Integration of individualized and population-level molecular epidemiology data to model COVID-19 outcomes.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants with enhanced transmissibility and immune escape have emerged periodically throughout the coronavirus disease 2019 (COVID-19) pandemic, but the impact of these variants on disease severity has remained unclear. In this single-center, retrospective cohort study, we examined the association between SARS-CoV-2 clade and patient outcome over a two-year period in Chicago, Illinois. Between March 2020 and March 2022, 14,252 residual diagnostic specimens were collected from SARS-CoV-2-positive inpatients and outpatients alongside linked clinical and demographic metadata, of which 2,114 were processed for viral whole-genome sequencing. When controlling for patient demographics and vaccination status, several viral clades were associated with risk for hospitalization, but this association was negated by the inclusion of population-level confounders, including case count, sampling bias, and shifting standards of care. These data highlight the importance of integrating non-virological factors into disease severity and outcome models for the accurate assessment of patient risk.

Development of a workflow for the detection of vancomycin-resistant Enterococcus faecium and Enterococcus faecalis from rectal swabs using the spectra VRE medium.

BACKGROUND: Spectra™ VRE agar (Remel, Lenexa, KS) is a chromogenic agar that is FDA approved for screening patients for VRE colonization. The package insert recommends confirming isolates with identification and susceptibility testing, but confirming every culture delays time to result. Given the agar's historic high specificity for E. faecium isolates, we theorized the agar could be utilized as a stand-alone screening to minimize reagents and time. AIM: Our laboratory sought to develop a workflow to optimize the use of the medium. METHODS: We plated 3,815 rectal swabs to the Spectra VRE agar and compared results to traditional identification and susceptibility testing. RESULTS: Dark blue or purple colonies on the agar demonstrated a sensitivity of 98% and specificity of 85% for detection of VRE faecium, but light blue colonies were significantly less specific for E. faecalis. CONCLUSIONS: We streamlined our workflow to accept dark blue or purple colonies as VRE faecium and plan to perform additional testing only on light blue colonies. Interestingly, higher quantity of growth increased the accuracy of the agar. In the future, growth quantity may be used to further streamline the workflow once more data is obtained.

Machine Learning To Stratify Methicillin-Resistant Staphylococcus aureus Risk among Hospitalized Patients with Community-Acquired Pneumonia.

Methicillin-resistant Staphylococcus aureus (MRSA) is an uncommon but serious cause of community-acquired pneumonia (CAP). A lack of validated MRSA CAP risk factors can result in overuse of empirical broad-spectrum antibiotics. We sought to develop robust models predicting the risk of MRSA CAP using machine learning using a population-based sample of hospitalized patients with CAP admitted to either a tertiary academic center or a community teaching hospital. Data were evaluated using a machine learning approach. Cases were CAP patients with MRSA isolated from blood or respiratory cultures within 72 h of admission; controls did not have MRSA CAP. The Classification Tree Analysis algorithm was used for model development. Model predictions were evaluated in sensitivity analyses. A total of 21 of 1,823 patients (1.2%) developed MRSA within 72 h of admission. MRSA risk was higher among patients admitted to the intensive care unit (ICU) in the first 24 h who required mechanical ventilation than among ICU patients who did not require ventilatory support (odds ratio [OR], 8.3; 95% confidence interval [CI], 2.4 to 32). MRSA risk was lower among patients admitted to ward units than among those admitted to the ICU (OR, 0.21; 95% CI, 0.07 to 0.56) and lower among ICU patients without a history of antibiotic use in the last 90 days than among ICU patients with antibiotic use in the last 90 days (OR, 0.03; 95% CI, 0.002 to 0.59). The final machine learning model was highly accurate (receiver operating characteristic [ROC] area = 0.775) in training and jackknife validity analyses. We identified a relatively simple machine learning model that predicted MRSA risk in hospitalized patients with CAP within 72 h postadmission.

Vancomycin-resistant Enterococcus outbreak in a pre- and post-cardiothoracic transplant population: Impact of discontinuing multidrug-resistant organism surveillance during the coronavirus disease 2019 pandemic.

INTRODUCTION: Many institutions suspended surveillance and contact precautions for multidrug-resistant organisms (MDROs) at the outset of the coronavirus disease 2019 (COVID-19) pandemic due to a lack of resources. Once our institution reinstated surveillance in September 2020, a vancomycin-resistant Enterococcus (VRE) faecium outbreak was detected in the cardiothoracic transplant units, a population in which we had not previously detected outbreaks. METHODS: An outbreak investigation was conducted using pulsed-field gel electrophoresis for strain typing and electronic medical record review to determine the clinical characteristics of involved patients. The infection prevention (IP) team convened a multidisciplinary process improvement team comprised of IP, cardiothoracic transplant nursing and medical leadership, environmental services, and the microbiology laboratory. RESULTS: Between December 2020 and March 2021, the outbreak involved thirteen patients in the cardiothoracic transplant units, four index cases, and nine transmissions. Of the 13, seven (54%) were on the transplant service, including heart and lung transplant recipients, patients with ventricular assist devices, and a patient on extracorporeal membrane oxygenation as a bridge to lung transplantation. Four of 13 (31%) developed a clinical infection. DISCUSSION: Cardiothoracic surgery/transplant patients may have a similar risk for VRE-associated morbidity as abdominal solid organ transplant and stem cell transplant patients, highlighting the need for aggressive outbreak management when VRE transmission is detected. Our experience demonstrates an unintended consequence of discontinuing MDRO surveillance in this population and highlights a need for education, monitoring, and reinforcement of foundational infection prevention measures to ensure optimal outcomes.

Impact of early antimicrobial stewardship intervention in patients with positive blood cultures: results from a randomized comparative study.

BACKGROUND: Antimicrobial stewardship intervention (ASI) appears to be necessary to realize the full benefits of rapid diagnostic technologies in clinical practice. This study aimed to compare clinical outcomes between early ASI paired with matrix-associated laser desorption ionization-time of flight mass spectrometry (MALDI-TOF) compared with MALDI-TOF with standard of care (SOC) reporting in patients with positive blood cultures. METHODS: Adult patients with positive blood cultures and organism speciation via MALDI-TOF admitted between February 2015 and September 2015 were randomized to ASI or SOC in a 1:1 fashion. Patients admitted for at least 48 h following positive culture were included in analyses. ASI was defined as a clinical assessment by a stewardship team member with non-binding treatment recommendations offered to the primary team. The primary outcome was time to definitive therapy. Secondary outcomes included post-culture length of stay (LOS), time to first change in antibiotics, and in-hospital mortality. RESULTS: In total, 149 patients were included in the analyses (76 in the ASI group and 73 in the SOC group). ASI and SOC arms did not differ according to age, sex, comorbidities or severity of illness. Gram-positive organisms were common in both SOC and ASI arms (74.0 vs. 61.8%, P=0.11). Time to definitive therapy was reduced, on average, by 30.3 h in the ASI group (71.6 vs. 41.3 h, P=0.01). Hospital LOS following the first positive blood culture was significantly shorter in the ASI group (8.7 vs. 11.2 days, P=0.049). CONCLUSIONS: ASI combined with MALDI-TOF reduced the time to definitive therapy and time to first change in antibiotics, and was associated with a shorter post-culture LOS.

Diagnostic performance of Ion 16S metagenomics kit and Ion reporter metagenomics workflow for bacterial pathogen detection in culture-negative clinical specimens from sterile sources.

PCR-based deep sequencing of 16S rRNA gene allows for detection of a wide array of bacterial pathogens in culture-negative specimens. Ion 16S metagenomics kit and Ion Reporter metagenomics workflow (Ion 16S mNGS) provides an end-to-end solution with integrated workflow. Ninety-eight clinical samples with the diagnosis generated with 16S rRNA gene PCR/chain termination (Sanger) sequencing (16S CS) was used to assess the performance of Ion 16S mNGS. Compared to species level detection of 16S CS, the Ion 16S mNGS had 88% sensitivity and 76% specificity. When accounting for genus level of detection, the Ion 16S mNGS had 100% sensitivity. Notably, Ion 16S mNGS generated diagnosis in 13% of 16S CS and culture-negative samples. In addition, Ion 16S mNGS had the advantage of detecting more than 1 pathogen in 16S CS positive samples. We showed that the workflow had high reproducibility.

Optimizing a real-time PCR assay for rapid detection of Candida auris in nasal and axillary/groin samples.

Introduction. Candida auris is an emerging fungal pathogen. The organism can cause invasive infections associated with high mortality, has been implicated in outbreaks in healthcare settings and is frequently resistant to multiple antifungal agents, making it a significant challenge to infection prevention and patient treatment.Aim. To implement a real-time PCR assay for detection of C. auris in patient surveillance samples collected with the Copan Liquid Amies elution swab (ESwab) collection and transport system.Methodology. We optimized a real-time PCR testing procedure based on the sample collection device used in our institution.Results . ESwab transport medium was strongly inhibitory to the real-time PCR. Removing the medium with centrifugation, followed by suspending the pellet in PBS-BSA buffer (concentration 1 %), sufficiently eliminated the inhibition. The manual sample preparation method, freeze-thaw followed by mechanical disruption, allowed the detection of C. auris at the lowest cell concentration.Conclusion . The optimized procedure was used to test 1414 patient surveillance samples. The real-time PCR detected all culture-positive samples with 100 % sensitivity and 100 % specificity.

The intersection of hand hygiene, infusion pump contamination, and high alarm volume in the health care environment.

BACKGROUND: Researchers have found that lack of hand hygiene and environmental contamination are sources of infection transmission in the health care environment. One factor that may lead to lack of hand hygiene is alarm fatigue, the sensory overload that results when clinicians are exposed to an excessive number of alarms, causing them to silence alarms without taking proper precautions. In this study, we report hand hygiene compliance and infusion pump contamination in the context of infusion pump alarm prevalence. METHODS: Health care worker hand hygiene audits were conducted to determine percent compliance. Cultures were obtained from infusion pumps to determine environmental contamination. The frequency of alarms from August 4, 2019 to September 7, 2019 was determined. RESULTS: Hand hygiene compliance ranged from 50% to 87%. Pump contamination ranged from 20% to 70% per unit. A total of 116, 872 infusion pump alarms sounded in the hospital. DISCUSSION: Pumps were contaminated primarily with skin flora. This was demonstrated in the context of poor hand hygiene compliance and a high number of alarms, indicative of alarm fatigue. CONCLUSIONS: The intersection of a high prevalence of infusion pump alarms and poor hand hygiene resulting in bacterial contamination of pumps could be a source of health care-associated infection transmission for patients.

Development of a protocol for detection of SARS-CoV-2 in sputum and endotracheal aspirates using Cepheid Xpert Xpress SARS-CoV-2.

Sputum and endotracheal aspirates (ETs) are not among the vendor-approved specimens for the Cepheid Xpert SARS-CoV-2 assay. However, they are the common lower respiratory tract specimens submitted for laboratory diagnosis. Testing for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in lower respiratory tract samples is required for the discharge of patients from coronavirus disease (COVID) units at some institutions. We developed a protocol used for testing unliquified viscous sputum or tracheal aspirate with the Cepheid Xpert SARS-CoV-2 assay.

Evaluation of the impact of the Accelerate Pheno™ system on time to result for differing antimicrobial stewardship intervention models in patients with gram-negative bloodstream infections.

BACKGROUND: Initiating early effective antimicrobial therapy is the most important intervention demonstrated to decrease mortality in patients with gram-negative bacteremia with sepsis. Rapid MIC-based susceptibility results make it possible to optimize antimicrobial use through both escalation and de-escalation. METHOD: We prospectively evaluated the performance of the Accelerate Pheno™ system (AXDX) for identification and susceptibility testing of gram-negative species and compared the time to result between AXDX and routine standard of care (SOC) using 82 patient samples and 18 challenge organisms with various confirmed resistance mechanisms. The potential impact of AXDX on time to antimicrobial optimization was investigated with various simulated antimicrobial stewardship (ASTEW) intervention models. RESULTS: The overall positive and negative percent agreement of AXDX for identification were 100 and 99.9%, respectively. Compared to VITEK® 2, the overall essential agreement was 96.1% and categorical agreement was 95.4%. No very major or major errors were detected. AXDX reduced the time to identification by an average of 11.8 h and time to susceptibility by an average of 36.7 h. In 27 patients evaluated for potential clinical impact of AXDX on antimicrobial optimization, 18 (67%) patients could potentially have had therapy optimized sooner with an average of 18.1 h reduction in time to optimal therapy. CONCLUSION: Utilization of AXDX coupled with simulated ASTEW intervention notification substantially shortened the time to potential antimicrobial optimization in this cohort of patients with gram-negative bacteremia. This improvement in time occurred when ASTEW support was limited to an 8-h coverage model.

Rapid Detection of Methicillin-Resistant Staphylococcus aureus in BAL: A Pilot Randomized Controlled Trial.

BACKGROUND: Guidelines recommend empirical vancomycin or linezolid for patients with suspected pneumonia at risk for methicillin-resistant Staphylococcus aureus (MRSA). Unneeded vancomycin or linezolid use may unnecessarily alter host flora and expose patients to toxicity. We therefore sought to determine if rapid testing for MRSA in BAL can safely decrease use of vancomycin or linezolid for suspected MRSA pneumonia. METHODS: Operating characteristics of the assay were initially validated against culture on residual BAL. A prospective, unblinded, randomized clinical trial to assess the effect of antibiotic management made on the basis of rapid diagnostic testing (RDT) compared with usual care was subsequently conducted, with primary outcome of duration of vancomycin or linezolid administration. Secondary end points focused on safety. RESULTS: Sensitivity of RPCR was 95.7%, with a negative likelihood ratio of 0.04 for MRSA. The clinical trial randomized 45 patients: 22 to antibiotic management made on the basis of RDT and 23 to usual care. Duration of vancomycin or linezolid administration was significantly reduced in the intervention group (32 h [interquartile range, 22-48] vs 72 h [interquartile range, 50-113], P < .001). Proportions with complications and length of stay trended lower in the intervention group. Hospital mortality was 13.6% in the intervention group and 39.1% for usual care (95% CI of difference, -3.3 to 50.3, P = .06). Standardized mortality ratio was 0.48 for the intervention group and 1.18 for usual care. CONCLUSIONS: A highly sensitive BAL RDT for MRSA significantly reduced use of vancomycin and linezolid in ventilated patients with suspected pneumonia. Management made on the basis of RDT had no adverse effects, with a trend to lower hospital mortality. TRIAL REGISTRY: ClinicalTrials.gov; No. NCT02660554; URL: www.clinicaltrials.gov.

Factors contributing to vancomycin-resistant Enterococcus spp. horizontal transmission events: exploration of the role of antibacterial consumption.

BACKGROUND: The relationship between antibiotic consumption and resistance is relatively well defined at the population/ecologic level. Increases in antimicrobial consumption correlate with increased antibiotic resistance for clinical and surveillance isolates. However, the impact of antimicrobial consumption on nosocomial transmission of resistant bacteria is less well defined. This study explores the association between antimicrobial consumption, hand hygiene, and horizontal resistant organism transmission. METHODS: A retrospective cohort pilot study was conducted. Vancomycin-resistant Enterococcus spp. (VRE) horizontal transmission events during a 2-year period were identified. VRE transmission events were defined as isolation of genetically similar VRE strain-types (determined using pulsed field gel electrophoresis) from patients who were temporally and geographically co-localized within our hospital. The Centers for Disease Control and Prevention Antimicrobial Use and Resistance Module was utilized to collect antibacterial consumption data of commonly utilized agents. Hand hygiene was quantified using floor-by-floor peer audit data. Regression techniques were employed to assess population-level relationships between study variables and transmission events. RESULTS: One hundred nineteen transmission events were identified. Hand hygiene estimates were homogeneous and did not correlate with VRE transmission rates. Stepwise-multivariate linear regression revealed that aztreonam consumption was associated with a lower rate of transmissions in the medical intensive care unit (P=0.031), and carbapenem consumption was associated with a higher rate of VRE transmission events on one of two oncology floors (P=0.033). DISCUSSION/CONCLUSION: Consumption of aztreonam and carbapenems was associated with VRE horizontal transmission rates. Further studies are necessary to identify other associations and elucidate the full clinical significance of this finding.

Unacceptably high error rates in Vitek 2 testing of cefepime susceptibility in extended-spectrum-ß-lactamase-producing Escherichia coli.

While a lack of concordance is known between gold standard MIC determinations and Vitek 2, the magnitude of the discrepancy and its impact on treatment decisions for extended-spectrum-ß-lactamase (ESBL)-producing Escherichia coli are not. Clinical isolates of ESBL-producing E. coli were collected from blood, tissue, and body fluid samples from January 2003 to July 2009. Resistance genotypes were identified by PCR. Primary analyses evaluated the discordance between Vitek 2 and gold standard methods using cefepime susceptibility breakpoint cutoff values of 8, 4, and 2 µg/ml. The discrepancies in MICs between the methods were classified per convention as very major, major, and minor errors. Sensitivity, specificity, and positive and negative predictive values for susceptibility classifications were calculated. A total of 304 isolates were identified; 59% (179) of the isolates carried blaCTX-M, 47% (143) carried blaTEM, and 4% (12) carried blaSHV. At a breakpoint MIC of 8 µg/ml, Vitek 2 produced a categorical agreement of 66.8% and exhibited very major, major, and minor error rates of 23% (20/87 isolates), 5.1% (8/157 isolates), and 24% (73/304), respectively. The sensitivity, specificity, and positive and negative predictive values for a susceptibility breakpoint of 8 µg/ml were 94.9%, 61.2%, 72.3%, and 91.8%, respectively. The sensitivity, specificity, and positive and negative predictive values for a susceptibility breakpoint of 2 µg/ml were 83.8%, 65.3%, 41%, and 93.3%, respectively. Vitek 2 results in unacceptably high error rates for cefepime compared to those of agar dilution for ESBL-producing E. coli. Clinicians should be wary of making treatment decisions on the basis of Vitek 2 susceptibility results for ESBL-producing E. coli.

Outcomes of an enhanced surveillance program for carbapenem-resistant Enterobacteriaceae.

Optimal surveillance strategies for identifying patients colonized with and at risk for transmitting carbapenem-resistant Enterobacteriaceae (CRE) are urgently needed. We instituted an enhanced surveillance program for CRE that identified unrecognized CRE-colonized patients but failed to identify possible CRE transmissions. We also identified risk factors associated with transmitting CRE.

Quantifying the clinical virulence of Klebsiella pneumoniae producing carbapenemase Klebsiella pneumoniae with a Galleria mellonella model and a pilot study to translate to patient outcomes.

BACKGROUND: Previous studies may have overestimated morbidity and mortality due to Klebsiella pneumoniae producing carbapenemase (KPC) Klebsiella pneumoniae infections because of difficulties in modeling patient comorbidities. This pilot study sought to evaluate KPC virulence by combining clinical and Galleria mellonella models in patients with K. pneumoniae blood stream infections (BSIs). METHODS: G. mellonella were inoculated using KPC(+) and KPC(-) isolates from these patients. Extent and rapidity of insect mortality was analyzed. Patients were stratified by KPC BSI status. Clinical outcomes of mortality and length of stay post-infection for survivors (LOS) were analyzed. Median virulence scores calculated from the insect studies were imputed in the clinical model. RESULTS: The in-vivo model revealed greater mortality in KPC(-) isolates (p < 0.001). Fifteen patients with KPC(+) BSI were matched with 60 patients with KPC(-) BSI. Hospital mortality was greater in the KPC(+) group versus the KPC(-) group (OR 3.79, 95% CI 1.00 - 14.34). LOS was longer in the KPC(+) group (p < 0.01). Conversely the virulence score attenuated the association between KPC(+) status and mortality and LOS in the final translational models. CONCLUSIONS: KPC(+) status was associated with decreased virulence in GM. Opposite findings were observed in patients. This pilot study demonstrates that measured virulence from GM may differ from human estimates of virulence.

Pathogenicity of clinical Acinetobacter baumannii isolates in a Galleria mellonella host model according to bla(OXA-40) gene and epidemiological outbreak status.

Clinical studies have suggested that blaOXA-40-positive Acinetobacter baumannii isolates are associated with poor patient outcomes; however, reasons for unfavorable outcomes are difficult to discern in clinical studies. The objective of this study was to assess the virulence of carbapenem-resistant A. baumannii according to blaOXA-40 and epidemiological outbreak status in a Galleria mellonella model. Eight isolates of A. baumannii were studied. Nonoutbreak isolates and blaOXA-40-negative isolates more rapidly killed infected G. mellonella (P < 0.01).

Correlations of antibiotic use and carbapenem resistance in enterobacteriaceae.

Epidemiological studies have shown a link between carbapenem use and resistance; however, the clinical relationship between antibiotic consumption and the epidemiology of carbapenem-intermediate or -resistant Enterobacteriaceae (CIRE) remains unclear. This study sought to analyze temporal antibiotic consumption trends for relationships with incident CIRE. In total, 310,892 days of therapy and 55 deduplicated CIRE were analyzed. When conservative corrections were applied for multiple comparisons, carbapenem class use and piperacillin-tazobactam use retained significant positive and negative relationships with the incidence of CIRE, respectively.

Characterization of ciprofloxacin resistant Escherichia coli isolates among men undergoing evaluation for transrectal ultrasound guided prostate biopsy.

PURPOSE: We determine the prevalence of ciprofloxacin resistant gram-negative bacilli in patients scheduled for transrectal ultrasound guided prostate biopsy, characterize the Escherichia coli strains recovered from this patient population, and characterize the mechanisms responsible for ß-lactam and ciprofloxacin resistance. MATERIALS AND METHODS: Rectal swabs from 991 patients were cultured for ciprofloxacin resistant gram-negative bacilli with a selective medium. Recovered E. coli isolates were further analyzed with susceptibility testing, pulsed field gel electrophoresis, plasmid isolation and sequencing. RESULTS: A total of 193 ciprofloxacin resistant gram-negative bacilli were recovered and of these isolates 167 (87%) were E. coli. The prevalence of ciprofloxacin resistant E. coli in the study population was 17%. Only 38 (26%) of the 149 E. coli isolates that received susceptibility testing were susceptible to ampicillin and ampicillin-sulbactam. In select isolates transferrable plasmids carrying ß-lactamase were responsible for the resistance to the ß-lactam agents and other nonß-lactam antimicrobials. Diverse combinations of gyrA and parC mutations associated with fluoroquinolone resistance were identified. Strain typing and plasmid typing indicated that the E. coli isolates did not share a common origin. CONCLUSIONS: Of the patients in our study 17% carried ciprofloxacin resistant E. coli. Analysis of resistance mechanisms and plasmid analysis along with strain typing demonstrated that this patient population harbored organisms with heterogeneous phenotypic susceptibility, indicating that universal prophylaxis would not provide optimal coverage for patients undergoing transrectal ultrasound guided prostate biopsy.

Virulence of vancomycin-resistant Enterococcus faecium according to linezolid resistance and clinical outbreak status.

Assessing clinical virulence differences between vancomycin-resistant Enterococcus faecium (VREF) strains resistant to linezolid (LRVRE) and linezolid-susceptible VRE (LSVRE) strains is difficult due to confounding patient variables. Galleria mellonella is a validated host interaction model allowing straightforward organism virulence assessment. The objective of this study was to assess the virulence of VREF in G. mellonella according to linezolid resistance and clinical outbreak status. A genetically related pair of VREF strains with and without genotypically confirmed linezolid resistance was selected for analysis. Additionally, six strains of LSVRE and two strains of LRVRE were selected according to epidemiologic outbreak status. Mortality of G. mellonella was assessed daily over a 5-day period and analyzed using Kaplan-Meier survival curves and log rank tests. Linezolid resistance did not have a significant effect on G. mellonella mortality in the genetically related pair (P = 0.93). There was no significant difference in mortality over time between strains (non-outbreak [i.e., no patient transmissions were recorded] [n = 2] versus outbreak [i.e., transmission occurred between 3 or more patients in a period of 30 days] [n = 6], P = 0.84; extensive transmission [i.e., the isolate was transmitted between at least 80 patients] [n = 2] versus limited transmission [i.e., the isolate was transmitted between fewer than 10 patients] [n = 4], P = 0.78). These results suggest that patients infected with LRVRE or outbreak strains of VREF are at no greater risk of poor outcomes mediated by organism virulence than those infected with LSVRE or non-outbreak strains.

Temporal changes in pulsed-field gel electrophoresis banding in vancomycin-resistant Enterococcus faecium and implications for outbreak investigations.

BACKGROUND: Patients are often screened with surveillance cultures to discern transmissions vs transformation of an isolate to vancomycin-resistant Enterococcus faecium. To determine the amount of time between which isolates could be considered genetically similar by pulsed-field gel electrophoresis, isolate change over time within single patients was studied. METHODS: A minimum of 4 isolates per patient, separated by at least 2 months, were collected from previously frozen stores. Visual comparison of banding patterns was conducted, and percent relatedness was calculated. RESULTS: Twenty-eight isolates from 6 patients were studied. No isolate differed by more than 3 bands before 150 days, and the average percent difference per band was 3.7%. The isolates diverged genetically as a linear function of number of bands over time (good model fit intrapatient r(2) = 0.42; poor model fit interpatient r(2) = 0.0062). CONCLUSION: Trajectory of genetic variation appears to be isolate/patient specific; however, commonalities exist and tested isolates were relatively stable out to 150 days.