Paul P. Thompson

Persistent Pneumocystis Colonization Leads to the Development of Chronic Obstructive Pulmonary Disease in a Nonhuman Primate Model of AIDS

Human immunodeficiency virus (HIV)-infected patients are at increased risk for development of pulmonary complications, including chronic obstructive pulmonary disease (COPD). Inflammation associated with subclinical infection has been postulated to promote COPD. Persistence of Pneumocystis is associated with HIV infection and COPD, although a causal relationship has not been established. We used a simian/human immunodeficiency virus model of HIV infection to study pulmonary effects of Pneumocystis colonization. Simian/human immunodeficiency virus-infected/Pneumocystis-colonized monkeys developed progressive obstructive pulmonary disease characterized by increased emphysematous tissue and bronchial-associated lymphoid tissue. Increased levels of T helper type 2 cytokines and proinflammatory mediators in bronchoalveolar lavage fluid coincided with Pneumocystis colonization and a decline in pulmonary function. These results support the concept that an infectious agent contributes to the development of HIV-associated lung disease and suggest that Pneumocystis colonization may be a risk factor for the development of HIV-associated COPD. Furthermore, this model allows examination of early host responses important to disease progression, thus identifying potential therapeutic targets for COPD.

Validation of Real-Time PCR for Laboratory Diagnosis of Acanthamoeba Keratitis

ABSTRACT Confirmation of Acanthamoeba keratitis by laboratory diagnosis is the first step in the treatment of this vision-threatening disease. Two real-time PCR TaqMan protocols (the Rivière and Qvarnstrom assays) were developed for the detection of genus-specific Acanthamoeba DNA but lacked clinical validation. We have adapted these assays for the Cepheid SmartCycler II system (i) by determining their real-time PCR limits of detection and amplification efficiencies, (ii) by determining their ability to detect trophozoites and cysts, and (iii) by testing a battery of positive and negative samples. We also examined the inhibitory effects of a number of commonly used topical ophthalmic drugs on real-time PCR. The results of the real-time PCR limit of detection and amplification efficiency of the Rivière and Qvarnstrom assays were 11.3 DNA copies/10 μl and 94% and 43.8 DNA copies/10 μl and 92%, respectively. Our extraction protocol enabled us to detect 0.7 Acanthamoeba cysts/10 μl and 2.3 Acanthamoeba trophozoites/10 μl by both real-time PCR assays. The overall agreement between the assays was 97.0%. The clinical sensitivity and specificity of both real-time PCR assays based on culture were 100% (7 of 7) and 100% (37 of 37), respectively. Polyhexamethylene biguanide was the only topical drug that demonstrated PCR inhibition, with a minimal inhibitory dilution of 1/640 and an amplification efficiency of 72.7%. Four clinical samples were Acanthamoeba culture negative and real-time PCR positive. Our results indicate that both real-time PCR assays could be used to diagnose Acanthamoeba keratitis. Polyhexamethylene biguanide can inhibit PCR, and we suggest that specimen collection occur prior to topical treatment to avoid possible false-negative results.

Cyclic AMP negatively regulates prodigiosin production by Serratia marcescens.

Many Serratia marcescens strains produce the red pigment prodigiosin, which has antimicrobial and anti-tumor properties. Previous reports suggest that cyclic AMP (cAMP) is a positive regulator of prodigiosin production. Supporting this model, the addition of glucose to growth medium inhibited pigment production in rich and minimal media. Unexpectedly, we observed highly elevated levels of prodigiosin production in isogenic strains with mutations in genes involved in cAMP production (cyaA and crr) and in cAMP-dependent transcriptional signaling (crp). Multicopy expression of the Escherichia coli cAMP-phosphodiesterase gene, cpdA, also conferred a striking increase in prodigiosin production. Exogenous cAMP decreased both pigment production and pigA-lacZ transcription in the wild-type (WT) strain, and pigA-lacZ transcription was significantly increased in a crp mutant relative to WT. Suppressor and epistasis analysis indicate that the hyperpigment phenotype was dependent upon pigment biosynthetic genes (pigA, pigB, pigC, pigD and pigM). These experiments establish cAMP as a negative regulator of prodigiosin production in S. marcescens.

A simple method for automated lung segmentation in x-ray CT images

We developed and tested an automated scheme to segment lung areas depicted in CT images. The scheme includes a series of six steps. 1) Filtering and removing pixels outside the scanned anatomic structures. 2) Segmenting the potential lung areas using an adaptive threshold based on pixel value distribution in each CT slice. 3) Labeling all selected pixels ingo segmented regions and deleting isolated regions in non-lung area. 4) Labeling and filling interior cavities (e.g., pleural nodules, airway wall, and major blood vessels) inside lung areas. 5) Detecting and deleting the main airways (e.g., trachea and central bronchi) connected to the segmented lung areas. 6) Detecting and separating possible anterior or posterior junctions between the lungs. Five lung CT cases (7-10 mm in slice thickness) with variety of disease patterns were used to train or set up the classification rules in the scheme. Fifty examinations of emphysema patients were then used to test the scheme. The results were compared with the results generated from a semi-automated method with manual interaction by an expert observer. The experimental results showed that the average difference in estimated lung volumes between the automated scheme and manually corrected approach was 2.91%±0.88%. Visual examination of segmentation results indicated that the difference of the two methods was larger in the areas near the apices and the diaphragm. This preliminary study demonstrated that a simple multi-stage scheme had potential of eliminating the need for manual interaction during lunch segmentation. Hence, it can ultimately be integrated into computer schemes for quantitative analysis and diagnosis of lung diseases.

A 13-Year Retrospective Review of Polymerase Chain Reaction Testing for Infectious Agents from Ocular Samples

PURPOSE Polymerase chain reaction (PCR) is a molecular technique for the diagnosis of ocular infectious disease. In this large patient sample and multiyear study, the impact of PCR for detecting infectious agents from ocular samples was reviewed in comparison with nonmolecular diagnostic techniques. DESIGN A retrospective laboratory review of PCR testing. PARTICIPANTS Three thousand fifty-six patient samples with a differential of ocular infection. METHODS The daily laboratory logs for diagnostic testing were reviewed for PCR, cell culture isolation, shell vial isolation, and Acanthamoeba isolation from January 1997 through May 2010 for herpes simplex virus (HSV), adenovirus, varicella zoster virus (VZV), Chlamydia trachomatis, Acanthamoeba, and infrequent pathogens of intraocular inflammation. MAIN OUTCOME MEASURES Incidence of the positive presence of ocular infectious agents. RESULTS Polymerase chain reaction results were positive more often than culture results for HSV (P = 0.0001), VZV (P = 0.00001), C. trachomatis (P = 0.00005), and Acanthamoeba (P = 0.04). For adenovirus, cell culture isolation results were positive more often than PCR results (P = 0.001). Polymerase chain reaction was the primary diagnostic test for detecting cytomegalovirus and Toxoplasma. CONCLUSIONS The current study demonstrated the importance of PCR as a routine diagnostic test for detecting both common and infrequent ocular pathogens. Cell culture isolation is still a definitive test for adenovirus and a confirmatory test for HSV and Acanthamoeba.

An Independent In Vitro Comparison of Povidone Iodine and SteriLid

AIM Povidone iodine (PI) and SteriLid (SL) are biocides used to decrease bacterial load on the eyelid margin. We compared the antibacterial activity of PI and SL to determine the better antiseptic. METHODS Time-kill studies of PI and SL against a battery of bacteria that included Staphylococcus epidermidis, methicillin-susceptible Staphylococcus aureus, methicillin-resistant Staphylococcus aureus, Streptococcus pneumoniae, Bacillus cereus, Pseudomonas aeruginosa, Escherichia coli, Serratia marcescens, and Branhamella catarrhalis were conducted at time points 1, 2, 10, and 30 min. The comparative outcome measures were based on 90% and 99.9% decreases in bacterial colony counts. RESULTS PI was more effective at a 90% kill than SL at time points, 1 min (8/9 vs. 7/9), 2 min (9/9 vs. 6/9), 10 min (9/9 vs. 8/9), and 30 min (9/9 vs. 8/9). PI was more effective at a 99.9% kill than SL at time points, 1 min (5/9 vs. 3/9), 2 min (7/9 vs. 4/9), 10 min (9/9 vs. 4/9), and 30 min (9/9 vs. 6/9). CONCLUSIONS Our study supports PI as an effective antiseptic for decreasing the bacterial load that can exist on the eyelid margin. SL appears to be a less effective alternative.

Cell culture isolation can miss the laboratory diagnosis of HSV ocular infection.

AIM We compared polymerase chain reaction (PCR) to cell culture isolation for the laboratory diagnosis of ocular herpes simplex virus (HSV) disease. METHODS Laboratory and medical records of consecutive patients were reviewed for results of 1) HSV PCR testing, 2) HSV cell culture isolation, and 3) clinical diagnosis. PCR results were statistically compared to cell culture isolation and patients initially diagnosed for ocular HSV infection. RESULTS Of 581 cases submitted for laboratory testing, 520 were PCR negative, cell culture negative (89.6%); 0 were PCR negative, cell culture positive (0%); 27 were PCR positive, cell culture negative (4.6%); and 34 were PCR positive, cell culture positive (5.8%). PCR tested more positive than cell culture isolation (McNemars, P=0.0001). Of 47 HSV PCR positive cases with complete medical records, 19 were cell culture negative for HSV and 28 were cell culture positive for HSV. Fourteen of 19 cell culture negative cases (74%) (Without PCR, 5 cases of HSV would be missed) and 25 of the 28 cell culture positive cases (89%) (Laboratory testing was necessary for diagnosing 3 cases) were clinically diagnosed with HSV at the initial examination. CONCLUSION PCR was a more definitive test for diagnosing HSV ocular infection than cell culture isolation. Cell culture isolation alone can miss an atypical presentation of HSV ocular infection.

Evaluation of the new OxyPlate™ Anaerobic System for the isolation of ocular anaerobic bacteria.

AIM Anaerobic bacteria can cause ocular infections. We tested the OxyPlate™ Anaerobic System (OXY) to isolate pertinent anaerobic bacteria that can cause ocular disease. METHODS OXY, which does not require direct anaerobic conditions (i.e. bags, jars), was compared to conventional isolation of incubating culture media in anaerobic bags. Standard colonies counts were performed on anaerobic ocular bacterial isolates under aerobic and anaerobic conditions (anaerobic bags) using agar media: 1) OXY (aerobic only), 2) 5% sheep blood (SB), 3) Chocolate, and 4) Schaedler. The bacteria tested were de-identified ocular isolates cultured from endophthalmitis and dacryocystitis that include 10 Propionibacterium acnes and 3 Actinomyces species. The colony counts for each bacteria isolate, on each culturing condition, were ranked from largest to smallest, and non-parametrically compared to determine the best culturing condition. RESULTS All anaerobic conditions were positive for all of the anaerobic isolates. SB and Schaedlers agar under aerobic conditions did not support the growth of anaerobic bacteria. Sparse growth was noted on chocolate agar with Propionibacterium acnes. As an anaerobic system, SB in an anaerobic bag isolated higher colony counts than OXY (P=0.0028) and chocolate agar (P=0.0028). CONCLUSION Although OXY did not test to be more efficient than other anaerobic systems, it appears to be a reasonable alternative for isolating anaerobic bacteria from ocular sites. The use of an agar medium in a specially designed plate, without the requirement of an anaerobic bag, rendered OXY as an advantage over other anaerobic systems.