phase III trial.
doi:10.1186/1471-2334-2-19
PMCID: PMC126272  PMID: 12223114
14.  Development of real-time NASBA assays with molecular beacon detection to quantify mRNA coding for HHV-8 lytic and latent genes 
Background
Human herpesvirus-8 (HHV-8) is linked to the pathogenesis of Kaposi's sarcoma (KS), and the HHV-8 DNA load in peripheral blood mononuclear cells (PBMC) is associated with the clinical stage of KS. To examine the expression of HHV-8 in PBMC, four HHV-8 mRNA specific NASBA assays were developed
Methods
We have developed four quantitative nucleic acid sequence-based amplification assays (NASBA-QT) specifically to detect mRNA coding for ORF 73 (latency-associated nuclear antigen, LANA), vGCR (a membrane receptor), vBcl-2 (a viral inhibitor of apoptosis) and vIL-6 (a viral growth factor). The NASBA technique amplifies nucleic acids without thermocycling and mRNA can be amplified in a dsDNA background. A molecular beacon is used during amplification to enable real-time detection of the product. The assays were tested on PBMC samples of two AIDS-KS patients from the Amsterdam Cohort.
Results
For all four assays, the limit of detection (LOD) of 50 molecules and the limit of quantification (LOQ) of 100 molecules were determined using in vitro transcribed RNA. The linear dynamic range was 50 to 107 molecules of HHV-8 mRNA. We found HHV-8 mRNA expression in 9 out of the 10 tested samples.
Conclusion
These real-time NASBA assays with beacon detection provide tools for further study of HHV-8 expression in patient material.
doi:10.1186/1471-2334-2-18
PMCID: PMC126271  PMID: 12207829
15.  Fast relapse and high drop out rate of 48 weeks daily interferon monotherapy in HIV-infected patients with chronic hepatitis C 
Background
The standard of care for HCV Hepatitis is the combination of interferon (IFN) plus Ribavirin. In HIV patients the use of this combination therapy may induce drug interactions, and reduces the adherence to HAART.
The aim of this study is to evaluate safety and efficacy of a 48 weeks daily dose IFN schedule.
Methods
We evaluated 50 coinfected patients; alpha IFN 2a was administered at a dose of 3 MU daily. The baseline values were the following : CD4+ 515 cells/mmc (mean); HIV-RNA <50 copies/ml in all patients; HCV-RNA 28, 3 × 106 copies/ml.
Results
At 48 weeks, 10 patients (20%) achieved a biochemical and virological response according to an intention to treat analysis.
Twenty four patients (48%) underwent a drop-out mainly by side effects related to overlapping toxicity of interferon and antiretroviral therapy. All the patients, who responded to the treatment, showed a fast relapse one month after the end of treatment.
Conclusion
Although our results demonstrated a very poor outcome and a bad tolerance to interferon monotherapy, this approach should not be dropped out, mainly in patients at high risk for side effects and in those with cirrhosis who do not tolerate or are at increased risk for the use of ribavirin.
doi:10.1186/1471-2334-2-17
PMCID: PMC128825  PMID: 12199910
HIV; HCV; Coinfection; Interferon; Ribavirin
16.  Shorter courses of parenteral antibiotic therapy do not appear to influence response rates for children with acute hematogenous osteomyelitis: a systematic review 
Background
Acute hematogenous osteomyelitis (AHO) occurs primarily in children and is believed to evolve from bacteremia followed by localization of infection to the metaphysis of bones. Currently, there is no consensus on the route and duration of antimicrobial therapy to treat AHO.
Methods
We conducted a systematic review of a short versus long course of treatment for AHO due primarily to Staphylococcus aureus in children aged 3 months to 16 years. We searched Medline, Embase and the Cochrane trials registry for controlled trials. Clinical cure rate at 6 months was the primary outcome variable, and groups receiving less than 7 days of intravenous therapy were compared with groups receiving one week or longer of intravenous antimicrobials.
Results
12 eligible prospective studies, one of which was randomized, were identified. The overall cure rate at 6 months for the short course of intravenous therapy was 95.2% (95% CI = 90.4, 97.7) compared to 98.8% (95% CI = 93.6, 99.8) for the longer course of therapy. There was no significant difference in the duration of oral therapy between the two groups.
Conclusions
Given the potential increased morbidity and cost associated with longer courses of intravenous therapy, this finding should be confirmed through a randomized controlled equivalence trial.
doi:10.1186/1471-2334-2-16
PMCID: PMC128824  PMID: 12181082
17.  Antitubercular therapy decreases nitric oxide production in HIV/TB coinfected patients 
Background
Nitric oxide (NO) production is increased among patients with human immunodeficiency virus (HIV) infection and also among those with tuberculosis (TB). In this study we sought to determine if there was increased NO production among patients with HIV/TB coinfection and the effect of four weeks chemotherapy on this level.
Methods
19 patients with HIV/TB coinfection were studied. They were treated with standard four drug antitubercular therapy and sampled at baseline and four weeks. 20 patients with HIV infection, but no opportunistic infections, were disease controls and 20 individuals were healthy controls. Nitrite and citrulline, surrogate markers for NO, were measured spectrophotometrically.
Results
The mean age of HIV/TB patients was 28.4 ± 6.8 years and CD4 count was 116 ± 36.6/mm. Mean nitrite level among HIV/TB coinfected was 207.6 ± 48.8 nmol/ml. This was significantly higher than 99.7 ± 26.5 nmol/ml, the value for HIV infected without opportunistic infections and 46.4 ± 16.2 nmol/ml, the value for healthy controls (p value < 0.01). The level of HIV/TB coinfected NO in patients declined to 144.5 ± 34.4 nmol/ml at four weeks of therapy (p value < 0.05). Mean citrulline among HIV/TB coinfected was 1446.8 ± 468.8 nmol/ml. This was significantly higher than 880.8 ± 434.8 nmol/ml, the value for HIV infected without opportunistic infections and 486.6 ± 212.5 nmol/ml, the value for healthy controls (p value < 0.01). Levels of citrolline in HIV/TB infected declined to 1116.2 ± 388.6 nmol/ml at four weeks of therapy (p value < 0.05).
Conclusions
NO production is elevated among patients with HIV infection, especially so among HIV/TB coinfected patients, but declines significantly following 4 weeks of antitubercular therapy.
doi:10.1186/1471-2334-2-15
PMCID: PMC119853  PMID: 12147177
18.  Increased risk of tuberculosis in health care workers: a retrospective survey at a teaching hospital in Istanbul, Turkey 
Background
Tuberculosis (TB) is an established occupational disease affecting health care workers (HCWs). Determining the risk of TB among HCWs is important to enable authorites to take preventative measures in health care facilities and protect HCWs. This study was designed to assess the incidence of TB in a teaching hospital in Istanbul, Turkey. This study is retrospective study of health records of HCWs in our hospital from 1991 to 2000.
Results
The mean workforce of the hospital was 3359 + 33.2 between 1991 and 2000. There were 31 cases (15 male) meeting the diagnostic criteria for TB, comprising eight doctors, one nurse and 22 other health professionals. Mean incidence of TB was 96 per 100,000 for all HCWs (relative risk: 2.71), 79 per 100,000 for doctors (relative risk: 2.2), 14 per 100,000 for nurses and 121 per 100,000 (relative risk: 3.4) for other professionals. The mean incidence of TB in Turkey between 1991 and 2000 was 35.4 per 100,000. Incidence of TB was similar in the Departments of Chest Diseases and Clinical Medicine but there were no TB cases in the Basic Science and Managerial Departments.
Conclusion
HCWs in Turkey who work in clinics have an increased risk for TB. Post-graduate education and prevention programs reduce the risk of TB. Control programs to prevent nosocomial transmission of TB should be established in hospitals to reduce risk for HCWs.
doi:10.1186/1471-2334-2-14
PMCID: PMC122064  PMID: 12144709
19.  A quantitative PCR (TaqMan) assay for pathogenic Leptospira spp 
Background
Leptospirosis is an emerging infectious disease. The differential diagnosis of leptospirosis is difficult due to the varied and often "flu like" symptoms which may result in a missed or delayed diagnosis. There are over 230 known serovars in the genus Leptospira. Confirmatory serological diagnosis of leptospirosis is usually made using the microscopic agglutination test (MAT) which relies on the use of live cultures as the source of antigen, often performed using a panel of antigens representative of local serovars. Other techniques, such as the enzyme linked immunosorbent assay (ELISA) and slide agglutination test (SAT), can detect different classes of antibody but may be subject to false positive reactions and require confirmation of these results by the MAT.
Methods
The polymerase chain reaction (PCR) has been used to detect a large number of microorganisms, including those of clinical significance. The sensitivity of PCR often precludes the need for isolation and culture, thus making it ideal for the rapid detection of organisms involved in acute infections. We employed real-time (quantitative) PCR using TaqMan chemistry to detect leptospires in clinical and environmental samples.
Results and Conclusions
The PCR assay can be applied to either blood or urine samples and does not rely on the isolation and culture of the organism. Capability exists for automation and high throughput testing in a clinical laboratory. It is specific for Leptospira and may discriminate pathogenic and non-pathogenic species. The limit of detection is as low as two cells.
doi:10.1186/1471-2334-2-13
PMCID: PMC117785  PMID: 12100734
Leptospirosis; TaqMan; real-time PCR; diagnosis
20.  Smoking, season, and detection of chlamydia pneumoniae DNA in clinically stable COPD patients 
Background
The prevalence and role of Chlamydia pneumoniae in chronic obstructive pulmonary disease (COPD) remain unclear.
Methods
Peripheral blood mononuclear cells were obtained from 100 outpatients with smoking-related, clinically stable COPD, and induced sputum was obtained in 62 patients.
Results
Patients had mean age (standard deviation) of 65.8 (10.7) years, mean forced expiratory volume in one second of 1.34 (0.61) L, and 61 (61.0%) were male. C. pneumoniae nucleic acids were detected by nested polymerase chain reaction in 27 (27.0%). Current smoking (odds ratio {OR} = 2.6, 95% confidence interval {CI}: 1.1, 6.6, P = 0.04), season (November to April) (OR = 3.6, 95% CI: 1.4, 9.2, P = 0.007), and chronic sputum production (OR = 6.4, 95% CI: 1.8, 23.2, P = 0.005) were associated with detection of C. pneumoniae DNA.
Conclusions
Prospective studies are needed to examine the role of C. pneumoniae nucleic acid detection in COPD disease symptoms and progression.
doi:10.1186/1471-2334-2-12
PMCID: PMC117240  PMID: 12098361
21.  Increased risk of traffic accidents in subjects with latent toxoplasmosis: a retrospective case-control study 
Background
The parasite Toxoplasma gondii infects 30–60% of humans worldwide. Latent toxoplasmosis, i.e., the life-long presence of Toxoplasma cysts in neural and muscular tissues, leads to prolongation of reaction times in infected subjects. It is not known, however, whether the changes observed in the laboratory influence the performance of subjects in real-life situations.
Methods
The seroprevalence of latent toxoplasmosis in subjects involved in traffic accidents (N = 146) and in the general population living in the same area (N = 446) was compared by a Mantel-Haenszel test for age-stratified data. Correlation between relative risk of traffic accidents and level of anti-Toxoplasma antibody titre was evaluated with the Cochran-Armitage test for trends.
Results
A higher seroprevalence was found in the traffic accident set than in the general population (Chi2MH = 21.45, p < 0.0001). The value of the odds ratio (OR) suggests that subjects with latent toxoplasmosis had a 2.65 (C.I.95= 1.76–4.01) times higher risk of an accident than the toxoplasmosis-negative subjects. The OR significantly increased with level of anti-Toxoplasma antibody titre (p < 0.0001), being low (OR = 1.86, C.I.95 = 1.14–3.03) for the 99 subjects with low antibody titres (8 and 16), higher (OR = 4.78, C.I.95 = 2.39–9.59) for the 37 subjects with moderate titres (32 and 64), and very high (OR = 16.03, C.I.95 = 1.89–135.66) for the 6 subjects with titres higher than 64.
Conclusion
The subjects with latent toxoplasmosis have significantly increased risk of traffic accidents than the noninfected subjects. Relative risk of traffic accidents decreases with the duration of infection. These results suggest that 'asymptomatic' acquired toxoplasmosis might in fact represent a serious and highly underestimated public health problem, as well as an economic problem.
doi:10.1186/1471-2334-2-11
PMCID: PMC117239  PMID: 12095427
22.  Kinetics of maternal immunity against rabies in fox cubs (Vulpes vulpes) 
Background
In previous experiments, it was demonstrated that maternal antibodies (maAb) against rabies in foxes (Vulpes vulpes) were transferred from the vixen to her offspring. However, data was lacking from cubs during the first three weeks post partum. Therefore, this complementary study was initiated.
Methods
Blood samples (n = 281) were collected from 64 cubs (3 to 43 days old) whelped by 19 rabies-immune captive-bred vixens. Sera was collected up to six times from each cub. The samples were analysed by a fluorescence focus inhibition technique (RFFIT), and antibody titres (nAb) were expressed in IU/ml. The obtained data was pooled with previous data sets. Subsequently, a total of 499 serum samples from 249 cubs whelped by 54 rabies-immune vixens were fitted to a non-linear regression model.
Results
The disappearance rate of maAb was independent of the vixens' nAb-titre. The maAb-titre of the cubs decreased exponentially with age and the half-life of the maAb was estimated to be 9.34 days. However, maAb of offspring whelped by vixens with high nAb-titres can be detected for longer by RFFIT than that of offspring whelped by vixens with relatively low nAb-titres. At a mean critical age of about 23 days post partum, maAb could no longer be distinguished from unspecific reactions in RFFIT depending on the amount of maAb transferred by the mother.
Conclusions
The amount of maAb cubs receive is directly proportional to the titre of the vixen and decreases exponentially with age below detectable levels in seroneutralisation tests at a relatively early age.
doi:10.1186/1471-2334-2-10
PMCID: PMC116597  PMID: 12069694
23.  Dimethyl sulfoxide blocks herpes simplex virus-1 productive infection in vitro acting at different stages with positive cooperativity. Application of micro-array analysis 
Background
Dimethyl sulfoxide (DMSO) is frequently used at a concentration of up to 95% in the formulation of antiherpetic agents because of its properties as a skin penetration enhancer. Here, we have analyzed the effect of DMSO on several parameters of Herpes Simplex Virus replication.
Methods
Productive infection levels of HSV-1 were determined by plaque assay or by reporter gene activity, and its DNA replication was estimated by PCR. Transcript levels were evaluated with HSV-specific DNA micro-arrays.
Results
DMSO blocks productive infection in vitro in different cell types with a 50% inhibitory concentration (IC50) from 0.7 to 2% depending upon the multiplicity of infection. The concentration dependence exhibits a Hill coefficient greater than 1, indicating that DMSO blocks productive infection by acting at multiple different points (mechanisms of action) with positive cooperativity. Consistently, we identified at least three distinct temporal target mechanisms for inhibition of virus growth by DMSO. At late stages of infection, DMSO reduces virion infectivity, and markedly inhibits viral DNA replication. A third mode of action was revealed using an oligonucleotide-based DNA microarray system for HSV. These experiments showed that DMSO reduced the transcript levels of many HSV-1 genes; including several genes coding for proteins involved in forming and assembling the virion. Also, DMSO markedly inhibited some but not all early transcripts indicating a previously unknown mode for inhibiting the early phase of HSV transcription-replication cycle.
Conclusion
These observations suggest that DMSO itself may have a role in the anti-herpetic activity of formulations utilizing it as a dispersant.
doi:10.1186/1471-2334-2-9
PMCID: PMC116584  PMID: 12052246
24.  Lack of microbiological concordance between bone and non-bone specimens in chronic osteomyelitis: an observational study 
Background
Prognosis of chronic osteomyelitis depends heavily on proper identification and treatment of the bone-infecting organism. Current knowledge on selecting the best specimen for culture is confusing, and many consider that non-bone specimens are suitable to replace bone cultures. This paper compares the microbiology of non-bone specimens with bone cultures, taking the last as the diagnostic gold standard.
Methods
Retrospective observational analysis of 50 patients with bacterial chronic osteomyelitis in a 750-bed University-based hospital.
Results
Concordance between both specimens for all etiologic agents was 28%, for Staphylococcus aureus 38%, and for organisms other than S. aureus 19%. The culture of non-bone specimens to identify the causative organisms in chronic osteomyelitis produced 52% false negatives and 36% false positives when compared against bone cultures.
Conclusions
Diagnosis and therapy of chronic osteomyelitis cannot be guided by cultures of non-bone specimens because their microbiology is substantially different to the microbiology of the bone.
doi:10.1186/1471-2334-2-8
PMCID: PMC115844  PMID: 12015818
25.  Assessing the contribution of the herpes simplex virus DNA polymerase to spontaneous mutations 
Background
The thymidine kinase (tk) mutagenesis assay is often utilized to determine the frequency of herpes simplex virus (HSV) replication-mediated mutations. Using this assay, clinical and laboratory HSV-2 isolates were shown to have a 10- to 80-fold higher frequency of spontaneous mutations compared to HSV-1.
Methods
A panel of HSV-1 and HSV-2, along with polymerase-recombinant viruses expressing type 2 polymerase (Pol) within a type 1 genome, were evaluated using the tk and non-HSV DNA mutagenesis assays to measure HSV replication-dependent errors and determine whether the higher mutation frequency of HSV-2 is a distinct property of type 2 polymerases.
Results
Although HSV-2 have mutation frequencies higher than HSV-1 in the tk assay, these errors are assay-specific. In fact, wild type HSV-1 and the antimutator HSV-1 PAAr5 exhibited a 2–4 fold higher frequency than HSV-2 in the non-HSV DNA mutatagenesis assay. Furthermore, regardless of assay, HSV-1 recombinants expressing HSV-2 Pol had error rates similar to HSV-1, whereas the high mutator virus, HSV-2 6757, consistently showed signficant errors. Additionally, plasmid DNA containing the HSV-2 tk gene, but not type 1 tk or LacZ DNA, was shown to form an anisomorphic DNA stucture.
Conclusions
This study suggests that the Pol is not solely responsible for the virus-type specific differences in mutation frequency. Accordingly, it is possible that (a) mutations may be modulated by other viral polypeptides cooperating with Pol, and (b) the localized secondary structure of the viral genome may partially account for the apparently enhanced error frequency of HSV-2.
doi:10.1186/1471-2334-2-7
PMCID: PMC113270  PMID: 12019036

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