A Fatal Case of Disseminated Histoplasmosis by Histoplasma capsulatum var. capsulatum Misdiagnosed as Visceral Leishmaniasis-Molecular Diagnosis and Identification.

Histoplasmosis is an endemic mycosis in the Americas. However, its diagnosis is challenging due to the complexity and limited availability of conventional laboratory techniques-antigen tests, culture, and staining. Microscopic preparations often confuse with other pathogens, such as Leishmania spp. The genus Histoplasma capsulatum comprises three varieties: var. capsulatum, var. duboissi, and var. farciminosum, which cannot be distinguished using conventional techniques. An infant from a tropical region of Ecuador was hospitalized for fever, bloody diarrhea, and anemia persisting for two months. Upon admission, he received antibiotics and immunosuppressants. Histopathological examination of the lymph nodes, intestines, and bone marrow aspirate reported the presence of Leishmania-like amastigotes, and treatment was initiated with meglumine antimoniate and conventional amphotericin B. However, subsequent analysis of samples using PCR and DNA sequencing identified H. capsulatum var. capsulatum but not Leishmania. Despite fluconazole and amphotericin B, the infant succumbed to the disease. The delay in clinical and laboratory diagnosis of histoplasmosis and the use of nonspecific and ineffective drugs such as fluconazole led to disease dissemination and, ultimately, death. Implementing molecular diagnosis and antigen tests in laboratories located in endemic regions and reference hospitals is crucial.

The Development of a Multiplex PCR Assay for Fast and Cost-Effective Identification of the Five Most Significant Pathogenic Prototheca Species.

A multiplex PCR system (m-PCR) has been developed to accurately differentiate the five most important pathogenic Prototheca species, including the three species associated with infection in dairy cattle (P. ciferrii, P. blaschkeae, and P. bovis) and the two species associated with human infections (P. wickerhamii and P. cutis). The method is low-cost since it employs a simple "heat-shock" method in a TE buffer for DNA extraction. Furthermore, it requires only primers, a Taq polymerase, an agarose gel, and a molecular weight marker for identification. The method was based on published Prototheca cytochrome B sequences and was evaluated using reference strains from each of the five Prototheca species. The validity of the method was confirmed by identifying 50 strains isolated from milk samples. The specificity was tested in silico and with experimental PCR trials, showing no cross-reactions with other Prototheca species, as well as with bacteria, fungi, cows, algae, animals, or humans. The method could detect mixed infections involving two or three Prototheca species, providing a rapid test that delivers results within three hours.

Molecular Tracking of the Origin of Vesicular Stomatitis Outbreaks in 2004 and 2018, Ecuador.

The Vesicular Stomatitis Virus (VSV) is an arbovirus causing vesicular stomatitis (VS) in livestock. There are two serotypes recognized: New Jersey (VSNJV) and Indiana (VSIV). The virus can be transmitted directly by contact or by vectors. In 2018, Ecuador experienced an outbreak of Vesicular Stomatitis (VS) in cattle, caused by VSNJV and VSVIV, with 399 cases reported distributed over 18 provinces. We determined the phylogenetic relationships among 67 strains. For the construction of phylogenetic trees, the viral phosphoprotein gene was sequenced, and trees were constructed based on the Maximum Likelihood method using 2004 outbreak strains from Ecuador (GenBank) and the 2018 sequences (this article). We built a haplotype network for VSNJV to trace the origin of the 2004 and 2018 epizootics through topology and mutation connections. These analyses suggest two different origins, one related to the 2004 outbreak and the other from a transmission source in 2018. Our analysis also suggests different transmission patterns; several small and independent outbreaks, most probably transmitted by vectors in the Amazon, and another outbreak caused by the movement of livestock in the Andean and Coastal regions. We recommend further research into vectors and vertebrate reservoirs in Ecuador to clarify the mechanisms of the reemergence of the virus.

High Prevalence of Prototheca bovis Infection in Dairy Cattle with Chronic Mastitis in Ecuador.

The genus Prototheca, a unicellular, non-photosynthetic, yeast-like microalgae, is a pathogen of concern for the dairy industry. It causes bovine mastitis that currently cannot be cured, and hence generates significant economic losses in milk production. In this study, for the first time in Ecuador, we identify Prototheca bovis as the etiologic agent of chronic mastitis in dairy cattle. Milk samples (n = 458) of cows with chronic mastitis were cultured on Sabouraud Dextrose Agar (SDA). Microscopy and cytB gene sequencing were used to identify Prototheca, whereby Prototheca bovis was isolated from 15.1% (n = 69) of the milk samples, one of the highest infection rates that can be found in the literature in a "non-outbreak" situation. No other Prototheca species were found. We were unable to isolate the alga from environmental samples. We showed that P. bovis was relatively resistant to disinfectants used to sterilize milking equipment on the cattle farms where it was isolated. We discuss how to avoid future infection and also hypothesize that the real prevalence of Prototheca infection in bovine mastitis is probably much higher than what was detected. We recommend a protocol to increase the diagnostic yield in the bacteriology laboratory.