Modified Kinyoun Stain Research Articles

Cyclosporiasis in immunocompetent and immunocompromised patients - A Twelve years experience from a tertiary care centre in Northern India.

Cyclosporiasis is an emerging enteric coccidian parasitic disease worldwide, caused by the parasite Cyclospora cayetanensis. There is scanty data from India, especially among immunocompetent patients. The aim is to evaluate the occurrence of Cyclosporiasis in immunocompetent and immunocompromised patients. It is a prospective cohort study conducted from June 2006 to May 2018 at our tertiary care center. Stool samples were collected from the 900 patients with diarrhea (both immunocompetent and immunocompromised) and 170 healthy controls to look for Cyclospora by modified Kinyoun staining. Mann-Whitney U test/Fisher exact test were used for statistical analysis. Oocysts of C. cayetanensis were detected in 10/900 patients and none of the healthy controls. The median age of patients was 38.5 years (10-65 years) and males (6/10) outnumbered the females in harboring the parasite. Eight patients were immunocompromised (five postrenal transplant cases and one-one patient each with HIV, non-Hodgkin's lymphoma, and juvenile polyarthritis), and two patients were immunocompetent. Cyclospora infection was more common in immunocompromised patients (8/300, 2.67%) than the immunocompetent patients (2/600, 0.33%); P < 0.001. Eight patients responded well to trimethoprim-sulfamethoxazole, one died, and one was lost to follow-up. Coinfection with Cryptosporidium spp. was seen in one patient. Cyclospora causes diarrhea in both immunocompromised and immunocompetent persons. Its burden may be underestimated due to a lack of awareness and appropriate diagnostic methods. Special staining techniques are important for diagnosis as they may be missed by routine microscopy.

A Road Less Travelled: Clinical Comparison of HIV Seropositive and Seronegative Patients with Cystoisosporiasis – An 11-Year Experience from a Tertiary Care Centre in Northern India

Background:Cystoisospora is a well-known opportunistic enteric parasite among human immunodeficiency virus (HIV) seropositive patients but there is a paucity of data among HIV negative patients. This study investigated Cystosporiasis on both HIV positive and negative patients, with or without diarrhea, presenting to a tertiary care and super specialty center of northern India. Methodology:Oocysts of Cystoisospora were detected on light microscopy, by modified Kinyoun staining of stool specimens, over an 11-year study period. Results: Of the 10,233 stool specimens evaluated, Cystoisospora was detected in 64 patients, 37 (57.81%) of whom were HIV positive. Year-wise analysis showed an overall declining trend of cystoisosporiasis. Maximum cases were detected in May and June in HIV positive patients and February and September among HIV negative patients. Among HIV positive patients, the mean CD4 count was 152.04 ± 81.12cells/μL, mean absolute eosinophil count (AEC) was 229.16 ± 175.62 cells/μL and 12.5% patients had mild eosinophilia. Tuberculosis was the most common co-morbidity. Dual infections of Cystoisospora with Cryptosporidium and Giardia were also seen. Among HIV negative patients, eight had primary autoimmune disorders, seven were solid organ transplant recipients and the rest had chronic bowel diseases. The mean AEC was 485.47 ± 414.88 cells/μL, with 14.81% patients showing mild and 11.11% showing marked eosinophilia. Dual infection with Giardia was seen. Recurrent cystoisosporiasis was noted, despite cotrimoxazole treatment in a single case. Conclusion: The epidemiology of cystoisosporiasis differs between HIV seropositive and seronegative patients in terms of year-wise and month-wise trends, co-infections and most importantly, AECs.

English

Primary cutaneous nocardiosis can present as an abscess or cellulitis with sporotrichoid morphology. Deep inoculation in the subcutaneous tissue can present as a mycetoma. Mycetoma over the elbow region is rare. We herein describe a case of primary cutaneous nocardiosis presenting as a mycetoma, caused by Nocardia asteroides. The patient had extensive keloidal lesions with few sinuses extruding serous discharge over the right forearm around elbow joint. Initially pus and later, the deep tissue biopsy samples were sent for culture and sensitivity, without suspecting nocardial infection. Gram stain of the pus showed few WBCs and occasional Gram positive branching filaments. Modified Kinyoun stain of the same revealed acid-fast filamentous structures. Culture of the tissue specimen yielded Nocardia asteroides. The patient responded well to a modified Ramam's regime. On follow-up, the patient showed no recurrence at 1 year. Our case demonstrates the need for focused clinical search with a high degree of suspicion, and appropriate laboratory procedures in the diagnosis and management of cutaneous nocardiosis.

Cryptosporidium spp. infection in mares and foals of the northwest region of São Paulo State, Brazil

The present study aimed to analyze the occurrence of infection by Cryptosporidium spp. in mares and their respective foals. This study was carried out in 11 farms located in the municipalities of Araçatuba, Birigui, Guararapes and Santo Antônio do Aracangua, in the northwest region of the State of Sao Paulo, from November 2010 to March 2011. A total of 98 mares and 98 foals of several breeds were analyzed; among foals, 59 were males and 39 females, aged from three to 330 days. Feces were collected directly from the rectal ampulla, purified and processed according to modified Kinyoun stain. Occurrence of Cryptosporidium spp. was 21.4% (21/98) for foals and 18.4% (18/98) for mares. Occurrence of Cryptosporidium spp. had significant association with breeds and age of animals. Results obtained led to the conclusion that foals older than two months and Mangalarga animals are less susceptible to the occurrence of Cryptosporidium spp.

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.

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.

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.

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.

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.

Growth of acid fast L forms from the blood of patients with sarcoidosis.

Acid fast cell wall deficient forms (CWDF) of bacteria have been grown from blood, bronchial washings, and ocular anterior chamber fluid from patients with sarcoidosis. A monoclonal antibody raised against Mycobacterium tuberculosis whole cell antigen (H37RV) was used to characterise further CWDF grown from the blood of patients with sarcoidosis. Blood from 20 patients with active sarcoidosis and from 20 controls was cultured using methods favourable for the growth of CWDF. Isolates were further characterised by indirect fluorescent antibody analysis using a monoclonal antibody highly reactive with M tuberculosis. CWDF were grown from the blood of 19 of 20 subjects with sarcoidosis. All isolates stained positively with the monoclonal antibody and with a modified Kinyoun stain. No organisms were grown from the blood of controls. These data demonstrate that CWDF can be grown from the blood of nearly all patients with active sarcoidosis. The results confirm that the organisms are mycobacterial in origin and are similar, if not identical, to M tuberculosis. Their role in the pathogenesis of sarcoidosis is unknown.