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  Issues in Mycobacterium tuberculosis Complex (MTBC) Drug Susceptibility Testing: Pyrazinamide (PZA) 
 

Pyrazinamide (PZA) is a critical component of first-line drug combination therapy for

Mycobacterium tuberculosis complex (MTBC) including both susceptible and multi-drug resistant

tuberculosis (MDR TB). Inclusion of PZA has shortened the previous 9–12 month chemotherapy

regimen to 6 months.  PZA has also become an essential part of MDR TB treatment regimens

that include novel compounds now clinically available, such as bedaquiline.  PZA is inactive

against organisms in the growth phase during standard culture conditions at neutral pH. PZA has

a sterilizing effect due to its significant activity against non-replicating “persister” organisms or

semi-dormant slowly replicating bacilli at acid pH conditions (pH 5.5), killing bacilli that are not

eliminated by other TB drugs, such as those found in acidic regions of acute inflammation. 

The mechanism of action of PZA and resistance to PZA by Mycobacterium tuberculosis (MTBC)

is not well understood. Pyrazinamide is a pro-drug which requires conversion to its active form

of pyrazinoic acid (POA) by MTBC. Pyrazinamide enters mycobacteria by passive diffusion and is

then transformed in the cytoplasm by a nicotinamidase that has pyrizinamidase (PZase) activity,

encoded by the pncA gene of MTBC. Pyrazinoic acid accumulates in the cytoplasm and is actively

expelled by a putative efflux pump. Outside of the bacilli, POA is protonated and then re-enters

the organism and release of the protons occurs, resulting in an increasingly acidic cytoplasm

and the accumulation of POA. This disrupts membrane permeability and transport, resulting

in cellular damage.  Recently, the ribosomal protein S1 (translated from the rpsA gene) was

identified as a target of POA, which interferes with trans-translation activity, which is required for

efficient protein synthesis. 

The primary mechanism of PZA resistance is due to mutations in the pncA gene resulting in loss

of PZase activity, thus preventing conversion of PZA to POA.   In particular, mutations in specific

amino acid locations in the protein affecting catalytic sites of the PZase enzyme and Fe2+ ion

binding site cause loss of PZase activity and are associated with MTBC PZA resistance.  
   
  Issues in Mycobacterium tuberculosis Complex (MTBC) Drug Susceptibility Testing: Ethambutol (EMB) 
 

Ethambutol (EMB) is an antituberculosis drug used as part of the first line treatment against

Mycobacterium tuberculosis complex (MTBC).  A primary role of EMB when used in initial

combination antituberculosis therapy is to minimize the risk of drug resistance development

to companion first line drugs, particularly isoniazid.  Resistance to EMB will not decrease the

effectiveness or increase the length of treatment for MTBC susceptible to the other first-line

drugs. Within current dosing recommendations, EMB is bacteriostatic against actively replicating

bacteria but can be bactericidal when serum concentrations are over 10 μg/mL.   Ethambutol acts

through disruption of MTBC cell wall synthesis, targeting and inhibiting the function of arabinosyl

transferases, encoded by the embCAB operon, and responsible for biosynthesis of the cell wall

components arabinogalactan and lipoarabinomannan.  This disruption may lead to increased

permeability of the cell wall.  Ethambutol has also been found to inhibit RNA biosynthesis.

 

The recommended dose for EMB is 15-20 mg/kg once daily in combination with other

antituberculosis medications. It is used most often in the US in the initial multi-drug treatment

regimen if drug susceptibility results are not yet available and background drug resistance in the

community exceeds 4% or where isolates from a given case are determined to be resistant to other

medications.  Common side effects include optical neuritis, gastrointestinal upset, nausea, fever

dizziness and rash. According to information provided from the NIH on hepatotoxicity, the addition

of EMB to the drug regimen does not appear to increase serum aminotransferase elevations.

Ethambutol has only been associated with rare instances of acute, symptomatic liver injury.  

   

Determination of Mycotoxin and Mycotoxin Metabolites using LC/MS/MS
  Mycotoxins are the secondary by-products of fungal mold metabolism. The toxicity and potential weaponization threat of mycotoxins demands the need for sensitive, robust and rugged analytical methodologies. Current methods used by agriculture and food laboratories are often unable to detect a suite of these toxins. In addition, these methods do not address exposure to these compounds, where detection of the metabolites would be necessary for clinical diagnosis and treatment. Our laboratories have developed both a GC/MS/MS and an LC/MS/MS protocol for the determination of a select group of mycotoxins and their metabolites (Figure 1) in both environmental and clinical matrices.
   
Analysis of Organophosphorous Nerve Agent Metabolites by LC/MS/MS
  Nerve agents are the most well known and publicized chemical warfare agents. The chemical properties, lethality and history of these agents are well documented. Because of the potential use of nerve agents, methods for the detection and quantitation of these compounds are necessary. Of special interest is the development of methods to determine exposure levels to decrease the burden on the healthcare system. Our laboratory has developed and validated a method to measure the exposure to these agents by determination of the major metabolite the methyl phosphonic acids. This protocol uses solid phase extraction followed by electrospray negative ion LC/MS/MS analysis.
   
LC/MS/MS Determination of Urinary Concentrations of Insecticides and Herbicides in Professional Applicators
  The objective of this work was the development of a robust, high-throughput, and sensitive method for the determination of specific pesticides in urine of professional turf applicators following occupational exposures. The compounds of interest in this study are: the herbicides dicamba, mecoprop (MCPP), 4-chloro-2-methylphenoxyacetic acid (MCPA), and 2,4-D; the insecticides imidacloprid and bifenthrin, and their urinary metabolites 6-chloronicotinic acid (6- CNA) and 2-methyl-3-phenylbenzoic acid (MPA), respectively. Quantitative analysis was performed using solid-phase extraction (SPE) followed by negative ion electrospray ionization HPLC/MS/MS from approximately 120 professional applicators. In 2003, twenty (20) applicators provided 19 consecutive 24-hour urine samples. In 2004, approximately 100 applicators will provide a total of six, 24 hour urine samples over three different time periods (spring, summer and fall). Measured concentrations of these analytes are to be used in conjunction with pesticide application information to predict the total absorbed dose following multiple exposures in a large occupational field study. This information, along with information collected from each subject by questionnaire, will be used to conduct an external validation of previously developed epidemiologic prediction models. Data obtained in this biomonitoring study will also be used to develop recommendations to reduce occupational exposure and for health risk assessments.
   
Rapid Sample Preparation and Identification of Infectious Microorganisms using Matrix-assisted Laser Desorption/Ionization and Time-of-flight Mass Spectrometry
  The rapid detection of unknown infectious microorganisms in biological and environmental samples is a public health priority. The current Polymerase Chain Reaction (PCR) detection methods used in Public Health Laboratories are time consuming and results are not available for 24-48 hours after sample preparation. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF/MS) has been shown to be a much more powerful technology for the rapid screening for infectious microorganisms in biological samples. Previous work in this area has shown that it is possible to make distinctions at the strain level of microorganisms using a variety of MALDI-TOF/MS approaches. Studies have been performed to identify Escherichia coli strains based on ribosomal subunits obtained from whole cell lysates [1]. Whole cell approaches to differentiating between strains of microorganisms have been utilized with E. coli spp [2,3], Haemophilus spp[4], and Staphylococcus spp[5]. Screening for unknown infectious microorganisms is aided by the development of libraries of known microorganism spectra as well as pattern recognition software [6] and algorithm improvements [7] in recent years. The use of MALDI-TOF/MS is ideally suited for rapid screening of unknown infectious microorganisms due to the minimal amount of time needed for sample preparation prior to analysis.
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