Ziehl-Neelsen Research Articles

Case report of pulmonary nocardiosis in a patient of right renal transplantation

Nocardiosis results from infection with bacteria of genus Nocardia, which are saprophytic aerobic actinomycetes that commonly reside in soil relatively and are inactive on standard biochemical tests. Pulmonary and systemic nocardiosis is common in adult males, almost all being sporadic, common in tuberculosis endemic regions and usually seen in individuals with deficient cell mediated immunity. It may present as pneumonia, empyema, pericarditis, mediastinitis, laryngitis, peritonitis, and meningitis. Diagnosed with sputum or pus for branching beaded gram positive filaments which are 1 micron meter wide and 50 micron meter long.A male immunocompromised patient presented with respiratory symptoms. Patient was evaluated elsewhere and received multiple courses of antibiotics. On admission in our facility HRCT thorax followed by fibre optic bronchoscopy was done. Bronchoalveolar lavage was sent for microbiological analysis. Acid-fast in kinyoun and Ziehl Neelsen stain came positive for Nocardia and was managed with trimethoprim- sulfamethoxazole which is the treatment of choice.

Cryptosporidiosis in HIV patients: A case report for timely diagnosis and prompt intervention

Cryptosporidium is one of the major causes of diarrhea in HIV-positive patients. Infection is related to the ingestion of oocyst-contaminated drinking water or food. In HIV infected individuals, this infection could have a higher mortality and vary in clinical manifestation. We report a case of cryptosporidiosis in a recently detected HIV patient who was suffering from intermittent diarrhea for last 2 months. Stool samples were collected and examined by modified Kinyoun’s acid fast staining. On microscopy of smear, we found pink, spherical oocyst 4-6µm in diameter. The absolute CD4+ count of patients was 85 cells/µl and viral load was 560 copies/ml. Patient was treated with an antiparasitic drug Nitazoxanide for 3 days and anti-retroviral Treatment started in ART clinic. On follow up there was resolution of symptoms and no complaints of intermittent diarrhea.

Assessment of the Occupational Risk of Tuberculosis & Air Borne Infection Control in High-Risk Hospital Wards and Its Implications on Healthcare Workers in a Tertiary Care Hospital in South India.

Introduction The indoor air in hospitals could play a significant role in the transmission of a wide array of infections, especially in respiratory intensive care units, pulmonary outpatient departments, and other areas. Unprotected coughing and sneezing may facilitate the release of aerosols and contaminate the indoor environment. The majority of infections transmitted through these modes include viral diseases, including tuberculosis (TB), influenza, and measles, among several others. Moreover, the possibility of direct and indirect transmission of microbes by air has been underestimated in hospital settings, especially in developing countries. This studytherefore was carried out to assess the burden of microbes in the air of selected wards in a tertiary care hospital and evaluate the occupational risk of some infections among healthcare workers (HCWs). Methods This study was carried out between September 2019 and February 2021 at a tertiary care teaching hospital in South India. A total of 30 symptomatic healthcare workers (HCWs) were included in the study and were screened for present and past tuberculosis (TB) as well as other lower respiratory tract infections. A tuberculin skin test, chest X-ray, and sputum acid-fast staining were performed on all the HCWs who were negative for other bacterial infections and were symptomatic. The study was conductedin coordination with the pulmonology department. Active monitoring of air was performed by microbiological air sampler in the respiratory intensive care unit (RICU) and other high-risk areas including the pulmonology outpatient department (OPD), the radiology OPD, and the microbiology department. Results Sputum for tuberculousbacteriawas positive in four (16.6%) HCWs. The chest X-ray showed radiological findings suggestive of TB in five (20.8%) HCWs. Three (12.5%) HCWs who were screened for extrapulmonary TBrevealed one (33.3%) was positive for TB of the hip joint. Among the HCWs, eight (33%) returned positive tuberculin tests. Assessment of the hospital air in theRICU revealedthe bacterial count (288 CFU/m3) exceeded the normal limit (≤50 CFU/m3). The COVID-19 isolation ward showed the lowest bacterial count (06 CFU/m3) and no fungi. The predominant bacterial isolates were gram-positive cocci in clusters (Methicillin-sensitive Staphylococcus aureus). After proper disinfection and correction of ventilation techniques, the resampling results noted microbial colonies under normal limits. Conclusion A high burden of TB was noted among the HCWs. The airborne infection control strategies are essential to minimize the risk ofnosocomial infections and occupational TB risk to HCWs. Most microbes are transmitted through the airborne route and therefore it is extremely important to take measures to control the transmission of such pathogens in hospital settings.

Tuberculous peritonitis in patients on peritoneal dialysis: a 35-year experience from a large medical center in Northern Taiwan

Introduction Tuberculous peritonitis (TBP) is a rare but fatal complication in patients on peritoneal dialysis (PD). In this study, we aimed to determine the demographic features, clinical features, laboratory parameters, and clinical outcomes of PD patients with TBP and to clarify possible risk factors for mortality. Materials and methods We retrospectively reviewed 2084 PD patients from January 1985 to December 2019. The diagnosis of TBP was established by positive peritoneal fluid culture for Mycobacterium tuberculosis. Results 18 patients were diagnosed with TBP. The incidence was 2.029 episodes per 1000 patient-years. The most common symptom was fever (94.4%), followed by cloudy effluent (83.3%) and abdominal pain (83.3%). The average peritoneal dialysis effluent (PDE) white blood cell (WBC) count was 172.7 cells/μL. Nine patients (50%) had WBC counts lower than 100 cells/μL and 13 patients (72.2%) had neutrophilic predominant WBC counts. Acid fast stain (AFS) was positive in 7 patients (38.9%). Only 2 patients (11.1%) continued with PD after TB infection, while 10 patients (55.6%) changed to hemodialysis. Seven patients (38.9%) died within 1 year. Significant differences were observed in sex (p = 0.040), the presence of diabetes mellitus (p = 0.024), and PD catheter removal (p < 0.001) between TBP patients with and without mortality. However, none of them was a significant factor for 1-year mortality in multivariate Cox regression model. Conclusion Physicians should pay attention to the unusual presentations of peritonitis, especially if symptoms include fever or an initial low PDE WBC count. Catheter removal is not mandatory if early diagnosis and appropriate therapy are available.

Approaches to increase recovery of bacterial and fungal abortion agents in domestic ruminants.

Abortions in domestic ruminants cause significant economic losses to farmers. Determining the cause of an abortion is important for control efforts, but it can be challenging. All available diagnostic methods in the bacteriology laboratory should be employed in every case due to the many limiting factors (autolysis, lack of history, range of samples) that complicate the investigation process. The purpose of this study was to determine whether the recovery of diagnostically significant isolates from domestic ruminant abortion cases could be increased through the use of a combination of the existing aerobic culture andBrucellaselective method with methods that are commonly recommended in the literature reporting abortion investigations. These methods are examination of wet preparations and impression smears stained by the modified Ziehl-Neelsen method, anaerobic, microaerophilic,Leptospira, Mycoplasmaand fungal culture. Samples of placenta and aborted foetuses from 135 routine clinical abortion cases of cattle (n= 88), sheep (n= 25) and goats (n= 22) were analysed by the new combination of methods. In 46 cases, bacteria were identified as aetiological agents and in one case a fungus. Isolation ofBrucellaspecies increased to 7.4% over two years compared with the previous 10 years (7.3%), as well asCampylobacter jejuni(n= 2) andRhizopusspecies (n= 1).Salmonellaspecies (5.9%) andTrueperella pyogenes(4.4%) were also isolated more often. In conclusion, the approach was effective in removing test selection bias in the bacteriology laboratory. The importance of performing an in-depth study on the products of abortion by means of an extensive, combination of conventional culture methods was emphasised by increased isolation ofBrucella abortusand isolation ofC. jejuni. The combination of methods that yielded the most clinically relevant isolates was aerobic, microaerophilic,Brucellaand fungal cultures.

An outbreak of tuberculosis in endangered northern pig-tailed macaques (Macaca leonina) and milu deer (Elaphurus davidianus) from a zoo in China.

Tuberculosis (TB) is a zoonotic disease that affects humans and domesticated and wild animals. Animals in zoos are potentially an important source of TB for humans; however they are often neglected in routine disease surveillance programs. This investigation reports an outbreak of TB in milu deer and northern pig-tailed macaques in a zoo in Wuhan, China, which highlighted the need for improved prevention and control of TB in China. Between 24 November and 9 December 2020 two milu deer and a northern pig-tailed macaque that were displaying signs of wasting died. Post-mortem, histopathological diagnosis and acid fast staining were used for the dead animals. Multiple PCR for Mycobacterium tuberculosis complex (MTBC) was performed to identify the bacterial in both milu deer and northern pig-tailed macaque. The serum antibody iELISA for MTBC was then performed for all the surviving milu deer and northern pig-tailed macaques. Six seropositive milu deer and a seropositive northern pig-tailed macaque were subsequently euthanised and, along with two other dead milu deer, necropsied. DNA from these tissue samples was extracted and detected MTBC using PCR and Real-time PCR. Subsequently bacterial isolation was used to confirm the infection. The lungs of the dead animals displayed gross and histological TB-like lesions and changes, and red staining bacilli were detected in smears of the lesions by microscopy after acid fast staining. Mycobacterium bovis (M. bovis) was detected in the two milu deer and Mycobacterium tuberculosis (M. tb) in the northern pig-tailed macaque using multiple PCR for MTBC. 35.3% surviving milu deer and 50% surviving northern pig-tailed macaques MTBC serologically positive. Six of the euthanised milu deer were also positive on a DNA test for M. bovis and the euthanised northern pig-tailed macaque was positive to M. tb. This is the first report of tuberculosis in the endangered species, milu deer and northern pig-tailed macaques, in China, and warrants urgent attention by researchers and conservation authorities. These cases highlight the need for expanding surveillance for MTBC to zoos in China.

Role of Bleach Concentration Method for Detection of Acid-fast Bacilli (AFB) in Sputum using Conventional Ziehl-Neelsen (ZN) Staining Technique

Sputum microscopy is the most important tool for the detection of Mycobacterium tuberculosis in peripheral laboratories. Sodium hypochlorite concentration technique prior to sputum microscopy may improve the detection of AFB bacilli over direct Ziehl Neelsen’s staining method. A prospective study was conducted at Kasturba Medical College, Manipal. Consecutive sputum samples were collected for 3 months. ZN stained smears were made directly of fresh specimens and of specimens that were processed with 3.5% sodium hypochlorite (NaOCl), from each patient. Then, ZN stained smears were observed under light microscopy. A total of 239 samples were collected. The yield of positive smears with ZN staining after NaOCl centrifugation was higher than that with microscopy after direct ZN stain. The percentage increase yield of ZN staining after NaOCl centrifugation over direct ZN staining was 3.3% (8/239). Bleach centrifugation can help in increasing the yield of positivity in sputum smear.

The Presence of Potentially Pathogenic Protozoa in Lettuce (Lactuca sativa) Sold in Markets in the Central Peruvian Andes.

Peru is currently one of the world’s leading culinary destinations, whose world-renowned cuisine uses vegetables, mainly lettuce, as frequent ingredients. Vegetable consumption is promoted worldwide as a part of a healthy diet. However, vegetables, more frequently lettuce, have been implicated as a vehicle of infection for several foodborne parasites. This study aimed to determine the presence of potentially pathogenic parasites in lettuce marketed in the Central Andes of Peru. A total of 75 lettuce samples were collected from the two largest wholesale markets and the main open-air market in Jauja province, in the central Peruvian Andes. The province of provenance (coast vs. highlands), lettuce variety (“curly-leaf”, “iceberg”, and “butter”), and type of market were recorded. The samples were microscopically examined for detection of parasitic life forms using standard parasitological methods including direct slide smear, Lugol’s iodine staining, and Modified Ziehl−Neelsen staining. The overall positivity of parasitic contamination in lettuces was 45.3% (CI 95%: 34−56.6%). Cryptosporidium spp., Isospora belli, Giardia lamblia, Balantidium coli, and Entamoeba spp. were detected in twenty-six (34.7%), six (8%), four (5.3%), two (2.7%), and two (2.7%) lettuces, respectively. I. belli was found in a significantly (p < 0.01) lower proportion in the “butter” variety, and significantly (p < 0.05) higher contamination with G. lamblia was found in lettuce sold at the open-air market. B. coli, G. lamblia, and E. histolytica/E. dispar/E. moshkovskii were detected only in lettuce from the highlands (Tarma province). This study provides important data for health authorities to develop food safety programs. This information is also of interest to the international community because of the increased visibility that Peru has gained as a tourist destination.

Epidemiological studies of gastrointestinal parasites infecting dogs in Kwara Central, North Central, Nigeria

Dogs are the most cosmopolitan pets of humans and as such a means of transmitting zoonotic parasites to their owners. This study was designed to investigate the diversity, prevalence, pattern of infection, intensity of infections, and the risk factors associated with gastrointestinal parasites of dogs in Kwara Central, North Central, Nigeria. Three hundred and five clinically healthy dogs were sampled. Faecal samples were subjected to the direct smear, simple faecal centrifugation flotation, formol-ether concentration, and the Modified Ziehl-Neelsen staining techniques. Oocysts/eggs per gram of faeces were counted using the modified McMaster technique. Data were analysed using univariate logistic regression, multivariate logistic regression, and the one-way analysis of variance (ANOVA). A p -value of < 0.05 was considered significant for all analyses. One hundred and sixty-six dogs were positive for at least one species of gastrointestinal parasite, representing 54.43% (95% CI: 44.81 – 59.96) of the sampled population. The study identified Cystoisospora species (15.41%), Cryptosporidium species (25.25%), Ancylostoma species (25.25%), Toxocara canis (19.02%), Strongyloides stercoralis (7.54%), Uncinaria stenocephala (6.89%), and Dipylidium caninum (2.30%) as the gastrointestinal parasites infecting dogs in the study area. Coinfection with more than one species of gastrointestinal parasites was a common finding in dogs. The intensity of Cystoisospora spp. among infected dogs ranged between 40 and 980 oocysts per gram of faeces, while that of helminth parasites was 40 – 1560 eggs per gram of faeces. Age, sex, breeds, body condition score, presence of ticks on dogs, the purpose of keeping dog(s), types of housing, types of feed consumed, vaccination status, and treatment with antiparasitics were predators associated with the prevalence and intensity of gastrointestinal parasites infections. Due to the zoonotic nature of most of the encountered gastrointestinal parasites, there is need for regular antiparasitic treatment, proper dog management, and adequate personal hygiene to prevent zoonosis.

The utility of immunohistochemistry for detecting mycobacterial infections in bronchoalveolar lavage & bronchial washings.

Tuberculosis, most commonly caused by Mycobacterium tuberculosis (MTB), is an infectious bacterial disease, with a major impact on global health. In this study, immunohistochemistry (IHC), acid-fast bacilli (AFB) culture and Ziehl-Neelsen (ZN) staining, techniques were compared on bronchoalveolar lavage (BAL) and bronchial washings (BW) with respect to sensitivity and specificity for detecting mycobacteria, taking culture as the gold standard. Consecutive BAL and BW specimens were included in the study, over a period of one year for which AFB cultures were available. Samples with diagnosis other than inflammatory pathology such as malignancies or inadequate samples were excluded. A total of 203 BAL and BW specimens from patients with age ranging from 14 to 86 yr were analyzed for the presence of mycobacteria. The utility and efficacy of ZN stain and IHC in detecting mycobacteria was tested using AFB culture as a gold standard. Out of 203 cases, 10.3 per cent (n=21) were positive on AFB culture. Of these, 5.9 per cent (n=12) smears were positive for ZN stain, whereas IHC positivity was seen in 8.4 per cent (n=17) of the cases. ZN staining had a sensitivity of 57.1 per cent and a specificity of 100 per cent whereas, IHC had a sensitivity of 81 per cent and a specificity of 81.9 per cent. Comparison with AFB culture (gold standard), IHC was found to be superior to ZN stain in terms of sensitivity, whereas ZN stain was found to be superior to IHC in terms of specificity. These findings therefore suggest that IHC may be a useful adjunct to ZN stain in the detection of mycobacteria in specimens from the respiratory tract.

A simple heat-based alternative method for deparaffinization of histological sections significantly improves acid-fast staining results for Mycobacteria in tissue

Histopathology is the study of how disease alters human and animal tissue and is based on the microscopic examination of stained tissue sections. To maintain tissue integrity, preserving it from degradation, it is initially fixed, primarily with formalin, before being treated with alcohol and organic solvents, allowing the infiltration of paraffin wax. The tissue can then be embedded in a mold and sectioned, usually at a thickness between 3 and 5 μm, before staining with dyes or antibodies to demonstrate specific components.As the paraffin wax is insoluble in water, it is necessary to remove it from the tissue section before applying any aqueous or water-based dye solution, to allow the tissue to successfully interact with the stain. This deparaffinization/hydration step is normally carried out using xylene, an organic solvent, followed by hydration using graded alcohols.However, this use of xylene has been shown to have detrimental effects on acid-fast stains (AFS), such as those employed to demonstrate Mycobacterium, including the causative agent of tuberculosis (TB), as the integrity of the lipid-rich wall present in these bacteria may be compromised using xylene.A simple, novel method, Projected Hot Air Deparaffinization (PHAD) removes the solid paraffin from the tissue section without the use of any solvents, which produces significantly improved staining results using AFS. PHAD relies on the projection of hot air onto the histological section to melt and remove paraffin from the tissue, which can be achieved using a common hairdryer.•PHAD relies on the projection of hot air onto the histological section which can be achieved using a common hairdryer.•The blowing force is such that melted paraffin is removed from the tissue in 20 min.•Subsequent hydration allows for using aqueous histological stains with success, such as the fluorescent auramine O acid-fast-stain.

Tuberculoid granuloma in bone marrow biopsy can be a rare cause of pancytopenia

Tuberculoid granuloma in bone marrow biopsy can be a rare cause of pancytopenia

Efficient Removal of Basic Fuchsin from Synthetic Medical Wastewater and Competitive Adsorption in the Mixture

The Ziehl-Neelsen stain is a mixture of Basic Fuchsin (BF), phenol (Ph), and methylene blue. It is used to stain the cell walls of Mycobacterium species. In this study, Basic Fuchsin was efficiently removed from synthetic wastewater using natural clay of Gankawa (GC) from Sulaimanyah city, and the effect of the presence of high concentrations of phenol in the adsorption mixture is demonstrated. In addition, X-ray diffraction (XRD), X-ray fluorescence (XRF), N2 gas adsorption analyzer, and field-emission scanning electron microscopy (FESEM) were used to characterize the natural clay. The clay was found to be mostly calcite, with a minor percentage of smectite, and contaminated with low percentages of illite. The adsorption kinetics show a relatively fast equilibration time (60-70 minutes). A second-order pseudokinetic model better fits the experimental kinetic data. The effect of the initial pH of the solution mixture was negligible at the experimental concentration range of the study. Freundlich and Langmuir’s adsorption isotherm models were applied to the equilibrium experimental data using nonlinear regression curve fitting. Both kinetics and isotherm studies point to a chemical adsorption mechanism for the process. For adsorption in the mixture, phenol molecules were found to compete with BF molecules for the active adsorption sites, while a synergetic effect of BF exists on phenol adsorption. As a naturally abundant cheap material, GC shows a superior adsorption capacity toward BF (287.0 mg g-1) over all natural materials and most of the synthetic or modified materials found in the literature.

Detection of the Mycobacterium avium complex in dogs with lymphadenitis.

Mycobacterium avium complex (MAC) is connected to human immunosuppressive diseases, including HIV-AIDS, and may pose a zoonotic threat. MAC causes lymphadenopathy in children, respiratory infection in adults, and generalized infection in immunocompromised individuals. Infection with nontuberculous Mycobacteria (NTM) in humans is now primarily brought on by MAC. Recently, MAC members have emerged as pathogenic organisms for animals and humans. While dogs are generally resistant to mycobacterial infections, there have been some cases of infection that result in systemic or disseminated diseases. The organisms can be transmitted to dogs through oral contact, and their faeces can be a possible source of infection for dog owners. It is important to note that this ailment is zoonotic, especially if infected pet dogs are in prolonged contact with their humans. The study was planned to demonstrate the occurrence of MAC organisms and other Mycobacteria in dogs associated with lymphadenopathy cases with special emphasis on lymphadenitis. A total of 123 samples (100 lymph node aspirates, 15 lymph node tissues, and 8 blood samples) from 83 dogs suspected of lymphadenitis accompanied by gastroenteritis, chronic skin infections, immunosuppression, chronic pulmonary diseases, and other chronic undiagnosed diseases were studied. The samples were processed for cytological and microscopic examination by Ziehl-Neelsen staining. Following the decontamination procedure, the aspiration and lymph node tissue samples were inoculated into Middlebrook 7H11 media for up to 8 weeks. The aspirated material was also directly used for molecular detection by triplex-nested polymerase chain reaction (nPCR) assay. A cytological study revealed pyogranulomatous inflammation of the lymph node tissue. Impression smears from lymph node tissues displayed the presence of acid-fast organisms. Out of 83 cases of dogs, 8 were found to be positive for Mycobacterium spp. Among those 8 positive cases, 3 were confirmed to belong to MAC, and 5 belonged to the Mycobacterium tuberculosis complex (MTB complex). MAC and MTB are the underestimated bacteria that could be the causative agents of lymphadenitis in animals.

Occurrence of gastrointestinal parasites in dogs in a rural area of Santa Catarina, Brazil.

We investigated the occurrence of gastrointestinal parasites in dogs in rural areas of the municipality of Painel, Santa Catarina, Brazil. For this, 91 canine feces samples were collected from 82 farms between August, 2017 and January, 2018. These fecal materials were processed using the techniques of spontaneous sedimentation, centrifugation-floatation in zinc sulfate and Ziehl-Neelsen staining. A questionnaire in the form of an interview was administered to the dogs' owner and information about the farm and the main care provided for the dogs was obtained. Among 91 sampled dogs, 27 (29.7%) were positive for at least one parasite species. Ancylostoma was the most prevalent genus (16.5%), followed by Giardia duodenalis (14.3%), Trichuris vulpis (6.6%), Toxocara canis (5.5%), Entamoeba spp. (4.8%), Cryptosporidium spp. (3.3%) and Taeniidae (1.1%). Most dogs do not receive veterinarian care and rarely received antiparasitic treatment. They were free to roam and had free access to animal remains and garbage, which was reflected in the significant associations with the occurrence of parasites that were found. We conclude that rural dogs harbor gastrointestinal parasites, but that their owners are unaware of the risks that these parasites can bring to human health.

Diagnosis of tuberculous pleural effusion in a tertiary care hospital of central India: The role of xpert Mycobacterium tuberculosis/rifampicin.

In India, 15%-20% of tuberculosis (TB) cases are categorized as extra-pulmonary TB, and tuberculous pleural effusion (TPE) is the second-most common type after tuberculous lymphadenitis. However, the paucibacillary nature of TPE makes its diagnosis challenging. As a result, relying on empirical anti-TB treatment (ATT) based on clinical diagnosis becomes necessary for achieving the best possible diagnostic outcome. The study aims to determine the diagnostic utility of Xpert Mycobacterium tuberculosis/rifampicin (MTB/RIF) for the detection of TB in TPE in high incidence setting of Central India. The study enrolled 321 patients who had exudative pleural effusion detected through radiological testing and were suspected of having TB. The medical procedure of thoracentesis was conducted to collect the pleural fluid, which was then subjected to both the Ziehl-Neelsen staining and Xpert MTB/RIF test. The patients who showed improvement after receiving anti-tuberculosis treatment (ATT) were considered the composite reference standard. The sensitivity of smear microscopy was found to be 10.19%, while that of the Xpert MTB/RIF method was 25.93% when compared to the composite reference standard. The accuracy of clinical diagnosis was measured using receiver operating characteristics based on clinical symptoms, and it was found to be 0.858 (area under the curve). The study shows that Xpert MTB/RIF has significant value in diagnosing TPE, despite its low sensitivity of 25.93%. Clinical diagnosis based on symptoms was relatively accurate, but relying on symptoms alone is not enough. Using multiple diagnostic tools, including Xpert MTB/RIF, is crucial for accurate diagnosis. Xpert MTB/RIF has excellent specificity and can detect RIF resistance. Its quick results make it useful in situations where a rapid diagnosis is necessary. While it should not be the only diagnostic tool, it has a valuable role in diagnosing TPE.

Isolation and Molecular Identification of Mycobacterium bovis from Slaughtered Cattle in Nekemte Municipality Abattoir, Ethiopia.

Tuberculosis (TB) is a zoonotic disease that can spread from animals to humans as well as from human to human. Little research has been conducted on bovine tuberculosis prevalence and molecular characterization in the western part of Ethiopia. To investigate this, a cross-sectional study was conducted on slaughtered cattle at the Nekemte municipal abattoir between January 2020 and June 2021. A detailed postmortem examination, culture, acid-fast staining technique, molecular characterization using RD4 deletion, and spoligotyping were all carried out. Based on a detailed postmortem examination, the overall prevalence of bovine tuberculosis was 7.8% (80 of 1020). Mycobacterium isolation confirmed only 12.5% (10/80) of the suspected tuberculosis tissue lesions. With acid-fast bacilli staining, all Mycobacterium spp. isolates (n = 10) were positive. However, only 9/10 isolates were confirmed to be M. bovis with RD4 molecular deletion typing. Spoligotyping revealed that 55.6% (5/9) of the isolate patterns had previously been reported, but 44.4% (4/9) of the isolates were new. In the current investigation, it was discovered that 80% (4/5) of the M. bovis strains circulating in the cattle population of study regions were SB2233 (2/5) and SB0134 (2/5), whereas 20% (1/5) of the strains corresponded to SB1176, which is compatible with previously documented M. bovis spoligotypes. These findings suggested that M. bovis was the main cause of bovine tuberculosis in the study area and posed a risk of disease transmission from cattle to humans due to low levels of public health awareness. As such, improved awareness among citizens and the development of control policies are warranted.

A case of melioidosis with hemoptysis: Strategies to isolate and identify Burkholderia pseudomallei

Background: Melioidosis is an infectious disease caused by Burkholderia pseudomallei with diverse clinical manifestations. Mortality is due to septic shock and pneumonia. Burkholderia pseudomallei is a Gram-negative rod with bipolar staining. Culture is the gold standard diagnostic test. Gram stain, biochemical reactions, and Poymyxin resistance aid in the identification of the organism. Case Description: A 52-year-old female with type 2 diabetes mellitus was hospitalized for a chronic cough. A sputum sample was collected for Culture and Acidfast staining. Burkholderia pseudomallei was isolated and the patient was prescribed appropriate antibiotic. On the 15th day of admission, patient expired following a bout of hemoptysis despite antibiotic therapy. Conclusion: Culture is the gold standard diagnostic method in melioidosis. Burholderia pseudomallei can be easily differentiated from other organisms by Gram stain, methylene blue stain, and screening for polymyxin sensitivity. Early diagnosis and appropriate antibiotic therapy are warranted in melioidosis.

Non-Tuberculous Mycobacteria with Enlarged Mediastinal Lymph Node by Next Generation Sequencing: Case Report

A young patient characterized by rapid enlargement of mediastinal lymph nodes was diagnosed as non-tuberculous mycobacterial pulmonary disease (NTM-PD) by bronchoalveolar lavage fluid Next Generation Sequencing (NGS). Laboratory examination, Chest CT scan, electronic bronchoscopy, and endobronchial ultrasound-guided transbronchial needle aspiration (EBUS-TBNA) were performed to diagnose non-tuberculous mycobacterium pulmonary disease. Detection of bird-intracellular mycobacterium complex in bronchoalveolar lavage fluid by NGS. Chest CT scan showed multiple enlarged lymph nodes in mediastinum. 4R region TBNA: chronic granulomatous inflammation, positive bacilli were found by acid-fast staining. After the anti-NTM treatment, the symptoms of the patients were relieved. When the patient shows mediastinal lymph node enlargement of unknown cause, NTM-PD can be considered and NGS can be used to assist in the diagnosis.

Comparative analysis of cartridge-based nucleic acid amplification test (CBNAAT) with conventional methods in the diagnosis of pediatric tuberculosis at a tertiary care center

BackgroundTuberculosis is an important cause of morbidity and mortality among children. Early diagnosis and treatment in children are challenging, more so in resource-limited, tuberculosis-endemic countries. In 2017, the WHO endorsed the use of CBNAAT for tuberculosis diagnosis. We have undertaken this study to evaluate the diagnostic value of CBNAAT in pediatric tuberculosis in comparison to other methods like microscopic detection of acid-fast bacilli and detection of mycobacteria-by-mycobacteria growth indicator tube (MGIT). Material and methodsThis hospital-based, cross-sectional, observational prospective study was conducted in the department of pediatrics, at a tertiary care center. A detailed history, general physical examination, and relevant physical examination were performed systematically and the findings were noted in the proforma. All necessary basic investigations like CBC, ESR, X-Ray, etc., and advanced investigations like MRI, CT, and FNAC were done as per the requirement of the subjects and the results were mentioned in the study proforma. Sensitivity, specificity, positive and negative predictive value, and diagnostic accuracy were calculated for various methods. A comparison between the two methods was done using the Mc Nemar test. p-value ≤0.05 was taken as statistically significant. All statistical analyses were done using Epi info version 7.2.1.0 statistical software. ResultsAmong 102 children suspected to be suffering from tuberculosis, the maximum number of TB cases were found in the age group of 11–16 years (43.2%), there were 58.2% of females, 58.8% belonged to the rural population, fever (78.4%) was the most common presenting symptom and 35.3% had a history of contact. In the present study, CBNAAT and ZN staining had equal sensitivity (60.8%) and specificity (100%) while the yield for MGIT culture was quite low (sensitivity 37.3%, specificity 100%). ConclusionsCBNAAT as a test was found to be useful, especially for early diagnosis and detection of rifampicin resistance in pediatric tuberculosis against MGIT culture. Since MGIT results become available only after 42 days and have a relatively lower yield so they can be utilized only in a selected clinical situation or in patients with high suspicion of tuberculosis where another test is not able to detect the organisms.