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PubMed2001

Assessing excess nurse work load generated by multiresistant nosocomial bacteria in intensive care.

OBJECTIVE: To compare three methods for assessing the excess nurse work load related to recommended procedures for managing nosocomial infections (NI) due to multiresistant bacteria (MRB): two activity scores, the Omega score and the Projet de Recherche en Nursing (PRN) system, and a specific evaluation based on functional analysis of nursing procedures. SETTING: 10 beds in a medical intensive care unit (MICU). PATIENTS: Patients admitted from November 15, 1995, to June 15, 1996, were included and divided in two groups based on presence of MRB colonization or infection (MRB+ and MRB-groups). METHODS: Data were collected regarding length of stay (LOS) in days; Omega score for the entire stay; PRN score for the entire stay and per day; and time required to perform correctly four nursing procedures related to MRB NI, as evaluated specifically by the nursing staff, using a detailed functional analysis document that described all elementary nursing tasks in chronological order and all material needed to carry out those tasks. RESULTS: The LOS and total Omega and PRN scores were higher in the MRB+ group than in the MRB- group: LOS, 23 +/- 20.6 versus 12 +/- 15.3 days, (P<.001); Omega score, 164 +/- 103.4 versus 123 +/- 93.7 points (P<.001); PRN score, 3,606 +/- 3,187 versus 1,854 +/- 2,356 points (P<.001), respectively. The daily PRN score was also higher in MRB+ group (PRN, 160 +/- 25 vs 146 +/- 34 points in the MRB- group; P<.028). Four nursing procedures made necessary by MRB acquisition were identified: isolation precautions, with two levels according to whether the risk of contamination was mild-moderate or high; bathing the patient with antiseptic solution; bedpan management; and microbiological screening. The functional analysis indicated that the time needed to carry out these four procedures correctly was 245 minutes per patient per day, as compared to 85 minutes according to the PRN system. CONCLUSIONS: Our data confirm that MRB NIs are responsible for an increase in nurse work load, as estimated by LOS, Omega, and PRN scores. However, the daily excess nurse work load related directly to recommended procedures for managing MRB NIs in MICUs is underestimated by these activity scores, as compared to a specific functional analysis of nursing tasks. This may be of importance in evaluating potential links between nurse work load and MRB NIs and in determining the number of nurse hours needed to comply with infection control recommendations.

Cross Infection↗

PubMed2004

[A study on inoculum density and reproducibility of drug susceptibility testing by BACTEC MGIT 960].

OBJECTIVE: The BACTEC MGIT 960 drug susceptibility system (MGIT AST) has been recently introduced in Japan. The issue of discordant MGIT results compared with the conventionally used Ogawa method has been raised. It has been speculated that discordant results might be due to MGIT inoculum density since there is no standardization step other than dilution of growth for tubes beyond 2 days after MGIT turns out to be positive. In this study, we examined the reproducibility of the MGIT AST system. MATERIALS AND METHODS: Nineteen sputum specimens from drug-resistant and susceptible pulmonary tuberculosis patients were processed with CCE pretreatment reagent (Japan BCG), inoculated into 3 MGIT tubes, and loaded into the MGIT 960. Inocula for MGIT AST were prepared 1, 3, and 5 days after MGIT tubes became positive. Cultures on day 3 and 5 were diluted 1: 5 with saline. Ten-fold dilutions from each positive culture were plated on Middlebrook 7H11 agar plates for CFU determination. MGIT AST results were compared with those of the conventional proportion method on Ogawa egg and Vite-spectrum (Kyokuto), or Pyrazinamidase (Pzase) assay and Kyokuto PZA test. RESULTS AND CONCLUSION: A total of 15 specimens were culture positive in all 3 tubes. Four of 19 cases were removed from the analysis because of negative cultures in one or more tubes. Three of 4 culture negative cases were MDR-TB. Colony counting showed the mean CFU/ml of inocula prepared from tubes 1, 3, and 5 days after MGIT tube became positive were 3.6 x 10(6), 1.6 x 10(6), 3.1 x 10(6), respectively. There was no significant difference although the CFU range was wide (8 x 10(4)-2 x 10(7)). MGIT AST results were consistent among 3 inocula. Moreover, overall concordance rates between MGIT AST and the conventional methods were over 90% for 5 first-line antituberculosis drugs. These results indicate that the BACTEC MGIT 960 system is very useful for rapid diagnosis of drug resistant tuberculosis.

Humans↗

PubMed1999

Analysis of HIV cross-resistance to protease inhibitors using a rapid single-cycle recombinant virus assay for patients failing on combination therapies.

OBJECTIVE: To assess the patterns of HIV phenotypic cross-resistance to protease inhibitors (PI) in patients experiencing viral load rebound on combination therapy including a PI. METHODS: Phenotypic analysis of sensitivity to indinavir, nelfinavir, saquinavir, ritonavir and amprenavir was carried out using a single-cycle recombinant virus assay. Viral protease was sequenced by automated dideoxynucleotide chain termination. RESULTS: Of the 108 patients studied, 68 had received indinavir, 50 ritonavir, 25 saquinavir and eight nelfinavir. The majority (71%) had received only one PI. The incidence of cross-resistance between indinavir, nelfinavir, ritonavir and saquinavir was high (60-90%). Cross-resistance to amprenavir was less frequent (37-40%). However there was some correlation between levels of sensitivity to amprenavir and indinavir (r2 = 0.34; P < 0.01). Conversely, the correlation between levels of sensitivity to indinavir and saquinavir was poor (r2 = 0.25), particularly for patients who had not received saquinavir. The degree of cross-resistance correlated with the level of resistance and with the total number of mutations in the protease gene (P < 0.05, chi square test) but could not be significantly correlated to any one particular mutation or combination of mutations. Mutation 184V was significantly associated with cross-resistance to amprenavir, with no mutations at codon 50 observed, while mutations associated with cross-resistance to saquinavir differed according to the treatment received. CONCLUSIONS: These results suggest that, although the total number of protease mutations correlates with the degree of cross-resistance, the specific mechanisms accounting for primary resistance and for cross-resistance may be different.

Anti-HIV Agents↗

PubMed2004

Escherichia coli O157 prevalence and enumeration of aerobic bacteria, Enterobacteriaceae, and Escherichia coli O157 at various steps in commercial beef processing plants.

The effectiveness of current antimicrobial interventions used in reducing the prevalence or load of Escherichia coli O157 and indicator organisms on cattle hides and carcasses at two commercial beef processing plants was evaluated. Sponge sampling of beef cattle was performed at five locations from the initial entry of the animals to the slaughter floor to the exit of carcasses from the "hotbox" cooler. For each sample, E. coli O157 prevalence was determined and total aerobic bacteria, Enterobacteriaceae, and E. coli O157 were enumerated. E. coli O157 was found on 76% of animal hides coming into the plants, but no carcasses leaving the cooler were identified as contaminated with E. coli O157. A positive relationship was seen between the incidence of E. coli O157 in hide samples and that in preevisceration samples. Aerobic plate counts and Enterobacteriaceae counts averaged 7.8 and 6.2 log CFU/100 cm2, respectively, on hides, and 1.4 and 0.4 log CFU/100 cm2, respectively, on chilled carcasses. Aerobic plate counts and Enterobacteriaceae counts on preevisceration carcasses were significantly related to the respective levels on the corresponding hides; the carcasses of animals whose hides carried higher numbers of bacteria were more likely to carry higher numbers of bacteria. Implementation of the sampling protocol described here would allow processors to evaluate the efficacy of on-line antimicrobial interventions and allow industrywide benchmarking of hygienic practices.

Animals↗

PubMed2001

Phase 1 evaluation of a local delivery device releasing silver ions in periodontal pockets: safety, pharmacokinetics and bioavailability.

A new local delivery device (LDD) capable of releasing silver in periodontal pockets has been developed and tested pre-clinically. Silver has potent antimicrobial effects on Gram-negative periodontal pathogens with a mean in vitro minimum bactericidal concentration (MBC) < or =0.5 microg/ml. This phase 1 study assessed the safety, pharmacokinetics and bioavailability of silver ions delivered intracrevicularly with a resorbable LDD (PocketGuard) in a group of 9 volunteers affected with periodontitis. In each subject, a PLGA/PEG LDD loaded with 12% silver nitrate (w/w) was inserted in each of 4 selected pockets > or =5 mm. Serum, gingival fluid and subgingival plaque samples were evaluated before and at various time points after LDD placement for 21 days. At each time point, the concentration of silver in gingival crevicular fluid (GCF) was quantified with an Inductively Coupled Plasma-Mass Spectrometer. Subgingival plaque samples were processed for evaluation of total anaerobic and aerobic counts (CFU/ml). The maximum mean silver concentration in GCF was 1,493 +/- 709 microg/ml (range 589-2,245). It decayed exponentially with a half-life of 7.1 +/- 6.1 days (2.7-20.4). Average silver concentrations in excess of 10 microg/ml were detected in each patient for 14 days after LDD placement with the average concentration for all patients in excess of 25 microg/mL at day 21. Total anaerobic counts decreased an average of 1.7 +/- 1.9 x 10(6) CFU/ml (p= 0.0078) from baseline to day 7, indicating that the silver was biologically active. A mild increase in cervical root discoloration was observed at day 21:0.25 +/- 0.31 stain index units. Discoloration that did not resolve spontaneously could be removed at the end of the study with polishing. No systemic effects were observed. It is concluded that local silver concentrations above the MBC in serum were maintained for at least 21 days. A specific microbiologic effect was also observed.

Absorbable Implants↗

PubMedDate not supplied

Mild hypotension and body burns synergistically increase bacterial translocation in rats consistent with a "two-hit phenomenon".

Wistar rats were burned over 15% of their total body surface area and suffered hemorrhagic hypotension (mean blood pressure 80 mm Hg) 72 hours after the thermal injury (burn + hypotension group). Rats in other groups were treated in the same way except sham hypotension in burn group, sham burn in hypotension group, and sham hypotension and sham burn in control group. At the end of the study period, the mesenteric lymph nodes (MLN), liver, and spleen were cultured for translocated bacteria. The cecal bacterial burden was not affected by the insults. The number of bacteria translocated to the MLN after the thermal injury was much greater in the burn + hypotension group (156.19 +/- 42.29 colony-forming units [CFUs]/gm tissue) rather than in the other groups (5.24 +/- 8.34, 9.86 +/- 16.56, 40.21 +/- 45.8 in the control, hypotension, and burn groups, respectively). This indicates that bacterial translocation (BT) is the result of a two-hit phenomenon. The gut-MLN BT ratio, obtained by dividing the bacterial CFUs in the MLN by those in the cecum, was higher in the burn + hypotension group than in the other groups (p < 0.01). This ratio allows standardized measurements of BT to the MLN despite differences in the absolute bacterial load in the gut, and the elevated gut mesenteric lymph nodes BT ratio of the burn + hypotension group suggests that the two-hit phenomenon of BT may be the result of alterations in the gut mucosal barrier function.

Animals↗

PubMed2006

Mucosal immunization with a urease B DNA vaccine induces innate and cellular immune responses against Helicobacter pylori.

BACKGROUND: Helicobacter pylori is recognized as a major risk factor for recurrent gastroduodenal inflammatory diseases and gastric adenocarcinoma. The high prevalence of H. pylori infection worldwide, the risks of side-effects from antibiotic therapy, and increasing resistance to antibiotics are the main primers for the development of improved H. pylori vaccines. The antigenic potential of its urease enzyme, a critical virulence factor required for colonization of the gastric mucosa, has been demonstrated in animal and human studies. An important but controversial issue in H. pylori vaccine studies is the type of immune response required to control infection. A new approach in H. pylori vaccinology is the administration of DNA vaccines, which has included heat-shock protein and catalase DNA vaccines. MATERIALS AND METHODS: The H. pylori urease subunit B construct or vector alone was administered to mice via the intranasal route. Spleens and stomachs were examined on day 0 and weeks 3, 6, and 12 after immunization. Proliferation of spleen cells was assessed using the carboxyfluorescein diacetate succinimidyl ester-based flow cytometry assay and cytokine secretion from cultured spleen cells was detected by ELISA, after stimulation with the urease subunit B recombinant antigen. Total RNA was isolated from stomach and spleen tissue and the expression of beta-defensin and cytokine genes was monitored by reverse transcription followed by polymerase chain reaction (RT-PCR). Immunized mice were challenged with H. pylori and bacterial DNA quantified by TaqMan PCR. RESULTS: The urease B subunit DNA vaccine increased INF-gamma secretion and splenocyte proliferation without inducing adverse effects in the spleen. Increase in gastric beta-defensin 1 and marked induction in local IL-10 : IFN-gamma ratio up to 12 weeks post-immunization suggest a potential role for local innate immune responses in protection at the site of infection. Although significant bacterial reduction in the stomachs of urease B subunit DNA-immunized mice was observed, intermediate reduction was also noted in the vector group. Increased defensin expression and adjuvant effects of the cytosine preceding guanosine motifs may contribute to this phenomenon. Our data confirm that cytosine preceding guanosine motifs, even without coadministration with antigen, can reduce extracellular bacterial load. CONCLUSIONS: In this study, a DNA construct encoding the urease B subunit was assessed for its immune profile and its ability to reduce bacterial colonization in the murine stomach. Our studies suggest that local innate immune responses may play a greater role than previously supposed in limiting H. pylori colonization in the gastric mucosa.

Animals↗

PubMed2006

Net benefits of resistance testing directed therapy compared with standard of care in HIV-infected patients with virological failure: A meta-analysis.

BACKGROUND: We incorporated the latest available information to evaluate the net benefit of using resistance testing in HIV-infected patients with virological failure. METHODS: Meta-analysis of randomized controlled trials comparing the clinical impact of selecting antiretroviral therapy according to results of resistance testing (phenotype or genotype) or according to the standard of care. The population studied included HIV-infected patients with virological failure. The outcome measures were the proportion of patients with HIV-RNA below the detection limit, and the decline in HIV-RNA and increase in CD4 lymphocyte count at the end of follow-up (< or = 24 weeks). Clinical trials were identified through searches in MEDLINE, EMBASE and proceedings from major infectious diseases meetings. RESULTS: Eight trials including a total of 1810 patients were eligible. Therapy guided by resistance testing resulted in a higher percentage of patients with HIV-1 RNA below the detection limit at the end of follow-up (< or = 24 weeks) as compared with the standard of care (40.2% vs. 32.9%). The pooled risk ratio was 1.23; 95% CI 1.09-1.40, p = 0.0009; test for heterogeneity I(2)=0%; p = 0.46). The number needed to treat [NNT] was 13 (95% CI: 9-25). Subgroup analysis showed greater benefits in therapy guided by genotype testing with expert interpretation, when compared with standard of care (NNT: 5; 95% CI: 3-9; p = 0.06). The heterogeneity among trials for evaluating HIV-1 RNA decline and CD4 lymphocyte cell count increase made unfeasible pooling the results across studies. CONCLUSION: Genotype testing with expert interpretation showed the greatest benefit for guiding therapy in patients with HIV infection and virological failure.

Anti-HIV Agents↗

PubMed2006

Molecular characterization of Escherichia coli O157:H7 hide contamination routes: feedlot to harvest.

This study was conducted to identify the origin of Escherichia coli O157:H7 contamination on steer hides at the time of harvest. Samples were collected from the feedlot, transport trailers, and packing plant holding pens and from the colons and hides of feedlot steers. A total of 50 hide samples were positive for E. coli O157:H7 in two geographical locations: the Midwest (25 positive hides) and Southwest (25 positive hides). Hide samples were screened, and the presence of E. coli O157: H7 was confirmed. E. coli O157:H7 isolates were fingerprinted by pulsed-field gel electrophoresis and subjected to multiplex PCR procedures for amplification of E. coli O157:H7 genes stx1, stx2, eaeA, fliC, rfbEO157, and hlyA. Feedlot water trough, pen floor, feed bunk, loading chute, truck trailer side wall and floor, packing plant holding pen floor and side rail, and packing plant cattle drinking water samples were positive for E. coli O157:H7. Pulsed-field gel electrophoresis banding patterns were analyzed after classifying isolates according to the marker genes present and according to packing plant. In this study, hide samples positive for E. coli O157:H7 were traced to other E. coli O157:H7-positive hide, colon, feedlot pen floor fecal, packing plant holding pen drinking water, and transport trailer side wall samples. Links were found between packing plant side rails, feedlot loading chutes, and feedlot pens and between truck trailer, different feedlots, and colons of multiple cattle. This study is the first in which genotypic matches have been made between E. coli O157:H7 isolates obtained from transport trailer side walls and those from cattle hide samples within the packing plant.

Animal Husbandry↗

PubMed1991

Incidence of molds and mycotoxins in commercial animal feed mills in seven midwestern states, 1988-1989.

A total of 82 feed manufacturers located within seven midwestern states (Iowa, Nebraska, Minnesota, Illinois, Indiana, Ohio, Michigan) participated in a survey of mold and mycotoxin contamination of corn. Samples were submitted from a composite of the grading samples taken from each incoming load of corn. The survey was initiated in July 1988. During the 12-mo period, moisture content of the corn samples upon receipt at the laboratory ranged from 10.5 to 13.3%. The greatest variation occurred in the springtime. Iowa's corn samples were driest (11.2%), and samples submitted from Ohio were wettest (12.8%). Mold counts averaged 2.63 x 10(4) per gram during the year. The predominant mold found was Fusarium sp. Samples were checked by black light and averaged 25.4% positive during the period. When assayed for mycotoxins, 19.5% of the samples were positive for at least one of the following: aflatoxin, zearalenone, T2 toxin and deoxynivalenol (vomitoxin). Aflatoxin and T2 toxin made up the majority of these samples containing toxin. The highest incidence of mycotoxin-contaminated corn (48%) occurred in samples submitted in July of 1988. Over the 12-mo period, the highest mycotoxin contamination occurred in Iowa, Illinois and Michigan. When samples were subjected to 90% relative humidity and 32 degrees C, an average of 3.9 d was required for mold growth to appear. After incubation, 24.7% of the samples contained one of the four toxins. The data indicate that mold and mycotoxin contamination of mixed samples of corn is widespread, even in the midwestern corn belt of the U.S.

Aflatoxins↗

PubMed2005

Predictive value of drug levels, HIV genotyping, and the genotypic inhibitory quotient (GIQ) on response to saquinavir/ritonavir in antiretroviral-experienced HIV-infected patients.

Plasma levels of HIV protease inhibitors (PI) are often close to IC50 values of wild-type viruses when administered without ritonavir boosting. The impact of drug levels, resistance mutations, and the genotypic inhibitory quotient (GIQ) were examined on the response to saquinavir/ritonavir (SQV/r)-based salvage therapy. Patients with prior exposure to PI other than SQV and currently failing virologically were recruited prospectively in a multicenter trial. All patients began SQV/r 1000/100 mg bid plus another two antiretrovirals. A total of 139 patients was enrolled. At month 12, virological response (VR), defined as plasma HIV-RNA decline >1 log, was recorded in 68.2% of patients on treatment (41.7% in the intent-to-treat analysis). The median baseline number of protease resistance mutations was three. The VR was significantly lower in patients with >5 protease resistance mutations and in those with plasma SQV Cmin<0.1 microg/ml. At week 48, the VR was seen in 77.1% of patients with a GIQ>0.04 compared to 18.2% of those with a lower GIQ (P=0.001). In the multivariate analysis, 0.1 microg/ml were independently associated with VR. Interestingly, drug levels had their highest predictive value of the VR at week 12, since low SQV plasma levels often permitted ruling out poorly adherent patients. In contrast, the number of protease resistance mutations had the highest impact on the VR at week 24, suggesting that for those taking the drugs, the VR is highly dependent of the presence of resistance mutations. At any time, nevertheless, the GIQ provided the most accurate prediction of the VR.

Antiretroviral Therapy, Highly Active↗

PubMed2003

The influence of Lentinus edodes (Shiitake mushroom) preparations on bacteriological and morphological aspects of the small intestine in piglets.

Among substances intended to replace growth promoting antibiotics in pig nutrition, non-digestible oligosaccharides or polysaccharides could be potential alternative compounds. Therefore, the influence of beta-1,3-1,6 glucans on bacteriological, biochemical and morphological aspects of the small intestine in weaned piglets was investigated. As sources of beta-glucans, Lentinan (extract of Lentinus edodes mycelium) or dried L. edodes mycelium were added to the diet. Four homogenous groups of 5 newly weaned piglets (4 weeks of age) received one of four diets: control diet (C), C supplemented with Avilamycin (50 mg/kg, positive control), C supplemented with 0.1% of Lentinan and C supplemented with 5% of dried L. edodes mycelium powder. A first group of 10 piglets was euthanized after 11 days and the remaining 10 on day 12 of the experiment. The gastrointestinal tract was divided in segments and samples taken from digesta (stomach, proximal and distal jejunum, caecum), mucosal scrapings (jejunum) and ring shaped tissue samples (1 cm) of proximal and distal jejunum. Bacterial counts were made with digesta and mucosal samples, and short-chain fatty acids (SCFA), lactic acid and ammonia concentrations were determined. Tissue samples of both jejunal sites were embedded in paraffin wax for morphometrical (villus length, crypt depth) and histological observations (numbers of intraepithelial lymphocytes (IEL), goblet cells, apoptotic enterocytes on villi, mitotic cells in crypts). Only the diet containing 5% of dried L. edodes consistently resulted in lower viable counts (ca. 1-2 log10 CFU) of total bacteria, E. coli, streptococci and lactic acid bacteria, and luminal and mucosal effects agreed very well. With this diet, acetate and butyrate concentrations in the distal jejunum were doubled, which is favourable in view of the trophic effect on enterocytes and colonocytes. Villus length (V) was increased with both diets containing beta-glucans while crypt depth (C) was not altered, but V/C was higher. IEL counts were decreased by both diets although bacterial numbers, which is only one parameter of bacterial load, were only diminished with the L. edodes feed. The three supplemented feeds lowered the number of apoptotic enterocytes on the villi, but these numbers were very low (control diet : 44 cells per 100 villi), making clear interpretation difficult. The mitotic index was slightly lower with the L. edodes feed, although not statistically significant. Decreased viable counts observed with the latter diet is a favourable effect as it is accepted that a lower bacterial load causes lower turnover rates of the intestinal epithelial cells, while there is also less competition for specific substrates. A higher V/C ratio, a smaller number of IEL in the epithelium and a lower apoptotic index also indicate slower turnover rate of the mucosa when Lentinan and L. edodes diets were fed. The inconsistent effects observed with Lentinan were probably due to the low amount added to the diet. It should be taken into account that the influence of L. edodes mycelium powder was more likely due to the presence of antibacterial compounds (eg. lenthionine, lentinamycin, terpenoids, polyphenols), rather than to an immunostimulating action of beta-glucans with increased release of IgA onto the mucosa surface.

Adaptation, Physiological↗

PubMed1999

Longitudinal study of dental implants in a periodontally compromised population.

BACKGROUND: The purpose of this longitudinal study was to determine the clinical status and the composition of the subgingival microbiota of dental implants and natural teeth in patients with a history of periodontitis. METHODS: Twenty-five partially edentulous patients treated for moderate to advanced adult periodontitis and having a total of 42 implants participated in this 3-year study. The assessment of clinical status was done 1, 2, and 3 years after prosthetic loading (T1, T2, and T3, respectively). Clinical parameters evaluated included probing depth (PD), clinical attachment level (CAL), gingival index (GI), and plaque index (PI). The subgingival microbiota at peri-implant and periodontal sites were analyzed at T1 and T2. RESULTS: No significant difference in clinical parameters between implants and teeth and within the 2 groups between different time points was observed through the study. PD and CAL measurements of sampled periodontal and peri-implant sites did not show any statistically significant difference through the study and between the 2 groups. PI of sampled periodontal sites showed a statistically significant improvement during the study. From the morphological observation of the subgingival microbiota, a significant difference in the composition of motile rods between implants and teeth was found at T1. There were no differences detected in the subgingival microbiota, culturally identified at peri-implant and periodontal sites for the duration of the study. CONCLUSIONS: In conclusion, implants were colonized by the indigenous periodontal microbiota and were well maintained in patients with a history of periodontitis. No significant association between progressing or non-progressing periodontal or peri-implant sampled sites in terms of loss of attachment and infection with at least one of the searched periodontal pathogens was found, suggesting that the presence of putative periodontopathogens at peri-implant and periodontal sites may not be associated with future attachment loss or implant failure.

Adult↗

PubMed1999

A randomized trial comparing regimens of four reverse transcriptase inhibitors given together or cyclically in HIV-1 infection--the Quattro Trial. Quattro Steering Committee.

OBJECTIVES: To assess if cyclical therapy with four reverse transcriptase inhibitors is less toxic and as active, based on virological and immunological markers, as the same four drugs given together over a 64-week period. METHODS: An open randomized trial comparing concurrent therapy (T4) with zidovudine, lamivudine, loviride and zalcitabine with the same four drugs given cyclically each for 8 weeks (C4) and with concurrent zidovudine and lamivudine (T2), all given for a total of 64 weeks. The primary endpoint was the change in plasma HIV RNA level from baseline at weeks 32 and 64. Phenotypic and genotypic resistance, CD4+ cell counts, and clinical and laboratory assessments of safety were also compared. Patients were followed for up to a further 32 weeks beyond 64 weeks. Eligible patients had CD4+ cell counts between 50 and 350 x 10(6)/l and no prior antiretroviral therapy. RESULTS: One hundred individuals (34 T4, 34 C4, 32 T2) were recruited between 31 July 1995 and 11 July 1996, of whom 22 had AIDS; the mean (SD) HIV RNA at baseline was 4.9 (0.7) log10 copies/ml and the median (interquartile range) CD4+ cell count was 170 (100-260) x 10(6)/l. A total of 28 T4, 19 C4 and 26 T2 participants were still on the allocated regimen at week 64. A new AIDS event or death was reported in three T4, seven C4 and five T2 participants (P = 0.7). Serious adverse events that were likely to be drug related were observed in three T4, one C4 and four T2 participants. The reduction from baseline in HIV-1 RNA (log10 copies/ml) was greatest in the T4 arm; at 32 weeks the mean reduction (SD) was 1.45 (0.72), 0.42 (0.45) and 1.05 (0.70) in T4, C4 and T2 respectively (global P = 0.0001) and at week 64 1.24 (0.86), 0.73 (0.91) and 0.78 (0.55) respectively (P = 0.02). The pattern of CD4+ change mirrored the changes in HIV RNA. Very few mutations associated with resistance to loviride or zalcitabine were seen. The mutation at codon 215 associated with zidovudine resistance was detected (> 5% of population mutant) in 11 out of 24 T4 participants compared with three out of 21 C4 and 11 out of 20 T2 participants at week 64 (P = 0.02). Further assays of viral resistance including phenotypic assays are ongoing and results will be reported separately.

Adolescent↗

PubMed2002

Murine model of BCG lung infection: dynamics of lymphocyte subpopulations in lung interstitium and tracheal lymph nodes.

C57Bl/6 female mice were infected with an intrapulmonary dose of 2.5 x 10(4) BCG (Mycobacterium bovis Bacillus Calmette-Guerin). Lymphocyte populations in lung interstitium and lung-associated tracheal lymph nodes (LN) were examined at 1, 2, 4, 5, 6, 8 and 12 weeks after infection. BCG load in lungs peaked between 4-6 weeks post-infection and declined to very low levels by the 12th week of infection. Lung leukocytes were obtained over the course of infection by enzyme digestion of lung tissue followed by centrifugation over Percoll discontinuous density gradients. By 4 to 6 weeks after infection, numbers of lung leukocytes had more than doubled but the proportions of lymphocytes (about 70%), macrophages (about 18%) and granulocytes (about 12%) remained essentially unaltered. Flow cytometric studies indicated: (i) the total number of CD3+ T cells in lungs increased by 3-fold relative to uninfected controls at 5 to 6 weeks post-infection, but the relative proportions of CD4 and CD8 cells within the T cell compartment remained unaltered; (ii) relative proportion of NK cells in lungs declined by 30% but the total number of NK cells (NK1.1+) per lung increased by about 50%, 5-6 weeks post infection; (iii) tracheal LN underwent marked increase in size and cell recoveries (6-10-fold increase) beginning 4 weeks after infection. While both T and B cells contributed to the increase in cell recoveries from infected tracheal LNs, the T/B ratio declined significantly but CD4/CD8 ratio remained unaltered. In control mice, IFNgamma producing non-T cells outnumbered T cells producing IFNgamma. However, as the adaptive response to infection evolves, marked increase occur in the number of IFNgamma producing T cells, but not NK cells in the lungs. Thus, T cells are the primary cell type responsible for the adaptive IFNgamma response to pulmonary BCG infection. Few T cells in tracheal LN of BCG infected mice produce IFNgamma, suggesting that maturational changes associated with migration to the lungs or residence in the lungs enhance the capability of some T cells to produce this cytokine.

Animals↗

PubMed1999

Clinical cross-resistance between the HIV-1 protease inhibitors saquinavir and indinavir and correlations with genotypic mutations.

OBJECTIVES: To determine the clinical efficacy of the HIV-1 protease inhibitor indinavir (IDV) in saquinavir (SQV)-experienced patients and delineate the developing drug-resistance patterns. DESIGN: Open-label prospective clinical trial. SETTING: University hospital research center. PATIENTS: Ten patients who had completed a SQV monotherapy study in which they had received SQV at a dose of 3600 or 7200 mg daily (two and fourfold the standard dose). INTERVENTIONS: At enrollment patients received IDV for 4 weeks as monotherapy, after which zidovudine (ZDV) and lamivudine (3TC) were added to their drug regimen. Patients then received combination therapy (IDV-ZDV-3TC) for an additional 20 weeks to complete a total of 24 weeks of therapy. MAIN OUTCOME MEASURES: Plasma HIV RNA viral load and CD4+ T-cell counts were monitored. Sequencing of the HIV protease gene was performed to determine the development of resistance mutations. Plasma samples for sequencing were taken before initial SQV therapy, after SQV therapy before starting IDV, and after 24 weeks of IDV therapy. RESULTS: The average duration of high-dose SQV before starting IDV was 58+/-29.2 weeks. A 0.58 log10 RNA copies/ml increase was noted during the 3-week washout phase followed by a mean reduction in plasma HIV RNA viral load of 1.2 log10 RNA copies/ml after 4 weeks of IDV. After the addition of ZDV and 3TC at week 4, HIV RNA continued to fall reaching a mean reduction of 1.96 log10 RNA copies/ml at week 24. Plasma HIV RNA was below 400 RNA copies/ml in six out of nine patients at week 24. CD4+ T-cell counts showed a gradual rise from 328 x 10(6)/l to 453 x 10(6)/l by week 24. SQV therapy had resulted in multiple mutations in the protease gene. Six of the patients had developed five or more mutations: L90M in two, G48V in four (of which three also contained L101), and V82A in three. Patients in whom plasma HIV RNA was not durably suppressed by subsequent IDV combination therapy developed multiple (up to four) additional mutations within 24 weeks, including codons 54, 82 and 93 amongst others. No clear correlation was found between the mutations that had developed in individual patients after SQV and the subsequent efficacy of IDV. CONCLUSION: Prolonged use of SQV at potent doses in the presence of elevated viral load levels resulted in the development of multiple resistance mutations. Individual resistance patterns varied greatly between patients, as did their virological response to therapy. Resistance assays may be useful in identifying which patients will benefit from salvage therapy with a second protease inhibitor.

CD4 Lymphocyte Count↗

PubMed2026

Whole-genome surveillance supports hazard profiling of Escherichia coli lineages in recycled water treatment systems.

UNLABELLED: The use of treated wastewater is increasingly important for sustainable water management under a changing climate, yet conventional monitoring based on Escherichia coli enumeration provides limited insight into strain diversity and associated public health hazards. Here, we applied longitudinal whole-genome sequencing (WGS) to 180 E. coli isolates collected across the treatment continuum of a recycled water facility, from influent to final effluent. Genomic analysis revealed extensive strain-level heterogeneity, comprising 88 sequence types across eight phylogroups, with greater diversity in influent than in treated effluent. Phylogenetic comparisons with contextual Australian genomes indicated clustering with strains associated with companion animals, wild birds, humans, and livestock, suggesting multiple potential source reservoirs rather than a single dominant origin, although source contributions were not definitive. Despite a >90% reduction in total E. coli loads, isolates recovered from upstream and downstream stages exhibited broadly comparable virulence factor and antimicrobial resistance gene (ARG) profiles, suggesting that, within the cultured isolate collection, reductions in abundance exceeded shifts in genomic composition. To assess operational relevance, we prototyped a genomics-informed hazard framework integrating virulence determinants, ARGs, plasmid-associated mobility, and reuse-specific exposure context. Using this framework, 92.8% of isolates were classified as low hazard, and 7.2% as moderate hazard, with no isolates meeting criteria for high or critical hazard classifications. These findings demonstrate that genomic profiling of indicator organisms can reveal population structure and hazard heterogeneity not captured by conventional enumeration alone, and can provide a practical basis for incorporating genomic information into hazard-informed monitoring of recycled water systems. IMPORTANCE: Routine recycled water monitoring relies largely on culture-based E. coli counts, which indicate regulatory compliance but provide limited insight into strain diversity, persistence, and genomic characteristics relevant to public health. Using longitudinal whole-genome sequencing, we show that genetically distinct E. coli lineages, including isolates carrying combinations of virulence and antimicrobial resistance determinants, can persist through advanced treatment despite substantial reductions in overall E. coli loads. While most isolates were classified as low genomic hazard and no high- or critical-hazard isolates were detected, these findings demonstrate that conventional enumeration alone cannot distinguish between genetically diverse lineages with differing hazard potential in highly treated systems. By integrating genomic data into a hazard classification framework, this study demonstrates an applied approach to contextualize E. coli detections and distinguish low-risk background populations from isolates with elevated genomic hazard profiles. This work supports the use of genomic profiling of indicator organisms to improve surveillance, inform treatment performance assessment, and enable more risk-based management of recycled water systems.

Escherichia coli↗

PubMed2001

Absence of rapid selection for acyclovir or penciclovir resistance following suboptimal oral prodrug therapy of HSV-infected mice.

BACKGROUND: Acyclovir (ACV) resistant herpes simplex virus (HSV) isolates can be readily selected in animal infection models receiving suboptimal ACV treatment, however no comparative studies of the emergence of resistance following suboptimal treatment with valacyclovir (VCV) or famciclovir (FCV), the prodrugs of acyclovir and penciclovir, respectively, have been reported. METHODS: Mice (n = 30) were infected with HSV type 1 or 2 in the ear pinnae and administered oral prodrugs at one fifth a dose previously shown to be effective. To select and amplify drug-resistant HSV, a total of seven consecutive in vivo passages with suboptimal treatment were performed for each virus sample and progeny virus from each passage was characterized by the plaque reduction (PRA) and plating efficiency assays (PEA). RESULTS: No drug-resistant HSV-2 and only a single drug-resistant HSV-1 variant were identified. Virus recovered from the first three sequential passages of this HSV-1 sample was susceptible by PRA, although the proportion of resistant virus recovered gradually increased upon passage. The resistant HSV-1 phenotype was confirmed by PRA after four sequential passages in mice. Unexpectedly, this in vivo-selected drug-resistant HSV-1 failed to yield an infection completely refractory to treatment in subsequent passages. CONCLUSIONS: Sub-optimal therapy of immunocompetent mice with either VCV or FCV did not readily select for HSV-mutants resistant to either ACV or PCV, suggesting that selection of resistance with either prodrug remains difficult using this system. Futhermore, this study suggests that the PEA may represent a useful adjunct to the PRA for monitoring alterations in the proportion of drug-resistant virus even when no change in IC50 is apparent.

Acyclovir↗