Detección y genotipificación de Pneumocystis jirovecii en muestras de pacientes mexicanos VIH positivos y negativos / Detection and genotyping of Pneumocystis jirovecii in samples from HIV positive and negative Mexican patients

Resumen Introducción: Pneumocystis jirovecii es un hongo atípico detectado particularmente en pacientes VIH-positivos o con trasplante. Objetivo: Detectar y genotipificar Pneumocystis jirovecii en muestras de pacientes de dos hospitales de la ciudad de México. Método: Fueron procesadas 89 muestras respiratorias, correspondientes a 53 pacientes (30 VIH positivos y 23 VIH negativos) con sintomatología respiratoria y 11 personas sanas incluidas como control negativo. El DNA fue extraído y amplificado por PCR anidada de la región del espaciador transcrito interno, obteniendo un fragmento en cada ronda (de 693 y 550 pb). Los genotipos y su relación filogenética fueron determinados por secuenciación del fragmento de 550 pb. Resultados: Cuarenta y ocho muestras de 30 pacientes VIH-positivos provenían de un solo hospital, de las cuales 11 (36.6 %) fueron positivas a Pneumocystis jirovecii. Ninguna fue positiva en pacientes VIH-negativos o personas sanas. Los haplotipos detectados con mayor frecuencia fueron Eg y Em. Conclusiones: La frecuencia de infección por Pneumocystis jirovecii fue alta en la población mexicana estudiada. El genotipo más frecuente fue diferente a los reportados en otros países. Es necesario encauzar este problema de salud hacia la detección temprana de esta infección.

Abstract Introduction: Pneumocystis jirovecii is an atypical fungus particularly detected in HIV-positive or transplant patients. Objective: To detect and genotype Pneumocystis jirovecii in patient samples from two hospitals in Mexico City. Method: Eighty-nine respiratory tract samples, corresponding to 53 patients (30 HIV-positive and 23 HIV-negative) with respiratory symptoms and to 11 healthy individuals included as negative control, were processed. DNA was extracted from the ITS region and amplified by nested polymerase chain reaction from the internal transcribed spacer, with one fragment being obtained at each round (693 and 550 bp). Genotypes and their phylogenetic relationship were determined by sequencing the 550 bp fragment. Results: Forty-eight samples from 30 HIV-positive patients were received from a single hospital, out of which 11 (36.6 %) were positive for Pneumocystis jirovecii. No sample was positive in HIV-negative patients or healthy subjects. The most frequently detected haplotypes were Eg and Em. Conclusions: The frequency of Pneumocystis jirovecii infection was high in the studied Mexican population. The most common genotype was different from those reported in other countries. It is necessary to address this health problem through early detection of this infection.

Sensitive detection of Pneumocystis jirovecii DNA in gastric wash using nested polymerase chain reaction.

One hundred and five samples of gastric washes were obtained from 52 pediatric patients. Eleven of the 105 samples (10%) gave positive results using immunofluorescence antibody test (IFA) for Pneumocystis jirovecii. Single-step polymerase chain reaction (PCR) produced 13% (14 samples), whereas detection by nested PCR was increased to 65 samples (62%). Moderate agreement (kappa = 0.5) was found between test results of IFA and single-step PCR, but no agreement was found between the results of IFA and nested PCR (kappa = 0.1).

Genetic diversity of Pneumocystis jirovecii in Thailand.

A fungus Pneumocystis jirovecii, which causes a diffuse bilateral pneumonia called Pneumocystis pneumonia (PcP) is one of the most common opportunistic infections in HIV-infected patients in Thailand. Molecular techniques have demonstrated diversity among isolates of P. jirovecii by comparison of DNA-sequence variation at the internal transcribed spacer region 1 (ITS1) and region 2 (ITS2) of the nuclear ribosomal RNA genes. The studies confirm that a high diversity of P. jirovecii ITS types exists in different populations from different geographical areas. Type Eg is found globally from represent countries in Europe, North America, South Africa and Asia. Among the 23 types of P. jirovecii observed in Thailand, type Ir is present at the highest frequency (28.6 %), followed by type Eb (21.4%) and types Eg and Rp (14.3 %), respectively. Ir and Rp are unique types observed in Thailand. Mixed infections of more than one types of P. jirovecii are commonly observed in all studies with prevalence of 25-82 %. Moreover, unique types of P. jirovecii can be found in a specific group of populations. These types may be used as genetic markers to study the evolution of the organism in each geographical area.

Pneumocystis pneumonia: an update.

Pneumocystis pneumonia is a major cause of illness and death in immunocompromised hosts. The numbers of pneumocystis pneumonia cases in Thailand have increased each year from 1992 to 2000 and peaked in 2000 at 6,255 cases. The microbe that causes pneumocystis pneumonia in humans is called Pneumocystis jirovecii. Pneumocystis sp. was discovered nearly a century ago, but the knowledge of Pneumocystis sp. remained poorly understood, until the molecular biology techniques help scientists verify it fungus nature. In the past, Pneumocystis sp. was misclassified as protozoan due to its morphologic features. Later, it was reclassified as fungus due to DNA analysis. Cotrimaxazole, the combination of trimethoprim-sulfamethoxazole, is the drug of choice for treatment and prophylaxis of pneumocystis pneumonia. However, increasing evidence of mutations in the enzyme dihydropteroate synthase (DHPS), the target of sulfa drugs represent emergence of sulfa resistance.

Partial sequence of the rRNA operon of Pneumocystis carinii isolates from Puerto Rico

Several reports indicate geographic variation of isolates of Pneumocystis carinii hominis. We have sequenced the internal transcribed spacer (ITS) region and large subunit Group I intron of rRNA genes from P. carinii DNA obtained from two patients from Puerto Rico. Both can be subclassified as Type II, according to the sequence of the ITS region. A system capable of identifying individual isolates will be an essential tool for epidemiological studies of the organism. The amplification of DNA from fixed tissues may facilitate the processing of a large number of samples.