Jamwal Ashima^1*, Swami Anjali M², Baveja Sujata³

¹Speciality Medical Officer, ²Associate Professor, ³Professor and HOD, Department of Microbiology, Lokmanya Tilak Municipal Medical College and General Hospital, Sion, Mumbai, Maharashtra, INDIA.

Email: Ashi.jamwal@gmail.com

METHODOLOGY

The economic variant of the proportion method is recommended by the RNTCP guidelines for DST testing. Drug concentrations added to LJ Media and critical proportion for interpretation for the economic variant of proportion method were:

Table 1

One set of LJ media bottles for testing were taken. One culture consists of five LJ slopes, one for neat, two for 10^-2 and two for 10^-4; eight LJ drug-containing slopes i.e. two each for drugs H, R, E and S (one each for 10^-2 and 10^-4 suspensions) and one for PNB slope, a total 14 LJ slopes were inoculated. The following procedure was adopted according to RNTCP guidelines (NTEP now);

1 ml SDW (Sterile Distilled Water) with six 3 mm glass beads + 1 loop-full (3 mm loop) of culture

 ↓

Was Vortexed for 20 – 30 seconds

 ↓

Added 4 ml of SDW to the above suspension of culture isolate

 ↓

Turbidity matched with McFarland std.1

 ↓

S2-10^–2 two loop-full of neat + 2ml of SDW

 ↓

S4-10^–4 two loop-full of S2 + 2ml of SDW

LJ media slopes were then labelled and inoculated respectively. The inoculated slopes were incubated at 37⁰C. The growth was seen at 28 days and again at 42 days. Dividing the number of colonies in drug-containing slopes by that in drug-free slopes gave the proportion of resistant bacilli existing in the strain. Below 1%, the critical proportion, the strain was classified as sensitive; above that value, it was classified as resistant. If the result of the reading made on the 28th day was “resistant”, no further reading of the test for that drug was required: the strain was classified as resistant. If the result at the 28th day was “sensitive”, a second reading was made on the 42nd day only for the sensitive strain. The final definitive results for all the four drugs were reported on the 42nd day. If the strain was resistant to all the four drugs on the 28th day, then the report was given on the same day. Otherwise, incomplete reports were not be given before the 42nd day. Growth was interpreted as : Confluent growth = 3+ , More than 100 colonies = 2+ ,Actual number of colonies = 1-100 cols. When the number of colonies on a given dilution was less than 5, the number of colonies was counted with the next larger inoculum or estimated if more than 100. No attempt was made to estimate the number of colonies if the growth is 3+. The data obtained was subjected to statistical analysis using computer software (SPSS version 23; Chicago Inc., USA). The qualitative data were expressed in proportion and percentages and the quantitative data expressed as mean and standard deviations. Statistical analysis was done using descriptive statistics.

RESULTS

All 150 patient samples were included in the present study. 21-30 years (40.6%) age was the most common age group, followed by 31-40 years (22.7%). The mean age among the distribution of cases was 28.16 ±10.12 yrs. There was a higher Male (53.3%) preponderance observed in 21-30 yrs age and male to female ratio was 1.14:1. The maximum sample collected from patients was pus (30.8%), followed by pleural fluid (16%), CSF (15.3%), tissue (12%), lymph node biopsy (10%), ascitic fluid (9.3%), urine (3.3%) and Knee aspirate/Synovial fluid (3.3%). AFB smear positivity by ZN staining was seen in 31 specimens (20.7%). Using LJ medium. growth on culture was observed in 25 (16.7%) specimens whereas 125 did not show any growth. The sensitivity of ZN staining was 48% specificity and 78.7% accuracy. On comparing smear and culture positivity, in 25 culture positive specimens AFB smear positivity on ZN staining was seen in 12 specimens whereas 13 were smear negative. Five specimens being smear positive did not show any growth on LJ and 125 were smear as well as culture negative. Out of 25 culture positive specimens, 2 were NTM’s while 23 were confirmed M. tb when tested on Mpt64 rapid antigen detection kit. Among the 23 M. tuberculosis positive patients, 11 (47.8%) showed resistance to the four first-line anti-tubercular drugs, 3 (13.0%) isolates showed monoresistance to Rifampicin and 2 (8.7%) to Streptomycin. MDR was observed in 5 (21.7%) isolates while polyresistance to HES was noted in a single isolate (table given below).

Table 2: Patterns of resistance of MTB isolates to the first-line anti-tubercular drugs:(n= 23)

{*Resistant to two or more drugs but not both isoniazid and rifampicin; EMB=Ethambutol; INH=Isoniazid; MDRTB=Multidrug-resistant tuberculosis (resistant to at least both isoniazid and rifampicin with or without resistance to other drugs); RIF=Rifampicin; SM=Streptomycin} Overall, the distribution of samples showing resistance were as; 6 (54.5%) pyogenic samples (Pus) which showed resistance i.e. 4 with MDR and 2 with monoresistance to Streptomycin, followed by 2 tissue samples (18.2%) i.e. 1 MDR and 1 with monoresistance to Rifampicin. Two lymph node biopsy (18.2%) showing polyresistance and monoresistance respectively. Single pleural fluid sample (9.1%) reported monoresistance to Rifampicin. Among 12 new cases, only 2 (16.7%) cases showed monoresistance to Streptomycin each and the rest 10 were susceptible to all drugs as compared to 9 (81.8%) in previously treated cases which showed resistance to HR (5 isolates; 45.5%) followed by Rifampicin monoresistance (3 isolates; 27.3%); and HES (1 isolate; 9.1%).

DISCUSSION

Isolation of mycobacteria from clinical specimens is considered to be the best method for the diagnosis of extrapulmonary tuberculosis (EPTB). Culture isolation followed by reliable and accurate DST is essential to determine an effective treatment regimen and to avoid further development of drug resistance. In the present study, 21-30 years (40.6%) was the most common age group, followed by 31-40 years (22.7%) and male to female ratio was 1.14:1. Sharma et al.⁶ reported the mean age similar to the present study and 15-41 years age group was most common (71%) in a study by Sethi et al.⁷ In the present study the sensitivity of ZN staining was 48% specificity and 78.7% accuracy, similar findings were observed by Ozkutuk et al.⁸ while Munir et al.⁹ presented the findings which were discordant with present study. These differences in observations are quite likely due to the paucibacillary nature of the sample and large inter-observer variability. We observed the culture positivity in 25 (16.7%) isolates. Comparable results were reported by Dusthackeer et al.¹⁰ and Maurya et al.¹¹ Patients with drug-resistant tuberculosis are increasing rapidly due to high population density, high prevalence of TB and an overstretched public, coupled with an unregulated private health sector. The overall prevalence of drug resistance was found to be 47.8%. Dusthackeer et al.¹⁰ reported an overall drug resistance of 41% from southern India. Overall sensitivity to all four anti TB drugs was 52.17% in the present study. Maurya et al.¹¹ reported similar results. The overall prevalence of monoresistance in the present study was observed to be 21.7%, 13 % to only Rifampicin and 8.7% to Streptomycin respectively. Menon et al. noted overall monoresistance concordant with the present study. No case of monoresistance to Isoniazid was recorded in the present study. The emergence of MDR TB has been attributed to factors such as nonadherence to treatment, inappropriate treatment regimens, drug malabsorption, poor drug quality and poor health infrastructure for effective delivery of treatment. Overall, the prevalence of MDR TB (resistance to Isoniazid and Rifampicin) was observed to be 21.7% in the present study. Menon et al.¹⁴ reported a similar outcome of multidrug resistance. In the present study, it was observed that among 12 new cases only 2 (16.7%) cases showed resistance, i.e. to Streptomycin only as compared to 9 (81.1%) in previously treated cases which showed resistance to HR (45.5%) followed by Rifampicin only (27.7%) and HES (9.1%). Similarly, D’Souza et al.¹⁷ and Sethi et al.⁷ reported concordant results. The present study had a limitation of including a small number of cases since the results may vary with a larger sample size. Also, the clinical outcomes of the drug-resistant TB cases were not studied.

CONCLUSION

In a city like Mumbai, with increasing population density and poor living conditions, Tb burden is on the rise. Although time consuming, LJ culture DST remains a gold standard in the diagnosis of TB. The difficulty in the identification of the emerging heteroresistance is a matter of concern when we totally rely on molecular diagnostics. Also the paucibacillary nature of EPTB samples make it difficult to diagnose on smear microscopy (5000-10,000 bacilli/ml), so the culture positivity holds a great value as it can detect 10-100 bacilli of concentrated material. The expensive set-ups and the requirement of an expertise hand is a drawback in the molecular diagnostics compared to culture DST, therefore its cost-effectiveness is an advantage which cannot be ignored especially in the developing countries. Also the studies with larger subsets of population should be undertaken.

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