We recently reported the development of a 5-hour multiplex PCR test for the detection of tinea unguium and the optimization of this test by the inclusion of an inhibition control. Here we report the performance of this procedure as used in a routine clinical laboratory as compared to conventional microscopy and culture-based techniques performed in a mycology reference laboratory. We found in processing 109 samples that 22 (20.2%) yielded fungi in culture while the suspected etiologic agents were noted microscopically in 15 (13.8%) that were negative in culture. Fungi were detected by PCR in 37 (33.9%) samples, of which only three were positive in culture. Since the majority of PCR positive but culture negative samples were positive in microscopic examinations, the increased sensitivity was not due to contamination. PCR inhibitors were present in 5% of the samples, but this was overcome by re-running the samples with a 50% reduction of sample DNA. In conclusion, the PCR test performance in the routine setting was excellent and provided a markedly reduced time to diagnosis with a higher sensitivity.

To determine the distribution of Blastocystis sp. subtypes from Blastocystis cyst excreters, 1,000 fecal samples from patients suspected of enteroparasitic disease were scored for stool consistency, submitted to xenic in vitro culture (XIVC), formol ethyl acetate concentration (FECT) with subsequent isopycnic centrifugation, and polymerase chain reaction (PCR) with subtype (ST) analysis. Blastocystis was significantly more prevalent in specimens from patients with travel-associated diarrhea (15.6%) than those with persistent diarrhea (8.3%) (P = 0.005). Overall, 115 (11.5%) and 35 (3.5%) specimens were positive by XIVC and FECT, respectively. Blastocystis cysts were detected in 33 (28.7%) of the XIVC-positive specimens. A positive FECT result was associated with ST3 (P = 0.05). The presence of Blastocystis in general or Blastocystis cysts was independent of stool consistency, and no particular ST was significantly associated with cyst identification. In view of these data, the present study indicates that Blastocystis cyst formation is independent of Blastocystis sp. subtype and gastrointestinal transit time.

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Host-parasite interactions may be modulated by host-or parasite-associated microbes, but the role of these are often overlooked. Particularly for parasites with intestinal stages (either larval or adult), the host gut microbiome may play a key role for parasite establishment; moreover, the microbiome may change in response to invading parasites. Hypothesis testing at the organismal level may be hampered, particularly in mammalian definitive hosts, by ethical, logistical, and economical restrictions. Thus, invertebrates naturally serving as intermediate hosts to parasites with complex life cycles may inform the development of mammalian models as an early-stage host-parasite model. In addition, several important pathogens are vectored by insects, and insect gut microbiome-pathogen interactions may provide essential base-line knowledge, which may be used to control vectorborne pathogens. Here, we used the grain beetle, Tenebrio molitor, a host of the tapeworm Hymenolepis diminuta, to explore interactions between infection status and resident gut microbiota at two pre-determined time points (day two and seven) post infection. Using 16S/18S microbial profiling, we measured key parameters of the composition, relative abundance, and diversity of the host gut bacteriome and mycobiome. In addition, we quantified the systemic beetle immune response to infection by Phenoloxidase activity and hemocyte abundance. We found significant changes in the gut bacteriome and mycobiome in relation to infection status and beetle age. Thus, the relative abundance of Proteobacteria was significantly higher in the gut of infected beetles and driven mostly by an increased abundance of Acinetobacter. In addition, the mycobiome was less abundant in infected beetles but maintained higher Shannon diversity in infected compared with non-infected beetles. Beetles treated with a broad-spectrum antibiotic (Tetracycline) exhibited significantly reduced parasite establishment compared with the untreated control group, indicating that the host microbiome may greatly influence hatching of eggs and subsequent establishment of H. diminuta larvae. Our results suggest that experimental work using invertebrates may provide a platform for explorative studies of host-parasite-microbe interactions and their underlying mechanisms.

So far codon-optimized HIV-1 envelope genes have been investigated for the T cell line-adapted strain MN, which differs in several aspects from primary isolates. Envelopes of primary isolates may be more relevant for vaccine purposes. This article describes for the first time the engineering and characterization of four "humanized" genes encoding the secreted gp120/gp140, or the membrane-bound gp150/gp160, of a primary CCR5 tropic, clade B, clinical isolate HIV-1(BX08). The genes were built in fragments for easy cassette exchange of regions important for immunogenicity, function, and expression. The transcription and expression of the synthetic genes in mammalian cell lines were Rev independent and highly increased. Increased expression of membrane-bound gp160 induced a high cytopathic effect in U87.CD4.CCR5 cells. Gene gun and intramuscular DNA vaccination in mice induced a strong specific cytotoxic T lymphocyte response independent of the gene construct, expression level, or DNA immunization route. In contrast, the highest anti-gp120 antibody levels were induced by synthetic genes encoding the secreted glycoproteins followed by gp160/gp150. Unlike HIV-1(MN), HIV-1(BX08) V3 was not immune dominant. Despite the high antibody response only low and inconsistent neutralizing titers to the homologous HIV-1 isolate were measured. However, neutralization of SHIV89.6P could be obtained. Thus, the neutralizing epitopes on the cell line-adapted SHIV89.6P and HIV-1(MN) may be more antigenically available for the cross-neutralizing antibodies induced. In conclusion, complete "humanization" of the DNA vaccine genes failed to induce a consistent neutralizing antibody response, albeit expression and immunogenicity of the primary HIV-1 glycoproteins were greatly improved. Optimization in terms of improving neutralization may require further modifications of the DNA vaccine gene. The synthetic cassette construct described is a convenient tool developed to investigate this further.