Post Surgical Non-tuberculous Mycobacterium: A Case Series

Background There has been an increase in non-tuberculous mycobacteria (NTM) infection reports in humans. Surgeons are concerned about the link between them and surgical site infections. As a result, it has been challenging to determine just how common this illness is. A two-year study by the authors examines the occurrence of NTM infections after different surgical procedures. Design and methods Researchers at a tertiary care hospital in central India performed this prospective study over two years. NTM was found in six of the 25 instances of post-surgical wound infections. Ziehl-Neelsen staining, Auramine O-Rhodamine fluorescence staining, Multiplex Real-Time PCR, and Genotyping were used to identify the species. Results M. fortuitum, M. chelonae and M.kansasii were isolated from discharge in 2 cases each respectively. NTM isolates evaluated for antibiotic susceptibility pattern were all sensitive to Clarithromycin (100%). One case with prolonged healing had to be intervened with amikacin along with clarithromycin. Conclusion NTM is an infection of uncommon nature which can occur following surgical procedures. Identification of these organisms through sensitive techniques and appropriate therapeutic regimen formulation must be done to tackle this growing menace in health care setups.


Introduction
Mycobacteria that do not cause tuberculosis (NTM) date back to the time of Robert Koch when he first discovered them [1]. These microorganisms are known to live in various environments, including water, animals, soil, and dairy products [2]. The Mycobacterium chelonae, Mycobacterium abscessus, Mycobacterium fortuitum and Mycobacterium smegmatis groups are most often linked with infections after NTM surgical intervention worldwide [3].
NTM is often linked to the presence of a foreign substance. According to conventional wisdom, infections are more likely when compromised skin's integrity. Antiseptic and disinfectant resistance is making these illnesses a growing threat. Surgical wound and skin biopsy, Ziehl-Neelsen staining, and mycobacterial cultures are not regularly performed; hence this burden is exacerbated [4].
These organisms can produce biofilm because of their hydrophobic nature. When these microorganisms shed, they produce biofilms that increase the likelihood of infection [5]. The infection from surgical wounds can be prevented by adequately sterilizing tools and endoscopes. The literature depicts the contamination of instruments and surgical site infections [6].
Globally, nosocomial infection outbreaks and cases have increased in recent years [6]. There have been reports of NTM in post-operative wounds in India [7][8][9][10][11]. Literature suggests that NTM is becoming more common, yet it may be difficult to diagnose. Long-term medication and lowered tolerance make it more

Inclusion criteria
Delay in wound healing, breakdown of wounds, discharge or inflammation in the wound site, presence of nodule within the vicinity of wounds and those resistant to antibiotic intervention were included.

Exclusion criteria
Emergency surgical cases and wounds developed within seven days after surgery were excluded. Immunocompromised individuals and participants not consenting to the study were not included.

Data collection
A case sheet elaborating on pre-operative and post-operative history and clinical examination was recorded for all cases.

Sample collection
A total of six swab discharge samples were collected from the deepest possible point of the wound. The swabs under strict aseptic measures were evaluated in our dedicated Biosafety Level II (BSLII) laboratory for mycobacterium evaluation.

Microbiological investigation
The samples were taken one by one as they were recruited. Gram stain smear was prepared from the first sample. Similarly, Ziehl Neelsen (ZN) and Auramine O -Rhodamine fluorescent staining (Acridine Orange) were done from the second and third samples. Genotype NTM-DR Version 1.0 to detect NTM used the fourth sample. Culture and Antimicrobial Susceptibility tests were processed from the fifth and sixth samples.

Sample processing
Using N-Acetyl-L-Cysteine -Sodium Hydroxide as a decontaminant, samples were centrifuged at 3000 rpm for 15-20 minutes before being suspended in 2 ml phosphate buffer for further analysis. The obtained supernatant and the decontaminated sample were then processed by real-time PCR test to demonstrate NTM at the infection site. Specimens obtained were subsequently cultured on Culture Media -7H11, 7H9, and LJ (Lowenstein-Jensen) media to identify Rapidly Growing Mycobacteria (RGM) isolates.

Results
Twenty-five patients with post-surgical infections were included in this research, with an average age of 41.23 years. Patients that tested positive for NTM were on average 37.33 years old, with a ratio of four men to two women. Surgical interventions are enumerated in Table 1.  All isolates were sensitive to clarithromycin (CLR), while only one NTM confirmed case required Amikacin added along with CLR, which was coincidentally associated with diabetic comorbid conditions. No surgical treatment was given along with clarithromycin to facilitate the healing of the wounds. Clarithromycin was given for two months in these six cases.

Variables
The mean duration of follow-up of the patients after the initiation of clarithromycin therapy was a minimum of six months for all cases. The lesions did not recur in any of the patients after stopping clarithromycin. The mean healing of wounds was 11.283 days.
The patients treated are recovering at different rates, which is why species that have tested positive have hampered the healing process in various ways and to varying degrees.

Discussion
Previous studies report an incidence of NTM ranging from 3.4% to 24.7% in India. An increase in reporting is due to better adaptation of histopathology, adherence to culture technique, and species diagnosis with DNA sequencing. Surgical predisposition varied amongst cases, with no tendency for either closed or open surgeries [13].
Culture sensitivity to ZN staining was detected to 83.33% of confirmed NTM infections. This was in nonconcordance to the study of Ghosh et al. [14], who reported sensitivity in a lesser number of cases. This was attributed to inappropriate sampling technique, which inhibited the transfer of highly hydrophobic microbe to solid media or inadequate pretreatment before sample collection.
Generally, the ZN stain and culture are extensively used for diagnosing mycobacterial infections. AFB staining is a fast and inexpensive approach; however, it has poor sensitivity (22-78%), making it difficult to distinguish between NTM and M. tuberculosis [15]. Our study had 83% sensitivity to ZN staining. Advanced PCR techniques have 100% sensitivity and specificity for the DNA isolated from cultured specimens and can detect NTM faster than conventional PCR. The current study employed real-time PCR which targeted the 16S rRNA, rpoB, and hsp65 genes providing a success rate of 65%, 82%, and 87%, respectively [16]. M. kansasii NTM isolates were found in our study, which is scarcely reported in published literature. First isolated in 1953, this yellow species colony carries a hereditary predisposition or shared environmental susceptibility. Conflicting reports of prevalence are found, suggesting significant regional variability. The enhanced sensitivity of Genotyping sequence (CM kitty) utilized in the present study played a pivotal role in identifying this rare colonizing organism [18]. Antibiotic susceptibility testing in the present study was by the disc diffusion method. RGM susceptibility pattern varies significantly with different species across the geographical location. It is recommended by the Infectious Diseases Society of America and the American Thoracic Society that all-important NTM isolates be tested for susceptibility before therapy can begin. Though micro broth dilution is the recommended choice by the Clinical and Laboratory Standards Institute (CLSI), the disc diffusion technique remains a reliable method [19][20][21].
Clarithromycin was effective against all NTM isolates. Amikacin was added along with clarithromycin in only one case of M. fortuitum infection. Hundred percent sensitivity of NTM to these drugs was also demonstrated in previous studies [22][23][24]. Our study did not require the usage of fluoroquinolones or multidrug regimen comprising macrolide or aminoglycoside. Literature documents intralesional injections as well, the need for which did not arise in the current study [23]. All cases responded to Clarithromycin 500 mg BD. The only exception was the NTM infection in the shoulder implant surgery case, which took longer to heal and had lesions at two different sites, the elbow and shoulder location. The post-surgical wound of the elbow was sensitive to clarithromycin (CLR), while the shoulder lesion had to be treated with Amikacin four doses per week and took a long time to heal (181 days). In this context, emphasis is placed on reporting NTM infection and sensitivity patterns.
NTM infection increases due to inappropriate disinfection or sterilization of reusable medical equipment [25].
The multifactorial nature of infection cannot be overlooked. Though all surgical interventions followed similar infection control protocols during surgery and were exposed to the same environmental situation, the sporadicity of cases manifesting in only a few cases remains unclear. Our study addressed confounding issues by rigid inclusion of cases not to include immunosuppressed. The response of the individual human host, variation of NTM pathogenicity, and virulence are certain factors to be borne in mind.
In the context of the study, the limitations included the exclusion of comorbid condition patients, and the sample size was minimal, which can be dealt with just as a pilot trial. There is a significant limitation concerning the species tested that could be tested for more species.

Conclusions
NTM infection is vastly implicated in surgical wound infections. Difficulty in diagnosing and prolonged morbidity caused by these microorganisms further add to the burden of the existing health care setup. The need of the hour is to increase awareness regarding the rising occurrence of NTM post-surgically, as it plays a significant role in the therapeutic regimen. Culture sensitivity and species identification are mandatory to provide optimal antimicrobial intervention. Surgical care for patients with recurring skin and soft-tissue infections necessitates understanding the risk.

Additional Information Disclosures
Human subjects: Consent was obtained or waived by all participants in this study. Peoples College of Medical Sciences and Research Centre's Research Ethics Committee issued approval IEC No. PCMS/OD/2019/470. Animal subjects: All authors have confirmed that this study did not involve animal subjects or tissue. Conflicts of interest: In compliance with the ICMJE uniform disclosure form, all authors declare the following: Payment/services info: All authors have declared that no financial support was received from any organization for the submitted work. Financial relationships: All authors have declared that they have no financial relationships at present or within the previous three years with any organizations that might have an interest in the submitted work. Other relationships: All authors have declared that there are no other relationships or activities that could appear to have influenced the submitted work.