Comparison of NucliSens and Amplicor Monitor Assays for Quantification of Human Immunodeficiency Virus Type 1 (HIV-1) RNA in Plasma of Persons with HIV-1 Subtype A Infection in Abidjan, Côte d’Ivoire

We compared the sensitivity and accuracy of the NucliSens assay and those of both the standard and modified (addition of a new primer set, primer mix 1, supplied by Roche) Amplicor HIV Monitor assays to quantify human immunodeficiency virus type 1 (HIV-1) RNA in persons infected with HIV-1 subtype A in Abidjan, Côte d’Ivoire. Seventy-one plasma samples from HIV-1-seropositive persons at different stages of HIV infection and 15 samples from HIV antibody-negative persons were analyzed. The HIV-1 genetic subtype was determined either by DNA sequencing or by a restriction fragment length polymorphism assay. Of the 71 samples, 70 (98%) were subtype A and 1 was subtype G. Of the 70 subtype A samples, the proportion of RNA-positive plasma samples and mean HIV-1 RNA levels were significantly higher by the modified HIV Monitor assay (n = 67 [96%]; mean RNA levels, 5.2 log₁₀ HIV-1 RNA copies/ml) than the NucliSens assay (n = 56 [80%]; 4.3 log₁₀ HIV-1 RNA copies/ml) or the standard HIV Monitor assay (n = 44 [63%]; mean RNA levels, 3.8 log₁₀ HIV-1 RNA copies/ml) (all P values were <0.05). The HIV-1 RNA levels by the modified HIV Monitor assay correlated significantly with those by the NucliSens assay (r = 0.76; P < 0.001) and the standard HIV Monitor assay (r = 0.57; P < 0.001), as did the RNA levels by the NucliSens and the standard HIV Monitor assays (r = 0.60; P < 0.001). Lower CD4 cell counts were significantly correlated with higher HIV-1 RNA levels by all three assays (r = −0.47 for the NucliSens assay, −0.45 for the standard HIV Monitor assay, and −0.62 for the modified HIV Monitor assay). These results indicate that the modified HIV Monitor assay has the highest sensitivity and efficiency at quantifying the levels of RNA in persons infected with HIV-1 subtype A and thus constitutes a valuable tool for the monitoring of RNA levels in areas of Africa were HIV-1 subtype A is predominant.     

Virulence Properties of Shiga Toxin-Producing Escherichia coli (STEC) Strains of Serogroup O118, a Major Group of STEC Pathogens in Calves

Shiga toxin-producing Escherichia coli (STEC) strains of serogroup O118 are the most prevalent group among STEC strains in diarrheic calves in Germany (L. H. Wieler, Ph.D. thesis, University of Giessen, 1997). To define their virulence properties, 42 O118 (O118:H16 [n = 38] and O118:H− [n = 4]) strains were characterized. The strains displayed three different Stx combinations (Stx1 [36 of 42], Stx1 and Stx2 [2 of 42], and Stx2 [4 of 42]). A total of 41 strains (97.6%) harbored a large virulence-associated plasmid containing hly_EHEC (hly from enterohemorrhagic E. coli). The strains’ adhesive properties varied in relation to the eukaryotic cells tested. Only 28 of 42 strains (66.7%) showed localized adhesion (LA) in the human HEp-2 cell line. In contrast, in bovine fetal calf lung (FCL) cells, the number of LA-positive strains was much higher (37 of 42 [88.1%]). The locus of enterocyte effacement (LEE) was detected in 41 strains (97.6%). However, not all LEE-positive strains reacted positively in the fluorescence actin-staining (FAS) test, which indicated the attaching and effacing (AE) lesion. In HEp-2 cells, only 22 strains (52.4%) were FAS positive, while in FCL cells, the number of FAS-positive strains was significantly higher (38 of 42 [90.5%; P < 0.001]). In conclusion, the vast majority of the O118 STEC strains from calves (41 of 42 [97.6%]) have a high virulence potential (stx, hly_EHEC, and LEE). This virulence potential and the high prevalence of STEC O118 strains in calves suggest that these strains could be a major health threat for humans in the future. In addition, the poor association between results of the geno- and phenotypical tests to screen for the AE ability of STEC strains calls the diagnostic value of the FAS test into question.     

Detection of Antibodies to Human Immunodeficiency Virus Type 1 in Oral Fluids: A Large-Scale Evaluation of Immunoassay Performance

Paired serum and oral-fluid (OF) specimens (n = 4,448) were collected from blood donors and patients attending local sexually transmitted disease clinics in Trinidad and Tobago and the Bahamas and were tested for the presence of human immunodeficiency virus type 1 (HIV-1) antibodies. Sera were tested by Abbott AB HIV-1/HIV-2 (rDNA) enzyme immunoassay (EIA), and positive specimens were confirmed by Cambridge HIV-1 and HIV-2 Western blotting (WB). OF specimens were collected with the OraSure collection device and were tested by Murex GACELISA and by two EIAs from Organon Teknika (the Oral Fluid Vironostika HIV-1 Microelisa System [OTC-L] and the Vironostika HIV-1 Microelisa System [OTC-M]). EIA-reactive OF specimens were confirmed by miniaturized WB (OFWB). GACELISA detected all 474 HIV-1 seropositive specimens (sensitivity, 100%). OTC-L detected 470 positive specimens (sensitivity, 99.2%), while OTC-M detected 468 positive specimens (sensitivity, 98.8%). Specificities ranged from 99.2 to 100% for the three assays. Concordance of OFWB with serum WB was 99.4%, and banding patterns determined by the two methods were similar. The immunoglobulin G (IgG) concentration of OF specimens ranged from 0.21 to 100 μg/ml, with a mean of 17.1 μg/ml. Significant differences in OF IgG concentrations were observed between HIV antibody-positive and HIV antibody-negative persons (31.94 versus 15.28 μg/ml, respectively [P < 0.0001]). These data further confirm the suitability of OF specimens for detection of HIV-1 antibodies. Currently available HIV-1 antibody assays provide sensitivities and specificities with OF specimens comparable to those achieved with serum specimens.     

Direct Comparison of Xpert MTB/RIF Assay with Liquid and Solid Mycobacterial Culture for Quantification of Early Bactericidal Activity

The early bactericidal activity of antituberculosis agents is usually determined by measuring the reduction of the sputum mycobacterial load over time on solid agar medium or in liquid culture. This study investigated the value of a quantitative PCR assay for early bactericidal activity determination. Groups of 15 patients were treated with 6 different antituberculosis agents or regimens. Patients collected sputum for 16 h overnight at baseline and at days 7 and 14 after treatment initiation. We determined the sputum bacterial load by CFU counting (log CFU/ml sputum, reported as mean ± standard deviation [SD]), time to culture positivity (TTP, in hours [mean ± SD]) in liquid culture, and Xpert MTB/RIF cycle thresholds (C_T, n [mean ± SD]). The ability to discriminate treatment effects between groups was analyzed with one-way analysis of variance (ANOVA). All measurements showed a decrease in bacterial load from mean baseline (log CFU, 5.72 ± 1.00; TTP, 116.0 ± 47.6; C_T, 19.3 ± 3.88) to day 7 (log CFU, −0.26 ± 1.23, P = 0.2112; TTP, 35.5 ± 59.3, P = 0.0002; C_T, 0.55 ± 3.07, P = 0.6030) and day 14 (log CFU, −0.55 ± 1.24, P = 0.0006; TTP, 54.8 ± 86.8, P < 0.0001; C_T, 2.06 ± 4.37, P = 0.0020). The best discrimination between group effects was found with TTP at day 7 and day 14 (F = 9.012, P < 0.0001, and F = 11.580, P < 0.0001), followed by log CFU (F = 4.135, P = 0.0024, and F = 7.277, P < 0.0001). C_T was not significantly discriminative (F = 1.995, P = 0.091, and F = 1.203, P = 0.316, respectively). Culture-based methods are superior to PCR for the quantification of early antituberculosis treatment effects in sputum.     

Diversity of Helicobacter pylori vacA and cagA Genes and Relationship to VacA and CagA Protein Expression, Cytotoxin Production, and Associated Diseases

The vacuolating cytotoxin and the cytotoxin-associated protein, encoded by vacA and cagA, respectively, are important virulence determinants of Helicobacter pylori. Sixty-five H. pylori strains were isolated from dyspeptic patients (19 with peptic ulcer disease, 43 with chronic gastritis, and 3 with gastric cancer) and studied for differences in the vacA and cagA genes and their relationship to VacA and CagA expression, cytotoxin activity, and the clinical outcome of infection. By PCR, fifty-four (83.1%) of 65 strains had the vacA signal sequence genotype s1 and only 10 (15.4%) had the type s2. After primer modification, the vacA middle-region types m1 and m2 were detected in 24 (36.9%) and 41 (63.1%) strains, respectively. The combinations s1-m2 (31 [47.7%]) and s1-m1 (23 [35.4%]) occurred more frequently than s2-m2 (10 [15.4%]) (P = 0.01). No strain with the combination s2-m1 was found. All 19 patients with peptic ulcers harbored type s1 strains, in contrast to 32 (74.4%) of 43 patients with gastritis (P = 0.02). The vacA genotype s1 was associated with the presence of cagA (P < 0.0001), VacA expression (P < 0.0001), and cytotoxin activity (P = 0.003). The cagA gene was detectable in 48 (73.8%) of 65 isolates and present in 16 (84.2%) of 19 ulcer patients and 29 (67.4%) of 43 patients with gastritis (P = 0.17). The vacA genotypes of German H. pylori isolates are identical to those previously reported. H. pylori strains of vacA type s1 are associated with the occurrence of peptic ulceration and the presence of cagA, cytotoxin activity, and VacA expression.     

Immunoblot Profile as Predictor of Toxoplasmic Encephalitis in Patients Infected with Human Immunodeficiency Virus

In order to define more accurately human immunodeficiency virus-infected patients at risk of developing toxoplasmic encephalitis (TE), we assessed the prognostic significance of the anti-Toxoplasma gondii immunoglobulin G (IgG) immunoblot profile, in addition to AIDS stage, a CD4⁺ cell count <50/mm³, and an antibody titer ≥150 IU/ml, in patients with CD4 cell counts <200/mm³ and seropositive for T. gondii. Baseline serum samples from 152 patients included in the placebo arm of the ANRS 005-ACTG 154 trial (pyrimethamine versus placebo) were used. The IgG immunoblot profile was determined using a Toxoplasma lysate and read using the Kodak Digital Science 1D image analysis software. Mean follow-up was 15.1 months, and the 1-year incidence of TE was 15.9%. The cumulative probability of TE varied according to the type and number of anti-T. gondii IgG bands and reached 65% at 12 months for patients with IgG bands of 25 and 22 kDa. In a Cox model adjusted for age, gender, Centers for Disease Control and Prevention (CDC) clinical stage, and CD4 and CD8 cell counts, the incidence of TE was higher when the IgG 22-kDa band (hazard ratio [HR] = 5.4; P < 0.001), the IgG 25-kDa band (HR = 4.7; P < 0.001), or the IgG 69-kDa band (HR = 3.4; P < 0.001) was present and was higher for patients at CDC stage C (HR = 4.9; P < 0.001). T. gondii antibody titer and CD4 cell count were not predictive of TE. Thus, detection of IgG bands of 25, 22, and/or 69 kDa may be helpful for deciding when primary prophylaxis for TE should be started or discontinued, especially in the era of highly active antiretroviral therapy.     

Blood monocyte activation in rheumatoid arthritis: increased monocyte adhesiveness, integrin expression, and cytokine release

Infiltration of the synovium by mononuclear cells, namely lymphocytes and monocytes, is one of the main features of rheumatoid arthritis (RA) and is considered to be responsible for the development of the disease. In this study in 31 consecutive patients with RA, we investigated whether peripheral blood monocytes exhibited markers of cellular activation related to cell migration. Using flow cytometry with the respective specific antibodies, we studied the expression of integrins CD11a, CD11b, CD11c, CD49d (VLA-4), and CD49e (VLA-5) on monocytes from patients with RA and from normal (N) subjects. IL-1β, IL-6, and tumour necrosis factor-alpha (TNF-α) production by cultured monocytes was measured by immunoassay. Adhesiveness of monocytes was studied on various surfaces (plastic, human fibronectin, gelatin-coated plasma, subendothelial matrix) and on cultured endothelial cells under basal conditions or after stimulation by IL-1β. An increased number of CD14⁺ monocytes (Mo) from RA patients expressed the CD11b molecule (RA Mo = 90·3%, N Mo = 83·4%, P < 0·005). The expression of CD11b on CD14⁺ monocytes was significantly increased in RA patients (median fluorescence intensity (FI): RA Mo = 145 (range 80–466) units; normal Mo = 95 (range 24–164) units; P < 0·003). Production of extracellular IL-1β and IL-6 by RA monocytes was significantly enhanced compared with monocytes from normal subjects (IL-1β: RA = 2·65 ± 0·91 ng/ml versusN = 1·35 ± 0·85 pg/ml, P < 0·05; IL-6: RA = 4·83 ± 0·90 ng/ml versusN = 2·40 ± 0·95 ng/ml, P < 0·05). Compared with normal monocytes, RA monocytes exhibited increased adhesion to the various surfaces studied (plastic, P < 0·01; fibronectin, P < 0·01; and gelatin-coated normal or RA plasma, P < 0·01) as well as to unstimulated (P < 0·01) and IL-1β-stimulated endothelial cells (IL-1β for 4 h, P < 0·05; IL-1β for 24 h, P < 0·05). In our study, blood monocytes from RA patients exhibited features of activation related to cell adhesion.     

Short-Term Storage Does Not Affect the Quantitative Yield of Mycobacterium tuberculosis in Sputum in Early-Bactericidal-Activity Studies

Early-bactericidal-activity (EBA) studies measure the change in mycobacterial load in sputum over time to evaluate antituberculosis drugs. We investigated whether a delay in sputum processing influences the quantitative results of sputum mycobacterial culture. We identified pretreatment smear-positive sputum samples collected overnight and processed at a single laboratory. Sputum volume, time from sputum collection to processing, CFU counts/ml of sputum, and time to culture positivity (TTP) data were retrieved. We obtained 817 TTP and 794 CFU results from a total of 844 sputum samples. Contamination did not occur more frequently with prolonged storage (TTP, 2.0%; CFU, 2.4%). Sample volumes were <5 ml in 5%, 5 to 10 ml in 46%, and >10 ml in 49%. Delays to processing were 0, 1, 2, and 3 days in 696 (43.2%), 722 (44.8%), 128 (7.9%), and 65 (4.0%) samples, respectively. TTP and CFU did not significantly differ between days of delay to processing (P = 0.098 and P = 0.908, respectively), but there was a nonsignificant trend toward a prolonged TTP over time (P = 0.052, Jonckheere-Terpstra trend test). Sputa of <5 ml in volume showed a significantly prolonged TTP compared to sputum of >5 ml (113 h versus 99 h; P < 0.01) but no significant decrease in CFU. Sputum can be stored under refrigerated conditions for deferred processing for at least 3 days. This means that central laboratories can be used for quantitative mycobacterial study endpoints when delays to processing are not expected to exceed a few days. Care should be taken to collect sputum of sufficient volume.     

Diagnosis of Human Immunodeficiency Virus Type 1 Infection with Different Subtypes Using Rapid Tests

We evaluated six rapid tests for their sensitivity and specificity in diagnosing human immunodeficiency virus type 1 (HIV-1) infection using 241 specimens (172 HIV-1 positive, 69 HIV-1 negative) representing different HIV-1 subtypes (A [n = 40], B [n = 47], C [n = 28], E [n = 42], and F [n = 7]). HIVCHEK, Multispot, RTD and SeroStrip were 100% sensitive and specific. Capillus failed to identify two of eight subtype C specimens (overall sensitivity of 98.85%), while the SUDS test (the only test approved by the Food and Drug Administration) gave false-positive results for 5 of 69 seronegative specimens (specificity of 93.24%). Our results suggest that although rapid tests perform well in general, it may be prudent to evaluate a rapid test for sensitivity and specificity in a local population prior to its widespread use.     

Association of Borderline Oxacillin-Susceptible Strains of Staphylococcus aureus with Surgical Wound Infections

Staphylococcus aureus isolates which produce type A staphylococcal β-lactamase have been associated with wound infections complicating the use of cefazolin prophylaxis in surgery. To further evaluate this finding, 215 wound isolates from 14 cities in the United States were characterized by antimicrobial susceptibility and β-lactamase type and correlated with the preoperative prophylactic regimen. Borderline-susceptible S. aureus isolates of phage group 5 (BSSA-5), which produce large amounts of type A β-lactamase and exhibit borderline susceptibility to oxacillin, comprised a greater percentage of the 120 wound isolates associated with cefazolin prophylaxis than they did of the 95 isolates associated with other prophylactic regimens (25% versus 12.6%, respectively; P < 0.05). In contrast, methicillin-resistant S. aureus isolates were distributed evenly between the two groups (8.3% versus 11.6%, respectively). In vitro assays demonstrated that cefazolin was hydrolyzed faster by BSSA-5 strains than by other β-lactamase-producing, methicillin-susceptible strains (1.54 versus 0.50 μg/min/10⁸ CFU, respectively; P < 0.0001). These data demonstrate that BSSA-5 strains are a distinct subpopulation of methicillin-susceptible S. aureus which frequently cause deep surgical wound infections. Cefazolin use in prophylaxis is a risk factor for BSSA-5 infection.     

Interpretations of Antibody Responses to Salmonella enterica Serotype Enteritidis gm Flagellin in Poultry Flocks Are Enhanced by a Kinetics-Based Enzyme-Linked Immunosorbent Assay

Many regulatory and diagnostic programs for the detection of Salmonella enterica serotype Enteritidis infection in commercial poultry flocks have relied on rapid Pullorum agglutination tests to screen birds because of the shared antigens of S. enterica Enteritidis and S. enterica Pullorum and Gallinarum; however, the use of the enzyme-linked immunosorbent assay (ELISA) format affords better analytical sensitivity than crude agglutination tests. In this study, we adapted our earlier conventional indirect ELISA, using gm flagellin as the antigen, to a kinetics-based, computer-controlled ELISA (KELA). The KELA was used to screen for flagellin antibody from three commercial flocks: (i) a large flock involved in a U.S. Department of Agriculture trace back from a human S. enterica Enteritidis foodborne outbreak (n = 3,209), (ii) a flock infected with the endemic S. enterica Enteritidis serotype but which also had multiple other salmonella serotypes (n = 65), and (iii) an S. enterica Pullorum-infected flock (n = 12). The first flock (S. enterica Enteritidis prevalence of 2.45% based on culture) provided a field test of the KELA and allowed the calculation of diagnostic sensitivity (D-Sn) and diagnostic specificity (D-Sp). With a cutoff of 10 (used for screening flocks [i.e., high sensitivity]), the KELA has a D-Sn of 95.2% and a D-Sp of 18.5%; with a cutoff of 140 (used in confirmatory flock testing [i.e., high specificity]), the KELA has a D-Sn of 28.0% and a D-Sp of 99.1%. We found that with a cutoff of 60 (D-Sn = 63.1%; D-Sp = 91.6%), we could eliminate reactions in the KELA caused by other non-S. enterica Enteritidis salmonellae. The KELA was also compared to two commercial rapid Pullorum tests, the Solvay (D-Sn = 94.9%; D-Sp = 55.5%) and the Vineland (D-Sn = 62.0%; D-Sp = 75.3%).     

Antimicrobial Resistance and Serotype Distribution of Streptococcus pneumoniae Strains Causing Childhood Infections in Bangladesh, 1993 to 1997

Three hundred sixty-two Streptococcus pneumoniae strains were isolated from children under 5 years of age at Dhaka Shishu (Children) Hospital from 1993 to 1997. The strains were isolated from blood (n = 105), CSF (n = 164), ear swab (n = 61), eye swab (n = 20), and pus (n = 12). Of the 362 isolates, 42 (11.6%) showed intermediate resistance (MIC, <0.1 μg/ml) and only 4 (1.1%) showed complete resistance (MIC, >2.0 μg/ml) to penicillin. Penicillin resistance exhibited a strong relationship with serotype 14; 47.8% of the penicillin-resistant strains belonged to this type. A remarkably high (64.1%) resistance to co-trimoxazole was observed, along with a significant increase during the time period studied; there was no relationship to capsular type. By way of contrast, penicillin resistance did not show any significant change during the study period. Resistance to chloramphenicol (2.2%) and erythromycin (1.1%) was rare. The high resistance to co-trimoxazole and its increasing trend demand elucidation of the clinical impact of pneumonia treatment by this antimicrobial and reconsideration of the World Health Organization recommendation for co-trimoxazole administration to children with community-acquired pneumonia at the health care worker level in Bangladesh.     

Salivary Nitrate,Nitrite and Nitrate Reductase Activity in Relation to Risk of Oral Cancer in Egypt

It has been suggested that nitrate and nitrite may play a role in the etiology of human oral cancer. We investigated whether salivary nitrate and nitrite and the activity of nitrate reductase (NRase) may affect the risk of oral cancer in Egypt, an area with high levels of environmental nitrosating agents. Levels of salivary nitrite (8.3 ± 1.0 μg/ml) and nitrate (44 ± 3.7 μg/ml) and activity of NRase (74 ± 10 nmol/ml/min) were significantly (P < 0.05) higher in oral cancer patients (n = 42) compared to control Egyptian healthy individuals (n = 40, nitrite = 5.3 ± 0.3 μg/ml, nitrate = 27 ± 1.2 μg/ml, and NRase activity = 46 ± 4 nmol/ml/min). The adjusted odds ratio (OR) and the 95% confidence intervals (C.I.) for risk of oral cancer, categorized by the levels of salivary nitrate and nitrite and NRase activity, showed a higher cancer risk associated with nitrite > 7.5 μg/ml (OR: 3.0, C.I.: 1.0–9.3), nitrite > 40 μg/ml (OR: 4.3, C.I.: 1.4–13.3) and NRase activity > 50 nmol/ml/min (OR: 2.9, C.I.: 1.1–7.4). Our findings suggest that increased consumption of dietary nitrate and nitrite is associated with elevated levels of salivary nitrite. Together with the increased activity of salivary NRase, these observations may explain, at least in part, the role of nitrate and nitrite in the development of oral cancer in individuals from an area with a high burden of N-nitroso precursors.     

Comparison of Sample Sequences of the Salmonella typhi Genome to the Sequence of the Complete Escherichia coli K-12 Genome

Raw sequence data representing the majority of a bacterial genome can be obtained at a tiny fraction of the cost of a completed sequence. To demonstrate the utility of such a resource, 870 single-stranded M13 clones were sequenced from a shotgun library of the Salmonella typhi Ty2 genome. The sequence reads averaged over 400 bases and sampled the genome with an average spacing of once every 5,000 bases. A total of 339,243 bases of unique sequence was generated (approximately 7% representation). The sample of 870 sequences was compared to the complete Escherichia coli K-12 genome and to the rest of the GenBank database, which can also be considered a collection of sampled sequences. Despite the incomplete S. typhi data set, interesting categories could easily be discerned. Sixteen percent of the sequences determined from S. typhi had close homologs among known Salmonella sequences (P < 1e⁻⁴⁰ in BlastX or BlastN), reflecting the proportion of these genomes that have been sequenced previously; 277 sequences (32%) had no apparent orthologs in the complete E. coli K-12 genome (P > 1e⁻²⁰), of which 155 sequences (18%) had no close similarities to any sequence in the database (P > 1e⁻⁵). Eight of the 277 sequences had similarities to genes in other strains of E. coli or plasmids, and six sequences showed evidence of novel phage lysogens or sequence remnants of phage integrations, including a member of the lambda family (P < 1e⁻¹⁵). Twenty-three sample sequences had a significantly closer similarity a sequence in the database from organisms other than the E. coli/Salmonella clade (which includes Shigella and Citrobacter). These sequences are new candidate lateral transfer events to the S. typhi lineage or deletions on the E. coli K-12 lineage. Eleven putative junctions of insertion/deletion events greater than 100 bp were observed in the sample, indicating that well over 150 such events may distinguish S. typhi from E. coli K-12. The need for automatic methods to more effectively exploit sample sequences is discussed.     

Comparison of Two Rapid Whole-Blood Tests for Helicobacter pylori Infection in Chinese Patients

Consecutive Chinese patients undergoing endoscopy for dyspepsia were tested for Helicobacter pylori infection by two rapid whole-blood tests: FlexPack HP (Abbott Laboratories) and Helisal One-Step (Cortecs Diagnostics). Biopsy-based tests (rapid urease test and histology) and the [¹³C]urea breath test were used as the “gold standard.” One hundred sixty-one consecutive patients were studied, and 88 (54.7%) were confirmed to have H. pylori infection. The sensitivities, specificities, and positive and negative predictive values were 81.8%, 83.6% (P = 0.008), 85.7% (P = 0.04), and 79.2% for FlexPack HP and 84.1%, 63.0% (P = 0.008), 73.3% (P = 0.047), and 76.7% for Helisal One-Step, respectively.     

Sputum Volume Predicts Sputum Mycobacterial Load during the First 2 Weeks of Antituberculosis Treatment

Disease severity in patients with pulmonary tuberculosis is associated with mycobacterial sputum load. To ascertain whether reduced sputum production during treatment is a useful clinical sign of improvement, we analyzed the mycobacterial loads of 5,552 sputum samples collected from 439 newly diagnosed sputum smear-positive tuberculosis patients who participated in six 14-day studies of antituberculosis treatment. Sputum volumes were categorized as low (<6 ml), medium (6 to 10 ml), or large (>10 ml), and mycobacterial load was measured by the time to positivity in liquid culture and the CFU counts on solid culture. The association of sputum volume with mycobacterial load was estimated with multiple linear regression models adjusted for repeated measures. The predictor variables were sputum volume category, treatment day, specific study , and the interaction of sputum volume category and treatment day. Mycobacterial load was significantly associated only with the day on treatment and sputum volume, which tended to decrease with ongoing treatment. With the volume held constant, each day on treatment decreased the log CFU by 0.082 (P < 0.001) and increased the time to positivity (TTP) by 1.04 h (P < 0.001). From low to medium and from medium to large sputum volumes, the log CFU/ml increased by 0.265 (P < 0.003) and 0.490 (P < 0.001), respectively, and the TTP decreased by 1.17 h (P < 0.001) and 1.30 h (P < 0.001), respectively, for a given day of treatment. The variability of the sputum load measurements increased with the day of treatment and lower sputum volumes. The significant association of sputum volume and mycobacterial load validates decreasing sputum production as a clinical sign of improvement during early antituberculosis treatment.     

Use of the BacT/Alert Blood Culture System for Culture of Sterile Body Fluids Other than Blood

Studies have demonstrated that large-volume culture methods for sterile body fluids other than blood increase recovery compared to traditional plated-medium methods. BacT/Alert is a fully automated blood culture system for detecting bacteremia and fungemia. In this study, we compared culture in BacT/Alert standard aerobic and anaerobic bottles, BacT/Alert FAN aerobic and FAN anaerobic bottles, and culture on routine media for six specimen types, i.e., continuous ambulatory peritoneal dialysate (CAPD), peritoneal, amniotic, pericardial, synovial, and pleural fluids. Specimen volumes were divided equally among the three arms of the study. A total of 1,157 specimens were tested, with 227 significant isolates recovered from 193 specimens. Recovery by method was as follows: standard bottles, 186 of 227 (82%); FAN bottles, 217 of 227 (96%); and routine culture, 184 of 227 (81%). The FAN bottles recovered significantly more gram-positive cocci (P < 0.001), Staphylococcus aureus (P = 0.003), coagulase-negative staphylococci (P = 0.008), gram-negative bacilli (P < 0.001), Enterobacteriaceae (P = 0.005), and total organisms (P < 0.001) than the routine culture. There were no significant differences in recovery between the standard bottles and the routine culture. The FAN aerobic bottle recovered significantly more gram-positive cocci (P < 0.001), S. aureus isolates (P < 0.001), coagulase-negative staphyococci (P = 0.003), and total organisms (P < 0.001) than the standard aerobic bottle, while the FAN anaerobic bottle recovered significantly more gram-positive cocci (P < 0.001), S. aureus isolates (P < 0.001), Enterobacteriaceae (P = 0.03), and total organisms (P < 0.001) than the standard anaerobic bottle. For specific specimen types, significantly more isolates were recovered from the FAN bottles compared to the routine culture for synovial (P < 0.001) and CAPD (P = 0.004) fluids. Overall, the FAN bottles were superior in performance to both the standard bottles and the routine culture for detection of microorganisms from the types of sterile body fluids included in this study.     

An assessment of peripheral immunity in patients with sarcoidosis using measurements of serum vitamin D3, cytokines and soluble CD23

The aetiology of the peripheral anergy in sarcoidosis is unclear. To investigate this further we measured the serum levels of several factors important in different aspects of immune regulation to obtain a profile of those factors which promote and inhibit immune activation in sarcoidosis. Thirty-seven patients with sarcoidosis and 20 healthy controls of similar sex and age comprised the study group. Serum IL-10, interferon-gamma (IFN-γ), soluble CD23 (sCD23), IL-8, granulocyte-macrophage colony-stimulating factor (GM-CSF), IL-1β and tumour necrosis factor-alpha (TNF-α) were measured using in-house ELISAs. Vitamin D3 was measured using a radioreceptor assay. Serum levels of sCD23 and IL-10 were significantly elevated in patients with sarcoidosis relative to controls (median 13.9 versus 9.5 arbitrary units/ml, P < 0.01 for sCD23, and 9.6 versus 5.0 pg/ml, P < 0.04 for IL-10). Regardless of steroid therapy or disease activity, serum levels of IFN-γ, TNF-α, IL-1β, GM-CSF and IL-8 were no different in patients with sarcoidosis and controls. Vitamin D3 levels were significantly higher in patients with sarcoidosis versus normal controls (medians 78.0 versus 56.0, P < 0.001), active sarcoidosis (n = 20) versus inactive disease (n = 17) (medians 81.5 versus 66.0, P < 0.03) and active sarcoidosis versus controls (medians 81.5 versus 56.0, P < 0.0002). The levels were no different between patients with inactive sarcoidosis and controls. We suggest that IL-10 and vitamin D3 may contribute to the peripheral anergy in sarcoidosis. The elevated serum sCD23 suggests an increase in peripheral humoral immunity. Consistent with a quiescent peripheral immune system, factors capable of monocyte/macrophage activation (TNF-α, IFN-γ, GM-CSF and IL-8) were not elevated in the peripheral circulation.     

Intestinal Secretory Factor Released by Macrophages Stimulated with Clostridium difficile Toxin A: Role of Interleukin 1β

Clostridium difficile toxin A is associated with enterocolitis in animals and humans. However, the mechanisms of its secretory and damaging effects are not totally understood. In this work, we examined the intestinal secretion of electrolytes and water caused by supernatants from macrophages stimulated with toxin A in rabbit ileal mucosa mounted in Üssing chambers. We also investigated the mechanism by which the intestinal secretory factor (ISF) is released from stimulated macrophages. Supernatants from macrophages stimulated with toxin A caused potent intestinal secretion (change in short-circuit current [ΔIsc], 76 μA · cm⁻²; P < 0.01). The release of the ISF was pertussis toxin sensitive (reduction, 61%; P < 0.01) and was also reduced (P < 0.05) by a protein synthesis inhibitor (67%), protease inhibitors (57%), a phospholipase A₂ inhibitor (54%), a cyclo-oxygenase inhibitor (62%), a dual cyclo- and lipoxygenase inhibitor (48%), a platelet-activating factor (PAF) receptor antagonist (55%), and tumor necrosis factor alpha (TNF-α) synthesis inhibitors (48%). However, this release was not inhibited by a lipo-oxygenase inhibitor. Monoclonal anti-interleukin 1β (IL-1β) but not anti-IL-1α antibody blocked (72%; P < 0.01) the secretory action of the ISF, as did recombinant human IL-1 receptor antagonist (80%; P < 0.01). High levels of IL-1β (3,476 pg/ml) were detected by an enzyme-linked immunosorbent assay in the above supernatants. Furthermore, the addition of IL-1β to the serosal side caused a potent secretory effect (ΔIsc, 80 μA · cm⁻²; P < 0.01). These results show that macrophages stimulated with toxin A release an ISF capable of provoking intestinal secretion. The regulation of this factor is dependent upon the activation of the G protein. In addition, prostaglandins, PAF, and TNF-α are involved in the release of the ISF. We conclude that IL-1β is probably the ISF released by macrophages in response to toxin A.     

Receiver Operating Characteristics Analyses of Food and Drug Administration-Cleared Serological Assays for Natural Rubber Latex-Specific Immunoglobulin E Antibody

Receiver operating characteristics (ROC) analyses to evaluate and compare the diagnostic accuracy of Food and Drug Administration (510K)-cleared natural rubber latex (NRL)-specific immunoglobulin E (IgE) antibody immunoassays have not been performed using well-characterized skin-testing reagents. Sera were collected from 311 subjects (131 latex puncture skin test [PST] positive and 180 PST negative). All masked, coded sera were analyzed for latex-specific IgE antibodies in the Diagnostic Products Corporation microplate AlaSTAT, HYCOR HY-TEC RAST, and Pharmacia-Upjohn CAP System RAST FEIA (CAP). Diagnostic accuracy was evaluated using GraphRoc for Windows software to construct and analyze ROC curves in relation to the subjects' PST status and the results of the immunoassays. The ROC areas under the curve (AUCs) ± standard error based on PST for the three diagnostic tests were 0.858 ± 0.024, 0.869 ± 0.024, and 0.924 ± 0.017, respectively, for AlaSTAT, CAP, and HY-TEC. The HY-TEC system had a significantly greater AUC based on PST than those observed for AlaSTAT (P < 0.05) and CAP (P < 0.05) analyses. When the diagnostic tests were probed as to the cutoffs giving maximal diagnostic efficiency compared to PST, CAP and AlaSTAT yielded values of <0.35 kU of allergen IgE (kU_A)/liter and <0.35 kU/liter while the HY-TEC assay yielded 0.11 kU/liter. The diagnostic efficiencies based on PST in our cohort at these cutoffs were 87.1, 88.1, and 88.7%, respectively. The HY-TEC assay had a significantly greater AUC than CAP and AlaSTAT using PST as a diagnostic discriminator in our cohort. When the HY-TEC system was probed at its maximally efficient cutoff (0.11 kU/liter) versus HYCOR's recommended cutoff of 0.05 kU/liter, a loss of sensitivity of 8.4% was observed with a gain in specificity of 19.5%.