Genomic surveillance of SARS-CoV-2 and emergence of XBB.1.16 variant in Rajasthan.

PURPOSE: Genomic surveillance of positive SARS-CoV-2 samples is important to monitor the genetic changes occurring in virus, this was enhanced after the WHO designation of XBB.1.16 as a variant under monitoring in March 2023. From 5th February till May 6, 2023 all positive SARS-CoV-2 samples were monitored for genetic changes. METHODS: A total of 1757 samples having Ct value <25 (for E and ORF gene) from different districts of Rajasthan were processed for Next Generation Sequencing (NGS). The FASTA files obtained on sequencing were used for lineage determination using Nextclade and phylogenetic tree construction. RESULTS AND CONCLUSIONS: Sequencing and lineage identification was done in 1624 samples. XBB.1.16 was the predominant lineage in 1413 (87.0%) cases while rest was other XBB (207, 12.74%) and other lineages (4, 0.2%). Of the 1413 XBB.1.16 cases, 57.47% were males and 42.53% were females. Majority (66.53%) belonged to 19-59 year age. 84.15% of XBB.1.16 cases were infected for the first time. Hospitalization was required in only 2.2% cases and death was reported in 5 (0.35%) patients. Most of the cases were symptomatic and the commonest symptoms were fever, cough and rhinorrhea. Co-morbidities were present in 414 (29.3%) cases. Enhanced genomic surveillance helped to rapidly identify the spread of XBB variant in Rajasthan. This in turn helped to take control measures to prevent spread of virus and estimate public health risks of the new variant relative to the previously circulating lineages. XBB variant was found to spread rapidly but produced milder disease.

Genomic profile of SARS-CoV-2 Omicron variant and its correlation with disease severity in Rajasthan.

Background: Omicron, a new variant of Severe Acute Respiratory Syndrome-Coronavirus 2 (SARS-CoV-2), was first detected in November 2021. This was believed to be highly transmissible and was reported to evade immunity. As a result, an urgent need was felt to screen all positive samples so as to rapidly identify Omicron cases and isolate them to prevent the spread of infection. Genomic surveillance of SARS-CoV-2 was planned to correlate disease severity with the genomic profile. Methods: All the SARS-CoV-2 positive cases detected in the state of Rajasthan were sent to our Lab. Samples received from 24 November 2021 to 4 January 2022 were selected for Next-Generation Sequencing (NGS). Processing was done as per protocol on the Ion Torrent S5 System for 1,210 samples and bioinformatics analysis was done. Results: Among the 1,210 samples tested, 762 (62.9%) were Delta/Delta-like and other lineages, 291 (24%) were Omicron, and 157 (12.9%) were invalid or repeat samples. Within a month, the proportion of Delta and other variants was reversed, 6% Omicron became 81%, and Delta and other variants became 19%, initially all Omicron cases were seen in international travelers and their contacts but soon community transmission was seen. The majority of patients with Omicron were asymptomatic (56.7%) or had mild disease (33%), 9.2% had moderate symptoms, and two (0.7%) had severe disease requiring hospitalization, of which one (0.3%) died and the rest were (99.7%) recovered. History of vaccination was seen in 81.1%, of the previous infection in 43.2% of cases. Among the Omicron cases, BA.1 (62.8%) was the predominant lineage followed by BA.2 (23.7%) and B.1.529 (13.4%), rising trends were seen initially for BA.1 and later for BA.2 also. Although 8.9% of patients with Delta lineage during that period were hospitalized, 7.2% required oxygen, and 0.9% died. To conclude, the community spread of Omicron occurred in a short time and became the predominant circulating variant; BA.1 was the predominant lineage detected. Most of the cases with Omicron were asymptomatic or had mild disease, and the mortality rate was very low as compared to Delta and other lineages.

Comparison of Ziehl Neelsen & Auramine O staining methods on direct and concentrated smears in clinical specimens.

BACKGROUND: In developing countries like ours with a large number of tuberculosis (TB) cases and limited resources, the diagnosis of TB relies primarily on smear microscopy for Acid Fast Bacilli (AFB) but its sensitivity is limited in paucibacillary cases. AIM: To evaluate the increase in efficacy of smear microscopy when smears are prepared from clinical samples after concentration by Petroff's method and stained by Auramine O (AO) fluorescent dye as against Ziehl Neelsen (ZN) staining of similar taking culture as the gold standard. METHODS: Smears were prepared from 393 clinical samples both by direct and after Petroff's concentration and examined by fluorescent microscopy and Ziehl Neelsen method .The concentrated material was also cultured on Lowenstein Jensen media and the results of the two microscopy methods were compared with the culture results taken as the gold standard. RESULTS: Mycobacterial growth was detected in 137 (35.77%) specimens, out of which three were non-tubercular mycobacteria. Using culture as the reference method, the sensitivity of direct staining was 55.55% for ZN and 71.85% for AO. Direct fluorescent microscopy detected 9.29% paucibacillary sputum samples that were missed on ZN staining. On concentration, the sensitivity increased by 6.67% for ZN and 11.11% for AO. The sensitivity of AFB smear microscopy increased by 27.41% and was statistically significant (p = < .001) when both methods were combined. The specificity was 99.19% for both ZN and AO. CONCLUSION: Fluorescent microscopy has higher sensitivity and comparable specificity which is further enhanced by concentration. Now with the advent of newer inexpensive Light Emitting Diode (LED) based fluorescent microscopes (FM), which are easier to use, fluorescent microscopy can be widely used even in peripheral laboratories where culture facilities are not available.

Comparison of MGIT 960 & pyrazinamidase activity assay for pyrazinamide susceptibility testing of Mycobacterium tuberculosis.

BACKGROUND & OBJECTIVES: Pyrazinamide is an important front line antimycobacterial drug, which is also being used in the treatment of multi drug resistant tuberculosis along with second line drugs in DOTS plus programme. Conventional testing of pyrazinamide on solid medium is difficult as it is active at acidic pH. Therefore, there is a need for a rapid and simple method for susceptibility testing of pyrazinamide. This study was carried out to compare pyrazinamide susceptibility testing by MGIT 960 and two rapid pyrazinamidase activity tests. METHODS: Pyrazinamide susceptibility was tested in 136 clinical isolates of Mycobacterium tuberculosis by MGIT 960 and pyrazinamidase activity was tested by classical Wayne's method and modified PZase agar method. RESULTS: There was 88.9 per cent concordance between MGIT 960 and classical Wayne's method and 93.38 per cent with modified method for pyrazinamidase activity. Using MGIT 960 results as gold standard the sensitivity and specificity of Wayne's method was 88.15 and 90 per cent respectively and that of modified method was 89.4 and 98.3 per cent. INTERPRETATION & CONCLUSIONS: Our study demonstrates that the modified pyrazinamidase activity test can be used as a screening test to detect resistance to pyrazinamide specially in resource limited settings but confirmation of susceptibility should be done by standard methods like MGIT 960.

Evaluation of a Rapid Differentiation Test for Mycobacterium Tuberculosis from other Mycobacteria by Selective Inhibition with p-nitrobenzoic Acid using MGIT 960.

Tuberculosis is caused by Mycobacterium tuberculosis (M.tb) as well as Non-tubercular mycobacterium (NTM) with similar clinical presentation. Infections due to NTM are reported to have increased in the past few years. Growth of M.tb is inhibited by p-Nitrobenzoic acid (PNB), whereas, NTM are resistant. One hundred and nine isolates from various clinical samples were identified up to species level by their growth rate, pigmentation, and a battery of biochemical tests, including niacin accumulation, nitrate reduction, and heat-stable catalase (68°C) reactions. Para-nitrobenzoic acid (PNB) inhibition test was performed to differentiate between M.tb and NTM. PNB was added to the Lowenstein-Jensen (LJ) medium and BACTEC™ MIGIT (Mycobacteria Growth Indicator Tube)960 medium to a final concentration of 500 µg/ml. All the M.tb isolates, including Mycobacterium tuberculosis H37Rv (standard strain), were inhibited by PNB on both LJ and MGIT 960. Of the NTM isolates, all were resistant to PNB on MGIT 960 and on LJ PNB, except one isolate of Mycobacterium marinum that was resistant to MGIT 960 PNB, but was susceptible to LJ PNB. The reporting time for M.tb ranged from 4-11 days (median 5.9 days) by MGIT 960 and for NTM it was 2-10 days with an average of 4.5 days. This study was carried out to establish the accuracy and efficiency of MGIT 960 PNB and to differentiate between M.tb and NTM.

Transport and storage of sputum specimen by using cetylpyridinium chloride for isolation of mycobacteria.

Of the 191 sputum specimens that were collected from pulmonary tuberculosis patients, 78.65% (140/178) specimens were culture positive when processed within 48 h by the NaOH method. The culture positivity in the same specimen that were stored with cetylpyridinium chloride (CPC) and processed after 7-8 days was 70.22% (125/178), whereas those stored without CPC and processed by the NaOH method was 46.62% (83/178). The difference in number of positive cultures obtained before storage and after storage (without CPC) was statistically significant (P = 0.001). Culture positivity by the CPC method was comparable with that of NaOH method before storage and the difference was not statistically significant (P = 0.35).

Optimum Time to Detection of Bacteria and Yeast Species with BACTEC 9120 Culture System from Blood and Sterile Body Fluids.

The culture results of 4,807 blood and 383 sterile body fluid specimens received in our laboratory during a 54-month period, were analyzed to determine the time required for culture to become positive, time at which a culture could safely be considered negative, and the spectrum of isolated organisms. The specimens were processed by automated BACTEC 9120 culture system. A total of 1,677 clinically significant microorganisms were isolated. Gram positive and negative bacterial isolation rates were found to be 62.55% and 32.20%, respectively. Yeasts were recovered in 5.24%. False positivity rate was 1.5%. Clinically significant isolates recovered on day four and five were 97.81% and 99.88%, respectively. At day five, the sensitivity was 99.94% and negative predictive value 99.96%. Our data support a five-day incubation protocol for recovery of all clinically significant organisms with sensitivity reduced by 0.06%, when compared with a six-day protocol.