The usefulness of the buffy coat smear and panbacterial polymerase chain reaction in early diagnosis of neonatal sepsis.

OBJECTIVE: In this study are evaluated the usefulness of the buffy coat smear and panbacterial polymerase chain reaction (PCR) as diagnostic tests in the early detection of neonatal sepsis. MATERIAL AND METHODS: It was studied 49 patients aged up to 28 days who were hospitalized in the Intensive Care Unit (ICUs) of the Neonatology, with a clinical diagnosis of neonatal sepsis and 49 umbilical cord samples of healthy newborns. Blood cultures and 50 microL of plasma were taken for the DNA and performance of the broad-range PCR primer system (panbacterial PCR). Simultaneously, were taken three capillaries with blood for the leukocyte layer (buffy coat) smear, we performed three stains: Gram; Löeffler blue methylene (LBM), and acridine orange (AO). Statistical analysis included sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) against the clinical diagnosis. RESULTS: With respect to stains of buffy coat smear, they resulted very specific, from 90-97%, with 64-75% sensitivity, 87-94% PPV, and 77-82% NPV. In inverse fashion, PCR resulted very sensitive at 96%, with 91% specificity, 92% PPV, and 96% NPV. CONCLUSIONS: Buffy coat smear stains are easy, fast, and specific, while that of PCR was highly sensitive. Thus, both can be utilized as diagnostic tests.

[Identification of Mycobacterium avium-intracellulare complex by PCR of AIDS and disseminated mycobacteriosis]. / Identificación del complejo Mycobacterium avium-intracellulare por PCR en SIDA y micobacteriosis diseminada.

OBJECTIVE: The aim of this study is to differentially identify MAC by PCR in patients with AIDS and disseminated mycobacteriosis. METHODS: A cross sectional study was conducted in Mexico to identify MAC by Molecular Biology. Two sets of primers were synthesized: MAV and MIN, for M. avium and M. intracellulare, respectively. Whole-cell DNAs obtained from 29 clinical isolates and clinical serum specimens from other 24 patients with AIDS and disseminated mycobacterial infection were extracted and amplified by PCR with the MAV and MIN primers. The MAV and MIN primers each amplified one highly specific 1.3-kb segment of the homologous DNA, respectively. RESULTS: Twenty-nine DNAs from MAC clinical isolates identified by Gen-Probe AccuProbes were amplified with the MAV primers. Of the 24 clinical samples, 3 were positive for M. avium and 6 for M. tuberculosis. CONCLUSIONS: Our results demonstrated that PCR technique could be applied for the differentiation of M. avium and M. intracellulare by specific 16S rRNA primers. In patients with advanced stage AIDS and in whom disseminated mycobacteriosis is suspected, the presence of anemia (even with negative cultures), elevated alkaline phosphatase and a median CD4 count of 15.9/mL, the diagnosis of infection by MAC should be strongly considered; we suggest that in accordance with our findings, a more precise stratification of patients in terms of their CD4 T cell counts is warranted.

Introducción: el objetivo de este artículo es Identificar y diferenciar el complejo MAC por PCR en pacientes con SIDA y micobacteriosis diseminada. Métodos: se llevó a cabo un estudio transversal para identificar MAC por biología molecular. Se sintetizaron dos conjuntos de iniciadores: MAV y MIN, para M. avium y M. intracellulare, respectivamente. El ADN total de células obtenidas de 29 aislados clínicos y muestras de suero de otros 24 pacientes con SIDA e infección micobacteriana diseminada fue extraído y se amplificó por PCR con los iniciadores MAV y MIN. Cada uno de los iniciadores MAV y MIN amplificó un segmento altamente específico de 1.3 kb del ADN homólogo, respectivamente. Resultados: veintinueve ADN de los aislados clínicos de MAC identificadas por Gen-Probe AccuProbes se amplificaron con los iniciadores MAV (M. avium). De las 24 muestras clínicas, 3 fueron positivas para M. avium y 6 para M. tuberculosis. Conclusiones: nuestros resultados demostraron que la técnica de PCR se puede aplicar para la diferenciación de M. avium y M. intracellulare por iniciadores específicos 16S rRNA. En pacientes con estadio avanzado de SIDA y en quienes se sospecha micobacteriosis diseminada, la presencia de anemia (incluso con cultivos negativos) fosfatasa alcalina elevada y una mediana de CD4 de 15.9/ml, se debe considerar seriamente el diagnóstico de infección por MAC; sugerimos que, de acuerdo con nuestros resultados, se justifica una estratificación más precisa de los pacientes en términos de sus recuentos de células T CD4.

Molecular analysis of mycobacteria isolated in Mexican patients with different immunodeficiencies in a tertiary care hospital.

BACKGROUND AND AIMS: The prevalence of infections with Mycobacterium tuberculosis (MTB) and non-tuberculous mycobacteria (NTM) species in patients with immunodeficiencies in Mexico is unknown. The aim of this study was to identify, at the molecular level, the mycobacterial species most frequently affecting patients with immunodeficiencies and evaluate the genotypic diversity of MTB complex strains. METHODS: We conducted a retrospective study of 97 strains in patients with the diagnosis of pulmonary (all isolates were of pathological significance) or extrapulmonary tuberculosis. PCR analysis was performed to determine whether they belonged to the MTB complex (MTC) or the Mycobacterium avium complex (MAC). Noncharacterized NTM were sequenced and, finally, MTC were genotyped by MIRUs-VNTR and spoligotyping. RESULTS: Of the 97 mycobacterial strains isolated, 53% were M. tuberculosis, 10% M. bovis, 24% M. avium, 9% M. simiae, 2% M. kansasii and 2% M. gordonae. A great genetic diversity was found by MIRU-VNTR with the greatest polymorphism in MIRU 10, 16, 23 and 27. By spoligotyping, the predominant family was T1. Combining both methods, the association of 13 strains in four different groups was found. CONCLUSIONS: This is the first molecular analysis of mycobacteria isolated from patients with immunodeficiencies in Mexico, describing the prevalence of different mycobacterial species in this population. A great genetic diversity of MTB strains was identified. This is also the first report in Mexico describing clinically important isolates of M. simiae.