Ziehl-Neelsen Acid-fast Staining Research Articles

DETECTION OF NON-GRANULOMATOUS LESIONS OFMYCOBACTERIUMSPECIES IN CULTURED FRESHWATER HYBRID STURGEON

A disease outbreak with 71% (cumulative) mortality was reported in a freshwater hybrid sturgeon farm located in Hualien County, Taiwan. The moribund fish showed signs of lethargy, anorexia, diffuse external hemorrhages around mouth and anus, and on the base of fin and abdomen surface accompanied with abdomen swelling. Post-mortem examination revealed multiple red patches ranging from 1 to 5 cm in size on the liver. The histopathology revealed extensive coagulative to liquefied necrosis of various sizes in the liver and spleen. Focal necrosis of kidney was also evident. The lesions were positive with Ziehl-Neelsen acid fast staining. A rapid growth of nontuberculous mycobacteria (NTM) was isolated from liver of moribund sturgeon and the polymerase chain reaction (PCR) studies using primer sets derived from Mycobacterium sp. suggested this NTM was closely related to Mycobacterium marinum. There was only one bacterial colony been isolated in brain heart infusion agar (BHI Agar) and been identified as Streptococcus iniae. These findings suggest that a bacterium identified as belonging to M. marinum might cause heavy mortality.

Expression pattern of Mycobacterium tuberculosis Ag85B and its value in pathological diagnosis

To detect the expression of Mycobacterium tuberculosis secreted protein Ag85B in paraffin-embedded tissues by immunohistochemistry (IHC), and to evaluate its application in the pathological diagnosis of tuberculosis. One hundred and five tuberculosis specimens (54 pulmonary tuberculosis, 51 lymph nodal tuberculosis) and 51 specimens of other diseases (8 lung cancer, 10 pulmonary abscess, 10 bronchiectasis, 7 lymphoma, 5 necrotizing lymphadenitis, 4 reactive hyperplasia lymphoid, and 7 sarcoidosis) were collected from January 2012 to July 2013 from Beijing Chest Hospital, Capital Medical University. One-step IHC was performed on paraffin-embedded tissues using antibody directed against Ag85B. IHC and Ziehl-Neelsen (ZN) acid-fast staining showed that distribution and intensity of Ag85B expression were concordant with the distribution and number of acid-fast bacilli. IHC showed significantly higher sensitivity than ZN staining (50.5%, 53/105 vs. 31.4%, 33/105; χ² = 7.877, P = 0.005). The combined sensitivity of IHC and ZN staining was 59.0%. Moreover, oil immersion was not necessary for IHC, allowing more rapid diagnosis. IHC detection of Ag85B is a simple method with higher sensitivity than ZN staining, and demonstrated good value in the pathological diagnosis of tuberculosis.

Comparative evaluation of ziehl neelsen staining and knowledge, attitudes and practices of laboratory personnel in relation to ziehl nielsen.

To describe laboratory personnel's attitude and practices toward phenol exposure during Ziehl Neelsen (ZN) acid fast staining method and to evaluate the feasibility of an alternate modified Kinyoun cold (MKC) stain. A total of 187 sputum samples were collected from suspected tuberculosis cases and stained by the MKC method and ZN stain and were read by an experienced microscopist and a researcher. A cross-sectional questionnaire survey of 35 laboratory personnel was also conducted. Modified Kinyoun cold stain gave sensitivity, specificity, positive and negative predictive values of 100%, 99.4%, 94.1% and 100%, respectively. Both stains corresponded with an agreement rate of 99.5%. Almost 94.7% of respondents reported that they worked in a closed area when staining and 57.1% did the staining method without ventilation. Material safety data sheet (MSDS) of phenol was not known to 77.1% of laboratory personnel. All of the participants (100%) in this study welcomed a similar, non heating method for acid-fast bacillus (AFB). There was significant association between those not comfortable with phenol exposure (77.1%) and complaints of irritation (48.6%) and headache (2.9%) [c2 = 10.98, r = 0.55, p = 0.001]. The MKC is suitable for use as a substitute for the ZN method for the demonstration of AFB in the primary diagnosis and treatment assessment of pulmonary tuberculosis. Focus should be given on educating laboratory staff on the hazards, risks and precautions associated with the phenol/ZN method.

Crypto-Giardia antigen rapid test versus conventional modified Ziehl-Neelsen acid fast staining method for diagnosis of cryptosporidiosis

ObjectiveTo evaluate the validity of Crypto-Giardia antigen rapid test (CA-RT) in comparison with the conventional modified Ziehl-Neelsen acid fast (MZN-AF) staining method for the diagnosis of cryptosporidiosis. MethodsFifteen preserved stool samples from previously confirmed infections were used as positive controls and 40 stool samples from healthy people were used as negative control. A total of 85 stool samples were collected from suspected patients with cryptosporidiosis over 6 months during the period from January till June, 2011. The study was conducted in the department of parasitology, central laboratory, Alnoor Specialist Hospital, Makkah, Saudi Arabia. All samples were subjected to CA-RT and conventional MZN-AF staining method. Validation parameters including sensitivity (SN), specificity (SP), accuracy index (AI), positive predictive value (PPV), and negative predictive value (NPV) were evaluated for both tests. ResultsOut of 15 positive controls, CA-RT detected 13 (86.7%) while MZN-AF detected 11(73.3%) positive cases. However, CA-RT detected no positive case in 40 normal controls but MZN-AF detected 2(5%) as positive cases. Based on the results, the SN, SP, AI, PPV and NPV were high in CA-RT than MZN-AF staining method, ie., 86.7%vs. 73.3%, 100%vs. 95%, 96.4%vs. 89.1%, 100%vs. 84.6% and 95.2%vs. 90.5%, respectively. Out of a total of 85 suspected specimens, CA-RT detected 7(8.2%) but MZN-AF detected 6(7.1%) cases as positive. ConclusionsCA-RT immunoassay is more valid and reliable than MZN-AF staining method.

Prevalence of Cryptosporidium Parvum among Children in Iraq

Cryptosporidiosis is a parasitic disease caused by an apicomplexan protozoa of. Cryptosporidium parvum is the specific infective agent in human . The present study aimed to search for the presence of C. parvumand to determine the prevalence of this parasite among children in Kut city, Iraq. Six hundred stool samples were collected from children less than twelve years old from October 2011 to May 2012. Stool samples were inspected by modified Ziehl-Neelsen acid fast stain and ELISA. Results indicated that 203 cases gave positive results (33.83 %) and 397 cases gave negative results (66.17%) with Ziehl-Neelsen acid fast stain . The higher infection, 115 (19.17%) appeared in age (<1) year while the lower infection 37 (6.17%) appeared in age (1-6) years. There was association between anemia 66.01% (134/203), Packed Cell Volume (PCV) 66.01% (134/203), White Blood Cells Count (WBC's) 66.01% (134/203) that showed increase in number, and infection with cryptosporidiosis, respectively. The high percentage of positive cases (100%) was recorded in microscopic examination compared to 72.5% (129/178) of positive cases detected by ELISA assay. The present study is the first record of cryptosporidiosis among children in Wasit Province, Iraq. It demonstrated clearly a high prevalence rate of C. parvum among children of less than 12 years old in Iraq. ELISA technique will be of great value in the rapid and accurate diagnosis of C. parvum in human fecal materials.

A modified acid-fast staining method for rapid detection of Mycobacterium tuberculosis

A modified acid-fast staining method was developed for rapid detection of Mycobacterium tuberculosis and its L forms, wherein carbol fuchsin and dioxogen were mixed into the sputum smear. With this method, the dyeing time is shortened and heating is not required. The sensitivity, specificity, positive predictive value, negative predictive value, positive rate, and diagnostic efficiency of the new method were compared to those obtained by PCR using 50 clinical samples. Further, 468 clinical samples were analyzed using the new method, the modified intensified Kinyoun (IK) acid-fast staining method, and the traditional Ziehl–Neelsen acid-fast staining method. Differences among the positive detection rates of the three methods were analyzed using Student's t-test, and no significant differences were found between the new method and the modified IK acid-fast staining method, while the rates of both these methods were higher than that of the traditional acid-fast staining method. Additionally, the dyeing time in the new method was markedly less than that in the modified IK acid-fast staining method (5min and 24h, respectively).

The first reported cases of human cryptosporidiosis caused by Cryptosporidium hominis in Slovak Republic

Cryptosporidiosis belongs to the important parasitic infections with zoonotic potential and the occurrence in European countries is rare. The first cases of cryptosporidiosis caused by Cryptosporidium hominis detected in the Slovak republic were described here. Collection of examined humans consisted of five family members. Faecal specimens were examined by formalin sedimentation, by the Sheather's sugar flotation and by immunochromatography and visualised by the Ziehl-Neelsen acid fast stain. A fragment of the Cryptosporidium small subunit ribosomal RNA gene was amplified by nested polymerase chain reaction and species was determined by restriction fragment length polymorphism analysis with the endonucleases SspI and VspI. C. hominis was found in faeces of two immunocompetent siblings (a 7-year-old boy and a 2-year-old girl). The symptoms occurred only in the boy as gastrointestinal disorders lasting 5 days, and manifested by abdominal pain, an elevated body temperature (37.2 °C), mild diarrhoea, accompanied by lassitude, depression and anorexia. Ultrasonic scan revealed enlarged spleen and mezenteric lymph nodes. Microscopic examination of the stool sample revealed numerous Cryptosporidium oocysts. The DNA typing identified C. hominis subtype IbA10G2. Cryptosporidium was also detected in the boy's sister without any complications and symptoms. Their father, mother and grandmother were parasitologically negative. The source of infection remained unknown. Human cases in present study reflect necessity of systematic attention on intestinal parasites diagnostic inclusive of cryptosporidia.

Genetic Characterization of Cryptosporidium spp. in Diarrhoeic Children from Four Provinces in South Africa

The diversity of Cryptosporidium at species, subtype family and subtype level in diarrhoeic children was investigated in four provinces in South Africa. A total of 442 stool samples from children <5 years of age were collected under a large rotavirus surveillance programme and analysed by Ziehl-Neelsen acid-fast staining. Fifty-four (12.2%) were positive for Cryptosporidium, of which 25 were genotyped by polymerase chain reaction (PCR)-restriction fragment length polymorphism (RFLP) and DNA sequence analyses of the 18S rRNA gene. The majority of genotyped specimens were identified as C. hominis (76%), and a high genetic diversity was found with five different C. hominis subtype families (Ia, Ib, Id, Ie and If). Cryptosporidium parvum was found in 20% of the isolates, and three subtype families were identified (IIc, IIe and IIb), with subtype family IIc being the most common. One specimen was identified as C. meleagridis of the subtype family IIId. These results are in accordance with findings from other developing countries and report for the first time the presence in South Africa of C. meleagridis, various subtypes of C. parvum and the subtype family Ie of C. hominis. The results suggest that C. hominis and anthroponotic C. parvum subtypes are the major cause of cryptosporidiosis in South Africa. Further molecular studies are needed to better understand the epidemiology and public health importance of Cryptosporidium in humans in South Africa.

Detection of Cryptosporidium oocysts in calves and children in Mosul, Iraq

This study was carried out during May-October 2010 to determine the prevalence of Cryptosporidium spp. in calves and children in rural areas around Mosul, north of Iraq. A total of 78 faecal samples were taken from calves which were no more than one year old. Information about age, consistency of faeces (as diarrheic or normal) and contact with human were recorded. At the same time, 95 stool samples were taken from children aged one month to 12 years old suffering from diarrhea and living in the same farms where the samples of calves have been collected. All samples were stained with modified Ziehl-Neelsen acid-fast stain and Auramine O techniques to detect the presence of Cryptosporidium oocysts. Results revealed that the prevalence of Cryptosporidium in calves and children were 43.56% and 18.9%, respectively. Comparison of results obtained with the two stain techniques showed no significant difference and being sensitive.

Blastocystis hominis as a contributing risk factor for development of iron deficiency anemia in pregnant women

Intestinal parasitic infection increases the risk of developing iron deficiency anemia (IDA) during pregnancy. The objective of this study was to assess Blastocystis hominis as a contributing risk factor for development of IDA in pregnant women. A total of 200 fecal specimens from 120 pregnant women with IDA (mean Hb = 9.6 g/dl), and 80 non-anemic controls were examined for Blastocystis. Fecal specimens were examined by the formalin/ethyl-acetate concentration technique, iron hematoxylin staining, modified Ziehl-Neelsen acid-fast staining, and by the in vitro cultivation technique for Blastocystis. Frequency of Blastocystis infection, detected microscopically and by the in vitro culture technique, was significantly higher in IDA study group (n = 48; 40%) compared to non-anemic controls (n = 5; 6.3%; P < 0.0001), and 26.5% (n = 53) in all study subjects. Among the 48 cases, Blastocystis without other intestinal parasitic infections was detected in 41 cases (34.2%), while seven cases (5.8%) with Blastocystis were coinfected with other intestinal parasites which included Giardia and Cryptosporidium (1.7% each), and Entamoeba sp., Ascaris, and Trichuris (0.8% each). The mean Hb level of the 48 Blastocystis-infected cases was 9.2 g/dl (mild anemia). While the other 72 IDA cases with no infection had mean Hb of 10.0 g/dl (mild anemia), with a significant difference in mean Hb level between Blastocystis-infected and the non-infected IDA cases (P < 0.0001). Furthermore, among the 48 Blastocystis-infected IDA cases, the mean Hb of the 41 Blastocystis-infected cases without other intestinal parasitic co-infection was 9.1 g/dl (mild anemia), while the mean Hb level of the 7 Blastocystis-infected cases with other intestinal parasitic co-infection was 8.7 g/dl (moderate anemia). Findings of the current study showed that B. hominis infection contributes to the development of IDA in pregnant women. Hence, parasitological diagnostic tests are recommended in routine examination at all antenatal clinics.

Diagnosis of paratuberculosis in goats by cell mediated immune response, conventional and molecular diagnostic techniques

In the present study efficacy of single intradermal Johnin test, acid fast staining of faecal smear and IS 900 faecal polymerase chain reaction tests was evaluated in 200 goats for detection of Mycobacterium avium subsp paratuberculosis. Two hundred goats comprising 150 goats from an organised farm in Trichur district and 50 goats reared under field condition at farmers premise from Malappuram district of Kerala state formed the study population. Faecal smear from all the 200 goats was stained by Ziehl-Neelsen acid fast stain and faecal polymerase chain reaction (PCR) specific for M. avium subsp paratuberculosis (MAP); IS 900 was performed on all samples. All the animals were subjected to single intradermal Johnin test. Out of 200 goats screened for paratuberculosis, six goats (3%), 11 goats (5.5%) and 42 goats (21%) were found positive by Ziehl-Neelsen acid fast staining of faecal smear, single intradermal Johnin test and IS 900 PCR respectively. Results of the present study indicate that amplification of IS 900 insertion element was the most specific and sensitive diagnostic detection method. Single intradermal Johnin test and Ziehl-Neelsen acid fast staining did not show any significant difference.

Genotyping of Cryptosporidium spp. in clinical samples: PCR-RFLP analysis of the TRAP-C2 gene.

The aim of the present study was to determine the species and genotypes of Cryptosporidium spp. among children with diarrhea by PCR-RFLP using the TRAP-C2 gene. Cryptosporidium is a globally distributed protozoan parasite and one of the most common causes of infection and diarrhea in humans. Four hundred and sixty nine stool samples were collected from children less than 12 years with diarrhea who had been referred to Pediatrics Medical Centers in Gazvin provinces. The presence of Cryptosporidium oocysts was determined by Ziehl-Neelsen acid fast staining, then, genomic DNA was extracted from positive samples and nested PCR-RFLP was performed to amplify the TRAP-C2 gene. The overall prevalence of Cryptosporidium infection in children was 2.5 %. Results of nested PCR amplification showed that of 12 positive children samples, 10 (83.3%) were belonged to C. parvum, followed by C. hominis in 1 (8.3%) and mixed infection in 1 isolate (8.3%). This study showed that Cryptosporidium parvum (the zoonotic genotypes) is more prevalent than other Cryptosporidium species in children from this area. This suggests that zoonotic transmission is the main mode of transmission of Cryptosporidium infection in Iran.

Pathological study of mycobacterium abscessus keratitis

To characterize the cytopathology, histopathology, immunohistochemistry and ultrastructural organization features of Mycobacterium abscessus keratitis. This retrospective case report included six cases with the lesions tissues scraped from acute phase of mycobacterium abscessus keratitis after corneal foreign body removal. Among the 6 scrapings, 4 were tested for Ziehl-Neelsen acid-fast staining and 3 were examined by transmission electron microscopy (TEM). The corneal tissues of 2 cases in chronic phase of the disease were collected during keratoplasty and were examined by pathological examination including hematoxylin-eosin (HE) staining, acid-fast staining, immunohistostaining and TEM. A single or fascicular red bacilli were observed after acid-fast staining in the four cases of corneal scrapings. The smear samples contained a large number of neutrophils and necrotic corneal epithelium and stroma tissue accompanying with a large number of mycobacteria that were positive for acid-fast staining scattered in and out of inflammatory cells. The corneal buttons obtained at keratoplasty in the 2 patients demonstrated corneal epithelial hyperplasia, stroma degeneration, necrosis and exelcosis, neutrophil and lymphocyte infiltration, neovascularization and fibrous hyperplasia, but no positive mycobacteria by Ziehl-Neelsen acid-fast staining. There were no CD4(+) or CD8(+) cells from immunohistochemistry examination. TEM of the corneal scrapings of the 3 cases showed phagocytosis of mycobacterium abscessus in mononuclear macrophage or neutrophils, inclusion compounds of small vacuoles and metachromatic granules in the thallium. TEM of corneal buttons of 2 cases showed slender rod-shaped mycobacteria with several to dozens of finely ground particles with high electron-dense, but no cell wall (L-form) or inclusion compounds. Mycobacterium abscessus keratitis presents acute purulent inflammatory changes in early stage and advances to chronic and non-specificity inflammation changes in late stage. L-formed mycobacterium abscessus make the disease protraction.

Spontaneous bladder perforation: a rare complication of tuberculosis

Spontaneous bladder perforation secondary to tuberculosis (TB) is very rare. Only three cases have been reported so far in the literature. Due to its rarity, the diagnosis of spontaneous bladder perforation is often missed. Confirmation of TB via culture takes a long time and starting empirical treatment for TB is necessary. We relate our experience with a young woman who presented with clinical features of a perforated appendix and was only diagnosed with bladder perforation during laparotomy. She also had distal right ureteral stricture and left infundibular stenosis. The provisional diagnosis of TB was attained via typical histopathological features and a positive Mantoux test. She was started empirically on anti-TB treatment and recovered without any complications. Urine culture after 6 weeks confirmed the diagnosis of TB.

Diagnosis of Avian Tuberculosis in Swinhoe's Pheasants Using Conventional and Molecular-Based Techniques

Avian tuberculosis was diagnosed via histopathology, microbiology, and molecular biology in two of six pheasants from a local sanctuary bird house in Taiwan. Swinehoe's pheasant (Lophura swinhoii) is a near-threatened species in Taiwan. The infected birds showed clinical signs such as fatigue, inappetence, diarrhea, and fluffing of feathers. On postmortem, nonmineralized caseogranulomas were present in the brain, heart, lung, liver, spleen, costal membranes, and intestinal tracts. The presence of granulomas in the lungs of the infected pheasants may suggest that exposure to the infective agent was via the respiratory route rather than the alimentary route. Histopathologic findings were typical of avian tuberculosis, including acid-fast bacilli and centrally located caseous necrosis surrounded by epitheloid macrophages, lymphocytes, and multinucleated giant cells. Laboratory confirmation was made based on lesions and via Ziehl-Neelsen acid-fast stain, polymerase chain reaction, nucleic acid sequencing, and a reliable assay protocol for identification of diseases bioactive amplification with probing assay.

Detection and identification of mycobacteria by mycolic acid analysis of sputum specimens and young cultures

Ziehl–Neelsen acid-fast staining and mycolic acid analysis of concentrated samples and Middlebrook 7H9 cultures were carried out on 127 sputum specimens to evaluate a rapid method for detecting and identifying mycobacteria by analyzing fluorescent derivatives of mycolic acids in concentrated sputum specimens and in Middlebrook 7H9 cultures and compare with mycobacterial detection using Lowenstein–Jensen (LJ) cultures. All samples were classified into five groups according to the number of acid-fast bacilli observed in the smear. The group of samples with 3+ acid-fast bacilli in the smear had the highest number of positive detections of mycolic acids in the concentrated samples and the Middlebrook 7H9 cultures (81.8 and 100%, respectively). The overall percentages of mycolic acid detection for both sample types were 43.2 and 91.3%, respectively. The mycolic acid analysis of the Middlebrook 7H9 cultures had the fewest false negative detections with respect to the LJ cultures. The analysis of fluorescent derivatives of mycolic acids, using HPLC, is useful for concentrated sputum samples with large number of bacilli (3+) and is preferred for Middlebrook 7H9 cultures, even for clinical specimens with a low number of bacilli. Furthermore, with this analytical method, the simultaneous detection and identification of mycobacteria is usually possible.

Narrow‐spectrum staining pattern of Pityrosporum*

Broad-spectrum fungal stains are used to detect fungal organisms, but narrow-spectrum stains can assist in fungal differential diagnosis. These stains include mucicarmine and Fontana-Masson (FM) for Cryptococcus, Alcian Blue for Cryptococcus and Blastomyces, Congo Red for Blastomyces and Coccidioides, and Ziehl-Neelsen for some examples of Blastomyces and Histoplasma. Pityrosporum is increasingly being recognized as a pathogen capable of significant cutaneous and systemic infections, but the narrow-spectrum staining pattern of Pityrosporum has not yet been systematically studied and reported. Paraffin-embedded archival tissue from 10 skin biopsies containing Pityrosporum was stained with Alcian Blue, Congo Red, Ziehl-Neelsen acid-fast stain, and FM stain. In addition to the 60-min staining time, a modified FM stain with 15-, 30-, and 45-min staining times was also performed. All cases stained positive with Alcian Blue, Congo Red, and FM stains and negative with the Ziehl-Neelsen stain. The modified FM stain with the 15-min staining time showed significant staining in more than 50% of the cases. The narrow-spectrum staining pattern of Pityrosporum distinguishes it from Cryptococcus, Blastomyces, Candida, Histoplasma, and Coccidioides.

Disseminated Mycobacteriosis Due to Mycobacterium Avium in Captive Bengal Tiger (Panthera Tigris)

A 2-year-old captive female Bengal Tiger (Panthera tigris) died after prolonged anorexia in the Gwangju Uchi Park Zoo, Gwangju, Republic of Korea. Necropsy revealed multiple nodules of varying sizes in the lung, liver, kidney, and spleen. Histopathologic examination revealed a typical granuloma composed of caseous necrotic areas surrounded by lymphocytes with a few giant cells and foamy macrophages. Periodic acid-Schiff stain and Gomori methenamine silver stain did not reveal any fungal bodies. The Ziehl-Neelsen acid-fast stain revealed few acid-fast organisms in the lung, liver, kidney, and spleen. A polymerase chain reaction assay of the lung, liver, kidney, and spleen yielded a positive result for Mycobacterium avium subsp. avium. This is an unusual case of disseminated infection of a wild mammal with avian mycobacteriosis, and is believed to be most likely associated with the feeding of tigers with culled chickens infected with M. avium.

Comparison of Diagnostic Detection Methods for Mycobacterium avium subsp. paratuberculosis in North American Bison

Tissues and fecal material were collected from 14 North American bison (Bison bison) that were suspected of having Johne's disease and analyzed for the presence of Mycobacterium avium subsp. paratuberculosis (M. paratuberculosis). Sections of ileum, ileal-cecal lymph node, and three sequential sections of jejunum with their associated mesenteric lymph nodes were taken from each animal. Fecal culture indicated that 5 of 14 (35.7%) animals were infected, whereas cultures from tissues detected 12 of 14 (85.7%) animals as infected and 59 of 111 (53.2%) of the tissues as positive for M. paratuberculosis. Polymerase chain reaction analysis identified infection in 14 of 14 (100%) animals and in 91 of 112 (81.2%) tissues. In addition, tissues were processed for Ziehl-Neelsen acid-fast staining, auramine O/acridine orange fluorescent staining, and immunohistochemical staining. Ziehl-Neelsen and auramine O staining identified 7 of 14 (50%) and 5 of 14 (35.7%) animals as infected and 24 of 112 (21.4%) and 28 of 112 (25%) tissues as positive, respectively. Immunohistochemical analyses of bison tissues, using antisera collected from rabbits immunized with four different preparations of M. paratuberculosis, identified a greater percentage of infected animals (ranging from 57 to 93%) and positive tissues (ranging from 28 to 46%). Collectively, these data indicate that DNA-based detection of M. paratuberculosis was more sensitive than bacterial culture or staining, identified infection in all the bison, and detected the greatest number of positive tissues within each animal.

Molecular analysis of the 18S rRNA gene of Cryptosporidium parasites from patients with or without human immunodeficiency virus infections living in Kenya, Malawi, Brazil, the United Kingdom, and Vietnam.

An 840-bp fragment of the 18S rRNA gene was used to identify Cryptosporidium spp. recovered from human immunodeficiency virus (HIV)-infected and -uninfected patients from Kenya, Malawi, Brazil, the United Kingdom, and Vietnam. Initial identification was by Ziehl-Neelsen acid-fast staining. Confirmation was by nested PCR, targeting the most polymorphic region of the 18S rRNA gene. Genotyping was by restriction endonuclease digestion of the PCR product followed by nucleotide sequencing. Among 63 isolates analyzed, four genotypes of Cryptosporidium were identified; 75% of the isolates were of the C. parvum human genotype, while the potentially zoonotic species were of the C. parvum bovine genotype (21.7%), the C. meleagridis genotype (1.6% [one isolate]), and the C. muris genotype (1.6% [one case]). HIV-infected individuals were more likely to have zoonotic genotypes than the HIV-uninfected individuals. Among the C. parvum group, strains clustered distinctly into either human or bovine genotypes regardless of the geographical origin, age, or HIV status of the patients. The intragenotypic variation observed in the C. parvum human genotype was extensive compared to that within the C. parvum bovine genotype group. The variation within genotypes was conserved in all geographical regions regardless of the patients' HIV status. The extensive diversity within genotypes at the 18S rRNA gene locus may limit its application to phylogenetic analyses.