Acid-fast bodies in faecal smears stained by the modified Ziehl-Neelsen technique

The modified Ziehl-Neelsen (ZN) stain has proved useful in the laboratory diagnosis of cryptosporidiosis and, more recently, for the laboratory diagnosis of cyclospora. Apart from cryptosporidia and cyclospora, many other organisms and artefacts are present in faeces, and may be seen in faecal smears stained by the modified ZN method. Described here is the presence of such organisms and artefacts in faecal samples submitted to the routine microbiology laboratory of a district general hospital. Over 6000 faecal smears were examined using the modified ZN method, with an incidence of cyclospora and cryptosporidium of approximately 0.1% and 1%, respectively. Other organisms and artefacts were observed, with an incidence ranging from 0.1% to 1%. It is emphasised that the identification of known gastrointestinal tract pathogens should not rely solely on the results of their staining reactions. It is essential that criteria such as morphology and size be taken into account to differentiate organisms with similar staining reactions.     

Efficacy of fluorochrome stain in the diagnosis of pulmonary tuberculosis co-infected with HIV

The resurgence of Mycobacterium tuberculosis in association with HIV infection has focussed much attention in the rapid diagnosis of high risk cases. Infection with HIV is known to alter the presentation of pulmonary tuberculosis. The present study was under taken to compare the efficacy of fluorochrome stain (Fl) with conventional Ziehl Neelsen (ZN) stain in the diagnosis of pulmonary tuberculosis. Two hundred cases of pulmonary tuberculosis were included in the study. Sputum smears were screened for acid fast bacilli (AFB) by ZN and Fl methods and blood samples were screened for HIV. Sputum positive cases detected by Fl stain were higher in number (69%) when compared to ZN stain (50%). Of the total cases studied 15.5% were HIV seropositive. CONCLUSIONS: Fluorochrome staining was found to be more efficient (45%) when compared to ZN staining (29%) in detecting cases associated with HIV seropositivity, especially paucibacillary cases.     

Comparison of conventional bacteriology with nucleic acid amplification (amplified mycobacterium direct test) for diagnosis of tuberculous meningitis before and after inception of antituberculosis chemotherapy

The role of nucleic acid amplification techniques in the rapid diagnosis of tuberculous meningitis remains uncertain. We compared the performance of Ziehl-Neelsen (ZN) staining, the Gen-Probe amplified Mycobacterium tuberculosis direct test (MTD), and culture with 341 cerebrospinal fluid specimens from 152 adults (73 with and 79 without tuberculous meningitis) before and after inception of antituberculosis chemotherapy. The sensitivity, specificity, and positive and negative predictive values of ZN staining before treatment were 34/66 (52%), 79/79 (100%), 34/34 (100%), and 79/111 (71%), compared with 25/66 (38%), 78/79 (99%), 25/26 (96%), and 79/120 (66%) for MTD. The sensitivity of combined ZN staining and MTD (either positive) was 45/66 (68%). The sensitivity of staining and culture fell more rapidly than that of MTD after the start of treatment: after 5 to 15 days of treatment, MTD was more sensitive than ZN staining (12/43 [28%] versus 2/43 [2%]; P = 0.013). Slower bacterial clearance was observed if M. tuberculosis was resistant to isoniazid and/or streptomycin: resistant organisms were more likely to be cultured from cerebrospinal fluid after 2 to 5 days of treatment than fully sensitive organisms (P < 0.001). The sensitivities of ZN staining, MTD, and the two tests combined were improved by repeated sampling to 38/59 (64%), 35/59 (59%), and 49/59 (83%), respectively. In conclusion, ZN staining of the cerebrospinal fluid is at least as good as MTD for the rapid diagnosis of tuberculosis and is much faster and less expensive. However, the combination of these methods on serial samples detects more cases. Alternative tests are still urgently required.     

Modified detergent Ziehl-Neelsen technique for the staining of Cyclospora cayetanensis.

Cyclospora cayetanensis is a cause of prolonged diarrhoea, mainly in travellers. Laboratory diagnosis may be achieved by a number of methods such as the staining of faecal smears by the modified Ziehl-Neelsen (ZN) technique. Safer methods using this technique have been described for the staining of acid fast bacilli and cryptosporidia by replacing the phenol content of the carbol fuschin stain with various concentrated detergents. In this report the technique was modified slightly using a non-concentrated detergent and applied to the staining of oocysts of C cayetanensis. It was found that oocysts of C cayetanensis do not stain using the modified detergent ZN method when compared with similar preparations containing oocysts of Cryptosporidium spp.     

Autofluorescence—an important ancillary technique for the detection of Mycobacterium tuberculosis: Revisited

Tuberculosis is one of the oldest diseases known to affect humans and is caused by Mycobacterium tuberculosis. Lymph nodes are the most common site of extrapulmonary involvement. Tuberculous lymphadenitis can be presumptively diagnosed morphologically on fine‐needle aspiration biopsy of lymph nodes. Additional tests like Ziehl‐Neelsen (ZN) stain for detection of acid fast bacilli, fluorescence, and molecular markers are often employed for confirmation of diagnosis. However, culture is essential for obtaining a definitive diagnosis. Unfortunately, culture is time consuming and expensive. Newer investigative methods are required. The efficacy of autofluorescence in the diagnosis of tuberculous lymphadenitis was evaluated for this purpose. Fine‐needle aspiration biopsies were collected from patients with a clinical diagnosis of tuberculous lymphadenitis. Cytomorphological examination, ZN staining, autofluorescence, and culture were performed on all specimens. ZN staining was positive in 37.5% (30/80) of cases, while autofluorescence was positive in 57.5% (46/80) of patients and was found to have 81.8% specificity, 95% sensitivity, and a positive predictive value of 82.6%. This technique allowed rapid and early diagnosis of tuberculous lymphadenitis in a number of patients, permitting early institution of appropriate therapy. Diagn. Cytopathol. 2013;41:330–334. © 2012 Wiley Periodicals, Inc.     

The comparative evaluation of immunohistochemical and acid fast staining with histopathological changes in naturally occurring paratuberculosis in sheep

Histopathological changes and degrees of positivity by Ziehl–Neelsen (ZN) and avidin–biotin complex peroxidase (ABC) techniques were studied in 29 adult sheep grossly or clinically suspected to have paratuberculosis. Of these, 16 (55.1%) had microscopic lesions associated with paratuberculosis, which were classified into 4 categories. Focal–multifocal lesions (6.9%) consisted of small granulomas in the lamina propria of the ileum especially in the ileocecal Peyer’s patches and also in the mesenteric lymph nodes. Diffuse multibacillary lesions (31%) characterized by a diffuse granulomatous enteritis with epithelioid macrophages infiltration as main inflammatory cells that arranged in mosaic-like appearance, filled with numerous acid fast bacilli in ZN, and highly positive in ABC. Diffuse paucibacillary (lymphocytic) lesions (6.9%) composed of lymphocytes as main inflammatory infiltrate with some epithelioid macrophages or giant cells containing few if any mycobacteria. Diffuse intermediate (mixed) lesions (10.3%) characterized by a diffuse granulomatous enteritis with infiltration of large numbers of lymphocytes and also epithelioid macrophages with varying degrees of positivity in ZN and ABC. In the diffuse lesions, other lesions such as granulomatous or lymphocytic lymphangitis, lymphangiectasis, intravascular aggregates of epithelioid macrophages, lymphocytic infiltration around the myenteric nerve ganglia, severe granulomatous arteritis, atherosclerotic-like lesions and vascular thrombosis especially in the capsule of mesenteric lymph nodes, and serosal layer of intestine were observed. We could not find any necrotic or calcified lesion in the intestinal tissues of affected sheep but we found a few small foci in the mesenteric lymph nodes of two cases. High numbers of multinucleated langhans giant cells were seen only in mesenteric lymph node of one affected sheep. In conclusion, the present study showed four categories of lesions in which multinucleated giant cells, necrosis, and calcification were uncommon particularly in the intestine. Both immunohistochemistry (IHC) and ZN methods can detect IHC with more positivity in almost all the sheep with diffuse lesions; more cases with focal and paucibacillary forms were positive by IHC than by ZN. The importance of sampling the ileum (especially ileocecal valve), jejunum, and mesenteric lymph nodes to find microscopic lesions of paratuberculosis in sheep is emphasized.     

The role of fine-needle aspiration cytology and Ziehl Neelsen staining in the diagnosis of cutaneous tuberculosis

The present study highlights the role of fine-needle aspiration cytology (FNAC) and Ziehl Neelsen (ZN) staining in diagnosis of cutaneous tuberculosis and correlates the cytomorphological features with histopathology.FNAC and biopsy was performed on 30 cases of cutaneous tuberculosis and along with the routine stains, ZN and periodic acid Schiffs staining was carried out in all cases.On cytology, out of 9 cases of lupus vulgaris, 89% showed cohesive epithelioid cell granulomas with or without chronic inflammatory infiltrate; however, acid fast bacilli (AFB) could be demonstrated only in 22.2% on cytology while none on histopathology. Of 19 cases diagnosed as Scrofuloderma, 79% showed caseation necrosis with or without granulomas, 10.5% revealed granulomas with acute inflammatory infiltrates. AFB was demonstrated in 78.9% cases on cytology when compared with 15.8% on histopathology. No conclusion could be drawn in one case each of TBVC and lichen scrofulosorum.Hence, correlating cytomorphological patterns with clinical presentations often yields diagnostic information in cases of cutaneous tuberculosis and frequently obviates the need for biopsy especially in cases of scrofuloderma.     

Simultaneous detection and strain differentiation of Mycobacterium tuberculosis complex in paraffin wax embedded tissues and in stained microscopic preparations.

AIMS: To detect and differentiate Mycobacterium tuberculosis simultaneously by polymerase chain reaction (PCR) in clinical samples prepared for histopathological analysis and for microscopic detection of acid fast bacteria. METHODS: Paraffin wax embedded tissue samples and Ziehl-Neelsen (ZN) and auramine stained microscopic preparations from culture positive tuberculosis patients were subjected to DNA extraction and amplification by PCR. PCR was performed with primers specific for direct repeats and the product was detected by hybridisation to a set of 43 different oligonucleotides, a procedure designated as "spoligotyping". RESULTS: Mycobacterium tuberculosis complex DNA was detected in all of the 23 paraffin wax embedded tissues analysed. Strain differentiation was possible in 20 of the 23 paraffin wax embedded tissues. Mycobacterium tuberculosis complex DNA was also detected and typed in eight of 10 ZN stained microscopic preparations. The hybridisation patterns obtained from virtually all of these samples were identical to those obtained from DNA extracted from cultures. CONCLUSION: Simultaneous detection and strain differentiation of M. tuberculosis complex bacteria is possible in clinical samples prepared by current methods for microscopic and histopathological analysis, without the need to culture. The methodology described opens the way to rapid disclosure of outbreaks in high risk settings, such as hospitals and prisons, where dissemination of tuberculosis might be very fast as a result of a high prevalence of human immunodeficiency virus infected patients.     

Immunocytochemical detection of mycobacterial antigen in extrapulmonary tuberculosis

The aim of the study is to determine whether immunostaining for mycobacterial antigen can contribute to the cytological diagnosis of extrapulmonary tuberculosis (EPTB). The study was carried out on aspirated material of lymph nodes, and other accessible sites, from 65 patients with clinical diagnosis of tuberculosis (TB). Twenty patients, diagnosed by fine‐needle aspiration, with non‐tuberculous granulomas served as controls. The diagnosis of TB was based on the demonstration of acid‐fast bacilli (AFB), culture positivity for Mycobacterium tuberculosis (M. tuberculosis), or response to treatment with standard anti‐tubercular therapy. Immunostaining was done using polyclonal antibody to mycobacteria. AFB positivity by Ziehl Neelsen (ZN) staining was 21%, 65.38%, and 68% respectively in Pattern 1 (granulomas alone), in Pattern 2 (granulomas with necrosis), and in Pattern 3 (necrosis alone). Overall AFB positivity was 56.92%. Twenty‐eight of 65 cases were negative for AFB on direct smear. Culture was positive in 46% (13/28). Sensitivity and specificity of immunostaining were 96.92% (63/65) and 95%, respectively. Immunoreactivity was seen in 26 (92.8%) of 28 cases which were negative by ZN staining. Except in the case of leprosy, in which cross reactivity was seen, there was no immunoreactivity in the control group. Immunocytochemistry (ICC) had high sensitivity (96.2%) and specificity (95%) in the diagnosis of EPTB. ICC may be a useful adjunct to evaluation of cytomorphology and ZN staining. Diagn. Cytopathol 2014;42:391–395. © 2013 Wiley Periodicals, Inc.     

Mycobacterial culture of fine needle aspirate - a useful tool in diagnosing tuberculous lymphadenitis

A prospective study was undertaken on suspected lymph node TB (LNTB) patients, to evaluate the diagnostic utility of mycobacterial culture of fine needle aspirate (FNA), in comparison with the cytological examination and acid fast staining. Eighty percent of 157 aspirates studied were positive by cytological examination; 18% by ZN smear and 45% were positive by culture. Twelve aspirates which were negative by cytological features yielded positive mycobacterial cultures; four out of these were from HIV positive patients. Our observations suggest that supplementing FNA cytology with mycobacterial culture would increase the sensitivity of diagnosing LNTB; in addition to giving a highly specific diagnosis.     

Nontuberculous mycobacterial infections: comparison of the fluorescent auramine-O and Ziehl-Neelsen techniques in tissue diagnosis

Nontuberculous (i.e., atypical) mycobacterial infections are increasing among pediatric and immunosuppressed patients, who commonly present with subcutaneous inflammatory masses or adenitis, which is often surgically excised. Since the most frequently isolated species also grow slowly in culture, early diagnosis may depend on histologic detection of a mycobacterial organism in the biopsy specimen. However, the histologic methods used for this purpose are of uncertain value in the diagnosis of these infections. Biopsy specimens from 22 patients with clinical histories highly consistent with nontuberculous mycobacteriosis in which part of the tissue was cultured were selected for study. Coded tissue blocks and control specimens were stained by the Ziehl-Neelsen (ZN) or auramine-O (A0) fluorescent technique and examined blindly for the presence of characteristic organisms. Results of these studies were compared with the culture results, and predictive values were calculated. This experience showed that the AO technique is technically simpler, allowing faster screening at lower power and showing greater sensitivity and predictive value of a negative result although less specificity than the ZN technique. The lower specificity of AO may be factitious and due to the detection in the tissue of organisms that did not grow in culture. Previous observations that nontuberculous mycobacterial infections may elicit tissue reactions that simulate cat-scratch disease, sarcoidosis, and nonspecific chronic inflammation were also confirmed.     

Application of bleach method in diagnosis of extra-pulmonary tuberculosis

Extra pulmonary tuberculosis (TB) comprises 15% of the total tuberculosis cases. Bleach concentration method for demonstration of Acid fast bacilli (AFB) has been recently described for sputum. The aim of this study is to apply this method for demonstration of AFB in material obtained from extra-pulmonary sites and to correlate with cytology and conventional Ziehl Neelsen (Z N) staining. A total of 55 samples were studied from clinically suspected cases of extrapulmonary TB which included, FNA lymph nodes (17), abscesses drained from various body parts (18), body fluids (18) and skin scrapping (2). All the samples were processed for routine cytology, conventional ZN staining and bleach method followed by ZN staining. Out of 55 samples, 24(43.40%) were indicative of tuberculosis on cytology, 12(21.8%) were positive for AFB on conventional ZN staining, while the positivity increased to 39(70.90%) by Bleach method. Bleach solution is inexpensive and readily available in hospitals and its application has been proved in pulmonary tuberculosis. However to the best of our knowledge this is a pioneer study applied to the extra-pulmonary samples and the results of the present study shows improved detection of AFB.     

Breast cancer with associated granulomatous axillary lymphadenitis: a diagnostic and clinical dilemma in regions with high prevalence of tuberculosis

Intratumoral granulomas and granulomas in lymph nodes draining breast carcinomas have been reported previously. However, in regions like Pakistan, where the incidence of tuberculosis (TB) is high, it is sometimes difficult to distinguish between TB and a non-specific granulomatous response especially if there is an association with focal necrosis. We present a series of cases of invasive breast carcinoma with an associated granulomatous reaction in lymph nodes with or without necrosis, which were further analyzed for a possible coexisting tuberculosis using special stains and PCR-based assays for the identification of Mycobacterium tuberculosis. Twenty-two cases were examined using ZN stain for AFB and PAS stain for fungal organisms. Nested PCR assays for M. tuberculosis DNA were performed on formalin-fixed, paraffin-embedded tissue. In all the cases, ZN stain for AFB and PAS stain for fungus were negative. M. tuberculosis DNA was detected in 11 (50%) out of the 22 cases. Six of 12 cases which had granulomas in association with necrosis were positive for MTB-DNA, while 5 of 10 cases without necrosis were also positive for MTB-DNA. It is concluded that the presence of granulomas with or without necrosis in association with malignancies should be further evaluated, particularly in regions with a high prevalence of tuberculosis, for the possibility of coexistent tuberculosis, as this may alter the postoperative management of the patient. PCR-based assays are recommended for the diagnosis of TB in cases where ZN is unhelpful for demonstrating AFB or no tissue is submitted for microbiological studies.     

Use of conventional PCR and smear microscopy to diagnose pulmonary tuberculosis in the Amazonian rainforest area

The diagnostic usefulness of Ziehl-Neelsen (ZN)-stained sputum smears combined with conventional polymerase chain reaction (ZN/PCR) to amplify IS6110 region DNA extracted from ZN slides was evaluated. The objective was to verify if this association could improve tuberculosis (TB) diagnosis in patients at remote sites. The study was carried out in 89 patients with culture-confirmed pulmonary TB as defined by the Brazilian Manual for TB Treatment. The participants were recruited in a reference unit for TB treatment in Rondônia, a state in the Amazonian area in northern Brazil. ZN, PCR, and culture performed in the sputum samples from these patients were analyzed in different combinations (i.e., ZN plus PCR and ZN plus culture). The prevalence rates of pulmonary TB in these patients were 32.6 and 28.1% considering culture and ZN/PCR, respectively. The sensitivity and specificity of ZN/PCR were 86 and 93%, respectively. ZN/PCR was able to detect more TB cases than ZN alone. This method could offer a new approach for accurate tuberculosis diagnosis, especially in remote regions of the world where culture is not available.     

Same day sputum smear microscopy approach with modified ZN staining for the diagnosis of pulmonary tuberculosis in a microscopy centre at Rajahmundry

Background: Sputum smear microscopy is the main-stay in the diagnosis of pulmonary tuberculosis in many developing countries. To overcome the drop outs, same day diagnosis is ideal. Materials and Methods: In the current study, two spot sputum samples (SS₂ approach) are collected within a gap of one hour (same day sputum smear microscopy) in addition to the standard spot morning (SM) approach. The smears were stained with standard Ziehl Neelsen (ZN) and modified ZN staining techniques. Results: Out of 1537 patients, sputum smear positivity (SSP) was 9.43% (146 patients) in SM approach with standard ZN staining. Smear positivity was increased to 9.8% (151 patients) with modified ZN staining. For SS₂ approach, SSP was 9.37% (144 patients) and 9.8% (151 patients) with standard and modified ZN staining procedures, respectively. Conclusions: Diagnosis of lung tuberculosis is possible with two spot sputum samples with modified ZN staining.     

Optimisation of acid fast smears for the direct detection of mycobacteria in clinical samples

AIMS: Despite its long history, the acid fast smear remains unstandardised. Technical variations in both the preparation of clinical material and subsequent staining mean that smear sensitivity relative to culture may vary from 50% to over 80%. This study assessed the sensitivity of acid fast microscopy at each of five stages of sample preparation and by both commonly used staining methods. METHODS: Sputum samples thought for varying reasons to be highly likely to be culture positive were used to prepare a series of smears in which the effects of digestion (liquefaction), concentration (centrifugation), and decontamination (sodium hydroxide) could be assessed, together with a comparison of staining by the auramine/phenol and Ziehl-Neelsen techniques. RESULTS: The most effective method for the demonstration of acid fast organisms in sputum was found to be an auramine phenol stain applied to a liquefied, concentrated sample and examined before the decontamination process. CONCLUSIONS: The auramine phenol stain applied to a liquefied, concentrated sample and examined before the decontamination process is the most effective method for the demonstration of acid fast organisms in sputum.     

Acid-fast actinomyces in a child with pulmonary actinomycosis.

Open-lung biopsy in a child with chronic pneumonia revealed branched gram-positive acid-fast organisms, later identified as Actinomyces israelii. Like Nocardia asteroides, A israelii can be acid fast by the commonly used Putt stain. The pneumonia was cured with 12 weeks of penicillin treatment.     

Cytomorphological patterns of M. bovis BCG and M. tuberculosis on fine needle aspiration biopsies: Does HIV make a difference?

To determine if fine needle aspiration (FNAB) of mycobacterial lymphadenopathy can differentiate infection with M. bovis BCG (BCG) from M. tuberculosis (TB) and whether HIV status affects discriminatory cytological features. A retrospective study of culture positive, fine needle aspiration biopsies of lymph nodes in children (<13 years) between 2003 and 2008. A total of 77 aspirates were available for evaluation with 67 (87%) patients having known HIV status. BCG occurred at a younger age (6 months), predominantly axillary lymph nodes (90%) compared with TB (5 years and 20% axillary lymph nodes). Amorphous necrosis was only seen in aspirates from TB lymph nodes, while in HIV negative children with TB, foamy macrophages were absent. On ZN staining there were more organisms in the BCG group and in HIV positive patients the organisms were present in both extra- and intracellular locations, whereas in the HIV negative patients the organisms were predominantly extracellular in location. Demographic and cytomorphologic features that can assist in distinguishing between the two mycobacterial species include: age of patient, location of the lymph node, and presence/absence of amorphous necrosis and foamy macrophages on FNAB. However the only reliable method to identify the mycobacterial species is by mycobacterial culture and/or PCR.     

HIV-related incremental yield of bleach sputum concentration and fluorescence technique for the microscopic detection of tuberculosis

Bleach sputum concentration and fluorescence microscopy (FM) are reportedly more sensitive than direct Ziehl-Neelsen (ZN) sputum smears for tuberculosis detection, and might be particularly valuable for human immunodeficiency virus (HIV)-positive patients excreting fewer bacilli. This study, implemented in Yaoundé, Cameroon, determined the yield from both direct and bleach-concentrated FM and ZN duplicate smears against culture on Löwenstein-Jensen medium, with HIV testing from the sputa. From 418 HIV-positive and 518 HIV-negative tuberculosis suspects, 185 (44.3%) and 243 (46.9%) cultures, respectively, grew Mycobacterium tuberculosis. Direct ZN was positive for, respectively, 87 (47.0%) and 202 (83.1%) of the culture-positive cases. Proportional incremental yield over direct ZN from ZN and FM bleach smears was 14.9% (P?–3) and 17.2% (P?–4) for HIV-positive versus 4.9% (P?–2) and 2.0% (non-significant) for HIV-negative cases. There was no gain from direct FM. Bleach FM showed 2% excess false positives. The bleach concentration, therefore, increases the yield of ZN and FM, particularly from HIV-positive patients, but with a higher risk for false positives with bleach FM. With excellent baseline direct ZN, the gain remains modest. Field studies under real-life conditions are needed to determine whether it is worth the risks and operational challenges in HIV high-prevalence populations. FM was not more sensitive than ZN in this study, probably because of sub-optimal objective power and background staining. Culture on solid media with sparing laurylsulfate decontamination was clearly superior for HIV-positives, but it remains to be seen if culture also leads to more cases started on treatment routinely.     

A Combination Stain for Acid Fast Bacilli and Fungi

Abstract

A combination stain for acid fast bacilli and fungi is described. The method is simple and it provides a reliable method of recognizing both organisms in tissue sections simultaneously in one setting. Basically it is a combination of Beamer's modification of Fites for acid fast bacilli and Gridley's stain for fungi.