The Diagnostic Laboratory Hub: A New Health Care System Reveals the Incidence and Mortality of Tuberculosis, Histoplasmosis, and Cryptococcosis of PWH in Guatemala.

BACKGROUND: A Diagnostic Laboratory Hub (DLH) was set up in Guatemala to provide opportunistic infection (OI) diagnosis for people with HIV (PWH). METHODS: Patients newly presenting for HIV, PWH not receiving antiretrovirals (ARVs) for >90 days but returned to care (Return/Restart), and PWH on ARVs with symptoms of OIs (ARV treatment) were prospectively included. Screening for tuberculosis, nontuberculous mycobacteria (NTM), histoplasmosis, and cryptococcosis was done. Samples were couriered to the DLH, and results were transmitted electronically. Demographic, diagnostic results, disease burden, treatment, and follow-up to 180 days were analyzed. RESULTS: In 2017, 1953 patients were included, 923 new HIV infections (an estimated 44% of all new HIV infections in Guatemala), 701 on ARV treatment, and 315 Return/Restart. Three hundred seventeen (16.2%) had an OI: 35.9% tuberculosis, 31.2% histoplasmosis, 18.6% cryptococcosis, 4.4% NTM, and 9.8% coinfections. Histoplasmosis was the most frequent AIDS-defining illness; 51.2% of new patients had <200 CD4 cells/mm3 with a 29.4% OI incidence; 14.3% of OIs in new HIV infections occurred with CD4 counts of 200-350 cells/mm3. OIs were the main risk factor for premature death for new HIV infections. At 180 days, patients with OIs and advanced HIV had 73-fold greater risk of death than those without advanced disease who were OI-free. CONCLUSIONS: The DLH OI screening approach provides adequate diagnostic services and obtains relevant data. We propose a CD4 screening threshold of <350 cells/mm3. Mortality remains high, and improved interventions are required, including expansion of the DLH and access to antifungal drugs, especially liposomal amphotericin B and flucytosine.

Burden of serious fungal infections in Spain.

Estimates of the incidence and prevalence of serious fungal infections, based on epidemiological data, are essential in order to inform public health priorities given the lack of resources dedicated to the diagnosis and treatment of these serious fungal diseases. However, epidemiology of these infections is largely unknown, except for candidaemia and cryptococcosis. The aim of this work is to calculate the burden of serious fungal infections in Spain. All published epidemiology papers reporting fungal infection rates from Spain were identified. Where no data existed, we used specific populations at risk and fungal infection frequencies in those populations to estimate national incidence or prevalence, depending on the condition. Around 8.1 million people suffer a fungal infection every year. Most of them are skin or mucosal infections causing no deaths. Candidaemia is more common than in other European countries and has risen by 1.88-fold in frequency in the last decade (8.1 cases × 100,000). Good estimates of invasive aspergillosis (2.75 cases × 100,000) and mucormycosis (0.04 × 100,000) are available. Fungal infections with a high mortality such as invasive aspergillosis, candidaemia, Pneumocystis pneumonia and mucormycosis are not numerous in Spain, but they affect those with severe underlying diseases and are therefore linked to poor outcomes. Additional studies are required, especially for high burden diseases such as recurrent thrush in women (∼9000 cases × 100,000 women), allergic bronchopulmonary aspergillosis (126 cases × 100,000) and severe asthma with fungal sensitisation (198 cases × 100,000).

Performance of panfungal--and specific-PCR-based procedures for etiological diagnosis of invasive fungal diseases on tissue biopsy specimens with proven infection: a 7-year retrospective analysis from a reference laboratory.

A retrospective analysis of real-time PCR (RT-PCR) results for 151 biopsy samples obtained from 132 patients with proven invasive fungal diseases was performed. PCR-based techniques proved to be fast and sensitive and enabled definitive diagnosis in all cases studied, with detection of a total of 28 fungal species.

DNA barcoding in Mucorales: an inventory of biodiversity.

The order Mucorales comprises predominantly fast-growing saprotrophic fungi, some of which are used for the fermentation of foodstuffs but it also includes species known to cause infections in patients with severe immune or metabolic impairments. To inventory biodiversity in Mucorales ITS barcodes of 668 strains in 203 taxa were generated covering more than two thirds of the recognised species. Using the ITS sequences, Molecular Operational Taxonomic Units were defined by a similarity threshold of 99 %. An LSU sequence was generated for each unit as well. Analysis of the LSU sequences revealed that conventional phenotypic classifications of the Mucoraceae are highly artificial. The LSU- and ITS-based trees suggest that characters, such as rhizoids and sporangiola, traditionally used in mucoralean taxonomy are plesiomorphic traits. The ITS region turned out to be an appropriate barcoding marker in Mucorales. It could be sequenced directly in 82 % of the strains and its variability was sufficient to resolve most of the morphospecies. Molecular identification turned out to be problematic only for the species complexes of Mucor circinelloides, M. flavus, M. piriformis and Zygorhynchus moelleri. As many as 12 possibly undescribed species were detected. Intraspecific variability differed widely among mucorealean species ranging from 0 % in Backusella circina to 13.3 % in Cunninghamella echinulata. A high proportion of clinical strains was included for molecular identification. Clinical isolates of Cunninghamella elegans were identified molecularly for the first time. As a result of the phylogenetic analyses several taxonomic and nomenclatural changes became necessary. The genus Backusella was emended to include all species with transitorily recurved sporangiophores. Since this matched molecular data all Mucor species possessing this character were transferred to Backusella. The genus Zygorhynchus was shown to be polyphyletic based on ITS and LSU data. Consequently, Zygorhynchus was abandoned and all species were reclassified in Mucor. Our phylogenetic analyses showed, furthermore, that all non-thermophilic Rhizomucor species belong to Mucor. Accordingly, Rhizomucor endophyticus was transferred to Mucor and Rhizomucor chlamydosporus was synonymised with Mucor indicus. Lecto-, epi- or neotypes were designated for several taxa.

Population-based survey of filamentous fungi and antifungal resistance in Spain (FILPOP Study).

A population-based survey was conducted to investigate the epidemiology of and antifungal resistance in Spanish clinical strains of filamentous fungi isolated from deep tissue samples, blood cultures, and respiratory samples. The study was conducted in two different periods (October 2010 and May 2011) to analyze seasonal variations. A total of 325 strains were isolated in 29 different hospitals. The average prevalence was 0.016/1,000 inhabitants [corrected]. Strains were identified by sequencing of DNA targets and susceptibility testing by the European Committee for Antimicrobial Susceptibility Testing reference procedure. The most frequently isolated genus was Aspergillus, accounting for 86.3% of the isolates, followed by Scedosporium at 4.7%; the order Mucorales at 2.5%; Penicillium at 2.2%, and Fusarium at 1.2%. The most frequent species was Aspergillus fumigatus (48.5%), followed by A. flavus (8.4%), A. terreus (8.1%), A. tubingensis (6.8%), and A. niger (6.5%). Cryptic/sibling Aspergillus species accounted for 12% of the cases. Resistance to amphotericin B was found in 10.8% of the isolates tested, while extended-spectrum triazole resistance ranged from 10 to 12.7%, depending on the azole tested. Antifungal resistance was more common among emerging species such as those of Scedosporium and Mucorales and also among cryptic species of Aspergillus, with 40% of these isolates showing resistance to all of the antifungal compounds tested. Cryptic Aspergillus species seem to be underestimated, and their correct classification could be clinically relevant. The performance of antifungal susceptibility testing of the strains implicated in deep infections and multicentric studies is recommended to evaluate the incidence of these cryptic species in other geographic areas.

Efficacy of DNA amplification in tissue biopsy samples to improve the detection of invasive fungal disease.

The performance of a pan-fungal PCR-based technique was evaluated to assess the aetiology of invasive fungal diseases (IFDs). A total of 89 formalin-fixed paraffin-embedded biopsy samples from 84 patients with proven IFD were studied. Culture of tissue was performed in 68 (81%) patients. The sensitivities of the PCR-based technique and microbiological culture of tissues were 89% and 56%, respectively (p <0.01). According to PCR results, Aspergillus species accounted for 67%, Candida species for 13%, zygomycetes for 11%, and rare and emerging fungi for 9%. Aspergillus species were significantly associated with lung samples (79.6%, p <0.01), Mucorales were associated with skin/subcutaneous samples, and Candida species were associated with gastrointestinal samples. Regarding biopsy samples with Aspergillus species, Aspergillus fumigatus DNA was detected in 43 of 50 (86%), and Aspergillus flavus in six of 50 (12%). PCR was positive in 24 of 30 (80%) cases with negative culture. In nine of the 84 patients, the PCR technique failed to amplify the DNA. Six also had negative cultures, and in the remaining three cases culture was positive (Rhizopus microsporus, Rhizopus arrhizus, and Sakseneae vasiformis), suggesting that the PCR technique was not as effective in amplifying the DNA of some species of Zygomycetes. In five cases, there was no correlation between culture results and those obtained with DNA amplification, indicating the possibility of a mixed infection or the presence of colonizer/contaminant microorganisms. In summary, PCR-based techniques for DNA amplification should be implemented in histopathology and microbiology departments, as they appear to be complementary to conventional methods for IFD detection.

Development of a single tube multiplex real-time PCR to detect the most clinically relevant Mucormycetes species.

Mucormycetes infections are very difficult to treat and a delay in diagnosis could be fatal for the outcome of the patient. A molecular diagnostic technique based on Real Time PCR was developed for the simultaneous detection of Rhizopus oryzae, Rhizopus microsporus and the genus Mucor spp. in both culture and clinical samples. The methodology used was Molecular beacon species-specific probes with an internal control. This multiplex real-time PCR (MRT-PCR) was tested in 22 cultured strains and 12 clinical samples from patients suffering from a proven mucormycosis. Results showed 100% specificity and a detection limit of 1 fg of DNA per microlitre of sample. The sensitivity was 100% for clinical cultured strains and for clinical samples containing species detected by the PCR assay. Other mucormycetes species were not detected in clinical samples. This technique can be useful for clinical diagnosis and further studies are warranted.

HPLC/UV or bioassay: two valid methods for posaconazole quantification in human serum samples.

In this paper we report on the development and validation of two different methods for posaconazole quantification from serum samples, HPLC/UV and bioassay. Both methods have been validated according to international guidelines and were also applied to the analysis of 61 trough serum samples from treated patients. A good correlation between both methods was observed. The HPLC method, more laborious and expensive, was demonstrated to be more accurate, precise and faster (analytical range 0.125-16 µg/mL, accuracy between -2.48 and 3.70% and precision between 2.77 and 5.93%, with an analytical run time of 11 min), making it a valuable tool for reference laboratories that centralize high numbers of samples. The microbiological method, however, is simple and offers sufficient precision and accuracy (analytical range 0.125-16 µg/mL, accuracy between -8.10 and 3.77% and a precision between 4.52 and 10.07%), to be used to monitor posaconazole. It may be a valid alternative to chromatographic methods in clinical laboratories without specialized facilities.

EUCAST technical note on Amphotericin B.

The European Committee on Antimicrobial Susceptibility Testing-Subcommittee on Antifungal Susceptibility Testing (EUCAST-AFST) has determined breakpoints for amphotericin B for Candida spp. This Technical Note is based on the EUCAST amphotericin B rationale document (available on the EUCAST website: http://www.eucast.org). Species-specific breakpoints for C. albicans, C. glabrata, C. krusei, C. parapsilosis and C. tropicalis are S: MIC ≤1 mg/L, R: MIC > 1 mg/L. There are insufficient data to set breakpoints for other species. The breakpoints are based upon pharmacokinetic data, epidemiological cut-ff values and clinical experience. Breakpoints will be reviewed regularly.

EUCAST technical note on anidulafungin.

The European Committee on Antimicrobial Susceptibility Testing-Subcommittee on Antifungal Susceptibility Testing has determined breakpoints for anidulafungin for Candida spp. This Technical Note is based on the EUCAST anidulafungin rationale document (available at: http://www.eucast.org). Species-specific breakpoints for C. albicans are S ≤0.03 mg/L and R >0.03 mg/L and for C. glabrata, C. tropicalis and C. krusei S ≤0.06 mg/L and R >0.06 mg/L. C. parapsilosis was not regarded a good target for anidulafungin. There are insufficient data to set breakpoints for other species. The breakpoints are based upon pharmacokinetic data, epidemiological cut-off values and clinical experience. Breakpoints will be reviewed regularly.

EUCAST technical note on posaconazole.

The European Committee on Antimicrobial Susceptibility Testing-Subcommittee on Antifungal Susceptibility Testing (EUCAST-AFST) has determined breakpoints for posaconazole for Candida spp. This Technical Note is based on the EUCAST posaconazole rationale document (available on the EUCAST website: http://www.eucast.org). Species-specific breakpoints for C. albicans, C. parapsilosis and C. tropicalis are S: MIC ≤0.06 mg/L, R: MIC >0.06 mg/L. There are insufficient data to set breakpoints for C. glabrata and C. krusei as well as non-species-related breakpoints. The breakpoints are based upon pharmacokinetic data, epidemiological cut-off values and clinical experience. Breakpoints will be reviewed regularly.

Cutaneous infection by Phomopsis longicolla in a renal transplant recipient from Guinea: first report of human infection by this fungus.

We report the case of a 72-year-old female renal transplant recipient with a nodular lesion in the distal phalange of the third left finger produced by a dematiaceous fungus that was identified as Phomopsis longicolla. She was treated with itraconazole and terbinafine and later with voriconazole, without response. The patient underwent a surgical resection with lesion-free edge and continued on voriconazole. One year later she was asymptomatic and had not developed new lesions.

Guidelines for the prevention of invasive mould diseases caused by filamentous fungi by the Spanish Society of Infectious Diseases and Clinical Microbiology (SEIMC).

Invasive fungal infections (IFIs) caused by filamentous fungi still have high rates of mortality, associated with difficulties in early detection of the infection and therapeutic limitations. Consequently, a useful approach is to prevent patients at risk of fungal infection from coming into contact with conidia of Aspergillus and other mould species. This document describes the recommendations for preventing IFI caused by filamentous fungi worked out by Spanish experts from different medical and professional fields. The article reviews the incidence of IFI in different risk populations, and questions related to environmental measures for prevention, control of hospital infections, additional procedures for prevention, prevention of IFI outside of hospital facilities and antifungal prophylaxis are also analysed.

Frequency of voriconazole resistance in vitro among Spanish clinical isolates of Candida spp. According to breakpoints established by the Antifungal Subcommittee of the European Committee on Antimicrobial Susceptibility Testing.

A total of 4,226 Spanish clinical isolates of Candida spp. were analyzed to assess resistance to voriconazole according to breakpoints established by the European Committee for Antimicrobial Susceptibility Testing (where susceptibility [S] to voriconazole corresponds to a MIC of ≤ 0.12 mg/liter). Resistance was uncommon among Candida albicans (5%), C. parapsilosis (1.2%), and C. tropicalis (11%) isolates. Voriconazole MICs of >0.12 mg/liter were more frequent among Candida glabrata and C. krusei isolates. A significant percentage of voriconazole-resistant strains came from oropharyngeal infections and exhibited high MICs of other azoles.

Prevalence of Candida bracarensis and Candida nivariensis in a Spanish collection of yeasts: comparison of results from a reference centre and from a population-based surveillance study of candidemia.

Two new species related to Candida glabrata, i.e., Candida nivariensis and Candida bracarensis, have been proposed. The occurrence of these species among isolates collected in a Spanish mycology reference laboratory in 2008-2009 was reviewed. In addition, strains recovered as part of an active population-based surveillance of candidemia conducted in Barcelona between 2002 and 2003 were also analyzed. Among 143 clinical isolates received in 2008-2009, three (2%) were identified as C. bracarensis and none as C. nivariensis through sequencing of their ribosomal DNA. Of the 31 strains initially identified as C. glabrata in the 2002-2003 population-based study (0.38 cases/100,000 population), none were found to belong to these related new species. Results from in vitro susceptibility studies of C. bracarensis isolates were comparable to those found with C. glabrata. Since new and cryptic species have been described, periodic surveillance including the use of molecular identification methods seems to be necessary in order to determine their frequency, geographical distribution and susceptibility profile.

Zygomycosis in Europe: analysis of 230 cases accrued by the registry of the European Confederation of Medical Mycology (ECMM) Working Group on Zygomycosis between 2005 and 2007.

Zygomycosis is an important emerging fungal infection, associated with high morbidity and mortality. The Working Group on Zygomycosis of the European Confederation of Medical Mycology (ECMM) prospectively collected cases of proven and probable zygomycosis in 13 European countries occurring between 2005 and 2007. Cases were recorded by a standardized case report form, entered into an electronic database and analysed descriptively and by logistic regression analysis. During the study period, 230 cases fulfilled pre-set criteria for eligibility. The median age of the patients was 50 years (range, 1 month to 87 years); 60% were men. Underlying conditions included haematological malignancies (44%), trauma (15%), haematopoietic stem cell transplantation (9%) and diabetes mellitus (9%). The most common manifestations of zygomycosis were pulmonary (30%), rhinocerebral (27%), soft tissue (26%) and disseminated disease (15%). Diagnosis was made by both histology and culture in 108 cases (44%). Among 172 cases with cultures, Rhizopus spp. (34%), Mucor spp. (19%) and Lichtheimia (formerly Absidia) spp. (19%) were most commonly identified. Thirty-nine per cent of patients received amphotericin B formulations, 7% posaconazole and 21% received both agents; 15% of patients received no antifungal therapy. Total mortality in the entire cohort was 47%. On multivariate analysis, factors associated with survival were trauma as an underlying condition (p 0.019), treatment with amphotericin B (p 0.006) and surgery (p <0.001); factors associated with death were higher age (p 0.005) and the administration of caspofungin prior to diagnosis (p 0.011). In conclusion, zygomycosis remains a highly lethal disease. Administration of amphotericin B and surgery, where feasible, significantly improve survival.

Predictors of candidaemia caused by non-albicans Candida species: results of a population-based surveillance in Barcelona, Spain.

Although Candida albicans (CA) is the most common cause of Candida bloodstream infections (BSIs), recent studies have observed an increasing percentage of candidaemias caused by non-albicans Candida species (NAC). In the present study, we attempted to identify the predictors of candidaemia due to NAC compared to CA. We analyzed data from an active population-based surveillance in Barcelona (Spain) from January 2002 to December 2003. Factors associated with NAC fungaemia were determined by multivariate analysis. A total of 339 episodes of Candida BSI, in 336 patients (median age 63 years, interquartile range: 41-72 years), were included. CA was the most commonly isolated (52%), followed by Candida parapsilosis (23%), Candida tropicalis (10%), Candida glabrata (8.6%), Candida krusei (3.4%) and other NAC spp. (3%).Overall, 48% of cases were due to NAC spp. Multivariate logistic regression analysis identified factors associated with a risk of BSI due to NAC spp.: having received a haematologic transplant (OR 10.8; 95% CI 1.31-90.01; p 0.027), previous fluconazole exposure (OR 4.47; 95% CI 2.12-9.43; p <0.001) and neonatal age (OR 4.42; 95% CI 1.63-12.04; p 0.004). Conversely, previous CA colonization (OR 0.33; 95% CI 0.19-0.57; p 0.001) and previous antibiotic use (OR 0.42; 95% CI 0.21-0.85; p 0.017) were associated with CA fungaemia compared to NAC. In conclusion, NAC candidaemia comprised 48% of cases in our series. Predictors of NAC include having received a haematologic transplant, neonatal age and previous fluconazole use.

In vitro activity of antifungals against Zygomycetes.

To date, no reference standard for therapy for zygomycosis has been established because there are insufficient clinical data with which to make such a judgement. Knowledge of the species responsible for the infection and its antifungal susceptibility profile has become increasingly important in the management of patients. Amphotericin B is the most active drug against all the species involved, followed by posaconazole, whereas voriconazole has no activity. Echinocandins are completely inactive in vitro, but may be an interesting option when used in combination with other drugs.

Utility of real-time PCR for the detection of Paracoccidioides brasiliensis DNA in the diagnosis of imported paracoccidioidomycosis.

An increase in immigration from endemic regions has resulted in a number of cases of paracoccidioidomycosis (PCM) being imported into Spain. A molecular diagnostic technique based on real-time PCR was developed for the detection of Paracoccidioides brasiliensis DNA in both culture and patients' clinical samples. A Molecular Beacon probe was used, labelled with FAM and directed at the ITS1 region of ribosomic DNA. The detection limit of the technique developed was 1 fg of fungal DNA per microl of sample. This procedure proved to be very reproducible and specific. The technique was tested with cultures of 12 clinical strains and on samples from two patients with proven PCM. Real-time PCR was positive for all the culture strains, as well as those from both patients. By samples, the technique was positive in sputum and tissue biopsies but less useful on blood samples. Samples were analyzed several months after patient treatment, detecting a small amount of fungal DNA in one respiratory sample. This technique of real-time PCR is a sensitive method for rapid diagnosis of paracoccidioidomycosis and could serve to monitor patients after treatment has begun.