A sensitive and inexpensive high-resolution melting-based testing algorithm for diagnosis of transient abnormal myelopoiesis and myeloid leukemia of Down syndrome.

Patients with Down syndrome (DS) are commonly affected by a pre-leukemic disorder known as transient abnormal myelopoiesis (TAM). This condition usually undergoes spontaneous remission within the first 2 months after birth; however, in children under 5, 20%-30% of cases evolve to myeloid leukemia of Down syndrome (ML-DS). TAM and ML-DS are caused by co-operation between trisomy 21 and acquired mutations in the GATA1 gene. Currently, only next-generation sequencing (NGS)-based methodologies are sufficiently sensitive for diagnosis in samples with small GATA1 mutant clones (≤10% blasts). Alternatively, this study presents research on a new, fast, sensitive, and inexpensive high-resolution melting (HRM)-based diagnostic approach that allows the detection of most cases of GATA1 mutations, including silent TAM. The algorithm first uses flow cytometry for blast count, followed by HRM and Sanger sequencing to search for mutations on exons 2 and 3 of GATA1. We analyzed 138 samples of DS patients: 110 of asymptomatic neonates, 10 suspected of having TAM, and 18 suspected of having ML-DS. Our algorithm enabled the identification of 33 mutant samples, among them five cases of silent TAM (5/110) and seven cases of ML-DS (7/18) with blast count ≤10%, in which GATA1 alterations were easily detected by HRM. Depending on the type of genetic variation and its location, our methodology reached sensitivity similar to that obtained by NGS (0.3%) at a considerably reduced time and cost, thus making it accessible worldwide.

Cervical Cytology of Samples with Ureaplasma urealyticum, Ureaplasma parvum, Chlamydia trachomatis, Trichomonas vaginalis, Mycoplasma hominis, and Neisseria gonorrhoeae Detected by Multiplex PCR.

INTRODUCTION: Despite increasing application of molecular diagnostic methods for the detection of sexually transmitted infections, the cytological findings in pap smears of patients with pathogens that can be identified only by PCR are not yet well described. The aim of this study was to describe the most common cytological features in cervical pap smears of patients with Chlamydia trachomatis, Neisseria gonorrhoeae, Mycoplasma genitalium, Trichomonas vaginalis, Mycoplasma hominis, Ureaplasma urealyticum, and Ureaplasma parvum detected by multiplex PCR. METHODS: Cervical samples for conventional and liquid-based cytology and for multiplex PCR were collected from women ranging from 23 to 54 years old, who underwent routine screening at a gynecological Unit. RESULTS: Multiplex PCR was positive in 36.2% of the samples: Ureaplasma parvum 14.9%, Chlamydia trachomatis 10.6%, Trichomonas vaginalis 10.6%, Mycoplasma hominis 8.5%, Ureaplasma urealyticum 4.2%, Neisseria gonorrhoeae 2.1%, and Mycoplasma genitalium (0). Multiple pathogens were observed in 12.8% of samples. Microscopic cervicitis (≥10 polymorphonuclear leukocytes/epithelial cell) and normal (predominantly lactobacillary) microbiota were the most frequent findings in the samples in which the pathogens were detected alone or in multiple infections, except for samples with Trichomonas vaginalis in which the coccobacillary microbiota was the most common. In samples with microscopic cervicitis and normal microbiota, those with at least one pathogen identified by multiplex PCR were significantly more frequent than those with no pathogen, 66.6% versus 33.3%. CONCLUSION: Failure to identify an inflammatory agent in pap smear with intense neutrophil exudate may suggest the presence of Ureaplasma parvum, Ureaplasma urealyticum, Chlamydia trachomatis, or Trichomonas vaginalis. A remark on the intensity of inflammation should be made in the reports of cervical pap smears so that this cytological finding can be correlated with clinical and PCR results.