Impact of heteroresistance on treatment outcomes of people with drug-resistant TB.

BACKGROUND: Poor treatment outcomes among people with drug-resistant TB (DR-TB) are a major concern. Heteroresistance (presence of susceptible and resistant Mycobacterium tuberculosis in the same sample) has been identified in some people with TB, but its impact on treatment outcomes is unknown. METHODS: We used targeted deep sequencing to identify mutations associated with DR-TB and heteroresistance in culture samples of 624 people with DR-TB. We evaluated the association between heteroresistance and time to unfavorable treatment outcome using Cox proportional hazards regression. RESULTS: The proportion of drug-resistant isolates with a known mutation conferring resistance was lower for streptomycin (45.2%) and second-line injectables (79.1%) than for fluoroquinolones (86.7%), isoniazid (93.2%) and rifampin (96.5%). Fifty-two (8.3%) had heteroresistance, and it was more common for fluoroquinolones (4.6%) than rifampin (2.2%), second-line injectables (1.4%), streptomycin (1.7%), or isoniazid (1.3%). There was no association between heteroresistance and time to unfavorable outcome among people with multidrug-resistant TB (adjusted hazard ratio [aHR] 1.74, 95% CI 0.39-7.72) or pre-extensively DR-TB (aHR 0.65, 95% CI 0.24-1.72). CONCLUSIONS: Heteroresistance was relatively common (8.3%) among people with DR-TB in the Philippines. However, we found insufficient evidence to demonstrate an impact on unfavorable treatment outcomes.

CONTEXTE: Les résultats médiocres du traitement chez les personnes atteintes de TB résistante aux médicaments (DR-TB, pour l'anglais « drug-resistant TB ¼) constituent une préoccupation significative. L'hétérorésistance, caractérisée par la coexistence de souches sensibles et résistantes de Mycobacterium tuberculosis dans un même échantillon, a été observée chez certains patients, mais les conséquences de cette situation sur l'efficacité des traitements demeurent incertaines. MÉTHODES: Nous avons recouru au séquençage profond ciblé afin d'identifier les mutations liées à la DR-TB et à l'hétérorésistance dans les échantillons de culture provenant de 624 personnes atteintes de DR-TB. Nous avons analysé le lien entre l'hétérorésistance et le délai jusqu'à l'issue défavorable du traitement en utilisant une régression des risques proportionnels de Cox. RÉSULTATS: La proportion d'isolats, présentant une mutation connue associée à la résistance, était inférieure pour la streptomycine (45,2%) et les médicaments injectables de deuxième ligne (79,1%) par rapport aux fluoroquinolones (86,7%), à l'isoniazide (93,2%) et à la rifampicine (96,5%). Parmi les isolats, cinquante-deux (8,3%) manifestaient une hétérorésistance, plus courante pour les fluoroquinolones (4,6%) que pour la rifampicine (2,2%), les médicaments injectables de deuxième ligne (1,4%), la streptomycine (1,7%) ou l'isoniazide (1,3%). Aucune association n'a été observée entre l'hétérorésistance et un délai d'évolution défavorable chez les patients atteints de TB multirésistante (rapport de risque ajusté [aHR] 1,74 ; IC à 95% 0,39­7,72) ou de DR-TB pré-extensive (aHR 0,65 ; IC à 95% 0,24­1,72). CONCLUSIONS: L'hétérorésistance a été observée de manière relativement fréquente (8,3%) chez les personnes atteintes de DR-TB aux Philippines. Néanmoins, nous n'avons pas identifié de preuves suffisantes pour établir un lien avec des résultats de traitement défavorables.

Understanding patient-level costs of weekly isoniazid-rifapentine (3HP) among people living with HIV in Uganda.

BACKGROUND: Twelve weeks of weekly isoniazid and rifapentine (3HP) prevents TB disease among people with HIV (PWH), but the costs to people of taking TB preventive treatment is not well described.METHODS: We surveyed PWH who initiated 3HP at a large urban HIV/AIDS clinic in Kampala, Uganda, as part of a larger trial. We estimated the cost of one 3HP visit from the patient perspective, including both out-of-pocket costs and estimated lost wages. Costs were reported in 2021 Ugandan shillings (UGX) and US dollars (USD; USD1 = UGX3,587)RESULTS: The survey included 1,655 PWH. The median participant cost of one clinic visit was UGX19,200 (USD5.36), or 38.5% of the median weekly income. Per visit, the cost of transportation was the largest component (median: UGX10,000/USD2.79), followed by lost income (median: UGX4,200/USD1.16) and food (median: UGX2,000/USD0.56). Men reported greater income loss than women (median: UGX6,400/USD1.79 vs. UGX3,300/USD0.93), and participants who lived further than a 30-minute drive to the clinic had higher transportation costs than others (median: UGX14,000/USD3.90 vs. UGX8,000/USD2.23).CONCLUSION: Patient-level costs to receive 3HP accounted for over one-third of weekly income. Patient-centered approaches to averting or defraying these costs are needed.

The importance of histidine residues in human ecto-nucleoside triphosphate diphosphohydrolase-3 as determined by site-directed mutagenesis.

Most ecto-nucleoside triphosphate diphosphohydrolases (eNTPDases) are inhibited by the histidine reagent diethyl pyrocarbonate (DEPC), while being resistant to inhibition by many other chemical modification agents. We used site-directed mutagenesis to investigate the sites of modification responsible for DEPC inhibition. First, we constructed the mutations H135A and R67H in eNTPDase-3 to address the possibility that, in eNTPDase-3, histidine 135 compensates for the lack of a histidine in apyrase conserved region (ACR) 1, present in all other membranous eNTPDases (but replaced by R67 in ACR1 of eNTPDase-3). We found histidine 135 is a major, but not the sole, target for DEPC-induced inhibition in eNTPDase-3. In addition, analysis of the R67H mutant led us to conclude that this site is important for DEPC inactivation of other eNTPDases. We also mutated singly and collectively three of the most conserved histidine residues present in eNTPDase-3 (129, 257 and 447) to alanine. None of the single, conserved histidine mutations nor the triple histidine mutation inactivated the enzyme or decreased susceptibility to DEPC inhibition. However, changes in the tendency of monomers to self-associate were noted, and the triple histidine mutant exhibited a higher nucleotidase specific activity than the wild-type.

Site-directed mutagenesis of human nucleoside triphosphate diphosphohydrolase 3: the importance of residues in the apyrase conserved regions.

Ecto-nucleoside triphosphate diphosphohydrolase 3 (eNTPDase-3, also known as HB6 and CD39L3) is a membrane-associated ecto-apyrase. Only a few functionally significant residues have been elucidated for this enzyme, as well as for the whole family of eNTPDase enzymes. Four highly conserved regions (apyrase conserved regions, ACRs) have been identified in all the members of eNTPDase family, suggesting their importance for biological activity. In an effort to identify those amino acids important for the catalytic activity of the eNTPDase family, as well as those residues mediating substrate specificity, 11 point mutations of 7 amino acid residues in ACR1-4 of eNTPDase-3 were constructed by site-directed mutagenesis. Mutagenesis of asparagine 191 to alanine (N191A), glutamine 226 to alanine (Q226A), and arginine 67 to glycine (R67G) resulted in an increase in the rates of hydrolysis of nucleoside diphosphates relative to triphosphates. Mutagenesis of arginine 146 to proline (R146P) essentially converted the eNTPDase-3 ecto-apyrase to an ecto-ATPase (eNTPDase-2), mainly by decreasing the hydrolysis rates for nucleoside diphosphates. The Q226A mutant exhibited a change in the divalent cation requirement for nucleotidase activity relative to the wild-type and the other mutants. Mutation of glutamate 182 to aspartate (E182D) or glutamine (E182Q), and mutation of serine 224 to alanine (S224A) completely abolished enzymatic activity. We conclude that the residues corresponding to eNTPDase-3 glutamate 182 in ACR3 and serine 224 in ACR4 are essential for the enzymatic activity of eNTPDases in general, and that arginine 67, arginine 146, asparagine 191, and glutamine 226 are important for determining substrate specificity for human ecto-nucleoside triphosphate diphosphohydrolase 3.

Expression and characterization of human ecto-ATPase and chimeras with CD39 ecto-apyrase.

Human ecto-ATPase (ecto-nucleoside triphosphate diphosphohydrolase 2 [eNTPDase2], also known as CD39L1) has been expressed and characterized in COS cells. It exhibits some unusual enzymology that is similar to a few members of this class of proteins but different from the majority of the family members. Hydrolysis of ATP by human ecto-ATPase is nonlinear with time, and its activity is stimulated/stabilized by both the lectin concanavalin A and the chemical cross-linking agent disuccinimidyl suberate. Like other members of the eNTPDase family, the human ecto-ATPase is a tetramer, the activity of which depends on its glycosylation. Chimeras of this protein with human CD39 (eNTPDasel) were constructed to test the hypothesis that the N-terminal half of these proteins regulates nucleotide specificity. The two chimeras generated demonstrated that the N-terminal half of these proteins is crucial for determining the relative activities of the nucleoside di- and triphosphatases. Chemical cross-linking of the two chimeras suggests that disuccinimidyl suberate interacts with the C-terminal half of ecto-ATPase in a manner that results in an increase of activity for both the ecto-ATPase and the ecto-apyrase/ecto-ATPase chimera.

Expression and characterization of soluble and membrane-bound human nucleoside triphosphate diphosphohydrolase 6 (CD39L2).

Ecto-nucleoside-triphosphate diphosphohydrolase-6 (eNTPDase6(1), also known as CD39L2) cDNA was expressed in mammalian COS-1 cells and characterized using nucleotidase assays as well as size exclusion, anion exchange, and cation exchange chromatography. The deduced amino acid sequence of eNTPDase6 is more homologous with the soluble E-type ATPase, eNTPDase5, than other E-type ATPases, suggesting it may also be soluble. To test this possibility, both the cell membranes and the growth media from eNTPDase6-transfected COS-1 cells were assayed for nucleotidase activities. Activity was found in both the membranes and the media. Soluble eNTPDase6 preferentially exhibits nucleoside diphosphatase activity, which is dependent on the presence of divalent cations. Western blot analysis of eNTPDase6 treated with PNGase-F indicated both soluble and membrane-bound forms are glycosylated. However, unlike some membrane-bound ecto-nucleotidases, the eNTPDase6 activity was not specifically inhibited by deglycosylation with peptide N-glycosidase F. Soluble eNTPDase6 hydrolyzed nucleoside triphosphates poorly and nucleoside monophosphates not at all. Analysis of the relative rates of hydrolysis of nucleoside diphosphates (GDP = IDP > UDP > CDP >> ADP) suggests that soluble eNTPDase6 is a diphosphatase most likely not involved in regulation of ADP levels important for circulatory hemostasis.

Acute high-dose methotrexate neurotoxicity in the rat.

Intravenous high-dose methotrexate chemotherapy may produce acute, subacute, or chronic neurotoxicity in patients with cancer. Acute encephalopathies following high-dose methotrexate treatment are recognized with increasing frequency. This study describes a model of acute high-dose methotrexate neurotoxicity in the rat characterized by a profound dose-dependent depression of cerebral glucose metabolism in association with behavioral and electroencephalographic abnormalities. Alterations in the amino acid profile, similar to those described in cancer patients after high-dose methotrexate treatment, were observed in the absence of biochemical evidence of systemic organ toxicity. This model facilitates the study of the biochemical mechanisms of antifolate neurotoxicity in humans and permits the evaluation of potential therapeutic interventions.

Pyrimidine dimers block simian virus 40 replication forks.

UV light produces lesions, predominantly pyrimidine dimers, which inhibit DNA replication in mammalian cells. The mechanism of inhibition is controversial: is synthesis of a daughter strand halted at a lesion while the replication fork moves on and reinitiates downstream, or is fork progression itself blocked for some time at the site of a lesion? We directly addressed this question by using electron microscopy to examine the distances of replication forks from the origin in unirradiated and UV-irradiated simian virus 40 chromosomes. If UV lesions block replication fork progression, the forks should be asymmetrically located in a large fraction of the irradiated molecules; if replication forks move rapidly past lesions, the forks should be symmetrically located. A large fraction of the simian virus 40 replication forks in irradiated molecules were asymmetrically located, demonstrating that UV lesions present at the frequency of pyrimidine dimers block replication forks. As a mechanism for this fork blockage, we propose that polymerization of the leading strand makes a significant contribution to the energetics of fork movement, so any lesion in the template for the leading strand which blocks polymerization should also block fork movement.

A symbol calculus for Toeplitz operators.

We give a complete characterization of those functions on 2n-dimensional Euclidean space for which the Berezin-Toeplitz quantizations admit a symbol calculus modulo the compact operators. The functions in question are characterized by a condition of "small oscillation at infinity."