Efficiency of neutral honey as a tissue fixative in histopathology

In the routine laboratory, 10% neutral buffered formalin (NBF) is the fixative of choice. However, formalin is a human carcinogen. To the best of our knowledge, neutral honey, not natural or artificial honey, has not been tested to fix histological tissues. This study aimed to examine the efficiency of neutral buffered honey and other types of honey fixatives to fix histological tissues. The most two natural common Omani honey were used as fixatives, namely Sumar and date. We tested samples of rat liver, kidney, and stomach. Nine types of fixatives were used. All tissues were treated equally. The evaluation was performed blindly by three senior biomedical scientists who work in a histopathology laboratory. Hematoxylin and eosin showed adequate staining in all groups when compared to 10% NBF. The intensity and specificity of Jones Methenamine silver stain in 10% Sumer and Date honey and 10% alcoholic Sumer honey showed similar findings of 10% NBF. The specificity and intensity of all groups for Periodic acid–Schiff were comparable with 10% neutral buffered formalin accepts for 10% Sumer honey and 10% Alcoholic Date honey. However, all honey groups showed weak staining for the reticulin fibers using Gordon and Sweets method. Vimentin showed comparable findings with 10% NBF as there were no significant differences. The findings of this study are promising. Further in depth research on honey as a possible safe substitute fixative for formalin should be conducted.

Segmental Enteritis Associated with Pythium insidiosum Infection in a Horse

Background: Pythiosis is an infectious disease caused by the oomycete Pythium insidiosum, with higher occurrence in wetlands and hot climate regions. This microorganism develops its cycle in aquatic plants, and most cases happen because of the contact of animals or people with water containing the motile zoospores (infectious form). Horses are the principal species affected and develop principally cutaneous and subcutaneous lesions, but the gastrointestinal tract is seldom affected. Humans develop various forms of pythiosis, such as a vascular form. The objectives of the current study are to describe an unusual case of intestinal pythiosis, its clinical signs, aspects of pathogenesis, and diagnosis.Case: A 13-year-old Crioula mare, from Santa Maria, RS, Brazil, presented with reduced food and water intake, apathy, restlessness, rolling, nasal reflux, firm abdomen upon palpation, and tachypnea during 2 days. The horses of this farm were fed native pasture and horse feed, and they had access to a nearby pond. Two days following the start of the clinical signs, the horse died and was necropsied on the farm. During necropsy, there was around 400 mL of reddish effusion in the abdominal cavity (modified transudate). A 15 cm segment of jejunum was firm upon palpation and had a severe transmural thickening. The wall of the affected area was up to 3 cm in thickness and firm, with small yellowish and irregular masses that stood out and looked friable, interpreted as kunkers. Microscopically, the yellowish masses (kunkers) were characterized by dense accumulations of intact and degenerate eosinophils (eosinophilic necrosis). Within these kunkers, and also on their periphery, there were multiple negatively stained hyphal profiles. Hyphae were also seen on the wall of small arteries inside the kunkers. These hyphae had nearly parallel walls and were occasionally septate, with a diameter of about 10 μm. The hyphae stained black with Grocott’s methenamine silver stain (GMS) and were positive on immunohistochemistry (IHC) using specific anti-P. insidiosum polyclonal antibody.Discussion: Colic is the principal clinical presentation in horses suffering from gastrointestinal tract disorders. In spite of the presence of colic in this horse, intestinal pythiosis was not clinically suspected in this case, principally because of its rare occurrence in horses. Even though it is persistent to observe horses ingesting water with suspected contamination by P. insidiosum, the cutaneous form of pythiosis is a lot more common than the intestinal form. This is the only case of intestinal pythiosis in a horse in 52 years of routine diagnosis in our laboratory. It is suspected that this horse got infected by drinking contaminated water from the nearby pond and microlesions in the intestinal mucosa due to plant material or some unknown pathogen may have favored zoospore adhesion, encysting and starting the colonization of the tissue by emitting a germ tube. A macroscopic observation of kunkers in the intestine is rare, but when observed it is highly indicative of pythiosis. The immunohistochemistry technique using anti-P. insidiosum antibody, in accordance with the literature, validates the diagnosis of pythiosis. In this case, the presence of hyphae in the blood vessel wall inside the kunkers propose that this finding may be involved in the spread of the lesion but needs more detailed studies.

Granulomatous pneumonia due to pythiosis in captive South American coatis (Nasua nasua)

ABSTRACT: Pythiosis is an emerging infectious disease affecting captive and free-ranging wild animals. We report granulomatous pneumonia due to Pythium insidiosum in two South American coatis (Nasua nasua), who were found dead without any clinical records. Severe granulomatous pneumonia associated with pleural effusion was revealed in the necropsy. Microscopically, variably sized granulomas and pyogranulomas presented negative hyphae profiles at the periphery of their necrotic cores. Grocott methenamine silver stain highlighted these structures, and immunostain (anti- P. insidiosum) was strongly positive. Molecular analysis by polymerase chain reaction amplified P. insidiosum specific DNA. These findings characterized P. insidiosum as a cause of granulomatous pneumonia in coatis and proved that pythiosis needs to be considered in the differential diagnosis of respiratory diseases affecting this species in endemic areas.

Pneumocystis jirovecii Pneumonia in a HIV-Infected Patient with a CD4 Count Greater Than 400 Cells/μL and Atovaquone Prophylaxis

We describe a rare case of Pneumocystis jirovecii pneumonia (PCP) in a heterosexual man with a pertinent medical history of well-controlled human immunodeficiency virus (HIV) on highly active antiretroviral therapy (HAART) and PCP prophylaxis with atovaquone. The patient presented with recurrent shortness of breath, worsening malaise, and fever, following treatment for hypersensitivity pneumonitis one month prior, including a twenty-four-day course of 40 milligrams daily glucocorticoid with taper. However, transbronchial biopsies, lavage, and cytology from prior admission were inconclusive. The patient refused video-assisted thoracic surgery (VATS) at that time. Upon readmission, bronchoscopy with right VATS and lung biopsy were performed. Grocott’s methenamine silver stain of right lung biopsy was positive for Pneumocystis jirovecii. This case is a rare example of PCP in a patient with a normal CD4 count (>487 cells/μL) and a low viral load (<20 copies/mL) despite PCP prophylactic antibiotics in the setting of recent iatrogenic immunosuppression.

Mixed Actinomyces israelii and Aggregatibacter actinomycetemcomitans infection causing empyema necessitatis and multiple skin abscesses in an immunocompetent patient

A 45-year-old- man presented with left chest wall pain, swelling and cough. Over a 2-month period he developed abscesses in the right foot, right anterior thigh, left buttock and left chest. Incision and drainage of the soft tissue abscesses and video-assisted thoracoscopic surgery to drain the loculated empyema contiguous with the chest wall abscess were performed as surgical management. Gram stain showed beaded Gram-positive rods and the culture initially grew Aggregatibacter actinomycetemcomitans and Eikenella corrodens. Pathological evaluation of the pleura showed sulfur granules and organisms consistent with Actinomyces spp. on Gomori methenamine silver stain; Actinomyces israelii was recovered in culture with extended incubation. The patient was treated for 3 weeks with ceftriaxone and oral metronidazole, followed by oral amoxicillin. Culture of A. actinomycetemcomitans with other findings consistent with actinomycosis warrants 6–12 months of antibiotic therapy.

Actinomycotic Endomyometritis Associated with a Long-Term Use of Intrauterine Device Lasting for 42 Years

In women, pelvic actinomycosis is closely associated with prolonged use of the intrauterine devices (IUD). A 70-year old female presented with intermittent blood-stained vaginal discharge. An analysis of her history revealed, she was inserted with an IUD 42 years ago, but it has remained in situ untill now. Curettage of the uterus was done, but an IUD was firmly attached inside the cavity and there was not able to remove it. A biopsy material consisted of the large round and oval granules of filamentous and mycelium-like microorganisms. They showed strong positivity with Periodic acid–Schiff stain and Gömöri methenamine silver stain. Histopathology was consisted with uterine actinomycosis. A total abdominal hysterectomy with bilateral adnexectomy was performed. The uterus contained a retained plastic IUD. Microscopic investigation revealed a diffuse chronic active endomyometritis with sporadic Actinomycetes colonies. Wearing an IUD continuously for very long periods of time can lead to actinomycotic infection, which may manifest for many years after its application. All IUD users have to keep in mind regular gynecological check-ups to avoid the complications of a retained and “forgotten” IUD.

Do Pentraxins Bind to Fungi in Invasive Human Gastrointestinal Candidiasis?

Tissue from 13 autopsy cases with invasive gastrointestinal candidiasis was studied for the binding of the pentraxins, C-reactive protein (CRP), pentraxin 3 (PTX3), and serum amyloid P component (SAP) to fungal surfaces. Invasive candidal infection was demonstrated using a hematoxylin and eosin stain and a Gomori methenamine silver stain (GMS). Immunohistochemistry was performed with CRP and PTX3 monoclonal antibodies and did not demonstrate CRP or PTX3 bound to fungi (0 of 13 cases), although CRP was extensively deposited on human tissue. A polyclonal antibody to SAP showed that SAP was bound to fungi in 12 of 13 cases. Although all three pentraxins have been reported to bind to fungi or bacteria, only SAP was bound to filamentous and yeast forms of Candida in human tissue, as detected by immunohistochemistry. SAP was abundantly present on fungi and may have affected the host innate immune response to the invading fungi.