A cautionary tale of pseudo- Cryptococcus infection in neonates

Introduction

We report two cases of neonates, where India Ink preparations of cerebrospinal fluid (CSF) received in glass vials were suggestive of Cryptococcus. No correlation with clinical signs and similar sequential reports from the same unit raised alarm bells. Repeat smears using routine in-house and newly prepared stains were prepared and sent in newly sterile specimen containers, confirmed negative microscopy results (both uncentrifuged and centrifuged). The source of the contamination was ultimately found to be non-sterile glass vials used for CSF collection. The requisite to maintain an aseptic chain from sample collection to processing is mandatory to prevent reporting false positives.

Case reports

Case 1: Our first case was a 1-day old female neonate, delivered by lower segment Caesarian section (LSCS) on account of foetal tachycardia. Her mother had a history of oligohydramnios and preterm rupture of membranes six days beforehand. She had been started on intravenous vancomycin and gentamicin post-LSCS. The newborn was delivered at 33 weeks gestation, had respiratory distress at birth, and weighed 1.07 kg. To rule out sepsis, CSF was sent for microbiological diagnosis in a glass vial (an injection vial with a rubber stop). Microbiological processing was done following the standard operating procedures, under aseptic conditions. India Ink, wet mount, and Gram stain of CSF were prepared, which revealed round budding yeasts with a surrounding halo, suggestive of Cryptococcus (Fig. 1A). In the absence of any suggestive predisposing factors, a detailed history was taken; the HIV status of the mother and neonate were tested and both were found to be negative, with no significant past and personal history. A repeat sample was requested.

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Figure 1.

(A) and (B) India ink showing round budding yeast, surrounded by a halo, suggestive of cryptococcus (40×) (A: case 1 and B: case 2).

Case 2: Another case with the same diagnostic dilemma was noted the next day, in a male neonate, who had been delivered by LSCS in view of ruptured membranes, intrauterine breech presentation, and intracardiac foci. The neonate did not cry at birth, weighed 1.57 kg, was in respiratory distress, was diagnosed with hyaline membrane disease, and was intubated. The mother was started on intravenous ampicillin and amikacin as empirical therapy. A CSF sample from the neonate was sent for microbiological diagnosis in a glass vial (similar to the previous one) (Fig. 2). Sample processing continued as before. India Ink, wet mount, and Gram stain of CSF revealed round yeasts with surrounding halo, suggestive of Cryptococcus (Fig. 1B).

The presence of similar findings as in the first case rang alarm bells; a detailed neonatal history revealed no significant history nor predisposing factors. The HIV status of both the mother and neonate was negative.

The smear was therefore repeated using a strictly sterile specimen container for sample collection and transport. Freshly prepared India Ink, using a new set of slides and cover-slips, and sterile specimen containers and samples processed under strict aseptic conditions were carried out.

All smears were duly negative.

Discussion

Cryptococcal meningitis is one of the substantial causes of morbidity and mortality among both immunocompetent and immunocompromised individuals. Infection is known to be endemic in Africa, Brazil, the Mediterranean, and Southeast Asia, especially among immunocompromised, transplant patients and people living with HIV. Infection is encountered more commonly in adults than children. Paediatric cryptococcosis is quite uncommon, and rare in neonates. ¹ CSF, being a sterile sample is rarely prone to contamination, so a microscopic finding is rarely questioned. In the absence of a suggestive history, however, analysis of the sample collection to reporting is mandatory.

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Figure 2.

Glass vial used for cerebrospinal fluid (CSF) collection, which was later traced as the source of contamination in the present cases.

Contamination of glass vials proved to be the factor; the need to abandon an age-old practice of using glass vials in lumbar puncture set for CSF collection is therefore presented. Collection and transport of all microbiological samples must be in sterile containers. ² Many cases of pseudo-infections and pseudo-outbreaks owing to the usage of non-sterile containers have been reported.^3–7 Where usage of glass vials cannot be prevented, stringent adherence to sterilisation practices is essential. Repeat sampling should be undertaken whenever in doubt, especially when the chain of aseptic precautions is not perfect.