Mycobacterium Tuberculosis Diagnosis

A policy must be decided for determining which specimens of sputum are to be examined for tubercle bacilli. Pulmonary tuberculosis is both a threat to the life of the patient and a dangerous source of infection to others. It is therefore that cases should be correctly diagnosed at the earliest possible stage and that drug therapy should be based on sensitivity tests made on a culture grown from the patient.
In communities where tuberculosis is moderately or very common, every specimen of sputum received in the laboratory should be screened for tubercle bacilli, regardless of whether the physician requests the examination. At least microscopy of a Ziehl-Neelsen or auramine-stained smear should be done and the positive specimens then cultured.
Dangerous suspected patients are all patients with unexplained cough continuing for more than 4-6 weeks, elderly persons with supposed ‘smoker’s cough’, coughing patients with haemoptysis or cachexia, persons who have been in close contact with patients diagnosed as tuberculous, and patients with illnesses that make them specially vulnerable (e.g. AIDS).
Laboratory staff must be protected against the risk of becoming infected from specimens containing tubercle bacilli. If, therefore, there nay be cases of pulmonary tuberculosis in the patient population served by the laboratory, all specimens of sputum should be regarded as possibly dangerous and the procedures of making smears and seeding cultures should be Performed in a safety cabinet.
Methods
-- Samples processing: All samples ARE subjected to decontamination by NaOH -NALC (N-acetyl-L-cysteine) method and then concentrated by centrifugation for 20 min at 10,000 g . Then the sediments were resuspended in sterile normal saline and subjected to Ziehl Neelsen (Z.N) smear and culture. Sterile samples like CSF, blood samples for Mycobacterium tuberculosis culture are not subjected to decontamination process.
- Loewenstein-Jensen (LJ) culture: Ready to use bottles of Loewenstein-Jensen media (Hispan Lab, Medicopharmatrade. Co, Madrid, Aspen) are inoculated with 0.l ml of concentrated sputum sediments and will be kept in CO2 incubator for 8 weeks. Bottles are inspected twice a week for visible colonies and suspicion growth is subjected to Z.N staining. Negative culture is discarded after 8 weeks (biohazard wate handling). Resulting growth is left in light for 2 hours and examined for yellow pigments to identify photo-chromogen species.
-Culture using radiometrie BACTEC 460 TB system: BACTEC 460 TB system and BACTEC 12B vials (Becton Dickinson Microbiology system, Cockeysville, Md.) were used for radiometrie culture of MTB . BACTEC 12B vials are prepared by addition of antimicrobial agents "PANTA" which contains polymyxin, amphotericin B, nalidixic acid, trimethoprim, and azlocillin. One half ml of concentrated sputum sediments are inoculated into each BACTEC 12B vial and kept at 37°C and then tested 3 times a week. Positive cultures as indicated by BACTEC460 are subjected to further identification by NAP test (P-Nitro-acetyl amino hydroxyl-Proionophenone)for the presence of typical Myucobacterium tuberculosis. The system is obsealate now
The usually used system now is MGIT Bactec 960

-Antimicrobial Drug susceptibility testing (AST): Isolated TB colonies from BACTEC 12B media and from LJ media will be subjected to AST using both Agar proportion method and BACTEC 460 TB system .
a) Agar proportion method: The modified agar proportion method using Middlebrook 7H10 agar plates are used to determine the susceptibility of MTB isolates(Inderlied1996). Briefly, antibiotic stock solutions are diluted and added to Middlebrook 7H10 agar containing 10% oleic acid-albumin-dextrose (OADC) to give the following critical concentrations in quadrant plates: isoniazid (INH) 0.2 µg/ml; rifampicin (RIF) 1µg/ml; ethambutol (EMB) 5.0 µg/ml; streptomycin (SM) 2.0 µg/ml. One hundred microliter aliquots of diluted bacterial samples are inoculated to quadrants of drug containing or drug free media. Drug resistance in 7H10 method of proportion was defined as 1% or more growth of colonies on the drug containing agar quadrant compared to growth on the drug free quadrant. Results were recorded at 21 days after inoculation.
b) BACTEC 460 TB system: The recommendation of the manufacture for sensitivity testing, the modified critical concentrations of provided drugs (SIRE, Becton Dickinson) are adopted: 2 µg/ml for SM; 0.1 µg/ml for INH; 2.0 µg/ml for RIF; and 2.5 µg/ml for EMB.
-Bacteriophage methods or PCR using primers for the target sequences.
-Direct PCR to detect Mycobacterium tuberculosis in clinical samples.
-Tuberculin skin test. Inject 5-10 Iu/ml PPD reagent intradermal in forearm by insulin syringe an surround the area of injection by pen. Read result after 24-48 hours. Interpretate positive if diameter if induration is 10mm or more.

Natural Killer Activity and Mycobacterium tuberculosis Infection

There is recent increase in tuberculosis with multidrug resistant strains. On the other hand little information is available about the role of natural killer cells (N.K) in the immunity to tuberculosis and antituberculous sensitivity.
In this work 25 chest patients and 6 controls were studied for N.K activity by release of Cr from erythroleukaemia cell line. T.B diagnosis was performed by culture and sensitivity with Bactec system . specific serology (1gA , 1gG, 1gM) and tuberculin test.
All patients have typical mycobacteria tuberculosis with sensitivity 72% to isoniazid, 56% to rifampicin, 36 % to ethambutol, 32% to streptomycin and 16 % were resistant to all drugs . Tuberculin was positive in 56%, 1gG and 1gM in 16 % and 1gA in 8%. There was significant decrease (P < .0001) in N.K activity in patients compared to control. The results suggest that the N.K cells play role in defense mechanism to T.B.
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Hybridization and automated Molecular Methods for Laboratory assessment of Mycobacterium tuberculosis

lRapid detection and identification of Mycobacterium tuberculosis (M. tuberculosis) complex  in respiratory samples are extremely important for optimal diagnosis and effective treatment, as well as for prevention and control of tuberculosis transmission

lDiagnostic means of tuberculosis (TB), have considerably improved. Available standardized nucleic acid-based amplification techniques were shown to yield reliable results within 5 to 7 hours of sample processing.

lIn spite of their theoretical ability to detect even a single mycobacterial cell, nucleic acid amplification tests (NAATs) are not sufficiently reliable to replace conventional diagnostic methods for pulmonary tuberculosis (TB)

l Both inherent test characteristics and errors in testing procedures may account for their inaccuracy

lOn the other hand, false positive results arise most often from contamination of negative samples with either organisms or target DNA from samples containing large numbers of mycobacteria or from amplicons contaminating the laboratory room

lTo overcome these problems, automated commercial systems were developed which were made more robust by the use of standardized procedures and reagents for sample processing, amplification, and detection.

lOn the other hand, false positive results arise most often from contamination of negative samples with either organisms or target DNA from samples containing large numbers of mycobacteria or from amplicons contaminating the laboratory room

lTo overcome these problems, automated commercial systems were developed which were made more robust by the use of standardized procedures and reagents for sample processing, amplification, and detection.

lThese procedures, which allow different steps of the process to take place in a single sealed tube, were intended to reduce the risk of contamination.

lAt the same time, the use of larger sample volumes or the introduction of internal amplification controls to detect inhibitors was adopted to cut down the rate of false negative results

lThe US Centers for Disease Control (CDC) recommend that commercial NAATs be used with microscopy to improve diagnostic certainty (pending culture results and/or patient’s response to treatment) and that clinicians should rely on their clinical judgment in the interpretation of results.

lAccording to the CDC, the diagnosis of pulmonary TB can be presumed in smear positive (acid-fast bacilli (AFB)+) patients with a positive NAAT result and in smear negative (AFB–) patients with two subsequent positive NAAT results.

lHere we described  our experience in Egypt for diagnosis of Mycobacterium tuberculosis with molecular techniques

lWe use  fully automated PCR system that use seminested PCR  (Acu-Gene M. tuberculosis quantitative test ) for determination of Mycobacterium tuberculosis determine positive clinical samples with low level of bacilli doses.

lAlso we asses the presence of rifampicin resistant tuberculosis strains by cultural method by BACTEC 460 system and   proportional agar method compared to determination  the type of  mutation pattern of rpoB gene in those strains by line probe  assay  and DNA sequence analysis

 Acu-Gene M. tuberculosis quantitative test (Amplisen­sor assay for quantitative TB)

lDNA Amplisensor assay for M. tuberculosis is based on the principle of amplisensor  assay to determine the initial target ­dosage of M. tuberculosis through PCR amplification.

lThe test involves a two-stage amplification scheme: (1) an initial asymmetric amplification to overproduce one strand of the target sequence and (2) a subsequent heminested amplification to monitor AmpliSensor (a fluorogenic primer duplex) as it is converted into amplification product.

lThe assay quantifies the amount of the target sequence by correlating the initial target dosage to a “transition” cycle number, which marks the beginning of signifi­cant AmpliSensor conversion. To prevent false negative, the assay calls for a polymerase activity check all the re­agents for sample preparation and complete reaction mixes for amplification and detection.

l All the procedures of assay set up and reagent preparation should be per­formed in an UV irradiated clean hood.

 Line probe assay

lTest principle: It is based on amplification of the rifampicin resistance region of gene encoding for the subunit of RNA polymerase (rpoB). Amplified biotinylated DNA material was hybridized with specific oligonucleotide probes immobilized as parallel lines on membrane based strips.

l After hybridization, streptavidin labeled with alkaline phosphatase was added and bound to any biotinylated hybrid previously formed. Incubation with BCIP/NBT chromogen resulted in purple/brown precipitate.

lThe presence of MTB complex in the sample was monitored by MTB complex-specific probe. The reactivity of an amplified fragment with one or more of the 5 wild type probes (Sl to S5) was prevented by the presence of a mutation within the probe region. Four additional probe; R2, R4a, R4b and R5 were expected to hybridize to mutant sequences of the most commonly observed mutations

 

 

 

Mycobacterium Tuberculosis Clinical Aspects

Tuberculosis (TB) represents a major public health problem, especially in low-resource countries where the burden of the disease is more important. According to estimates of the World Health Organization (WHO), two billion people, roughly one third of the world’s population is latently infected with Mycobacterium tuberculosis the causative agent of the disease. In 2005 there were 8.9 million new cases of TB and 1.6 million deaths were attributed to the diseas. This scenario is aggravated by the human immunodeficiency virus (HIV) pandemic with roughly one third of the 40 million people currently infected with HIV also coinfected with TB.

The world can be divided into two parts based on the extent of tuberculosis epidemics. One part is the low-prevalence areas. They are composed of countries that experienced serious tuberculosis epidemics after the 18th century but have gradually overcome them and have finally reduced the incidence rate to 100 per 100 000 or less. The other part is the high-prevalence areas comprising countries with an incidence rate exceeding 100 per 100 000 that have suffered tuberculosis epidemics after the turn of the 20th century. The low-prevalence countries are industrialized countries, while the high-prevalence countries are mostly developing counties or areas. The latter accounts for two-thirds of the world population, but as much as 95% of the estimated number of newly occurring tuberculosis patients (of all forms) globally. Furthermore, 98% of tuberculosis deaths occur in these high-prevalence areas.¹ Tuberculosis accounts for 2.7% of the total disability-adjusted life-years in low- and middle-income countries.⁴ In addition to the difference in its level, there are clear differences in characteristics of tuberculosis disease. In high-prevalence countries, most tuberculosis patients are in their 20s to 40s, resulting in tremendous socioeconomic loss as this is the most productive generation. In contrast, among low-prevalence countries, tuberculosis is drifting to involve the elderly, socioeconomically marginalized people, medical high-risk groups (e.g. diabetics⁵ and those treated with immunosuppressive agents, such as TNF-alpha blockers⁶), which presents a challenge to both medical and welfare services.

As a consequence of the global efforts in tuberculosis control under the Directly Observed Treatment Short-course strategy since the 1990s, the incidence of tuberculosis is estimated to have started to decline for the first time around 2003, although very slowly.¹ At the same time, issues that had been given only lower priority in the developing world have emerged as unavoidable challenges. One of these issues is multi-drug resistant (MDR) tuberculosis that strikes a half million people annually and is a malignant burden to the patients and community, as well as to national tuberculosis programmes with its poor treatment outcome.⁷ In line with this problem, extensively drug resistant (XDR) strains of M. tuberculosis are emerging recently.⁸ The use of effective secondary drugs based on the result of high-performance drug sensitivity tests is necessary in order to address these issues, which requires technical innovation.⁷

A second newly emerging issue is co-infection of the HIV and M. tuberculosis. Currently, 15% of the new tuberculosis patients are infected with HIV, and in some areas or countries this proportion exceeds 50%. One quarter of the global tuberculosis deaths are due to HIV, and this is equal to one-third of new HIV-positive tuberculosis cases and to 23% of the estimated two million HIV-related deaths in 2007.¹ Diagnosing tuberculosis in these subjects with sputum smear examination alone cannot prevent their infectiousness and save their lives; more aggressive case-finding and treatment of smear-negative cases are required. Another issue is tuberculosis in children for whom Mycobacterium bovis Bacille Calmette Guerin (BCG) vaccination has been virtually the only control measure in developing countries. This also requires accurate diagnosis in the early stage of tuberculosis.⁹

As seen above, tuberculosis appears as a typical south-north problem of health, but currently in many developed countries over half of the new tuberculosis cases are foreign-born, that is, immigrants from high-prevalence areas, or spill-over of tuberculosis.^10,11 It is actually argued that to further reduce tuberculosis in the low-prevalence countries it is necessary to strengthen control efforts in the high-prevalence, developing countries.¹²

Clinical Manifestation of Mycobacterium tuberculosis

In general, clinical manifestations of illness are the first clue to the diagnosis of tuberculosis. However, they are often non-specific and misleading, and therefore the diagnosis is not always easy. This is especially the case for tuberculosis in children and in elderly subjects, as seen below. Further, extrapulmonary tuberculosis often poses a challenge to early diagnosis, especially pertaining to its variety of presentations. Clinical signs and symptoms of tuberculosis of the organs other than the lungs are summarized in ^134,302

Pleuricy

Primary infection cases have generally an acute course of symptoms than reactivation-type TB. Cough (non-productive) and chest pain (pleuritic—sharp, stabbing, associated with respiration). Feverishness, dyspnea, chills, sweats, weight loss in more advanced cases. TB pleural effusions are generally exudates.

Lymphadenopathy

Most often in the neck and head region, rare in the axillary & inguinal region. Right predominates but 1/4 have biliateral & 78% have multiple lesions. 41% have pulmonary TB. In superficial LN, lesion starts as a painless enlargement, with no inflammation over the skin, then may undergo pustulation and fistulation over several weeks or months. In a case with limited lesion, general symptoms are rare

Bone & Joint

Commonest in vertebral column, followed by hip and knee. Fever and wasting may appear in large inflammatory collections, but the local manifestation predominates. Pain is commonest. Soft tissue collection (cold abscess) at/near the bone or joint focus. Neurological signs (weakness or numbness from compression of the spinal cord).

Disseminated or miliary TB

Diverse depending on the organs involved. Feverishness, weakness or debility, anorexia, weight loss, headache (meningeal complication), abdominal pain/swelling (peritoneal involvement), cough.^39,40

Central nervous system

The presentation depends on the size and location of the tuberculoma and the pressure it produces.Early symptoms (feverishness, malaise, anorexia, irritability, headache) followed by neurological symptoms (progressive headache, lethargy, personality changes, memory disturbance, impaired cognition, confusion), and then stupor-coma with or without neurological deficit.

Cerebrospinal fluid for pressure, cellularity, protein, glucose,⁴⁶ MTB (microscopy, culture^47,48 and PCR^49,50), immunology (ELISA, IgG immune complex, antibody assays and IGRA^51–56), and ADA. Radiography, CT, MRI.^57–61 Meningeal biopsy (histology, MTB).

Other infections (fungal, viral, trypanosomal, bacterial), vascular (multiple emboli, SBE, thrombosis of sagittal vein), collagen vascular (SLE, polyarteritis, and others).

Abdominal

Frequency: peritonitis, followed by ileocaecal, anorectal and mesenteric lymph node infection.In peritoneal TB, abdominal swelling, fever, ascites, pain, anorexia/weight loss are common.^62,63

Peritonitis: ultrasound,^64–66 laparoscopy (with guided biopsy),^67–70 paracentesis of ascites for culture and IGRA^71,72 and ADA.⁷³

Malignant ascites, cirrhosis with spontaneous bacterial peritonitis, starch peritonitis, sarcoidosis, NTM peritonitis.

Pericarditis

Dyspnoea, tachycardia, neck vein distension, oedema, hepatomegaly, paradoxical pulse, pericardial rub, fever.^74,75

Pericardial tissue/fluid for bacteriology, histology,^76,77 IGRA,^78,79 and ADA.^80–82 Echocardiography,^83–85 CT and MRI (pericardial effusion and thickening),⁸⁶ ECG (low voltage, inversion of T).⁸⁷

Bacterial (e.g. Pneumococcus), viral (e.g. CMV, HSV, Coxsackievirus) or fungal (e.g. Aspergillus) infections; collagen vascular diseases; uremia; post-myocardial infarction or post-pericardiotomie; malignancy; trauma.

Genitourinary

Dysuria, frequency, nocturia, urgency, pain in the back, flank or abdomen, tenderness/swelling of the testis or epididymis, haematuria. Superimposed urinary tract infection with other bacteria in urinary stasis cases.^88–93

Tuberculosis in children

Because of its paucibacillary nature, tuberculosis of children is difficult to diagnose. Bacteriological confirmation seldom exceeds 30–40% among children in developed as well as developing countries.^106,107 Consequently, the diagnosis of tuberculosis in children in resource-poor settings is largely dependent on a combination of a history of contact with a known tuberculosis patient, clinical signs and symptoms, and special examinations, such as chest radiography and the TST when available. Edwards and colleagues observed a total of 91 tuberculosis cases younger than 15 years, of whom about half were HIV-infected, and found the following frequency of symptoms and signs in the HIV-seronegative children: weight loss 69%, fever 100%, cough 83%, night sweat 43%, fatigue 21%, tuberculosis contact 60%, malnutrition 57%, lymphadenopathy 88%, organomegaly 31%, positive TST 89%, elevated erythrocyte sedimentation rate 79%, and chest X-ray infiltration 100%.¹⁰⁸ Based on these observations, several point-scoring systems, diagnostic classifications and diagnostic algorithms have been developed to support an objective diagnostic judgment. Marais et al. tested such an approach and found that combining a persistent non-remitting cough lasting over 2 weeks, documented deterioration of health (in the preceding 3 months) and fatigue provided reasonable diagnostic accuracy in HIV-uninfected children (sensitivity 62.6%; specificity 89.8%; positive predictive value 83.6%). The performance was poorer in HIV-infected children than in the low-risk group, which offers a serious challenge in resource-poor settings with high HIV epidemics.¹⁰⁹ However, given this set of sensitivity and specificity, the positive predictive value is calculated as only 24% in a patient population with a prevalence of tuberculosis of as high as 5%.

Tuberculosis in old ages

In low-prevalence situations, tuberculosis is a problem predominantly of the aged population and includes many more cases of clinical development in immunologically compromised subjects. This is why there are many tuberculosis cases with ‘atypical’ clinical presentation(s) in older persons.^110,111 Elderly patients are more likely to have extrapulmonary tuberculosis, including miliary disease.¹¹¹ The proportion of bacteriologically confirmed pulmonary tuberculosis patients was higher in the elderly than in the younger patients as reported in a meta-analysis.¹¹²

Fever, sweating and haemoptysis are less frequent in older patients, but dyspnoea is more frequent.¹¹² Laboratory findings, such as the TST-positive rate, serum total protein level and white blood cell counts, were lower in elderly patients. Also, cavity formation was less common in elderly patients, while lesions in the upper lung were similar for both age groups.¹¹² The most common chest X-ray findings in the elderly or immunocompromized tuberculosis patients are lesion in the lower zone accompanied by basal effusion or thickening.¹¹⁰ Such atypical clinical presentation of tuberculosis in the elderly can often cause delay in diagnosis, which can be further complicated due to underlying illnesses.

One of the basic indicators of quality in diagnosing tuberculosis is the delay in diagnosis (‘doctor's delay’, or ‘health system's delay’), that is, the time from the first visit of a patient until the establishment of tuberculosis diagnosis. Figure 2 depicts the delay separately for high-, intermediate- and low-prevalence settings, together with the patient's delay, that is, the time from the onset of clinical symptoms until the first visit to a health facility, based on published studies (T. Mori, pers. comm.). It is remarkable that these delays in the low-prevalence settings are always longer than those in the high-prevalence settings.

Sasaki et al. reviewed the diagnostic process of private practitioners with Japanese patients and concluded that insufficient medical work-ups, including AFB examinations of sputa and chest X-rays of subjects with a high suspicion for tuberculosis, was the principal cause of delayed diagnoses.^113,114 In Hong Kong, general practitioners' practice was reviewed, and it became clear that they depend too much on X-rays rather than sputum examinations, and that they were slow in referring tuberculosis patients to the government tuberculosis service.¹¹⁵ Rozovsky-Weinberger et al. compared the management of suspect tuberculosis cases at three public hospitals and seven not-for-profit private hospitals in the USA in terms of their rates of ordering acid-fast smears and isolations, and urged private hospitals to be more alert to tuberculosis.¹¹⁶ Similar reviews of hospital management were reported by several other studies,^117–119 the results of which illustrate the need for improved education of doctors. All of these studies urge a higher index of suspicion for tuberculosis in medical staff in low-prevalence countries.

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