Diversity Of Ntm Genotypes And Their Clinical Significance Biology Essay

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There has been an increase in the number and prevalence of Non-Tuberculous Mycobacteria (NTM) as well as advances in diagnostic methods and drug therapies for disease caused by these agents.1 Improved diagnostics have allowed enhanced detection and more-accurate species identification of Mycobacteria isolated from clinical specimens, including the classification of species not recognized earlier.2

Although often regarded as environmental contaminants with no evidence of human to human transmission, NTM are increasingly being associated with human illness. Mycobacterium avium complex (MAC) causes a tuberculosis-like disease (primarily in adults) and cervical adenitis (primarily in children). Bacteraemia is common in individuals with Acquired Immunodeficiency Syndrome (AIDS), resulting in the recovery of these organisms from various foci.3, 4 When NTM are isolated from normally sterile sites such as blood and bone marrow, diagnosis is usually easy. However isolates from non-sterile sources pose a challenge because they may be from environmental contamination and colonisation.5

In recent years NTM has emerged as a major cause of opportunistic infections in those who have AIDS. NTM disease in AIDS is caused primarily by M. avium.6 The isolation of NTMs generally raises questions of their clinical significance, especially in an African setting. It is recommended that assessment of this significance of NTM should be guided by the diagnostic criteria of the American Thoracic Society. According to these criteria, the diagnosis of NTM pulmonary disease must be based on solid clinical, radiographic, and bacteriologic factors.1, 7 The capacity for identification of mycobacteria is generally limited in many African countries. Clinically relevant infection due to NTM seems to occur in HIV-positive as well as in HIV-negative patients in Zambia. The role of NTM in human disease in a low-income and high HIV burdened setting such as Zambia may well be underestimated.7

Zambia is in the middle of a region where clinicians and program managers are increasingly confronted by Multidrug- Resistant Tuberculosis (MDR-TB) complex.8 The difficulty associated with the management of MDR-TB is worsened by the fact that NTM can have a drug susceptibility pattern similar to MDR-TB. Various conventional and molecular methods exist for the identification of Mycobacterium Tuberculosis complex (MTBc). However, it is thought that in many MDR-TB programmes, a substantial proportion of the patients actually have NTM rather than MDR-TB especially in high-HIV settings.9, 10 This proposed study intends to evaluate the identification methods used in Zambia to distinguish MTB from NTM and conduct genotypic analysis to establish the genetic diversity of these isolates in relation to the clinical significance.

Main objective

To determine the diversity of NTM genotypes common in clinical isolates in Zambia as well as their clinical significance in MDR-TB infections

Specific objectives

To Identify the common species of NTM in clinical isolates in Zambia

To determine the clinical manifestations of NTM disease in Zambia

To evaluate usefulness of the American Thoracic Society Criteria for diagnosis of NTM in a low income setting

To determine the extent to which NTM contributes to MDR-TB diagnoses in the National TB Programme

To determine the genotypic diversity of the NTMs isolated from clinical cases in Zambia.

Hypothesis

Genotyping of suspected MDR-TB cases proves that they are in fact due to NTM.

Justification

Although NTM is a group of environmental bacteria and rarely causes clinical disease, it has gained increasing importance in AIDS patients. Little is known about the significance of these organisms in Zambia. The extent to which they are responsible for clinical disease, their occurrence in cases of MDR-TB as well as the diversity of genotypes found in Zambia will be studied and contribute important clinical and scientific knowledge in the fight against TB.

Methods

Study design

This study will be a cross sectional survey of all clinical isolates of NTM archived by both the Zambart laboratory as well as the national and regional TB reference laboratories in Lusaka and Ndola. Further, we will investigate some patients whose sputum will be found to have NTM in order to determine the presence of NTM clinical disease according to the American Thoracic Society criteria (ATS).1 Patients suspected to have MDR-TB will be investigated to establish the presence of NTM.

Laboratory investigations

All the samples with positive smears for acid alcohol fast bacilli [AAFB] will be cultured at the TDRC Regional tuberculosis Reference laboratory, the Zambart laboratory as well as the Chest Diseases Laboratory (CDL) in Lusaka using the Mycobacteria Growth Indicator Tube (MGIT). Species identification will be done using the HAIN GenoType® MTBDRplus for identification of MTB and MDR-TB status. We will also use the GenoType® Mycobacteria CM to identify species of NTM. All the samples shown to be NTM will result in follow up of the patient so as to document clinical cases associated with NTM disease. To have complete first-line treatment drug susceptibility test (DST) results for MTBc, MGIT method will be used to determine MTBc drug resistance against Streptomycin and Ethambutol. Eligible and consenting patients will be requested to undergo voluntary counselling followed by HIV testing and CD4 count tests. Laboratory information already collected and documented by the CDL, TDRC and Zambart laboratories will be used in the characterisation of NTM species.

Study population

The study will be conducted in Ndola and Lusaka. . Thus the recruitment entry point will be the TB culture laboratory register from where all patients in whom NTM was isolated will be followed up through the TB focal point persons and treatment supporters. The patients identified as having positive culture of NTM by the TB culture laboratories will be eligible for recruitment from all districts in Zambia. A questionnaire will be administered to patients once they give consent to participate in the study. Data will be collected on demographic characteristics, risk factors, and evaluate whether the patients meet the criteria for the diagnosis of NTM clinical disease. A sputum sample will be collected to confirm the reported isolation of NTM.

All the cases identified as MDR-TB through the routine TB services will be evaluated to determine whether the MDR could be attributed to the non-tuberculous mycobacteria.

Sample size estimation

Based on preliminary data from the CDL lab, the prevalence of NTM isolates from sputum cultures was about 2.1%. Using the following formula:

n≥ (1.96)2 p(1-p)

δ2

n is the minimum sample size required

p is the proportion of interest (proportion of NTM isolates in sputum cultures)

δ is the precision required

From the foregoing, the Minimum sample size will be 180 in each arm

Data Management

Questionaire and laboratory data entry, cleaning and analysis will be performed using EPI-INFO and SAS software available at TDRC. Monovariate and multivariate analysis to relate the clinical and laboratory data will be applied.

Outcomes

The study will reveal novel information on the genetic diversity of NTM isolated in Zambia and their clinical significance in NTM disease. It will also determine the proportion of MDR-TB that can be attributed to NTM. We will also be able to determine the treatment outcomes of patients who will be diagnosed as having NTM and treated with the recommended antibiotics.

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