Unanticipated Mycobacterium tuberculosis complex culture inhibition by immune modulators, immune suppressants, a growth enhancer, and vitamins A and D: clinical implications.

BACKGROUND: The development of novel antibiotics to treat multidrug-resistant (MDR) tuberculosis is time-consuming and expensive. Multiple immune modulators, immune suppressants, anti-inflammatories, and growth enhancers, and vitamins A and D, inhibit Mycobacterium avium subspecies paratuberculosis (MAP) in culture. We studied the culture inhibition of Mycobacterium tuberculosis complex by these agents. METHODS: Biosafety level two M. tuberculosis complex (ATCC 19015 and ATCC 25177) was studied in radiometric Bactec or MGIT culture. Agents evaluated included clofazimine, methotrexate, 6-mercaptopurine, cyclosporine A, rapamycin, tacrolimus, monensin, and vitamins A and D. RESULTS: All the agents mentioned above caused dose-dependent inhibition of the M. tuberculosis complex. There was no inhibition by the anti-inflammatory 5-aminosalicylic acid, which causes bacteriostatic inhibition of MAP. CONCLUSIONS: We conclude that, at a minimum, studies with virulent M. tuberculosis are indicated with the agents mentioned above, as well as with the thioamide 5-propothiouricil, which has previously been shown to inhibit the M. tuberculosis complex in culture. Our data additionally emphasize the importance of vitamins A and D in treating mycobacterial diseases.

Use of dried clinical samples for storing and detecting influenza RNA.

BACKGROUND: Most clinical samples collected for diagnostic influenza testing and monitoring require refrigerated or frozen storage or shipment, which imparts logistic and cost burdens. The ability to store and ship dried clinical specimens under ambient conditions for influenza testing would significantly reduce costs and protect samples from improper storage or equipment failure, especially in remote or resource-limited areas. OBJECTIVES: To evaluate the collection and storage of dried clinical samples on a transport matrix (ViveST™, ST) for influenza RNA testing by real-time reverse-transcription PCR (RT-PCR). METHODS: Viral transport medium from swab or sputum samples was applied to ST, dried, and stored under ambient conditions from 2 days to 6 months. Additional aliquots of samples were frozen. Testing of frozen and ST-stored samples was performed using the WHO/CDC real-time influenza A (H1N1) RT-PCR protocol and compared to the Luminex xTAG RVP assay. RESULTS: ST-stored samples yielded slightly higher threshold cycle values (median 2·54 cycles) compared to frozen samples tested in parallel. This difference was consistent regardless of viral input. There was no significant difference in signal recovery between samples stored for 1 week versus samples stored for 3 weeks, or from three samples stored for 6 months. Qualitatively, clinical specimens stored on ST were 100% concordant (36/36) with frozen samples for detecting the presence of influenza A RNA. CONCLUSION: ST-processed dried specimens produced similar rates of seasonal or novel 2009 HIN1 influenza RNA detection compared to conventional sample processing and thus presents a viable alternative to refrigerated or frozen samples.

Rhodotorula mucilaginosa lymphadenitis in an HIV-infected patient.

We report a case of lymphadenitis due to Rhodotorula mucilaginosa in a man with well-controlled HIV infection. The diagnosis was established microbiologically by positive lymph tissue cultures, and clinically by responses of lymphadenitis to antifungal therapy. The patient was asymptomatic and was treated with itraconazole 200mg orally once daily as an outpatient. Clinical response was evident within three weeks with improvement of lymphadenopathy on serial computed tomography scans. Lymphadenopathy resolved completely after 8 months of itraconazole therapy and had not recurred 9 months after treatment was stopped.

On the action of 5-amino-salicylic acid and sulfapyridine on M. avium including subspecies paratuberculosis.

BACKGROUND: Introduced in 1942, sulfasalazine (a conjugate of 5-aminosalicylic acid (5-ASA) and sulfapyridine) is the most prescribed medication used to treat "inflammatory" bowel disease (IBD.) Although controversial, there are increasingly compelling data that Mycobacterium avium subspecies paratuberculosis (MAP) may be an etiological agent in some or all of IBD. We have shown that two other agents used in the therapy of IBD (methotrexate and 6-MP) profoundly inhibit MAP growth. We concluded that their most plausible mechanism of action is as antiMAP antibiotics. We herein hypothesize that the mechanism of action of 5-ASA and/or sulfapyridine may also simply be to inhibit MAP growth. METHODOLOGY: The effect on MAP growth kinetics by sulfasalazine and its components were evaluated in bacterial culture of two strains each of MAP and M. avium, using a radiometric ((14)CO(2) BACTEC(R)) detection system that quantifies mycobacterial growth as arbitrary "growth index units" (GI). Efficacy data are presented as "percent decrease in cumulative GI" (%-DeltacGI). PRINCIPAL FINDINGS: There are disparate responses to 5-ASA and sulfapyridine in the two subspecies. Against MAP, 5-ASA is inhibitory in a dose-dependent manner (MAP ATCC 19698 46%-DeltacGI at 64 microg/ml), whereas sulfapyridine has virtually no effect. In contrast, against M. avium ATCC 25291, 5-ASA has no effect, whereas sulfapyridine (88%-DeltacGI at 4 microg/ml) is as effective as methotrexate, our positive control (88%-DeltacGI at 4 microg/ml). CONCLUSIONS: 5-ASA inhibits MAP growth in culture. We posit that, unknowingly, the medical profession has been treating MAP infections since sulfasalazine's introduction in 1942. These observations may explain, in part, why MAP has not previously been identified as a human pathogen. We conclude that henceforth in clinical trials evaluating antiMAP agents in IBD, if considered ethical, the use of 5-ASA (as well as methotrexate and 6-MP) should be excluded from control groups.

On the action of methotrexate and 6-mercaptopurine on M. avium subspecies paratuberculosis.

BACKGROUND: Clinical improvement in inflammatory bowel disease (IBD) treated with methotrexate and 6-mercaptopurine (6-MP) is associated with a decrease in pro-inflammatory cytokines. This has been presumed to indicate the mechanism of action of methotrexate and 6-MP. Although controversial, there are increasingly compelling data that Mycobacterium avium subspecies paratuberculosis (MAP) may be an etiological agent in some or all of IBD. We hypothesized that the clinical efficacy of methotrexate and 6-MP in IBD may be to simply inhibit the growth of MAP. METHODOLOGY: The effect on MAP growth kinetics by methotrexate and 6-MP were evaluated in cell culture of two strains each of MAP and M. avium using a radiometric ((14)CO(2) BACTEC detection system that quantifies mycobacterial growth as arbitrary "growth index units" (GI). Efficacy data are presented as "percent decrease in cumulative GI" (% -DeltacGI). PRINCIPAL FINDINGS: The positive control antibiotic (clarithromycin) has >or=85% -DeltacGI at a concentration of 0.5 microg/ml. The negative control (ampicillin) has minimal inhibition at 64 microg/ml. MAP ATCC 19698 shows >or=80% -DeltacGI for both agents by 4 microg/ml. With the other three isolates, although more effective than ampicillin, 6-MP is consistently less effective than methotrexate. CONCLUSIONS: We show that methotrexate and 6-MP inhibit MAP growth in vitro. Each of the four isolates manifests different % -DeltacGI. These data are compatible with the hypothesis that the clinical improvement in patients with IBD treated with methotrexate and 6-MP could be due to treating a MAP infection. The decrease in pro-inflammatory cytokines, thought to be the primary mechanism of action, may simply be a normal, secondary, physiological response. We conclude that henceforth, in clinical studies that evaluate the effect of anti-MAP agents in IBD, the use of methotrexate and 6-MP should be excluded from any control groups.

Detection of extrahepatic hepatitis C virus replication by a novel, highly sensitive, single-tube nested polymerase chain reaction.

We established a cell culture system for the replication of hepatitis C virus (HCV) by using human T and B leukemia cell lines. These 2 cell lines were infected in vitro by using HCV-positive pooled patient serum samples. HCV RNA was extracted from infected cell lines at different times after infection, and a sequence of the virus 5' untranslated region was analyzed. Hepatitis C minus-strand RNA was detected in the infected cell lines by highly strand-specific rTth (recombinant Thermus thermophilus DNA polymerase)-based reverse transcription followed by a novel, highly sensitive, single-tube nested polymerase chain reaction (PCR) method. PCR products were analyzed by direct DNA sequencing. These results indicate that the HCV can replicate in T and B lymphocytes. This model should represent a valuable tool for the detailed study of the initial steps of the HCV replication cycle and for the evaluation of antiviral molecules.

Prevalence of hepatitis C and coinfection with HIV among United States veterans in the New York City metropolitan area.

OBJECTIVES: The aims of this study were to determine the prevalence of hepatitis C virus (HCV) infection and its risk factors, as well as the prevalence of coinfection with HIV and its risk factors, among patients with confirmed HCV infection. METHODS: In a 1-day cross-sectional HCV survey at six Veterans Affairs Medical Centers in the New York City metropolitan area, all 1943 patients undergoing phlebotomy for any reason were asked to be tested for HCV antibody by enzyme immumoassay (EIA). A total of 1098 patients (57%) agreed to HCV testing, 1016 of whom also completed a questionnaire on demographics and HCV risk factors. All HCV EIA(+) samples were confirmed by HCV RNA and HCV recombinant immunoblot assay (RIBA) antibody testing and were also tested for HCV viral load, HCV genotype, and antibodies to HIV in a blinded fashion. RESULTS: The prevalence of confirmed HCV infection was 10.6% (95% CI = 8.7-12.4%), and the prevalence of HCV viremia was 8.2% (95% CI = 6.6-9.8%). The rate of HCV viremia among anti-HCV(+) patients was 77.6%, and HCV genotype 1 was present in 87.5% of viremic patients. Independent risk factors for HCV infection were injection drug use (OR = 35.6, 95% CI = 16.9-75.2), blood exposure during combat (OR = 2.6, 95% CI = 1.2-5.7), alcohol abuse (OR = 2.4; 95% CI = 1.2-4.8), and service in the Vietnam era (OR = 2.1; 95% CI = 1.0-4.5). Coinfection with HIV was present in 24.8% of anti-HCV(+) patients. The only independent risk factor for coinfection was age <50 yr (OR = 3.7, 95% CI = 1.1-12.1). CONCLUSIONS: U.S. veterans who are receiving medical care at VA medical centers in the New York City metropolitan area have a much higher rate of chronic hepatitis C than the general population, with a high frequency of genotype 1. Coinfection with HIV is very common in patients with confirmed HCV infection, and these patients should routinely be offered HIV testing.