Nested polymerase chain reaction in the diagnosis of negative Ziehl-Neelsen stained Mycobacterium tuberculosis fistula-in-ano: report of four cases

PURPOSE: Mycobacterium is one of the causes of granulomatous diseases within the anorectal region. Early diagnosis of Mycobacterium infection is important before the use of antituberculosis chemotherapy. Clinical diagnosis is usually dependent on microscopic detection using Ziehl-Neelsen stain and mycobacterial culture, but the sensitivity and specificity of these two methods are low. In this study nested polymerase chain reaction was used to detect mycobacterial infection in anal fistulas. METHODS: Paraffin-embedded specimens from three patients and discharge from one patient were used. DNA extraction was performed using phenol/chloroform techniques. IS6110-based nested polymerase chain reaction, yielding a 259-bp amplicon, for the diagnosis of Mycobacterium infection was done to facilitate treatment. RESULTS: Four cases of suspected Mycobacterium tuberculosis fistulas-in-ano presented with persistent fistula or unhealed wound. Histopathologic examination revealed granulomatous inflammation with failed microscopic detection of acid-fast bacilli using Ziehl-Neelsen stain. Nested polymerase chain reaction confirmed the presence of M. tuberculosis in all cases. The anal lesions healed rapidly following a course of antituberculosis therapy. CONCLUSION: Molecular diagnosis of M. tuberculosis fistula-in-ano by nested polymerase chain reaction is useful for clinically highly suspected Mycobacterium infection despite a negative Ziehl-Neelsen stain.     

Diagnosis of malaria aided by polymerase chain reaction in two cases with low-level parasitaemia

Abstract
Light microscopy of thick and thin blood smears is the mainstay of malaria diagnosis. In situations of low-level parasitaemia such as drug-modified disease, however, this may be difficult making clinical management problematic. Polymerase chain reaction (PCR) methods have shown high sensitivity for the diagnosis of malaria and are able to differentiate the Plasmodium species involved. Two cases are presented in the present study, which illustrate how a PCR method can aid light microscopic malaria diagnosis and species differentiation in returned travellers with low-level parasitaemia. Plasmodium vivax was detected by PCR prior to the light microscopy becoming positive in one case, and in the second case Plasmodium malariae was detected when light micro­s­copy was unable to speciate the causative Plasmodium species. (Intern Med J 2003; 33: 613−615)     

聚合酶链技术检测慢性前列腺炎病原体的评价(附2071例报告)

目的探索慢性前列腺炎的病因，提高诊治水平。方法应用聚合酶链技术(PCR)对门诊疑诊慢性前列腺炎患者的前列腺液行淋球菌(NG)、沙眼衣原体(CT)及解脲支原体(UU)检测。结果检出单纯NG感染33例(9．09％)，CT感染32例(5．15％)，UU感染250例(23％)，CT与UU合并感染13例，NG合并UU感染5例，NG合并CT感染4例，并应用PCR监测治疗效果。结论PCR检测慢性前列腺炎病原体具有快速、敏感性高、特异性强等优点，值得临床推广应用。     

Cardiac echinococcosis with negative serologies: a report of two cases

Cardiac involvement in hydatid cyst disease is uncommon, occurring in approximately 2% of hydatid cases. Two cases of cardiac hydatid cysts with negative serologic tests are reported herein. In Case 1, the patient underwent surgery to remove cysts from the liver and 10 years later presented with symptoms and signs of ischaemic heart disease. In Case 2, the patient first underwent surgery to remove cysts from the brain and 3 years later for cyst removal from the breast. In both cases the diagnosis was established by transthoracic two-dimensional echocardiography and then confirmed by surgery and histological examination. These cases are of particular interest because of the rarity of cardiac localisation as a new site of the hydatid cyst after one or more previous surgeries for hydatid cyst removal, and stress the need for frequent reevaluation to detect new hydatid cyst formation in the heart and elsewhere caused by the Echinococcosis organism.     

Use of polymerase chain reaction in the diagnosis of abdominal tuberculosis

BACKGROUND: Early diagnosis and prompt treatment of abdominal tuberculosis is vitally important as it greatly reduces disease and treatment related morbidity and even mortality in extreme cases. A polymerase chain reaction (PCR) test was evaluated for its feasibility as a diagnostic tool in abdominal tuberculosis (TB) in the Indian scenario. METHODS: PCR for the identification of M. tuberculosis amplified a 340 bp nucleotide sequence located within the 38 kDa protein gene of M. tuberculosis. Tissues for processing were obtained from patients suspected to have abdominal TB. These were from various sources such as abdominal lymph nodes, segments of intestine and bowel obtained at various times and in different ways such as laparoscopy, colectomy, bowel and lymph node resection. Fifty such patients had their tissues sent for PCR. RESULTS: PCR results were compared with histopathology (HP). Of the 50 samples, 31 were positive for abdominal TB by HP whereas 30 were positive by PCR. Twenty-four of these were positive for both HP and PCR while of the seven samples positive for HP, five were negative and two gave inhibition by PCR. Six samples negative by HP were positive by PCR. CONCLUSION: This study demonstrates that PCR can be used as an effective tool to diagnose abdominal TB.     

Evaluation of a polymerase chain reaction for the diagnosis of tuberculosis

A polymerase chain reaction for the specific detection of Mycobacterium tuberculosis has been developed and evaluated for clinical applicability. Primers were designed to amplify a 240 base pair region in the MPB 64 protein coding gene (nts 460-700). From among 15 different DNA templates tested (including 10 species of mycobacteria) PCR amplified the DNA from M. tuberculosis complex only, demonstrating its exquisite specificity. Sensitivity studies using serial ten-fold dilutions of M. tuberculosis bacilli determined the limit of detectability to be 10 organisms. A total of 143 clinical specimens were analysed. This consisted of 26 known non-tuberculous specimens (control group) and 117 specimens received at the Tuberculosis Diagnostic Service of AIIMS (test group). None of the specimens in the control group was positive by PCR. Out of 117 specimens in the test group, 19 were culture positive for mycobacteria and 17 of these isolates were identified as M. tuberculosis. All the specimens from which M. tuberculosis was grown were also PCR positive. The remaining two isolates were identified as mycobacteria other than M. tuberculosis and these two specimens were PCR negative. An additional 14 culture negative specimens were PCR positive yielding an overall M. tuberculosis positivity rate of 26.5% (31/117) compared to 14.5% (17/117) by culture. The superior sensitivity of PCR over culture was more evident in non-pulmonary cases where PCR picked up 10 cases in addition to three culture positives out of 69 specimens. On the other hand, out of 48 pulmonary specimens only four cases in addition to 14 culture positives were picked up by PCR.     

Acromioclavicular joint tuberculosis: A report of two cases

Acromioclavicular joint tuberculosis is an extremely rare presentation with only 16 cases reported so far and has a relatively high propensity to be misdiagnosed. India being a tuberculosis endemic region accounts for almost 27% of cases worldwide (global index of 2018 was 10 million). With a higher index of suspicion an earlier diagnosis can be made. We report two patients of AC joint tuberculosis, treated with multidrug chemotherapy resulting in a good functional outcome.     

Detection of Mycobacterium tuberculosis DNA from peripheral blood in patients with HIV-seronegative and new cases of smear-positive pulmonary tuberculosis by polymerase chain reaction

Tuberculosis is still one of the most important cause of mortality and morbidity in many countries and there is a need for new methods for accurate and rapid diagnosis of tuberculosis. To determine the sensitivity and specificity of polymerase chain reaction (PCR) method, we have evaluated Mycobacterium tuberculosis DNA in peripheral blood samples with PCR technique in adult patients with human immunodeficiency virus (HIV)-negative and new cases of smear-positive pulmonary tuberculosis. We investigated the relationship between characteristic of the patients, radiological extension of the disease, sputum smear grade, presence of cavity, body-mass index (BMI) serum albumin level, total delay time and PCR positivity. Forty patients (33 male and 7 female; mean age 37.8 +/- 14.1) and 20 healthy control subjects (13 male and 7 female; mean age 35.6 +/- 7.3) were enrolled in this study. PCR was positive in 16 of 40 (40%) patients with pulmonary tuberculosis and negative in 24 of 40 (60%). None of the healthy controls had positive PCR results. The overall sensitivity specificity and accuracy of the PCR assay was 40, 100 and 60%, respectively. We found the positive correlation between PCR positivity and sputum smear grade (r=0.46, P=0.003) radiological extension of the disease (r=0.69, P=0.001), presence of cavity (r=0.90, P=0.001). We conclude that the detection of M. tuberculosis DNA from peripheral blood by PCR technique is useful for the rapid diagnosis of tuberculosis patients with HIV-negative. Hematogenous dissemination was important in tuberculosis patients and peripheral blood samples were suitable and easy materials. However, standardization of the PCR method must be ensured for the diagnosis of tuberculosis.     

Diagnosis of tuberculosis in children using a polymerase chain reaction.

We investigated the value of the polymerase chain reaction (PCR) in the diagnosis of active tuberculosis in children and evaluated the relationship between PCR results in children with tuberculous infections and mediastinal adenopathies detected by computerized tomography (CT-Scan). This was a controlled, blinded, prospective study comparing nested PCR, mycobacterial cultures and the clinical diagnosis based on 350 clinical specimens from 117 children referred for evaluation of suspected pulmonary tuberculosis. All children with tuberculous infection but without active disease underwent a chest CT-scan to detect the presence of mediastinal adenopathies not evident on chest x-ray. The sensitivity of PCR was 56.8% in children with clinically active disease (culture: 37.8%; smears: 13.5%). A major advantage of PCR over cultures was noted when there was no parenchymal involvement on chest radiograph and when the patient was undergoing anti-tuberculous treatment. There were nine specimens with false-negative PCR results due to the presence of amplification reaction inhibitors. PCR was positive in five children with tuberculous infection without active disease and these children presented mediastinal adenopathies on the CT-scan that were not evident on chest radiography. There were no false-positive PCR results in the control groups of children. We conclude that nested PCR is a rapid and sensitive method for the early diagnosis of tuberculosis in children. It is especially useful when the diagnosis of active tuberculosis is difficult. In our study children with tuberculous infection without apparent disease who have positive PCR results have mediastinal adenopathies on CT-scan.     

巢式聚合酶链反应检测支气管内膜结核患者活检组织中结核 …

目的 了解巢式聚合酶链反应（ＮＰＣＲ）技术对支气管内膜结核的诊断价值。方法 应用巢式聚合酶链反应（ＮＰＣＲ）对６７份支气管内膜活检组织进行结核分支杆菌ＤＮＡ检测。并与病理检查,刷检涂片,支气管镜检后痰涂片和痰培养结果比较,对照为４３例支气管肺癌患者,结果６７例支气管内膜结核活检组织病理检查,刷检涂片,支气管镜检“激惹”后痰涂片,镜检术后痰培养及ＮＰＣＲ检测阳性率分别为１３％,１９％,２２％,１５％     

Spinal tuberculosis presenting with abdominal symptoms--a report of two cases

We present two unusual cases of tuberculosis of the spine presenting with abdominal symptoms; both patients underwent exploratory laparotomy before the true nature of the diagnosis became known. The diagnosis of spinal tuberculosis was evident on the pre-operative abdominal radiographs but this was overlooked.     

Detection of Mycobacterium tuberculosis in sputum samples using a polymerase chain reaction

A polymerase chain reaction (PCR) assay for the rapid detection of Mycobacterium tuberculosis in sputum samples is described. The target DNA is a 123-base pair (bp) segment of IS6110, which is repeated in the M. tuberculosis chromosome and is specific for the M. tuberculosis complex. Methodology used to lyse the mycobacteria, extract the DNA, and amplify the 123-bp target DNA is presented. The amplified PCR product is detected by examination of ethidium-bromide-stained acrylamide gels. An internal control using the same primers as the target DNA has been constructed to assess the efficacy of each individual reaction. Of 162 sputum samples tested, 82 were smear-positive for acid-fast bacilli. Of the 94 specimens from patients in whom pulmonary tuberculosis was diagnosed, 51 were culture-positive, smear-positive, or both. Fifty of these were PCR positive. Of the 42 specimens from patients with nontuberculous mycobacterial pulmonary disease, 41 were PCR negative. All 26 specimens from patients without mycobacterial infection were PCR negative. This assay provides a sensitive and specific means for the laboratory diagnosis of tuberculosis within 48 h that is relatively simple to perform.     

A dengue shock patient with negative serology and polymerase chain reaction (PCR) tests

A 6-month-old Thai girl presented with clinical manifestations of dengue shock syndrome (DSS) with encephalopathy and urinary tract infection. Serology and PCR tests were negative whereas dengue virus type 2 was isolated. In cases of highly suspected dengue infections, viral isolation should be done even when serological and PCR tests are negative.