Evaluation of nitrate reduction assay, resazurin microtiter assay and microscopic observation drug susceptibility assay for first line antitubercular drug susceptibility testing of clinical isolates of M. tuberculosis

Background

Drug resistant tuberculosis (TB) is a growing concern worldwide. Early detection of multidrug-resistant Mycobacterium tuberculosis is of primary importance for both patient management and infection control. Optimal method for identifying drug-resistant M. tuberculosis in a timely and affordable way in resource-limited settings is not yet available.

Aim

This study evaluated; nitrate reductase assay (NRA), resazurin microtiter assay (REMA) and microscopic observation drug susceptibility assay (MODS) against the conventional 1% proportion method (PM) for the detection of resistance to first line antitubercular drugs, in M. tuberculosis clinical isolates.

Methods

A total of one hundred and five clinical isolates of M. tuberculosis; 50 pan sensitive and 55 pan resistant were tested with NRA, REMA and MODS. The 1% proportion method on Lowenstein-Jensen medium was used as reference test.

Results

Of all three methods which were tested NRA was found to be most sensitive and specific. Sensitivity for rifampicin resistance detection was 100%, 94.55% and 92.73% by NRA, REMA and MODS respectively. NRA and REMA were found to be 100% specific, while the MODS was 98% specific for detection of rifampicin resistance. Test results with all these methods were obtained within 8-14 days.

Conclusion

Rapid non-conventional and inexpensive methods may serve as a replacement for 1% proportion method in resource limited settings.     

Evaluation of rapid alternative methods for drug susceptibility testing in clinical isolates of Mycobacterium tuberculosis

A study was carried out to compare the performance of a commercial method (MGIT) and four inexpensive drug susceptibility methods: nitrate reductase assay (NRA), microscopic observation drug susceptibility (MODS) assay, MTT test, and broth microdilution method (BMM). A total of 64 clinical isolates of Mycobacterium tuberculosis were studied. The Lowenstein-Jensen proportion method (PM) was used as gold standard. MGIT NRA, MODS, and MTT results were available on an average of less than 10 days, whereas BMM results could be reported in about 20 days. Most of the evaluated tests showed excellent performance for isoniazid and rifampicin, with sensitivity and specificity values > 90%. With most of the assays, sensitivity for ethambutol was low (62-87%) whereas for streptomycin, sensitivity values ranged from 84 to 100%; NRA-discrepancies were associated with cultures with a low proportion of EMB-resistant organisms while most discrepancies with quantitative tests (MMT and BMM) were seen with isolates whose minimal inhibitory concentrations fell close the cutoff MGIT is reliable but still expensive. NRA is the most inexpensive and easiest method to perform without changing the organization of the routine PM laboratory performance. While MODS, MTT, and BMM, have the disadvantage from the point of view of biosafety, they offer the possibility of detecting partial resistant strains. This study shows a very good level of agreement of the four low-cost methods compared to the PM for rapid detection of isoniazid, rifampicin and streptomycin resistance (Kappa values > 0.8); more standardization is needed for ethambutol.     

Direct nitrate reductase assay versus microscopic observation drug susceptibility test for rapid detection of MDR-TB in Uganda

The most common method for detection of drug resistant (DR) TB in resource-limited settings (RLSs) is indirect susceptibility testing on Lowenstein-Jensen medium (LJ) which is very time consuming with results available only after 2-3 months. Effective therapy of DR TB is therefore markedly delayed and patients can transmit resistant strains. Rapid and accurate tests suitable for RLSs in the diagnosis of DR TB are thus highly needed. In this study we compared two direct techniques--Nitrate Reductase Assay (NRA) and Microscopic Observation Drug Susceptibility (MODS) for rapid detection of MDR-TB in a high burden RLS. The sensitivity, specificity, and proportion of interpretable results were studied. Smear positive sputum was collected from 245 consecutive re-treatment TB patients attending a TB clinic in Kampala, Uganda. Samples were processed at the national reference laboratory and tested for susceptibility to rifampicin and isoniazid with direct NRA, direct MODS and the indirect LJ proportion method as reference. A total of 229 specimens were confirmed as M. tuberculosis, of these interpretable results were obtained in 217 (95%) with either the NRA or MODS. Sensitivity, specificity and kappa agreement for MDR-TB diagnosis was 97%, 98% and 0.93 with the NRA; and 87%, 95% and 0.78 with the MODS, respectively. The median time to results was 10, 7 and 64 days with NRA, MODS and the reference technique, respectively. The cost of laboratory supplies per sample was low, around 5 USD, for the rapid tests. The direct NRA and MODS offered rapid detection of resistance almost eight weeks earlier than with the reference method. In the study settings, the direct NRA was highly sensitive and specific. We consider it to have a strong potential for timely detection of MDR-TB in RLS.     

Latent nitrate reductase activity is associated with the plasma membrane of corn roots

Latent nitrate reductase activity (NRA) was detected in corn (Zea mays L., Golden Jubilee) root microsome fractions. Microsome-associated NRA was stimulated up to 20-fold by Triton X-100 (octylphenoxy polyethoxyethanol) whereas soluble NRA was only increased up to 1.2-fold. Microsome-associated NRA represented up to 19% of the total root NRA. Analysis of microsomal fractions by aqueous two-phase partitioning showed that the membrane-associated NRA was localized in the second upper phase (U₂). Analysis with marker enzymes indicated that the U₂ fraction was plasma membrane (PM). The PM-associated NRA was not removed by washing vesicles with up to 1.0 M NACl but was solubilized from the PM with 0.05% Triton X-100. In contrast, vanadate-sensitive ATPase activity was not solubilized from the PM by treatment with 0.1% Triton X-100. The results show that a protein capable of reducing nitrate is embedded in the hydrophobic region of the PM of corn roots.Abbreviations L₁ first lower phase - NR nitrate reductase - NRA nitrate-reductase activity - PM plasma membrane - T:p Triton X-100 (octylphenoxy polyethoxyethanol) to protein ratio - U₂ second upper phase     

A rapid detection of multidrug-resistant Mycobacterium tuberculosis by a nitrate reductase assay on blood agar

The susceptibility of 49 Mycobacterium tuberculosis clinical isolates to isoniazid (INH) and rifampisin (RIF) (28 multi-drug resistant-tuberculosis samples) was determined by a nitrate reductase assay (NRA) on blood agar. Agreement between the NRA and other testing methods was found to be 93.8% for both INH and RIF. The sensitivity, specificity, positive predictive value and negative predictive value for INH were 92.8%, 94.2%, 86.6% and 97%, respectively. The sensitivity, specificity, positive predictive value and negative predictive value for RIF were 90.4%, 96.4%, 95% and 93.1%. In conclusion, we show here that blood agar can be used effectively for the NRA test.     

Rapid detection of resistance against rifampicin in isolates of Mycobacterium tuberculosis from Brazilian patients using a reverse-phase hybridization assay

The main objective of this study was to evaluate INNO-LiPA Rif.TB and to determine the frequency of mutations in rpoB in rifampicin-resistant Mycobacterium tuberculosis isolates of Brazilian tuberculosis patients. We used the reverse hybridization assay on 113 resistant and 15 sensitive clinical isolates of M. tuberculosis and on reference strains belonging to 37 different species. All MTB complex strains and none of the other strains reacted with the MTB complex-specific probe, meaning that the assay is 100% specific and 100% sensitive for detection of strains of the MTB complex. In 80 resistant strains, mutations causing S531L (n=55), H526Y (n=9), H526D (n=12) or D516V (n=9) were detected while in 30 strains, mutations were present but their exact nature was not determined by the assay (DeltaS patterns). All sensitive strains had the sensitive genotype while among resistant isolates, a sensitive genotype was obtained in three due to the absence of mutations in the hot spot region, demonstrating an assay accuracy of 97.6% for detection of drug susceptibility. In 10 resistant cultures, two or more mutations were detected and in five, mixed sensitive and resistant genotypes were observed. The sensitivity of the assay for detection of resistant organisms in a mixture with sensitive ones were 2% and 70%, respectively, considering the appearance and disappearance of the R2 and S2 bands. The sensitivity to detect heteroresistance is similar to that of the proportion method when a specific probe for the mutation is present but the performance of the assay in the patient population will depend on the frequency of mutation distribution.     

Molecular identification and characterization of rifampicin-resistant Mycobacterium tuberculosis isolates by line probe assay: an approach for rapid diagnosis of multidrug-resistant tuberculosis

Aim:  Early identification and characterization of rifampicin-resistant (R^r) Mycobacterium tuberculosis isolates recovered from the samples of tuberculosis (TB) patients in the Aegean (West Anatolian) Region was intended.
Methods and Results:  Sixty isolates [47 (78·3%) multidrug-resistant (MDR)], which were identified as M. tuberculosis complex and phenotypically resistant to rifampicin by both BACTEC mycobacteria growth indicator tube (MGIT) 960 and 460 systems were analysed by a commercial line probe assay (INNO-LiPA Rif TB). The concordance of LiPA with the in vitro susceptibility test was found as 98·3%. Among the isolates, S531L (R5 pattern; 46·7%) and L511P/R, S512T, Q513L/K (ΔS1 pattern; 11·7%) were the most frequent mutation patterns. As compared with the BACTEC systems and conventional techniques for cultivation, identification and in vitro susceptibility testing, INNO-LiPA Rif TB after cultivation in BACTEC MGIT 960 system provided an average of 20 days early diagnosis of R^r M. tuberculosis isolates.
Conclusions:  Rapid molecular identification and characterization of R^r M. tuberculosis isolates after BACTEC MGIT 960 cultivation would be useful for faster diagnosis, infection control and planning of accurate treatment in MDR-TB patients.
Significance and Impact of the Study:  Patients with MDR-TB need a specified treatment and efficient follow-up strategies. Rapid and practical methodologies to diagnose and follow these patients should be applied in routine use.     

Evaluation of BACTEC MGIT 960 System for Testing Susceptibility of Mycobacterium tuberculosis to First-Line Drugs in China

Background

The purpose of this study was to evaluate the performance of the BACTEC MGIT 960 (M960) system compared with the proportion method (PM) on Löwenstein-Jensen (L-J) medium in a peripheral laboratory in China for the testing of Mycobacterium tuberculosis (MTB) susceptibility to streptomycin (SM), isoniazid (INH) rifampicin (RIF) and ethambutol (EMB) a combination known as SIRE.

Methods

The susceptibility of 205 clinical isolates of MTB to SM, INH, RIF and EMB was performed with the M960 system. The drugs were tested at the following concentrations: 1.0 µg/ml for SM, 0.1 µg/ml for INH, 1.0 µg/ml for RIF, and 5.0 µg/ml for EMB. The results were compared with those obtained by the L-J PM. The L-J PM at an arbiter site was used to resolve any discordant results.

Results

The overall consistency was 96.6% and concordance values were 95.6% for SM, 97.6% for INH, 98.0% for RIF and 95.1% for EMB. The overall sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) of the M960 system for PM (the standard method) was 95.6%, 97.3%, 96.2% and 96.9% respectively, and the sensitivity were 93.3% for SM, 96.9% for INH, 97.4% for RIF and 94.6% for EMB, the specificity were 96.9% for SM, 98.2% for INH, 98.4% for RIF and 95.5% for EMB, the PPV were 94.6% for SM, 97.9% for INH, 97.4% for RIF and 94.6% for EMB, the NPV were 96.2% for SM, 97.3% for INH, 98.4% for RIF and 95.5% for EMB. The turnaround time with the M960 system (median 8.0 days, ranged from 5 to 14 days) was significantly shorter than that with the PM (28 days or 42 days).

Conclusion

There was a substantial degree of agreement between the two methods. The M960 system was a reliable and rapid method for SIRE susceptibility testing of tuberculosis in China.     

三磷酸腺苷发光法快速检测结核分枝杆菌药敏技术的研究

目的 建立一种利用三磷酸腺苷( ATP) 与荧光素酶反应测定结核分枝杆菌( 简称结核杆菌) 释放的ATP 来判断结核杆菌药敏的技术。方法 ATP 生物发光法( 简称ATP 法) 通过裂解液体培养基中的结核杆菌, 释放活菌中的ATP, 加入荧光素酶使之发光以检测结核杆菌的活性。共采用H37Rv 标准株和10 株临床分离菌株, 用ATP 法与BACTEC 3D 法同步平行进行利福平药敏检测, 连续7 d 检测结核杆菌释放的ATP, 观察其生长曲线, 并以此判断对药物的敏感性。结果 在生长5 ～7 d的培养基中ATP法可以检测到敏感菌释放的ATP, 并且显著高于耐药菌所释放的ATP, 通过与BACTEC 3D 法相比确定其判断药敏的临界值, 检测结果与L-J 法及同步平行的BACTEC 3D 法对照组符合率达100% 。结论 ATP法可用于结核杆菌对抗结核药物敏感性的检测, 且因其价格较低, 无放射性元素的存在, 作为一种新型的结核杆菌药敏检测技术具有巨大的临床应用潜力。     

Rapid colorimetric testing for pyrazinamide susceptibility of M. tuberculosis by a PCR-based in-vitro synthesized pyrazinamidase method

Pyrazinamide (PZA) is an important first-line anti-tuberculosis drug. But PZA susceptibility test is challenging because PZA activity is optimal only in an acid environment that inhibits the growth of M. tuberculosis. For current phenotypic methods, inconsistent results between different labs have been reported. Direct sequencing of pncA gene is being considered as an accurate predictor for PZA susceptibility, but this approach needs expensive sequencers and a mutation database to report the results. An in-vitro synthesized Pyrazinamidase (PZase) assay was developed based on PCR amplification of pncA gene and an in vitro wheat germ system to express the pncA gene into PZase. The activity of the synthesized PZase was used as an indicator for PZA susceptibility. Fifty-one clinical isolates were tested along with pncA sequencing and the BACTEC MGIT 960 methods. The in-vitro synthesized PZase assay was able to detect PZA susceptibility of M. tuberculosis within 24 h through observing the color difference either by a spectrometer or naked eyes. This method showed agreements of 100% (33/33) and 88% (14/16) with the pncA sequencing method, and agreements of 96% (27/28) and 65% (15/23) with the BACTEC MGIT 960 method, for susceptible and resistant strains, respectively. The novel in-vitro synthesized PZase assay has significant advantages over current methods, such as its fast speed, simplicity, no need for expensive equipment, and the potentials of being a direct test, predicting resistance level and easy reading results by naked eyes. After confirmation by more clinical tests, this method may provide a radical change to the current PZA susceptibility assays.     

Characterization of the association of nitrate reductase with barley (Hordeum vulgare L.) root membranes.

The nature of the association between nitrate reductase (NR) and membranes was examined. Nitrate reductase activity (NRA) associated with the microsomal fraction of barley (Hordeum vulgare L.) roots amounted to 0.6 to 0.8% of soluble NRA following sonication in the presence of 250 mM KI and repeated osmotic shock. This treatment removed all contaminating soluble NRA from microsomes of uninduced barley roots that had been homogenized in a soluble extract from roots of NO3(-)-induced plants. On continuous sucrose gradients, NRA co-migrated specifically with VO4(-)-sensitive ATPase activity, a plasma membrane (PM) marker; activity of glucose-6-phosphate dehydrogenase, assayed as cytosolic marker, co-migrated with NRA. Microsomal NRA was absent in barley deficient in soluble NR. Perturbation and trypsinolysis experiments with PM vesicles isolated by aqueous two-phase partitioning indicated that NR is associated with the periphery of the cytoplasmic face of the bilayer. These results demonstrate that PM and soluble NRs are essentially the same protein but that the membrane-associated form is tightly bound. Although it is possible that PM-associated NR exists in vivo, unequivocal evidence for this has yet to be shown. However, PM NR is definitely present in vitro.     

A new Multi-PCR-SSCP assay for simultaneous detection of isoniazid and rifampin resistance in Mycobacterium tuberculosis

Prompt detection of drug resistance in Mycobacterium tuberculosis is essential for effective control of tuberculosis (TB). We developed a Multi-PCR-SSCP method that detects more than 80% commonly observed isoniazid (INH) and rifampin (RIF) resistance M. tuberculosis in a single assay. The usefulness of the newly developed method was evaluated with 116 clinical isolates of M. tuberculosis. Distinct SSCP patterns were observed for different mutations and the correlation between Multi-PCR-SSCP results and DNA sequencing data was strong. Using the culture-based phenotypic drug susceptibility testing as a reference, the sensitivity of the newly developed Multi-PCR-SSCP assay was determined to be 80% and 81.8% for INH and RIF, respectively. The specificity of the assay was 100% and 92%, for INH and RIF, respectively. Multi-PCR-SSCP provides a rapid and potentially more cost-effective method of detecting multidrug-resistant TB.     

Utility of nitrate reductase assay for detection of multidrug-resistant Mycobacterium tuberculosis in a low resource setting

Introduction. The performance of a drug susceptibility test may change when moving from the research stage to implementation on a population level in actual public health practice. Objective. The performance of a rapid drug susceptibility test was described for detecting multidrug-resistant Mycobacterium tuberculosis when implemented in the routine workflow of a low-resource reference laboratory. Materials and methods. A prospective study was done comparing the performance of the nitrate reductase assay with the conventional proportion method for rifampicin and isoniazid on 364 isolates were obtained from multidrug-resistant tuberculosis risk patients referred from diffrent Colombian laboratories. Results. When compared with the proportion method, the nitrate reductase assay sensitivity was 86.8% and 84.9% for rifampicin and isoniazid, respectively, whereas nitrate reductase assay specificity was 100% for isoniazid and rifampicin. Nitrate reductase assay sensitivity was significantly higher when the age of isolate was less than 70 days. A sensitivity of 94.4% dropped to 78.1% for rifampicin resistance for fresh and old isolates, respectively (Fisher exact test, p=0.05). For isoniazid resistance using fresh and old isolates, 94.7% vs.74.3% sensitivities, were achieved (chi square test, p=0.03). The proportion of nitrate reductase assay ambiguous results was significantly higher in multidrug-resistant than in non-multidrug-resistant isolates (17.6% vs. 4.0%, chi square test, p<0.005). Conclusions. The nitrate reductase assay demonstrated provided reliable results for antibiotic resistance. However, using old cultures leds to a higher proportion of false sensitive results; furthermore, the nitrate reductase assay capability to detect multidrug-resistant tuberculosis decreased due to a higher proportion of non-interpretable results.     

Potential application of dot-immunobinding assay as a rapid diagnostic test in tuberculous meningitis

A simple dot-immunobinding assay (Dot-Iba) in nitrocellulose paper was developed for the detection of specific IgG antibody to Mycobacterium tuberculosis antigen 5 and mycobacterial antigen in cerebrospinal fluid of patients with tuberculous meningitis (TBM). The assay gave 77.1% sensitivity for the detection of IgG antibody to M. tuberculosis antigen 5 and 48.6% sensitivity for the detection of mycobacterial antigen in patients with TBM.     

Detection of anti-tuberculosis activity in some folklore plants by radiometric BACTEC assay

Aims: The anti‐tubercular drugs are less effective because of the emergence of multi‐drug resistant (MDR) and extensively drug resistant (XDR) strains of M. tuberculosis, so plants being an alternative source of anti‐microbial compounds. The aim of this study was to investigate anti‐tuberculosis potential of the plants using Mycobacterium smegmatis as a rapid screening model for detection of anti‐mycobacterial activity and further to evaluate the active plants for anti‐tuberculosis activity against M. tuberculosis using radiometric BACTEC assay. Methods and Results: The 15 plants were screened for anti‐mycobacterial activity against M. smegmatis by the disk diffusion assay. The ethanolic extracts of Mallotus philippensis, Vitex negundo, Colebrookea oppositifolia, Rumex hastatus, Mimosa pudica, Kalanchoe integra and Flacourtia ramontchii were active against M. smegmatis in primary screening. The anti‐tuberculosis potential was identified in the leaves extracts of Mallotus philippensis by radiometric BACTEC assay. The ethanolic extract of M. philippensis showed anti‐tuberculosis activity against virulent and avirulent strains of M. tuberculosis H₃₇Rv and M. tuberculosis H₃₇Ra with minimum inhibitory concentration 0·25 and 0·125 mg ml^?1, respectively. The inhibition in growth index values of M. tuberculosis was observed in the presence of ethyl acetate fraction at a minimum concentration of 0·05 mg ml^?1. Conclusion: We found that BACTEC radiometric assay is a valuable method for detection of anti‐tuberculosis activity of the plant extracts. The results indicate that ethanolic extract and ethyl acetate fraction of M. philippensis exhibited significant anti‐mycobacterial activity against M. tuberculosis. Significance and Impact of the Study: These findings provide scientific evidence to support the traditional medicinal uses of M. philippensis and indicate a promising potential of this plant for the development of anti‐tuberculosis agent.     

A bispecific antibody based assay shows potential for detecting tuberculosis in resource constrained laboratory settings

The re-emergence of tuberculosis (TB) as a global public health threat highlights the necessity of rapid, simple and inexpensive point-of-care detection of the disease. Early diagnosis of TB is vital not only for preventing the spread of the disease but also for timely initiation of treatment. The later in turn will reduce the possible emergence of multi-drug resistant strains of Mycobacterium tuberculosis. Lipoarabinomannan (LAM) is an important non-protein antigen of the bacterial cell wall, which is found to be present in different body fluids of infected patients including blood, urine and sputum. We have developed a bispecific monoclonal antibody with predetermined specificities towards the LAM antigen and a reporter molecule horseradish peroxidase (HRPO). The developed antibody was subsequently used to design a simple low cost immunoswab based assay to detect LAM antigen. The limit of detection for spiked synthetic LAM was found to be 5.0 ng/ml (bovine urine), 0.5 ng/ml (rabbit serum) and 0.005 ng/ml (saline) and that for bacterial LAM from M. tuberculosis H37Rv was found to be 0.5 ng/ml (rabbit serum). The assay was evaluated with 21 stored clinical serum samples (14 were positive and 7 were negative in terms of anti-LAM titer). In addition, all 14 positive samples were culture positive. The assay showed 100% specificity and 64% sensitivity (95% confidence interval). In addition to good specificity, the end point could be read visually within two hours of sample collection. The reported assay might be used as a rapid tool for detecting TB in resource constrained laboratory settings.     

Direct rapid diagnosis of rifampicin-resistant M. tuberculosis infection in clinical samples by line probe assay (INNO LiPA Rif-TB)

M. tuberculosis is one of the leading causes of death worldwide and Multi Drug Resistant Tuberculosis (MDR-TB) is associated with a high case-fatality rate. Rapid identification of resistant strains is crucial to institute prompt appropriate therapy, and prevent the development of further resistance and spreading of MDR strains. The INNO-LiPA Rif. TB is a commercial reverse hybridisation line probe assay designed for rapid detection of rpoB gene mutations in clinical isolates. We applied this test directly to 44 smear-positive and 45 smear-negative clinical specimens collected from patients suspected of active TB. The capability of this technique to correctly identify local MDR-TB strains was tested on 50 MDR strains isolated in Italy. Results of the test were compared to conventional antibiogram performed on isolated strains. The concordance rate of the LiPA test results on clinical specimens with those obtained with "in vitro" sensitivity was 100%. These results show that the LiPA test can be useful in rapid detection and prompt management of tuberculosis when MDR disease is suspected.     

Impact of drug resistance on fitness of Mycobacterium tuberculosis strains of the W-Beijing genotype

Mycobacterium tuberculosis strains of the W-Beijing genotype became a common cause of tuberculosis during the past years and they are often associated with drug resistance. The biological factors facilitating the selection and wide dissemination of these strains are not known. To determine how acquisition of drug resistance affected growth of strains of the W-Beijing genotype, the growth of 55 M. tuberculosis isolates were studied using the BBL MGIT Mycobacteria Growth Indicator Tube and the BACTEC MGIT 960 System. Susceptible strains of non-Beijing genotypes were found to be the most fit strains. Drug-resistant strains of non-Beijing genotypes were more likely to grow slower than susceptible strains (P=0.001). Drug-resistant strains of the W-Beijing genotype had two tendencies of growth: some of them showed reduced growth compared to susceptible strains, while others did not show loss of fitness measured as growth.     

A dot-immunobinding assay (dot-Iba) for rapid diagnosis of pulmonary tuberculosis

IgG antibody to Mycobacterium tuberculosis from the sera of patients with 'definite' pulmonary tuberculosis (PT) was isolated and coupled with Cyanogen bromide-Sepharose 4B. Using an immunoabsorbent affinity chromatography, 14 kDa antigen was recovered from the culture filtrates of M. tuberculosis. With this mycobacterial antigen, a dot immunobinding assay (Dot-Iba) was developed for the detection of specific antibody to M. tuberculosis in the sera of patients with PT and controls. The assay gave positive results in all the 12 sputum-smear positive [acid fast bacilli (AFB)] patients with PT and gave negative results in the 50 sera from control groups. The Dot-Iba as described in this study, is simple, rapid and specific for laboratory diagnosis of PT.