Molecular detection, not extended culture incubation, contributes to diagnosis of fungal infection

Background Despite its low sensitivity, fungal culture remains one of the key methods for diagnosis and treatment of fungal infections, as it identifies the etiology at the genus and species level and affords the opportunity for susceptibility testing. The Manual of Clinical Microbiology recommends that fungal culture screening for all pathogens should routinely be held for 4 weeks to maximize the recovery of slow-growing species. Information on the optimal fungal culture time in this era of expansion of immunocompromised populations and availability of molecular diagnostics is lacking. We reviewed our experience with fungal culture to determine the optimal culture incubation time. In addition, our experience of broad-range ITS PCR for diagnosis of culture-negative fungal infections was also reviewed. Methods Fungal culture and ITS PCR results from January 1, 2013, to December 31, 2017, were reviewed. Results This study included 4234 non-duplicated positive cultures. Ninety-six percent (4058) of the positive cultures were detected in the first 7 days of incubation. During the second week of incubation, 111 (2.8%) positives were detected from day 8 to day 10, and 71 (1.7%) were detected from day 11 to day 14. Only 6 (0.1%) positive cultures were detected in the third week of incubation, and no positive culture was detected in the fourth week of incubation. No clinically significant fungal isolates were recovered after 14 days. Clinically significant pathogens were detected in 16 (0.2%) culture-negative samples by ITS PCR. Conclusion Extending culture incubation beyond 2 weeks did not generate clinically relevant results. When culture failed to make a laboratory diagnosis, broad-range internal transcribed spacer (ITS) rRNA gene PCR followed by sequencing produced clinically significant results.

Manejo in vitro de antracnosis (Colletotrichum acutatum Simmonds) en aguacate mediante el uso de principios activos botánicos

La antracnosis es una enfermedad causada por el hongo fitopatógeno del género Colletotrichum, que disminuye la calidad y rendimiento del aguacate. El objetivo del estudio fue determinar la actividad antifúngica de D-limoneno, β-citronelol y eucaliptol en Colletotrichum acutatum de aguacate. Se realizó el aislamiento del patógeno, la identificaciónmorfológica, y molecular mediante técnica (ITS-PCR). Posteriormente, se realizaron pruebas in vitro con D-limoneno, eucaliptol y β citronelol. Los datos se analizaron mediante análisis probit, con ANVA y prueba de Tukey (p ≤ 0.05). Se midió crecimiento micelial, porcentaje de esporulación y germinación de conidios. Se identificó a la especie Colletotrichum acutatum. En la evaluación, β citronelol mostró la mayor efectividad, con intervalos de inhibición de 29 a 89% en el crecimiento micelial, de 61 a 100% en la esporulación y 96 a 100% en la germinación. Por tanto, β citronelol representa una alternativa botánica para el control in vitro de la antracnosis.

Challenges in the Diagnostic Performance of Parasitological and Molecular Tests in the Surveillance of African Trypanosomiasis in Eastern Zambia

African animal trypanosomiasis (AAT) control programs rely on active case detection through the screening of animals reared in disease endemic areas. This study compared the application of the polymerase chain reaction (PCR) and microscopy in the detection of trypanosomes in cattle blood in Mambwe, a rural district in eastern Zambia. Blood samples were collected from 227 cattle and tested for infection with trypanosomes using microscopy and Ribosomal RNA Internal Transcribed Spacers (ITS)-PCR. Microscopy on the buffy coat detected 17 cases, whilst thin and thick smears detected 26 cases and 28 cases, respectively. In total, microscopy detected 40 cases. ITS-PCR-filter paper (FP) on blood spots stored on FP detected 47 cases, and ITS-PCR-FTA on blood spots stored on Whatman FTA Classic cards detected 83 cases. Using microscopy as the gold standard, ITS-PCR-FTA had a better specificity (SP) and sensitivity (SE) (SP = 72.2%; SE = 77.5%; kappa = 0.35) than ITS-PCR-FP (SP = 88%; SE = 60%; kappa = 0.45). The prevalence of Trypanosoma brucei s.l. was higher on ITS-PCR-FTA (19/227) than on ITS-PCR-FP (0/227). Our results illustrate the complexities around trypanosomiasis surveillance in rural Africa and provide evidence of the impact that field conditions and staff training can have on diagnostic results, which in turn impact the success of tsetse and trypanosomiasis control programs in the region.

Molecular Detection, not Extended Culture Incubation, Contributes to Diagnosis of Fungal Infection

Background. Despite its low sensitivity, fungal culture remains one of the key methods for diagnosis and treatment of fungal infections, as it identifies the etiology at the genus and species level and offers susceptibility testing. The Manual of Clinical Microbiology recommends that fungal culture screening for all pathogens should routinely be held for 4 weeks to maximize the recovery of slow-growing species. Information on the optimal fungal culture time in this era of expansion of immunocompromised populations is lacking. We reviewed our experience with fungal culture to determine the optimal culture incubation time. In addition, our experience of broad-range ITS PCR for diagnosis of culture-negative fungal infections was also reviewed.Method. Fungal culture and ITS PCR results from January 1, 2013, to December 31, 2017, were reviewed.Results. Ninety-six percent of positive cultures (4058) were detected in the first 7 days of incubation. During the second week of incubation, 2.8% of positives (111) were detected from day 8 to day 10, and 1.7% (71) were detected from day 11 to day 14. Only 0.1% of positive culture were detected in the third week of incubation, and no positive culture was detected in the fourth week of incubation. No clinically significant fungal isolates were recovered after 14 days. Clinically significant pathogens were detected in 0.2% of culture-negative samples by ITS PCR.Conclusion. Extending culture incubation beyond 2 weeks did not generate clinically relevant results. When culture failed to make a laboratory diagnosis, broad-range internal transcribed spacer (ITS) rRNA gene PCR followed by sequencing produced clinically significant results.

Challenges in the Diagnostic Performance of Parasitological and Molecular Tests in the Detection of African Trypanosomiasis in Cattle in Mambwe District in Eastern Zambia

Background: Trypanosomiasis is a Neglected Tropical Disease with serious health and economic implications. Disease eradication and control programs rely on active case detection through mass population screening. Screening tools and techniques therefore need to be adequately sensitive, practically quick to perform, and affordable. This study compared the field application of the polymerase chain reaction, the loop mediated isothermal amplification technique, and microscopy, in the detection of trypanosomes in cattle blood in Mambwe district in eastern Zambia. Methods: Blood samples were collected from 227 cattle into three heparinised micro capillary tubes, and tested for trypanosomiasis infection using microscopy, ITS-PCR and RIME-LAMP. The comparative diagnostic performance of each of the methods was evaluated using the chi-square test, kappa test and receive operator curves. Results: Microscopy on buffy coat detected 17 cases (n=227), by thin smears detected 26 cases (n=227), and by thick smears detected 28 cases (n=227). In total, microscopy detected 40 cases (n=227). ITS-PCR- on blood spots stored on filter paper detected 47 cases (n=227), ITS-PCR- on blood spots stored on FTA cards detected 83 cases (n=227) and RIME-LAMP-FTA detected 18 cases (n = 131). Using microscopy as gold standard, sensitivity and specificity of ITS-PCR was compared. ITS-PCR-FTA had a better specificity and sensitivity (SE=77.5%; SP=72.2%; k = 0.35) than ITS-PCR-FP (SP = 88%; SE = 60%; kappa = 0.45). Prevalence of Trypanosoma brucei s.l. was higher on RIME-LAMP-FTA (18/131) than ITS-PCR-FTA (19/227). Conclusion: Our results are not perfect but are a good illustration of the current diagnostic challenges in rural Africa. Findings showed that none of the diagnostic tests could be taken as having performed better than the others and that each of the tests offered some advantages and limitations. In endemic rural areas of Africa, the use of PCR and LAMP requires specialised staff, laboratory supplies and infrastructure which is often not available. For this reason, microscopy remains the most practical option for field diagnosis of trypanosomes but understanding its limitations is critical particularly when applied for surveillance purposes.

Fungus used for germination is supplanted after reintroduction of Hadrolaelia jongheana (Orchidaceae)

The great diversity in colors and forms become the orchids a business with high economic value. The habitat fragmentation contributes to the extinction of orchids. Inoculation of orchid with mycorrhizal fungi for seedlings can guarantee the success of reintroduction. For this purpose, seeds of Hadrolaelia jongheana were germinated using an isolate of Tulasnella sp. Seedlings were transferred to the natural field. Roots samples were collected before re-introduction, and 120th and 240th days. The diversity of mycorrhizal fungi was performed by ITS-PCR-DGGE. The ecological succession occurred in the field and the diversity was higher after 240th d. This work comprises the first study using tropical orchids for reintroduction for approaching to ecological aspects of mycorrhizal fungi association in Brazil with conservation purposes.

Molecular Detection, not Extended Culture Incubation, Contributes to Diagnosis of Fungal Infection

Background. Despite of low sensitivity, fungal culture remains one of the key methods for diagnosing and treatment of fungal infections as it identifies etiology at genus and species level and offers susceptibility testing. The Manual of Clinical Microbiology recommends that fungal cultures screening for all pathogens should routinely be held for 4 weeks to maximize the recovery of slow growing species. Information on the optimal fungal culture time in the era of expansion of immunocompromised populations is lacking. The goal is to review our experience with fungal culture in order to determine the optimal culture incubation time; to review our experience of broad-range ITS PCR for diagnosis of culture negative fungal infections. Methods. Fungal culture and ITS PCR results from January 1, 2013 to December 31, 2017 were reviewed.Results. Ninety six percent of positive cultures (4058) were detected in the first seven days of incubation. During the second week of incubation, 2.8% of positives (111) were detected from day 8 to 10, and 1.7% (71) were detected from day 11 to 14. Only 0.1% of positive culture were detected in the third week of incubation, and no positive culture was detected in the fourth week of incubation. No Clinical significance of fungal isolates recovered after 14 days. Clinical significant pathogens were detected in 0.2% culture negative samples.Conclusion. Extending culture incubation beyond two weeks did not generate clinical relevant results. When culture failed to make laboratory diagnosis, ITS PCR produced clinical significant results.