Background

Fungal load quantification is a critical component of fungal community analyses. Limitation of current approaches for quantifying the fungal component in the human microbiome suggests the need for new broad-coverage techniques.

Methods

We analyzed 2,085 18S rRNA gene sequences from the SILVA database for assay design. We generated and quantified plasmid standards using a qPCR-based approach. We evaluated assay coverage against 4,968 sequences and performed assay validation following the Minimum Information for Publication of Quantitative Real-Time PCR Experiments (MIQE) guidelines.

Results

We designed FungiQuant, a TaqMan® qPCR assay targeting a 351 bp region in the fungal 18S rRNA gene. Our in silico analysis showed that FungiQuant is a perfect sequence match to 90.0% of the 2,617 fungal species analyzed. We showed that FungiQuant’s is 100% sensitive and its amplification efficiencies ranged from 76.3% to 114.5%, with r2-values of >0.99 against the 69 fungal species tested. Additionally, FungiQuant inter- and intra-run coefficients of variance ranged from <10% and <20%, respectively. We further showed that FungiQuant has a limit of quantification 25 copies and a limit of detection at 5 copies. Lastly, by comparing results from human-only background DNA with low-level fungal DNA, we showed that amplification in two or three of a FungiQuant performed in triplicate is statistically significant for true positive fungal detection.

Conclusions

FungiQuant has comprehensive coverage against diverse fungi and is a robust quantification and detection tool for delineating between true fungal detection and non-target human DNA.

Determination of the prevalence of accumulated antiretroviral drug resistance among persons infected with human immunodeficiency virus (HIV) is complicated by the lack of routine measurement in clinical care. By using data from 8 clinic-based cohorts from the North American AIDS Cohort Collaboration on Research and Design, drug-resistance mutations from those with genotype tests were determined and scored using the Genotypic Resistance Interpretation Algorithm developed at Stanford University. For each year from 2000 through 2005, the prevalence was calculated using data from the tested subset, assumptions that incorporated clinical knowledge, and multiple imputation methods to yield a complete data set. A total of 9,289 patients contributed data to the analysis; 3,959 had at least 1 viral load above 1,000 copies/mL, of whom 2,962 (75%) had undergone at least 1 genotype test. Using these methods, the authors estimated that the prevalence of accumulated resistance to 2 or more antiretroviral drug classes had increased from 14% in 2000 to 17% in 2005 (P < 0.001). In contrast, the prevalence of resistance in the tested subset declined from 57% to 36% for 2 or more classes. The authors’ use of clinical knowledge and multiple imputation methods revealed trends in HIV drug resistance among patients in care that were markedly different from those observed using only data from patients who had undergone genotype tests.

Background

Immune responses to Pneumocystis jirovecii are not well understood in HIV infection, but antibody responses to proteins may be useful as a marker of Pneumocystis risk or presence of Pneumocystis pneumonia (PcP).

Design

Retrospective analysis of a prospective cohort

Methods

Enzyme-linked immunosorbent assays of antibodies to recombinant Pneumocystis proteins of major surface glycoprotein fragments (MsgC1, C3, C8, and C9) and of antibody titers to recombinant kexin protein (KEX1) were performed on three sequential serum samples up to 18 months prior to and three samples after first AIDS-defining illness from Multicenter AIDS Cohort Study participants and compared between those who had PcP or a non-PcP AIDS-defining illness.

Results

Fifty-four participants had PcP and 47 had a non-PcP AIDS-defining illness. IgG levels to MsgC fragments were similar between groups prior to first AIDS-defining illness, but the PcP group had higher levels of IgG to MsgC9 (median units/ml 50.2 vs. 22.2, p=0.047) post-illness. Participants with PcP were more likely to have an increase in MsgC3 (OR 3.9, p=0.02), MsgC8 (OR 5.5, p=0.001), and MsgC9 (OR 4.0, p=0.007). The PcP group was more likely to have low KEX1 IgG prior to development of PcP (OR 3.6, p=0.048) independent of CD4 cell count and to have an increase in high IgG titers to KEX1 after PcP.

Conclusion

HIV-infected individuals develop immune responses to both Msg and kexin proteins after PcP. Low KEX1 IgG titers may be a novel marker of future PcP risk before CD4 cell count has declined below 200 cells/μl.

Background

In the early highly active antiretroviral therapy (HAART) era, kidney dysfunction was strongly associated with death among HIV-infected individuals. We re-examined this association in the later HAART period to determine whether chronic kidney disease (CKD) remains a predictor of death after HAART-initiation.

Methods

To evaluate the effect of kidney function at the time of HAART initiation on time to all-cause mortality, we evaluated 1415 HIV-infected women initiating HAART in the Women’s Interagency HIV Study (WIHS). Multivariable proportional hazards models with survival times calculated from HAART initiation to death were constructed; participants were censored at the time of the last available visit or December 31, 2006.

Results

CKD (eGFR <60 ml/min/1.73 m2) at HAART initiation was associated with higher mortality risk adjusting for age, race, hepatitis C serostatus, AIDS history and CD4+ cell count (hazard ratio [HR]=2.23, 95% confidence interval [CI]: 1.45–3.43). Adjustment for hypertension and diabetes history attenuated this association (HR=1.89, CI: 0.94–3.80). Lower kidney function at HAART initiation was weakly associated with increased mortality risk in women with prior AIDS (HR=1.09, CI: 1.00–1.19, per 20% decrease in eGFR).

Conclusions

Kidney function at HAART initiation remains an independent predictor of death in HIV-infected individuals, especially in those with a history of AIDS. Our study emphasizes the necessity of monitoring kidney function in this population. Additional studies are needed to determine mechanisms underlying the increased mortality risk associated with CKD in HIV-infected persons.

This study was an investigation of the effect of ultraviolet B light exposure on the risk of cortical cataract as a function of the region of the lens. The degree to which the lower nasal predominance of cortical cataract is a result of UVB exposure was assessed.

Purpose.

In studies of cortical cataract, a severity score representing the area covered by cataract is often used as the primary outcome. However, additional disease information may exist in the spatial distribution of opacities. Further, it has been hypothesized that the lower nasal region of the lens is the most susceptible to damage by environmental ultraviolet light exposure.

Methods.

In a sample of 107 lens images from the Salisbury Eye Evaluation Study, a digital cortical cataract grading algorithm was used to capture the location of opacities in binary images. These images were used to estimate the severity of cataract in 16 regions around the lens. The effect of individual cumulative lifetime ocular exposure to ultraviolet B light on cortical cataract risk for each lens region was examined, as estimated by using an empiric model and baseline occupation and leisure activities data, in a linear mixed-effects model.

Results.

The lower nasal regions had the highest cortical cataract severity in both the right and left eyes. In the combined data, region 9 (the lower nasal corner of the lens) was estimated to have the highest severity. In an assessment of the high- and low-exposure ultraviolet light groups (dichotomized at the median exposure level), higher exposure had the most effect in the lower regions of the lens.

Conclusions.

These results indicate that there are regional lens differences in the association between cataract and exposure to ultraviolet light but that ultraviolet light may not entirely explain the variations in cortical cataract severity across the lens.

Measurement of iohexol plasma disappearance GFR in children require optimization of duration and sampling times Shortening the plasma disappearance study may overestimate GFR. We examined iohexol plasma disappearance curves in 27 children to determine the degree of overestimation in GFR due to shortening studies from 6 to 5 and to 4 hours. GFR measured after 5 hours was comparable to that after 6 hours, but shortening to 4 hours resulted in a 3% (p< 0.01) overestimation of GFR. Another simplification would be reducing the number of blood samples used to determine GFR. We followed the lead of Brochner-Mortensen and derived the relationship between a single compartment (slowGFR) disappearance curve and that from a double exponential analysis (two compartments sampled at 10, 30, 120, and 300 minutes), using 489 GFR measurements (median= 44 ml/min per1.73m2) in visit 1 of the Chronic Kidney Disease in Children (CKiD) study. Using polynomial regression methods, we developed coefficients to accurately measure GFR from a single compartment: GFR2 = K1*slowGFR + K2*(slowGFR)2. The coefficients (K1=1.0019 ; K2=-0.001258) were then used to generate GFR2 to be compared with 361 four point GFRs in visit 2. There was excellent correlation (r=0.999) and no bias or change in between-individuals’ dispersion. Conclusion: GFR can be accurately measured in children with CKD using the slow component of the iohexol plasma disappearance curve, provided the duration of study is at least 5 hours.

Background

The optimal time for the initiation of antiretroviral therapy for asymptomatic patients with human immunodeficiency virus (HIV) infection is uncertain.

Methods

We conducted two parallel analyses involving a total of 17,517 asymptomatic patients with HIV infection in the United States and Canada who received medical care during the period from 1996 through 2005. None of the patients had undergone previous antiretroviral therapy. In each group, we stratified the patients according to the CD4+ count (351 to 500 cells per cubic millimeter or >500 cells per cubic millimeter) at the initiation of antiretroviral therapy. In each group, we compared the relative risk of death for patients who initiated therapy when the CD4+ count was above each of the two thresholds of interest (early-therapy group) with that of patients who deferred therapy until the CD4+ count fell below these thresholds (deferred-therapy group).

Results

In the first analysis, which involved 8362 patients, 2084 (25%) initiated therapy at a CD4+ count of 351 to 500 cells per cubic millimeter, and 6278 (75%) deferred therapy. After adjustment for calendar year, cohort of patients, and demographic and clinical characteristics, among patients in the deferred-therapy group there was an increase in the risk of death of 69%, as compared with that in the early-therapy group (relative risk in the deferred-therapy group, 1.69; 95% confidence interval [CI], 1.26 to 2.26; P<0.001). In the second analysis involving 9155 patients, 2220 (24%) initiated therapy at a CD4+ count of more than 500 cells per cubic millimeter and 6935 (76%) deferred therapy. Among patients in the deferred-therapy group, there was an increase in the risk of death of 94% (relative risk, 1.94; 95% CI, 1.37 to 2.79; P<0.001).

Conclusions

The early initiation of antiretroviral therapy before the CD4+ count fell below two prespecified thresholds significantly improved survival, as compared with deferred therapy.

Background

Diagnostic images are often assessed for clinical outcomes using subjective methods, which are limited by the skill of the reviewer. Computer-aided diagnosis (CAD) algorithms that assist reviewers in their decisions concerning outcomes have been developed to increase sensitivity and specificity in the clinical setting. However, these systems have not been well utilized in research settings to improve the measurement of clinical endpoints. Reductions in bias through their use could have important implications for etiologic research.

Methods

Using the example of cortical cataract detection, we developed an algorithm for assisting a reviewer in evaluating digital images for the presence and severity of lesions. Available image processing and statistical methods that were easily implementable were used as the basis for the CAD algorithm. The performance of the system was compared to the subjective assessment of five reviewers using 60 simulated images. Cortical cataract severity scores from 0 to 16 were assigned to the images by the reviewers and the CAD system, with each image assessed twice to obtain a measure of variability. Image characteristics that affected reviewer bias were also assessed by systematically varying the appearance of the simulated images.

Results

The algorithm yielded severity scores with smaller bias on images where cataract severity was mild to moderate (approximately ≤ 6/16ths). On high severity images, the bias of the CAD system exceeded that of the reviewers. The variability of the CAD system was zero on repeated images but ranged from 0.48 to 1.22 for the reviewers. The direction and magnitude of the bias exhibited by the reviewers was a function of the number of cataract opacities, the shape and the contrast of the lesions in the simulated images.

Conclusion

CAD systems are feasible to implement with available software and can be valuable when medical images contain exposure or outcome information for etiologic research. Our results indicate that such systems have the potential to decrease bias and discriminate very small changes in disease severity. Simulated images are a tool that can be used to assess performance of a CAD system when a gold standard is not available.