On September 2010, a 21-days-old female baby with a two-day history of fever was admitted to Fray Bartolomé de las Casas (FBC) District Hospital. FBC is a rural low-transmission malaria endemic area (population 55,073 inhabitants) in Northern Guatemala. FBC was a high-risk area where malaria transmission has been greatly reduced over the last years due to a scale-up of control interventions. Long-lasting insecticide-treated nets (LLITNs) and the improvement of the case management system implemented from 2006, resulted in a dramatic reduction in malaria parasite incidence from 91/1,000 population in 2006 to 1.9/1,000 population in 2010. Since 2009, no case of P
has been reported in the FBC area (National Malaria Control Program, Guatemala; unpublished data). Malaria transmission is sustained in the population by the temporal succession of the main vectors, Anopheles albimanus
, Anopheles vestitipennis
and Anopheles darlingi
On admission physical examination of the baby revealed paleness, hepatosplenomegaly (1 cm), dusky skin appearance and desquamation, and a body temperature of 36.7°C which increased to 38.9°C in the next 24 hours. Neurological, respiratory and cardiac examinations were unremarkable. Main laboratory findings were anaemia (Hb 7.2 g/dL), severe thrombocytopaenia (45,000 platelets/μL on the 1st day of admission, 24,000 platelets/μL on the 2nd day of admission), and leucopaenia (white blood cells 3.5x103/μL) with relative lymphocytosis (66.8%). Yet, the newborn did not show any signs or symptoms of haemodynamic failure nor evidence of bleeding throughout admission or after discharge. Biochemical and microbiological examinations were not available in the hospital. Based on initial suspicion of neonatal sepsis, the baby was started on intravenous (IV) ampicillin along with IV fluids and antipyretics. The thick blood smear examination showed P. vivax infection (parasite density 2,079 asexual parasites/μL), and then she was started on anti-malarial treatment with oral chloroquine (CQ) (¼ tablet of CQ 150 mg base, daily for five days). The neonate was treated with CQ only, following national guidelines in Guatemala that do not recommend the administration of primaquine to infants less than six months. Clinical progress was adequate and the baby was discharged after treatment completion. National guidelines also recommend therapy with ¼ tablet of CQ 150 mg base every 21 days until the patient is six months of age, so the infant kept on with CQ prophylaxis until that age after treatment completion. Follow-up during household visits two and eight months after hospital discharge was also favourable.
The 23-year-old primigravid mother was a FBC permanent resident. She was enrolled at the first antenatal clinic (ANC) visit, at 16 weeks of gestational age, in a multi-centre descriptive study –PregVax study- aimed to determine the burden and impact of P. vivax infection in pregnancy. As part of the PregVax study, maternal peripheral blood was collected at each ANC visit and at delivery for active detection of Plasmodium infection by microscopy and molecular methods. Samples were also collected from cord, placental, and newborn’s peripheral blood by heel prick at delivery for parasitaemia determination by microscopy and molecular methods, and placental impression smear and biopsy were taken for microscopic and histological examination, respectively.
This is a retrospective congenital malaria case report that mainly builds on the hospital clinical data available during its management. Molecular analyses, which were available due to the mother’s participation in the main study and carried out after the detection of CM infection, allowed the confirmation of the malaria case.
Plasmodium infection was not detected in the mother, either actively or passively, during pregnancy, neither by microscopy nor by polymerase chain reaction (PCR) at any of the ANC at 16, 21 and 26 weeks of gestational age. Delivery was at term (40 weeks), and the birth weight was 2,839 g. At delivery, the mother reported fever within the last 24 hours. Physical examination showed an axillary temperature of 38°C, paleness, and laboratory tests showed thrombocytopaenia (46,000 platelets/μL) and haemoglobin of 12.4 g/dL. Plasmodium vivax asexual parasites (6,429 parasites/μL) were detected in peripheral blood (see Table ). She was treated with oral CQ (1,500 mg given over three days) and oral primaquine (15 mg daily for 14 days), following national guidelines. A follow-up home visit was conducted by the National Malaria Control Programme staff to assess treatment compliance and confirm parasite clearance.
Plasmodium vivax parasitaemia and molecular results from the different compartments and time points
A placental biopsy sample was collected at delivery from the maternal side of the placenta, kept at 4°C in neutral buffer formalin, processed for histological examination, and stained with haematoxylin and eosin as previously described [18
]. No evidence of parasites or malaria pigment deposition was found in the histological examination. Placental impression smears were not assessable due to lack of quality of the samples.
Immediately after birth, physical examination of the newborn revealed no clinical abnormalities. No Plasmodium infection was detected in the blood thick smear collected at delivery from the newborn. Examination of the cord blood thick smear showed P. vivax asexual parasites (50 parasites/μL). Cord blood parasitaemia determination is not routinely done at this health facility. In this case, cord blood parasitaemia determination was done later after birth at the study referral laboratory as part of the study objectives.
DNA extraction (Purelink TM Genomic DNA Kit – Invitrogen) was done on one whole blood-spot on filter paper from each of the following compartments: maternal peripheral blood at delivery, placental and cord blood, and neonate’s blood at the time of birth and during hospital admission. Plasmodium vivax
infection was detected by conventional PCR for specific P
merozoite surface protein 1 gene (Pvmsp1
) in all samples except in the newborn’s blood at the time of birth (Figure ) [19
]. Also, confirmation of the presence of P
and absence of P
infection was carried out on all samples by Real-Time PCR with a LightCycler® 480 system (Roche). Species-specific primers and probes were used as previously described by Veron et al
], selected from sequence of the small subunit of 18S rRNA, according to the following steps: pre-incubation at 95°C for 10 min; amplification at 95 °C for 10 sec, 50°C for 20 sec and 72°C for 5 sec for 50 cycles. All reactions were done in duplicate in a final volume of 20 μL.
Figure 1 Molecular detection of Plasmodium vivax in blood samples from mother and newborn by the amplification of Pvmsp1 gene. PCR products were separated by electrophoresis through a 2.2% agarose gel stained with ethidium bromide and flanked by a 100 bp DNA ladder (more ...)
microsatellites (MS) were amplified from P
positive samples as described in Karunaweera et al
]. Briefly, PCR amplifications were performed in a 10-μL reaction mixture containing 2 μL of DNA, 1x reaction buffer, 1.75 mM MgC12
, 200 μM of each deoxynucleotide triphosphate, 5 picomoles of each primer and 2.5U of FastStar Taq polymerase (Roche). To determine the amplicon length variation, PCR products were analyzed by CEQ 8000 Genetic Analysis System (Beckman Coulter) using CEQ 8000 software for fragment analysis. Identical allelic profiles were obtained at the seven MS loci tested (MS1:231 bp, MS2:210 bp, MS3:177 bp, MS7:139 bp, MS8:243 bp, MS10:222 bp, MS20:187 bp), confirming that the same genotype was present in all compartments.