Does the Recovery of Respiratory Viruses Impact Pulmonary Function at Baseline and 1-, 6-, and 12-Month Follow-Up in People Living with HIV and Pneumonia?

The frequency of respiratory viruses in people living with HIV (PLHIV) and their impact on lung function remain unclear. We aimed to determine the frequency of respiratory viruses in bronchoalveolar lavage and induced sputum samples in PLHIV and correlate their presence with lung function. A prospective cohort of adults hospitalized in Medellín between September 2016 and December 2018 included three groups: group 1 = people diagnosed with HIV and a diagnosis of community-acquired pneumonia (CAP), group 2 = HIV, and group 3 = CAP. People were followed up with at months 1, 6, and 12. Clinical, microbiological, and spirometric data were collected. Respiratory viruses were detected by multiplex RT-PCR. Sixty-five patients were included. At least 1 respiratory virus was identified in 51.9%, 45.1%, and 57.1% of groups 1, 2 and 3, respectively. Among these, 89% of respiratory viruses were detected with another pathogen, mainly Mycobacterium tuberculosis (40.7%) and Pneumocystis jirovecii (22.2%). The most frequent respiratory virus was rhinovirus (24/65, 37%). On admission, 30.4% of group 1, 16.6% of group 2, and 50% of group 3 had airflow limitation, with alteration in forced expiratory volume at first second in both groups with pneumonia compared to HIV. Respiratory viruses are frequent in people diagnosed with HIV, generally coexisting with other pathogens. Pulmonary function on admission was affected in patients with pneumonia, improving significantly in the 1st, 6th, and 12th months after CAP onset.

Etiology and the challenge of diagnostic testing of community-acquired pneumonia in children and adolescents.

BACKGROUND: Pneumonia is the leading cause of mortality in pediatric population. The etiology of pneumonia in this population is variable and changes according to age and disease severity and where the study is conducted. Our aim was to determine the etiology of community-acquired pneumonia (CAP) in children aged 1 month to 17 years admitted to 13 Colombian hospitals. METHODS: Prospective cohort study. Hospitalized children with radiologically confirmed CAP and ≤ 15 days of symptoms were included and followed together with a control group. Induced sputum (IS) was submitted for stains and cultures for pyogenic bacteria and Mycobacterium tuberculosis, and multiplex PCR (mPCR) for bacteria and viruses; urinary antigens for pneumococcus and Legionella pneumophila; nasopharyngeal swabs for viruses, and paired serology for atypical bacteria and viruses. Additional cultures were taken at the discretion of primary care pediatricians. RESULTS: Among 525 children with CAP, 71.6% had non-severe pneumonia; 24.8% severe and 3.6% very severe pneumonia, and no fatal cases. At least one microorganism was identified in 84% of children and 61% were of mixed etiology; 72% had at least one respiratory virus, 28% pyogenic bacteria and 21% atypical bacteria. Respiratory syncytial virus, Parainfluenza, Rhinovirus, Influenza, Mycoplasma pneumoniae, Adenovirus and Streptococcus pneumoniae were the most common etiologies of CAP. Respiratory syncytial virus was more frequent in children under 2 years and in severe pneumonia. Tuberculosis was diagnosed in 2.3% of children. IS was the most useful specimen to identify the etiology (33.6%), and blood cultures were positive in 3.6%. The concordance between all available diagnostic tests was low. A high percentage of healthy children were colonized by S. pneumoniae and Haemophilus influenzae, or were infected by Parainfluenza, Rhinovirus, Influenza and Adenovirus. CONCLUSIONS: Respiratory viruses are the most frequent etiology of CAP in children and adolescents, in particular in those under 5 years. This study shows the challenges in making an etiologic diagnosis of CAP in pediatric population because of the poor concordance between tests and the high percentage of multiple microorganisms in healthy children. IS is useful for CAP diagnosis in pediatric population.

Induced sputum as an adequate clinical specimen for the etiological diagnosis of community-acquired pneumonia (CAP) in children and adolescents.

OBJECTIVES: This study aimed to evaluate the utility of induced sputum (IS) for the diagnosis of community-acquired pneumonia (CAP) in pediatric population. METHODS: This cross-sectional study included pediatric population aged between 1 month and 17 years who were hospitalized with a diagnosis of CAP in 13 hospitals in Colombia, in whom an IS sample was obtained. Gram staining, aerobic bacterial and mycobacterial culture tests, and polymerase chain reaction (PCR) for 6 atypical bacteria and 15 respiratory viruses were performed. We evaluated the quality of IS samples. RESULTS: IS samples were collected in 516 of 525 children included in this study. The median age was 32 months, 38.6% were younger than 2 years, and 40.9% were between 2 and 5 years. Two patients had transient hypoxemia during the procedure. The quality of the IS obtained was good in 48.4% and intermediate in 24.5%. Identification of a respiratory pathogen was achieved with an IS sample (with Gram staining, culture test, and PCR) in 372 of 516 children with CAP. CONCLUSION: Our study shows that IS is an adequate sample for the diagnosis of CAP in pediatric population that required hospitalization. The procedure was safe, well tolerated, and with better diagnostic yields compared with the rest of the samples obtained.

Is It Possible to Differentiate Pneumocystis jirovecii Pneumonia and Colonization in the Immunocompromised Patients with Pneumonia?

Respiratory sample staining is a standard tool used to diagnose Pneumocystis jirovecii pneumonia (PjP). Although molecular tests are more sensitive, their interpretation can be difficult due to the potential of colonization. We aimed to validate a Pneumocystis jirovecii (Pj) real-time PCR (qPCR) assay in bronchoscopic bronchoalveolar lavage (BAL) and oropharyngeal washes (OW). We included 158 immunosuppressed patients with pneumonia, 35 lung cancer patients who underwent BAL, and 20 healthy individuals. We used a SYBR green qPCR assay to look for a 103 bp fragment of the Pj mtLSU rRNA gene in BAL and OW. We calculated the qPCR cut-off as well as the analytical and diagnostic characteristics. The qPCR was positive in 67.8% of BAL samples from the immunocompromised patients. The established cut-off for discriminating between disease and colonization was Ct 24.53 for BAL samples. In the immunosuppressed group, qPCR detected all 25 microscopy-positive PjP cases, plus three additional cases. Pj colonization in the immunocompromised group was 66.2%, while in the cancer group, colonization rates were 48%. qPCR was ineffective at diagnosing PjP in the OW samples. This new qPCR allowed for reliable diagnosis of PjP, and differentiation between PjP disease and colonization in BAL of immunocompromised patients with pneumonia.

Highly sensitive scent-detection of COVID-19 patients in vivo by trained dogs.

Timely and accurate diagnostics are essential to fight the COVID-19 pandemic, but no test satisfies both conditions. Dogs can scent-identify the unique odors of volatile organic compounds generated during infection by interrogating specimens or, ideally, the body of a patient. After training 6 dogs to detect SARS-CoV-2 by scent in human respiratory secretions (in vitro diagnosis), we retrained 5 of them to search and find the infection by scenting the patient directly (in vivo screening). Then, efficacy trials were designed to compare the diagnostic performance of the dogs against that of the rRT-PCR in 848 human subjects: 269 hospitalized patients (COVID-19 prevalence 30.1%), 259 hospital staff (prevalence 2.7%), and 320 government employees (prevalence 1.25%). The limit of detection in vitro was lower than 10-12 copies ssRNA/mL. During in vivo efficacy experiments, our 5 dogs detected 92 COVID-19 positive patients among the 848 study subjects. The alert (lying down) was immediate, with 95.2% accuracy and high sensitivity (95.9%; 95% C.I. 93.6-97.4), specificity (95.1%; 94.4-95.8), positive predictive value (69.7%; 65.9-73.2), and negative predictive value (99.5%; 99.2-99.7) in relation to rRT-PCR. Seventy-five days after finishing in vivo efficacy experiments, a real-life study (in vivo effectiveness) was executed among the riders of the Metro System of Medellin, deploying the human-canine teams without previous training or announcement. Three dogs were used to examine the scent of 550 volunteers who agreed to participate, both in test with canines and in rRT-PCR testing. Negative predictive value remained at 99.0% (95% C.I. 98.3-99.4), but positive predictive value dropped to 28.2% (95% C.I. 21.1-36.7). Canine scent-detection in vivo is a highly accurate screening test for COVID-19, and it detects more than 99% of infected individuals independent of key variables, such as disease prevalence, time post-exposure, or presence of symptoms. Additional training is required to teach the dogs to ignore odoriferous contamination under real-life conditions.

Diagnóstico microbiológico en lavado broncoalveolar. Revisión de la literatura / Microbiological diagnosis in bronchoalveolar lavage. Literature review

El lavado broncoalveolar (LBA) se describió hace aproximadamente 50 años, y desde ese momento se ha venido empleando cada vez con más frecuencia, llegando a ser uno de los métodos de elección para hacer el diagnóstico microbiológico de las infecciones respiratorias bajas, pues facilita la identificación de patógenos oportunistas y no oportunistas. Su uso se incrementó paralelamente con el número de pacientes inmunocomprometidos, sobre todo a causa del SIDA y los trasplantes, situaciones en las que con frecuencia los pacientes padecen infecciones pulmonares por gérmenes oportunistas. El LBA es un procedimiento seguro que permite obtener muestras que aportan información amplia de las características celulares y microbiológicas del tracto respiratorio inferior. Para garantizar su utilidad es fundamental que la recolección, transporte, almacenamiento y procesamiento de las muestras sean óptimos. El análisis de las muestras se hace por técnicas convencionales para identificación de microorganismos, como son las tinciones y el aislamiento en medios de cultivo, y por otros métodos tales como la inmunofluorescencia, pruebas inmunológicas para la detección de antígenos y anticuerpos, y pruebas de biología molecular. En la presente revisión, se hace una actualización sobre el procedimiento de obtención, almacenamiento y transporte de las muestras de LBA, así como de las técnicas de diagnóstico microbiológico más utilizadas para identificar los principales agentes infecciosos asociados con enfermedades del tracto respiratorio inferior

Bronchoalveolar lavage (BAL) was described approximately 50 years ago and since then it has been used with increasing frequency, becoming one of the methods of choice for making the microbiological diagnosis of lower respiratory infections, as it facilitates the identification of opportunistic and non-opportunistic pathogens. Its use increased in parallel with the number of immunocompromised patients, especially due to AIDS and transplantation, situations in which patients frequently suffer from lung infections due to opportunistic germs. BAL is a safe procedure that allows obtaining samples that provide comprehensive information on the cellular and microbiological characteristics of the lower respiratory tract. Optimal collection, transport, storage and processing of samples is essential to guarantee its usefulness. Analysis of the samples is done both by conventional techniques for the identification of microorganisms, such as staining and isolation in culture media, as well as by other methods such as immunofluorescence, immunological tests for the detection of antigens and antibodies, and molecular biology assays. In this review, an update in presented on the procedure for obtaining, storing and transporting BAL samples, as well as on the most widely used microbiological diagnostic techniques to identify the main infectious agents associated with lower respiratory tract diseases

Mycoplasma pneumoniae in Children With and Without Community-acquired Pneumonia. What do PCR and Serology Say?

BACKGROUND: IgM titers of Mycoplasma pneumoniae can remain high for months or years, and specific DNA can be detected in asymptomatic people. METHODS: We compared the performance of serology and PCR in children with and without community-acquired pneumonia (CAP) for the diagnosis of M. pneumoniae. RESULTS: In children with CAP, a positive test by M. pneumoniae (PCR and/or paired serology or both) were found in 13.9%. Of these, 10.3% were positive by multiplex PCR (Seeplex-Seegen), and 6.7% exhibited quadrupled titers (22 for IgG, 6 for IgM and 5 for both). Both tests were positive in 2.8% of cases. In the group without CAP, 3.3% were positive by PCR. Thirty-two percent of children with CAP and 38.3% of healthy children had IgM titers >11 in the acute phase. CONCLUSIONS: The detection of IgM is not useful for diagnosing acute M. pneumoniae infection, and a positive PCR result can be due to colonization and not infection. New and better diagnostic techniques are required.

Inflammatory Cell Differentiation and Chemotaxis and Extracellular Tissue Repair Markers Are Correlated with Pulmonary Dysfunction in HIV Infected Individuals Presenting with Community-Acquired Pneumonia.

Prior studies have shown that HIV patients develop permanent pulmonary dysfunction following an episode of community-acquired pneumonia (CAP). However, the mechanism causing pulmonary dysfunction remains an enigma. HIV patients experience chronic inflammation. We hypothesized that CAP exacerbates inflammation in HIV patients resulting in an accelerated decline in lung function. A prospective cohort pilot study enrolled HIV patients hospitalized in Medellin, Colombia, with a diagnosis of CAP. Sixteen patients were eligible for the study; they were split into 2 groups: HIV and HIV+CAP. Plasma, sputum, and pulmonary function test (PFT) measurements were retrieved within 48 h of hospital admission and at 1 month follow-up. The concentrations of 13 molecules and PFT values were compared between the 2 cohorts. The HIV+CAP group had lower lung function compared to the HIV group; forced vital capacity (FVC)% predicted and forced expiratory volume in 1 s (FEV1)% predicted decreased, while FEV1/FVC remained constant. APRIL, BAFF, CCL3, and TIMP-1 correlated negatively with FVC% predicted and FEV1% predicted; the relationships however were moderate in strength. Furthermore, the concentrations of BAFF, CCL3, and TIMP-1 were statistically significant between the 2 groups (P ≤ 0.05). Our results indicate that HIV patients with CAP have a different inflammatory pattern and lower lung function compared to HIV patients without CAP. BAFF, CCL3, and TIMP-1 were abnormally elevated in HIV patients with CAP. Future studies with larger cohorts are required to verify these results. In addition, further investigation is required to determine if BAFF, CCL3, and TIMP-1 play a role in the process causing pulmonary dysfunction.

Genotyping and macrolide resistance of Mycoplasma pneumoniae identified in children with community-acquired pneumonia in Medellín, Colombia.

OBJECTIVES: The aim of this study was to describe the genotypes and the main characteristics of community-acquired pneumonia (CAP) caused by Mycoplasma pneumoniae in hospitalized children in Medellín and neighboring municipalities during the period 2011-2012. METHODS: The M. pneumoniae genotype was determined by PCR and sequencing of the p1 and 23S rRNA genes from induced sputum samples and nasopharyngeal swabs (NPS). Samples were obtained from children with CAP who were hospitalized in 13 healthcare centers. In addition, a spatio-temporal analysis was performed to identify the potential risk areas and clustering of the cases over time. RESULTS: A variant of type 2 was the dominant genotype in the induced sputum (96.1%) and NPS (89.3%) samples; the type 1 variant was identified in 3.9% and 10.7% of these samples, respectively. No strains with mutations in the 23S rRNA gene associated with macrolide resistance were found. The cases in Medellín were mainly concentrated in the northeastern areas and western districts. However, no temporal relationship was found among these cases. CONCLUSIONS: A variant of type 2 of M. pneumoniae prevailed among children with CAP during the study period. No strains with mutations associated with macrolide resistance were found.

High transient colonization by Pneumocystis jirovecii between mothers and newborn.

The aim of the study was to explore the frequency and dynamics of acquisition and colonization of Pneumocystis jirovecii among neonates, as well as the epidemiological and genotypic characteristics in mother-child binomial. In a prospective enrolled cohort of women in their third trimester of pregnancy, nasopharyngeal swabs (NPS) and clinical and epidemiological data were collected at four different times: 17 days, 2nd, 4th, and 6th month of life of the newborn. P. jirovecii was detected by nested-PCR for the mtLSU-rRNA gene in each NPS; the genotypes were determined amplifying four genes. Forty-three pairs and 301 NPS were included. During the third trimester, 16.3% of pregnant women were colonized. The rate of colonization in mothers at delivery was 16, 6, 16, and 5% and in their children 28, 43, 42, and 25%, respectively. Within pregnant women, 53% remained negative throughout follow-up, and among these, 91% of their children were positive in at least one of their samples. In both, mothers and children, the most frequent genotype of P. jirovecii was 1. CONCLUSION: The frequency of colonization by P. jirovecii was higher in newborns than in their respective progenitors. Colonization of both mothers and children is transitory; however, the mother of the newborn is not necessarily the source of primary infection. What is Known: • We did not find studies comparing P. jirovecii colonization between mothers and children simultaneously, yet the frequency of colonization by serologic and molecular methods in pregnant women has been reported. What is New: • According to our findings, 3/4 of the children had transient colonization during the first 6 months of life, in only half in the mothers, without proof of mother-to-child transmission or vice versa.

Impact on Bacterial Resistance of Therapeutically Nonequivalent Generics: The Case of Piperacillin-Tazobactam.

Previous studies have demonstrated that pharmaceutical equivalence and pharmacokinetic equivalence of generic antibiotics are necessary but not sufficient conditions to guarantee therapeutic equivalence (better called pharmacodynamic equivalence). In addition, there is scientific evidence suggesting a direct link between pharmacodynamic nonequivalence of generic vancomycin and promotion of resistance in Staphylococcus aureus. To find out if even subtle deviations from the expected pharmacodynamic behavior with respect to the innovator could favor resistance, we studied a generic product of piperacillin-tazobactam characterized by pharmaceutical and pharmacokinetic equivalence but a faulty fit of Hill's Emax sigmoid model that could be interpreted as pharmacodynamic nonequivalence. We determined the impact in vivo of this generic product on the resistance of a mixed Escherichia coli population composed of ∼99% susceptible cells (ATCC 35218 strain) and a ∼1% isogenic resistant subpopulation that overproduces TEM-1 ß-lactamase. After only 24 hours of treatment in the neutropenic murine thigh infection model, the generic amplified the resistant subpopulation up to 20-times compared with the innovator, following an inverted-U dose-response relationship. These findings highlight the critical role of therapeutic nonequivalence of generic antibiotics as a key factor contributing to the global problem of bacterial resistance.

Comparison of serological methods with PCR-based methods for the diagnosis of community-acquired pneumonia caused by atypical bacteria.

BACKGROUND: The diagnosis of community-acquired pneumonia (CAP) caused by Legionella pneumophila, Mycoplasma pneumoniae, and Chlamydophila pneumoniae is traditionally based on cultures and serology, which have special requirements, are time-consuming, and offer delayed results that limit their clinical usefulness of these techniques. We sought to develop a multiplex PCR (mPCR) method to diagnosis these bacterial infections in CAP patients and to compare the diagnostic yields obtained from mPCR of nasopharyngeal aspirates (NPAs), nasopharyngeal swabs (NPSs), and induced sputum (IS) with those obtained with specific PCR commercial kits, paired serology, and urinary antigen. RESULTS: A total of 225 persons were included. Of these, 10 patients showed serological evidence of L. pneumophila infection, 30 of M. pneumoniae, and 18 of C. pneumoniae; 20 individuals showed no CAP. The sensitivities were mPCR-NPS = 23.1%, mPCR-IS = 57.1%, Seeplex®-IS = 52.4%, and Speed-oligo®-NPA/NPS = 11.1%, and the specificities were mPCR-NPS = 97.1%, mPCR-IS = 77.8%, Seeplex®-IS = 92.6%, and Speed-oligo®-NPA/NPS = 96.1%. The concordance between tests was poor (kappa <0.4), except for the concordance between mPCR and the commercial kit in IS (0.67). In individuals with no evidence of CAP, positive reactions were observed in paired serology and in all PCRs. CONCLUSIONS: All PCRs had good specificity but low sensitivity in nasopharyngeal samples. The sensitivity of mPCR and Seeplex® in IS was approximately 60%; thus, better diagnostic techniques for these three bacteria are required.

Usefulness of FTA® cards as a Pneumocystis-DNA extraction method in bronchoalveolar lavage samples.

BACKGROUND: FTA® cards (Fast Technology for Analysis of Nucleic Acids) are an alternative DNA extraction method in bronchoalveolar lavage (BAL) samples for Pneumocystis jirovecii molecular analyses. The goal was to evaluate the usefulness of FTA® cards to detect P. jirovecii-DNA by PCR in BAL samples compared to silica adsorption chromatography (SAC). METHODS: This study used 134 BAL samples from immunocompromised patients previously studied to establish microbiological aetiology of pneumonia, among them 15 cases of Pneumocystis pneumonia (PCP) documented by staining and 119 with other alternative diagnoses. The FTA® system and SAC were used for DNA extraction and then amplified by nested PCR to detect P. jirovecii. Performance and concordance of the two DNA extraction methods compared to P. jirovecii microscopy were calculated. The influence of the macroscopic characteristics, transportation of samples and the duration of the FTA® card storage (1, 7, 10 or 12 months) were also evaluated. RESULTS: Among 134 BAL samples, 56% were positive for P. jirovecii-DNA by SAC and 27% by FTA®. All 15 diagnosed by microscopy were detected by FTA® and SAC. Specificity of the FTA® system and SAC were 82.4% and 49.6%, respectively. Compared to SAC, positivity by FTA® decreased with the presence of blood in BAL (62% vs 13.5%). The agreement between samples at 7, 10 and 12 months was 92.5% for FTA®. Positive cases by FTA® remained the same after shipment by mail. CONCLUSIONS: Results suggest that FTA® is a practical, safe and economical method to preserve P. jirovecii-DNA in BAL samples for molecular studies.

Unique cytokine and chemokine patterns in bronchoalveolar lavage are associated with specific causative pathogen among HIV infected patients with pneumonia, in Medellin, Colombia.

We wanted to investigate the pro-inflammatory cytokine/chemokine profile associated with the etiological agents identified in HIV patients. Immunosuppressed patients admitted to two hospitals in Medellin, Colombia, with clinical and radiographic diagnosis of pneumonia were enrolled in the study. After consent, bronchoalveolar lavage (BAL) was collected for bacterial, mycobacterial and fungal diagnosis. All patients were followed for a year. A stored BAL sample was used for cytokine/chemokine detection and measurement using commercial, magnetic human cytokine bead-based 19-plex assays. Statistical analysis was performed by assigning cytokine/chemokine concentrations levels into <25 percentile (lower), 25-75 percentile (normal) and >75 percentile (higher). Principal component analysis (PCA) and Kruskal-Wallis analysis were conducted to identify the clustering of cytokines with the various infectious etiologies (fungi, Mycobacterium tuberculosis - MTB, and bacteria). Average age of patients was 35, of whom 77% were male, and the median CD4 count of 33cells/µl. Of the 57 HIV infected patients, in-hospital mortality was 12.3% and 33% died within a year of follow up. The PCA revealed increased IL-10, IL-12, IL-13, IL-17, Eotaxin, GCSF, MIP-1α, and MIP-1ß concentrations to be associated with MTB infection. In patients with proven fungal infection, low concentrations of IL-1RA, IL-8, TNF-α and VEGF were identified. Bacterial infections displayed a distinct cytokine pattern and were not misclassified using the MTB or fungi cytokine patterns (p-value<0.0001). Our results indicate a unique pattern of pro-inflammatory cytokine/chemokine, allowing differentiation between bacterial and non-bacterial pathogens. Moreover, we found distinct, if imperfectly discriminatory, cytokine/chemokine patterns associated with MTB and fungal infections.

[Microscopic diagnosis of Pneumocystis jirovecii pneumonia in bronchoalveolar lavage and oropharyngeal wash samples of immunocompromised patients with pneumonia]. / Diagnóstico microscópico de neumonía por Pneumocystis jirovecii en muestras de lavado broncoalveolar y lavado orofaríngeo de pacientes inmunocomprometidos con neumonía.

INTRODUCTION: The diagnosis of Pneumocystis jirovecii pneumonia is based on observation of the microorganism using several staining techniques in respiratory samples, especially bronchoalveolar lavage and induced sputum. Recently, the fungus also has been detected in oropharyngeal wash samples, but only using molecular tests. OBJECTIVE: The diagnostic yield of two microscopic stains, toluidine blue O and direct fluorescent antibody, was compared in bronchoalveolar lavage and oropharyngeal wash samples for the detection of P. jirovecii in immunocompromised patients with pneumonia. MATERIALS AND METHODS: Cross-sectional evaluation diagnostic tests were used in 166 immunosuppressed patients with suspected P. jirovecii. By protocol, bronchoscopic bronchoalveolar lavage and oropharyngeal wash samples were prepared by cytocentrifugation, and slides were stained with toluidine blue and fluorescent antibody. The proportion of positive results from each stain and concordance between them were determined. RESULTS: Twenty-four cases (14.5%) of P. jirovecii were detected in bronchoalveolar lavage samples. Of them, 21 were positive by both toluidine blue and fluorescent antibody stains, whereas 3 cases were detected by fluorescent antibody alone. None of the 166 oropharyngeal wash samples were positive by either of these techniques. No significant differences were found between proportions from positive results (p=0.63). Concordance (kappa coefficient) between both stains was 0.92 (95% CI: 0.84-1.00). CONCLUSIONS: Both techniques were useful to diagnose P. jirovecii in bronchoalveolar lavage samples. However, toluidine blue stain did not detect 12% of fluorescent antibody positive cases. Oropharyngeal wash samples do not provide sufficient material for the microscopic identification of this fungus.

Diagnóstico microscópico de neumonía por Pneumocystis jirovecii en muestras de lavado broncoalveolar y lavado orofaríngeo de pacientes inmunocomprometidos con neumoníA / Microscopic diagnosis of Pneumocystis jirovecii pneumonia in bronchoalveolar lavage and oropharyngeal wash samples of immunocompromised patients with pneumonia

Introducción. El diagnóstico de neumonía por Pneumocystis jirovecii se fundamenta en la visualización microscópica del hongo en secreciones respiratorias. Técnicas moleculares recientes también lo han detectado en muestras de orofaringe, pero su utilidad diagnóstica es discutible. En Colombia, hay poca información al respecto. Objetivo. Comparar el rendimiento de dos coloraciones, azul de toluidina O e inmunofluorescencia directa, en muestras de lavado broncoalveolar y lavado orofaríngeo en pacientes inmunocomprometidos con neumonía. Materiales y métodos. Se llevó a cabo un estudio transversal de evaluación de pruebas diagnósticas en 166 pacientes inmunocomprometidos con sospecha de neumonía por P. jirovecii. Por protocolo, las muestras de lavado broncoalveolar y orofaríngeo se citocentrifugaron y se colorearon con azul de toluidina e inmunofluorescencia. Se determinó la proporción de resultados positivos con ambas tinciones en cada una de las muestras, y la concordancia entre ellas. Resultados. Se detectaron 24 casos de neumonía por P. jirovecii en las muestras de lavado broncoalveolar (14,5 %), 21 de los cuales fueron positivos por ambas pruebas, mientras que tres casos se detectaron sólo por inmunofluorescencia. Ninguna de las 166 muestras de lavado orofaríngeo fue positiva por cualquiera de estas técnicas. Al comparar las proporciones de resultados positivos, no se encontraron desacuerdos significativos (p=0,63). La concordancia (coeficiente kappa) entre ellas fue de 0,92 (IC95%: 0,84-1). Conclusiones. Ambas coloraciones son útiles para diagnosticar neumonía por P. jirovecii en muestras de lavado broncoalveolar. Sin embargo, el azul de toluidina no detecta, aproximadamente, el 12 % de los casos positivos por inmunofluorescencia. Las muestras de lavado orofaríngeo no son apropiadas para detectar microscópicamente P. jirovecii.

Introduction. The diagnosis of Pneumocystis jirovecii pneumonia is based on observation of the microorganism using several staining techniques in respiratory samples, especially bronchoalveolar lavage and induced sputum. Recently, the fungus also has been detected in oropharyngeal wash samples, but only using molecular tests. Objective. The diagnostic yield of two microscopic stains, toluidine blue O and direct fluorescent antibody, was compared in bronchoalveolar lavage and oropharyngeal wash samples for the detection of P. jirovecii in immunocompromised patients with pneumonia. Materials and methods. Cross-sectional evaluation diagnostic tests were used in 166 immunossupressed patients with suspected P. jirovecii. By protocol, bronchoscopic bronchoalveolar lavage and oropharyngeal wash samples were prepared by cytocentrifugation, and slides were stained with toluidine blue and fluorescent antibody. The proportion of positive results from each stain and concordance between them were determined. Results. Twenty-four cases (14.5%) of P. jirovecii were detected in bronchoalveolar lavage samples. Of them, 21 were positive by both toluidine blue and fluorescent antibody stains, whereas 3 cases were detected by fluorescent antibody alone. None of the 166 oropharyngeal wash samples were positive by either of these techniques. No significant differences were found between proportions from positive results (p=0.63). Concordance (kappa coefficient) between both stains was 0.92 (95% CI: 0.84-1.00). Conclusions. Both techniques were useful to diagnose P. jirovecii in bronchoalveolar lavage samples. However, toluidine blue stain did not detect 12% of fluorescent antibody positive cases. Oropharyngeal wash samples do not provide sufficient material for the microscopic identification of this fungus.

[Molecular and immunological analyses suggest the absence of hydrophilic surface proteins in Leishmania (Viannia) panamensis]. / El análisis molecular y el inmunogénico sugieren la ausencia de las proteínas hidrofílicas de superficie en Leishmania (Viannia) panamensis.

INTRODUCTION: The genus Leishmania is divided into two subgenera: Leishmania and Viannia. The two subgenera present several important differences such as the pathology they cause in susceptible hosts, their in vitro growth behavior, their genetic characteristics, and the expression pattern of several proteins, including those of the hydrophilic surface protein family. OBJECTIVE: To characterize the hydrophilic surface protein family in Leishmania (Viannia) panamensis. MATERIALS AND METHODS: The hasp genes were amplified in L. (V.) panamensis, using specific primers previously designed to amplify this gene in Leishmania (Leishmania) major. The PCR products were cloned, sequenced, and the sequences analyzed using common bioinformatics tools. Secondly, a serological screening was undertaken with an enzyme-linked immunosorbent assay and Western blot to detect specific antibodies against the hydrophilic surface recombinant protein from L. (L.) major. RESULTS: A copy of a pseudogene was amplified in L. (V.) panamensis which was 60% homologous with the L. (L.) major orthologous gene. Antibodies responded to the hydrophilic surface recombinant proteins only in sera from patients with visceral leishmaniasis [Leishmania (Leishmania) chagasi]. CONCLUSION: These results suggest the lack of a functional hasp gene in L. (V.) panamensis, suggesting probably the loss of the complete gene family in this species of the Viannia subgenus.

El análisis molecular y el inmunogénico sugieren la ausencia de las proteínas hidrofílicas de superficie en Leishmania (Viannia) panamensis / Molecular and immunological analyses suggest the absence of hydrophilic surface proteins in Leishmania (Viannia) panamensis

Introducción. Los dos subgéneros en los cuales se divide el género Leishmania: Viannia y Leishmania, presentan diferencias significativas en las manifestaciones clínicas que causan, en su comportamiento de crecimiento en cultivos in vitro, en sus características genéticas y en la expresión de varias proteínas, entre ellas las de la familia hidrofílica de superficie superficie. Objetivo. Caracterizar las proteínas hidrofílicas de superficie en Leishmania (Viannia) panamensis. Materiales y métodos. Se amplificaron los genes hasp en L. (V.) panamensis usando cebadores específicos para la especie Leishmania (Leishmania) major. Los productos de la amplificación fueron clonados, secuenciados y analizados con herramientas bioinformáticas. Posteriormente, se realizó un análisis serológico por medio de ensayo inmunoabsorbente ligado a enzimas y Western blot para detectar la presencia de anticuerpos específicos contra las proteínas hidrofílicas recombinantes de superficie de L. (L.) major en sueros de pacientes con leishmaniasis de zonas endémicas de Colombia. Resultados. Se encontró una copia de un pseudogen en L. (V.) panamensis, el cual presentó una identidad del 60 por ciento con el gen haspa de L. (L.) major. Sólo se encontraron anticuerpos contra las proteínas recombinantes de superficie hidrofílicas en sueros de pacientes con leishmaniasis visceral. Conclusión. Estos resultados sugieren que no existe ninguna copia de un gen funcional hasp en L. (V.) panamensis, lo que indica una pérdida de la familia de genes en esta especie de Leishmania perteneciente al subgénero Viannia.