HIV screening among newly diagnosed TB patients: a cross sectional study in Lima, Peru.

BACKGROUND: Since 2006, the Peruvian National TB program (NTP) recommends voluntary counseling and testing (VCT) for all tuberculosis (TB) patients. Responding to the differential burden of both diseases in Peru, TB is managed in peripheral health facilities while HIV is managed in referral centers. This study aims to determine the coverage of HIV screening among TB patients and the characteristics of persons not screened. METHODS: From March 2010 to December 2011 we enrolled new smear-positive pulmonary TB adults in 34 health facilities in a district in Lima. NTP staff offered VCT to all TB patients. Patients with an HIV positive result were referred for confirmation tests and management. We interviewed patients to collect their demographic and clinical characteristics and registered if patients opted in or out of the screening. RESULTS: Of the 1295 enrolled TB patients, nine had a known HIV diagnosis. Of the remaining, 76.1% (979) were screened for HIV. Among the 23.9% (307) not screened, 38.4% (118) opted out of the screening. TB patients at one of the health care facilities of the higher areas of the district (OR = 3.38, CI 95% 2.17-5.28 for the highest area and OR = 2.82, CI 95% 1.78-4.49 for the high area) as well as those reporting illegal drug consumption (OR = 1.65, CI 95% 1.15-2.37) were more likely not to be screened. Twenty-four were HIV positive (1.9% of all patients 1295, or 2.4% of those screened). Of 15 patients diagnosed with HIV during the TB episode, ten were enrolled in an HIV program. The median time between the result of the HIV screening and the first consultation at the HIV program was 82 days (IQR, 32-414). The median time between the result of the HIV screening and antiretroviral initiation was 148.5 days (IQR 32-500). CONCLUSIONS: An acceptable proportion of TB patients were screened for HIV in Lima. Referral systems of HIV positive patients should be strengthened for timely ART initiation.

A triple mutant of Escherichia coli lacking secondary acyl chains on lipid A.

All possible combinations of insertion mutations in the three genes encoding the acyl carrier protein-dependent late acyltransferases of lipid A biosynthesis, designated lpxL(htrB), lpxM(msbB), and lpxP, were generated in Escherichia coli K12 W3110. Mutants defective in lpxM synthesize penta-acylated lipid A molecules and grow normally. Strains lacking lpxP fail to incorporate palmitoleate into their lipid A at 12 degrees C but make normal amounts of hexa-acylated lipid A and are viable. Although lpxL mutants and lpxL lpxM double mutants grow slowly on minimal medium at all temperatures, they do not grow on nutrient broth above 32 degrees C. Such mutants retain the ability to synthesize some penta- and hexa-acylated lipid A molecules because of limited induction of lpxP at 30 degrees C but not above 32 degrees C. MKV15, an E. coli lpxL lpxM lpxP triple mutant, likewise grows slowly on minimal medium at all temperatures but not on nutrient broth at any temperature. MKV15 synthesizes a lipid A molecule containing only the four primary (R)-3-hydroxymyristoyl chains. The outer membrane localization and content of lipid A are nearly normal in MKV15, as is the glycerophospholipid and membrane protein composition. However, the rate at which the tetra-acylated lipid A of MKV15 is exported to the outer membrane is reduced compared with wild type. The integrity of the outer membrane of MKV15 is compromised, as judged by antibiotic hypersensitivity, and MKV15 undergoes lysis following centrifugation. MKV15 may prove useful as a host strain for expressing late acyltransferase genes from other Gram-negative bacteria, facilitating the re-engineering of lipid A structure in living cells and the design of novel vaccines.

Relaxed acyl chain specificity of Bordetella UDP-N-acetylglucosamine acyltransferases.

Lipid A (endotoxin) is a major structural component of Gram-negative outer membranes. It also serves as the hydrophobic anchor of lipopolysaccharide and is a potent activator of the innate immune response. Lipid A molecules from the genus Bordetella are reported to exhibit unusual structural asymmetry with respect to the acyl chains at the 3- and 3'-positions. These acyl chains are attached by UDP-N-acetylglucosamine acyltransferase (LpxA). To determine the origin of the acyl variability, the single lpxA ortholog present in each of the genomes of Bordetella bronchiseptica (lpxA(Br)), Bordetella parapertussis (lpxA(Pa)), and Bordetella pertussis (lpxA(Pe)) was cloned and expressed in Escherichia coli. In contrast to all LpxA proteins studied to date, LpxA(Br) and LpxA(Pe) display relaxed acyl chain length specificity in vitro, utilizing C(10)OH-ACP, C(12)OH-ACP, and C(14)OH-ACP at similar rates. Furthermore, hybrid lipid A molecules synthesized at 42 degrees C by an E. coli lpxA mutant complemented with lpxA(Pe) contain C(10)OH, C(12)OH, and C(14)OH at both the 3- and 3'-positions, as determined by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. In contrast, LpxA from B. parapertussis did not display relaxed specificity but was selective for C(10)OH-ACP. This study provides an enzymatic explanation for some of the unusual acyl chain variations found in Bordetella lipid A.