Kinyoun Staining Research Articles

Evaluation of gram-chromotrope kinyoun staining technique: its effectiveness in detecting microsporidial spores in fecal specimens

This study was conducted to evaluate the modification of the usual Gram-chromotrope staining technique developed in-house known as Gram-chromotrope Kinyoun (GCK) in comparison with the Weber Modified Trichrome (WMT) staining technique; as the reference technique. Two hundred and ninety fecal specimens received by the Microbiology Diagnostic Laboratory of Hospital Universiti Kebangsaan Malaysia were examined for the presence of microsporidial spores. The sensitivity and specificity of GCK compared to the reference technique were 98% and 98.3%, respectively. The positive and negative predictive values were 92.5% and 99.6%, respectively. The agreement between the reference technique and the GCK staining technique was statistically significant by Kappa statistics ( K = 0.941, P < 0.001). It is concluded that the GCK staining technique has high sensitivity and specificity in the detection of microsporidial spores in fecal specimens. Hence, it is recommended to be used in the diagnosis of intestinal microsporidiosis.

Report of cryptosporidiosis in gray brocket deer (Mazama gouazoupira) in southern Brazil

The aim of this study was to report the occurrence of cryptosporidiosis in gray brocket deer in Brazil. Fecal samples of a male and a female Mazama gouazoupira kept in captivity in southern Brazil were evaluated. Samples collected at 10-d intervals were analyzed by the centrifugal flotation technique and stained by the modified Ziehl–Neelsen and by the Kinyoun staining methods. The first analysis showed a high number of Cryptosporidium parvum oocysts in the feces, associated with the presence of diarrhea in both animals. However, even without drug therapy, the number of oocysts of the protozoan decreased and the feces returned to normal after 10 days. This is the first report of cryptosporidiosis in M. gouazoupira.

Case report of disseminated nocardiosis during immunosppressive treatment

Infection caused by Nocardia spp. Is unusual and presents with a variety of clinical manifestations in both immunocompromised patients and in immunocompetent hosts.The diagnosis of Nocardia is often difficult, and further hampered by the absence of specific clinical symptoms.A man, 63 years old, caring for about a month from multiple erosions of the oral cavity and Inca intergluteale associated with blisters and erosions scattered in the face, scalp and trunk.The routine laboratory tests, including serological testing for HIV and the determination of circulating CD4 lymphocytes, were negative.The chest X-ray was also negative. Due to the diagnostic suspicion of Pemphigus vulgaris (PV), therapy with methylprednisolone and azathioprine was initiated. After 15 days the patient’s condition worsened and it became necessary hospitalization in intensive care. A CT scan found: pleural empyema, abscess, and paravertebral muscle abscess. The cultural examination of pleural fluid and synovial fluid gave positive results for Nocardia asteroides. On this basis, sulphamethoxazole was administered in association with trimethoprim for 2 months, with a complete remission of the symptoms.At a median follow up of 2 years the patient’s general condition was excellent, without residual signs of infection.The cultured samples from pleural and synovial fluids gave a clear positivity after 5 days of incubation at 37 ° C on blood agar and chocolate agar plates; the colonies showed chalky and thin filaments, slide as branched Gram-positives and appeared partially acid-alcohol resistant by modified Kinyoun staining (1% H2SO4). The presumptive identification of Nocardia spp. was additionally confirmed by biochemical tests for the identification of species with the following results: catalase test positive, oxidase test negative, aesculin hydrolysis, reduction of nitrite to nitrate and urease test positive. Given the low incidence of Nocardia spp. infection, and the non-typical clinical manifestations, microbiological diagnosis of this infection is critical for the selection of an appropriate antibiotic therapy that should be prolonged for several months.

A multiattribute utility evaluation of different methods for the detection of enteric protozoa causing diarrhea in AIDS patients

BackgroundEnteric protozoa and sporozoa have emerged as important opportunistic parasites and can cause fatal infections in AIDS patients. The line of treatment being different for them necessitates an accurate and prompt identification of these to avoid empirical treatment. In this study which is the first of its kind from India we did a comprehensive evaluation of different techniques, comparing them on the basis of the attributes like yield, cost, time taken, expertise and infrastructure. For the first time combination of Calcoflour White and DAPI, a nuclear stain, were used to identify Microsporidia spp. Thus, a diagnostic protocol was devised for rapid, sensitive and cost effective identification of the opportunistic enteric protozoa.ResultsThe organisms isolated from the stool samples of the cases (450 HIV patients) were predominantly Cryptosporidium spp., Microsporidia spp. and Cyclospora spp. Interestingly, the control group (200 relatives of the patients who were HIV negative) showed a high incidence (21%) of Cryptosporidium spp. We found a significant increase in the sensitivity of microscopy in detecting Cryptosporidium spp. and Cyclospora spp. after formol ether concentration. Kinyoun's staining was better compared to Modified safranin staining for Cryptosporidium spp. identification. Although ELISA had a sensitivity of 93.25% and specificity of 97% for Cryptosporidium spp. detection, we ranked Kinyoun's staining better than ELISA because it is not affordable to most of our patients. For detecting Cyclospora cayetanensis, autoflourescence was the easiest and most cost effective method followed by Safranin technique. Combination of Calcoflour White stain and DAPI gave good results for the identification of Microsporidia spp. We assessed the above techniques and graded the attributes in the following descending order: cost effectiveness, sensitivity, ease of use and interpretation, time taken for the procedure and batch testing.ConclusionThus, we conclude that a combination of minimum three procedures should be carried out for the screening of stool specimens of HIV positive patients. Kinyoun's staining should be made mandatory for every diarrheal stool sample from HIV patients. Also every laboratory should assign its own value to the attributes and apply Multiattribute utility theory or the Analytical hierarchy process to decide the most appropriate methodology.

Culture result of smear-positive sputum samples after 2 months of antituberculous treatment

To the Editors: The correlation between sputum smear for acid-fast bacilli (AFB) and culture for Mycobacterium tuberculosis is usually good before antituberculous treatment (ATT), but it is not so good after treatment has started. In previous studies, up to 23.5% of patients had sputum that remained smear positive after 2 months of ATT 1. Old age, high bacillary load, presence of pulmonary cavitary lesions, treatment interruption and emergence of resistant strains are risk factors of persistent smear positivity. Determining whether these sputum samples contain viable tuberculosis (TB) bacilli is crucial because it indicates higher rates of relapse 2, 3. Meanwhile, for patients without viable TB bacilli, modifying treatment regimens and isolation appears to be redundant. However, the results of mycobacterial culture are only available after 2 weeks, even when using fluorometric liquid culture technique. Despite having good sensitivity and specificity, commercialised nucleic acid amplification tests are expensive and do not discriminate between viable and dead bacilli and, therefore, are not recommended for TB patients receiving ATT. Thus, we conducted a study to identify clinical factors for predicting the culture results of the persistent smear-positive sputum samples in a 2,100 bed, tertiary-care referral centre in northern Taiwan and an 800 bed local teaching hospital in southern Taiwan. In the two hospitals, all sputum specimens were processed by adding NaOH-citrate- N -acetyl-l-cysteine at room temperature for 15 min. After centrifugation, the precipitate was resuspended 4. Before October 2006, smears for AFB of the processed samples were stained using the Kinyoun method. From November 2006, the smear was stained with auramine-rhodamine fluorochrome 5. Smears were graded according to guidelines established by the American Thoracic Society 6 and the highest smear grading was recorded. All collected …

Ocorrência de Giardia, Cryptosporidium e microsporídios em animais silvestres em área de desmatamento no Estado de São Paulo, Brasil

A ocorrência de Giardia, Cryptosporidium e microsporídios foi investigada por meio da análise de 98 amostras fecais de animais silvestres capturados em uma área de desmatamento para a construção das barragens de Paraitinga e Biritiba, localizadas nos Municípios de Mogi das Cruzes, Salesópolis e Biritiba-Mirim, no Estado de São Paulo. As amostras foram obtidas de 46 roedores, 21 marsupiais, 16 sapos, nove morcegos, três primatas e três lagartos. As técnicas de centrífugo-flutuação com sulfato de zinco, de Kinyoun e a coloração de Gram-Chromotrope foram utilizadas, respectivamente, para a pesquisa de Giardia, de Cryptosporidium e de microsporídios. O total de animais parasitados por um dos protozoários investigados foi de 17,35% (17/98). Cistos de Giardia foram encontrados em amostras fecais de dois pequenos roedores da espécie Coendou villosus (ouriço-cacheiro). Os três animais positivos para Cryptosporidium foram roedores das espécies Akodon montensis, Thaptomys nigrita (ambos conhecidos como ratos do mato) e Sciurus aestuans (serelepe ou caxinguelê). Esporos de microsporídios foram encontrados nas fezes de 12 animais, sendo seis roedores das espécies Oligoryzomys sp.(um), Akodon montensis (três) e Coendou villosus (dois), três marsupiais pertencentes às espécies Didelphis aurita (dois) e Marmosops incanus (um) e três morcegos da espécie Diphylla ecaudata. Este é o primeiro relato de microsporidiose em animais silvestres no Brasil. A presente investigação enfatiza a importância de animais silvestres, particularmente pequenos mamíferos, como potenciais fontes de infecção desses protozoários para outras populações animais, incluindo o homem, em áreas de desmatamento.

Disseminated Mycobacterium avium-intracellulare Infection Presenting as Multiple Ring-Enhancing Lesions on Brain MRI

A 33-year-old man in whom AIDS had been diagnosed in 2003 presented with a 6-week history of progressive generalized weakness, lethargy, dysphagia, slowing of speech, and left-sided weakness. Highly active antiretroviral therapy was initiated, but the patient was not adherent to this regimen. Disseminated Mycobacterium avium-intracellulare infection in the lungs and supraclavicular lymph nodes had been diagnosed 3 to 4 weeks before presentation. Magnetic resonance imaging (MRI) of the brain revealed multiple ring-enhancing lesions. A presumptive diagnosis of central nervous system toxoplasmosis was made, and an extensive work-up for ruling out other causes of multiple ring-enhancing lesions was initiated. Laboratory studies yielded a serum CD4 cell count of 2/μL and a human immunodeficiency virus RNA count of 6100 copies/μL. Results of the following were negative: serum cryptococcal antigen, Bartonella henselae IgM and IgG, cytomegalovirus antigen, rapid plasma reagin, and Toxoplasma IgM and IgG. The following cerebrospinal fluid (CSF) results were negative: Toxoplasma IgM, IgG, Toxoplasma by polymerase chain reaction and staining, acid-fast bacilli (AFB) stain, cryptococcal antigen, venereal disease research laboratory test, Gram stain, and bacterial and fungal cultures. Cytologic examination of the CSF showed numerous neutrophils but no malignant cells. Laboratory studies of the CSF yielded a protein value of 52 mg/dL (to convert to mg/L, multiply by 10) (reference range, 15-45 mg/dL) and a glucose value of 26 mg/dL (to convert to mmol/L, multiply by 0.0555) (reference range, 40-70 mg/dL); the CSF was clear, colorless with 5 red blood cells and 42 white blood cells (77% polymorphonuclear and 23% mononuclear cells). Brain biopsy with AFB stain revealed 3+ AFB (M avium) but showed no Nocardia-like organisms. A modified Kinyoun stain for Nocardia species was negative, as was a wetmount preparation for amebae. A liquid-based biopsy culture grew M avium after 10 days of incubation. A confirmatory DNA probe was performed to verify the diagnosis of central nervous system toxoplasmosis. Therapy with ethambutol, rifampin, and amikacin was initiated; however, the patient chose palliative care after talking with his family.

Detecting Mycobacterium tuberculosis in Bactec MGIT 960 Cultures by Inhouse IS 6110-based PCR Assay in Routine Clinical Practice

Diagnosis of tuberculosis is challenging because the current methods are time-consuming and laborious. We have developed a method combining the Bactec MGIT 960 rapid culture system with the IS6110-based PCR for rapid diagnosis of tuberculosis. A total of 1745 samples from 712 patients treated at the Tri-Service General Hospital, Taipei, Taiwan between June and August 2005 were tested. An aliquot of positive Bactec MGIT 960 culture fluids was Kinyoun stained, and the samples positive for Kinyoun staining were directly assayed by the IS6110-based PCR. The same samples were also examined by the conventional methods for identification of Mycobacterium tuberculosis complex. One hundred and four samples from 62 patients were positive according to the Bactec MGIT 960 system. Among these, 59 (56.7%) were positive and 45 (43.3%) were negative according to the IS6110-based PCR. Compared with the conventional identification methods, the IS6110-based PCR assay correctly identified all 59 M. tuberculosis isolates and gave negative results for all nontuberculosis mycobacteria. The mean turnaround time for M. tuberculosis identification by this combined method was 6.41 days for smear-positive and 14.33 days for smear-negative specimens. The sensitivity of the IS6110-based PCR assay was determined to be 5 cells or 0.1 pg of mycobacterial DNA. The combined use of the automated Bactec MGIT 960 system and the IS6110-based PCR assay is sensitive and rapid for the detection of M. tuberculosis complex, and we recommend that this method be used routinely for identification of mycobacteria in clinical laboratories.

A Case of Catheter-Related Bacteremia of Tsukamurella pulmonis

Tsukamurella pulmonis is an aerobic actinomycete. We report a catheter-related bacteremia of T. pulmonis. A 39 yr-old male with ALL was hospitalized to receive bone marrow transplantation (BMT). Although the patient developed a high fever at the 7th hospital day (HD), it subsided with vancomycin treatment, and he received BMT at 9th HD. Fever resurged at 16th HD despite sustained treatment with vancomycin, meropenem, and amphotericin B, but subsided with removal of Hickman catheter (HC) at 19th HD. Three sets of blood cultures comprising one from the HC and two from venipunctures were taken at 7th, 16th, and 19th HD, and the distal tip of the HC was also cultured. The aerobic vials of all 3 HC-withdrawn blood cultures and one peripheral blood culture taken at 19HD and the HC tip culture grew long, straight, thin gram-positive rods that were positive on modified Kinyoun stain. This organism showed tiny, rough, grey colonies after 3-day incubation and grew to large flat colonies when incubation was extended. It was catalase-positive, urease-positive, and alkaline-slant/alkaline-deep on triple sugar iron agar, and hydrolyzed hypoxanthine. The sequence of 1,296 base pairs of 16S rRNA of this organism showed a 100.0% homology with the published sequence of T. pulmonis DSM 44142T. To our knowledge, this is the first report of T. pulmonis bacteremia in Korea.

Evaluation of Different Staining Techniques (Ziehl Neelsen Stain, Kinyoun Stain, Modified Cold Stain, Fluorochrome Stain) for the Diagnosis of pulmonary Tuberculosis

Introduction Bacteriological examination of sputum is the cornerstone in the diagnosis of pulmonary tuberculosis in the developing world. The risk of spread of infection and emergence of drug resistant strain has created the need of rapid, sensitive and specific diagnostic test. Objectives This prospective study was performed in Tuberculosis research laboratory, Department of Microbiology, BPKIHS, Dharan with an objective to evaluate different staining techniques (Ziehl-Neelsen (ZN), Kinyoun, Modified cold (MC), Fluorochrome) for detection of acid fast bacilli (AFB) for diagnosis of pulmonary tuberculosis. Methods The study was cross-sectional study. All the samples were processed by modified Petroff’s method. From each sample four smears were prepared and were stained with four different staining techniques: ZN, Kinyoun, MC, and Fluorochrome respectively. Culture was employed as gold standard for tuberculosis diagnosis. Results A total of 1365 specimens from 500 patients were analyzed. One hundred nine patients (21.8%) were diagnosed as having TB by culture. The positive yield found with the staining techniques were 14.2 percent by ZN, 13.8 percent by Kinyoun, 14.4 percent by MC and 17.6 percent by Fluorochrome staining methods. With reference to culture, sensitivity of ZN, Kinyoun, MC and Fluorochrome were found to be 57.8 percent, 56 percent, 59.6 percent, and 71.6 percent respectively. The specificity in ZN, Kinyoun and Fluorochrome methods was 98 percent and that in MC as 98.2 percent. The positive predictive value of ZN, Kinyoun, MC, Fluorochrome was found to be 88.7 percent, 88.4 percent, 90.3 percent and 90.7 percent and the negative predictive value of ZN, Kinyoun, MC, Fluorochrome was found to be 89.3 percent, 88.9 percent, 89.7 percent and 92.5 percent respectively. Conclusion The fluorescent staining method was found most reliable out of the other staining techniques. The MC method also could be viable alternative to ZN and Kinyoun for primary diagnosis of tuberculosis.

Frequency of Blastocystis hominis and other intestinal parasites in stool samples examined at the Parasitology Laboratory of the School of Pharmaceutical Sciences at the São Paulo State University, Araraquara

Blastocystis homins is a protozoan that causes an intestinal infection known as human blastocystosis. This infection is diagnosed by means of parasitological examination of stools and by permanent staining techniques. The present study was developed to evaluate the frequency of Blastocystis hominis infection among inhabitants of the Araraquara region, State of São Paulo, and to compare different methods for investigating this protozoan in feces samples. Evaluations on 503 stool samples were performed by means of direct fresh examination and using the techniques of Faust et al., Lutz and Rugai et al. In addition, the iron hematoxylin, trichrome and modified Kinyoun staining techniques were used. Out of the 503 samples examined, 174 (34.6%) were found to be positive for the presence of intestinal parasites. The most frequent protozoa and helminths were Entamoeba coli (14.6%) and Strongyloides stercoralis (6.7%), respectively. Blastocystis hominis was present in 23 (4.6%) fecal samples, with a predominately pasty consistency and without characterizing a condition of diarrhea. Despite the low frequency of Blastocystis hominis found in the Araraquara region, compared with other regions of Brazil, it is important to perform laboratory diagnostic tests for this protozoan. Its finding in fecal material is indicative of food and drinking water contamination. Since the transmission route for this parasite is accepted to be oral-fecal, this implies that the population needs guidance regarding hygiene and basic sanitation measures as a means for controlling health problems caused by enteroparasites.

Blastocystis sp. and other intestinal parasites in hemodialysis patients

Chronic renal insufficiency disease (CRI) leads to uremia in hemodialysis patients and induces a state of immunodepression that results in higher frequencies of infections and diarrhea. Hemodialysis patients resident in the city of Campo Mourão, Paraná, Brazil were analyzed from April 2006 through September 2007 for Blastocystis sp. and other intestinal parasites and for associated diarrhea. Fecal samples from 86 hemodialysis patients and 146 healthy (reference) persons were examined by standard methods for detecting ova, larvae and cysts, which included preservation in 10% formalin and the Kinyoun method. Thirty-three hemodialysis patients (45.1%) and 36 reference individuals (25.7%) were found to be parasitized. The differences in the percentages of parasitism and polyparasitism between the reference group and the chronic renal patients was significant (p= 0.0318 and 0.0019, respectively). Blastocystis sp. (18%-20.1%), Endolimax nana (14%-16.3%), Cryptosporidium sp. (4%-4.7%) and Entamoeba coli (4%-4.7%) were the most frequent protozoa found in the hemodialysis patients. Parasitism was not significantly associated with diarrhea (p=0.9947) or with decreased white blood cell counts (p=0.7046) in these individuals. Because parasitic infections may be an important comorbidity factor in hemodialysis patients, we suggest that parasitological stool examinations, especially for Blastocystis sp. and Cryptosporidium sp., be included in routine medical follow-up examinations of these patients.

Quantitative toxoplasma gondii oocyst detection by a modified Kato Katz test using Kinyoun staining (KKK) in ME49 strain experimentally infected cats

We detected Toxoplasma gondii oocysts in feces of experimentally infected cats, using a Kato Katz approach with subsequent Kinyoun staining. Animals serologically negative to T. gondii were infected orally with 5 x 10(2) mice brain cysts of ME49 strain. Feces were collected daily from the 3rd to the 30th day after challenge. Oocysts were detected by qualitative sugar flotation and the quantitative modified Kato Katz stained by Kinyoun (KKK). In the experimentally infected cats, oocysts were detected from the 7th to 15th day through sugar flotation technique, but oocysts were found in KKK from the 6th to 16th day, being sensitive for a larger period, with permanent documentation. The peak of oocysts excretion occurred between the 8th to 11th days after challenge, before any serological positive result. KKK could be used in the screening and quantification of oocysts excretion in feces of suspected animals, with reduced handling of infective material, decreasing the possibility of environmental and operator contamination.

Tuberculosis: a study of 111 cases in an area of high prevalence in the extreme south of Brazil

Tuberculosis is an increasingly important public health problem in developing countries. We studied 111 tuberculosis patients confirmed by mycobacteria isolation between 1998 and 2000 in Rio Grande, in south Brazil. One-hundred-thirteen pulmonary and extrapulmonary clinical materials were examined through culture by the Ogawa-Kudoh method and through smear examination by the Ziehl-Nielsen or Kinyoun methods. The identification of Mycobacterium tuberculosis was done through the usual phenotypical methods. The proportion method (indirect technique) was used to determine the resistance of isolates. The man/woman ratio was 2.6:1, 75% were white, and the group had a mean age of 39.7+/-12.7. Risk factors were present in 38.7% (34 men and 9 women, p=0.2), with a mean age of 35 (p=0.002); infection by HIV was the most frequent (28.8%), followed by alcoholism (16.2%) and injected drug use (15.3%). The frequency of M. tuberculosis isolates was 99.1%. Pulmonary disease occurred in 88.3% of the cases, extrapulmonary in 9.9% and 1.8% in both. There was an association between extrapulmonary tuberculosis and the presence of risk factors (p=0.0001). Resistance to isoniazid was found in 4% of the isolates and to isoniazid and rifampin in 2%, all being patients with some risk factors. The profile of tuberculosis in the population of this study followed the pattern described for developing regions of the world.

Effectiveness of Cryptosporidium spp. oocysts detection and enumeration methods in water and milk samples

Cryptosporidium spp. oocyst recovery in water and milk samples was evaluated. Samples were inoculated with a suspension of 1.2×10(7) Cryptosporidium spp. oocysts and submitted to centrifugal flotation, using different solutions (sucrose, NaCl, MgSO4, ZnSO4, AlSO4, NH4SO4 40% and NH4SO4 80%). Centrifugation of the samples was carried out in two stages for concentration using two methods that differed in the order in which the saturated solutions were used, namely only in the first stage of method I and only in the second stage of method II. Oocyst identification was performed using the Kinyoun and Koster histochemical staining techniques. Samples analyzed by method I showed different degree of oocyst recovery, namely 10.9% with NaCl and 42.5% with MgSO4 in water and milk samples, while those samples analyzed by method II showed 10.6% with NaCl and 5.3% with sucrose in water and milk, respectively. Histochemical staining methods have no influence on the degree of oocysts recovery. The efficiency of Cryptosporidium spp. oocysts recovery methods depends on the nature and composition of the sample and on the methodology used for oocyst concentration.

Prevalencia de enteroparásitos en niños de la etnia Yukpa de Toromo, Estado Zulia, Venezuela

Yupkpa community in Perijá Sierra does not have drinking water or sewage elimination systems. Thus it becomes a propitious place for infection with intestinal parasites. To determine the prevalence of intestinal parasites in children, ages 0 to 14 years, in the Yukpa ethnic population of Toromo in the Perijá Mountains, Zulia State, Venezuela. Ninety one fecal samples were analyzed by the following coproparasitologic methods: saline solution and lugol fresh mount, formalin-ether concentration (Ritchie), and Kinyoun stain for intestinal coccidians. Overall parasite infection prevalence was 83.5%. The most frequently observed parasites were: Ascaris lumbricoides (57.1%), Trichuris trichiura (20.8%), Hymenolepis nana (14.2%), Blastocystis hominis (51.6%), Giardia lamblia (30.7%), and Entamoeba histolytica/E. dispar complex (21.9%). Coccidians were nont observed in any of the stool samples. The large number of infected people is directly related to the hygienic and sanitary conditions of the population studied.

Elevada prevalencia de Cyclospora cayetanensis en indígenas del estado Bolívar, Venezuela

The intestinal coccidioses caused by Cryptosporidium parvum, Isospora belli, and Cyclospora cayetanensis are parasitoses of major medical importance, but many epidemiological aspects of these infections are still unknown in Bolívar State, Venezuela. To determine the prevalence of intestinal parasites and especially of intestinal coccidiosis, an indigenous population in San Antonio de Morichal was evaluated from July 2003 to April 2004. Stool samples obtained by spontaneous evacuation were preserved in potassium dichromate 2.5% and examined by the formalin-ether concentration method and Kinyoun staining. Of the 160 indigenous people examined, 92.5% (148 cases) were infected. Entamoeba coli (58.8%), Ascaris lumbricoides (38.8%), and Giardia lamblia (18.8%) were the most prevalent intestinal parasites. Coccidiosis prevalence was 13.1%, and cyclosporiasis was the most prevalent with 11.9% (19 cases). One subject with C. parvum oocysts and another with I. belli oocysts were diagnosed. There was no difference according to sex (p > 0.05) and age (chi2 = 5.6; f.g. = 6) among individuals infected with C. cayetanensis. Of these, two cases had only diarrhea. In conclusion, a high prevalence of intestinal coccidiosis infection was found among indigenous people from this community, with C. cayetanensis as the most prevalent infection.

Cryptosporidium infection: diagnostic techniques

The aim of this study was to compare a rapid immunological test and a PCR method with the conventional morphological technique for the identification of Cryptosporidium in faecal samples. Cryptosporidium was found in five samples by Kinyoun acid-fast stain. Five samples yielded positive results on immunoassay, three of which yielded negative results on microscopy. Thus, only two patients were positive for Cryptosporidium according to both methods. PCR analysis confirmed only one sample as positive. Non-homogeneous distribution of parasites in stool samples, lack of oocysts in the tested sample and antigenic diversity among Cryptosporidium species may explain the poor agreement among the three tests. Based on our experience, microscopy test with Kinyoun stain is the best and cheapest way to detect Cryptosporidium spp. in faecal samples. With this method, we have found a 5.4% prevalence of Cryptosporidium infection in our area, similar to those reported for other regions of Italy and Europe.

Assessment of possible tuberculous lymphadenopathy by PCR compared to non-molecular methods

Tuberculosis is a major public health problem in India and other developing countries and has formed a lethal partnership with AIDS. It often presents a diagnostic challenge especially when clinical presentation is suggestive but bacteriological proof is lacking. The objective of this study was to compare the various diagnostic techniques in clinically suspected cases of tubercular lymph nodes and to find a suitable, cost-effective but sensitive and specific method for diagnosis. A total of 100 cases were recruited for the study. Fine needle aspiration cytology was done in all cases and the smears prepared were processed for Giemsa, Ziehl-Neelsen's, Kinyoun and Papanicolaou stains. Parts of the aspirated materials were assessed by fluorescent staining, culture and PCR. Seventy-four percent of aspirates were positive by fluorescent stain while only 22% were positive by culture. PCR could be performed in 55 cases, out of which 22 (40%) were positive. When compared to culture, the sensitivity and specificity of PCR were found to be 89.5% and 86.1%, respectively. Fluorescent stain was found to be the most sensitive (81.8%) of the conventional methods but showed poor specificity (28.2%). Interestingly, PCR detected 80% of smear-negative but culture-positive cases.

Sporotrichoid nocardiosis caused by Nocardia nova in a patient infected with human immunodeficiency virus

A23‐year‐old agricultural worker presented with multiple nodulo‐ulcerative lesions in a linear pattern along the right upper extremity. Two months prior, he had sustained an injury at the dorsal aspect of the base of the right thumb while working in the field. A nodular lesion appeared at the site after 3 weeks and subsequently suppurated, with discharge of purulent material and ulcer formation. Over the past 1 month, he had developed several other similar lesions along the right forearm up to the elbow. He was afebrile but complained of loss of weight in the recent past. He had a history of repeated unprotected sexual intercourse with commercial sex workers.General physical examination revealed a mild pallor. On local cutaneous examination, there was found to be an ulcer of about 2 cm × 2 cm at the base of the right thumb on the dorsal aspect (Fig. 1). The ulcer had a ragged margin with dirty slough at the floor, and the base was indurated and tender. On pressure, a purulent discharge was seen exuding from the surface. Similar nodulo‐ulcerative lesions of varying size were seen along the flexor aspect of the right forearm in a linear pattern (Fig. 2). Tender cord‐like structures were palpable between the lesions. The right epitrochlear and axillary lymph nodes were significantly enlarged and tender. Other cutaneous and systemic examinations were normal. A clinical diagnosis of nodular lymphangitis was made and the patient was investigated for etiological confirmation.Noduloulcerative lesion at the base of the thumbimageLesions in a sporotrichoid pattern over the forearmimageThe patient's hemoglobin was 10 mg dL−1, with hypochromic, microcytic peripheral blood. Erythrocyte sedimentation rate (ESR) was 100 mm in the first hour. Routine biochemical tests and chest X‐ray were within normal limits. A venereal disease research laboratory (VDRL) test was nonreactive. An enzyme‐linked immunosorbent assay (ELISA) for human immunodeficiency virus (HIV) was positive and the CD4+ T‐cell count was 550 cells/µL. A Gram‐stained smear from the discharge of a nodulo‐ulcerative lesion revealed numerous pus cells and a few irregularly stained, Gram‐positive, thin branching filaments. Modified Kinyoun staining demonstrated acid‐fast, branching filamentous structures (Fig. 3). Culture of the aspirated material from an unruptured nodule on Sabouraud's dextrose agar media grew small, whitish, dry, wrinkled colonies after 8 days.Photomicrograph showing thin branching filaments of Nocardia in the specimen of pus (modified Kinyoun stain, magnification ×100)imageBiochemically, the species showed urease positivity and reduced nitrate. Citrate utilization, gelatin liquefaction, casein hydrolysis and acetamide utilization tests were negative. An antibiogram showed sensitivity to erythromycin and broad‐spectrum cephalosporins. The species was identified as Nocardia nova on the basis of biochemical properties and the antibiogram. A skin biopsy was taken from the margin of an ulcer and histopathological examination revealed chronic granulomatous inflammation without demonstration of any organism.The patient was started on cotrimoxazole double strength (DS) tablets, twice daily. At follow‐up after 1 month, significant reductions in both inflammation and the size of the lesions were observed.The appearance of new nodular lesions was stopped. Follow‐up at 3 months showed complete healing of the nodulo‐ulcerative lesions, leaving few scars. The patient was advised to continue the same treatment for a further 3 months and has been monitored with regular follow‐up.