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Purpose: The aim of this study was to find out Bacterial etiology and antimicrobial susceptibility pattern of bacteria causing infective keratitis at a tertiary care hospital of LahoreStudy Design:Descriptive cross sectional studyStudy Place and Study Period: PGMI/AMC/LGH, Lahore, in 2015 - 2018. Sample Collection: Non-probability convenience samplingMaterial and Methods: The corneal scrapings taken by the ophthalmologist was immediately inoculated on Chocolate agar, Blood agar and MacConkay agar culture plates with the help of sterilized wire loop in the operation theater.These plates were taken to laboratory for incubation and further microbiological processing. After inoculating on the culture agar plates, the scrapings from cornea were smeared on glass slides with the help of sterilized wire loop. The smears were air-dried stained with Gram’s stain, Kinyoun stain and Giemsa stain. The culture and staining were further processed for microbiological analysis of bacterial etiology and antimicrobial susceptibility.Results:7 of each cases of Staphylococcus aureus and Streptococcus pneumoniae were isolated accounting 28% of total bacteria. Gram negative bacteria are relatively sensitive to all groups of antibiotics as compared to Gram positive bacteria who were resistant of major groups of antibiotic like Fluoroquinolones and aminoglycosides. Conclusion:We concluded that a cumulative antimicrobial data should be published from laboratory on antimicrobial susceptibiblity pattern of bacterial keratitis for its empirical treatment.

Stool samples from 150 immunocompromised patients (including 120 Gastrointestinal [GI] symptomatic and 30 GI asymptomatic) suspected to have Cyclospora cayetanensis infection were examined to evaluate the validity of Milacek-Vitovec staining method in comparison with kinyoun stain for the diagnosis of cyclosporiasis. All results were confirmed with nested PCR as a gold standard. Validation parameters included sensitivity, specificity, accuracy index, positive predictive value and negative predictive value. In addition, infection intensity was compared among all positive samples. DNAs of Cyclospora oocysts were detected among 30/120 (25%) of GI symptomatic patients versus 1.7 % of GI asymptomatic (5/30). Milacek-Vitovec stain showed more validity than kinyoun with a sensitivity reached 80% versus 73.4% (28 versus 25 from 35 cases) while the specificities of showed 90.4% (104/115) versus 81.7% (94/115), respectively. Mild infection intensity was detected among 10 patients only three of them (30%) were detected by Milacek-Vitovec stain while only one (10%) was detected by Kinyoun, Moderate infection were identified among 10 patients all of them were diagnosed by Milacek-Vitovec stain and 9 (90%) was detected by Kinyoun stain. Heavy infection was detected among 15 patients all diagnosed with Milacek-Vitovec stain while 14 were detected with kinyoun.

Highlights • We present a case of Nocardia beijingensis infection in an immunocompetent patient. • Magnetic resonance imaging (MRI) scan showed countless miliary-like ring-enhancing cerebrospinal lesions. • A brain biopsy is essential to confirm the diagnosis and to identify the etiological species of Nocardia. • 16S ribosomal RNA sequencing is useful for species identification., The genus Nocardia usually infect immunocompromised patients. Pulmonary nocardiosis is the most common Nocardia-induced infection while central nervous system (CNS) is the most common extrapulmonary site to develop nocardial disease. Approximately 54 % cases of previously reported nocardial brain abscesses were solitary lesions, while 38 % cases had multiple lesions; but miliary-like ring enhancing lesions have not been reported previously. We present a case of immunocompetent patient with miliary-like cerebrospinal lesions caused by Nocardia beijingensis. A 68-year-old Japanese man presented with acute-onset headache, vomiting, and progressive disturbance of consciousness. A contrasted magnetic resonance imaging (MRI) scan showed countless miliary-like ring-shaped enhancing lesions on the patient’s whole brain, brain stem, and cervical spinal cord. A brain biopsy was performed and Kinyoun stain suggested that the brain lesions were abscesses caused by Nocardia species. 16S ribosomal RNA sequencing identified Nocardia beijingensis as the causative microorganism. The trimethoprim/sulfamethoxazole regimen was continued for 7 months and the patient showed good improvement with recovery of consciousness. Clinical manifestation of Nocardia beijingensis infection is poorly studied. Further cases are needed to be accumulated.

The following fictional case is intended as a learning tool within the Pathology Competencies for Medical Education (PCME), a set of national standards for teaching pathology. These are divided into three basic competencies: Disease Mechanisms and Processes, Organ System Pathology, and Diagnostic Medicine and Therapeutic Pathology. For additional information, and a full list of learning objectives for all three competencies, see http://journals.sagepub.com/doi/10.1177/2374289517715040 . 1

Answer: Cutaneous infection due to Mycobacterium chelonae. The presence of acid-fast bacilli in a Kinyoun stain of the tissue suggested that this infection was caused by Mycobacterium spp. Mycobacterial species capable of causing nodular skin lesions include Mycobacterium fortuitum, Mycobacterium

Nocardia species are strictly aerobic, gram-positive, branching, filamentous rods which are beaded appearing and stained variably with the modified acid-fast Kinyoun stain. They can fragment into pleomorphic, rod-shaped, or coccoid pieces. Nocardia are sometimes difficult to recognize and identify in the laboratory, leading to delays in clinical diagnosis. Their relatively slow growth can result in the cultures being discarded before the colonies can be seen. Nocardia are found most often in the environment in soil, water, and vegetation. More than half of the time, these organisms are opportunistic pathogens, causing disease in both solid organ and stem cell transplantations. Less recognized as opportunistic pathogens are Actinomyces species, gram-positive, anaerobic bacilli which are not acid-fast. These organisms are morphologically similar to Nocardia species and can be confused on Gram staining. Nocardia and Actinomycetes cause distinct syndromes which will be reviewed here.

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.

A 35-yr-old woman, with acquired immunodeficiency syndrome, presented with right arm pain, erythema, and swelling. A lytic lesion of the ulna was found by radiograph. Ultrasonic and fluoroscopic guided fine-needle aspiration (FNA) yielded a neutrophilic and histiocytic exudate admixed with abundant long, bacillary forms that appeared as negative images on Papanicolaou stain and as very coarsely beaded acid-fast bacilli, resembling candy canes, on Kinyoun stain. These morphologic features permitted a preliminary diagnosis of mycobacteriosis, possibly of M. kansasii (MK) etiology. Appropriate therapy was initiated and resulted in not only marked symptomatic improvement of osteomyelitis and cellulitis, but resolution of chronic pulmonary infiltrates, presumed to be caused by fibrosis. Culture confirmed MK infection 3 wk after FNA. MK is one of the few mycobacteria that has a sufficiently characteristic morphology to permit presumptive diagnosis by smear. Culture, however, still remains the definitive method of speciation. Diagn. Cytopathol. 1998;19:94–97. © 1998 Wiley-Liss, Inc.

Background Microsporidia are obligate intracellular parasites that infect a broad range of vertebrates and invertebrates. They have been increasingly recognized as human pathogens in AIDS patients, mainly associated with a life-threatening chronic diarrhea and systemic disease. However, to date the global epidemiology of human microsporidiosis is poorly understood, and recent data suggest that the incidence of these pathogens is much higher than previously reported and may represent a neglected etiological agent of more common diseases indeed in immunocompetent individuals. To contribute to the knowledge of microsporidia molecular epidemiology in HIV-positive patients in Nigeria, the authors tested stool samples proceeding from patients with and without diarrhea. Methodology/Principal Findings Stool samples from 193 HIV-positive patients with and without diarrhea (67 and 126 respectively) from Lagos (Nigeria) were investigated for the presence of microsporidia and Cryptosporidium using Weber’s Chromotrope-based stain, Kinyoun stain, IFAT and PCR. The Weber stain showed 45 fecal samples (23.3%) with characteristic microsporidia spores, and a significant association of microsporidia with diarrhea was observed (O.R. = 18.2; CI: 95%). A similar result was obtained using Kinyoun stain, showing 44 (31,8%) positive samples with structures morphologically compatible with Cryptosporidium sp, 14 (31.8%) of them with infection mixed with microsporidia. The characterization of microsporidia species by IFAT and PCR allowed identification of Enterocytozoon bieneusi, Encephalitozoon intestinalis and E. cuniculi in 5, 2 and 1 samples respectively. The partial sequencing of the ITS region of the rRNA genes showed that the three isolates of E.bieneusi studied are included in Group I, one of which bears the genotype B. Conclusions/Significance To our knowledge, this is the first report of microsporidia characterization in fecal samples from HIV-positive patients from Lagos, Nigeria. These results focus attention on the need to include microsporidial diagnosis in the management of HIV/AIDS infection in Nigeria, at the very least when other more common pathogens have not been detected.

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.

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.

Newly available assays offer alternatives to conventional microscopic examination for Cryptosporidium spp. We compared two enzyme immunoassays, ProSpect Cryptosporidium microtiter assay (Alexon, Inc., Mountain View, Calif.) and Color Vue Cryptosporidium assay (Serady, Indianapolis, Ind.), and a direct immunofluorescent assay, Merifluor Cryptosporidium kit (Meridian Diagnostics, Cincinnati, Ohio), with acid-fast Kinyoun-staining for the detection of Cryptosporidium spp. Examinations were performed on 129 stool specimens received from patients during a recent waterborne outbreak. A specimen was considered positive when organisms could be identified visually by acid-fast and immunofluorescent stains or if organisms could be visualized by either acid-fast or immunofluorescent stain and detected by both enzyme immunoassays. The final number of positive specimens was 55. No single procedure detected all 55 positive specimens. Of these, ProSpect and Color Vue detected 52 (sensitivity, 94.5%), and the Kinyoun stain and Merifluor detected 53 (sensitivity, 96.4%). The final number of negative specimens was 74. One false-positive result was seen with both the Kinyoun stain and the ProSpect assay. The Color Vue and ProSpect assays required the most hands-on technologist time. The ProSpect assay and Merifluor kit were easiest to perform. The acid-fast stain was difficult to interpret. The Merifluor kit was easiest to read and was adaptable to both batch and single testing. Overall, the Kinyoun stain and the Merifluor test were preferable to both of the enzyme immunoassays because of the high reagent cost and hands-on time required for the enzyme immunoassays. The difficult interpretation of the Kinyoun stain smears made the Merifluor a more desirable test despite its higher cost. We conclude that all methods tested were equally sensitive and specific for the detection of Cryptosporidium spp. Ease of use, adaptability to batch testing, and cost are important criteria in determining the method of choice.

A 45-year-old man with acquired immune deficiency syndrome (AIDS) presented with an unusual peritonitis resulting from a microsporidian infection. An inflammatory mass formed by the omentum magnum was revealed at partial autopsy. This mass was infested with parasites measuring 2.5-2.6 microns by 1.2-1.5 microns, which appeared gram positive and stained dark blue with Giemsa stain. These organisms were pale pink with Kinyoun stain, and occasionally one of their poles was periodic acid-Schiff positive. Sporoblasts and spores contained in parasitophorous vacuoles were revealed by electron microscopic examination. These spores were mononucleated, without mitochondria, and had a polar filament with four or five coils. They were identified as Encephalitozoon cuniculi. Peritonitis resulting from this parasite has not been reported previously, however, it should be suspected among rare infections resulting from unusual parasites in patients with AIDS.

• Extrapulmonary foci of tuberculosis appear more frequently than in previous years, while the incidence of tuberculous otitis, a neglected and often difficult to diagnose entity, is unknown. We describe a case of an adult with miliary tuberculosis not initially suspected on clinical criteria, in whom the diagnosis of miliary tuberculosis was made by inspection of a modified Kinyoun stain of occult ear discharge. The significance and diagnostic problems associated with tuberculous otitis are briefly reviewed. ( Arch Otolaryngol Head Neck Surg 1988;114:1029-1030)