Detection of Mycoplasma pneumoniae in hospitalized children with pneumonia in Laos

TL;DR

The study found Mycoplasma pneumoniae in three hospitalized children in Laos, highlighting the need for better diagnostic systems and treatment guidelines.

Contribution

The study is the first to report M. pneumoniae detection in children with pneumonia in Laos using real-time PCR.

Findings

M. pneumoniae was detected in three children with pneumonia in Laos.

Two of the infected children also had other pathogens detected.

The findings suggest a need for improved diagnostics and understanding of M. pneumoniae in Laos.

Abstract

Mycoplasma pneumoniae has been described worldwide as an important cause of community-acquired pneumonia. From December 2013 to December 2014, 461 children admitted to Mahosot Hospital, Vientiane, Laos, with acute respiratory infection were investigated for upper respiratory microorganisms using probe-based real-time polymerase chain reaction (PCR) (FTD33).
M. pneumoniae was detected by FTD33 in the upper respiratory tract of three patients, two girls and one boy, 5.7 and 3.9 years old and 13.6 years old, respectively. They presented with clinical features compatible with
M. pneumoniae infection. The two girls were also positive for other potential pathogens. The boy had abnormal pulmonary auscultation, and one of the girls had significant anaemia. These results suggest that enhancement of diagnostic systems for
M. pneumoniae detection and analysis of its antibiotic resistance profile is needed to raise awareness and improve understanding of the epidemiology of
M. pneumoniae infection in Laos, enable targeted therapy, and inform treatment guidelines.

Tables

Table 1.. Characteristics of the three patients found PCR positive for Mycoplasma pneumoniae in upper respiratory tract.All three patients were of Lao loum ethnicity, all had documented fever and cough at enrolment, but none reported difficulty breathing, diarrhoea, chest indrawing, rash, cyanosis, respiratory distress, wheeze, stridor, nasal flaring, grunting, convulsion, conjunctival suffusion, lymphadenopathy, inability to drink, prostration or lethargy. None had known comorbidities at the time of presentation, or clinical evidence of pneumonia (WHO criteria 11 ), none required ICU admission and all were discharged alive with full recovery after two weeks.

	M. pneumoniae patients
Patient ID	64	70	396
Admission date	Feb 2014	Feb 2014	Sept 2014
Age (years)	5.7	13.6	3.9
Gender	female	male	female
Birth weight (g)	2,500	2,800	2,800
Ward	General Paediatrics	Infectious Disease Paediatric	Infectious Disease Paediatric
Clinical presentation
Duration of illness prior to hospitalization (days)	2	6	4
Rigors	no	no	yes
Sputum	no	yes	no
Runny nose	no	yes	yes
Sore throat	no	yes	yes
Vomiting	no	yes	yes
Nausea	unknown	yes	no
Physical examination
Abnormal pulmonary auscultation	no	yes	no
Oxygen saturation in room air (%)	99	90	unknown
Respiratory rate (breaths/min)	33	28	25
Duration of hospitalisation (days)	4	5	4
Other organisms detected by FTD33
Influenza B virus	yes	no	yes
Human cytomegalovirus	no	no	yes
Staphylococcus aureus	no	no	yes
Streptococcus pneumoniae	yes	no	yes
Moraxella catarrhalis	yes	no	yes
CBC Test of periperhal blood
White blood cells (10 3 cells/mm 3)	7.10	3.32	8.92
Lymphocytes (10 3 cells/mm 3)	1.04	1.55	1.98
Monocytes (10 3 cells/mm 3)	0.50	0.18	0.26
Granulocytes (10 3 cells/mm 3)	5.55	1.59	6.67
Lymphocytes (%)	14.7	46.8	22.2
Monocytes (%)	7.1	5.3	3
Granulocytes (%)	78.2	47.9	74.8
Hemoglobin (g/dL)	4.2	14.2	9.9
Mean corpuscular volume (fL)	59	83	79
Platelet count (10 3 cells/mm 3)	843	112	151
Treatment
	Paracetamol 250mg pro re nata	Paracetamol 500 mg pro re nata	Paracetamol 120mg x4 times/day
	Folic acid 5mg 1 time per day for 30 days	IV fluid	IV fluid
	Blood transfusion 2 times during admission (21 & 24 Feb 2014)	Vitamin C 500mg x3 times/day	Cefixime 50mg x 2 times/day, 1 day in hospital and continue at home
		Penicillin G 400,000 UI x 3 times/day for 5 days in hospital and continue for 5 days at home

Full text

Introduction

Mycoplasma pneumoniae, the smallest self-replicating microorganism, is associated with both upper and lower respiratory tract infections, most commonly tracheobronchitis, and diverse other clinical manifestations, especially of the skin and central nervous system. M. pneumoniae infection, which occurs worldwide with both endemic and epidemic patterns, may have been responsible for up to 20–40% of community-acquired pneumonia before the coronavirus disease 2019 (COVID-19) pandemic, especially in children aged 5–15 years old ^ 1, 2 ^. However, limited surveillance data hinder estimation of the true burden of M. pneumoniae infection. Similarities in presentation with other respiratory pathogens, and the absence of reliable point-of-care diagnostic tests, make it likely that the incidence of this infection is underestimated. For reasons that are not understood, M. pneumoniae appears to be more common in Asia than elsewhere. As far as is known M. pneumoniae only infects humans ^ 3– 5 ^. Diagnosis is important as conventional treatment of respiratory tract infections with β-lactam antibiotics will not be efficacious for M. pneumoniae, of which there is little awareness in Laos and adjoining countries.

Little is known about the diversity of etiologies of acute respiratory infection (ARI) in Laos. Few studies have been conducted, mainly on viruses ^ 6– 8 ^; M. pneumoniae has been reported in only one study, detected in 12 people with influenza-like illness from a community cohort study ^ 9 ^. We describe here three patients positive for M. pneumoniae, to raise awareness of the occurrence of this pathogen in Laos and the importance of including appropriate antimicrobial therapy in children with ARI.

Methods

From December 2013 to December 2014 we investigated the etiologies of ARI in hospitalized children at Mahosot Hospital, Vientiane ^ 10 ^ (Dubot-Pérès et al. unpublished). This is a ≈400-bed hospital providing primary, secondary, and tertiary care and receiving ≈2,000 inpatients/month.

Children aged <15-years-old admitted to paediatric wards with a clinical presentation compatible with ARI were included as previously described ^ 10 ^. Demographic, medical history, clinical and environmental data were collected by research physicians using a questionnaire, by interviews, physical examination and consulting medical charts (Dubot-Pérès et al. unpublished).

Written informed consent from the legal guardians of all patients was obtained before recruitment to the study. Ethical clearance was granted by the National Ethics Committee for Health Research, Ministry of Health, Vientiane, Laos, and the Oxford University Tropical Ethics Research Committee (Oxford, UK).

Nasal and throat swab specimens were collected from included patients using Sigma Virocult® (Medical Wire & Equipment), then 100 µL of each were pooled, extracted and tested by real-time polymerase chain reaction (RT-PCR) for 33 pathogens using the FTD® respiratory pathogens 33 kit (FTD33, Fast-track Diagnostics), as previously described ^ 10 ^. A PCR assay was considered as positive if the Cq value was <35. FTD33 targets the Adhesin P1 gene of M. pneumoniae and validation by the manufacturer showed a detection limit of 100 copies/mL of plasmid and no reaction with M. pneumoniae-negative samples by real-time PCR reference methods (communication from Fast-track Diagnostics). No cross-reaction was observed when clinical samples containing 66 different bacterial, viral or parasitic pathogens were tested with FTD33 (communication from Fast-track Diagnostics).

Results

From December 2013 to December 2014, of 472 patients eligible and consenting to the study, 461 hospitalized with an ARI presentation were included (11 patients had missing clinical or laboratory data). One or multiple potential pathogens were detected in respiratory specimens of 447/461 (97.0%) patients (Dubot-Pérès et al. unpublished) M. pneumoniae was detected in three patients, two girls and one boy, 5.7, 3.9 and 13.6-years-old, respectively ( Table 1). All lived in Vientiane City and all had fever and cough. One patient had low haemoglobin (4.2g/dL), low haematocrit (13.3%) and high platelet count (843×10 ^3^/mm ^3^) on presentation, without other respiratory or gastroenteric signs or symptoms. The two other patients presented with runny nose, sore throat, and vomiting, in addition to cough. The boy had abnormal pulmonary auscultation on presentation and was hypoxic (O 2 saturation of 90% on room air). None of these three patients had chest radiography performed.

**Table 1.: Characteristics of the three patients found PCR positive for Mycoplasma pneumoniae in upper respiratory tract.All three patients were of Lao loum ethnicity, all had documented fever and cough at enrolment, but none reported difficulty breathing, diarrhoea, chest indrawing, rash, cyanosis, respiratory distress, wheeze, stridor, nasal flaring, grunting, convulsion, conjunctival suffusion, lymphadenopathy, inability to drink, prostration or lethargy. None had known comorbidities at the time of presentation, or clinical evidence of pneumonia (WHO criteria

11 ), none required ICU admission and all were discharged alive with full recovery after two weeks.**

For the patient with abnormal pulmonary auscultation, M. pneumoniae was the only one of the 33 potential pathogens detected in the upper respiratory tract. Influenza B virus, Streptococcus pneumoniae and Moraxella catarrhalis were also detected in the two other patients. In addition, human cytomegalovirus and Staphylococcus aureus were also detected from one patient. Serology, culture and cold-aggultination assays were not available.

All patients received paracetamol to reduce fever. The patient with low haematocrit received a two unit blood transfusion, whereas the two other patients received penicillin G and cefixime, respectively, during hospitalization. None received a macrolide, tetracycline or fluoroquinolone. After 4–5 days of hospitalization, all three patients improved and were discharged and 2 weeks later the three patients had fully recovered.

Discussion

We describe three Lao children with M. pneumoniae infection, a pathogen that was first detected in Laos in 2019 ^ 9 ^. There is little awareness of the diagnosis, clinical features and management of this pathogen in Laos, as with the other causes of ‘atypical’ pneumonia, such as Chlamydia pneumoniae and Legionella pneumophila, that have not yet been described in the country. In the USA, the proportion of community-acquired pneumonia caused by M. pneumoniae increased following the introduction of pneumococcal vaccination ^ 3– 5 ^. The roll-out of pneumococcal vaccination in Laos since 2013 ^ 12 ^ therefore suggests that M. pneumoniae is a respiratory risk that warrants attention. However, there has been a very striking reduction in M. pneumoniae incidence in many countries during and since the COVID pandemic with as yet no evidence of a resurgence ^ 13 ^.

Attributing causality of M. pneumoniae infection to disease is complicated, as with so many other respiratory pathogens, as a result of its occurrence in asymptomatic people. The three patients described here all had illnesses consistent with the described clinical features of M. pneumoniae ^ 3, 5 ^, but two were also PCR-positive for other potential respirarory pathogens. In addition, one of the girls had significant anaemia, raising the possibility of M. pneumoniae cold-agglutinin associated haemolytic anaemia, although other potential causes of anaemia were not investigated and cannot be excluded ^ 5 ^. M. pneumoniae has been described from adjoining Cambodia, Vietnam and Thailand, but not, as far as we are aware, from Myanmar/Burma ^ 14, 15 ^. Further investigation of the importance of M. pneumoniae infection in Laos, and elsewhere where diagnostic services need reinforcing, is important for three key reasons. First, only one hospital in Laos has diagnostic facilities for M. pneumoniae, and these are not generally available for routine diagnosis in children with respiratory infection. Second, lacking a cell well, M. pneumoniae is not susceptible to β-lactam antibiotics that would most commonly be used for respiratory tract infections ^ 11 ^. Macrolides, tetracyclines and fluoroquinolones are most commonly used to treat M. pneumoniae infection but with a poor evidence-base for efficacy ^ 3, 4 ^. More information is needed to understand which patients should receive empirical macrolide therapy in addition to β-lactam antibiotics. The situation is analogous to the treatment of central nervous system infections in Laos and elsewhere in rural Asia, to cover not just the ‘conventional’ bacteria but also the common causes that are regarded wrongly as ‘atypical’, such as scrub typhus and murine typhus, that require non-β-lactam antibiotics for effective therapy ^ 16, 17 ^. The revised 2020 Lao Pediatric Antimicrobial Prescribing Guidelines recomend oral amoxicillin and intravenous ampicillin as first line therapy for pneumonia not needing and requiring hospital admission, respectively, with option for oral switch to azithromycin, erythromycin or clarithromycin ^ 18 ^ When to add these anti- Mycoplasma antibiotics is difficult to judge given the paucity of local data and the fact that all three children described here recovered without receiving such agents. As far as we are aware other causes of ‘atypical pneumona’, Chlamydophila pneumoniae and Legionella pneumophila, have not yet been described in Laos, giving an inadequate evidence base for when antibiotics for these pathogens should be recommended.

Third, macrolide-resistant M. pneumoniae has become increasingly prevalent worldwide, probably owing to the widespread use of this antibiotic class. Resistance frequencies in Asia are rising with reports that they represent the majority of isolates in some countries ^ 4, 19 ^. Mutations in domain V of the M. pneumoniae 23S rRNA is the major mechanism of macrolide resistance and should be monitored, especially given the difficulties with in vitro susceptibility testing of M. pneumoniae ^ 4, 19 ^. Isolates of M. pneumoniae with reduced susceptibility to tetracyclines and with a variety of mutations in 16S rRNA have been described after serial passages in subinhibitory concentrations of doxycycline in the laboratory ^ 20 ^. There are no data on the antibiotic resistance of M. pneumoniae from Laos in the public domain ^ 21 ^.

Hence, enhanced awareness and routine diagnostic systems for M. pneumoniae detection in Laos, and research into the local prevalence of antibiotic resistence in this pathogen, are needed, both for individual patient care and surveillance to understand the epidemiology of M. pneumoniae infection and to inform treatment guidelines.