Atypical mycobacterial infection at laparoscopic port site: Report of two cases indicating possibility of multi drug resistant strains in north-east India and review of literature.

A 45-year-old female presented with a painful indurated swelling in the anterior abdominal wall (right hypochondrium paraumbilical port site) 4 weeks after laparoscopic cholecystectomy. Ultrasonography showed two collections. One collection was located on anterior abdominal wall (3.5×2 cm) and appeared loculated, with evidence of a sinus tract extending into peritoneal cavity. Another collection was seen in the left iliac fossa (18 ×29 mm). Computed Tomography (CT) of the abdomen revealed an intramuscular collection measuring18x24x51 mm. Ultrasound-guided aspiration was performed, and the aspirated pus was sent for Gram stain, acid-fast bacilli (AFB) stain, atypical mycobacterial culture and the Mantoux test. Chest x-ray was normal. Both TRUNAAT and CBNAAT were negative. The erythrocyte sedimentation rate (ESR) was 140 mm/h, and the Mantoux test was positive. The patient was started on anti-tubercular therapy (ATT) comprising isoniazid, rifampicin, pyrazinamide and ethambutol for 6 months, along with amoxicillin-clavulanate 625 tablets, esomeprazole and domperidone. After 1 month, the ESR began to decrease, which was considered as sign of treatment response. After three months of ATT, the lesions continued to progress. Ultrasound-guided aspiration and Magnetic Resonance Cholangiopancreatography (MRCP) were due to the suspicion of bile duct leak. MRCP showed multifocal ill-defined T2FS hyperintense lesions in both lobes of liver with abscess formation, more in the segment VIII of liver likely hepatic tuberculosis. Smaller simple cysts seen in both lobes of liver. Loculated port site collections seen in anterior abdominal wall in right hypochondriac region. Ethambutol and rifampicin were continued, and clarithromycin 500 mg and ciprofloxacin were added because of the suspicion of atypical mycobacteria. As the lesions continued to progress after one month of the above therapy. MRI of the whole abdomen (plain and contrast) was performed. MRI revealed irregular loculated subcapsular/perihepatic collections with mild perilesional oedema. Hepatomegaly with cysts in both lobes was noted. The intrahepatic bile ducts were not dilated. The common hepatic duct and common hepatic bile duct were prominent, likely representing post cholecystectomy reservoir status. Loculated infective collections were identified in the left lower anterior abdominal wall and right upper anterolateral abdominal wall, without intraperitoneal extension. Compared with the MRCP performed one month earlier, a mild reduction in the size of right upper anterolateral abdominal wall collection was observed. The patient was started on Cefuroxime 500 mg twice daily. Aspiration of collection in left anterior abdominal wall showed 22000 cells/cumm of fluid with neutrophil 68%, lymphoid cells 12%, lymphoplasmacytoid cells 10% and histiocytoid cells 10%. Marked degenerative debris (with cell debris) were seen. Microbiology report showed no growth of organism after 48 h of aerobic incubation at 37℃. TB culture examination showed Mycobacterium fortuitum after 7days of incubation. Tablet ethambutol, rifampicin, clarithromycin 500 mg and ciprofloxacin 500 mg were continued as earlier. As the lesions continued to progress slowly, ultrasonography (USG) of the anterior abdominal wall was repeated after one month. It showed a loculated collection similar to that was observed earlier. However, a sinus tract extending into the peritoneal cavity was identified. Another similar collection (15x38mm) was noted in the subcutaneous plane of anterior abdominal wall in the left iliac fossa, with extension into underlying rectus muscle. However, further communication with the peritoneal cavity could not be ruled out. The patient was considered as a non-responder to clarithromycin and ciprofloxacin. The patient subsequently moved to another centre, where macrolide/fluoroquinolone resistant NTM-PSI was suspected. Surgical debridement with excision of the sinus tract was performed. The bacteriological examination of the excised tissue on KOH microscopy showed no fungal elements, AFB staining was negative, Gram stain revealed few pus cells with no bacteria. Biopsy from port site and anterior abdominal wall lesions showed necrotizing subacute inflammation with ill formed granulomas. In post-operative period, the patient received injection tigecycline 50 mg iv twice daily for two weeks, injection imipenem-cilastatin 1 g iv twice daily for two weeks, tablet linezolid 600 mg once daily for one month, injection amikacin 750 mg on thrice a week for two months and tablet azithromycin 500 mg once daily for four months. The patient subsequently recovered and remained well during 24 months of followup. A 32-year-old female underwent laparoscopic surgery for endometriosis. Six weeks later, she developed induration at the left paraumbilical port site (left hypochondrium) and the drain port site (left iliac fossa). Two weeks later, she developed a semi-purulent discharge from both the port sites. The patient was started on cefuroxime 500 mg orally twice daily for 5 days; however, the discharge persisted. Ultrasound revealed sinus tract formation measuring 18 mm depth at the left hypochondrium above the rectus sheath and 24 mm depth at the left iliac fossa drain port, without involvement of the underlying muscle layers. The patient was evaluated by an infectious disease specialist and empirically initiated on tablet clarithromycin 500 mg twice daily, tablet minocycline 100 mg twice daily and tablet cotrimoxazole (800 mg/160 mg) twice daily. Owing to intolerance, minocycline was replaced with doxycycline (100 mg twice daily). Subsequently, co-trimoxazole was discontinued due to adverse effects. Tablet linezolid 600 mg once daily was initiated along with clarithromycin and doxycycline. After one and half months of therapy, the left hypochondrial wound had healed, and the discharge had subsided. Repeat ultrasound showed resolution of the sinus tract in left hypochondrial region; however, the left iliac fossa wound had progressed to involve the underlying muscle layer and peritoneum. The patient subsequently presented to a higher centre where, MRI revealed a 4.3 cm linear STIR hyperintense sinus tract in the left iliac-fossa, extending from peritoneal cavity with adjacent mild fat stranding, traversing the internal and external oblique muscles and subcutaneous tissue, and opening externally onto the skin surface. Oral antibiotics were discontinued, and surgical excision was planned. The excised tissue was sent for bacteriology, histopathology and rapid-identification using Bactifast. Post operatively, the patient received injection imipenem-cilastatin 1 g IV twice daily for two weeks, injection amikacin 750 mg every alternate day and, tablet linezolid 600 mg once daily for two months, tablet azithromycin 500 mg once daily and tablet clofazimine 100 mg once daily for four months. Excision biopsy showed sinus tract with subacute inflammatory granulation tissue and well-formed granulomas. Fungus culture showed no fungal growth. Microbiological examination revealed growth of Pseudomonas aeruginosa . Following this management, patient recovered completely and remained disease free during seven months of followup.

Siva Kumar Shanmugam: Visualization, Validation, Supervision, Investigation. Daisy Hazarika: Writing – original draft, Resources, Methodology, Investigation, Conceptualization. K S Santhosh Anand: Visualization, Validation, Investigation. Sarma Dr Phulen: Writing – review & editing, Visualization, Validation, Supervision, Methodology, Investigation, Formal analysis, Data curation, Conceptualization. Anusuya Bhattacharyya: Visualization, Validation, Supervision, Methodology, Formal analysis, Conceptualization. Sankar Jyoti Bora: Writing – review & editing, Writing – original draft, Methodology, Formal analysis, Conceptualization. Mukesh Kumar Sathya Narayanan: Supervision, Investigation, Conceptualization. Abhilash Goyal: Visualization, Validation, Supervision, Investigation, Data curation, Conceptualization. Rajkumari Mazumdar: Visualization, Validation, Supervision. Sayan Chakraborty: Visualization, Validation, Investigation. Anirban Bhattacharjee: Investigation. Deb Kumar Boruah: Supervision, Investigation, Formal analysis. Pranjal Phukan: Visualization, Validation, Supervision, Investigation, Formal analysis. Bhupen Barman: Validation, Supervision, Methodology, Investigation. Deepjyoti Kalita: Visualization, Validation, Supervision, Investigation, Conceptualization. Kalyan Sarma: Visualization, Validation, Supervision, Investigation.

Written informed consent to take part in the study and to publish the article has been obtained from all participants or their legal representatives. The privacy rights of participants have been observed. This study was performed in compliance with relevant laws, regulatory frameworks and guidelines where the research took place. Ethics committee approval was not required under relevant laws and institutional guidelines. Ethics committee approval was not required for the publication of anonymized clinical case reports. This research follows the CARE guidelines and the CARE checklist.

Not applicable.

Written informed consent was obtained from the patient for publication of this case report and accompanying images. A copy of written consent is available for review by the Editor-in-chief of this journal on request.

Non tubercular mycobacterial infections are rare but serious complications of laparoscopic surgery and should always be considered in patients presenting with delayed PSI. Prevention relies on strict sterilization of laparoscopic instruments, the use of disposable trocars and adhering to infection control protocols in the operating rooms. The treatment consists of both antibiotic therapy and surgical excision, ideally guided by culture and drug susceptibility testing. In the view of increasing antimicrobial resistance, early surgical excision may lessen the morbidity. The prognosis is generally favourable when NTM infections are diagnosed early and treated adequately. However, delayed diagnosis and inadequate treatment may result in chronic infections and persistent wound related complications.

The advent of laparoscopy has transformed modern operative practice and has led to its widespread adoption across various specialties. These procedures are more commonly performed in females, and hence, port site infections (PSIs) also reported more frequently among women. Mycobacterium abscessus , Mycobacterium Chelonae , Mycobacterium fortuitum and Mycobacterium flaescens are among the most frequently isolated NTM species associated with PSIs [4] . The incidence of non-tuberculous mycobacterial (NTM) infections in India appears to be increasing. However, no established guidelines are currently available for the management of NTM-PSIs. In this context, we conducted a systematic literature review to understand the spectrum of laparoscopic surgery associated NTM infections reported from India. The search strategy is provided in supplementary table −1 and the details of the included cases are provided in supplementary table −2. Patients reported from India presented with similar postoperative complications following laparoscopic surgery and generally required prolonged combination antibiotics therapy for 3–6 months or longer (details in supplementary table 2 ). Both patient and procedure related factors have been associated with an increased risk of port-site infection. These include hypertension, diabetes mellitus, hypothyroidism, acute cholecystitis, and intraoperative spillage of bile, gallstones, or pus, particularly through the epigastric port [5] .Among patients undergoing laparoscopic cholecystectomy, the reported incidence of PSI is approximately 3.9%, whereas the prevalence of port site tuberculosis is around 0.2%, underscoring that even minimally invasive procedures meticulous careful infection control measures. Most PSIs have been reported in the context of laparoscopic cholecystectomy. NTM infection may involve a single or multiple port sites, most commonly the epigastric and umbilical ports. These are typically hospital-acquired and are associated with contaminated instruments endogenous contamination from infected tissue [6] . Clinically, NTM-PSIs usually present as erythematous swellings with mild pain that gradually progress to abscess formation and chronic sinus tracts discharging sterile pus, with little or no response to standard empiric antibiotics. Systemic manifestations are usually minimal. Because NTM infections mimic other bacterial or fungal infections, they are frequently misdiagnosed, particularly in countries with high tuberculosis (TB) burden, where diagnostic facilities and clinician awareness remain limited [7] . NTM infections pose significant diagnostic hurdles because of their diverse clinical manifestations, slow growth rate, and the requirement for specialized laboratory techniques for accurate identification [8] , [9] . The diagnosis is established using AFB staining, histopathology or FNAC (demonstrating granulomatous inflammation), mycobacterial culture and GeneXpert [5] . Conventional smear microscopy cannot differentiate NTMs from Mycobacterium tuberculosis , necessitating the use of culture and molecular tools, such as PCR, sequencing, or MALDI-TOF MS for accurate species identification [10] . Cultures should be performed using both liquid (e.g., MGIT) and solid media (e.g., Middlebrook 7H10, 7H11, or Lowenstein-Jensen), with liquid systems like MGIT−960 demonstrating high sensitivity and specificity for NTM detection. Recent advancements, such as NTM Elite agar, demonstrate improved sensitivity and selectivity over traditional media [11] . PCR assays such as Xpert MTB/RIF, can help exclude co-infection. Proper sample collection, transport using appropriate media, and a high-index of clinical-suspicion are crucial for timely and accurate NTM diagnosis [12] . Furthermore, NTMs may be difficult to detect in co-culture or mixed infections involving fast growing microorganism such as Pseudomonas aeruginosa because of several microbiological and diagnostic considerations [8] , [9] . NTM infections are challenging to manage as these organisms are difficult to isolate and identify, and they often exhibit resistance to multiple antimicrobial agents. In addition, host related factors, including immune-suppression, co-morbidities, treatment interactions, and toxicity further complicate the management of NTM infections. Empirical antibiotic therapy should be avoided before appropriate microbial investigation, as it may lead to the emergence of antimicrobial resistance [12] . Treatment with first-line ATT frequently fails due to resistance. The Mycobacterium fortuitum-chelonae complex are generally susceptible to macrolides, making macrolide-based regimens, such as clarithromycin an important component of therapy, as employed in our patients. Previous studies have also reported treatment with a three-month course of oral clarithromycin and ciprofloxacin in similar cases [13] . Treatment can begin with a combination of second-line antitubercular medications or other antibiotics depending on the local susceptibility of the mycobacterial species involved. Commonly used drugs include macrolides (e.g., clarithromycin), aminoglycosides (e.g., amikacin), and quinolones (e.g., ciprofloxacin). The duration of antibiotic therapy is often prolonged in such circumstances, ranging from 4 to 6 weeks to 6 months [14] . In both our cases, ciprofloxacin and clarithromycin showed partial/ complete resistance. Following surgical excision, both the patients were treated with antibiotics e.g. carbapenems (imipenem-cilastatin), aminoglycoside (amikacin), linezolid and macrolide (Azithromycin). Linezolid was associated with significant clinical improvement in both the patient, with therapeutic benefits becoming evident after one week of initiation. In the second patient, the drain port wound in left iliac fossa failed to heal despite linezolid therapy. In our report, both the patients were managed with surgical excision and prolonged oral and parenteral antibiotic therapy, which resulted in favourable clinical outcomes. Atypical mycobacteria are non-spore-forming bacilli that survive harsh environmental conditions primarily because of their thick, hydrophobic, mycolic acid-rich cell wall [15] . NTM infections following laparoscopic surgeries often arise due to inadequate sterilization of instruments. The insulated layers of the laparoscopic tools restrict the impact of autoclaving High-level disinfectant (HLD) is recommended for laparoscope, but the practice remains a subject of debate. Laparoscopic instruments are typically disinfected by immersion in 2–2.5% glutaraldehyde for 20 min. This provides disinfection, but does not achieve complete sterilization. The use of disposable laparoscopic instruments is the most effective strategy for preventing NTM infections. However, because of their high cost, its implementation is challenging in developing nations like India [16] . Current infection control guidelines recommend the use of 3.4% glutaraldehyde with an exposure time of 8–12 h of exposure. Glutaraldehyde solution can be reused up to 100 cycles over a period of 14 and 28 days at a concentration of 2.5% and 3.4% respectively. However, many healthcare facilities fail to adhere to replacement schedules or maintain records of the reused cycles, leading to reduced disinfectant potency. Additionally, rinsing instruments with boiled tap water after disinfection may result in recontamination. Sterilizing agents such as 0.55% orthophthaldehyde (OPA) and ethylene oxide (ETO) are reliable options for sterilizing [17] , [18] .

Minimally invasive procedures, such as laparoscopic surgery, represent one of the most groundbreaking advances in modern surgery, offering less pain, minimal scarring, abbreviated hospitalizations, and faster recovery relative to conventional open surgery. Although initially opposed, it soon became the preferred surgical approach, and today, laparoscopy is a routine procedure in various major surgical fields, including gastrointestinal surgery, urology, gynecology and oncosurgery [1] . Although laparoscopic surgery has transformed modern operative practice, port site infection (PSI) remains a significant postoperative complication that imposes a substantial direct, indirect, and intangible economic burden on patients and their families. PSIs are classifies as early (occurring within 7 days of surgery) or delayed (occurring within 3–4 weeks after surgery). Early PSIs are predominantly caused by Staphylococcus aureus or Pseudomonas aeruginosa . In contrast, delayed PSIs are often associated with nontuberculous mycobacteria (NTM), also known as atypical mycobacteria, which include all Mycobacterium species except Mycobacterium Leprae and Mycobacterium Tuberculosis complex. Delayed PSIs are more challenging to treat and prevent due to inherent resistance of NTM to multiple antimicrobial agents and disinfectants [2] . NTM port site infections (NTM-PSIs) are particularly challenging because of the ability of these organisms to survive routine sterilization procedures, inadequate sterilization practices, and the presence of heat-insulating components in laparoscopic instruments, which may hinder effective disinfection [3] . Currently, there are no established guidelines for the management of NTM-PSIs. Here, we report our experience in managing two cases of PSIs following laparoscopic surgery. We describe the clinical features, diagnostic challenges, and management strategies in both the patients. Additionally, we systematically reviewed all reported cases of laparoscopic NTM-PSIs from India.

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.